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CARIBBEAN REGIONAL MICROBIOLOGY STANDARD OPERATING PROCEDURE Genital Tract Specimens SOP No: CRM-SOP 6

STRENGTHENING OF MEDICAL LABORATORY SERVICES IN THE CARIBBEAN A CARIFORUM Project Funded by the European Union and Implemented by CAREC

STRENGTHENING OF MEDICAL LABORATORY SERVICES IN THE CARIBBEAN A CARIFORUM Project Funded by the European Union and Implemented by CAREC

Genital Tract Specimens


CARIBBEAN REGIONAL MICROBIOLOGY STANDARD OPERATING PROCEDURE Title: Genital Tract Specimens Prepared By: Caribbean Regional Standard Methods Drafting Group SOP No: CRM-SOP: 6 Version: 1 Page No: 2 of 28 Effective Date: 1st September 2007 Review Date: 1st September 2008

TA B L E O F C O N T E N T S
Acknowledgements Introduction Amendment Procedure Process Flow Chart 1. 2. 3. Title Purpose Introduction 3 5 6 7 8 8 8 8 9 9 9 9 9 10 10 10 11 11 11 12 13 14 19 19 19 19 21 22 23 23 24

4. Scope 5. Staff Competency Requirements 6. Safety Instructions 7. 8. 9. Pre-Examination Procedures 7.1 Sample Type 7.2 Sample Collection 7.3 Sample Transport & Storage 7.4 Rejection Critiria 7.5 Relevant Clinical Information Table of Media, Reagents, Materials & Equipment Examination Procedures 9.1 Quality Control 9.2 Microscopy 9.3 Culture 9.4 Identification 9.5 Susceptibility Testing 9.6 Sample Referral

10. Post-Examination Procedures 10.1 Interpretation of Results 10.2 Reporting 10.3 Sample Retention, Storage & Disposal 11. Limitations and Pitfalls of the Procedure 12. References 13. Appendices

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Genital Tract Specimens


CARIBBEAN REGIONAL MICROBIOLOGY STANDARD OPERATING PROCEDURE Title: Genital Tract Specimens Prepared By: Caribbean Regional Standard Methods Drafting Group SOP No: CRM-SOP: 6 Version: 1 Page No: 3 of 28 Effective Date: 1st September 2007 Review Date: 1st September 2008

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Genital Tract Specimens


CARIBBEAN REGIONAL MICROBIOLOGY STANDARD OPERATING PROCEDURE Title: Genital Tract Specimens Prepared By: Caribbean Regional Standard Methods Drafting Group SOP No: CRM-SOP: 6 Version: 1 Page No: 4 of 28 Effective Date: 1st September 2007 Review Date: 1st September 2008

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Genital Tract Specimens


CARIBBEAN REGIONAL MICROBIOLOGY STANDARD OPERATING PROCEDURE Title: Genital Tract Specimens Prepared By: Caribbean Regional Standard Methods Drafting Group SOP No: CRM-SOP: 6 Version: 1 Page No: 5 of 28 Effective Date: 1st September 2007 Review Date: 1st September 2008

INTRODUCTION
The Caribbean Regional Standard Methods include a variety of standard, validated methods, produced as a single standard operating procedure (SOP) for use in a variety of levels of microbiology laboratory service. It is intended that these methods provide detailed instructions for microbiology services for microbiological investigations, in order to provide accurate, reliable and reproducible results which will have clinical utility. These methods may be adopted by laboratories within the region, or adapted, provided that such adaptations use an evidence-based validation process. These methods have been developed by the Caribbean Regional Microbiology Standard Methods Drafting Group (CSMDG) in response to a request by the Caribbean Regional Microbiology Council (CRMC), which was set up by the CARIFORUM Project entitled Strengthening of Medical Laboratories in the Caribbean to strengthen specifically the microbiology services in the Caribbean Region. The Project was initiated in response to findings which indicated that there was an unacceptable level of error in laboratories within the region. External quality assessment results revealed that microbiology laboratories were not performing well and feedback from the region via laboratory staff, lab managers and directors was that they felt that guidance in microbiology requirements was required. The background for this initiative is a worldwide move to implement standards in all areas, which has now extended to include medical laboratories. As tourism is so vital to the regions economy, the need for accurate diagnosis and treatment is paramount. It was accepted that there is a requirement for validated methods for accreditation purposes and providing validated standard methods will assist in the move towards accreditation. The methods will be chosen for standardization by the Caribbean Regional Microbiology Council, and this selection will be based on a review of EQA results, most common and/ or critical tests. Part of the method standardization process will be an ongoing review and amendment procedure. The CSMDG consists of microbiology laboratory representatives from most of the CARIFORUM countries, all of whom were nominated to the task by the CRMC. This initiative should enable the region to implement a standardized and constructive method for ensuring that validated methods are available for the region, and that they are updated as required. Advantages of using regionally validated methods are to improve quality, make better use of resources, reduce costs, enable central procurement & media preparation, facilitate staff training and transfers due to horizontal integration, a reduction in variability of service provision, an improved quality of surveillance data, and the purchase of appropriate equipment. A major advantage is that the availability of regional standard methods would assist microbiology laboratories with documentation for accreditation. Although the CSMDG has taken every care with the preparation and issue of these standard procedures, and they have been validated regionally, nationally and internationally, the CSMDG, or any other organization, cannot be responsible for the accuracy of any statement or representation made or the consequences arising from the use of or alteration to any information contained in them. These procedures are intended solely as a resource for practicing microbiology professionals in the field, operating in the Caribbean region, and specialist advice should be obtained where necessary. If changes are made to the original publication, it must be made clear where changes have been made to the original document. When referring to these SOPs in successive documentation, the CSMDG should be acknowledged.

