Star Team

MMIC dPBL 2 overview of case

2008-01-23T10:32:00.007+08:00

Image: http://www.yahoo.com/ > Image> Indonesia

Indonesia [1] , in southeastern Asia, has a strategic location along major sea-lanes that connect the Indian and Pacific Oceans. The majority of the Indonesia’s islands consist of rugged volcanic mountains covered by dense tropical forests; however, some of the islands have swamps and plains. Indonesia is one of the most volcanically active regions on earth, with more than 100 active volcanoes. The region is subject to frequent earthquakes. The climate is tropical with an average daily temperature range of 21.1°C to 32.2° C with 80 to 90 percent humidity in the lowlands. Temperatures and humidity are lower as elevation increases, and temperatures below freezing can occur at elevations over 9,800 feet. Indonesia’s wet season occurs from November to March, and annual rainfall averages 40-78 inches.

Routes of transmission and entry of microorganism into host

Due to the humid and warm climate which is experienced in Indonesia all year round, bacterial, viral, protozoal and fungi infection can occur frequently in military soldiers.
Some pathogens are transmitted into huamns from wild animals and birds in the jungle. In the case of fungi infections, the infectious spores are carried from the soil to humans by air-borne routes. Food-borne diseases due contaminated water and food, un-cook food can also cause infections in these personnel. In addition, respiratory diseases and vector-borne diseases can also cause illness in these soldiers. Dirty living conditions such as not changing soiled clothes, not cleaning and airing of the feet frequently can also lead to microorganism infections. Other possible routes of transmission include aniaml bites (in the case of rabies), animal excretion, contaminated medications or fomites. [2]

References:
1) indonesia/">http://www.wikipedia.com>Indonesia
2) Jensen M. M. and Wright D. N. (1993) Introduction to Micobiology for Health Sciences. Prentices-Hall Inc., new Jersey

Bacteria

Presence of bacteria is extremely high in unhygienic conditions. Individuals infected by bacteria will present symptoms that may be infectious to other uninfected individual thus spreading the infection. [1] The soldiers having their army stint in Indonesia are exposed to numerous bacteria e.g. Shigella species, Salmonella species, E. coli, Campylobacter species as hygiene conditions are often overlooked. In addition, they are exposed to more bacteria during their jungle warfare training as soil, streams, rivers, animal feces, uncooked food which contain abundant amounts of bacteria. [1]

Food borne bacteria
In the jungle, military personnel have to prepare food themselves, thus the chances of consuming un-cook or contaminated food is relatively high. Therefore, they are at risk of be infected with various bacteria that are found on food when not cooked or cleaned properly. [5, 6]

Result from undercooking of food especially meat, organisms that are common include:
Campylobacter jejuni
Salmonella spp
Shigella spp
Listeria monocytogenes
Clostridium perfringens
Clostridium botulinium

Symptoms include:
Nausea
Vomitting
Abdominal cramping
Diarrhea
Fever

Shigella [3]

Transmission: Most individuals are infected with shigellae when they ingest food or water contaminated with human fecal material. Spread is always from a human source.

Causes: Shigellosis

Signs and symptoms: Fever, abdominal cramping, diarrhea, dehydration, tenesmus (straining of stool)

Salmonella species [2]

Transmission: Salmonella is spread from the carcasses to humans when the poultry is eaten undercooked. Other sources of Salmonellae include milk products, food, and water contaminated with animal feces or urine.

Causes: Salmonellosis, enteritis, Typhoid fever, primary septicemia

Signs and symptoms: Nausea, vomiting, abdominal pain, diarrhea, fever, weight loss, anemia, anorexia, headache.

Campylobacter [1]

Transmission: Campylobacter is usually acquired from food (especially poultry), milk, or contact with infected animals (e.g. dogs) through the fecal-oral route.

Causes: Acute enteritis

Signs and symptoms: Bloody diarrhea, fever, abdominal pain, headache.

Animal vector bacteria [1,3]
As the soldiers are training in the jungle, they will encounter many different forms of wildlife. Bacteria carried by them may be transmitted to the soldiers. Common ones include:

Brucella spp (contact with infected animals e.g. dogs, pigs, cattle, horse)
Francisella sp. (contact with wild animals that carry ticks, mites and lice)
Yersinia spp (contact with rodents that carry fleas that carry the bacteria)
Pasteurella multocida (cat and dog bites)

Soil vector bacteria [2, 3]
In addition to the exposure to various fauna and flora living in the jungle, soldiers may also come in contact with soil. Soil is a place where many bacteria flourished thus they chances of getting an infection casued by a soil organism is also relatively possible

Clostrodium tetani [2]

Transmission: Soils or materials in contact with animal wastes are usually heavily contaminated with C. tetani and offer excellent sources of infections. Soil-contaminated wounds are most frequent source of infection.

Causes: Tetanus

Signs and symptoms: Spasmodic contractions (tetany) in involved muscles that are affected by the toxin and muscle spasms.

Mode of control: Vaccines

References

1. Levinson, W. (2006). Review of Medical Microbiology & Immunology. San Francisco, California: Lange Medical Books/McGraw-Hill publishing company

2. Elliott T., Hasting M. and Desselberger U. (1997). Lecture Notes on Medical Microbiology. Blackwell Science, Oxford

3. Walker, T.S. (1998). Microbiology. Philadelphia, Pennsylvannia: W.B Saunders Company

4. http://aggie-horticulture.tamu.edu/ > Search > food poisoning

5. http://www.medicinenet.com/ > Search > food poisoning

Done by: Star team

Virus

2008-01-23T10:32:00.006+08:00

Virus is a sub-microscopic infectious agent requires a host to outside cell. [1] Each viral particle, or virion, consists of genetic material, DNA or RNA, within a protective protein coat called a capsid. [1] The capsid shape varies from simple helical and icosahedral (polyhedral or near-spherical) forms, to more complex structures with tails or an envelope.

When a virus infect a host cell, several changes occurs. [1] For example,
- death of cell due to inhibition of macromolecule synthesis
- fusion of cells to form multinucleated cells
- malignant transformation
- no morphological/functional changes

Pathogenesis in the infected patient involves [1]
- transmission of virus and its entry into host
- replication of virus (viral proteins) and damage to cells (lysis)
- immune responses against virus
- persistence of virus (unable to be remove from the body even after immune responses has been mount)

Virus can be transmitted from person-person or animal-person. [2] Person-person transmission can be due to exposure to respiratory secretion, saliva, blood (transfusion, sharing of needles), semen, fecal contamination of water or food. [1] This is called horizontal transmission. Person-person transmission can also be from maternal to fetal/child across the placenta or during breast-feeding. This is called vertical transmission. Animal-human transmission occurs directly when infected animal is exposed to human while indirect transmission occurs through the bite of an insect vector e.g. mosquito. [1]

Viral infections in human and animal hosts usually result in an immune response and disease.[5] Viruses are usually eliminated by immune responses in the body. Antibiotics have no effect on viruses, but antiviral drugs have been developed to treat life-threatening infections. Vaccines that produce lifelong immunity can prevent virus infections e.g. poliomyelitis.

Here are some of the viruses that have have shortlisted

Parainfluenza virus [2, 4]
Epidemiology: Parainfluenza 1, 2, 3 & 4, with Parainfluenza 1& 2 being commonly during autumn and Parainfluenza 3 infections occur throughout the year.

Transmission: Inhalation of large respiratory droplets, person-person contact. Re-infection occurs throughout life as body’s immunity to it is short.

Signs and symptoms: cold-like syndrome, croup, cough, chest pain

Causes: bronchiolitis, bronchitis, upper respiratory tract infections and pneumonia.

Modes of control: No vaccine is yet available for routine use

http://www.yahoo.com/ > Image > Parainfluenza virus

Influenza A virus [2, 4, 8]
Influenza A virus infects multiple species. Wild aquatic birds are the nature host to a variety of influenza A viruses. The virus is then transmitted to other species and causing devastating outbreaks in domestic poultry or give rise to human influenza infections. There are several types of influenza A virus:

· H1N1 caused "Spanish Flu."
· H2N2 caused "Asian Flu."
· H3N2 caused "Hong Kong Flu."
· H5N1 is a pandemic threat in 2007–8 flu season; Avian Flu
· H7N7 has unusual zoonotic potential.
· H1N2 is endemic in humans and pigs.
· H9N2, H7N2, H7N3, H10N7

Avian Flu [2, 4, 8, 9]
Epidemiology: Bird populations especially in Southeast Asia

Transmission: Avian Flu caused by H5N1 virus infects bird populations. The infected birds then spread its infection through saliva, nasal secretion, blood and droppings to other animals and humans with extensive physical contact e.g. direct contact with these bodily fluids or through contact with surfaces contaminated with them

Causes: Avian Flu

Signs and symptoms: Fever, cough, sore throat, pneumonia, breathing difficulties and sometimes diarrhea.

Modes of control: H5N1 Vaccines but they are not always effective due to continuous mutation of the virus, culling of infected bird population, protection used when dealing with droppings.

http://www.yahoo.com/ > Image > Avian Flu Virus

Influenzavirus B [2, 4, 8]
Epidemiology: Worldwide occurrence: epidemics are local and pandemics are worldwide

Transmission: Spread by inhalation of small aerosol droplets expelled for infected individual, saliva, blood, feces, nasal secretion.

Causes: Pneumonia, lower respiratory infection, acute influenza infection in adults and complications influenza virus infections.

Signs and symptoms: Fever, sore throat, muscle aches, coughing, nausea, vomiting

Modes of control: amanatdine, rimantadine etc, killed vacinne contains predicted yearly strains of influenza A & B virus

SARS coronavirus [6, 8]
Epidemiology: Most number of SARS cases occurred in People’s Republic of China, Hong Kong, Taiwan, Singapore and Canada with 7%, 17%, 14% & 17% fatality respectively

Transmission: Spread mainly by close person-to-person contact. When an infected person coughs or sneezes, droplets of mucus or saliva that contain the virus are sent through the air. Once these droplets land on the mouth, enter the nose or eyes, an infection can occur. However, the airborne spread of SARS does not seem to be a major route of transmission. In addition, kissing, touching, sharing utensils for eating and drinking, or talking with an infected person are also a risk factor for infections. The presence of virus in the stool also suggests the possibility of oral-fecal transmission

Causes: Severe acute respiratory syndrome (SARS)

Signs and symptoms: Fever, Chills, muscle soreness, headache and a general feeling of discomfort also are common. Severe pneumonia, leading to an insufficient amount of oxygen in the blood (hypoxemia) can also occur

Modes of control: Wash your hands frequently with soap and hot water, Cover your mouth and nose when sneezing and coughing. Use soap and hot water to wash the utensils, towels, bedding and clothing of someone with SARS. Don't use these items yourself until they're clean.

http://www.yahoo.com/ > Image> SARS coronavirus

Alphavirus [2, 4, 5]

Chikungunya Virus (It is a virus under the category of alphavirus)
Epidemiology: The disease was first recognized in Eastern Africa in the 1950s. Outbreaks were also reported in Malaysia, India, Sri Lanka and Indonesia in 2006. In 2008, 6 infections caused by chikungunya virus were reported in Singapore.

Transmission: Chikungunya is spread by the bite of an infected mosquito. The main vector is mosquito Aedes aegypti. Other mosquito likes Aedes albopictus and Culex can also transmit the disease. Mosquito becomes infected when they feed on a person infected with chikungunya. Monkeys and other wild animals may also serve as a reservoir of the virus. Infected with mosquito can then spread the virus to other humans when they bite. There is no direct person to person spread.

Causes: Chikungunya fever

Signs and symptoms: Chills, fever, headache, vomiting, fatigue, nausea, rash and muscle and joint pain, some patients may also develop a rash affecting the trunk

Modes of control: The most effective means of prevention are those that protect against any contact with the disease-carrying mosquitos. These include the usage of insect repellents with substances like DEET (Diethyl-3-methylbenzamide), icaridin (also known as picaridin and KBR3023), PMD (p-menthane-3, 8-diol, a substance derived from the lemon eucalyptus tree), or IR3535. Wearing bite-proof long sleeves and trousers also offers protection. In addition, garments can be treated with pyrethroids, a class of insecticides that often has repellent properties. Vaporized pyrethroids (for example in mosquito coils) also have a certain special repellency.

Of all the alphavirus, Chikungunya virus is the only virus in this category that is distributed in South East Asia (Indonesia), thus it is the most probably virus from this category to infect soldiers who are training in the forest of Indonesia

http://www.yahoo.com/ > Image > Chikungunya Virus

Flavivirus [2, 4, 5]

Epidemiology: Aedes mosquito, which carries dengue and yellow fever, is found in urban areas, pools of water and jungles while Culex mosquito is found in the forest, urban areas. Disease is more common in summer.

Transmission:

Signs and symptoms: Flu like symptoms (chills, fever, rash, aches), dengue yellow fever

Mode of control: Mosquito breeding sites should be eliminated. Live attenutaed vaccines are available for yellow fever virus

Adenovirus [2, 4, 10]
Epidemiology: Worldwide endemic, frequent observations in transplant patients and military recruits.

Transmission: Adenovirus can be human-to-human transmission mainly by fecal-oral or respiratory route, by fingers, by fomites (including towels and medical instruments). In addition, close interaction among people such as military barracks promotes the spread of the virus

Causes: Adenoviral pneumonia, due to adenovirus serotype 4 & 7, is a complication of acute respiratory diseases in military recruits, conjuctivitis

Signs and symptoms: Fever, cough, pharynitis
Modes of control: Live vaccine for serotype 4 and 7 are avaliable for military use

http://www.yahoo.com/ > Image > Adenovirus

Enterovirus [2, 3, 5]
Polioviruses (causes poliomyelitis)
Coxsackieviruses (A and B) (causes hand-foot-mouth disease)
Echoviruses
Enteroviruses e.g. Enterovirus 70

Epidemiology: Worldwide distribution except for poliovirus which has been eradicated, only a few random cases occurs.
Polioviruses – humans, ape, monkeys
Coxsackieviruses – human, most common in summer and autumn
Echoviruses – human
Enteroviruses - human

Infections most common in early childhood but adults get infected when their bodies do not possess the immunity against the enterovirus invading.

Transmission:
Transmitted by oral-fecal route, poor hygiene, inhalation of aerosol, ingestion via contaminated food and water, contact with infected hands and fomites, poor sanitation, crowded living conditions

Causes:
Polioviruses - poliomyelitis
Coxsackieviruses (A and B), Echoviruses and Enteroviruses - causes hand-foot-mouth disease, herpangina,aseptic meningitis, myocarditis and can cause serious disease especially in infants and the immunocompressed.
Enterovirus 71 causes respiratory infection.

Signs and symptoms: Rash disease, respiratory diseases, undiferentiated fever

Modes of control: Practice good hygiene, improve living condition

Poliovirus is an extinct disease due to vaccination except in undeveloped countries, in addition, it is manifested since young, the army soldiers are unlikely to be infected by it.

Molluscum contagiosum [1, 3, 4]
Epidemiology: Worldwide distribution.

Transmission: Transmitted by direct contact with infected human e.g. sexual contact, wrestling, or sharing of fomites e.g. towel, bed, clothing.

Signs and symptoms: skin lesion most commonly on trunk, genitalia and proximal extremities.

Causes: DNA poxvirus Molluscum contagiosum virus. There are 4 types of MCV, MCV-1 to -4, with MCV-1 being the most prevalent and MCV-2 usually seen in adults.

Modes of control: Hygiene - no sharing of baths and articles of clothing (towels), no scratching of lesion, bandage bumps.

http://www.yahoo.com/ > Imgae > Molluscum contagiosum

Bunyaviridae [2, 4]

Epidemiology: Common during summer. Found in South America, Southeast Asia, southeastern Europe and Africa.

Transmission: Transmitted by infected mosqutio, or ticks to vertebrates. The animal then becomes the resvoirs for the virus which in turn infect human. Also, humans get infected when they enter the environment of insect vector.

Signs and symptoms: Febrile illness and rash, headache, vomitting

Causes: Hantavirus pulmonary syndrome, Hemorrhagic fever

Mode of control: Elimination of vector or vector’s habitat

http://www.yahoo.com/ > Image>Bunyavirus

http://www.wikipedia.com/ > Hantavirus > Image

Hepatitis [2, 4, 5, 11]

Hepatitis A and E [2, 4, 5, 11]
Epidemiology: Worldwide, most common in tropical and subtropical regions.

Transmission: Fecal-oral route, direct contact with contaminants e.g. uncooked food, water, contact with infected individual e.g. sharing of food, drinks.

Signs and symptoms: Fever, loss of appetite, nausea, headache, pain in abdomen

Causes: Hepatitis A, Hepatitis E

Mode of control: Vaccination, good hygiene, consume thoroughly cooked food and water.

Considering that the soldiers are not exposed to blood products or any transfusion process, Hepatitis B, C and D are not possible. The lack of clean food and water in the jungle could have caused Hepatitis A and/or E infection is the soldiers.

http://www.yahoo.com/ > Image> hepatitis C

Rabies [2, 12]
Epidemiology: Animal reservoir e.g. foxes, bats, racoons. Most commonly found in Asia, Africa and South America.

Transmission: Spread from animals especially wild animals to human, Inhalation of aerosolized virus, saliva bite by a rabid animal.

Signs and symptoms: Fever, malasie, itching at the site of the bite, fear of water (hydrophobia).

Causes: infection to central nervous system (especially brain)

Modes of control: Avoid contact with wild animals as they are not vaccinated, immediate washing of wound with soap, detergent or other substances which inactivate the virus, Instill anitrabies serum with the adminstration of huamn rabies immunoglobulin (HRIG)

http://www.yahoo.com/ > Image > Rabies virus

Rhinovirus [2, 3, 4]
Epidemiology: occurs worldwide with high prevalence during winter

Transmission: crowding and close contact favors transmission via respiratory route.

Causes: chronic bronchitis, sinusitis

Sign and symptoms: coryza (inflammation of nasal membrane), fever (however not common in adults)

There is likelihood for soldiers to be infected with rhinovirus as it is transmitted by crowding and close contact, all these are common occurrence in the barrack.

http://www.yahoo.com/ > Image > rhinovirus

Human Immunodeficiency Virus (HIV) [2, 5]
Epidemiology: occurs worldwide

Transmission: Sexual contact, blood, maternal to fetal, breast milk.

Causes: HIV, other opportunistic infections as HIV-infected individual are immunocompromised

Sign and symptoms: flu-like symptoms after 2 weeks of manifestation

Assuming the soldiers visited the neighboring villages and had sexual contact/blood contact with an infected individual, there is a high chance that they are infected with HIV. However, if the soldiers had not venture out of their barrack and the jungle, the likelihood of them infected with HIV is very low as an HIV-infected individual would have been detected during their military health screening.

http://www.yahoo.com/ > Image > aids HIV virus

Norovirus [2, 4, 5]
Epidemiology: Worldwide, all ages

Transmission: faecally contaminated food or water, person-to-person transmission

Causes: stomach flu, gastroenteritis

Sign and symptoms: nausea, vomiting, diarrhea, weakness, fever.

Virus can be transmitted when contaminated food and water has been consumed, the likelihood increases when food/water is not thoroughly cooked. In army, due to large amount of food required to prepare and the lack of fire source during jungle warfare, food/water may not be thoroughly cooked.

http://www.yahoo.com/ > Image> Norovirus

Astrovirus [2, 3, 5]
The genus "Astrovirus" within the familiy of Astroviridae is divided into eight human species:
- human astrovirus 1 (HAstV-1) - human astrovirus 2 (HAstV-2) - human astrovirus 3 (HAstV-3) - human astrovirus 4 (HAstV-4) - human astrovirus 5 (HAstV-5) - human astrovirus 6 (HAstV-6) - human astrovirus 7 (HAstV-7) - human astrovirus 8 (HAstV-8)

Epidemiology: All ages especially the young and elderly. Adults in military barracks are most likely to develop the disease due to poor hygiene control.Worldwide endemic, common in tropical with rainy season

Transmission: contaminated food and water

Causes: gastroenteritis

Sign and symptoms: Diarrhea, vomiting, pain in stomach, dehydration

Virus can be transmitted when contaminated food and water has been consumed, the likelihood increases when food/water is not thoroughly cooked. In army, due to large amount of food required to prepare and the lack of fire source during jungle warfare, food/water may not be thoroughly cooked.

Filovirus [2]
There are two genera: the Ebola virus and Marburg virus. These viruses cause viral hemorrhagic fevers, characterized by bleeding and coagulation abnormalities, often leading to death. Natural reservoir of both the Marburg virus and the Ebola virus appears to be zoonotic. Infected individual symptoms may be mistaken for influenza, malaria, typhoid fever.

Marburg Virus [2]
Epidemiology: Africa, commonly transmitted by bats especially fruit bats

Transmission: disease is spread through bodily fluids, including blood, excrement, saliva, and vomit

Causes: Marburg haemorrhagic fever.

Sign and symptoms: Early stage: fever, headache and myalgia maculopapular rash present on the trunk. Later-stage: is acute and causes jaundice, pancreatitis, weight loss, delirium and neuropsychiatric symptoms, haemorrhaging, hypovolemic shock and multi-organ dysfunction, with liver failure.

Ebola Virus [2]
Epidemiology: Africa, commonly transmitted by animals e.g. monkeys, bats especially fruit bats

Transmission: airborne, from infected animals to human, human to human transmission is rare.

Causes: Ebola haemorrhagic fever.

Sign and symptoms: Early stage: high fever, severe headache, muscle, joint, or abdominal pain, severe weakness and exhaustion, sore throat, nausea, and dizziness. Later-stage: diarrhea, dark or bloody feces, vomiting blood, red eyes due to distention and hemorrhage of sclerotic arterioles, petechia, maculopapular rash, and purpura.

During jungle warfare, the soldiers are exposed to wide variety of wild animals e.g. monkey and bats. The animals may be infected with the virus and transmit it to the soldiers thus infecting them. Therefore, the occurrence of filovirus infection is rather high.

References

1. Levinson, W. (2006). Review of Medical Microbiology & Immunology. San Francisco, California: Lange Medical Books/McGraw-Hill publishing company

2. Murray, P.R. (2002). Medical Microbiology. St. Louis, Missouri: Mosby Inc.
3. Elliott T., Hasting M. and Desselberger U. (1997). Lecture Notes on Medical Microbiology. Blackwell Science,Oxford

4. Walker, T.S. (1998). Microbiology. Philadelphia, Pennsylvannia: W.B Saunders Company

5. Brook G. F., Butel J. S. and Morse S. A. (1998). Jawetz, Melnick & Adelberg’s Medical Microbiology. Prentice Hall International Inc., United States of America

6. http://www.mayoclinic.com/ > search> SARS
7. http://www.sarsreference.com/

8. http://www.wikipedia.org/ > search influenza
9. http://www.wikipedia.org/ > search > avian flu
10. http://www.wikipedia.org/ > search > adenovirus
11. http://www.wikipedia.org/ > search hepatitis

12. http://www.wikipedia.org/ > search > rabies

Done by:
Eugene and Phuiyuen, TG02

Fungi

2008-01-23T10:32:00.003+08:00

Introduction
Fungi are eukaryotic cells that lack chlorophyll. They have cell walls and filamentous structures and produce spores, or conidia. These organisms grow as saprophytes that obtain nutrients from dead organic matter. Most of the fungi that cause systemic infections may infect incidental hosts (i.e. humans and other animals) who are exposed to fungal elements or spores.

