MMIC - PBL2. Avian Flu

Avian Influenza (Bird Flu):

Pathogen involved: Avian influenza viruses (RNA viruses)

commons.wikimedia.org > wiki > Image

· Typically carried by wild migratory birds in their intestines, which are immune to them. However, it is highly contagious amongst birds, and can infect domesticated birds, that usually fall severely ill.
· Several strains exist:
Denoted by “H" and “N”. “H” stands for hemagglutinin and “N” stands for neuraminidase, two proteins on the surface of the virus that allow it to enter and exit host cells.
o Sixteen different hemagglutinins and nine different neuraminidases have been identified to date.
o Typically, most avian flu is restricted to bird to bird transmission. The most deadly being H5N1 strain as it can spread from bird to human.

Indonesia has had outbreaks of H5N1 viruses, mainly from the poultry breeding farms. Typically, H5N1 transmission is from bird to humans. However in 2006, there was a case of human to human transmission of H5N1 versus between a group of small families, but the spread appears to have died off. H5N1 can last indefinitely at a temperature dozens of degrees below freezing.

Pathogenesis:
Spread to humans via contact, potentially airborne (incubation period is longer, less adapted to droplet transmission):
· Directly from birds or from avian virus-contaminated environments to people (consumption of poultry products, direct contact with live poultry – bodily fluids such as blood, salvia etc and contaminated food sources).
· Through an intermediate host, such as a pig.
Typically develops 1 to 5 days following exposure.

Signs and symptoms:

Common:
- Cough
- High fever (typically > 38°C)
- Headaches
- myalgia (muscle ache/pain)
- malaise (general discomfort)
- Sore throat
- Shortness of breath
- Diarrhoea
- A relatively mild eye infection (conjunctivitis), sometimes the only indication of the disease.

Severe signs and symptoms:
- Viral pneumonia
- Acute respiratory distress (the most common cause of bird flu-related deaths)
- Seizures

Necessary precautions:
· Avoid contact with live birds, and all forms of poultry:
- Chickens, ducks, turkeys and geese and their feces, feathers and pens if at all possible.
· Avoid poultry products in Indonesia, as cases of H5N1 have been reported there.
· All foods from poultry, including eggs should be cooked thoroughly. Egg yolks should not be runny or liquid. Influenza viruses are destroyed by heat, hence cooking temperature for poultry meat should be 74oC (165 F).
· Avoid cross contamination of other foods by use of separate kitchen utensils and surfaces exposed to raw poultry.
· Wash hands with soap and water after any poultry contact.
· Avoid live food markets.

Treatment:
A) Antivirals:
Suppress virus, keep it from replicating and infecting within the host. Must be taken soon (often within 48 hours following infection).
· Neuraminidase inhibitor:
Mode of action consists of blocking the function of the viral neuraminidase protein, preventing the virus from reproducing.
1. Relenza
2. Tamiflu

B) Vaccination:
Vaccines expose an individual to a weakened/dead virus to stimulate antibody production against it, so that immune system can fight off infections should it arise.
· There are at least 15 different strains of avian flu and they are constantly mutating, hence vaccination may not prove to be effective for long.
1. Live vaccines (attenuated, weakened):
- Requires less antigen (active ingredient) than killed vaccine.
- Live vaccine may contain too few copies of the weakened virus to trigger an immediate immune response.
- However, once inside the host, the virus can replicate to render it detectable by the immune system and trigger an immune response.
- Does not require injection – oral consumption will do.
2. Killed virus (inactivated):
- Must be injected – only route to administer them that will bring them into contact with the immune system.
Requires larger dosage than live vaccines due to its lack of ability to multiply within host.
3. Recombinant vaccine:
- Genetically engineered vector (usually a low virulence virus) to express H5N1 protein antigen on surface, to stimulate immune response (production of antibodies).

References:
sciencedaily.com > releases > 2007 > 08/070828154944.htm
cidrap.umn.edu > cidrap > content > influenza > avianflu > news > dec3005halvorson.html
evolution > berkeley.edu > evolibrary > news > 51115_birdflu
http://www.cbc.ca/> news > background > avianflu > protection.html

Cheers,

Debra, TG02

MMIC - Follow up on Specimen 3.