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Genital Tract Specimens


CARIBBEAN REGIONAL MICROBIOLOGY STANDARD OPERATING PROCEDURE Title: Genital Tract Specimens Prepared By: Caribbean Regional Standard Methods Drafting Group SOP No: CRM-SOP: 6 Version: 1 Page No: 6 of 28 Effective Date: 1st September 2007 Review Date: 1st September 2008

AMENDMENT PROCEDURE
Controlled Document Reference Controlled Document Title CRM-SOP 6 Standard Operating Procedure for Genital Tract Specimens

Each Regional Standard Method should be reviewed annually by the Caribbean Standard Methods Drafting Group. Any amendments should be validated and authorized by an agreed process, and referenced. Each Regional Standard Method has an individual record of amendments. The current amendments are listed on this page. On issue of revised or new pages, each controlled document should be updated by the copyholder in the laboratory. Amendment Issue Number Insert Page Number / Date Discarded Issue Number Section(s) involved Amendment

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Genital Tract Specimens


CARIBBEAN REGIONAL MICROBIOLOGY STANDARD OPERATING PROCEDURE Title: Genital Tract Specimens Prepared By: Caribbean Regional Standard Methods Drafting Group SOP No: CRM-SOP: 6 Version: 1 Page No: 7 of 28 Effective Date: 1st September 2007 Review Date: 1st September 2008

PROCESS FLOW CHART


GENITAL TRACT SPECIMENS

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Genital Tract Specimens


CARIBBEAN REGIONAL MICROBIOLOGY STANDARD OPERATING PROCEDURE Title: Genital Tract Specimens Prepared By: Caribbean Regional Standard Methods Drafting Group SOP No: CRM-SOP: 6 Version: 1 Page No: 8 of 28 Effective Date: 1st September 2007 Review Date: 1st September 2008

1. Title
Genital Tract Specimen Processing.

2. Purpose
To ensure the correct, validated procedure is followed for processing genital tract specimens, and to provide accurate, reliable and reproducible results having clinical utility.

3. Introduction
Infections of the genital tract fall into two categories: sexually transmitted infection (STI) Infections other than STIs For this reason, the provision of clinical data is critical as the method of processing specimens, and interpretation of bacterial growth are usually based upon both the type of specimen and the clinical data provided. A range of sexually transmitted organisms can cause infections which are responsible for a large number of clinical syndromes, therefore when a specific sexually transmitted infection (STI) is diagnosed, it is recommended to screen for other STIs. Normal vaginal flora consists of a wide range of organisms, including Lactobacillus species, streptococci, enterococci and coagulase negative staphylococci. Yeasts are often present as commensal organisms in the vagina, but can proliferate when alterations occur in the vaginal environment. eg after antimicrobial therapy, during pregnancy, use of oral contraceptives, diabetic patients. Children are also at risk of acquiring STIs. The presence of STIs in children is highly suggestive of sexual abuse, although exceptions do exist, and this possibility should always be investigated. Specimens from females presenting with clinical syndromes such as cervicitis, vulvo-vaginitis, urethritis, bacterial vaginosis (BV), pelvic inflammatory disease or genital ulcers are received for detection of microorganisms. Specimens from males exhibiting urethritis, epididymitis, and prostatitis can also be received. Accurate diagnosis of genital infections from the male and female genitalia, depends on the isolation of microbial pathogens from the normal genital microbiota.

4. Scope
This procedure provides detailed instructions for processing genital specimens, as offered by regional laboratories. It may be adopted or adapted by any laboratory as needed, provided that such adaptations use an evidence-based validation process. This SOP is intended to be used for the isolation of Neisseria gonorrhoeae, yeasts, -haemolytic streptococci and other specific target organisms (including Trichomonas vaginalis), and the microscopic diagnosis of bacterial vaginosis (BV). It does not include the isolation of Haemophilus ducreyi and Chlamydia trachomatis. STRENGTHENING OF MEDICAL LABORATORY SERVICES IN THE CARIBBEAN A CARIFORUM Project Funded by the European Union and Implemented by CAREC


Genital Tract Specimens


CARIBBEAN REGIONAL MICROBIOLOGY STANDARD OPERATING PROCEDURE Title: Genital Tract Specimens Prepared By: Caribbean Regional Standard Methods Drafting Group SOP No: CRM-SOP: 6 Version: 1 Page No: 9 of 28 Effective Date: 1st September 2007 Review Date: 1st September 2008

5. Staff Competency Requirements


Laboratory personnel, trained and assessed to be competent to perform this procedure.