Systemic mycoses

Histoplasma capsulatum

Epidemiology
Dimorphic fungus (exists as mold in soil and yeast in tissue) that is found worldwide and in temperate, subtropical and tropical climates. Grows in soil and material contaminated with bat or bird droppings.

Causes
Histoplasmosis. Infection occurs resulting from inhalation of spores.

Transmission
Infection resulting from inhalation of spores of the fungi in their mold forms in soil. Within the lungs, the spores differentiate into yeasts.

Signs & symptoms
Fever
Headache
Malaise
Myalgia (muscle pain)
Abdominal pain
Chills

Mode of control
No means of prevention except avoiding exposure in areas of endemic infection.

Dermaphytes

Trichophyton species
Trichophyton ajelloi (geophilic)
Trichophyton concentricum (anthrophilic)
Trichophyton flavescens (geophilic)
Trichophyton gloriae (geophilic)
Trichophyton interdigitale (anthrophilic)
Trichophyton megnini (anthrophilic)
Trichophyton mentagrophytes (zoophilic, anthrophilic) (rodents)
Trichophyton phaseoliforme (geophilic)
Trichophyton rubrum (anthrophilic) (most common)
Trichophyton schoenleinii (anthrophilic)
Trichophyton simii (zoophilic) (monkey, chicken)
Trichophyton soudanese (anthrophilic)
Trichophyton terrestre (geophilic)
Trichophyton tonsurans (anthrophilic)
Trichophyton vanbreuseghemii (geophilic)
Trichophyton violaceum (anthrophilic)
Trichophyton yaoundei (anthrophilic)

Epidermophyton floccosum

Epidemiology
Found worldwide, more common in countries with hot and humid climates with crowding or debilitation

Cause
Dermatophytosis
Tinea corporis (ringworm)
Tinea cruris (jock itch)
Tinea pedis (athlete’s foot)
Tinea manuum (hand)
Tinea favosa (scalp)
Folliculitis (inflammation of hair follicles)
Tinea capitis (hair infection)
Onychomycosis (nail infection)
Invasive infection for immunocompromised host

Transmission
Direct or indirect contact with infected host (human or animal)
Direct or indirect contact with skin or hair
Geophilic species are acquired via contact with spores in the soil
Skin trauma (burns, cuts), high temperature and humidity and increase susceptibility to infection

Signs & symptoms
Itching, stinging and burning between the toes, on the soles
Excessive dryness of the skin on the bottom of the feet
Thick, crumbly, discoloured, brittle and ragged nails
Blisters
Hair loss
Scabs
Tinea of the nails

Mode of control
No means of prevention except avoiding exposure in areas of infection

Microsporum species
Microsporum audouinii (anthrophilic)
Microsporum canis (zoophilic) (cats and dogs)
Microsporum cooeki (geophilic, zoophilic) (fur from cats, dogs and rodents)
Microsporum ferrugineum (anthrophilic)
Microsporum gallinae (zoophilic) (chicken)
Microsporum gypseum (geophilic)
Microsporum nanum (geophilic, zoophilic) (pig)
Microsporum persicolor (zoophilic) (rodents)

Epidemiology
Found worldwide, more common in countries with hot and humid climates with crowding or debilitation

Cause
Dermatophytosis
Tinea corporis (ringworm)
Tinea cruris (jock itch)
Tinea pedis (athlete’s foot)
Tinea manuum (hand)
Tinea favosa (scalp)
Tinea capitis (scalp)
onychomycosis (rare)
Invasive infection for immunocompromised host
Note: Microsporum persicolor does not infect hair

Transmission
Direct or indirect contact with infected host (human or animal)
Direct or indirect contact with skin or hair
Geophilic species are acquired via contact with spores in the soil
Skin trauma (burns, cuts), high temperature and humidity and increase susceptibility to infection

Signs & symptoms
Itching, stinging and burning between the toes, on the soles
Excessive dryness of the skin on the bottom of the feet
Thick, crumbly, discoloured, brittle and ragged nails
Blisters
Hair loss
Scabs
Tinea of the nails

Mode of Control
No means of prevention except avoiding exposure in areas of infection

Malassezia furfur

Epidemiology
Worldwide, part of normal skin flora, infections likely to occur in hot and humid conditions

Cause
Pityriasis versicolor (skin rash)
Seborrheic dermatitis (flaky scalp, face, trunk)
Folliculitis (inflammation of hair follicles)
Blepharitis (inflammation of the eyelids)
Tinea Blanca (hair)
Severe systemic infections in immunocompromised host

Transmission
Normal skin flora, that causes infection when it becomes an opportunistic infection

Signs and Symptoms
Itching
Scaling
However sometimes the infection is asymptomatic

Sporothrix schenckii

Epidemiology
Worldwide, isolated from soil, living and decomposing plants, wood and moss

Cause
Sporotrichosis (Rose handler’s disease)
Invasive sinusitis
Meningitis
Osteoarticular infection
Bursal infection

Transmission
Infection by entry of infecting fungus through the skin via minor trauma (eg. cuts)

Signs & symptoms
Large ulcerations
Small painless skin bump on the arms, fingers or hand
Boil-like lesions
Firm skin nodules
Suppurative nodules

EXCLUSION OF FUNGI

1. Opportunistic mycoses
These fungi cause infections only in immunocompromised people.

Candida species
Candida albicans
Candida guilliermondii
Candida parapsilosis
Candida tropicalis

Epidemiology
This fungus is found worldwide. For some of the fungi, they are part of the normal microbial flora of the mucous membranes of the mouth and intestinal tract.

Cryptococcus species
Cryptococcus neoformans
Cryptococcus gattii

Epidemiology
Cryptococcus neoformans is found in soil, especially those enriched by pigeon droppings
Cryptococcus gattii can be found in eucalyptus trees and decaying wood forming hollows in living trees

Aspergillus species
Aspergillus fumigatus
Aspergillus niger
Aspergillus flavus
Aspergillus terreus
Aspergillus nidulans

Epidemiology
Fungus is ubiquitous in the environment, growing in soil and decaying vegetation. The transmission of fungus is by airborne conidia.

2. Coccidioides immitis

Epidemiology
This fungus is found in arid regions of USA and South America

3. Blastomyces dermatitidis

Epidemiology
This fungus is endemic in North & South America, Africa and in the Middle East.

References
Martin
http://www.emedicine.com > search > Cryptococcosis
http://www.emedicine.com > search > Histoplasmosis
http://www.doctorfungus.org > search > Cryptococcus
http://www.mycology.adelaide.edu.au > search > Candida
Levinson, W. (2006). Review of Medical Microbiology & Immunology. San Francisco, California: Lange Medical Books/McGraw-Hill publishing company.
Murray, P.R. (2002). Medical Microbiology. St. Louis, Missouri: Mosby Inc.
Virella, G. (1997). Microbiology and Infectious Diseases. Pennsylvania, USA: Williams & Wilkins.

Randall
http://www.doctorfungus.org > search > Trichophyton
http://www.wrongdiagnosis.com > T > tinea
http://www.mayoclinic.com/ > health > search > athlete’s foot
http://www.emedicine.com/aaem/topic277.htm
http://www.doctorfungus.org > search > Epidermophyton
http://www.doctorfungus.org > search> Microsporum
http://www.doctorfungus.org > search > Malassezia furfur
http://www.doctorfungus.org > search > Sporothrix schenckii
http://www.wrongdiagnosis.com > S > sporotrichosis
Walker, T.S. (1998). Microbiology. Philadelphia, Pennsylvannia: W.B Saunders Company
Levinson, W. (2006). Review of Medical Microbiology & Immunology. San Francisco, California: Lange Medical Books/McGraw-Hill publishing company.

Done by: Martin Ng & Randall Chua (TG02)

Protozoa

2008-01-23T10:32:00.002+08:00

Parasites
Parasites occur in two distinct forms: single celled protozoa and multi-cellular metazoan called helminthes. [1]

Helminthes
Metazoan are subdivided into two groups: Platyhelminthes and the Nemathelminthes.
The phylum Platyhelminthes contains two important classes: Cestodes and Trematoda. [1]

Cestodes are tapeworms. It consists of two main parts: a rounded head called the scolex which has specialized means of attaching to the intestinal wall and a flat body of multiple segments called proglottids. The oldest proglottids at the distal end produce many eggs which are excreted in the feces and transmitted to various intermediate hosts such as cattle, pigs and fish. Humans usually acquire the infection when undercooked flesh containing the larvae is ingested. [1,2]

Taenia solium: T. solium is found worldwide. Because pigs are intermediate hosts of the parasite, completion of the life cycle occurs in regions where humans live in close contact with pigs and eat undercooked pork. Taeniasis and cysticercosis are very rare in predominantly Muslim countries, as Islam forbids the consumption of pork.[1]

Taenia saginata: T. saginata, also known as Taeniarhynchus saginata or the Beef tapeworm, is a parasite of both cattle and humans, but which can only reproduce in humans. T. saginata occurs where cattle is raised, human feces is improperly disposed of, meat inspection programs are poor, and where meat is eaten without proper cooking.[1]

Echinococcus multilocularis: The definitive hosts are mainly foxes and the intermediate hosts are various rodents. Humans are infected by accidental ingestion of food contaminated with fox feces. [2]

Trematodes are transmitted to humans either via penetration of the skin by the free-swimming cercariae of the schistosomes or via ingestion of cyst in undercooked fish or crabs in Clonorchis and Paragonimus infection, respectively. [1]

Schistosoma japonicum: It causes schistosomiasis. It is found only in Asia and domestic animals such as water buffalos and pigs acts as reservoirs. It is common in tropical and subtropical climates. Most patients are asymptomatic, but chronic infections may become symptomatic. [1]

Nemathelminthes are also known as nematodes. They are commonly classified under: intestinal nematodes and tissue nematodes. Infections with nematodes are associated with conditions of poor hygiene. Such infections are extremely common throughout the tropics and subtropics. [1]

Ancylostoma duodenale and Necator americanus: Humans are infected when filariform larvae in moist soil penetrate the skin. They are found especially in tropical areas. [2]

Strongyloides stercoralis: They are found in the soil. It penetrates the skin and occurs primarily in the tropics, especially SEA.[1]

Wuchereria bancrofti : It occurs in tropical and subtropical areas and is endemic in central Africa, along the Mediterranean coast, and in many parts of Asia, including China, Korea, Japan and the Philippines. [1]

Protozoa

Protozoa are eukaryotic cells and have many of the intracellular components of characteristic of higher forms of life. Most protozoa have some form of active locomotion. Protozoa are found in soil, in most bodies of water, and in many higher forms of life. Relatively few protozoa are able to cause diseases in humans and most are beneficial contributors to the various biological cycles in nature.

Protozoa and other parasitic diseases are common in underdeveloped, tropical, and sub tropical area. The overall effects of these diseases on the general well-being of the inhabitants of these areas are of major importance.

The diagnosis, treatment, and control of protozoal diseases differ in some ways from those used for other microbial diseases. Because of the large size, distinct shapes of the protozoa and that protozoa are difficult to grow on media, direct microscopy is the routine method of diagnosis. The symptoms of many protozoal diseases are very general and often are not used solely as a basis for a specific diagnosis.
Control of protozoal disease is often difficult since human are not the primary host, effective vaccines are not currently available, anti- protozoal drugs are often toxic to the host and close contact between humans and the disease reservoir is fostered by poverty and inadequate education.

Some disease-producing protozoa have a life cycle that involves more than one type of host. The parasite goes through sexual reproduction in the definitive host, whereas asexual reproduction occurs in the intermediate human host. The cell types which are present in a host may be the growing trophozoite (damages tissues) and the dormant, resistant cyst (the form which survives outside a host perpetuating transmission by indirect means.

Reasons for inclusion:

Indonesia is a tropical country with high humidity. In places with high humidity, Food and water can be easily contaminated with fecal products of humans and animals. In the jungle, lakes and water bodies are common. Wild animals’ and human feces may contaminate water and food that the soldiers eat thus infecting them. Swimming in lakes and water bodies may also cause infections as some protozoa can be transmitted through mucosal membranes, trauma and ingestion. Usually in the army, personal hygiene is always overlooked, combined with the poor sanitation of the jungle, it can cause severe infection. During military training, there is also personal time. Soldiers may explore neighboring villages. The soldiers may get infected with diseases during sexual intercourse with women in neighboring villages. In the villages, they may also come into contact with domestic animals.

Entamoeba histolytica [1]

Epidemiology: It is a cosmopolitan intestinal parasite. Organism is acquired by ingestion of cysts that are transmitted primarily by the fecal-oral route in contaminated food and water. Anal- oral transmission, e.g., among male homosexuals, also occurs. There is no animal reservoir. Infection is found worldwide, but occur most frequently in tropical countries, especially in areas with poor sanitation. There are no animal reservoirs.

Causes: Amebic dysentery and liver abscess

Signs and Symptoms:
Asymptomatic infection is common.

Acute intestinal amebiasis: Dysentry (bloody, mucus-containing diarrhea) accompanied by lower abdominal discomfort, flatulence, and tenesmus

Chronic amebiasis: occasional diarrhea, weight loss, and fatigue. Granulomatous lesion called an ameboma may form in the cecal or rectosigmoid areas of the colon.

Amebic abscess (liver): right upper-quadrant pain, weight loss, fever, and a tender, enlarged liver. Right lobe abscesses can penetrate the diaphragm and cause lung disease. Aspiration of the liver abscess yields brownish-yellow pus with the consistency of anchovy-paste.

Mode of Control: Improving basic hygiene and sanitation practices is most effective way of controlling outbreaks of amebiasis as E. histolytica thrives in conditions where standards of sanitation and hygiene are poor. Avoid fecal contamination of food and water and observing good personal hygiene such as hand washing. Water supplies should be purified before drinking. Vegetables should be cooked as it may have been contaminated with human feces.

Giardia lamblia [1.2]

Epidemiology: This organism is a cosmopolitan parasite, common in temperate latitudes and in developed countries, as well as in the tropics. Transmission occurs by ingestion of the cyst in food and water contaminated by feces. The source of infection may be infected human carriers or wild animals that act as reservoirs of infection and contaminate water with the cysts passes in their feces. They pass cyst in the stool which then contaminates the water supplies. Outbreaks are usually related to contaminate water supplies. Drinking of untreated stream water will lead to infection.

Causes: Giardiasis

Signs and Symptoms: Non bloody, foul- smelling diarrhea, nauseam anorexia, flatulence, and abdominal cramps that persist for weeks or months

Mode of Control: Water should be boiled, filtered, or treated with iodine. Frequent hand washing and separation of feeding areas from sanitization area. No vaccine or preventive drug is available.

Plasmodium Species [1]

Epidemiology: The four species are P. vivax, P. falciparum, P. ovale and P. malariae. P. vivax and P. falciparum are the most common causes. The vector and definitive host is the female Anopheles mosquito. Disease is transmitted primarily by the bite of the mosquito. This occurs mainly in the tropical and subtropical areas, especially in Asia.

Causes: Malaria

Signs and Symptoms: Abrupt onset of fever and chills, accompanied by headache myalgias (muscle pain), and aethralgias (joint pains). Fever can reach up 41°C. It is accompanies by shaking chills, nausea, vomiting and abdominal pain. Splenomegaly and hepatomegaly is commonly seen and anaemia is prominent.

P. falciparum can cause extensive brain (cerebral malaria) and kidney (blackwater) damage. (large scale intravascular lysis)

Mode of Control: Chemoprophylaxis of malaria. The use of mosquito netting, window screens, protective clothing and insect repellent can lower the probability of mosquito bites. Elimination of breeding site includes drainage of stagnant water in swamps and ditches, removal of standing water and improvement in land drainage.

Toxoplasma gondii [2]

Epidemiology: Infection by T. gondii occurs worldwide. It is an obligate intracellular parasite widespread among wild and domestic animals. The definitive host is the domestic cat and other felines; humans and other mammals are intermediate host. Cockroaches, flies and earthworms serve as non-infected transport host. Infection begins with the ingestion of cyst in undercooked meats or from contact with cat feces.

Causes: Toxoplasmosis

Signs and Symptoms: Resemble infectious mononucleosis (fever, muscle soreness, sore throat, fatigue)

Mode of control: Control is difficult because of the diversity of animal reservoir. To prevent toxoplasmosis, cook meat thoroughly to kill cyst. Cat should not be fed raw meat as it is the definitive host of the obligate intracellular parasite.

Acanthamoeba castellanii [2]

Epidemiology: They are free-living amoebae. They are found in warm freshwater lakes and in soil. Infection can occur when swimming in the lakes and when there is skin trauma

Causes: Meningeoncephalitis

Signs and Symptoms: eye infection

Mode of Controls: Do not swim in lakes. Avoid traumatized skin contact with lake water or soil.

Naegleria fowleri [1,2]

Epidemiology: N. fowleri thrives in warm water at lower oxygen tensions. It is often found in the mud at the bottom of puddles, ditches, and lakes. It enters the body trough mucous membranes or inhalation while an individual is swimming.

Causes: Meningeoncephalitis

Signs and Symptoms: headache, fever and deteriorates rapidly into coma

Mode of Controls: Do not swim in lakes. Avoid traumatized skin contact with lake water or soil.

Cyclospora cayetanensis [2]

Epidemiology: It is distributed worldwide. Infection appears to be acquired by ingestion of oocytes in water or with contaminated fruits or vegetables.

Causes: “Traveler’s diarrhea”

Signs and Symptons: Water diarrhea with low grade fever and abdominal cramps

Mode of control: Treatment of water supplies with chlorination and filtration. Improving basic hygiene and sanitation practices is most effective way of controlling outbreaks.

Cryptosporidium parvum [1]

Epidemiology: Infection is usually water borne or acquired from animals. Oocytes are present in animal feces and can be transmitted to the human through the fecal- oral route.

Causes: cryptosporidiosis

Signs and Symptoms: Diarrhea leading to fluid loss and malnutrition

Mode of control: Improving basic hygiene and sanitation practices is most effective way of controlling outbreaks. Swimming in lakes or any bodies of water is not advisable.

Trichomonas vaginalis [1,2]

Epidemiology: The primary location is the vagina and the prostate. Transmission of this organism is by sexual contact. It is predominantly a vaginal parasite, although urethritis may occur in the male consorts of infected women. Infection in women is more severe than in men.

Causes: Urinary tract infection

Signs and Symptoms: Pain during urination, discharge

Mode of Control: Abstinence or no unprotected sex

Reasons for exclusion:

Trypanosoma cruzi (Chagas’ disease) [1]
Vector is the reduviid bug. Animal reservoirs include wild species such as the armadillo, raccoon and rat. Rats are commonly found in jungles. However, this disease is common in America only.

Trypanosoma brucei rhodesiense (sleeping sickness) [1]
Wild animals as reservoirs for the tsetse fly, Glossina. Wild animals are abundant in jungles. But this disease occurs primarily in West Africa therefore it is ruled out.

Leishmania donovani [1]
Lifecycle involves the sandfly as the vector and a variety of mammals such as dogs, foxes and rodents as reservoirs. However, this is common in Middle East, Africa, Russia, India and China

Babesia [1,2]
Human infection is rare. Cases reported in Africa, New York, and Southern Europe. In immunocompetent individuals the disease is usually self limiting, so no specific treatment is required.

Isopora belli [2]
It causes self limiting diarrhea in immunocompetenet individuals, but in more severe cases, particularly in patients that are immunocompromised (e.g. AIDS), they will cause severe diarrhea. Medical screening is usually done before enlistment to determine if the individual is healthy. If the individual is immunocompromised, it can be detected during the screening.

Balantidium coli [1,2]
Domestic animals, especially pigs are the main reservoir for the organism. It is commonly associated with pig farming. Humans are infected after ingestion of the cyst in food or water contaminated with animal or human feces. However, it rarely causes disease in humans. Most individual are asymptomatic; diarrhea rarely occurs.

References:
1)Levinson, W. (2006). Review of Medical Microbiology and Immunology. San Francisco,California: Lange Medical Books/ McGraw-Hill Medical Publishing Division

2)Murray, P.R. (2002). Medical Microbiology. St. Louis, Missouri: Mosby Inc.

Done by:
Foo Yong Yang
Loh Mun Jo-anne
TG02

MMIC PBL 1

2007-12-10T00:51:00.000+08:00

A wound swab was obtained

The specimen is gram-stained. The shape, size, arrangement and whether they are gram-positive or gram-negative should be observed.

The specimen is cultured on appropriate media, to obtain ‘pure culture’. Plates should be incubated in aerobic or anaerobic environment.

Identification of the organism by means of biochemical tests.
Perform antibiotic susceptibility tests.

Note: Wound infection could be due to multiple organisms, so it’s important to culture the specimen on several different media under different atmospheric conditions e.g. aerobic or anaerobic. Gram staining provides useful information about the range of possible causative agents. [1]

Short listed bacteria [1, 2]:
Gram-positive cocci:
1) Streptococci Species (Facultative anaerobes)
- Enterococcus faecalis
- Streptococcus pyogenes

2) Staphylococcus aureus (Facultative anaerobes)

Gram-positive rod:
1) Bacillus anthracis (Strict aerobes; spore formation)

2) Clostridium perfringens (Stric anaerobes; spore formation)

Gram-negative rod:
1) Pseudomonas aeruginosa (Strict aerobes)

2) Enterobacteriaceae species (Facultative anaerobes)
- Escherichia coli
- Enterobacter cloacae
- Serratia marcescens
- Proteus mirabilis
- Klebsiella pneumoniae

Differential diagnosis for gram-positive cocci:

Streptococci Species
1] Enterococcus faecalis
2] Streptococcus pyogenes

Sheep blood agar:[1]

- Beta-hemolytic colonies: Streptococcus pyogenes
- Gamma-hemolytic or lack of change in the medium around colonies: Enterococcus faecalis

Catalase test: Both Negative [2]
This test differentiates Streptococci Species and Staphylococcus aureus.

- A drop of hydrogen peroxide is added to a sample from the bacterial colony
- Presence of catalase converts hydrogen peroxide into water and oxygen
- Results in the production of visible bubbles

Bile-esculi agar: [2]

- Positive: E. faecalis
- Negative: Strep. Pyogenes

E. faecalis hydrolyse esculin, which results in black discoloration of the agar.

Hypertonic (6.5%) NaCl solution:

- E. faecalis grows, while Strep. Pyogenes doesn’t

Antibody Susceptibility: [1]
1] Enterococcus faecalis:
- Penicillin
- Erythromycin
- Bacitracin
- Tetracycline

2] Streptococcus pyogenes:
- Penicillin
- Erythromycin
- Bacitracin
- Tetracycline

Staphylococcus aureus [1]

Sheep blood agar:
- Beta-hemolytic colonies indicate Staphylococcus aureus

Catalase test: Positive

Coagulase test: Positive

Coagulase test differentiates S. aureus, from other staphylococcus species such as S. epidermidis present in the normal flora.