Specimen 3:
Preliminary Information:

Patient: Maisy Hong (67 years)
Sample: Urine
Symptoms: Fever, chills, bladder distension; on indwelling catheter
Bladder distension: Inability to urinate. May be due to obstruction.
Indwelling catheter: prevent catheter from being pulled out of the bladder

Diagnosis: Urinary Track Infection

From week 1:

Possible causative agents: catheter- associated UTI

Most probable agents: according to incidence seen in UTI patients
1) Escherichia coli – 53-72%
2) Klebsiella spp. (pneumoniae) – 6-12%
3) Proteus spp. (mirabilis) – 4-6%
4) Enterococcus spp.(faecalis) – 2-12%


Additional suspected agents:
5) Pseudomonas spp. (aeruginosa)
6) Enterobacter spp. (aerogenes)
7) Serratia spp. (marcescens)
8) Candida spp. (albicans): may cause UTI, associated with use of catheter

Note: Chlamydia and Mycoplasma were excluded as these are typically sexually transmitted, highly unlikely to be the cause in a 67 year old woman with a catheter

Preliminary tests:
Specimen: Urine (dried on slide)

Stains:
Fungal stain
* Periodic acid-Schiff (PAS) can be used to detect if causative agent is fungi.
Gram stain
*Used to detect if bacteria is
- gram negative
- Escherichia coli (rod)
- Proteus Species (rod)
- Enterobacter Species (bacilli)
- Klebsiella Species (bacilli)
- Pseudomonas Species (bacilli)
*gram positive
- Enterococcus Species (cocci)

After the above test have been done, it would be clearer as to whether it is a fungi or bacteria. If it is a bacteria, it would have been narrowed down to gram type. Further screening as shown below for each individual suspected agent will then be performed if agent falls under the initial testing results.

Individual tests:

Escherichia coli
Morphology (microscopy): Gram negative bacilli, singly.
Pathogenesis: Adherence factor P fimbria binds specific galatose disaccharide found on uroepithelial cells surfaces. Siderophores produce cytotoxic alpha and beta hemolysins for lysis of urinary track cells for iron acquisition.
Key Characteristics: Rapidly ferment lactose, positive indole test, positive B-glucaronidase (using substrate MUG), facultative anaerobic (mixed-acid fermentation)
Tests:
1) EMB (Eosin Methylene Blue). Detect lactose fermenting colonies. Contains eosin and methylene blue.
· Black colonies with green sheen (positive, only E.coli can produce this colour due to acid production)
2) MacConkey Agar. Contains bile salts, crystal violet dye (to inhibit Gram-positive bacteria), neutral red dye (which stains microbes fermenting lactose).
· Red colonies (positive)
3) Indole test. Measures the ability of the microorganism to degrade tryptophan into indole, ammonia and pyruvic acid. Pure bacterial culture must be grown in sterile tryptophan or peptone broth for 24-48 hours prior to test. Following incubation, add 5 drops of Kovac's reagent.
· Red or red-violet color (positive)
4) Methyl Red (MR).
· Red (positive)
5) Citrate test. Utilizes Simmon's citrate media to determine if a bacterium can grow utilizing citrate as its sole carbon and energy source
· Green (negative)
6) MUG (on nutrient agar). Nutrient Agar with methylumbelliferyl-ß-D glucuronide (MUG) detects Escherichia coli (E.coli).
· Colonies with bright blue fluorescence
7) Blood agar plates (β-hemolytic)
· Hemolysis (Positive)
8) Urease Test
· Orange (Negative).
9) Triple Sugar Iron (TSI). Contains phenol red, high concentrations of lactose and sucrose, and a low concentration of glucose as well as sodium thiosulfate and ferric citrate . Provides aerobic and anaerobic conditions. Detects lactose fermentative bacteria – any bacteria that can ferment any of the three sugars.
· Red agar turns yellowish (positive). Acidic slant & butt (lactose and glucose fermenter)
· Stormy fermentation – production of hydrogen gas
10) Antibiotic Susceptibility :
· Amoxicillin, cephalosporins, carbapenems, aztreonam, trimethoprim-sulfamethoxazole, ciprofloxacin, nitrofurantoin and the aminoglycosides
11) Serology. Detection of O & H antigens (ELISA)
· Slide agglutination – Positive
· Tube agglutination - Positive
Biochemical summary:
IMViC
++--