6. Safety Instructions
Please refer to CRM-SOP 20: Safety In The Microbiology Laboratory. Keep all books, forms and papers away from technical work surfaces. Level II containment; require all personal protective equipment (PPE). Goggles should be used when performing processes that may give rise to aerosols. All microbiological samples for processing should be considered a potential source of transmissible infections so universal safety precautions should be observed. Hands should be thoroughly washed with soap and water before and after handling all specimens. Disinfect all work surfaces with 70% alcohol or a freshly prepared 10% bleach solution prior to testing and after processing All samples and reagents should be properly discarded according to the current standards for disposal of hazardous waste. Samples and culture plates should be autoclaved before finally discarding. Any procedure which is likely to produce aerosols should be performed in a biosafety cabinet.

7. Pre-Examination Procedures
7.1 Sample Type Urethral swabs (male & female) High vaginal swabs (HVS) discharge /Ante-natal screen Air-dried smears of vaginal discharge (for Bacterial Vaginosis) Endo cervical swabs (ECS) / Cervical Swabs (CS) Post operative specimens Intra Uterine Device (IUCD) Penile swabs Vulval swabs Operative fluids and aspirates

7.2 Sample Collection Urogenital specimens are usually collected by a physician or a trained, experienced nurse.

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Genital Tract Specimens


CARIBBEAN REGIONAL MICROBIOLOGY STANDARD OPERATING PROCEDURE Title: Genital Tract Specimens Prepared By: Caribbean Regional Standard Methods Drafting Group SOP No: CRM-SOP: 6 Version: 1 Page No: 10 of 28 Effective Date: 1st September 2007 Review Date: 1st September 2008

7. Pre-Examination Procedures continued

Appropriate specimens are often difficult to obtain and incorrect or sub-optimal specimens are often received. Cervical and high vaginal swabs should be taken with the aid of a speculum, and it is important to avoid vulval contamination of the swab. For Trichomonas, the posterior fornix, including any obvious candidal plaques should be swabbed. If pelvic infection, including gonorrhoea, is suspected, the cervical os should be swabbed. After the introduction of the speculum, the swab should be rolled firmly over the surface of the vaginal vault or rotated inside the endocervix. Endocervical swabs should be collected under direct vision. The swab should then be placed in Amies transport media with charcoal. Note: If Neisseriae gonorrhoeae or T. vaginalis is suspected, the swab must be transported to the laboratory within 2 hours of collection. Males should not pass urine for at least 1 hour prior to specimens being taken, and if a discharge is not apparent, attempts should be made to milk exudates from the penis. The swab should be passed through the urethral meatus and rotated. The swab should then be placed in Amies transport media with charcoal. 7.3 Sample Transport & Storage Processing of specimen should be done immediately wherever possible. If this is not possible, specimen should be placed at 2 8C for not more than 24 hours in transport medium. For T. vaginalis the specimen should be processed within 2 hours. Delays of greater than 48 hours before processing swabs are undesirable. If possible, inoculation of specimens for N. gonorrhoeae should be made directly on to the culture media at the bedside and incubated without delay. Transport time should be as short as possible. 7.4 Rejection Criteria Reject specimens: not received in transport medium, since the agents of genital infections lose viability easily. if significant delay (if > 24 hrs in transportation). that are not labelled or that are incorrectly labelled. that are sent to the Laboratory without an accompanying requisition form. Dry swabs should never be processed. If information on form and sample do not correlate exactly. If incorrect sample received. If expired transport media is used for swabs.

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CARIBBEAN REGIONAL MICROBIOLOGY STANDARD OPERATING PROCEDURE Title: Genital Tract Specimens Prepared By: Caribbean Regional Standard Methods Drafting Group SOP No: CRM-SOP: 6 Version: 1 Page No: 11 of 28 Effective Date: 1st September 2007 Review Date: 1st September 2008

7. Pre-Examination Procedures continued

7.5 Relevant Clinical Information This should include patient age, sample site, whether pre or postnatal if applicable, signs and symptoms, clinical history and provisional diagnosis, and information on any invasive/surgical procedures that have been performed. Details of any antimicrobial therapy. Clinical data will affect both the processing of the specimen and the interpretation of results.