Mannitol Salt Agar: Yellow colonies

Differential test for mannitol fermentors and non-fermentators; S. aureus ferments mannitol.

Antibody susceptiblity:
- Vancomycin
- Gentamicin
- Nafcillin
- Methicillin

Picture 1: Beta-hemolysis represented by beta sign and gamma-hemolysis represented by gamma sign
Picture: micrsweb>lab3grampostitive>lab3introtomethods.html/">rsweb>lab3grampostitive>lab3introtomethods.html/">http://www3.umdnj.edu>micrsweb>lab3grampostitive>lab3introtomethods.html

Picture 2: Catalase positive reaction indicating S. aureus infection
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Differential diagnosis for spore forming gram-positive rods:

Bacillus anthracis: [1, 2]

Blood agar (Culture aerobically): Gamma-hemolytic ;large and irregular colonies

Egg yolk agar: Lecithinase negative

In addition, it can be diagnosed by means of antigen and molecular tests.

Antibody susceptibility: [1]
- Ciprofloxacin
- Doxycycline

Clostridium perfringens: [2]

Blood agar (Culture anaerobically): Double zone of beta-hemolysis

Egg yolk agar: Lecithinase positive

Antibody susceptibility: [1]
- Penicillin

Picture 3: Left plate: Positive test for lecithinase; Right plate: Negative for lecithinase
Picture: kkiser>lecithpn.jpg/">kkiser>lecithpn.jpg/">kkiser>lecithpn.jpg/">http://users.stlcc.edu>kkiser>Lecithpn.jpg

Differential diagnosis for gram-negative rods:[1]

MacConkey agar: selective agar used for the isolation and differentiation of lactose-fermenting and non-fermenting gram-negative rods. Alternatively, Eosin Methylene Blue Agar (EMB) can be used.

Lactose fermenters: Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae; form red to pink colonies.

Non-fermenters: Proteus mirabilis, Pseudomonas aeruginosa and Serratia marcescens; form colourless colonies

Oxidase test: [1]
- Positive: Pseudomonas aeruginosa
- Negative: Enterobacteriaceae species

This test determines the presence of oxidase enzyme. An oxidase paper is touched onto colony, in the presence of oxidase, paper changes to purple (Pseudomonas aeruginosa).

Triple sugar iron agar: [1]

Biochemcial tests: Indole test, Methyl red test, Voges-Proskauer reaction test and Citrate utilization test (IMViC test)

Pseudomonas aeruginosa: [1]
- Colonies: Fluoresce under ultraviolet light; give fruity odor and grow at 42° C
- Indole test: Negative
- Methyl red test: Positive
- Voges-Proskauer reaction test: Negative
- Citrate utilization test: Positive
- Urease test: Negative

Antibody susceptibility: [1]
- Penicillin e.g. ticarcillin or piperacillin
- Aminoglyoside e.g. gentamicin or amikacin

Enterobacteriaceae species:

1] Escherichia coli [1]
- Motile
- Indole test: Negative
- Methyl red test: Positive
- Voges-Proskauer reaction test: Negative
- Citrate utilization test: Negative
- Urease test: Negative

Antibody susceptibility: [1]
- Cefotaxime
- Gentamicin
- Ampicillin
- Loperamide

2] Enterobacter cloacae [1]
- Indole test:Negative
- Methyl red test: Negative
- Voges-Proskauer reaction test: Positive
- Citrate utilization test: Positive
- Urease test: Negative

Antibody susceptibility: [1]
- Imipenem
- Gentamicin
- Cefotaxime

3] Serratia marcescens [1]
- Indole test: Positive
- Methyl red test: Negative
- Voges-Proskauer reaction test: Positive
- Citrate utilization test: Positive
- Urease test: Negative

Antibody susceptibility: [1]
- Imipenem
- Gentamicin
- Cefotaxime

4] Proteus mirabilis [1]
- Indole test: Positive
- Methyl red test: Positive
- Voges-Proskauer reaction test: Negative
- Citrate utilization test: Positive
- Urease test: Positive

Antibody susceptibility: [1]
- Ampicillin
- Cefotaxime

5] Klebsiella pneumoniae [1]
- Indole test: Positive
- Methyl red test: Positive
- Voges-Proskauer reaction test: Positive
- Citrate utilization test: Positive
- Urease test: Positive

Antibody susceptibility: [1]
- Imipenem
- Gentamicin
- Cefotaxime

Refereneces:

1)Levinson, W. (2006). Review of Medical Microbiology and Immunology. San Francisco,California: Lange Medical Books/ McGraw-Hill Medical Publishing Division

2)www.textbookofbacteriology.net>search

Foo Yong Yang
TG02
0503196H

Principles of Test used by the team

2007-12-09T22:00:00.000+08:00

Laboratory test Principles

Dear Mr Loh, my group has compile the principles of laboratory testings which are to done my the group members for the identification of the microorganism

MacConkey agar
MacConkey agar is a selective, differential platingmedium. It selects enterobacteriaceae and other gram-negative bacteria and differentiates them into lactose fermenters and non-lactose fermenters. The presence of bile salts and crystal violet will inhibit the growth of gram-positive microorganism. The incorporation of lactose serves as the sole carbohydrate source. Gram-negative bacilli which ferments lactose produces a dark red to pink colonies while non-lactose fermenting gram negative bacilli produces colourless or transparent colonies

http://www.yahoo.com/> Image search> MacConkey agar

Eosin-Methylene Blue Agar
Eosin-methylene blue agar is a selective, differential medium used for the isolation and identification of gram-negative bacteria. Eosin Y and methylene blue dyes inhibit the growth of gram-positive bacteria and allows the growth of gram-negative bacteria. Lactose and sucrose are incorporated to allow differentiation of isolates based on lactose fermentation. Fermentation is detected by colour changes and precipitation of dye such as pH drops. Sucrose is incorporated as it serves as another alternative source of carbohydrates for slow lactose fermenters.

http://www.yahoo.com/> Image search> Eosin-methylene blue agar

Blood Agar
Blood agar is used to cultivate a wide variety of fastidious bacterium. An infusion agar of tryptic soy agar base can be enriched by the addition of 5-10% of sheep’s, rabbit’s blood. The incorporation of blood not only provides enrichment for growth but also allows the detection and characterization of haemolytic patterns

micrsweb>lab3grampostitive>lab3introtomethods.html/">http://www3.umdnj.edu>micrsweb>lab3grampostitive>lab3introtomethods.html/

-alpha-hemolysis represented by alpha sign
alpha-hemolysis is the partial clearing of the blood around the colony that results in a green discoloration pf the medium

-Beta-hemolysis represented by beta sign
Beta-hemolysis is the complete clearing of blood around the bacterial colonies. This is due to the complete haemolysis of the surrounding RBCs

-Gamma-hemolysis represented by gamma sign
Gamma-hemolysis occurs when the RBCs surrounding the colonies are unaffected and there is no change in the colour of the bacterial colonies

Mannitol Salt Agar
Differential test for mannitol fermentors and non-fermentators; S. aureus ferments mannitol producing yellow colonies.

Left: Non- fermented mannitol Right: Fermented mannitol (S. aureus)

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Triple Sugar Iron Agar Test
TSI contains glucose, lactose and sucrose as well as a pH-sensitive color indicator. It also contains an iron ingredient for detecting hydrogen sulfide production, which blackens the medium if it occurs. Fermentation of the sugars by the test organism is interpreted by color changes in the butt
and the slant of the mediums.

Interpretation
Acid (yellow) butt – alkaline (pink) slant (K/A)
- glucose fermented
- sucrose or lactose not fermented

acid butt - acid slant (A/A)
- glucose fermented
- lactose and/or sucrose fermented

alkaline butt - alkaline slant (K/K)
- neither glucose, lactose or sucrose fermented
- organism probably not an enteric bacillus

Gas production - indicated by bubbles in the butt. With large amounts of gas production, the agar may be broken or pushed upward.
Hydrogen sulfide production - indicated by any amount of blackening in the butt.

TSI agar

http://www.yahoo.com/ > http://biology.uwsp.edu > faculty>TBarta > fermentation > TSI agar

Indole test
Bacteria that possess the enzyme tryptophanase are capable of hydrolyzing and deaminating tryptophan with the production of indole, pyruvic acid and ammonia. Indole production is an important characteristic in the identification of many species of microorganisms being particularly useful in separating E. coli (positive) from members of the Klebsiella-Enterobacter-Hafnia-Serratia group (mostly negative)

www.yahoo.com > http://www.mc.maricopa.edu >johnson >labtools >biochem

Interpretation Positive test: bright pink colour at the interface between the broth and the reagent Negative test: No colour change or a slight yellow ring

Methyl Red test
Methyl red is an acid sensitive dye that is yellow at pH above 4.5 and red at pH below 4.5. This test is used to indicate whether glucose has been broken down completely to highly acidic end products or only partially to less acidic end products.

www.yahoo.com > http://www.mc.maricopa.edu >johnson >labtools >biochem

Interpretation
Positive - bright red color, indicates pH of 4.5 or less
Negative - yellow or orange color, indicates pH of above 4.5

Voges Proskauer
The Voges-Proskauer test is used to detect the presence of acetylmethylcarbinol, one of the end products of glucose metabolism.

www.yahoo.com > http://www.mc.maricopa.edu >johnson >labtools >biochem

Interpretation
Positive - strong red color will develop
Negative - no color change

Citrate test
Organisms that are capable of utilizing citrate as their sole carbon source also utilize ammonium salts as their sole nitrogen source. This results in an alkaline environment that turns the indicator bromothymol blue to an intense blue when pH is above 7.6. This characteristic can used to differentiate members of the family Enterobacteriaceae and other gram-negative rods.

www.yahoo.com > http://www.mc.maricopa.edu >johnson >labtools >biochem

InterpretationPositive test: growth or a deep blue colour of the agarNegative test: no growth or no colour change of the agar (green)
Right tube- negative results; Left tube- positive results

Urease Test
Detects the enzyme urease, which breaks down urea into ammonia. Ammonia is a base and thus will raise the pH of the media if it is present. This change in pH is indicated by a pH indicator called phenol red which is present in the media.

InterpretationPositive test: A color change from yellow to bright pinkish-redNegative test: No colour change

Oxidation-Fermentation Test
The oxidative-fermentative approach permits the separation of bacteria by a simple method that differentiates anaerobic and aerobic degradation of carbohydrates in an environment responsive to weak production of acids. Agar is added to give a semisolid consistency so that acids that form on the surface of the agar may permeate throughout the medium, making interpretation off the pH shift of the indicator bromothymol blue easier to visualize.

Interpretation
Positive test: medium becomes yellow at the top indicating acid productionNegative test: medium remains green if no acid formed

Catalase Test
The catalase test is used to differentiate between bacterial species in the lab. The test is done by placing a drop of hydrogen peroxide on a microscope slide. Using an applicator stick, the colony is smeared into the hydrogen peroxide drop. If bubbles or froth form, the organism is said to be catalase-positive; if not, the organism is catalase-negative

Catalase positive reaction indicating S. aureus infection

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Oxidase Test
Use to determine the presence of oxidase enzymes. An oxidase paper is touched onto colony, in the presence of oxidase, paper changes to purple indicating Pseudomonas aeruginosa. The absence of oxidase result in no colour change, and is indicative of Enterobacteriaceae family

Coagulase Test
Coagualse test is performed to test the ability of the microorganism to clot plasma by the action of coagulase

Rabbit plasma is incubated with staphylococcus colony If staphylococci isolated from infection are able to clot blood, then they are coagulase positive S. aureus.

Coagulase test differentiates S. aureus, from other staphylococcus species such as S. epidermidis and S. saprophyticus present in the normal flora.

Right: Coagulase negative; Left: Coagulase positive

micrsweb>lab3grampostitive>lab3introtomethods.html/">micrsweb>lab3grampostitive>lab3introtomethods.html/">http://www3.umdnj.edu>micrsweb>lab3grampostitive>lab3introtomethods.html/

Bile esculin Test
The medium contains esculin and peptone that is to provide nutrition to the microorganism while bile inhibits Gram-postivie bacteria other than Group D streptococci and enterococci, and sodium azide to inhibit the Gram-negative bacteria. Ferric citrate is added as a color indicator.

Interpretation
Positive test: An agar slant that is more than half darkened after incubation.
Negative test: Less than half the slant has darkened.

Optochin Test
Optochin (ethylhydrocupreine hydrochloride), a quinine derivative, has a detergent-like action and causes selective lysis of pneumococci. A sterile disk impregnated with optochin is placed on the first sector of an isolation plate before incubation. A zone of inhibition (area with no growth) of 14 mm. or more in diameter will be seen around the disk after incubation if the organism is Streptococcus pneumoniae. Other alpha-hemolytic streptococci are resistant to (not killed by) optochin. Their colonies will thus grow right up to the disk of optochin or have zones of inhibition less than 14 mm. in diameter.

Bile Solubility Test
Bile will selectively lyse colonies of Streptococcus pneumoniae while other strep are immune to bile's activity. When a bile salt such as desoxycholate is added directly to Streptococcus pneumoniae growing on an agar plate or in a broth culture the bacteria will lyse and the area become clear. Other alpha-hemolytic streptococci are resistant to (not lysed by) bile and will stay visible or turbid (cloudy).

Bile-esculi agar
Bile-esculi agar is a selective, differential agar used to isolate and identify group D streptococci and enterococci. Bile salts is a selective component and esculin is the differential ingredient. All group D streptococci and enterococci hydrolyzes esculin. The products of esculin hydrolysis react with ferric citrate in the medium to produce insoluble iron slats. This results in the blackening of the agar.

micrsweb>lab3grampostitive>lab3introtomethods.html/">micrsweb>lab3grampostitive>lab3introtomethods.html/">http://www3.umdnj.edu>micrsweb>lab3grampostitive>lab3introtomethods.html/

6.5% NaCl Test
NaCl is sued to differentiate those gram-positive cocci that will grow in 6.5% NaCl from those that are inhibited by this salt concentration. Enterococcus species can withstand higher salt concentration than other gram-positive cocci

Egg Yolk Agar
Egg yolk agar is a differential medium used in the detection of lecithinase, lipase and protease activity. The egg emulsion in the agar provides the lecithin, lipase and proteins to be degraded by these enzymes. A given microorganism may produce one or all of the above enzymes.

Left plate: Positive test for lecithinase
Right plate: Negative for lecithinase

http://users.stlcc.edu>kkiser>Lecithpn.jpg

DNase Test
Extacellular DNases are only present in a few bacterial species such as Serratia marcescens, Pseudomonas aerugionsa. The biochemical and morphology of other Enterbacteriaceae are similar especially in Klebsiella-Enterobacter supp. The only difference which separates S. marcescens from Klebsiella-Enterobacter supp is the DNases that is produced.

8% sodium chloride broth
Test is done to differentiate vibrio cholerae and vibrio parahaemolyticus.
Unlike vibrio parahaemolyticus, vibrio cholerae cannot grow at 8% sodium chloride broth.

Spore stain (Bartholomew and mitters)
In spore stain test, hot malachite green is used to stain spores, while the safranin is used as a counterstain. In this test, spores would be stain green and the rest of the cell appear pink/red

References
http://www.mc.maricopa.edu/~johnson/labtools.html> Optochin sensitivity & bile solubility

http://en.wikipedia.org/wiki/Main_Page > search > catalase

http://en.wikipedia.org/wiki/Main_Page > search > oxidase

Mahon C. R. and Manuselis G, Jr (1995). Textbook of Diagnostic Microbiology. W.B. Saunders Company

MacFaddin J. F. (2000). Biochemical Test for Indentification of Medical Bacteria. Lippincott Williams & Wilkins

search>pseudomonas/">www.textbookofbacteriology.net>search>pseudomonas

Levinson, W. (2006). Review of Medical Microbiology and Immunology. San Francisco,California: Lange Medical Books/ McGraw-Hill Medical Publishing Division

med>topic2422/">http://www.emedicine.com>med>topic2422

http://www.yahoo.com/ > http://www2.umdnj.edu >labpix > E.coli > biochemical test

http://www.yahoo.com/ > http://people.uis.edu > Labs > Enterobacteriaceae > biochemical test

search>staph./">search>staph./">www.textbookofbacteriology.net>search>staph

Student attachment materials

Star Team

TG02

Mmic Case 3

2007-12-09T21:50:00.000+08:00

Patient : Maisy Hong
Age : 67
Urine Specimen 3

Diagnosis: UTI, indwelling catheter

E.coli on blood plate

E.coli on MacConkey plate - lactose fermentors

Serratia on blood plate - colonies appear as 'buff' coloured

Proteus on blood plate- swarming

All pictures obtained from:
http://www.microbelibrary.org/asmonly/details.asp?id=2038

Antibiotics

1. S. epidermidis
Highly resistant to antibiotics.
· Novobiocin
· Vancomycin+rifampin/aminoglycoside

2. Enterococci
· Ampicillin
· Nitrofurantoin

3. E.coli
· Ampicillin
· Aminoglycosides
· Cephalosporin
· SXT (Trimethoprim-sulfamethaxazole)

4. Proteus
· Penicillin-derivatives
· Aminoglycosides
· Cephalosporin
· Quinolones

5. Serratia
Mulit-drug resistant
· Third generation Cephalosporin

6. P. aeruginosa
Highly mulit-drug resistant therefore combination of antibiotics is necessary.
· Penicillin derivatives + aminoglycosides
· Third generation Cephalosporin
· Quinolones e.g. ciprofloxacin
· Monbactams e.g. aztreonam
· Carbepenam e.g. imipenam

7. Acinetobacter
· Aminoglycosides
· Cephalosporin

phuiyuen

2007-12-09T19:57:00.000+08:00

MMIC PBL 1 (Case 6) Blog Post 2

Name: Ong Fei Fei
Sex: Female
Age: 37 years old
IC NO: S210334X
Location: Clinic T (Outpatient)

Clinical Diagnosis: Urinary Tract Infection (UTI)
Antibiotic Treatment: Nil
Signs and Symptoms: Fever, pain during urination, virginal discharge
Specimen type: Vaginal Discharge

Urinary Tract Infection

Bacteria first travel to the urethra. An infection occurs when the bacteria multiply. When infection is limited only to the urethra, the infection is known as urethritis. If bacteria move to the lower urinary tract (bladder), an infection, called cystitis, results. If the infection is not treated promptly, bacteria may spread to the upper tract and multiply. This infects the kidneys causing infection known as pyelonephritis.

From the clinical diagnosis and symptoms, these are the possible causative agents:

Bacteria

Escherichia coli

Gram negative rod; Facultative anaerobe
Habitat is the human colon (fecal flora); it colonizes the vagina and urethra.
From the urethra it ascends and causes UTI.
Laboratory Diagnosis
Gram stained smear and culture on blood agar
Lactose fermenting colonies (pink) on EMB or MacConkey’s agar.
Green sheen on EMB agar
TSI agar show acid slant and acid butt with gas but no H2S
Motile
Indole +
Methyl red +
Voges- Proskauer reaction -
Citrate utilization -
Serologic tests for antibodies in patient’s serum not useful
Treatment
Ampicillin
Sulfonamides
Cephalosporin
Aminoglycosides

Picture 1: Green Sheen on EMB agar

www.google.com > EMB E.coli
http://www.microbelibrary.org/microbelibrary/files/ccImages/Articleimages/Atlas_EMB/E.coli_EMB_fig1.jpg

Staphylococcus saprophyticus

Gram positive cocci in clusters; Facultative Anaerobe
Inhabits the skin surrounding the genitourinary tract
Community acquired
Laboratory Diagnosis
Coagulase negative
Catalase Positive
Resistant to the antibiotic novobiocin
Treatment
Quinolone (norfloxacin)
Trimethoprim- Sulfamethoxazole

Psuedomonas aeruginosa

Gram negative rods; Obligate Aerobes
Produce water soluble pigments
Transmission via fecal contamination
Laboratory Diagnosis
Gram stain smear
Culture: Non Lactose fermenting colonies on MAC or EMB (colorless)
Blue green pigment on ordinary nutrient agar
TSI show alkaline slant and an alkaline butt
Oxidase +
Pyocyanin +
Fluorescein +
Citrate +
Urease +/-
Indole –
Serological test not useful
Treatment
Highly multidrug resistant
Combination therapy
Anit-pseudomonal penicillin and aminoglycoside are often used

Picture 2: Blue Green Pigment on Ordinary Nutrient agar

www.google.com > Pseudomonas aeruginosa nutrient agar
http://www.bmb.leeds.ac.uk/mbiology/ug/ugteach/icu8/images/uti/pyocyanin.jpg

Enterococcus faecalis

Gram positive cocci in chains; Facultative anaerobe
Habitat is the human colon; urethra and female genital tract can be colonized
Hospital acquired UTI
Laboratory Diagnosis
Gram stained smear and culture
Alpha-, Beta-, or non-hemolytic colonies on blood agar
Catalase negative
Coagulase negative
Grows in 6.5% NaCl and hydrolyzes esculin in the presence of 40% bile
Serological test not useful
Treatment
Combination Therapy
Penicillin or vancomycin plus an aminoglycoide such as gentamicin

Proteus-Providencia-Morganella Family

Community and hosipital acquired
Gram negative rod; Facultative anaerobe
Presence in colon and vigorous motility contribute to their ability to invade the urinary tract
Laboratory Diagnosis
Highly motile and produce a swarming overgrowth on blood agar
Growth on blood agar containing phenylethyl alcohol inhibits swarming, thus allowing isolated colonies of Proteus and other organisms to be obtained.
Produce non-lactose fermenting colonies on MacConkey or EMB agar
Urease producing
TSI agar shows an alkaline slant and acid butt with H2S
Indole +
Treatment
Trimethoprim-sulfamethoxazole
Ampicillin
3rd generation cephalosporin

Enterobacter-Klebsiella-Serrtia Family

Gram negative rod with large polysaccharide capsule; Facultative anaerobe
Transmitted to the urinary tract by ascending spread of fecal flora
Endotoxin causes fever
Laboratory Diagnosis
Gram stained smear and culture
Characteristic mucoid colonies are a consequence of the organism’s abundant polysaccharide capsule
Lactose fermenting colonies on MacConkey’s agar
Voges Prooskauer +
Serratia marcescens: Red-pigmented colonies
Treatment
Cephalosporins alone or with aminoglycosides, but antibiotic sensitivity testing must be done

Picture 3: Serratia marcescens: Red-pigmented colonies

http://www.google.com/ > Serratia Marcescens agar
http://microvet.arizona.edu/Courses/JC-MIC205/S08/Images/pour_Serratia.jpg