Klebsiella spp. (pneumoniae)
Morphology (microscopy): Encapsulated gram negative rod (pH 5, acidic) may be cocci in more alkaline conditions (pH 7)
Pathogenesis: Type 1 fimbriae consisting of building block protein (FimA) extend beyond the capsular matrix and mediate adhesion to host epithelial cells.
Capsular polysaccharide matrix encases cell surface to provide resistance against host defence mechanisms.
Key characteristics: Negative indole test. Positive Voges Proskauer (Vi). Lactose fermenting, facultative anaerobic.
Tests:
1) MacConkey Agar.
· Red colonies (positive)
2)Triple Sugar Iron (TSI).
· Red agar remains red (negative). Alkaline slant & butt. (non-fermenter)
3) Indole test.
· Yellow (negative)
4) Voges-Proskauer (VP) test. Determines if neutral products such as ethyl alcohol, acetoin and butanediol are formed
· Pink-burgundy color (positive), 30mins to develop colour
5) Methyl Red (MR) test:
· Yellow (negative)
6) Citrate test. Utilizes Simmon's citrate media to determine if a bacterium can grow utilizing citrate as its sole carbon and energy source
· Prussian blue color (positive)
7) Urease Test.
· Orange (Negative).
8) Antibiotic Susceptibility :
· Third generation cephalosporin
9) Serology. Capsular serotyping
· K antigens
Biochemical summary:
IMViC
--++


Proteus spp. (mirabilis)
Morphology (microscopy): Gram negative bacilli.
Key Characteristics: Mostly positive indole test, except P. mirabilis. Deaminate phenylalaine. Grow on potassium cyanide medium. Ferment xylose. Motile, swarming effect on agar plates. urease-positive. Facultative anaerobe.
Tests:
1) Oxidase test
· Negative
2) Triple Sugar Iron (TSI).
· Black precipitation (H2S production), red slant (alkaline). Glucose fermenter, non-lactose fermenter.
3) Indole Test.
· Most strains positive (red), mirabilis negative (yellow).
4) Methyl Red Test
· Red (positive)
5) Voges-Proskauer (VP) test.
· Negative.
6) Citrate test.
· Prussian blue color (positive)
7) Urease Test.
· Pink (Positive).
8) MacConkey Agar
· Light/colourless colonies (negative)
9) OILM medium. Test for ornithine decarboxylase utilization.
· Green/blue upper portion (Positive).
10) Blood agar plates
· Swarming observed
11) Antibiotic Susceptibility
· Penicillin derivatives, cephalosporins, quinolones, aminoglucosides
Biochemical summary:
IMViC
-+-+


Enterococcus spp.(faecalis)
Morphology (microscopy): Gram positive cocci. (diplococci)
Key Characteristics: catalase negative, colonies appear grey. Group D streptococci. gamma hemolytic (no hemolysis)
Tests:
1) Catalase test. Identifies organisms which produce the catalase enzyme; this enzyme converts hydrogen peroxide to water and oxygen gas
· Negative
2) Blood plate agar
· No hemolysis
3) Bile-Esculin Hydrolysis Test. Determine the ability of an organism to hydrolyze the glycoside esculin to esculatin and glucose in the presence of bile (10 - 40%).
· Black to dark brown slant (Positive)
4) 6.5% salt broth. Test salt tolerance.
· Positive, growth.
5) PYR (pyroglutamyl aminopeptidase) disc
· Positive
6) Xylose fermentation test. performed with D-xylose tablets.
· Positive
7) MacConkey Agar. Without crystal violet for inhibition of gram positive bacteria.
· Pink (positive)
8) Serology. Western blot (IgG from patient’s sera). ELISA.
· Polysaccharide antigen extracted from bacterial cell walls detected
9) Antibiotic Susceptibility
· Quinupristin, ampicillin and vancomycin


