P 12 Clinical microbiology III – examination in urogenital infections To study: special bacteriology from your own protocols Urinary tract infections Task 1: Sampling and transport of urine According to the teacher’s explanation, tick which sentences concerning urine sampling and transportation are true/false. Urine examination is recommended in non-complicated and necessary in complicated cystitis. r true r false Microbiologists recommend the use of catheterized urine as a routine way of sampling the urine for bacteriology. r true r false It is not important whether foreskin (prepuce – in men) or labia minora (in women) are in the way of urine stream during sampling the urine for bacteriology. r true r false External orifice of urethra should be carefully washed and eventually also disinfected before sampling the urine for bacteriology. r true r false The vessel into which the patient urinates should be sterile. r true r false The test tube used for urine transportation to the laboratory should have a yellow cap. r true r false If urine is not “routinely taken”, the order form should contain information whether it has been catheterized, punctured, or whether it is a specimen taken from a permanent catheter. r true r false Urine from a permanent catheter is equally important for bacteriological diagnostics as the catheterized urine (just for examination). r true r false Urine specimen should be delivered to the laboratory within 2 hours after sampling; if this is impossible, it should be kept in a refrigerator. r true r false Urine sample is better than urethral swab in gonorrhoea diagnostics. r true r false Task 2: Inoculation of sample of urine Observe your teacher demonstrating for you inoculation of sample of urine (or video with the same topic, if available). Fill in empty places in the following text: Urine sample is inoculated using calibrated loop, made of ______________________. The term “calibrated” means that it is set to specific volume, usually _______. The specimen of urine is inoculated to two media: __________________________ and ______________________________. Instead of the second medium we could also use ________________________________ or _____________________________. After inoculation, the specimen is incubated overnight in a thermostat at _____°C. Task 3: Evaluation of semiquantitative cultivation of urine After inoculation and incubation (see Task 2), the agar plates with result of urine specimen cultivation are evaluated. The number of colonies is counted (of estimated approximately) and recounted to number of bacteria in a mililitre of the original specimen of urine. Number of colonies on agar Number of bacteria in one microlitre of the original urine (µl) Number of bacteria in one mililitre of the original urine (ml) Interpretation <10 10–100 >100 Form for results of Enterotest 16: ONPG 1H 1G 1F 1E 1D 1C 1B 1A 2H 2G 2F 2E 2D 2C 2B 2A + black blue red blue red green black blue blue yellow yellow yellow yellow yellow yellow yellow – colourless green yellow green yellow yellow colourless yellow yellow green green green green green green green ? 1 2 4 1 2 4 1 2 4 1 2 4 1 2 4 1 2 Code: Identification Probability % T index Antibiotic susceptibility test Ampicillin AMP R < 14 S ≥ 14 Tetracyclin* TE R < 12 S ≥ 15 Cefalotin KF R < 14 S ≥ 18 Cefuroxime CXM R < 18 S ≥ 18 Co-trimoxazole SXT R < 13 S ≥ 16 Norfloxacin NOR R < 19 S ≥ 22 Nitrofurantoin F R < 11 S ≥ 11 write S = susceptible, R = resistant, eventually I = intermediary *result of this test is also valid for doxycycline Final conclusion and recommendation for treatment: _______________________________________________ Task 4: Interpretation and treatment of UTI In following table in each cell (except cells in the first column) one term is wrong. Add a dot to all terms you consider wrong. After that, check your choice with your teacher, and strike through all the really false terms. Clinical situation Most likely pathogens Drug of choice for initial therapy Alternative drugs (allergy etc.) Asymptomatic bacteriuria (ABU) pregnant women Escherichia coli Klebsiella pneumoniae Streptococcus pyogenes Enterococcus sp. nitrofurantoin* ofloxacin amoxicillin linezolide Asymptomatic bacteriuria (ABU) other situations no therapy nitrofurantoin no therapy