Mycoplasma genitalium

Small, wall-less organism
Reproduce by budding, fragmentation or binary fission
Normal flora of the genitourinary tract
Laboratory Diagnosis
Serologic testing
Cold agglutinin titre of 1:128 or higher indicate recent infection
4 fold rise in antibody titre in complement fixation test
Treatment
Resolves spontaneously
Penicillins and cephalosporins are inactive due to lack of cell wall
Macrolide ( Erythromycin/ Tetracycline)

Ureaplasma urealyticum

Small, wall-less organism
Reproduce by budding, fragmentation or binary fission
Normal flora of the genitourinary tract
Laboratory Diagnosis
Serologic testing
Cold agglutinin titre of 1:128 or higher indicate recent infection
4 fold rise in antibody titre in complement fixation test
Urease producing
Treatment
Resolves spontaneously
Penicillins and cephalosporins are inactive due to lack of cell wall
Macrolide ( Erythromycin/ Tetracycline)

Gardnerella vaginalis

Facultative gram variable rod associated with bacterial viginosis
Characterized by a malodorous vaginal discharge and clue cells, which hare vaginal epithelial cells covered with bacteria
Laboratory Diagnosis
“Whiff” test – consists of treating the vavginal discharge with 10% KOH and smelling a pungent, fishy odor is usually positive.
Trichomoniasis can also cause a positive whiff test and must be ruled out before a diagnosis of bacterial vaginosis can be made
Mobiluncus an anaerobic rod is often found in this disease as well
Treatment
Metronidazole

Parasite

Chlamydia trachomatis

Obligate intracellular parasites; Gram negative bacteria, possess rigid cell wall
Habitat is the human genital tract and is transmitted by sexual contact
Laboratory Diagnosis
Cytoplasmic inclusions seen on Giemsa-stained or fluorescent-antibody-stained smear
Glycogen-filled cytoplasmic inclusions can be visualized with iodine
PCR based assay and an ELISA using patient’s urine
Grown in cell cultures treated with cycloheximide, which inhibits host cell but not Chlamydia protein synthesis
Serologic test rarely helpful in diagnosis
Treatment
Tetracycline (Doxycycline)
Macrolide (Azithromycin)

Protozoa

Trichomonas vaginalis

Urogenital protozoan
Pear shape, flagellated trophozoites
No cysts or other forms
Transmitted sexually – attach to wall of vagina and cause inflammation and discharge
Laboratory Diagnosis
Visible in secretions
Wet mount - Motile
Treatment
Metronidazole

Fungi (Yeast)

Candida albicans

Oval yeast with a single bud
Part of normal flora of the female genital tracts
Laboratory Diagnosis
Microscopic examination reveals yeasts and pseudohyphae
Gram positive and can be visualized using calcofluor- white staining
Forms colonies of yeasts on Sabouraud’s agar
Germ tube formation and production of chlamydospores distinguish Candida albicans from other species of Candida
Serologic tests not useful
Treatment
Oral or topical antifungal agents such as nystatin or miconazole
Amphotericin B
Ketoconazole

Virus

Herpes Simplex Virus Type 2

Enveloped virus with icosahedral nucleocapsid and linear double stranded DNA
No virion polymerase
No herpes group specific antigen
Transmitted through sexual contact
Laboratory Diagnosis
Causes cytopathic effect in cell culture
Identify by antibody neutralization or fluorescent- antibody test
Rise in antibody titre can be used to diagnose a primary infection
Treatment
Acyclovir

Picture 4: Cytopathic Effect

http://www.google.com/ >cytopathic effect
http://pathmicro.med.sc.edu/lecture/images/multinuc.jpg

Identification of Bacteria

Identification of other Causative Agents

References

1)Levinson, W. (2006). Review of Medical Microbiology and Immunology. San Francisco,California: Lange Medical Books/ McGraw-Hill Medical Publishing Division

Loh Mun Jo-anne
TG02
0503324F

MMIC Case Study 4 Post 2

2007-12-09T17:03:00.000+08:00

Case Study 4

Patient Name: Tong Wei Hong
Sex: Male
Age: 68 years old
Complaints: Fever, chills, excessive phlegm, breathing problems
Diagnosis: Bronchitis

Bronchitis can also be caused by viruses such as Influenza virus, adenoviruses and rhinoviruses. The bacteria that have been shortlisted are:

Streptococcus pneumoniae
Klebsiella pneumoniae
Haemophilus influenzae
Moraxella catarrhalis
Bordetella pertussis
Mycoplasma pneumoniae
Chlamydia pneumoniae

Here are the summarized biochemical tests and antibiotic susceptibility tests for the suspected bacteria. Pictures to help for diagnosis of bacteria:
Haemophilus influenzae on blood agar
Picture taken from: http://en.wikipedia.org/ > search > Haemophilus Influenzae

Gram stain of Haemophilus influenzae
Picture taken from: http://www.textbookofbacteriology.net > search> Haemophilus Influenzae

Klebsiella pneumoniae on MacConkey’s agar (Pink)

Picture taken from http://www.microbelibrary.org/ASMOnly/details.asp?id=1976&Lang

Bile solubility test for Streptococcus pneumoniae (Right)
Picture taken from http://www.mc.maricopa.edu/~johnson/labtools/Dbiochem/opto.html

Optochin test for Streptococcus pneumoniae (Left)
Picture taken from http://www.mc.maricopa.edu/~johnson/labtools/Dbiochem/opto.html

Martin Ng TG02

2007-12-09T16:34:00.000+08:00

Blog 2

MMIC: Case 2
Name: Kwan Siew Yan
Sex: Female
I/C No: S000123X
HRN: OPD001
Age:29 years
Ward/Clinic: Clinic X

Clinical Diagnosis
Complaints: Diarrhea
Diagnosis: Enterocolitis
Antibotic Treatment (if any): Nil

Enterocolitis
Enterocolitis is the inflammation of the large and small intestines. The most common symptom of enterocolitis is diarrhea.

Causative Agents

The most common way a person can get enterocolitis is by infection. Infection includes Bacteria, Virus and Parasites. Below are the identified causative agents that can cause enterocolitis.

Bacteria

Campylobacter jejuni
Salmonella species
Shigella species
Escherichia coli
Vibrio cholerae
Vibrio parahaemolyticus
Clostridium perfringens
Clostridium difficile

Virus

Rotavirus
Norovirus

Parasites

Giardia lamblia/ Giardia intestinalis

Biochemical Tests

Since this PBL is on bacterial pathology I’ll only be discussing the biochemical properties of the microorganisms from the bacteria group.

Campylobacter jejuni
Domestic animals such as cattle, chickens and dogs serve as a source of the organisms for humans. Food and water contamination with infected animal feces is the major source of human infection.
· Laboratory Diagnosis
o Gram Negative Rod
o Motile
o Non-lactose fermenter
o Microaerophilic
o Incubation at 42°c
o Oxidase positive
o Catalase positive
o Hippurate positive
o TSI shows no H2S production
o Urease negative
o Culture on Charcoal based selective medium
o Methylene blue stain of a fecal sample
· Antibiotic Treatment
o Erythromycin
o Ciprofloxacin

www.yahoo.com> campylobacter culture pictures> http://www.flickr.com/photos/estherase/43320354/

Salmonella species
Humans and animals serve as reservoirs for the bacteria.
The ingestion of food and water contaminated by human and animal waste is the main mode of transmission.
· Laboratory Diagnosis
o Gram Negative Rods
o Non-Lactose Fermenting colonies on MacConkey’s agar or EMB agar
o Motile
o Facultative Anaerobe
o TSI shows alkaline slunt but acid butt with both gas and H2S produced
o Catalase positive
o Oxidase negative
o Urease negative
o Indole test negative
o Methyl Red Positive
o Voges-proskauer negative
o Citrate positive
o Lysine decarboxylase positive
o Ornithine decarboxylase positive
o Slide agglutination Test
o Commonly grown on Xylose Lysine Deoxycholate (XLD) agar
o Serological test such as widal test can be used
o Methylene Blue stain of fecal sample

· Antibiotic Treatment
o Antibiotic treatment is usually not prescribed, as salmonella is a self limiting disease that resolves without treatment
http://www.bact.wisc.edu> the microbial world> salmonella

Shigella Species
Enterocolitis caused by Shigella species is usually called bacillary dysentery. Shigellaosis is a human only disease and has no animal reservoir. Transmission is most common via the fecal-oral route
· Laboratory Diagnosis
o Gram Negative Rods
o Non-Lactose Fermenting colonies formed on MacConkey’s and EMB agar
o Non-motile
o Facultative Anaerobe
o Urease negative
o Lysine decarboxylase negative
o Indole negative
o Methyl red positive
o Voges-proskauer negative
o Citrate negative
o TSI shows alkaline slant and an acid butt, with no gas and no H2S produced
o Methylene Blue stain of fecal sample
o Commonly grown on Xylose Lysine Deoxycholate (XLD) agar
· Antibiotic Treatment
o In mild cases no antibiotics are prescribed, only fluid and electrolyte replacements
o In severe cases ciprofloxacin and Trimethoprim-sulfamethoxazole can be presribed

Escherichia coli
Escherichia Coli is frequently associated with traveler’s diarrhea. The reservoir of E.Coli includes both humans and animals. The source of the E.Coli that causes traveler’s diarrhea is by ingestion of food or water contaminated with human feces.
· Laboratory Diagnosis
o Gram Negative Rod
o Lactose Fermenting
o Motile
o Facultative anaerobe
o Indole positive
o Methyl-red positive
o Voges-proskauer negative
o Citrate negative
o Culture on blood agar plate followed by a differential medium either EMB or MacConkey’s agar
o Form lactose fermenting colonies on MacConkey’s agar
o A Characteristic green sheen is observed on EMB agar
o TSI shows acid slant and butt with gas produced and no H2S
o Methylene Blue on fecal smear to observe for neutrophils
· Antibiotic Treatment
o Trimethoprim-sulfamethoxazole
o Loperamide (Imodium)

www.yahoo.com> escherichia coli on blood agar> http://www.visualsunlimited.com/browse/vu303/vu303820.html

www.yahoo.com> escherichia coli on MacConkey's agar> http://www.visualsunlimited.com/browse/vu416/vu416843.html

Vibrio cholerae
Vibrio Cholerae is transmitted by fecal contamination of water and food, primarily from human sources.
· Laboratory Diagnosis
o Gram Negative Rod
o Motile
o Non lactose fermenting colonies form on MacConkey’s agar
o Facultative anaerobe
o Urease negative
o Oxidase Positive
o Lysine decarboxylase positive
o Ornithine decarboxylase positive
o Methyl Red positive/negative (depending on strain)
o Voges-proskauer positive/negative (depending on strain)
o TSI shows acid slant and acid butt without gas or H2S
o A retrospective diagnosis can be made serologically by detecting a rise in antibody titer in acute and convalescent-phase sera.
· Antibiotic Treatment
o Antibiotics are not necessary, as treatment usually consists of replacement of water and electrolytes.
o Antibiotics such as tetracyclines shorten the duration of symptoms and reduce the time of excretion of the organisms.

Vibrio parahaemolyticus
· Laboratory Diagnosis
o Gram Negative Rod
o Motile
o Beta-hemolysis colonies observed when cultured on blood agar plates
o Non-lactose fermenting colonies form on MacConkey’s agar
o Facultative anaerobe
o Urease negative
o Oxidase Positive
o Lysine decarboxylase positive
o Ornithine decarboxylase positive
o Methyl red negative
o Voges-proskauer negative
o TSI shows acid slant and acid butt without gas or H2S
o A retrospective diagnosis can be made serologically by detecting a rise in antibody titer in acute and convalescent-phase sera.
o Differentiated from V.Cholerae on the basis of growth in Sodium Chloride V.Parahaemolyticus grows in 8% sodium chloride while V.Cholerae does not
· Antibiotic Treatment
o Antibiotics are not necessary, as treatment usually consists of replacement of water and electrolytes.
o Antibiotics such as tetracyclines shorten the duration of symptoms and reduce the time of excretion of the organisms.

Clostridium perfringens
Spores located in soil can contaminate food. The heat resistant spores survive cooking and germinate. The organisms grow to large numbers in reheated food, especially meat dishes.
· Laboratory Diagnosis
o Gram Positive Rod
o Endospore-forming
o Anaerobic
o Spore staining positive ( Bartholomew and Mittwer)
o Nagler test
· Antibiotic Treatment
o Self-limiting disease
o Symptomatic treatment is given, no antimicrobial drugs are administered

Clostridim difficile
Normal flora in the intestines, that can become opportunistic pathogens in patients treated with broad-spectrum antibiotics such as penicillin and cephalosporin. The most common nosocomial cause of diarrhea.
· Laboratory Diagnosis
o Gram positive Rod
o Motile
o Anaerobic
o Endospore forming
o Spore staining positive (Bartholomew and Mittwer’s)
o ELISA testing
o Cytotoxicity testing
· Antibiotic Treatment
o Causative antibiotic should be withdrawn
o Metronidazole
o Vancomycin

Identification of Microorganism

Culture on Blood agar in anaerobic conditions

Microorganisms that grow in anaerobic conditions: Salmonella species, Shigella species, , Escherichia coli, Vibrio cholerae, Vibrio parahaemolyticus, Clostridium perfringens, Clostridium difficile

Microorganisms that do not grow in anaerobic conditions: Campylobacter jejuni,(microaerophilic)

Anaerobic Microorganisms

Perform Gram stain and observe under microscope: to differentiate gram positive and gram negative bacteria

Gram Negative Microorganisms: Salmonella species, Shigella species, Escherichia coli, Vibrio cholerae, Vibrio parahaemolyticus

Gram Positive Microorganisms: Clostridium perfringens, Clostridium difficile

Gram Negative Microorganism

Perform Wet mount: To differentiate motile and non-motile organisms

Motile: Salmonella species, Escherichia coli, Vibrio cholerae
Vibrio parahaemolyticus

Non-Motile: Shigella species

Motile Microrganisms

Culture on MacConkey's/ Eosin-Methylene Blue (EMB) agar:
To differentiate between lactose and non-lactose fermenting microorganisms

Lactose Fermenters: Escherichia coli

Non-Lactose Fermenters: Salmonella species, Vibrio cholerae
Vibrio parahaemolyticus

Non-Lactose Fermenting Microorganisms

Perform Oxidase Test: To differentiate between microorganisms containing the enzyme cytochrome oxidase and microorganisms that do not contain the enzyme.

Oxidase Positive: Vibrio cholerae, Vibrio parahaemolyticus

Oxidase Negative: Salmonella species

To differentiate between Vibrio cholerae and Vibrio parahaemolyticus, culture the microorganism into a 8% sodium choloride broth. Vibrio Parahaemolyticus can grow in 8% sodium chloride but Vibrio Cholerae cannot.

Gram Positive Microorganisms
Since the patient is not on any antibiotic treatment, we can rule out Clostridium difficile, as it is a nosocomial cause of diarrhea. Therefore if the microorganism is observed to be gram positive, a spore stain can be done to confirm that it is Clostridium perfringen

Randall

TG02
0503272G

References:
www.medicinenet.com>search> Diarrhea

www.wrongdiagnosis.com > search > Enterocolitis

http://www.cfsan.fda.gov/~mow/chap4.html

Levinson, W. (2006). Review of Medical Microbiology and Immunology. San Francisco,California: Lange Medical Books/ McGraw-Hill Medical Publishing Division

Bauman, R,W. (2004). Microbiology. San Francisco, California: Pearson Education inc/
Pearson Benjamin Cummings

Medical Microbiology [Case 1--Blog 2]

2007-12-08T11:16:00.001+08:00

Name of patient: Khong Fay Seah
Sex : Female
Age: 27 years old
Specimen: Urine [specimen 1]

Clinical Diagnosis
Complaints: Fever, Chills, Dysuria
Diagnosis: Urinary Tract Infection (UTI)

Interpretation of clinical diagnosis (complaints)

Fever [1]
Fever a common manifestation of infection and inflammation [such as influenza, urinary tract infection, bowel disease]. Fever can be caused by many bacterial products, most notably, endotoxin of gram-negative bacteria. Endotoxin would result in the release of endogenus pyrogen, which derived from monocytes and macrophages. Other substances that can cause fever are interlukin 1 and interferons.

Chills [2]
The word ‘chill’ refers to an episode of shivering, accompanied by paleness and feeling cold. Chills may occur at the beginning of an infection, and is usually associated with fever. Chills are caused by rapid muscle contraction and relaxation, and are the body's way of generating heat when the body feels that it is cold. Causes of chills includes exposure to cold environment and virus and bacterial infections [such as meningitis, urinary tract infection, pneumonia]

Dysuria [3]
Dysuria is a condition whereby there is discomfort/pain during urination. Causes of dysuria includes urinary tract infection, cystitis, prostatitis and urethritis

Although UTIs occur in both men and women, women tend to suffer more from it because of the anatomical difference. Women's urethra tube is shorter than men's, and the opening is just a short distance away from the vaginal tract and the rectal openings. Because of this, there is a higher risk of bacteria from these adjacent areas to scuttle up the tube into the bladder. [5]

Microorganism that causes urinary tract infection[4,6]
1) Bacteria [6]

a) Escherichia coli [About 75% of community acquired UTI and 40% of hospital acquired UTI are caused by E. coli]

E. coli http:///www.yahoo.com > Image> Search > Escherichia coli

b) Staphylococcus saprophyticus [About 5% of community acquired UTI and 1% of hospital acquired UTI are caused by S. saprophyticus]

S. saprophyticus http:///www.yahoo.com > Image> Search > Staphylococcus saprophyticus

c) Proteus mirabilis [About 3% of community acquired UTI and 10% of hospital acquired UTI are caused by P. mirabilis]

P. mirabilis http:///www.yahoo.com > Image> Search > Proteus mirabilis

d) Pseudomonas aeruginosa [About 1% of community acquired UTI and 5% of hospital acquired UTI are caused by P. aerginosa]

P. aeruginosa http:///www.yahoo.com > Image> Search > Pseudomonas aerugionsa

e) Enterobacter faecalis [About 5% of community acquired UTI and 8% of hospital acquired UTI are caused by E. faecalis]

E. faecalis http:///www.yahoo.com > Image> Search> Enterobacter faecalis

f) Staphylococcus epidermidis [About 2% of community acquired UTI and 3% of hospital acquired UTI are caused by S. epidermidis]

S. epidermidis http:///www.yahoo.com > Image> Search> Staphylococcus epidermidis

g) Other coliforms [About 5% of community acquired UTI and 25% of hospital acquired UTI are caused by other Enterobacteriaceae such as Klebsiella pneumonia, Serratia marcescens]

K. pneumonia http:///www.yahoo.com > Image> Search> Klebsiella pneumonia

S. marcescen http:///www.yahoo.com > Image> Search> Serrtia marcescen

In addition, Acinetobacter sp. causes nosocomial UTI. Other less common microorganism that causes UTI include Mycobacterium tuberculosis. [7]

2) Viruses [6]
a) Adenovirus
b) Human polyoma virus

3) Parasites [6]
a) Trichomonas vaginalis
b) Schistoma haematobium

4) Fungi [6]

a) Candida albicans

In this PBL, only bacteria is being dealt with thus viruses, parasites and fungus are eliminated.

1) Escherichia coli [4,6,8]
a) Facultative Anaerobe, gram negative bacilli
b) Confirmatory test bi)Microscopy and gram staining shows motile gram-negative rod
bii) Cultures shows dark pink to red, dry flat lactose-fermenting colonies on MacConkey agar, and blue black colonies w/wo green metallic sheen on eosin methylene blue (EMB) agar
biii) Biochemical test to be done/results for E. coli biii[a]) Citrate, H2S, Urea and Voges-Proskauer (VP) negative
biii[b]) Indole, Methyl-red positive
biii[c]) Ferments both glucose and lactose with gas and no H2S

biv) Treatment: Amplicllin, Cephalosporin [cefoxitin, cefotaxime, ceftazidme], Aminoglycosides [amikacin, gentamicin, tobramycin], Sulfonamides [bactrim]
Escherichia coli is the most common cause of UTI. It is a normal flora in the intestine and colon that enters and invades the urethra causing an infection.

2) Staphylococcus saprophyticus [4,6,8]
a) Facultative Anaerobe, gram positive cocci
b) Confirmatory test
bi) Microscopy and gram staining shows gram-positive cocci
bii) Absence of hemolysis on blood agar
biii)Biochemical test to be done/results for S. saprophyticus
biii[a]) Catalase positive
biii[b]) Oxidase, Coagulase, Mannitol fermentation negative

biv) Treatment: Quinilone [ norfloxacin, ciprofloxacin]
bv) Resistant: Bacitracin, Novobiocin

Staphylococcus saprophyticus is isolated in patient especially in sexually active women patient between age 16-35

3) Proteus mirabilis [4,6,8]
a) Facultative Anaerobe, motile, non-spore fermenting gram-negative rod
b) Confirmatory test
bi) Microscopy and gram staining shows motile gram-negative rod
bii) Pale colonies on MacConkey’s agar and colourless/light purple colonies on EMB agar
biii) Biochemical test to be done/results for P. mirabilis
biii[a]) Oxidase negative
biii[b]) H2S, Urea and Indole positive
biii[c]) Voges-Proskauer (VP) negative/positive biii[d]) Non-lactose fermenting

biv) Treatment: Cephalosporin [cefoxitin, cefotaxime, ceftazidme], Quinilone [ciprofloxacin], Ampicillin

bv) Resistant: Nitrofurantoin

Proteus mirabilis is a bacterium that is commonly infect recurrent patient with urinary tract abnormalities. It is also found in elderly who is undergoing long-term catheterization.

4) Pseudomonas aeruginosa [4,6,8]
a) Facultative Anaerobe, gram-negative rod
b) Confirmatory test
bi) Microscopy and gram staining shows motile gram-negative rod
bii) Flat and green pigmentation on MacConkey’s agar and , colourless/light purple colonies on EMB agar
biii) Biochemical test to be done/results for P. aerginosa
biii[a]) Citrate, Oxidase positive
biii[b]) Non-lactose fermenting
biii[c]) Indole negative
biv) Treatment: Ciprofloacin, Imipenam, Carbenicillin, Ticarcillin

bv) Resistant: Ampicillin, Cefloxitin, Nitrofurantoin, Trimethopriml-sulfamethoxazole
Pseudomonas aeruginosa is the most frequent colonizers of medical catheters. Since patient is not using such devices,Pseudomonas aeruginosa may not be the microorganism isolated

5) Klebsiella pneumonia [4,6,8]
a) Facultative Anaerobe, non-motile, gram-negative rod
b) Confirmatory test
bi) Microscopy and gram staining shows non-motile gram-negative rod
bii) Dark pink to red Mucoid colonies observed on MacConkey agar and blue black colonies w/wo green metallic sheen on eosin methylene blue (EMB) agar
biii)Biochemical test to be done/ results for K. pneumonia
biii[a]) Ferments both lactose and glucose with gas and no H2S
biii[b]) Urease, Voges-Proskauer, Citrate positive
biii[c]) Indole, methyl-red negative

biv) Treatment: Cephalosporin [cefoxitin, cefotaxime, ceftazidme], gentamicin
The chances of identifying Klebsiella pneumonia increases with the length of hospital stay. Since the patient is an out-patient, in this case, the bacteria would most probably not be the cause of UTI.