Pseudomonas spp. (aeruginosa)
Morphology (microscopy): Gram negative bacilli.
Key Characteristics: Aerobic (classified facultative anaerobe), rod-shaped bacterium with unipolar motility (due to single polar flagellum), opportunistic pathogen (seen in immunocompromised individuals). Pearlescent appearance and grape-like odor
Tests:
1) MacConkey Agar
· Colourless colonies - Negative (non glucose fermenter)
2) Catalase test. Identifies organisms which produce the catalase enzyme; this enzyme converts hydrogen peroxide to water and oxygen gas
· Positive
3) Blood plate agar
· swarming
4) Oxidase test
· Positive
5) Urease Test
· Positive
6) Indole Test
· Negative
7) Methyl Red
· Negative
8) Voges Proskauer Test
· Negative
9) Citrate test.
. Prussian blue color (positive)
10) TSI
· red slant (alkaline), red butt (alkaline) . Non fermenter of glucose
11) Antibiotic Susceptibility
· Aminoglycosides, Quinolones, Cephalosporins
12) Serology. Detect A, B , O antigens on cell wall
· Slide agglutination – Positive
· Tube agglutination - Positive
Biochemical summary:
IMViC
---+


Enterobacter spp. (aerogenes)
Morphology (microscopy): Gram negative bacilli.
Key Characteristics: Facultative anaerobe, oxidase negative, catalase positive. Typically caused by catheter insertions
Tests:
1) MacConkey Agar
· Pink colonies - Positive, may be weak (glucose fermenter)
2) Catalase test. Identifies organisms which produce the catalase enzyme; this enzyme converts hydrogen peroxide to water and oxygen gas
· Positive
3) Oxidase test
· Negative
4) Urease Test
· Variable
5) Indole Test
· Negative
6) Methyl Red
· Negative
7) Voges Proskauer Test
· Positive
8) Citrate test.
· Prussian blue color (positive)
9) TSI
· red slant (alkaline), yellow butt (acidic) . Fermenter of glucose
10) Antibiotic Susceptibility
. Ciprofloxacin, Tazobactem
Biochemical summary:
IMViC
--++


Serratia spp. (marcescens)
Morphology (microscopy): Gram negative bacilli.
Key Characteristics: Produces lactose-fermenting colonies on differential agar, but is a late lactose fermenter may seem like a negative reaction (considered negative). Forms red-pigmented colonies. Able to perform casein hydrolysis and degrates tryptophan and citrate.
Tests:
1) MacConkey Agar
· Red colonies. Negative (initial)
2) Catalase test. Identifies organisms which produce the catalase enzyme; this enzyme converts hydrogen peroxide to water and oxygen gas
· Positive
3) Oxidase test
· Negative
4) Urease Test
· Positive
6) Indole Test
· Negative
7) Methyl Red
· Negative
8) Voges Proskauer Test
· Positive
9) Citrate test.
· Prussian blue color (positive)
10) TSI
· red slant (alkaline), yellow butt (acidic) . Fermenter of glucose
11) Antibiotic Susceptibility
. Aminoglycoside , Amikacin, Gentamicin, Tobramycin and Quinolones
Biochemical summary:
IMViC
--++



Candida spp. (albicans)
Morphology (microscopy): chlamydospores.
Pathogenesis: Produces extracellular proteinases from SAP genes, phospholipases B enzymes and lipases that are secreted for host infection.

Tests:
1) Southern Blot. Detect SAP genes using SAP1 probe.
· Positive.
2) Serology. Detect cell wall antigens - ELISA (CWP, PPM, CW)
· Positive.
3) Oxoid OBIS albicans Test. Rapid card-based chromogenic test that detects the presence of two enzymes specific to C. albicans and C. dubliniensis: b-galactosaminidase and L-proline aminopeptidase
· Magenta colour (Positive).



References:
textbookofbacteriology.net > e.coli.html
http://www.cpg-biotech.com/ > userguides > tech1 > p35681
http://www.mc.maricopa.edu/ > johnson > labtools > Dbiochem > imvic.html
iai.asm.org > cgi > content > abstract > 73 > 8 > 4626
aem.asm.org > cgi > reprint > 28 > 4 > 534.pdf
http://www.blackwellpublishing.com/ > eccmid15 > abstract.asp?id=36567
http://www.clevelandchiropractic.edu/ > ClassFiles-LA > Microbiology > entero.htm
www2.austin.cc.tx.us > microbugz > html > catalase_test.html

Cheers,
Debra, TG02

Case 5 follow up

WFH's case of wound infection.