cefuroxime Acute non-complicated cystitis (community cystitis, that means „not-nosokomial“ one) Clostridium sp. Escherichia coli Staphylococcus saprophyticus Klebsiella pneumoniae ciprofloxacin nitrofurantoin co-trimoxazole (co-)amoxicillin vankomycin cefuroxime Accute pyelonephritis Escherichia coli Bacteroides fragilis Klebsiella pneumoniae Proteus sp. (co-)amoxicilin cefuroxime nitrofurantoin co-trimoxazole norfloxacin imipenem *except first trimester and the second haft of the third trimester Infections of genital system Task 5: Sampling methods in STIs and other infections of reproductive organs Find suitable swabs or other sampling methods for following clinical situations (suspicions for individual diseases). For some of them more than one sampling method is suitable. Use numbers 1 to 6 and mark your choice to individual situations. Correct yourself with help of your teacher. Bacterial vaginosis Aerobic vaginitis Vaginal mycosis Gonorrhoea Syphilis Mycoplasma infection Chlamydia infection Papillomavirus infection Numbers: 1 – Amies swab 2 – C. A. T. swab 3 – plain (dry) swab 4 – a smear on a slide 5 – clotted blood for indirect examination 6 – ulcus durum scraping for dark-field microscopy and PCR Task 6: Evaluation of vaginal smears ____________________________________ ____________________________________ Nugent score for BV: In diagnostics of vaginal infections, one very important method is microscopy. Cultivation results may be positive even when the amount of bacteria (e. g. Gardnerella sp.) is not significant. Therefore, microscopy is better, because we can see the ratio between various morphotypes of bacteria, and also other structures (epithelial cells including those with adhered bacteria – so called “clue cells”; white blood cells; yeast cells etc.). Sometimes, two smears are sent to the laboratory: one is stained by Giemsa staining (almost because of Trichomonas vaginalis diagnostics, as T. vaginalis cannot be Gram stained very well) and the other by Gram (especially for bacteriology). Observe a result of a vaginal smear and draw your result in the laboratory report. Try to count Nugent score of bacterial vaginosis with help of following table. (“Morphotype Lactobacillus” = robust and long G+ rods; “morphotype Gardnerella” = subtle Gram-negative or Gram-variable straight rods; “morphotype Mobiluncus” = subtle Gram-negative curved rods. “Morphotype” means “bacteria that look in microscope the same as”, so not all representativeness of “Gardnerella morphotype” really belong to Gardnerella genus.) The Nugent scoring system (adapted): Score Lactobacillus Morphotype per field Gardnerella morphotype per field Curved bacteria (Mobiluncus) per field 0 >30 0 0 1 5-30 <1 1-5 2 1-4 1-4 >5 3 <1 5-30 ----- 4 0 >30 ----- The criterion for bacterial vaginosis according to Nugent's criteria is a total score of 7 or more is labeled as Bacterial Vaginosis a score of 4 to 6 is called intermediate, and a score of 0 to 3 is considered normal. Reliability of diagnosing bacterial vaginosis is improved by a standardized method of gram stain interpretation. R P Nugent, M A Krohn, and S L Hillier, J Clin Microbiol. 1991 February; 29(2): 297–301. Task 7: Evaluation of vaginal swabs Vaginal swabs are usually cultured on blood agar, Endo agar, agar with 10 % NaCl, special blood agar for Gardnerella vaginalis, eventually also VL agar (anaerobic culture). As a normal flora, we can observe lactobacilli: very tiny colonies with viridation. There exist many species of lactobacilli, with different relations with oxygen, although they use to be microaerofilic. Nevertheless, sometimes they are able to grow on blood agar in normal atmosphere, sometimes in Gardnerella agar in an incubator with elevated CO[2] concentration, and sometimes under anaerobic conditions only. Besides lactobacilli, normal finding may contain small amounts of staphylococci, Enterobacteriaceae and some other bacteria. Sometimes lactobacilli are absent, especially in swabs taken from women after climacterium. Write your findings and try to make a conclusion. Blood agar + Endo agar Gardnerella agar Agar with 10 % NaCl VL agar (anaerobic) Conclusion: We have seen normal vaginal microflora only – normal vaginal microflora + a pathogen: ________________ (delete as appropriate)