6) Staphylococcus epidermidis [4,6,8]
a) Obligate Anaerobe, Gram-positive cocci
b) Confirmatory test
bi) Microscopy and gram staining shows cluster of gram-positive cocci
bii) Non-hemolytic colonies observed on sheep’s blood agar
biii) Biochemical test to be done/ results for S. epidermidis
biii[a]) Catalase, urease positive
biii[b]) Coagulase, mannitol fermentation negative

biv) Treatment: Novobiocin, Vancomycin + rifampin/ aminoglycoside
Staphylococcus epidermidis found in hospitalized patient older than 50 years of age with urinary tract complication.

7) Enterococcus faecalis [4,6,8]
a) Facultative Anaerobe, Non-motile, gram-positive Streptococci
b) Confirmatory test
bi) Microscopy and gram staining shows a gram-positive cocci
bii) Alpha, beta or no hemolysis on blood agar
biii) Biochemical test to be done/ results for E. faecalis
biii[a]) Bile esculin, 6.5% NaCl positive
biii[b]) Catalase negative

biv) Treatment: Vancomycin, Novobiocin
Enterococcus faecalis identification is usually associated with the usage of catheters. It is more commonly found in hospitalized patient. Since the patient is not required for any observation in the hospital [meaning that she is an out-patient], Enterococcus faecalis may not be the bacteria that is to be identified.

8) Serratia marcescens [4,6,8]
a) Facultative Anaerobe, motile, Gram negative bacilli
b) Confirmatory test
bi) Microscopy and gram staining shows a gram-negative bacilli
bii) Slight pink colonies on MacConkey Agar, colourless/light purple colonies on EMB agar
biii) Biochemical test to be done/ results for S. marcescens
biii[a]) Ferments both glucose and lactose and no H2S
biii[b]) Indole, methyl-red negative
biii[c]) Voges-Proskauer, Citrate positive
biii[d]) DNAse positive

biii[e]) Catalase positive

biv) Treatment: Cephalosporin [cefoxitin, cefotaxime, ceftazidme], gentamicin
The identification of Serratia marcescens increases with the length of hospital stay. In this case, since the patient is an out-patient, the bacteria would most probably not be the cause of UTI.

Mycobacterium tuberculosis is a rare bacteria that is being isolated in UTI cases, thus eliminated

Acinetobacter sp. is involved in hospital-acquired infection and commonly infect immunocomprised individuals.

Though either Escherichia coli or Staphylococcus saprophyticus may be the most probable microorganism isolated in this case, the microorganism above would be identified by a series of test

LABORATORY TEST to be done for the identification of causative agent

Facultative Anaerobe
-E.coli
-S. saprophyticus
-P. mirabilis
-P. aeruginosa
-E. faecelis
-S. marcescens
-K. pneumonia

Obligate Anaerobe
-S. epidermis

1) Culture on blood agar, in aerobic conditions

Organisms that can grow on blood agar in aerobic conditions:
-E.coli
-S. saprophyticus
-P. mirabilis
-P. aeruginosa
-E. faecelis
-S. marcescen
-K. pneumonia

Organisms that cannot grow on blood agar in aerobic conditions:
-S. epidermis

2) Perform Gram Stain

Gram negative organisms: Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa, Klebsiella pneumonia, Serratia marcescens

Gram positive organisms: Enterococcus faecalis, Staphylococcus saprophyticus

3) Gram negative organisms

3a) Perform Wet Mount

Motile organisms: Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa, Serratia marcescens

Non-motile organisms: Klebsiella pneumonia

3b) Culture on MacConkey

Microorganisms that form lactose fermenting colonies on MacConkey: Escherichia coli

E. coli(left) and P. mirabilis (right) on MacConkey agar http://www.yahoo.com/ > Image> Search> E. coli

Microorganisms that do not form lactose fermenting colonies on MacConkey: Proteus mirabilis,Pseudomonas aeruginosa, Serratia marcescens

Pseudomonas aeruginosa shown on the right side of the MacConkey agar http://www.yahoo.com/ > Image> search> Pseudomonas aeruginosa

3c) Oxidase Test

Oxidase positive organisms: Pseudomonas aeruginosa

Oxidase negative organisms: Proteus mirabilis, Serratia marcescens

3d) Perform TSI

H2S producing microorganisms: Proteus mirabilis

Non-H2S producing microorganisms: Serratia marcescens

4) Gram positive organisms

4a) Catalase test

Catalase positive organisms: Staphylococcus saprophyticus

Catalase negative organisms: Enterococcus faecalis

The image below show how the microorganism is being identified [flowchart]

References

[1] Coico, R., Sunshine, G. and Benjamini, E. (2003) Immunology: A Short Course. John Wiley & Sons, Inc., Hoboken, New Jersey.

[2] Medical Encyclopedia. (2007). Chills. Retrieved 31st November, 2007 from
http://www.nlm.nih.gov/ Search NLM Web Site> Chills

[3] Medicine Net. (2007). Dysuria. Retrieved 31st November, 2007 from
http://www.medterms.com/> MedTerms Dictionary> Dysuria> Medical Dictionary> Dysuria

[4] Spicer W. J. (2000). Clinical Bacteriology, Mycology and Parasitology: An illustrated colour text. Churchchill Livingstone

[5] Juicing for Health.com (20007). Urinary Tract Infection. Retrieved 31st November, 2007
http://www.juicing-for-health.com/> Google Search> www.juicing-for-health.com >Urinary Tract Infections

[6] Elliott, T., Hastings, M. and Desselberger, U. (1997). Lecture Notes on Medical Microbiology. Blackwell Science Ltd.

[7] Phage Therapy Center. (2005). Chronic and Acute Urinary Tract Infection. Retrieved 31st
http://www.phagetherapycenter.com/ >>Clinics>What we treat> Chronic and Acute Urinary Tract Infections (UTI) and Cystitis

[8] Mahon C. R. and Manuselis G, Jr (1995). Textbook of Diagnostic Microbiology. W.B. Saunders Company

Eugene Wong
TG02

MMIC PBL 1

2007-12-03T05:34:00.000+08:00

Name: Wong Fei Hong (Case 5)
Sex: Male
Age: 37 years old
Type of Specimen: Wound swab

Clinical diagnosis
Symptoms: Fever, swelling around operation wound
Diagnosis: Wound infection

Interpretation of symptoms
Fever:
Fever is a non-specific clinical manifestation for a wide range of viral or bacterial infections. In the case of bacterial infections, the effects of endotoxins from gram-negative bacteria, lead to the release of endogenous pyrogen from macrophage and cause fever. In addition, the release of various exotoxins from gram-positive bacteria can also cause fever. [1]

Swelling around operation wound:
A swell is a result of increased extravascular fluid and phagocyte infiltration to the damaged area. The swell around operation wound indicates localised inflammatory response due to the presence of bacteria at the site of wound. Thus, swab is collected and sent quickly for investigation. [1]

Interpretation of diagnosis
Wound infection:
A wound is generally defined as the break the continuity of the skin, which allows pathogenic microorganisms to gain access to tissues and cause infection. Operation wound infection known as surgical site infection (SSI) can be classified into 3 types, namely, superficial incisional SSI, deep incisional SSI, and organ SSI. [3]

Picture: http://www.emedicine.com/med/images/Large/22552255ppt1.jpg

Mode of transmission
Causative agents could be transmitted from non-human source such as fomite (i.e. towel), soil, animal, and also from the normal flora of the host, which lead to the symptoms described above. [1]

Possible causative agents for wound infection [1, 2]

Streptococci Species (facultative anaerobe, gram-positive cocci)
-Enterococcus faecalis
-Streptococcus pyogenes

Staphylococcus aureus (facultative anaerobe, gram-positive cocci)

Bacillus anthracis (aerobic, gram-positive rod, spore-forming)

Clostridium species (anaerobic, gram-positive rod, spore forming)
-Clostridium perfringens
-Clostridium tetani

Pseudomonas aeruginosa (aerobic, gram-negative rod)

Enterobacteriaceae species (facultative anaerobe, gram-negative rod)
-Escherichia coli
-Enterobacter
-Serratia marcescens
-Proteus mirabilis
-Klebsiella pneumoniae

Shortlisted

Enterococcus faecalis [2]
Reasons for inclusion: E. faecalis is part of the normal flora in colon but can cause serious disease when introduced into other part of the body. Patient could have been acquired the bacteria during his stay in hospital, or due to poor personal hygiene which led to wound infection.

Streptococcus pyogenes [2]
Reason for inclusion: S. Pyogenes is a common causative agent of skin infection for patient with skin trauma such as operation wound. Symptoms of infection include fever, inflammation around site of wound infection.

Staphylococcus aureus [1]
Reason for inclusion: Failure to follow aseptic surgical procedures could have lead to S. Aureus wound infection.

Bacillus anthracis [1]
Reason for inclusion: Spores could have entered wound from the soil;or animal products such as bristles, hives or wool.

Clostridium perfringens [1]
Reason for inclusion: Is present in normal flora of human colon, or soil, which could enter wound due to poor hygiene.

Clostridium tetani [1]
Reason for exclusion: Specific clinical feature such as lockjaw is absent.

Pseudomonas aeruginosa [2]
Reason for inclusion: P. aeruginosa is an opportunistic pathogen that cause wound infection, thus the assumption would be that patient is immunocompromised.

Enterobacteriaceae species [2]

-Escherichia coli
-Enterobacter
-Serratia marcescens
-Proteus mirabilis
-Klebsiella pneumoniae

Reason for inclusion: The Enterobacteriaceae species are opportunistic pathogens acquired from hospital,which can cause surgical wound infection. Similarly, the assumption would be that patient is immunocompromised.

P.S: In blog 2, I'll elaborate on various biochemical tests for differential/ confirmatory diagnosis, as well as antibody susceptibility test.

References:

1)Levinson, W. (2006). Review of Medical Microbiology and Immunology. San Francisco,California: Lange Medical Books/ McGraw-Hill Medical Publishing Division

2)www.textbookofbacteriology.net>search

3)med>topic2422/">http://www.emedicine.com>med>topic2422

Foo Yong Yang
TG02
0503196H

2007-12-03T01:00:00.000+08:00

MMIC PBL 1 (Case 6)
Name: Ong Fei Fei
Sex: Female
Age: 37 years old
IC NO: S210334X
Location: Clinic T (Outpatient)

Clinical Diagnosis: Urinary Tract Infection (UTI)
Antibiotic Treatment: Nil
Signs and Symptoms: Fever, pain during urination, virginal discharge
Specimen Type: Virginal Discharge

Bacteria first travel to the urethra. An infection occur when the bacteria multiply. When infection is limited only to the urethra, the infection is known as urethritis. If bacteria move to the lower urinary tract (bladder), an infection, called cystitis, results. If the infection is not treated promptly, bacteria may spread to the upper tract and multiply. This infects the kidneys causing infection known as pyelonephritis.

From the clinical diagnosis, I have short listed the possible bacteria causing UTI:

- Staphylococcus saprophyticus
§ Inhabits the skin surrounding the genitourinary tract
§ Particularly in sexually active young women
§ Second most common cause
§ Spherical gram positive cocci arranged in irregular grapelike cluster
§ Coagulase negative

- Escherichia coli
§ Major cause of UTI; Normal flora of the vagina and anterior urethra
§ Falcutative anaerobe
§ Gram-negative rod
§ Lactose fermenting
§ motile

- Pseudomonas aeruginosa
§ Inhabits the colon
§ Transmission is via fecal contamination
§ Gram negative rod
§ Non- lactose fermenting
§ Aerobic
§ Oxidase positive
§ Blue-green pigment (pyocyanin)

- Enterococcus faecalis
§ Habitat is the human colon; urethra and female genital tract can be colonized.
§ Gram-positive cocci in chains
§ Catalase negative
§
- Enterobacter-Klebsiella-Serrtia Family
§ Habitat is the human enteric tracts
§ Transmitted to the lungs by aspiration by ascending spread of fecal flora
§ Gram- negative rod
§ Lactose fermenting

- Proteus-Providencia-Morganella Family
§ Habitat is the human colon
§ Transmission to urinary tract is by ascending spread of fecal flora
§ Gram- negative rod
§ Produce urease
§ Highly motile
§ Non-lactose fermenting
§ Produce H2S
§ Indole positive

In my next blog entry, I will write about the investigation required to identify the bacteria.

References
http://adam.about.com>search: urinary tract infection

Levinson, W. (2006). Review of Medical Microbiology & Immunology. San Francisco, California: Lange Medical Books/McGraw-Hill publishing company

Loh Mun Jo-anne
TG02
0503324F

2007-12-03T00:10:00.000+08:00

MMIC: Case 2
Name: Kwan Siew Yan
Sex: Female
I/C No: S000123X
HRN: OPD001
Age:29 years
Ward/Clinic: Clinic X

Clinical Diagnosis
Complaints: Diarrhea
Diagnosis: Enterocolitis
Antibotic Treatment (if any): Nil

Enterocolitis
Enterocolitis is the inflammation of the large and small intestines. The most common symptom of enterocolitis is diarrhea.

Causative Agents

The most common way a person can get enterocolitis is by infection. Infection includes Bacteria, Virus and Parasites. Below are the identified causative agents that can cause enterocolitis.

Bacteria

Campylobacter Jejuni
Salmonella enteritidis
Shigella species
Escherichia ColiO157:H7 (ETEC)
Vibrio Cholerae
Vibrio Parahaemolyticus
Staphylococcus Aureus
Clostridium Perfringens

Virus

Rotavirus
Norovirus

Parasites

Giardia Lamblia/ Giardia intestinalis

Biochemical Tests

Since this PBL is on bacterial pathology I’ll only be discussing the biochemical properties of the microorganisms from the bacteria group.

Campylobacter Jejuni
Gram Negative Rod
Motile
Microaerophilic

Salmonella enteritidis
Gram Negative Rods
Non-Lactose Fermenting
Motile

Shigella Species
Gram Negative Rods
Non-Lactose Fermenting
Non-motile
Anaerobe

Escherichia Coli O157:H7
Gram Negative Rod
Lactose Fermenter
Facultative anaerobe
Anaerobe

Vibrio Cholerae
Gram Negative Rod
Oxidase Positive

Vibrio Parahaemolyticus
Gram Negative Rod

Staphylococcus Aureus
Gram Positive Cocci
Catalase- positive

Clostridium Perfringens
Gram Positive Rod
Spore-forming
Anaerobic

References

www.medicinenet.com>search> Diarrhea

www.wrongdiagnosis.com > search > Enterocolitis

http://www.cfsan.fda.gov/~mow/chap4.html

Levinson, W. (2006). Review of Medical Microbiology and Immunology. San Francisco,California: Lange Medical Books/ McGraw-Hill Medical Publishing Division

Randall

TG02

0503272G

MMIC PBL 1

2007-12-02T22:21:00.000+08:00

MMIC PBL 1 (Case 4)
Name: Tong Wei Hong
Sex: Male
Age: 68 years old

Complaints: Fever, chills, excessive phlegm, breathing problems
Diagnosis: Bronchitis

Bronchitis is inflammation of the large airways that branch off the trachea (bronchi), There are 2 types of bronchitis; acute and chronic. Acute bronchitis is usually caused by viral or bacterial infection, while chronic bronchitis is caused by irritation from a gas or particle.

Some symptoms include fever with chills, muscle aches, nasal congestion, breathing difficulties and sore throat.

Mycoplasma pneumoniae and Chlamydia pneumoniae often cause bacterial bronchitis in young adults. Among middle-aged and older people, Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis are the most common organisms causing bacterial bronchitis.

Possible bacteria that cause bronchitis and laboratory diagnosis to identify:

Mycoplasma pneumoniae

Cannot be identified by Gram-staining due to the lack of cell wall
Serologic testing is recommended. A cold-agglutinin titer of 1:128 or higher is indicative of recent infection.

Chlamydia pneumoniae

Gram-negative; possess rigid cell wall
Serologic testing is recommended. A four-fold rise in IgG or IgA antibody titer between paired sera is indicative of infection

Streptococcus pneumoniae

Gram-positive diplococci
Seen as alpha-hemolytic colonies on blood agar
Growth inhibited by bile and optochin
Quellung reaction occurs (capsular swelling in the presence of antisera)

Haemophilus influenzae

Gram-negative rod
Culture on chocolate agar (growth requires factors X[heme] and V[NAD]

Moraxella Catarrhalis

Gram-negative coccobacillus rod
Produce beta-lactamase (i.e. resistant to beta-lactam antibiotics such as penicillin, cephalosporins, etc.)

References

http://www.merck.com > search: bronchitis > Lung and Airway Disorders

http://www.pulmonologychannel.com > search: bronchitis> Acute bronchitis – pulmonologychannel

Levinson, W. (2006). Review of Medical Microbiology & Immunology. San Francisco, California: Lange Medical Books/McGraw-Hill publishing company

Martin Ng

TG02

0503312A

MMic Specimen 3

2007-12-02T22:03:00.000+08:00

Patient : Maisy Hong
Age : 67
Urine Specimen 3

Patient complains involve fever, chills, bladder distension; on indwelling catheter. This patient is diagnosed to be suffering from Urinary Tract Infection (UTI) by the doctor.

UTI is bacterial infection of the urinary tract. This infection stimulates the inflammation process thus explaining fever and chills suffered by the patient. Bladder distention is refered as abnormal enlargement of the bladder, resulting from an inability to excrete urine, causing its accumulation. Distention can be caused by a mechanical or anatomic obstruction, neuromuscular disorder, or the use of certain drugs. It is relatively common in all ages and both sexes. [1]

An indwelling urinary catheter can result in urine retention and bladder distention. While the catheter is in place, inadequate drainage due to kinked tubing or an occluded lumen may lead to urine retention. In addition, a misplaced urinary catheter or irritation with catheter removal may cause edema, thereby blocking urine outflow. [1]

Possible Microorganisms casing UTI [2]
· S. epidermidis
· S. saprophyticus
· E.coli
· Proteus
· Serratia
· P. aeruginosa
· Enterococci
· Acinetobacter
· Klebsiella

However, possible microorganisms that are causing the UTI in this patient are as follows [2]:
· S. epidermidis
· Enterococci
· E.coli
· Proteus
· Serratia
· P. aeruginosa
· Acinetobacter

S.saprophyticus and Klebsiella are removed as S. sapro is commonly found in young women (patient is a 67 year-old woman) while Klebsiella is commonly found in the bronchi rather than the urinary tract [2].

Laboratory tests to be carried out:

Urine samples collected from patient are cultured onto blood agar and MacConkey agar so that colonies will grow after incubation. Colonies on blood agar will be used for other tests that will confirm the identity of microorganism causing the UTI [4].

S. epidermidis [2]
· Gram-stain – gram-positive
· Coagulase test – coagulase negative, no agglutination appears

2. Enterococci [2,3]
· Gram-stain – gram-positive cocci that tend to appear in pairs

3. E.coli [2,3,4]
· Appears as lactose-fermenting colonies
· Gram-stain – gram-negative
· Indole test – positive
· MUG test – positive
· Methyl red test – positive
· Voges Proskauer – negative
· Citrate test - negative

Proteus [2,3,4]
· Producing swarming effect on blood agar with pungent odour
· Growth in potassium cyanide medium - positive
· Xylose fermentation - positive
· Phenylalaninie deamination - positive

Serratia [2,3]
· Gram-stain – gram-negative
· Methyl red test – negative
· Citrate test – positive
· Tryptophan - positive
· Oxidation - postive
· Fermentation - positive

P.aeruginosa [2,3,4]
· Green background around colonies in blood plate
· Non-lactose fermentors on MacConkey plate
· Gram-stain – gram-negative
· Oxidation – positive
· Citrate test – positive
· Indole test - neagtive

Acinetobacter [2,3]
· Generally non-lactose fermenting
· Gram-stain – gram-negative
· Oxidative – negative
· Nitrate - negative

References
1. Wrong Diagnosis.Com (2007) Bladder Distension. Retrieved 2nd December, 2007, from
http://www.wrongdiagnosis.com/symptoms/blood_in_urine/book-causes-16a.htm

2. Medical Microbiology Lecture Notes

3. Wikipedia.com (2007). Retrieved 2nd December, 2007, from
http://www.wikipedia.com/

4. Student Attachment materials

phuiyuen, TG02

Medical Microbiology [Case 1]

2007-12-02T20:22:00.000+08:00

Name of patient: Khong Fay Seah
Sex : Female
Age: 27 years old
Specimen: Urine [specimen 1]

Clinical Diagnosis
Complaints: Fever, Chills, Dysuria
Diagnosis: Urinary Tract Infection (UTI)

Interpretation of clinical diagnosis (complaints)
Fever [1]
Fever a common manifestation of infection and inflammation [such as influenza, urinary tract infection, bowel disease]. Fever can be caused by many bacterial products, most notably, endotoxin of gram-negative bacteria. Endotoxin would result in the release of endogenus pyrogen, which derived from monocytes and macrophages. Other substances that can cause fever are interlukin 1 and interferons.

Chills [2]
The word ‘chill’ refers to an episode of shivering, accompanied by paleness and feeling cold. Chills may occur at the beginning of an infection, and is usually associated with fever. Chills are caused by rapid muscle contraction and relaxation, and are the body's way of generating heat when the body feels that it is cold. Causes of chills includes exposure to cold environment and virus and bacterial infections [such as meningitis, urinary tract infection, pneumonia]

Dysuria [3]
Dysuria is a condition whereby there is discomfort/pain during urination. Causes of dysuria includes urinary tract infection, cystitis, prostatitis and urethritis

Interpretation of clinical diagnosis (urinary tract infection)
Urinary Tract Infection (UTI) is an infection that can occur along the urinary tract. [bladder, kidney, uretha, ureters] UTI can be classified into lower and upper tract infections. [4]

Although UTIs occur in both men and women, women tend to suffer more from it because of the anatomical difference. Women's urethra tube is shorter than men's, and the opening is just a short distance away from the vaginal tract and the rectal openings. Because of this, there is a higher risk of bacteria from these adjacent areas to scuttle up the tube into the bladder. [5]

Microorganism that causes urinary tract infection [4,6]

A) Bacteria [6]
1) Escherichia coli [About 75% of community acquired UTI and 40% of hospital acquired UTI are caused by E. coli]

2) Staphylococcus saprophyticus [About 5% of community acquired UTI and 1% of hospital acquired UTI are caused by S. saprophyticus]

3) Proteus mirabilis [About 3% of community acquired UTI and 10% of hospital acquired UTI are caused by P. mirabilis]

4) Pseudomonas aerginosa [About 1% of community acquired UTI and 5% of hospital acquired UTI are caused by P. aerginosa]

5) Enterobacter faecalis [About 5% of community acquired UTI and 8% of hospital acquired UTI are caused by E. faecalis]

6) Staphylococcus epidermidis [About 2% of community acquired UTI and 3% of hospital acquired UTI are caused by S. epidermidis]

7) Other Enterobactriaceae [About 5% of community acquired UTI and 25% of hospital acquired UTI are caused by other Enterobactriaceae such as Klebsiella, Enterobacter and Serratia]

In addition, Acinetobacter sp. causes nosocomial UTI. Other less common microorganism that causes UTI include Mycobacterium tuberculosis. [7]

B) Viruses [6]
1) Adenovirus
2) Human polyoma virus

C) Parasites [6]
1) Trichomonas vaginalis
2) Schistoma haematobium

D) Fungi [6]
1) Candida albicans

In this PBL, only bacteria is being dealt with thus viruses, parasites and fungus are eliminated.