Revised list of suspected microorganisms:
1. S. aureus
2. S. pyrogenes
3. Clostridium perfringens
4. Clostridium botulinum
5. Pseudomonas aeruginosa
6. S. faecalis (enterococcus)
7.Proteus mirabilis

Microorganism	Key characteristics	Lab diagnosis
S. aureus	Produce coagulase enzyme	Coagulase positive (agglutination)
	Produce catalase	Catalase positive [Frothing when react with H2O2 ]
	Ferments mannitol	Yellow colonies on mannitol salt agar
	Lyses rbc	B haemolysis on BA
	B lactamase	B lactam resistant. Sensitivity to naficin, methicillin, vancomycin
		Gram positive (purple) cocci, “grape-like”
S. pyogenes	Lyses rbc	B haemolysis on BA
	Does not produce catalase	Catalase negative
		Bacitracin sensitive
		Hippurate and cAMP negative
		Gram positive cocci in chains
C. perfringens	Double zone of haemolysis	B haemolysis on BA
	Alpha lecithinase	Lecithinase positive on egg yolk agar
		Gram positive, large pink rods
		Proteolysis on CM (meat turn black)
C. botulinum	Motile	Swarming effect on agar
	Lipase positive
	Lecithinase and protease neg	Meat at the bottom of CM remain brown
		Gram positive, oval and subterminal spores, bacilli
P. aeruginosa	Non fermentative	Pink colonies on macconkey agar
	Oxidase positive
	Pyocyanin pigment	Blue on pseudomonas agar P
	Pyoverdin pigment	Fluorescein on Pseudomonas agar F
	Utilizes citrate	Citrate + (green to blue on citrate agar slant)
		Urease+/-
	Unable to hydrolyse and deaminate tryptophan	Indole -
	Does not utilize carbohydrates	TSI-
S. faecalis	Non haemolytic	Gamma haemolysis on BA
	Tolerant to 6.5% NaCl	Growth on mannitol salt agar
	Resistant to bile
	Hydrolyses esculin	Blackening of esculin agar
	Does not produce B lactamase	B lactam sensitive
		Gram positive cocci in chains
P. mirabilis	Unable to metabolise lactose	Pink colonies on MAC
	Distinct ordor
	Utilises urea and citrate	Citrate +
	H2S production	Black precipitate on TSI
	Unable to hydrolyse and deaminate tryptophan	Indole -
		Nitrogen reductase -
	Mixed acid fermentation	Methyl red +
	Unable to ferment glucose	Voges- Proskauer -
	Produce catalase	Catalase +
		Oxidase -
		Phenylalanine deaminase +

Yeng Ting
Tg 02

MMIC blog 2 - case 4

Probable microorganisms:

Streptococcus pneumoniae
- gram-positive
- alpha-haemolytic diplococcus bacterium
- optochin sensitive

Pathogenesis:
- produce IgA protease that enhances organism’s ability to colonize the mucosa of the upper respiratory tract. It multiply in tissue and causes inflammation.

Lab investigation:
- Gram stain
- Culture on blood agar plates
- catalase negative
- antibiotic susceptibility – susceptible to penicillins and erythromycin.

Moraxella catarrhalis
- gram-negative
- aerobic
- diplococcus
- produce beta-lactamases

Pathogenesis:
- cause respiratory tract-associated infection in humans

Lab investigation:
- Gram stain
- oxidase negative
- antibiotic susceptibility – susceptible to cephalosporins, resistant to penicillin

Haemophilus influenzae
- gram-negative
- coccobacilli
- non-motile
- generally aerobic, but can grow as a facultative anaerobe.

Pathogenesis:
- Their capsule allows them to resist phagocytosis and complement-mediated lysis in the non-immune host. Unencapsulated strains are less invasive, but they are able to induce an inflammatory response that causes disease.

Lab investigations:
- Gram stain
- culture on blood agar
- catalase positive
- oxidase positive

Mycoplasma pneumoniae
- causes infectious bronchitis

Lab investigations:
- lack of bacteria in a gram stained sputum sample
- lack of growth on blood agar
- positive blood test for cold hemagglutinins.
- Antibiotic susceptibility – susceptible to erythromycin

Chlamydia pneumoniae
- causes infectious bronchitis

Lab investigations:
- Giemsa stain
- Serologic test for antibody in patient’s serum.
- Antibiotic susceptibility – susceptible to tetracycline such as doxycycline

Bordetella pertussis
- gram-negative
- coccobacillus
- aerobic
- non-motile

Lab investigation:
- Culture on Bordet-Gengou agar plate with added cephalosporin select for the organism
- Oxidase positive
- urease negative
- nitrate negative
- citrate negative
- Antibiotic susceptibility – susceptible to erythromycin