8. Table of Media, Reagents, Materials and Equipment


Reagents Gram Stain Reagents Sterile Normal Saline Oxidase Reagents Identification Reagents for G.C. Serum/Plasma for germ tube Acridine orange stain 70% alcohol/ 10% bleach solution (freshly prepared) Phadebact/Quadfern APINH Materials Cover Slips 22x22 Microscope Slides Well slides Pasteur pipettes Transfer pipettes Immersion oil Lens cleaner Lens Paper Paper Towels Paper Tissues Filter Paper Wooden applicator sticks Equipment CO2 atmosphere ANO2 incubator O2 incubator Light Microscope Inoculating loop Bunsen Burner Media Sheep Blood Agar (SBA) Modified Thayer Martin (MTM) OR New York City (NYC) MacConkey Chocolate Agar Sabouraud dextrose / Brilliant Cresyl Green (BCG) Trichosel broth SIMS/MIL (motility medium)

9. Examination Procedures
9.1 Quality Control Please refer to CRM-SOP 18: Media Preparation and Quality Control. Please refer to CRM-SOP 21: Quality Control of Reagents and Tests. Please refer to CRM-SOP 19: Propagation and Maintenance of Quality Control Organsims. Perform appropriate QC protocol for individual staining. Use Appropriate American Type Culture Collection (ATCC) controls for checking validity of media (for example TM / NYC) and identification tests. Check CLSI manual. ATCC Control strains should be used wherever possible.

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CARIBBEAN REGIONAL MICROBIOLOGY STANDARD OPERATING PROCEDURE Title: Genital Tract Specimens Prepared By: Caribbean Regional Standard Methods Drafting Group SOP No: CRM-SOP: 6 Version: 1 Page No: 12 of 28 Effective Date: 1st September 2007 Review Date: 1st September 2008

9. Examination Procedures continued

Results of all QC testing should be recorded on the appropriate QC forms available in the department. Results of all tests are invalid if the QC test results are not as expected. Ensure that reagents, stains and media are not used beyond the expiry date. Check all reagents and stains before use to ensure that they are free from contamination, debris or deposits. All media used should be examined visually just before use to ensure that there is no contamination or deterioration appearing as lysis, discolouration, drying, shrinkage, or cracking of the media. Refer to SOPs for media, Gram Stain and relevant biochemical tests for further details. 9.2 Microscopy The inoculation of plates must be done before the wet prep and Gram Stain is performed, to prevent contamination of the culture plates. 9.2.1 Gram Stain Prepare Gram film and examine under oil immersion for the presence of white cells (WBC), yeasts, clue cells, epithelial cells, and bacteria. Refer to CRM-SOP 7: Gram Stain. 9.2.2 Wet Preparation Examine wet preps for the presence of pus cells, red cells, yeast, T. vaginalis, clue cells, and epithelial cells as follows: Prepare by placing one drop of saline on a clean slide, roll swab into saline, place cover slip on top, taking care to avoid air bubbles, and examine immediately under 10x and 40x objectives of a light microscope. The preparation must not be too thick. Read wet preps immediately before the preparation starts to dry. To detect trophozoites of T. vaginalis, the preparation must be examined as soon as possible after collection. NB. Air-dried smears of vaginal discharge are the preferred specimen for the diagnosis of BV. 9.2.3 Broth Culture for Trichomonas. An alternative method to the direct wet prep is to use modified Trichosel broth (Tric broth). Prepare by breaking off the tip of the swab in a tube containing 5 ml of Tric broth, Cover the tube and incubate at 3537C for 18-24 hrs.

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CARIBBEAN REGIONAL MICROBIOLOGY STANDARD OPERATING PROCEDURE Title: Genital Tract Specimens Prepared By: Caribbean Regional Standard Methods Drafting Group SOP No: CRM-SOP: 6 Version: 1 Page No: 13 of 28 Effective Date: 1st September 2007 Review Date: 1st September 2008

9. Examination Procedures continued

After incubation, remove the swab from the tube using forceps and press swab against a clean glass slide to release enough broth culture to examine. Apply a cover slip and examine as above. T. vaginalis may be identified in the wet prep by its characteristic jerking and twisting movements, the thrashing of its flagella and the rippling of its undulating membrane. 9.3 Culture 9.3.1 Culture Procedure for Genital Specimens (Appendix 1). Refer to CRM-SOP 22: Inoculation of Culture Media. Female genital specimens for processing are placed into different categories depending upon the clinical data provided. Specimens where STIs only are suspected should be placed into Category 1 and treated accordingly. Other specimens, which have specific clinical data, e.g. post-operative, should be placed into Category 2, 3 or 4, depending on the clinical data, and have additional investigations performed. a. Inoculate specimens onto media selected according to clinical details. See Appendix 1.

b. Ensure that all plates are clearly labelled with a marker or grease pencil. Information required includes the date, patients name and number, sample number and sample type. c. Plates should be inoculated starting with the least inhibitory.

d. Half plates and quarter plates may be used for inoculation (care should be taken to avoid cross contamination when inoculating and streaking). This is more appropriate to selective media where the swarming of Proteus sp. is not likely. e. f. Place a metronidazole (MTZ 5ug) disk to one side of the primary inoculum area. on all anaerobic plates. Incubate pates in appropriate atmosphere (see Appendix 1) at 35-37C for 24 48 hours.