Of all the bacterium that causes urinary tract infection, E. coli is the most common organism that is being isolated and it may be the most probable microganism isolated in this specimen. Mycobacterium tuberculosis causing UTI is considered relatively rare.

Escherichia coli [4]

-Aerobic, gram negative bacilli
Confirmatory test
-Microscopy and gram staining shows motile gram-negative rod
-Cultures shows dry flat lactose-fermenting colonies on MacConkey agar and haemolytic colonies on blood agar
-Biochemical test to be done/results for E. coil a) Citrate, H2S, Urea and Voges-Proskauer (VP) negative
b) Indole positive
c) Ferments both glucose and lactose with gas

Staphylococcus saprophyticus [4,6]

-Aerobic, Facultatively Anaerobic, gram positive cocci Confirmatory test
-Microscopy and gram staining shows gram-positive cocci
- White, smooth and shiny colonies
-Absence of hemolysis on blood agar
-Biochemical test to be done/results for S. saprophyticus
a) Coagulase negative
b) Deoxyribonuclease negative c) Mannitol fermentation negative

Proteus mirabilis [4,6]

-Aerobic, motile, non-spore fermenting gram-negative rod
Confirmatory test
-Microscopy and gram staining shows motile gram-negative rod
-Pale colonies on MacConkey’s agar
-Biochemical test to be done/results for P. mirabilis
a) H2S, Urea and Indole positive
b) Voges-Proskauer (VP) negative c) non-lactose fermenting

Pseudomonas aerginosa [4,6]

-Aerobic, gram-negative rod
Confirmatory test
-Microscopy and gram staining shows motile gram-negative rod
-Flat and green pigmentation on MacConkey’s agar
-Biochemical test to be done/results for P. aerginosa
a) Citrate, Oxidase positive
b) Non-lactose fermenting Indole negative

References
[1] Coico, R., Sunshine, G. and Benjamini, E. (2003) Immunology: A Short Course. John Wiley & Sons, Inc., Hoboken, New Jersey.

[2] http://www.nlm.nih.gov/ Search NLM Web Site> Chills

[3] http://www.medterms.com/> MedTerms Dictionary> Dysuria> Medical Dictionary> Dysuria

[4] Spicer W. J. (2000). Clinical Bacteriology, Mycology and Parasitology: An illustrated colour text. Churchchill Livingstone

[5] http://www.juicing-for-health.com/> Google Search> www.juicing-for-health.com >Urinary Tract Infections

[6] Elliott, T., Hastings, M. and Desselberger, U. (1997). Lecture Notes on Medical Microbiology. Blackwell Science Ltd.

[7] http://www.phagetherapycenter.com/ >>Clinics>What we treat> Chronic and Acute Urinary Tract Infections (UTI) and Cystitis

Eugene Wong

TG02

Medical Microbiology

2007-11-04T16:17:00.000+08:00

HIV BLOT (Western Blot assay)

This assay is a qualitative enzyme immunoassay for in vitro detection of antibodies to HIV-1 and HIV-2 in human serum or plasma. It is intended for use as a more specific supplemental test on human serum or plasma specimens found repeatedly reactive using screening procedures such as ELISA. Screening tests are widely available for detecting antibodies against HIV-1 and HIV-2. However, screening tests can be extremely sensitive but can be potentially less specific, which leads to false positive interpretations. Therefore, western blot assay with its high specificity are necessary to further confirm the presence of HIV antibodies.

Test principle: Nitrocellulose strips are incorporated with separated, bound antigenic proteins from partially purified inactivated HIV-1 using electrophoretic blotting, plus specific HIV-2 synthetic peptide on the same strips. These nitrocellulose strips are commercially available. Individual strips are incubated with diluted serum and controls. Specific antibodies to HIV-1 and HIV-2 if present in specimens will bind to the HIV-1 proteins and HIV-2 peptide on the strips. The strips are washed to remove unbound materials. Antibodies that bind specifically to HIV proteins can be visualised using a series of reactions with goat anti-human IgG conjugated with alkaline phosphatase and substrate. This assay method is sensitive enough to detect small amount of HIV specific antibodies in serum or plasma.

Clinical interpretation and limitations: According to WHO criteria, two ENV bands with or without GAG or POL is indicative of HIV infection. A positive blot indicates an infection with HIV, but it does not constitute a diagnosis of AIDS. A diagnosis of AIDS can only be made clinically if a person meets the case definition of AIDS established by CDC or WHO. However, a negative blot is not a guarantee that HIV infection has not taken place. Patients who have just produced detectable HIV antibodies may show incomplete pattern (indeterminate) but increase reactivity i.e. the intensity and number of bands occurs when followed for a period of 2-6 months. Indeterminate interpretation refers to any viral specific bands present but pattern does not meet criteria for positive.

Pictures taken from:
http://www.mpbio.com

Yong Yang (TG02)

0503196H

Clinical Biochemistry

2007-10-30T20:18:00.000+08:00

Cholinesterase

Cholinesterase is an enzyme which catalyzes the hydrolysis of acetylcholine into choline and acetic acid, allowing a cholinergic neuron to return to its resting state after activation. It is crucial to the transmission of electrical impulses along the nerves and also indicates the liver capacity for synthesis.

There are 2 types of cholinesterase: plasma or pseudo-cholinesterase and acetylcholinesterase which are found in the blood (RBCs) and nerves.

Individuals exposed to organophosphorous compounds (e.g. insecticides) exhibit decreased levels of cholinesterase in RBCs and serum. Acetylcholine activity is a better marker for insecticide poisoning, because some individuals may have normal or near normal levels of total serum cholinesterase. However, others have recommended that the serum enzyme measurements reflect acute poisoning while RBC cholinesterase measurement is useful for long-term chronic exposure to insecticides.

Cholinesterase measurements are also used in the detection of patients with inherited abnormal variants of the enzyme. In the nerve endings, reduced cholinesterase activity results in accumulation of acetylcholine which triggers excessive stimulation.

Clinical manifestations for acute insecticide poisoning include excessive salivation, nausea, vomiting, diarrhea, abdominal cramping and asthmatic symptoms. For the cardiovascular system, bradycardia, arrthymias and pulmonary edema are some problems that may happen. Overstimulation of muscarinic sites in the CNS can cause confusion, ataxia and slurred speech. Death may occur.

Procedure for RBC cholinesterase

1. Patient’s blood in EDTA tube is poured into a glass test tube and filled to ¾ with 0.9% saline.
2. The suspension is mixed well by inverting several times and centrifuged at 3500rpm for 5 minutes.
3. Using the suction pump, the plasma and buffy coat (layer on top of RBCs) are removed.
4. 0.9% saline and spun cells are pipetted into a secondary tube to obtain a 1:1 dilution.
5. 0.5% saponin (lysing agent) and the suspension are pipetted into a new secondary tube.
6. The hemolysate is incubated in a refrigerator (2-8°C) for 10 minutes before analyzed on the Beckman Coulter LX20U analyzer.
7. The PCV (hematocrit) is measured using the suspension (obtained in Step 4). The blood is put into capillary tubes and centrifuged at 13000rpm for 5 minutes and the PCV is read off a calibrator rule.

As cholinesterase activity is expressed in activity units with respect to PCV, both enzyme activity and PCV for the hemolysate are determined. After getting the results, the formula [(Cholinesterase/PCV) X 100] is used to obtain the RBC cholinesterase level.

The reference interval for RBC cholinesterase is 15000-24000 U/L RBC.

Note: Serum cholinesterase is also measured in the laboratory, but on the Johnson & Johnson Vitros DT60 II. Information about this analyzer can be found more in 77th Medical Technologist Street blog post on the 5th week.

Martin Ng

TG02

0503312A

2007-10-21T22:54:00.000+08:00

Erythrocyte Sedimentation Rate – ESR
via Tapval and Sedi-rate System

Erythrocyte Sedimentation Rate is helpful in the diagnosis of conditions associated with acute and chronic inflammation, including infections, cancer, and autoimmune diseases. ESR is nonspecific because the increase in ESR does not indicate exactly where the inflammation is or what is the causative agent, it just shows there is inflammation in the body. Therefore, ESR is typically used in conjunction with other tests. ESR is helpful in diagnosing two specific inflammatory diseases, temporal arteritis and polymyalgia rheumatica. Also, ESR test is used for the diagnosis and monitoring of children with rheumatoid arthritis.
Blood is collected via veni-puncture and sent to the laboratory in EDTA tube. After inserting the blood into the EDTA tube, it should be homogenously mixed to ensure all of the blood is exposed to the anti-coagulant to prevent clotting. Clotted blood is rejected as it causes increased ESR.

Materials
Tapval tube with stopper containing Trisodic Citrate (Sodium Citrate)
Pressured Pipette with inner diameter of 1.25mm
Normal Pipette
Pipette Tips
Digital Timer

Methods
1. Patient’s particulars on the test request form are matched with the name on the blood tube.
2. Blood tube is inverted a few times to homogenously mix the blood
3. 320ml of blood is aliquot into the Tapval tube
4. The tube is flickered to attain homogenous mixture of blood with trisodic citrate
5. Pressured pipette is inserted into the tube. This must be done gently to prevent the blood from shooting up the pipette
6. After the pipette is in the tube, it further lowered into the blood until it travels to the top of the pipette
7. After ensuring there is no bubble or gap along the pipette, timer is set at 60 minutes and the pipette stands for the duration..
8. Patient’s label is then stuck to the pipette so as to prevent confusion when there’s a lot of ESR going on.
9. After 60 minutes, the level of red cells is read from its meniscus and recorded in the ESR logbook.

a
Sedimentation takes place in 3 stages:

A preliminary stage where rouleaux occurs and aggregates form.
Sinking of aggregates, which takes place at a constant speed.
Rate of sedimentation slows as the aggregated cells pack at the bottom of the tube.

Reference Values
1. Neonates
1-2 day = 0mm to 4mm
3days - 1month = 0mm to 10mm

2. Paediatric
2 months – 12 years old = 0mm to 12mm

3. Adult
> 12 years old = 0mm to 12mm

Discussion
Elevated ESR may be indicative of:
· Connective tissue diseases: systemic lupus erythematosus (SLE), giant cell arteritis and systemic sclerosis.
· Polymyalgia rheumatica (PMR): a condition particularly common among elderly women which causes chronic aching and stiffness of the muscles, especially in the shoulders and upper arms.
· Cancer
· Rheumatoid arthritis.
· Kidney disease.
· Sarcoidosis : disease of unknown cause in which inflammation occurs in the lymph nodes, lungs, liver, eyes, skin, or other tissues.
· Severe or chronic infection.

However, there are some factors that contribute to increased ESR which are independent of inflammation or infection. For example, increased fibrinogen, increased globulin, anaemia, decreased albumin and packed cell volume.
Also, females tend to have higher ESR, menstruation and pregnancy can cause temporary elevations. Drugs such as dextran, methyldopa (Aldomet), oral contraceptives, penicillamine procainamide, theophylline and vitamin A can increase ESR while aspirin, cortisone, and quinine may decrease it.

2007-10-16T15:57:00.000+08:00

Hey guys today im going to discuss with you people about glucose and how we detect it in our body.

Glucose is the major energy source for the human body. When the total daily energy intake exceeds daily expenditure, the excess is converted to fat and stored in adipose tissue. When daily energy needs are not met by sufficient calorie intake, glucose is produced from the berakdown of carbohydate stores and from non-carbohydrate sources (eg. gluconeogenic amino acids, lactate and glycerol) the glucose level in blood is kept within a fairly narrow range through a variety of influences. Feeding and fasting cause variation in the blood glucose levels..

Glucose measurement is performed for the diagnosis and follow-up of carbohydate metabolism abnormalities (eg. diabetes mellitus, hypoglycemia) and other conditions (such as cerebrospinal fluid glucose in suspected meningitis).

Glucose levels in the body increase due to:

Diabetes Mellitus including: Hemochromatosis, Cushing's syndrome, Acromegaly and gigantism
Increased circulating epinephrine: Adrenal injection, pheochromocytoma, stress (emotions, burns, shock)
Acute pancreatitis
Chronic pancreatitis
Wernicke's encephalopathy
CNS lesions
Effects of drugs

Glucose levels in the body decrease due to:

Pancreatic disorders: islet cell tumors
glucagon deficiency
carcinoma of adrenal gland
carcinomaof stomach
fibrosarcoma
Hepatic Disease: Poisoning, hepatitis, cirrhosis, primary/ metastatic
Endocrine Disorder: Hypopituitarism and Addison's disease, hypothyroidism
functional disturbances: post gastrectomy. gastroenterostomy, antonomic nervous system disorder
Pediatric Anomalies:

-prematurity,infant of diabetic mother, ketotic hypoglycemia, Zetterstrom's syndrome, idiopathic leucine sensitivity, spontaneous hypoglycaemia in infants

Enzyme diseases:

-Von Gerke's disease, galactosemia, maple syrup urine disease, fructose intolerance

The Analyser used for the glucose measurement is the Beckman Coulter LX20 PRO analyser, besides this test, the analyser can also be used to measure various electrolytes (eg. sodium, potassium) and some liver enzymes (GGT, ALT,AST etc)

The samples usually collected for the glucose test are serum, plasma, urine or cerebrospinal fluid.

Principle of Analysis

The analyser determines the glucose concentration by using a glucose reagent in conjunction with calibration standards done by the med tech. when the reagent is input into the machine.

The Analyser measures the oxygen consumption rate of the sample.

10 microlitre of sample is injected into the reaction cup containing the glucose oxidase solution. The ratio used is one part sample to 76 part reagent. The peak of the oxygen consumption is directly proportional to the concentration of glucose in the sample.

The machine then prints out the readout when the test has finished.

Thanks,

Randall

TG02

Identification and Quantitation of Stenotrophomonas maltophilia periplasmic proteins using 2-D Gel Electrophoresis and MALDI

2007-10-06T10:42:00.000+08:00

Hi guys, after talking about Biosafety for my 1st 2 postings, today I am going to talk about my MP work flow:Identification and Quantitation of Stenotrophomonas maltophilia periplasmic proteins using 2 Dimensional Gel Electrophoresis and Matrix Assisted Laser Desorption Ionization Mass Spectrometry (2D Gel-MALDI)

For my project, Stenotrophomonas maltophilia (S. maltophilia) was the bacteria used. It is a gram negative bacteria thus it has a relatively large periplasmic space(periplasm) as compared to gram positive bacteria.

Work flow:
S. maltophilia was streaked on the nurtient agar plate and incubated in a 37ºC incubator for 24 hours. After which all colonies will be scraped off the agar plate and reusepened in 5mL of PBS and the cell density is taken. 5X107 cells will then be inoculated into each LB broth and grown at 28ºC and 37ºC in 20mL LB- broth for 18 hours. Cells will be subjected to washing using PBS for the purpose of removing dead cells and proteins that are released due to cell lysis.
Picture taken from: http://rds.yahoo.com/_ylt=A9ibyiCO_gZHbVkAowyjzbkF/SIG=1300lqsrd/EXP=1191727118/**http://www.cofc.edu/~delliss/virtuallabbook/StreakPlates/StreakExamples.html
This is the procedure for cell washing:
1. Take out the cells from the incubator and spin at 3000g for 20 minutes at 10ºC
2. Discard the supernatant into a waste bottle
3. Pipette 10mL of PBS
4. Pipette well and vortex at low speed to resuspend the cells
5. Spin down centrifuge tubes at 3000g for 20 minutes at 10ºC
6. Repeat step 2 to step 5 twice
7. Resuspend the cells in 10mL of PBS
8. Pipette well and vortex at low speed to resuspend the cell pellet

Density of the live cells will then be determined. 6 eppendorf tubes cotaining each
10 to the power of 10 cells will be subjected to extraction using chloroform

This is the procedure for chloroform shock:
1. Spin down cells at 3000g for 20 minutes at 10oC
2. Resuspend pellet in remaining medium by brief vortexing
3. Add 20uL of chloroform to each eppenforf tube
4. Incubate at room temperature for 15 minutes
5. Combine the 6 eppendorf tubes together
6. Add 400uL of 0.1M Tris-HCl to the combined eppendorf tubes
7. Spin at 6000g for 20 minutes
8. Pipette out supernatant in a new eppendorf tube
9. Spin at 14000g for 10 minutes
10. Pipette supernatant in a new eppendorf tubes

Once periplasmic proteins are extracted, microcon purification is done. The purpose of microcon purification is to further concentrate the protein samples

Micron picture. Taken from: http://www.life.sci.qut.edu.au/epping/LSB607OLT/607concentrators.html

Then Bradford assay will be done to give a estimation of the protien concnetration
Taken from: http://www.biocompare.com/review/926/Quick-Start(tm)-Bradford-Protein-Assay-Kits-From-Bio-Rad.html

Once the protien concentration is determined, 2D gel electrophoresis will be done. Basically protiens will be 1st separated according to their pI by isolecetric focusing (IEF) after which according to their molecular weight (for information on how 2D gel electrophoresis is done, refer to Jia xin's blog on Aug 4, 2007)

BIO-RAD PROTEAN IEF CELL used for IEF (picture taken with permission)

BIO-RAD Criterion Dodeca™ Cell used to separate the protein sample according to molecular weight (picture taken with permission)

Once the proteins are separated, the gel is stained with SYPRO RUBY stain for 16 hours and scanned with PharosFX™ Plus Molecular Imager (for information pls refer to MIng Boon's 17th Aug posting)

This is how a SYPRO RUBY stained gel looks like ( picture taken with permission)

This is the PharosFX™ Plus Molecular Imager which I used ( picture taken with permission)

Once the gel is sacnned, quantitation will be done using a software program:PDQuest. PDQuest will allow us to quantitate the gel spots (for this research project, we want to quantify protein spots present in 28ºC and 37ºC with 2-folds) After Quantitation, gel spots will be slected, cut and automatic process by Xcise ( I won't dicsuss about how Xcise works, it was discussed in Ming Boon's posting on 07/07/07)

Xcise machine from Shimadzu Biotech, proteome systems, Japan (Picture taken with permission)

After processing by Xcise, the protein will be spotted on the MALDI plate and subjected to MALDI analysis. The picture below is the MALDI machine used

(picture taken with permission by my supervisor)

end of story=).......
...feel free to ask any question

Eugene Wong
TGO2

Biosafety Level II Cabinet

2007-10-06T10:30:00.000+08:00

Hi Guys, This is how a Biosafety Level II Cabinet looks like (in case you don't know how it looks like)

This pictures was taken by me with permission from my supervisor =)

Eugene Wong

TG02

Clinical Chemisty

2007-10-02T20:46:00.000+08:00

Sorry for the late update...busy with mp.

In order to prevent graft rejection, transplant recipients have to take immunosuppressive drugs for their whole life. Immunosuppressive drugs are intended to interrupt or prohibit the immunological mechanisms occurring during graft rejection by interference with the function and metabolism of activated cells.

Name of test: Cyclosporin testing

Cyclosporin is a potent immunosuppressive drug got combating tissue rejection following organ transplant. It has serious toxic side effects and is recommended to have regular drug monitoring. Cyclosporine has increased both the short- and long-term survival rates for transplant patients, especially in heart and liver operations. The rejection of grafted tissues occurs when white blood cells (lymphocytes) called T-helper cells stimulate the activity of cell-destroying (cytotoxic) T-killer cells. These T cells, along with other white blood cells like monocytes and macrophages, cause the tissue rejection of the implanted organs. Cyclosporine reduces the ability of accessory cells to produce interleukins, resulting in decreased replication of helper and killer T cells.
Testing cyclosporine levels in the blood can help ensure that drug levels are in a range that will be therapeutic for you.
Procedure of test:

Pre-treatment is needed. The drug is coated on RBCs, therefore the solubilization reagent is added to lyse the RBC and release it. Precipitation reagent is added to suspend the protein. It is then vortexed to ensure through mix and centrifuged to obtain clear supernatant. The clear supernatant is then pipetted out and place into a sample cup which is then placed into the TDX analyzer. The reagents needed are also placed inside the analyzer. TDX is used for quantitative measurement of drugs. It uses the Fluorescence Enzyme Immunoassay (FPIA) technology.

Clinical Interpretation:

If the level is too low, organ rejection may occur.
If the level is too high, it will result in toxicity. Toxic side effects produced are primarily nephrotoxicity and hepatotoxicity.

Loh Mun Jo-anne
TG02
0503324F

Sources:
http://www.rheumatology.org/public/factsheets/cyclosporine.asp?aud=pat
Clinical Diagnostics, Chapter 47: Therapeutic drug monitoring, page1155-1156

Medical Microbiology

2007-09-24T21:41:00.000+08:00

Name of Test: Detection of antibody to Hepatitis C virus (anti-HCV)

Principle of Test: The Anti-HCV assay is a two-step immunoassay, using Chemiluminescent Microparticle Immunoassay (CMIA) technology for the qualitative detection of anti-HCV in human serum and plasma. In the first step, patient sample, HCV coated microparticles and assay diluent are combined. Anti-HCV that is present in the sample binds to the HCV coated microparticles. After washing, anti-human acridinium-labeled conjugate is added in the second step. After washing again, Pre-trigger and trigger solutions are added to the mixture. The resulting chemiluminescent reaction is measured in relative light unit. The amount of anti-HCV in the sample is proportional to the relative light unit measured.

Test Result with reference range: Specimens with cut-off values <1.00 are considered nonreactive by the Anti-HCV assay and need not be tested further.

Specimens with cut-off value >/= 1.00 are considered reactive by the Anti-HCV assay.

Clinical interpretation: A positive anti-HCV test provides evidence of exposure to HCV. Specific antibodies against HCV may not always be detectable in early acute disease because antibody production is usually not detected until 7 to 31 weeks after infection. However, antibody detection doesn't differentiate between acute, chronic or past HSV infection. For diagnostic purposes, anti-HCV result should be used together with patient history. If Anti-HCV results are inconsistent with clincial evidence, further test would have to performed. Additional information can be obtained by testing for viral RNA in serum by PCR. A positive HCV RNA test is suggestive of viral replication in the liver and can confirm a diagnosis of either acute or chronic infection.