Klebsiella pneumoniae
- gram-negative
- facultative anaerobic
- non-motile
- lactose fermenting

Lab investigations:
- Culture – MacConkey’s agar with lactose fermenting colonies.
- oxidase negative
- TSI: slant & butt yellow (acidic)
- H2S negative
- indole negative
- methyl-red negative
- Voges-Proskauer positive
- citrate positive
- urease positive
- Antibiotic susceptibility – susceptible to cephalosporin, resistant to penicillin

References:
http://www.merck.com> Lung and Airway Disorders> Bronchitis
http://www.wikipedia.org> Streptoccocus pneumoniae
http://www.wikipedia.org> Moraxella catarrhalis
http://www.wikipedia.org> Haemophilus influenzae

eunice
tg02

Cheng Hong: MMic PBL Kuan Siew Yan follow up

There are some other organisms and viruses that are able to cause diarrhea like:

§ Clostridium perfringens
§ Staphylococcus aureus
§ Rotavirus
§ Norovirus
§ Vibrio cholerae


Clostridium perfringens
Characteristics: Rigid, thick walled cell, gram positive, spore forming anaerobic rods

Pathogenesis: Causes gas gangrene and food poisoning. C. perfringens is a normal flora in the colon but not found in the small bowels. If it is in the small bowels, the enterotoxin (super antigen on the C. perfringens) will cause diarrhea.

Why is not likely in this case: The diarrhea will only last for 24hrs and the disease will be resolved after 24hrs. This bacteria do not cause enterocolitis.


Staphylococcus aureus
Characteristics: Rigid, thick walled cell, gram positive cocci

Pathogenesis: Causes food poisoning. The enterotoxin causes food poisoning (vomiting/ watery, non-bloody diarrhea). The enterotoxin acts like a super antigen and stimulate the release of interleukin-1 and 2. May be transmitted through improperly cooked food as S. aureus is quite heat resistant.

Biochemical features: Coagulase positive, positive latex test, plate on Mannitol salt agar which acts as a selective medium and differential medium

Others: Smears will show gram positive cocci in clusters (purple).


Rotavirus
Characteristics:Reovirus family, non-enveloped, ssRNA virus, Icosahedral capsid

Pathogenesis: Causes diarrhea in young children, transmitted through fecal oral route. The virus multiplies in the small intestine and causing salt, glucose, water to be loss through diarrhea.

Diagnosis: Using ELISA techniques/ radioimmunology/ Rapid testing kits


Why it is not likely in this case: As Rotavirus infection mostly occurs in babies and very young children. Adults rarely will be infected as at the age of 6, children will have antibodies against at least 1 serotype of rotavirus.


Norovirus
Characteristics: Calicivirus family, non-enveloped, ssRNA virus, Icosahedral capsid

Pathogenesis: Transmitted through fecal oral route, or ingesting contaminated seafood/water. Virulent (low infectious dose). May cause vomiting, fever, diarrhea.

Diagnosis: PCR method or ELISA method



Vibrio cholerae
Characteristics: Rigid, thick walled cell, Gram negative, Facultative curved rods (comma shape)

Pathogenesis: Transmitted through fecal contamination in water and food, found in shellfish/ oysters. It causes watery diarrhea by causing the cells in the gut to lose water and ion. It does not cause bloody diarrhea or abdominal pain.

Biochemical features: oxidase positive, acid slant and acid butt, but no gas or H2S on TSI. Can be confirmed usingagglutination test (polyvalent O1 or non-O1 antiserum.

Please click the link below to view the biochemical test. (Maximise the table for better viewing)

http://www.geocities.com/alyssialam/biochemicalTest.gif

References from:Review of Medical Microbiology and Immunology(9th edition). WARREN LEVINSON
:Color Atlas of Medical Bacteriology. ASM PRESS

MMIC Blog 2 - Case 6

[A follow-up on my patient, Ong Fei Fei's investigation]


Laboratory Diagnosis

Since the patient has been previously diagnosed with UTI, the microbe could have move down and infect the vagina as well, hence urethra microbes are considered in this case too, for examples:

E. coli
Proteus mirabilis
S. saprophyticus
Group B streptococci (Alpha & non-hemolytic)
Coagulase-negative Staphylococci (S. aureus)
Klebsiella species
Proteus species
Pseudomonas aeruginosa
Enterobacteraceae