9.3.2 Plate Reading Procedure for Genital Specimens (Appendices 3 and 5). a. Examine plates at 24 hours (except ANO2 plate).

b. Perform identification and susceptibility testing on any significant isolates. c. Re-incubate all plates.

d. Re-examine again at 48 hours (including the ANO2 plate). Perform identification and sensitivities on any additional significant isolates. STRENGTHENING OF MEDICAL LABORATORY SERVICES IN THE CARIBBEAN A CARIFORUM Project Funded by the European Union and Implemented by CAREC

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CARIBBEAN REGIONAL MICROBIOLOGY STANDARD OPERATING PROCEDURE Title: Genital Tract Specimens Prepared By: Caribbean Regional Standard Methods Drafting Group SOP No: CRM-SOP: 6 Version: 1 Page No: 14 of 28 Effective Date: 1st September 2007 Review Date: 1st September 2008

9. Examination Procedures continued

9.3.3 Target Organisms Category 1 (Appendix 3) Trichomonas vaginalis Candida sp Neisseria gonorrhoea Bacterial vaginosis Group B -haemolytic streptococci (pregnant women only) Category 2 ( Appendix 5) Vulvo-vaginitis in pre-pubertal females may be caused by: Group A -haemolytic Streptococcus Staphylococcus aureus Candida albicans Haemophilus influenzae Neisseria gonorrhoea In elderly women, Coliforms and Pseudomonads may be considered significant only if isolated in PURE culture or post-operatively Category 3 (Appendix 5) As for Category 1, but including: Staphylococcus aureus -Hemolytic Streptococci Micro-aerophilic Streptococci Anaerobes: only if pure, heavy growth. Report only as Anaerobes with MTZ susceptibility Category 4 (Appendix 5) As Category 1,2 and 3, plus: Listeria sp. 9.4 Identification 9.4.1 Candida Perform a wet prep or Gram Stain on cream colonies growing on SAB. If budding yeast cells are seen, report as Candida sp. STRENGTHENING OF MEDICAL LABORATORY SERVICES IN THE CARIBBEAN A CARIFORUM Project Funded by the European Union and Implemented by CAREC

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CARIBBEAN REGIONAL MICROBIOLOGY STANDARD OPERATING PROCEDURE Title: Genital Tract Specimens Prepared By: Caribbean Regional Standard Methods Drafting Group SOP No: CRM-SOP: 6 Version: 1 Page No: 15 of 28 Effective Date: 1st September 2007 Review Date: 1st September 2008

9. Examination Procedures continued

If desired, further identification may be undertaken. Perform a germ tube test as follows: a. b. c. d. e. Emulsify a few colonies in 0.5 ml of serum or plasma in a tube. Incubate for at no longer than 2 hours. After incubation, place a drop of the emulsification on to a clean slide and apply a cover slip on top. Examine immediately (before the preparation starts to dry) under high power field for germ tube formation. If germ tubes are present report as Candida albicans.

Please refer to CRM-SOP 21: Quality Control of Reagents and Tests for detailed instructions for the germ tube test. NB. Ensure that incubation is no longer than 2hrs as pseudohyphae of Candida tropicalis may lead to misinterpretation. It may be necessary to identify further non-C. albicans yeasts that are persistent or resistant to treatment. Candida glabrata is second only to C. albicans in causing vaginal candidosis and infections in immunocompromised patients and is azole resistant. 9.4.2 -hemolytic Streptococci Perform a catalase test to help exclude Listeria sp. Perform a bile-esculin test (to exclude enterococci). If negative, perform the CAMP test or Lancefield group using a commercial Strep grouping kit. 9.4.3 Procedure for Camp Test. Please refer to CRM-SOP 21: Quality Control of Reagents and Tests for detailed instructions for the catalase and CAMP test. 5% SBA Staphylococcus aureus ATCC 25923 Control Group B Streptococcus ATCC 13813 Suspect organism

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CARIBBEAN REGIONAL MICROBIOLOGY STANDARD OPERATING PROCEDURE Title: Genital Tract Specimens Prepared By: Caribbean Regional Standard Methods Drafting Group SOP No: CRM-SOP: 6 Version: 1 Page No: 16 of 28 Effective Date: 1st September 2007 Review Date: 1st September 2008

9. Examination Procedures continued

Streak a known hemolytic strain of S. aureus across the centre of a 5% sheep blood agar plate. Inoculate a streak of the test organism onto the plate at right angles to the S. aureus. It is important that the test organism does not touch the S. aureus inoculum. Inoculate a streak of a strain of E. faecalis (negative control) in the same way as the test organism, at right angles to the S. aureus. e. Inoculate a streak of a known strain of S. agalactiae (positive control) in the same way as the test organism, at right angles to the S. aureus. f. Incubate plate at 35-37C overnight in 5-10% CO2. g. After incubation, examine for any interaction with the staphylococcal haemolysis, demonstrated by an arrowhead shaped area of enhanced hemolysis. a. b. c. d. Result: Arrow-shaped area of hemolysis No arrow-shaped hemolysis 9.4.4 Interpretation of Test Findings Arrow head zone of hemolysis is indicative of Group B -hemolytic streptococci.

= S. agalactiae = NOT S. agalactiae

Positive arrow head hemolysis 9.4.5 Staphylococcus aureus Perform Coagulase Test.