Yong Yang (TG02)
0503196H

Clinical Chemistry

2007-09-17T21:40:00.000+08:00

G6PD is the most common enzyme deficiency in the world, affecting approximately 400 million people. It is an X-linked inherited disorder which affects Africans, Asians, Mediterraneans and people of Middle-Eastern descent.

G6PD catalyzes NADP to its reduced form, NADPH, through the pentose phosphate pathway, which prevents oxidative damage to cells.

Diagnosis of G6PD deficiency is made through 2 ways, a rapid fluorescence spot test (screening), or a quantitative spectrophotometric analysis.

I will elaborate more on the screening test in the lab I am working at. In Singapore, all newborns are screened for G6PD deficiency.

Glucose-6 Phosphate Dehydrogenase (G6PD) screening test

Procedure for G6PD screening

1. Pipette 100µL of lysing agent into the wells of a titration plate. The first 4 wells are for the 3 levels of controls (Deficient, Intermediate and Normal) and for a blank. Patient samples are placed in rows of 4s below the controls.

2. Blood in the EDTA tube is mixed by gently inverting the tube several times.

3. Pipette 5µL of whole blood/controls into the wells and shake the plate to mix the solution.

4. Incubate at room temperature for 10 minutes for complete lysis of the RBCs to release the enzyme.

5. Using a stick, take 2 drops of reaction mix and spot on to the absorbent filter paper.

6. Dry the spots completely for about 3 minutes with the aid of a hair-dryer.

7. View the spots under long-wave (365nm) UV light. Deficient samples will not show any fluorescence. Intermediate samples will show little or some fluorescence. Normal samples will fluoresce brightly.

8. The screening test for G6PD is based on the following principle:

Glucose-6-Phosphate + NADP+ --(G6PD)--> Gluconate-6-phosphate + NADPH + H+

The NADPH produced in the reaction fluoresces under long-wave UV light (365nm). No fluorescence will be observed if there is marked deficiency or lack of the enzyme G6PD. This screening test might be tricky at times, especially when there is little fluorescence. Some people may think it is deficient, while others think it is normal (i.e very subjective to which level of enzyme activity). Thus, if a technician is unsure of the result, he/she will consult another technician to confirm the result.

Ng Wei Shen Martin

0503312A

TG02

Haematology

2007-09-09T20:23:00.000+08:00

Cell Dyn 3500 is automated Full Blood Counter. With tons of specimens to prcess everyday, it comes along very handy as it take less than a minutes to generate results for the following:

WBC Impedance Count (WIC)
WBC Optical Count (WOC)
Neutrophils
Lymphocytes
Monocytes
Eosinophils
Basophils
RBC
Haemoglobin
Hematocrit (HCT)
MCV
MCH
MCHC
RDW
Platelets
Mean Platelet Volume (MPV) : measurement of the average size of your platelets. New platelets are larger, and an increased MPV occurs when increased numbers of platelets are being produced. MPV gives information about platelet production in your bone marrow.

WBC Optical Count (WOC) and WBC Impedance Count (WIC) are just 2 different methods used by the analyzer to assure correct leukocyte count.

The Reagents involved:

1.Diluent
Dilute samples so the analyzer can measure the various components. It also rinses aspiration probes to reduce carry-over from the previous test.

2.Lysate
-Rapidly lyse RBCs
-Strip white cells cytoplasm leaving the nucleus membrane intact so the white cells’ nuclei can be enumerated
-converts haemaglobin to a single chromogen that is measurable at 540nm

3.Detergent

4.Sheath Reagent
-osmotically lyse RBCs
-maintain light scattering properties of WOC during the measuring period
- prevent accumulation of air bubble in WOC flow system.

5.Enzymatic Cleaner
-removes protein build-up within the analyzer

Blood can be collected via venipuncture or finger-prick and sent to the lab in EDTA tubes (big purple tube and small pink tubes). After the blood has been checked for clots and is well-mixed, it is loaded into the analyzer. Clotted blood tubes are rejected as they cause low platelet count.

CD 3500 is able to aspirate blood in 2 modes- OPEN and CLOSE. Open mode involves manual positioning of blood tube to the aspirating probe. In the case of Close mode, the blood tubes just need to be place in the cartridge and the probe will automatically mix and aspirate blood.

After blood is aspirated into the Shear Valve (inside the analyzer), the valve will cut the blood into different segment for various testing.

WBC
1. WIC
Blood will be diluted and mixed with lysis solution. The diluted sample passes through a thin aperture where electrical impedance is used to count the WBC as they transverse through the aperture.

2. WOC
Blood is diluted and transferred to the WOC flow cell by the peristaltic pump. A laser beam is focused on the flow cell, as the sample stream (one cell at a time) intersects the laser beam, the light scattered by the cells is measured.

RBCs and Platelets
Blood is diluted 10,812 times. Electrical impedance is used to count RBCs and platelets as they transverse through the aperture.

Haemaglobin
Hb concentration is measured spectrophotometrically. Lysis solution converts Hb to hemiglobinhydrozylamine complex. Light-emitting didode (LED) with a wavelength of 540nm determines the amount of Hb, by capturing the light emitted, by a photodetector.

The rest of the parameters are reported based on the calculation automatically done by the analyzer.

cheers,
phuiyuen

2007-09-02T15:04:00.000+08:00

Receiving of Specimen
Check that the name, identification number of the form matches that of the bottles or the bags
Once every item received tallies with the request form, sign your name, stamp the time and write the date.
Sort the specimens out according to type, some specimens such as placenta require trimming, and must be put into a special box.

Scribing of gross description

Samples such as product of conception (POC), Breast biopsies and endometrial samplings have to go through this.

For POC, the information to be written down on the form are:
-The amount of sample used in milliliters
-If there is no more sample left after putting into a cassette, NR or No Reserve is written on the -form.
-The number of cassettes used is also written down

For breast biopsy, the information to be written down on the form are:
-The number of strips received
-The length, Breadth and width of strips
-The number of cassettes used is also written down
-The number of strips in each cassette is written down

Since the histopathology laboratory has to generate many blocks of tissue a day,
In order to differentiate them, different specimens have different colours.

-For paediatrics specimens, a yellow cassette is used
-For adult specimens, a green cassette is used
-For urgent specimens, a purple cassette is used
-For Post Mortem cases, a pink casstte is used.

Tissue processing

The cassettes containing the tissues are put into the Shandon Excelsior tissue processor,
It is an automated processor that removes the job of having someone to manually do the tissue processing process which takes about 18 hours. With the Shandon Excelsior tissue processor, the histopathology laboratory does not need to be a 24-hour laboratory, all the medical technologists have to do is to put in the cassettes at the end of the day and allow it to run overnight
Processes that the samples undergo during tissue processing:

Fixation
-A process that fixes the tissue samples in their current state so that the tissues can be studied
-Fixation is done with Formalin

Dehydration
-A process done for the removal of water
-Dehydration is done usually with alcohol, although other laboratories have been known to use alcoholic variants

Clearing
-Clearing agents used in the process must be miscible with both the dehydrating agent and the infiltration medium
-Clearing is done, to remove the dehydrating agents from the tissue and to prepare the tissue for addition of infiltrating medium
-Clearing is done with Xylene

Infiltration
-done with paraffin wax
-done to remove the clearing medium from the tissue and to prepare the tissue sample for embedding

Embedding

-The embedding process is done with the aid of the machine Sakura Tissue-Tek
-Prepare 3 different kinds of forceps for aiding of the tissue embedding process
-Soak them in in melted paraffin wax
-On the left panel of the tissue embedder, molds of different sizes are kept there and ready for use for tissue samples of different sizes
-The right panel is used to put the cassettes after they have been removed from the tissue processing machine
-Once the tissue has been removed from the cassette, it is placed into the the mold of an appropriate size
-Fill the mold with wax and use the forcep to press down the tissue, to ensure that the tissue sample is level. This ensures that shaving and sectioning would be easier
-Once the tissue is leveled, attach the base of the cassette to the top of the mold and fill up with more wax
-A piece of colour coded paper is embedded into the wax before it cools, this is done for tracebility purposes.
-The molds are then placed in ice to cool down
-Do not force the cassettes out of the mold, but instead wait for the paraffin wax to contract and become loose on its own
-In the cases where the cassette is still stuck on the mold after a period of time use abit of force but remember to place the tissue block back on the ice

Shaving

-The tissue blocks are shaved at 10 microns, to expose the full surface of the tissue to make sectioning easier
-It makes sectioning easier, as the person doing sectioning requires less shaving of the tissue block
-When shaving the tissue block, always check how much of the tissue has been exposed, as if too much is shaved off some parts of the tissue may be lost
-Some tissue samples such as fibroid are hard to cut as they contain calcium deposits, therefore they must be placed in softerner for 5 mins to soften the tissue again to make tissue sectioning easier
-After the tissue blocks have been softened, wash them under running water until the soapy feel is removed
-Dry the blocks before placing them on a cooler set at between –10 to –20 degrees, this makes sectioning easier

Sectioning

-Sectioning is done with either the leica or sakura manufactured rotary microtomes
-After the tissue blocks have been placed on the cooler for a period of time, they can be ready for cutting
-Set the microtome to cut at 4 microns
-After the appropriate section has been obtained, transfer it from the microtome to a water bath
-Fish the section onto a clean glass slide, before transferring it to another water bath
-The section is then fished back onto the same slide and left to dry off
-After the slides have been dried, they are put into racks to prepare for staining by baking in the oven at around 80°c for about 15 minutes

Routine H & E staining

-In the laboratory, routine Haematoxylin & Eosin ( H&E) staining is done on an automated machine the leica ST5010
-All we have to do is insert the rack of prepared slides (each rack can accommodate 30 slides) into the machine, ensure that the correct program is selected and then press load.

The machine has 16 different stations, and starts of with:
Xylene (removes wax)
Xylene
Absolute alcohol (hydration)
95% alcohol (hydration)
Haematoxylin (staining of the nucleus)
Haematoxylin
Ammonium (blueing)
Acid Alcohol
Eosin
95% alcohol
absolute alcohol
absolute alcohol
absolute alcohol
absolute alcohol
xylene
xylene

Once the process has been completed, the machine will sound alerting the user

Attaching of coverslip

Once the slides have been removed from the autostainer, they will be transferred into the Leica CV 5030, a fully automated glass coverslipper.
The machine individually applies Depex onto each glass slide before covering it with a cover slip.
Once the process is done, the machine will also alert the user that the process has been completed.

Slide sorting

The slides are then sorted according to case number, and compared to the original tissue blocks. If overlapping or folding of the tissue is observed, the tissue block would have to be recut to obtain a new slide.
Once the slides have been double checked and confirmed as okay, the appropriate labels are stuck on to them before the cases are dispatched to their respective pathologists to read the slides.

thanks

Randall

Tg02, 0503272G

Laboratory Management and Quality Assurance

2007-08-25T14:24:00.000+08:00

Hi guys...how are u guys? Anyway sorry for this late posting..just finish my AIMM exam on friday =)

Anyway here's a summary of biosafety level 2+, 3 & 4 and details about Biosafety Cabinet Class II

Biosafety Level: BSL-2+

Agents:
Agents handled are associated with human disease[In the research lab I am in, we handle Stenotrophomonas maltophilia (S. maltophilia) of different strains] By the way, in the research lab I am working,it is a BSL 2+ lab

Practices:

1.Only people with access can enter
2.All activities are performed in a BSC cabinet
3.Biosafety manual defining any needed waste decontaminated or medical surveillance policies are all filed into a file which is kept in the

Safety Equipments:
1. Class II Biological Safety Cabinets are used
2. Personal Safety Equipments (PPE) are to be worn
3. Pipeting devices are used & pipeting is prohibited

Facilities:
1. A sink and disinfectant are present for hand washing
2. An autoclave machine within the BSL-2 lab is present
In addition, self closing double door access is implemented.
The air is not recirculated and Lab air pressure is lower than adjoining rooms

Biosafety Level: BSL-3

Agents:
Agents which are associated with human disease and cause illness by spreading through air or aerosol

Practices:
Practices which implies for BSL-2+ implies for BSL-3
In addition, there is controlled access and decontamination of lab clothing before laundering

Safety Equipments:
Safety equipments which are used in BSL-2 are also used
In addition, respiratory protection is needed

Facilities:

Facilities present is BSL-2+ are also found in BSL-3
Other facilities includes
Physical separation from access corridors
exhaust air is not recirculated
Negative air flow into the laboratory

Biosafety Level: BSL-4

Agents:
Agents which are associated with human disease and cause illness by spreading through air or aerosol or have unknown cause of transmission

Practices:

Practices which implies for BSL-3 also implies for BSL-4
In addition
1. Personnel must change their clothing before entering the lab
2. There is a shower, which is found at the exit of the lab. This shower is used when personnel enters and leaves the lab
3. All materials are decontaminated immediately before disposal
4. All experiments or activities are conducted in a BSC class III in combination with full- body, air-supplied, positive- pressure personnel suit

Facilities:
Facilities present is BSL-3 are also found in BSL-4
BSL-4 lab is established in a separate building or isolated from other areas of the hospital or research laboratory
Dedicated supply and exhaust, vacuum and decontamination systems are also present in the BSl-4 laboratory

Biosafety Cabinet Class II

Biosafety Class II cabinet are designed to protect the laboratory environmnet, work material & even protects the suer from the exposure to infectious aerosols and splashesthat may be generated when manipulating materials conatining infectious agents. All users must attend and complete a BSC II training before they are allowed to used the equipment. Just like what we have learnt in Drug Development & Clinical Trails, training must be documeneted =). Bunsen burners cannot be used in the BSC II cabinets as heat produced will distort the air flow. As a result, this damages the HEPA filters thus affecting the shelf-life of the cabinet. Traffice behind the user should be minimised to avoid any shifts in airflow

I guess that I don't need to tell you guys about the operation of BSC II [It's almost like the laminar flow hoods we use in Mammalian Cell Technology =) ]. But if you want to tell you...just tell me

Just a point to take note is that instead of those plastic covers that are used to cover the hood when the hood is not in use, instead, a glass viewing panel, which can be pulled down vertically is used to cover the hood. Please note that the glass viewing panel must not be lifted beyond the safety level when the cabint is in use, otherwise the alarm will be sound. I will try to get a picture of the BSC II on monday.

That's all for it now...See u in the next campus discussion =)

Eugene Wong

TG02

0502076C

2007-08-18T16:49:00.000+08:00

Subject Title: Clinical Chemistry

Name of Test: Paediatric Bilirubin Measurement

Principle of Test: Using LEICA UNISTAT Biliruninometer

This is a direct spectrophotometer that measures total bilirubin in neonatal serum samples. Light is passed through the sample cuvette. The beam is split by a dichroic mirror and directed towards the 460nm and 550nm filters. The transmittance of the light exiting of the filters is measured by the photodetectors. Total bilirubin is calculated based on the output from the photodetectors.

Procedure:

1. Specimen is collected in a heparinised capillary tube that is sealed at the end and firmly affixed onto a wooden spatula to prevent breakage. As it is light sensitive, it should be protected from light to avoid photolysis of the bilirubin.
2. The capillary tube is place in pre-labeled tube according to the number assigned and spun down to separate serum.
3. The serum is then expelled in the clean cuvette by safepette and measured in bilirubinometer.
4. Result is obtained directly from the display.
5. Results have to be reported immediately by calling up the wards.

- To avoid errors in readings, the outside surface of the cuvette must be free of serum, dust, dirt and smudges; clean if necessary.
- The sample seen in the clear rectangular area of the cuvette must be free of bubbles, tap the cuvette lightly to clear them.

Test results/Reference ranges:

0-1 days : 0-86 umol/L
1-2 days : 0-122 umol/L
3-5 days : 0-175 umol/L
>6 days : 3- 24 umol/L

Clinical Interpretation:

Jaundice is a common, temporary and usually harmless condition in newborn infants. It affects both full-term and premature babies, usually appearing during the first week of the baby's life.

Jaundice occurs when there is a build-up of a naturally bilirubin in the blood. Bilirubin is an orange/red pigment which is produced by the breakdown of haemoglobin. As bilirubin begins to build up, it deposits on the fatty tissue under the skin causing the baby's skin and whites of the baby's eyes to appear yellow.

Severe hyperbilirubinemia is of great concern because it may lead to bilirubin-related brain damage (Kernicterus).

Performing bilurubin test is the first step in making sure that normal degrees of jaundice do not become more severe and can be treated early.

Treatment:

Bilirubin is destroyed by u.v light, phototherapy.

Loh Mun Jo-anne
TG02
0503324F

Medical Microbiology

2007-08-12T20:11:00.000+08:00

Hi, I'm currently working in the tissue culture and viral isolation section of virology lab. The virology lab consists of tissue culture, viral isolation, immunofluorescence and HIV labs. Here, I'll be using the example of HSV to describe the principle of viral isolation.

Isolation of the Herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2)

The diagnosis of the HSV-1 & 2 can be made from the isolation of the virus from lesions found in patient, by growing in cell culture. Swabs from the penile, vaginal or oral lesions are commonly used. The swabs are sent in Hank's transport media to the laboratory, so that the viruses remain alive, in order for us to isolate them. Different viruses grow best in different cell lines. For example, the dengue virus is sensitive to C6/36 mosquito cell line, but not in mammalian cell lines.

For HSV-1 & 2, it is most sensitive to cell lines from Mink Lung (ML) and Human Embryonic Lung (HEL). Basically, the sample will be inoculated into healthy ML and HEL cell line and incubated in 36°C and observed for cytopathic effect (CPE) daily. Healthy cell line refers to the absence of cell degeneration and cell line with around 70% confluency, when viewed under light microscope is used. This can be vague at times, but with enough experience one should be able to gauge the confluency of the cell line. Uninoculated tube of the same batch of cell line for ML and HEL will be incubated, which acts as a control. This allows us to decide whether any morphological changes in the inoculated cell are due to viral infection or degeneration of cell line. Typically, CPE for HSV infected cells occurs within 7 days after inoculation, after which the virus is identified by fluorescent-antibody staining of the infected cells. However, some slow-growing viruses, such as enterovirus and measles would require a longer time to show CPE, 21 days and 14 days respectively.

CPE is a result of virus infection of cell, which leads to many changes to host cells. Some changes include, altered shape, cell lysis, membrane fusion, and apoptosis. In the case of HSV-1 & 2 infections, a 'ballooning' effect can be seen under the light microscope. The cells will round up and look like tiny balloons. This is indicative of viral infection. Thus, a confirmatory test using direct immunofluorescence test (IF) is carried out. Since there are two types of HSV, monoclonal antibody is used to identify the type of HSV. The principle of this test is similar to the CMV IF test I posted earlier on. The only difference is that the monoclonal antibody is conjugated with Fluorescein isothiocyanate (FITC), which exhibits green fluorescence when excited by ultraviolet light, indicating the presence of HSV infected cells.

Yea, I guess that's about it.

Yong Yang (TG02)
0503196H

Clincal Chemistry

2007-08-06T21:57:00.000+08:00

Yo. It's just 14 weeks to go to the end of SIP. 14 weeks! I will be describing the blood ketones tests here.

Blood Ketones Test

Blood ketones are tested using the Optium Xceed handheld meter. The basis for this meter is electrochemical. This is a very easy gadget to use. First, a new test strip is inserted into the strip-port. An ‘apply blood’ (it shows a blinking drop) icon will show. Then, apply 1 drop of blood onto the other end of the test strip. Wait for about 30 seconds and the results will be shown on the screen. However, it must be ensured that the blood samples are sent in lithuim heparin tubes (green cap). The heparin acts as an anticoagulant. Blood delivered in any other types of blood tubes are rejected. The reference range for this test is 0.0 – 0.6 mmol/L. Critical values (> 1.4 mmol/L) must be reported immediately to the appropriate personnel, so that action can be taken by the doctor to treat the patient.

Calibration is done for every new box of test strips by inserting a CODE key into the strip-port to check for the correct CODE number.

Controls are done only when there are requests for blood ketones, mainly because there are very few requests for blood ketones (about 1 a day). There are 2 levels of controls; one of a low value and the other high. Only 1 level of control is done when there is a blood ketone test, and the controls are tested alternatively (i.e. one day use control of high value, the next time use the control of low value).

Blood ketone testing is usually done on patients with diabetes. In diabetes, there is not enough insulin or insulin activity to help the muscles to absorb glucose from the blood. When there is not enough glucose, the body will burn fat for energy. Breakdown products, or ketones, are formed. Ketones are toxic to the body. Accumulation of these acids in the body may lead to ketosis. A more serious disease is known as ketoacidosis or DKA.

Why use blood and not urine for measurement?
There are 3 types of ketone bodies – acetone, acetoacetate and β-hydroxybutyrate. β-hydroxybutyrate makes up 75% of all ketones in DKA. Therefore, it is more relevant to measure β-hydroxybutyrate in blood rather than urine ketone tests based on the nitroprusside reaction which detects acetoacetate instead. Urine ketone tests also give false positive results in the presence of some drugs and false negative results when the test strips are exposed to air for too long.

That's it for now.

Martin Ng (TG02)

0503312A

Cytogenetics

2007-07-29T20:53:00.000+08:00

Sample collected: Aminotic Fluid (AF)

Purpose:
Check the fetus’ chromosomes at metaphase to see if it is normal.

A normal set of chromosome has
- a total chromosomal count of 46 consisting of a pair of each chromosome(1-22) and the sex chromosome- either XX or XY.

1) Detect abnormalities in fetus of women with advanced maternal age
With a maternal age of 35 and above, the chances of carrying a fetus with genetic abnormality increases. Common genetic abnormalities include Down’s syndrome (Trisomy 21) and Edward’s syndrome (Trisomy 18).

2) Doctors noticed abnormalities during ultrasound
During ultrasound, doctors notice something amiss about the baby’s condition and with may send AF out for test.

Why Aminotic Fluid?
AF is present all around in the placenta (which holds the baby). As the baby grows, it will shed cells in to the AF. Therefore, the AF contains baby cells that can be cultured and analysis for chromosomal abnormality.

How is it process?

Collection – Processing (Set-up) – Harvest – Drying – Baking – Staining -- Analysis

Firstly, the AF will be equally allocated into 3 tubes, one of which will be kept as a back-up. The tubes will then be centrifuged. After the supernatant is removed, culture media is added to resuspend the cells. Different media e.g. (will usually use) BioAMF and Chang’s Media will be used in the 3 tubes, this is part of quality control.

Secondly, the cell suspension will be aliquot into 4 petri dish containing cover-slips. They will then be incubated and the cells will eventually grow directly on the cover-slips.

After the cells have grown to form about 4 colonies, they will be harvested. Harvesting is done automatically using an auto-harvestor- Tecan. Tecan is able to carry out the entire harvesting process independently and during this period the technologist is able to carry out other duties.