(Follow how UTI microbes are identify in other cases - see debra's and/or elaine's)


As for more vaginal-specific microbes, they are the followings:


Trichomonas Vaginalis

• Wet mount preparation then Direct microscopy



• Motile characteristic



• Greater sensitivity: Fixed stained preparation (Giemsa/Papanicolaou) then Direct Immunofluorescence



• Culture (Diamond’s medium) then incubate at 37oC à microscopy



• Antibody detection techniques: high in false positive and false negative results



• Enzyme immunoassay for detection of T. vaginalis antigen

Candida Albicans

• Gram’s stain then Direct Microscopy



• Sabouraud’s agar then Incubate 24-48 hours. white, butyrous colonies observed if positive
  

Bacterial Vaginosis (anaerobic/non-specific)

• Clue cells (Vaginal epithelial cells with edges darkened by presence of numerous small bacteria adhering to their surface)



• pH (>4.5)



• Amine test: add few drops of KOH and presence of amines gives a fishy smell



• Gram’s stain



• Culture: for Gardnerella vaginalis and Mobiluncus species

1. Gardnerella vaginalis

Aerobic
Gram-variable
Bacillus
Slow growing
Non-motile
Catalase and oxidase negative
Beta-hemolysis on human blood agar but not on sheep blood agar
Selective blood agar: add gentamicin, nalidixic acid and amphotericin B
Hippurate hydrolysis: positive
Starch fermentation: positive
Metronizadole 50ug disc: sensitive
Sulphonamide 1000ug dsc: resistant


2. Mobiluncus species

Anaerobic
Gram-variable
Bacillus
Divided into: M. curtisii and M. mulieris
Fastidious; slow growing
Typically motile, catalase, oxidase, indole negative
Clear, colourless colonies (2mm) after 5 days incubation
Gas-liquid chromatography distinguish species
Commercial kit: detect enzyme activity (praline aminopeptidase and alpha-D-glucosidase)


3. Neisseria Gonorrhoea

Die readily outside human body
Fastidious
Use of rich media supplemented with yeast extract or iso-viatalex and blood (Chocolate agar)
Antibiotic inhibitors: vancomycin, nystatin, colistin, trimethoprim
Direct Microscopy (less sensitive for women – 50%)
Culture
Other technique: Direct immunofluorescence


4. Chlamydia trachomatis

[Direct examination of smear with fluorescein-conjugated monoclonal antibodies(Ab) (use of commercial kits)]

Roll specimen gently on slide
Fix with methanol for 4 min
2 Ab (one directed to the outer membrane – species specific, one specific to the genus lipopolysaccharide)
Subjective/ false positive
Suitable for small sample numbers and rapid screening


[Culture]

Uses McCoy’s cells treated with cyclohexamide
Centrifuged and incubate for 72 hours
Detection by Giemsa stain/iodine/fluorescein-labeled monoclonal Ab
Sensitivity: ~80%


[Enzyme-linked immunoabsorbent assays (ELISA)]

Uses polyclonal and monoclonal antibodies against lipopolysaccharide (Antigen detection)
Sensitivity: ~97%; Specificity: ~92.5% (improves by blocking tests)
False positive due to cross reactivity with other bacteria


[Nucleic acid probes]

DNA hybridization
Highly specific but lack sensitivity


[Serology]

Complement fixation tests are insensitive; difficult to differentiate the serotypes
Micro-immunofluorescence test detect specific IgG/IgM; difficult in sexually active populations



Summary Diagrams






























Other Investigation required


Urinalysis - for screening purpose; inexpensive and easy to perform (previously confirmed UTI)
Urine culture - for accurate diagnosis of infection to determine complications, such as antimicrobial susceptibility of infecting bacteria (previously confirmed UTI)


References

http://www.escriber.com/ > TrendsInUGSH > Features

http://www.cfps.org.sg/ > sfp > 23 > 232> articles > e232136.html

http://cks.library.nhs.uk/ > uti_lower_women > in_depth > goals_and_outcome_measures

http://www.merck.com/ > mmpe > sec17 > ch231 > ch231b.html

http://www.aafp.org/ > afp > 20020415 > 1589.pdf

http://books.mcgraw-hill.com/ medical > firstaidfortheboards > pdf > 0071443363 > 0071443363_282.pdf

http://classes.kumc.edu/ son > nrsg835 > gyninfect.htm



Posted by: Pei Shan, TG02

Elaine Blog2: case 1 Khong Fay seah

The entrobacteriaceae are heterogeneous group of gram-negative rods whose natural habitat is the intestinal tract of humans and animals. The family includes: Escherichia, shigella, salmonella, enterobacter, klebsiella, serratia.