Negative

Please refer to CRM-SOP 21: Quality Control of Reagents and Tests for detailed instructions for the coagulase test. If positive, report as S. aureus.

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CARIBBEAN REGIONAL MICROBIOLOGY STANDARD OPERATING PROCEDURE Title: Genital Tract Specimens Prepared By: Caribbean Regional Standard Methods Drafting Group SOP No: CRM-SOP: 6 Version: 1 Page No: 17 of 28 Effective Date: 1st September 2007 Review Date: 1st September 2008

9. Examination Procedures continued

S. aureus may be present as normal perineal flora and will depend on the quality of the specimen. It may be a cause of vaginal discharge but is more commonly associated with vulvo-vaginitus in children and pre-pubertal women and as an infectious agent in post-partum endometritis, Bartholins abscess and balanitis. The need to undertake susceptibility testing will depend on the specimen type, patient and clinical details. 9.4.6 Anaerobes A clear distinct zone of inhibition around the MTZ disk indicates the presence of anaerobes. (Rarely, anaerobes may be resistant to MZD). It is usually unnecessary to identify anaerobes unless in pure culture or from a normally sterile site such as amniotic fluid. If pure, perform Gram Stain on organism and identify. Disc diffusion susceptibility method is not recommended for anaerobes unless they grow rapidly within 24hrs. 9.4.7 Neisseria gonorrhoeae a. Perform the oxidase test on suspected colonies on NYC or TM media. If oxidase positive, perform Gram Stain on growth. b. If Gram-negative diplococci seen, subculture to CHOC for confirmatory testing and AST. c. Identify using Phadebact gonococcus test or Quadferm or any other confirmatory test for Neisseria gonorrhoeae. eg API NH. It is good practice to confirm N. gonorrhoeae using a minimum of two tests using different principles. d. Always ensure that the suspect colonies of Gram-negative diplococci are able to grow on the selective medium (NYC or TM). Commensal Neisseria sp. will not grow on selective media. e. A -lactamase test should be performed and reported. f. Perform susceptibility testing on all isolates as many strains are now resistant to penicillin. 9.4.8 Listeria Listeria sp. will grow on the SBA and may be confused with Group B -hemolytic streptococci. Colonies are of similar size and show weak hemolysis. If Listeria is suspected, perform Gram Stain. If Gram-positive cocco-bacilli seen perform a catalase test and a motility at both 37C and at room temperature. Listeria sp. are catalase positive and exhibit a tumbling motility at room temperature but not at 37C. If available, confirm with API Coryne.

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CARIBBEAN REGIONAL MICROBIOLOGY STANDARD OPERATING PROCEDURE Title: Genital Tract Specimens Prepared By: Caribbean Regional Standard Methods Drafting Group SOP No: CRM-SOP: 6 Version: 1 Page No: 18 of 28 Effective Date: 1st September 2007 Review Date: 1st September 2008

9. Examination Procedures continued

9.4.9 Motility Methods a. SIMS Inoculate organism using a sterile straight wire into semisolid motility medium in a straight line. Incubate tube at room temperature for 18 24 hours. After incubation observe for an umbrella shaped body along the line of inoculum.

Inoculum line Umbrella shaped body

b. Hanging Drop Method Result: Inoculate the test organism into two tubes containing either broth or peptone water. Incubate one at room temperature (approx. 25C) and the other at 35 37C , in air for 4 hours. After incubation, usng a slide with a well, place a small drop of the liquid culture on to a cover slip. Place the cover slip upside down on to the well of the slide and observe under high power for the characteristic tumbling motility.

Room temperature 35 37

= tumbling motility = little or no motility

} ]= Listeria sp.

If no motility is observed leave incubating overnight and examine the next day. Any atypical results should be referred to the Reference laboratory for confirmation. MINIMUM LEVEL OF IDENTIFICATION: Actinomyces Anaerobes -hemolytic streptococci Other Streptococci and enterococci Enterobacteriaceae Haemophilus Listeria Neisseria Actinomyces level Anaerobes level Lancefield group level genus level Coliforms level species level species level species level

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9. Examination Procedures continued

Pseudomonads Staph aureus Yeasts

Pseudomonads level species level yeasts level

Organisms may be further identified if clinically or epidemiologically indicated. 9.5 Susceptibility Testing Perform on clinically significant isolates. AST for different organisms should be performed on media recommended by CLSI.

9.6 Sample Referral If necessary for identification or further susceptibility testing. N. gonorrhoea for all antibiotic resistant isolates, and strains with a reduced susceptibility to penicillin. Isolates associated with outbreaks and where epidemiologically indicated. Organisms with unusual or unexpected resistance, and whenever there is a laboratory or clinical problem that requires elucidation. All referrals must be accompanied by appropriately completed laboratory requisition forms. First notify the Reference Laboratory before sending any samples, especially if an urgent specimen is included. When samples require overseas shipping, there should be strict adherence to IATA regulations.