Tecan will carry out the following processes:
-remove the culture media via a pump
-add hypotonic solution (KCl)
-timed an incubation period of 30mins in hypo
-remove hypo
-add fixative- made up of methanol and acetic acid.
-timed an incubation of 30mins in fixative
-change another round of fixative

When cells are exposed to hypotonic solution, they will swell and this allows the chromosomes to spread out and not clump together (which will be difficult for analysis). The addition of fixative removes water and preserves cells in its swelled-up state.

After the harvesting process is done, the Petri dish containing the cover-slip will be transferred into the Thermotron for further removal of water aka Drying. Drying involves the addition and removal of fixatives up to 3 times for complete dehydration of cell and to spreading the cells out. Thermotron is a incubator that facilitates the drying process, it is able control the humidity and temperature thus promoting optimal drying. This process is important as:

- if drying is too fast(low humidity, high temp), chromosomes won't be able to spread out effectively
-if drying is too slow(high humidity, low temp), chromosomes will spread so much that it goes beyond the cover-slip and thus will be washed away.

Next, the cover-slip will be remove from the Petri dish and be mounted on a glass slide. The glass slide will then be baked in the hot plate at 90°C for 11/2 hours; this is to fix the cells on the slide to prevent it from floating away during staining. Baking also causes the A-T bonds to dissociate thus allowing the dye to be taken up by the chromosome.

After which, cells will be stain in either Wright’s or Geimsa to give a visible/prominent light and dark bands on the chromosomes for analysis. The staining process involves the trysinisation of cells. Trypsin hydrolyses protein component of the chromatin thereby allowing the dye to react(dye) with the exposed DNA.

Finally, after staining, it's ANALYSIS time. Real-experienced med techs will study and identify the chromosomes and detect any deletion, addition or presence of extra chromosome with their naked eyes. They're real quick in identifying them!

Alrights, here's the end, if there's any question, just bring it on =)

phuiyuen

2007-07-22T13:12:00.000+08:00

Wow time sure flies, 4 weeks have gone by... and i've just ended my month long stint at the microbiology lab. I'm looking forward to cytogenetics! For a couple of reasons im sure you people can think of! =)
Alright basically i've learnt tons of new stuff at microbiology but i'm going to relate to you about my experiences of working the "Affirm VPIII Microbial Identification test" by Becton Dickinson. Basically the whole process of the experiment/test, involves both manual and automated parts.d Sample preparation is done manually, while the processing is done by the machine BD MicroProbe. I will elaborate more below.

Introduction
The Affirm VPIII Microbial Identification Test is a DNA probe test, used by the laboratory for the detection and identification of Candida species, Gardnerella vaginalis and Trichomonas vaginalis nucleic acid in vaginal fluid specimens from patients with symptoms of vaginitis.

Vaginitis is one of the most common problems observed in clinical medicine. There are 3 main categories of vaginitis, they are bacterial vaginosis (BV), yeast vaginitis (Candidiasis) and trichomoniasis vaginitis. BV is the most commmon form of vaginal infection, followed by vaginal candidiasis and trichomoniasis. This vaginal infections are clinically significant especially in pregnant woman, as they can lead to pre-term labor and birth and various adverse pregnancy outcomes.

Principles of the procedure
The Affirm VPIII Microbial Identification Test is based on the principles of nucleic acid hybridization. In nucleic acid hybridization tests, complementary nucleic acid strands join to form specific, double-stranded complexes called hybrids.

The test uses 2 specific single-stranded nucleic acid probes for each organism, a capture probe and a colour development probe. These 2 probes are complementary to the distinct genetic sequences of the target organism. The capture probes are immobilized on a bead embeded in a Probe Analysis Card (PAC). Each of the three organisms being tested has a separate bead on the PAC. The colour development probes are contained in a mutli-well reagent cassette.
The various components are put into the BD microprobe machine and run. At the end of the run, if there is even a slight colour change on any of the organism beads, it would indicate that that the specific organism is present in the patient's sample. Note that there are 2 beads that act as a positive and negative control. After the run, if the positive control bead does not show a change of colour or if the negative control shows a change of colour, it means that the results of the run are obsolete and that the sample must be rerun.

Procedure's after receiving patient sample

Label a sample collection tube (SCT) with the patients identification no.
Take the swab obtained from the patient and and cut it such that the SCT can be closed.
Drop 12 drops of lysis solution to the tube.
Mix the swab in the tube with the lysis solution.
Cover the tube with a cap and place in a lysis block of 85 degrees for 10 mins.
Remove the tube from the block and add 12 drops of buffer solution to the tube.
This time add a filter cap to the tube
Prepare the PAC and write down patients id no. in the space.

Prepare the reagent casette

Add 4 drops of substrate solution to well no.7 of the reagent cassette.
Drop the patients sample into well no.1 of the reagent cassette.
Place the PAC into well no.1 of the reagent cassette.
Press the run key to start the process and wait for about 32-35 minutes for the process to be done.

Reporting of results

Since we students do not have access to the hospitals system, we can only write down the test results on the test request form and wait for a medical technologist to come by verify the result and key the results in.

If the tests results show that the patient is negative for all 3 organisms, we write on the test request form "All -ve/ All Neg".

If the test results show that the patient tests results show 1 or more postive for the disease we write it on the test request form as eg. Gard +ve (Gardnerella positive)/ Cand +ve (candidiasis).

Thats all for my report! i'll get back to you all if you have any comments or queries.

Thanks

Randall Chua (TG02) 0503272G

Biosafety Laboratory level 1 and Biosafety Laboratory level 2

2007-07-14T09:46:00.000+08:00

Hi...Hope you guys have fun in the places you have been attached to. Basically I am attached to Temasek Polytechnic Reasearch Laboratory (AS4). Today I am going to talk about Biosafety Laboratory level 1 and 2.

Biosafety Laboratory level 1

Biosafety Laboratory level 1 commmoly known as BSL 1, can be used for micro-organism or chemicals classified as level 1.Basicall there are 4 classes ranging from class 1 to class 4. Micro-organism classified under class 4 are considered very dangerous. In AS4, class 3 or 4 micro-organism are not handled. BSL 1 is suitable for work involving well- characterized agents that are not known to cause disease in healthy adult humans and of minimal potential hazard to laboratory personnel and the environment. Work is done/ conducted on open bench top using standard microbiological practices.

The following standards, special practices, safety equipments and facilities apply to agents assigned to BSL1:

Acces to the lab is limited when experiments with culture are in progress
Eating, drinking, chewing gum or smoking are not permited in the lab
Mouth pipetting is prohibited
All procedures are performed carefully to minimize the creation of splashes &/or aerosol
In the BSL1, there are sinks for handwashing. All personnel must wash their hands, after they handle viable materials, after ther remove their gloves, before they leave the lab
Work surfaces are decontaminated at least once a day with 1:50 dilution of 5.25% NaOCl
If any spillage of micro-organism or chemicals occurs, the spillage area must be soak with 1:5 dilution of 5.25% NaCl
All cultures, stocks, contaminated needles, sharp equipments, broken glasses(such as glass pipettes), must be decontaminated before disposal, by autoclaving
Broken glasses must not be handled directly by hand but must be remove by a brush and dustpan
A biohazrad sign must be posted at the entrance of the lab. This only occurs if infectious agents are persent. The sign must include the name of agents i use, the name and phone no. of the investigator
Personnel Protective Equipments (PPE) such as gloves, lab coat must be worn in the lab at all times
Goggles should be worn, if the personnel wears contact lense or does not wear spectacles, when splashes of hazrads chemical or micro-organism is anticipated
Bench top in BSL1 are impervision to water and are resistant to organic solvent, acids, alkalis, moderate heat.
The bench top n BSL1 is also resistant to chemicals which are used to decontaminate the work surface and equipments
BSL 1 is a "pressured" laboratory. That is it prevents dirty air from entering the lab when the door is open

Biosafety Laboratory 2

Biosafety Laboratory 2 is similiar to BSL 1 lab. it is suitable for work involving agents of modertate potential hazard to the personnel and the environmnet. Nevertheless there are some differences. In BSL2, lab personnel have specific training in handing pathogenic agents and are directed by a trained personnel. Acces to the lab is restricted when work is being conducted. Extreme precautions are taken with contaminated sharp items and certain procedure in which infectious aerosol or splahes may be created in the biosafety cabinets.9details are below)
The standards, special practices, safety equipments and facilities apply to agents assigned to BSL1(point 1-15) also applies to BSL2. In AS4 , instead of BSL2,AS4 has 2 BSL 2 plus lab. One for micro-organism work while another one is for mammalian cell culture work

Apart from those ponits, there are a few more points personnel must follow

The laboratory director establishes polices and procedure whereby people who have been advised of potential hazards and meet specific entry requirements may enter the laboratory
Biosafety procedures are incorporated in to Standard Operating Procedures (SOPs) for the lab by the lab director
Personnel working in BSL2 are advised of specific hazards anf are required to read and follw instructions on practices and procedures
All personnel working in BSL2 must recieve appropriate training on the potential hazards associated with the work involved, the necessary precautions to prevent exposure to hazardous materials and evacuation procedures
Personnel Protective Equipments designated for BSL2 use are worn in BSL2. This protective clothing is removed and left in the lab before leaving for other lab or non-lab areas

I hope you guys had fun reading and understood everything. I hope I am able to answer all your questions. Good Luck and Good bye for now.

Eugene Wong TG02

2007-07-07T15:45:00.000+08:00

Hello. It is the end of the second week. I am having lots of fun in Biochem lab. =) ... we had a fire drill just yesterday and attended a staff meeting! and so far we have gone for two tutorials on LIS and Lab Safety. Not going to elaborate on that as i think everyone knows what it is about already. The five of us was split into 5 stations and we will be rotating every 2 weeks. For the last 2 weeks, i was posted to Station A (DD). Beckman Coulter Unicel DXI is one of the analyzer in Station A (DD). The other analyzer used is DTSCII and DT60II. In this post, I will elaborate on DXI.

Quantity can be determined for Serum ferritin, folate, RBC folate, Vitamin B12, Cortisol, Urine Cortisol, PTH, testosterone, D-HEAS and BHCG using DXI. It is a little similar to the analyzer in school that we used during practical. DXI has a continuous loading capability and approximately 120 samples can be loaded at once. Racks can be loaded directly from the centrifuge and is released in less than 5 minutes for other testing. This increases productivity and efficiency. When loading into analyzer, the sample must be checked for fibrin clots. Amount of plasma must be above 1cm, if not, it must be transferred to a plastic tube. DXI uses paramagnetic particle chemiluminescent immunoassay for quantitative determination. Light generated by the reaction is measured with a luminometer. Amount of analyte in the sample is determined from a stored, multipoint calibration curve. If results are too high, dilution is required. DXI has an on board dilution for ferritin. If after dilution, it is still high, do 3X manual dilution. Folate and B12 do not require dilution. The rest is manually diluted with either saline or SO calibrator.

Calibration is done once a month, when a new lot of reagent is used, on expiry, QC is unacceptable or after major maintenance procedures or replacement of parts. Calibration determines the relation between the result and the value of the input quantity.

Maintenance
1. Backup System
There is an auto system backup at 5am.
The cartridge is changed daily in case of breakdown.
2. Back up daily data system
3. Check inventory
Loading bulk supplies
-Substrate- equilibrated to room temperature under reagent load door, able to remain at room temp for 14days.
-Wash buffer- used to dilute samples/reagent, wash away unbound materials in RVs, wash pipettor probes and prime system.
-Reaction Vessels- store samples on board, prepare sample dilution, incubate sample with assay specific reagent during processing.
-Liquid waste generated when- pipettors are washed between processing steps and when unreactive fluid are washed into RV.
-Solid waste- collect empty reagent packs, used RVs, and condensation during processing.
-Reagent- If test not done so often, don’t keep adding reagent – not fresh.
4. Record test count of previous day
5. Shake solid waste container – radioactive wastebag is used
6. Run daily special clean routine – not the normal cleaning because Vitamin B12 reagent is sticky. Contrad, Citranox, Methanol 70%

Quality control is important and has to be done daily to keep the analyzers in top condition and results produced are accurate and precise. Charts are compared and if controls are not within the 2SD mean, QC must be done again.

Biorad Lyphocheck Immunoassay Plus Control Levels 1- 3 and PTH Control Levels 1-3 are done daily. PTH control is run twice as it is running 24hrs. Controls are parafilmed and stored in the fridge to prevent deterioration of the constituents. Liquickek Urine Chemistry Control Levels 1- 2 for Urine free Cortisol and RBC folate Control Levels 1- 3 are done when needed. If 2 out of 3 controls are in, patient samples can be loaded.

Principles
1. Ferritin- two site immunoenzymatic “sandwich” assay. Light production is directly proportional to concentration of ferritin.

2. Vitamin B12- competitive binding immunoenymatic assay. Photon production is inversely proportional to concentration of ferritin.

3. PTH – two site immunoenzymatic “sandwich” assay. Light production is directly proportional to concentration of ferritin.

4. DHEA- competitive binding immunoenzymatic assay. Light production is inversely proportional to concentration of ferritin.

5. Testosterone - competitive binding immunoenzymatic assay. Light production is inversely proportional to concentration of ferritin.

6. Cortisol- competitive binding immunoenzymatic assay. Light production is inversely proportional to concentration of ferritin.

- Serum cortisol
- 24hr Urine free Cortisol- Two sets of controls are used in case the first set is out of range. Ethyl acetate is added which produce two distinct layers as acetate density is lighter. It is used to extract cortisol from urine. 200ul is pipetted out from the top layer and left overnight in the fumehood to dry up. SO calibrator acts as a diluent to dissolve the precipitated cortisol. Run 1 set of control first. This reduces cost as a bottle of SO calibrator is very expensive. If not within range, use second set of control. If within range, run patient sample on DXI. Procedures are done in fume hood as acetate has a strong odour and is carcinogenic. Glass tubes are used as plastic react with acetate. As glass tubes are used, centrifuge speed is 2500 rpm to prevent breakage.

7. Folate- competitive binding receptor assay. Light production is inversely proportional to concentration of ferritin.

- Serum or Plasma (heparin) Folate – do not use hemolysed sample. Folate level in red cell is much greater than serum or plasma, lead to spuriously high results.
- RBC folate – contains EDTA- prevent clotting – whole blood or plasma. Specimen sent to Haem lab for HCT. Lysing solution added to release folate in RBC from endogenous binding proteins. Placed in cupboard for 1 ½ hrs as lysing solution and the lysing reaction is light sensitive. Run controls to see if they are within range before loading patient samples.

Thats all for now.

Jo-anne
TGO2
0503324F

Medical Microbiology

2007-06-30T13:01:00.000+08:00

Name of Test:
Light Diagnostic Cytomegalovirus (CMV) pp65 protein in periheral blood leukocytes

Principle of Test:
The test utilises the indirect immunofluorescence technique for identifying human CMV pp65 antigen in the blood leukocytes. Monoclonal antibody is added and bound to CMV pp65 antigen on formalin fixed leukocytes and incubated. Excess antibody is washed with phosphate buffered saline (PBS). Fluorescein isothiocyanate (FITC) conjugated antibody, also known as secondary antibody, is bound to the antigen-antibody complex and incubated. Excess antibody is washed with PBS. FITC exhibits green fluorescence when excited by ultraviolet light allowing visualization of antigen-antibody complex by fluorescence microscopy.

Test Result with reference range:
Control slides should be prepared from known positive and negative blood samples, for each batch of test. Positive result emits green light, while negative result emits dull red.

Clinical interpretation:
The test is used for the detection of CMV in blood leukocytes, which is closely associated with the clinical manifestation of the CMV disease. This will be useful in the diagnosis of CMV infection. The test is rapid and sensitive for detecting CMV in isolated leukocytes; this will prevent any delay in treatment. However, if negative and positive controls cannot be clearly distinguished, the test is considered invalid.

Student name: Foo Yong Yang

2007-05-22T20:56:00.000+08:00

After many hours of discussing and contemplating, the star team members came to an agreement that the list of 5 diseases should be cut down to 3.

the 3 diseases we will be shortlisting are:

Leptospirosis
Murine Typhus
Salmonellosis

2007-05-22T20:39:00.000+08:00

Sample Collection:

From the 85 year old man, we will be collecting faeces and blood samples from him.

Collection of the stool samples are for tests to determine the presence of diseases such as Salmonellosis and Campylobacteriosis.

For Salmonellosis, the faeces sample is cultured and a smear is created. Microscopic examination and gram staining is done.

For Campylobacteriosis, the faeces sample is also cultured and a smear is created. As with Salmonellosis, gram staining and microscopic examination is carried out. The only addition is dark field microscopy.

Collection of blood samples are for tests to determine the presence of the three diseases Murine Typhus, Leptospirosis and Lyme Disease.

For Murine Typhus, serum from the blood sample is tested for antibodies to the microorganism Rickettsia typhi using Immunofluorescence Assay (IFA).

For Leptospirosis, Microscopic Agglutination Test (MAT) is used, to detect Leptospira which agglutinates with commercial antibodies.

For Lyme Disease, serum is obtained to run through Enzyme Linked Immunosorbent Assay (ELISA). ELISA is done to test for IgG and IgM.

2007-05-22T20:21:00.000+08:00

After much discussion, we have finalized the 5 possible disease the 85 year old patient is suffering from.

(1) Salmonellosis

(2) Campylobacterosis

(3) Lyme Disease

(4) Murine Typhus

(5) Leptospirosis

2007-05-09T01:33:00.000+08:00

We think 2. Allegic Reaction cannot be classsify as a disease, it is just an adverse reaction to the bacteria he is exposed to. So, we've replaced it with Leptospirosis.

2. Leptospirosis is a disease cause by leptospira which is commonly found in rodents and dogs. They are capable of penetrating the skin through cuts or wounds. People are infected when exposed to water contaminated with infected animal's urine or when handling tissues of infected animals. Disease is most common in tropical months. Patients down with leptospirosis develop influenza-like illness with fever and myalgia(muscle pain) in the initial stage.

Reason: Patient's flat is infested with rats and he has a habit of feeding stray dogs and cats in which all these are the possible carriers of leptospira thus exposing himself to infection. There is also a high chance he may be exposed to the rats' urine and accidentally consumed water or food contaminated by them. Leptospirosis develop symptoms which are similar to what he is experiencing. Moreover, Singapore has a tropical climate which makes an optimum environment for Leptospirosis.

Source:
Medical Microbiology, 5th edition

2007-04-25T11:09:00.001+08:00

1. Disease: Campylobacteriosis is a disease caused by bacteria Campylobacter Jejuni. Symptoms of Campylobacteriosis include fever, diarrhea, cramping, abdominal pain and vomiting. The transmission of the Campylobacter Jejuni bacteria is most often through the handling of infected raw poultry, or the consumption of raw uncooked or partially uncooked poultry.

Reason: As the 85 year old man is living alone, he has to do all the housework himself. This includes preparing and cooking his own meals. He may have mishandled the preperation of the poultry meat or not cooked it thoroughly before eating it. Thus leading to him being infected with Campylobacteriosis.

Sources:
http://www.cdc.gov >Campylobacteriosis>Disease Listing, Campylobacter, General Information
http://en.wikipedia.org >Campylobacteriosis

2. Disease: Allergic reaction. Pet allergy is provoked by the fur of cats and dogs. Researchers have found that the major allergens are proteins secreted by oil glands in the animals' skin and shed in dander as well as proteins in the saliva, which sticks to the fur when the animal licks itself. Urine is also a source of allergy-causing proteins. When the substance carrying the proteins dries, the proteins can then float into the air. Cockroach calyx and dust mite excretion will also cause allergy leading to rashes.

Reason: He had contact with animals and spend time with them(feeding) thus the fur, urine and saliva of the animals caused the rash on the old man. As his living conditions was poor ( premise was infested with cockroach, fleas), cockroach calyx and dust mite excretion caused the rashes on him.

Sources:
http://www.wrongdiagnosis.com >symptom search>rash
http://www.wrongdiagnosis.com >animal allergies

3. Disease: Murine Typhus is a disease caused by the microorganism Ricketssia Prowazekii, the microorganism can be spread by rats, fleas, dogs and cats. Poor hygiene contributes highly to the spread of this disease. As shown by the feces and urine on the mattress.

Symptoms: Headache, chills, fever, prostration, confusion, photophobia, vomitting, rash (generally starting on trunk).

Source:
http://www2.ncid.cdc.gov/ >traveller's health> Search> Ricketssial Infection> CDC -yellow book: [4] Ricketssial Infections- CDC traveller
http://en.wikipedia.org/ >search> murine Typhus>

4. Disease: Lyme Disease is a disease caused by the bacteria Borrelia Burgdorferi, humans can get infected by being bitten by infected ticks found on animals, such as rats and mice.

Symptoms: skin rash, fever, headache, muscle pain, stiff neck and swelling of he joints
.Source:
http://www.cdc.gov > Search > Lyme> DVBID homepage:CDC Lyme disease
http://en.wikipedia.org > search> lyme> lyme disease

5. Salmonellosis is a bacterial disease caused by the bacterium Salmonella. Many different kinds of Salmonella can make people sick. Usually, people get salmonellosis by eating contaminated food, such as chicken or eggs. However, animals can carry Salmonella and pass it in their feces (stool). Therefore, people can also get salmonellosis if they do not wash their hands after touching the feces of animals.Dogs, cats, birds (including pet birds), horses, and farm animals can also pass Salmonella in their feces. A person's age and health status may affect his or her immune system, increasing the chances of getting sick.

Symptoms: Most people have diarrhea, fever, and stomach pain that start 1 to 3 days after they get infected.

Source:
http://www.cdc.gov/healthypets/diseases/salmonellosis.htm >search > Salmonellosis > 2nd link.

2007-04-25T10:40:00.002+08:00

Possible List of Diseases
Toxoplasmosis
Murine Typhus *
Rat Bite Fever
Leptospirosis *
Trichinellosis *
Campylobacteriosis *
Lyme Disease
Schistosomiasis
Plague(Black Death)
Salmonellosis *
Typhoid Fever
Hantavirus

* - denotes 1 of the 5 shortlisted disease...

2007-04-25T10:40:00.001+08:00

MMic Tutorials 1 to 4: A Stimulated Case

A 85 year old male was found to be staying alone in one-room flat. It is found that his mattress was soiled with human excreta and urine. The premise is infested with cockroaches, fleas and rats. It is known to the neighbors that the elderly man has the habit of feeding stray dogs and cats in the neighborhood.

The elderly man was referred to a nursing home and presented with high fever, rash and general weakness.

How would you approach this situation in order to provide final diagnosis of the suspected microorganisms?

Our Expectations...

2007-04-25T10:40:00.000+08:00

As a group we aim to be the best. Outdo everyone, be the leader of the pack... In other words "ULTIMATE SUPREMACY!!!"

As group members we aim to help each other overcome our individual weaknesses, sharing information and experiences that we may garner throughout the course of the academic year.
Starting from today: members of the group will work with this mantra:
"Aim for the stars, even if you miss you'll hit the moon".