E.coli form circular, convex, smooth colonies with distinct edges. Klebsiella colonies are large and very mucoid and tend to coalesce with prolonged incubation.
Salmonella and shigellae produce colonies similar to E.coli but they do not ferment lactose. However, salmonella and shigellae do not appear in the urinary tract.

E.coli is the most common cause of UTI and accounts for approximately 90% of first urinary tract infections in young women. The symptoms and signs include urinary frequency, dysuria, hematuria, and pyuria. However, none of these symptoms or signs is specific for E.coli.


procedure:

• Urine collected from clean-catch mid-stream or one obtained by bladder catherterization or suprapubic.


• common causative: E.coli; Enterobacteriaceae; other gram-negactive rods


• usual microscope: gram-negative rods seen on stained smear of uncentrifuged urine indicate more than 105/mL


• culture on blood agar and macConkey


• Comments: gray colonies that are B-hemolytic and give a positive spot indole test are usually E.coli; other require further biochemicsl tests

E.coli--> colonies show metallis on EMB agar

Klebsiella pneumoniae--> has large mucoid capsule and hence viscous colonies

Proteus mirabilis--> motility causes "swarming" on agar; produce urease

Pseudomonas aeruginosa--> blue-green pigment and fruity odor produced; causes nonsocomial infections and often drug-resistant

If unknown-form of colonies are observed (other than E.coli) , biochemical tests should be done to differentiate each micro-organism from each other.

Diagnosis: Biochemical Reaction:

IMViC (Indole Methy Re€d Voges-Proskauer simmons's Citrate) plus lactose fermentation & TSI

enterobacteriaceae

1. E.coli ++--+ acid slant/acid butt with gas production


2. Pseudomonas Aeruginosa -+--- alkaline slant/alkaline butt plus positive oxidation fermentation


3. Klebsiella Pneumoniae +-+++ acid slant/acid butt with gas production


4. Proteus Mirabilis -+++- alkaline slant/acid butt with H2S production

Other possible bacteria and tests required:

5. Enterococcus Faecalis

• growth in the presence of bile, hydrolyze esculin


• Alpha-hemolytic


• growth in 6.5% NaCl, PYR-positive

6. Staphylococcus Saprophyticus

• -ve mannitol


• -ve coagulase


• -ve Novobiocin sensitivity


• -ve alpha toxin

7. Neisseria Gonorrhoeae

• +ve glucose


• -ve maltose


• -ve Lactose


• -ve Sucrose

8. Mycoplasma Genitalium

• culture is difficult.


• Data obtained from PCR, molecular probes, serologic tests

9. Ureaplasma Urealyticum

• It required 10% urea for growth

Antibiotic Susceptibility Testing:

Escherichia coli (gram -ve rods enteric and related organisms)
drug choice: Cefotaxime, Fluoroquinolones, nitrofurantoin

Pseudomonas aeruginosa (gram -ve rods aerobic)
drug choice: Aminoglycoside, pencillin

klebsiella pneumoniae (gram -ve rods enteric and related organisms )
drug choice: A cephalosporin

proteus mirabilis (gram -ve rods enteric and related organisms)
drug choice: Ampicillin

Staphylococcus saprophyticus (gram +ve cocci)
drug choice: penicillin, tetracycline, vancomycin, gentamicin, rifampin

Neisseria gonorrhoeae (gram -ve cocci)
drug choice: ceftriaxone, ciprofloxacin, gatifloxacin,

Enterococcus faecalis (Pos cocci)
drug choice: Ampicillin, gentamicin

Mycoplasma genitalium (wall-less cells)
drug choice: erythromycin (ERY), clarithromycin (CLR), ciprofloxacin hydrochloride (CIP)

Ureaplasma urealyticum
drug choice: ciprofloxacin and ofloxacin, tetracycline and doxycycline, roxithromycin, erythromycin