10. Post-Examination Procedures


10.1 Interpretation of Results

10.1.1 Criteria for Positive Bacterial vaginosis films a. Presence of Gardnerella like organisms (slender Gram-variable bacilli). b. Absence of lactobacilli. c. Presence of clue cells these are epithelial cells from the surface tissues of the vaginal area, which are coated with bacteria. There are so many bacteria on these cells that their normal borders and surfaces are covered with bacteria. The presence of clue cells should be reported and whether or not the microscopy is suggestive of BV according to either Nugents or Hays criteria.

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Genital Tract Specimens


CARIBBEAN REGIONAL MICROBIOLOGY STANDARD OPERATING PROCEDURE Title: Genital Tract Specimens Prepared By: Caribbean Regional Standard Methods Drafting Group SOP No: CRM-SOP: 6 Version: 1 Page No: 20 of 28 Effective Date: 1st September 2007 Review Date: 1st September 2008

10. Post Examination Procedures continued

10.1.2 Nugents Criteria Numbers of Lactobacillus morphotypes seen Score Numbers of Gardnerella and Prevotella morphotypes seen >30/hpf 5-30/hpf 2-4/hpf 1/hpf none Score Numbers of Mobiluncus morphotypes seen >30/hpf 5-30/hpf 2-4/hpf 1/hpf none Score

>30/hpf 5-30/hpf 2-4/hpf 1/hpf none

0 1 2 3 4

4 3 2 1 0

4 3 2 1 0

Code each morphotype separately according to numbers of organisms seen as indicated in the table above and add individual scores together. Interpret scores as follows: Total score 0-3: normal. Total score 4-6: intermediate: suggestive of BV. Assess with clinical criteria and send repeat to confirm. Total score >6: abnormal: indicative of BV.

10.1.3 Hays Criteria Grade I Grade II Normal predominantly Lactobacillus morphotypes. Intermediate mixed Lactobacillus and other morphotypes. Assess with clinical criteria and send repeat to confirm if necessary.

Grade III Abnormal few or absent Lactobacillus morphotypes, but greatly increased number of G. vaginalis and other bacterial morphotypes. Suggestive of BV. Isolation of Neisseria gonorrhoeae, Streptococcus agalactiae (pregnant women), Candida albicans and Candida sp. is considered significant from any genital site. Anaerobic organisms which are sensitive to the 5 ug Metronidazole (MTZ) disk should be reported as Anaerobes. There is no need to fully identity or perform susceptibility testing unless isolated in pure culture or if requested to do so by the Consultant Microbiologist. If in doubt, refer to a senior member of staff. The presence of other organisms may or may not be indicative of infection and should be evaluated with caution. Consideration should be taken of the quantity of growth, patients symptoms and/or other clinical details and conditions (e.g. pregnancy). STRENGTHENING OF MEDICAL LABORATORY SERVICES IN THE CARIBBEAN A CARIFORUM Project Funded by the European Union and Implemented by CAREC

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Genital Tract Specimens


CARIBBEAN REGIONAL MICROBIOLOGY STANDARD OPERATING PROCEDURE Title: Genital Tract Specimens Prepared By: Caribbean Regional Standard Methods Drafting Group SOP No: CRM-SOP: 6 Version: 1 Page No: 21 of 28 Effective Date: 1st September 2007 Review Date: 1st September 2008

10. Post Examination Procedures continued

Coliforms and Pseudomonas sp. are rarely involved in genital infections and are assumed to be secondary invaders or colonizers except in certain clinical situations (e.g. normally sterile site such as amniotic fluid, peritoneal fluid). Only perform identification and susceptibility testing if: isolated in pure culture and isolated from either post-op. or from age > 50 yrs, or from Neo-natal Units (NNU) or Intensive Care Unit (ICU). If in doubt, refer to a senior member of staff. 10.2 Reporting 10.2.1 Wet Preparation Report the presence or absence of WBC, RBC, yeast, and T. vaginalis. 10.2.2 Gram Stain Report on yeast and WBCs if present, and on the presence or absence of intracellular gram negative diplococci. Report on organisms seen on smear (e.g. Gram-positive rods, Gram-negative bacilli, Gram-positive cocci etc.). Report on the presence of clue cells and interpret according to Nugent or Hays criteria. a. Positive: Microscopic evidence indicates Bacterial Vaginosis. b. Negative: Microscopic evidence does NOT indicate Bacterial Vaginosis. 10.2.3 Culture Category I (see Appendix 1). If Candida sp is isolated, report as Candida albicans or Candida sp. without susceptibility. If not isolated, report Candida sp. not isolated. For HVS Specimens: If Neisseria gonorrhoeae is suspected please send an endocervical swab. For other specimens processed > 24 hours after specimen is taken (unless an unrepeatable operative specimen): Specimens too old for testing N. gonorrhoeae. If unrepeatable specimen processed and cultures negative add a comment to the report stating that a delay of >24hrs may have compromised the isolation of N. gonorrhoeae. STRENGTHENING OF MEDICAL LABORATORY SERVICES IN THE CARIBBEAN A CARIFORUM Project Funded by the European Union and Implemented by CAREC

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Genital Tract Specimens


CARIBBEAN REGIONAL MICROBIOLOGY STANDARD OPERATING PROCEDURE Title: Genital Tract Specimens Prepared By: Caribbean Regional Standard Methods Drafting Group SOP No: CRM-SOP: 6 Version: 1 Page No: 22 of 28 Effective Date: 1st September 2007 Review Date: 1st September 2008

10. Post Examination Procedures continued

For Endo-Cervical Swabs / Cervical Swabs: Report the presence or absence of N. gonorrhoeae. For Category 2, 3 and 4 Report the presence of vaginal flora or mixed cultures without susceptibilities as No pathogens isolated, with the comment If Neisseria gonorrhoeae is suspected please send an endocervical swab for HVS specimens. Report the presence of major pathogens), Group A and Group B -hemolytic streptococci, S. aureus and N. gonorrhoeae with susceptibilities. Report the presence of anaerobes with MTZ susceptibility only. If isolated in pure culture, perform identification. Isolates of coliforms and Pseudomonas sp. are normally considered to be colonizers. They should be reported as: No pathogens isolated unless clinical conditions suggest significance. Identification and susceptibility testing should not be performed unless isolated in pure culture. Always report all clinically significant organisms isolated or Report the growth of only normal vaginal flora or Report the absence of specific pathogens or Report the absence of growth. Clinically urgent results should be telephoned or sent electronically as soon as they become available. Written reports should be sent out within 24 to 72 hours of receipt of specimens. If required, reports should indicate that a final one will be forthcoming. 10.2.4 Gram stained smear suggestive of Bacterial Vaginosis. Note: Clue cells within epithelial cells, and absence of lactobacilli.

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Genital Tract Specimens


CARIBBEAN REGIONAL MICROBIOLOGY STANDARD OPERATING PROCEDURE Title: Genital Tract Specimens Prepared By: Caribbean Regional Standard Methods Drafting Group SOP No: CRM-SOP: 6 Version: 1 Page No: 23 of 28 Effective Date: 1st September 2007 Review Date: 1st September 2008

10. Post Examination Procedures continued

10.2.5 Gram stained smear: Normal. Note: Predominance of lactobacilli from healthy vagina

10.3 Sample Retention, Storage & Disposal Keep samples for a minimum of 3 days, and preferably a week, in the refrigerator (2-8C) after results are sent out. Samples should be autoclaved before final discarding according to the current standards for disposal of hazardous waste.

11. Limitations and Pitfalls of the Procedure


a. Unless selective media is used, routine genital culture may not detect carriage of Group B -hemolytic streptococci in all cases. b. Staff must consult with senior technologist when necessary. c. Isolates must be assessed in conjunction with the clinical data provided. d. Untimely processing specimens received > 24 hours. e. Poor staining of slide, incorrect interpretation of slide. f. Pseudohyphae of C. tropicalis may be mistaken for germ tubes of C. albicans. g. Successful isolation and correct identification of pathogenic isolates depend on the skill, experience and knowledge of the technologist.

12. References
1. ASM Clinical Microbiology Procedure Handbook. 2. Health Protection Agency (2004). Investigation of Genital Tract and Associated Specimens. National Standard Method CRM-SOP 28 Issue 4. 3. Bailey and Scotts Diagnostic Microbiology, 8th Edition.

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Categorty

Clinical Data MAC Gram SAB Wet Prep

BA O2

ANO2 48 hrs + MZD disc CHOC in CO2 GC CO2 Significant / Target Organisms

1. Pregnant women * * * * *

Genital Tract Specimens

Vaginal Discharge: Vaginal Swab Age 12-50 vag discharge Ante-natal Pregnant No clinical data Endo-cervical/ cervical/ urethral/ penile swab * * * * (<11yrs only) * * As above +

Trichomonas vaginalis Candida sp. Bacterial Vaginosis -hemolytic streps GBS (pregnancy only) GC S. aureus

2.

<11 years >50 years

3.

H. influenzae (children) Coliforms & Pseudomonas sp (only if pure or post-op) Anaerobes (if heavy and pure) * * * * * * *

Post delivery Post operation Post operation gyne Diabetes mellitus Dyspareunia Post menstrual bleed (PMB) Post coital bleed (PCB) Pyrexia Pelvic inflammatory Disease (PID) Salpingitis; Urethritis; Cervicitis Lower abdo pain Acute vaginitis or vulvo-vaginitis Vaginal warts Post-natal or puerperal discharge IUCD in-situ, or removal of IUCD * * * * * * *

Normally sterile fluids: amniotic fluid; peritoneal fluid, deep operative fluid/tissue etc.

Any organism may be significant discuss with Medical Microbiologist As above +

APPENDIX 1: CULTURE REQUIREMENTS AND TARGET ORGANISMS FOR GENITAL SPECIMENS

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4.

Premature prolonged rupture of membranes (PROM) Spontaneous rupture of membranes (SROM) Spontaneous abortion / Listerosis IUCD

Listeria monocytogenes

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Genital Tract Specimens

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Genital Tract Specimens

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Genital Tract Specimens

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Genital Tract Specimens

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