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Protocol for Trichomonas vaginalis Resistance Monitoring in STD Clinics –

STD Surveillance Network (SSuN)

As of November 24, 2008

PROTOCOL SUMMARY

Collaborators:

Robert Kirkcaldy, MD, MPH

Lori Newman, MD

W. Evan Secor, PhD

Kim Workowski, MD

Emily Koumans, MD

Jane Schwebke, MD, Jefferson County Department of Health

Jennifer Donnelly, Colorado Department of Public Health and Environment

Preeti Pathela, DrPH, New York City Department of Health and Mental Health

Lenore Asbel, MD, Philadelphia Department of Public Health

Mark Pandori, PhD, San Francisco Department of Public Health

Paul Swenson, PhD, Washington State Department of Health

1. BACKGROUND

1.1. Literature Review and Current State of Knowledge about Project Topic

Trichomonas vaginalis is the most common STD worldwide.1 In the US, T. vaginalis infects 7.4 million men and women annually,2, with the highest prevalence of disease among young adults 3,4 and African-American women.5 The prevalence of trichomoniasis among women attending STD clinics is estimated as 13%.6 Risk factors include age greater than 20, African-American race, and low educational attainment.6

Trichomoniasis is recognized as a frequent cause of vaginal discharge, odor, and vaginal itching among women. Potential complications of infection include premature rupture of membranes during pregnancy, preterm labor, stillbirth, neonatal death, pelvic inflammatory disease and facilitation of HIV transmission.7-11

Current CDC STD Treatment Guidelines recommend the use of 5-nitroimidazole agents, either metronidazole or tinidazole, for the treatment of T. vaginalis infection.12 Both agents are associated with a 90-95% cure rate.13-15

Despite the high cure rates of current therapy, the availability of only a single class of antimicrobials for treatment has heightened concern about the emergence of T. vaginalis antimicrobial resistance. Metronidazole resistance has been reported since 1962 16, though the level of resistance has apparently remained low. Schmid et al found low-level metronidazole resistance in 2.4% of trichomonas isolates collected in 1997.17 Less than a decade later, however, Schwebke and colleagues tested T. vaginalis specimens (collected between 2003 and 2005) for resistance and found an overall resistance (MLC 50-200+ units) prevalence of 9.6%, with 3.4% of the entire sample demonstrating MLCs of at least 100 ug/mL.18 However, without ongoing systematic monitoring of resistance from differing geographic regions, it is difficult to draw firm conclusions about the emergence of resistance.

One of the challenges of evaluating and monitoring T. vaginalis antibiotic resistance is the lack of current standardization and variability between susceptibility testing results and clinical response. 16 Aerobic minimum lethal concentrations (MLCs) ranging from 50 to > 200 ug/mL have been considered consistent with resistance. Significant variability in the clinical response to metronidazole has been noted for isolates with MLCs 50 to 200 ug/mL. Of note, apparent clinical resistance has even been reported in patients with metronidazole MLCs of 12.5 ug/mL.19,20 Aerobic metronidazole MLCs of > 200 ug/mL seem to correspond well to clinical metronidazole treatment failures.16

1.2. Justification for Surveillance Activity

There is currently only one class of antibiotics with known effectiveness against T. vaginalis infection (5-nitroimidazoles). Emergence of moderate or high-level resistance to this class of agents has important public health and clinical implications. Inadequately treated trichomoniasis can lead to greater frequency of treatment failures, obstetric complications, T. vaginalis transmission to sexual partners, and HIV transmission. Increasing resistance may warrant changes to treatment guidelines.

1.3. Intended/Potential uses of surveillance findings

The data will be used to describe the prevalence of trichomoniasis and T. vaginalis antibiotic resistance. The data will be used to guide T. vaginalis treatment and screening recommendations.

1.4. Surveillance Locations

Under RFA #PS08-865, the Sexually Transmitted Disease Surveillance Network (SSuN), six grantees were selected to participate in the Part B Laboratory Component. These sites were:

▪ Jefferson County Health Department (JCHD) STD Clinic and the University of Alabama at Birmingham (UAB) laboratory, Birmingham, Alabama

▪ Denver Metro Health Clinic (DMHC) and the Colorado Department of Public Health and Environment (CDPHE) laboratory, Denver, Colorado

▪ One or more of the Bureau of STD Control (BSTDC) clinics and the New York City Public Health Laboratory (PHL), New York City, New York

▪ Philadelphia Department of Public Health District Health Center # 1 STD Clinic (DHC #1), Philadelphia, Pennsylvania

▪ The City Clinic and the San Francisco Department of Public Health (SFDPH) Public Health Laboratory (PHL), San Francisco, California

▪ Harborview STD Clinic, at the University of Washington’s Harborview Medical Center and the Public Health – Seattle & King County Laboratory (PHSKCL)

1.5. Objectives

▪ To improve control of T. vaginalis infection through:

► Describing the prevalence, sociodemographic, and clinical characteristics of women with trichomoniasis infection attending STD clinics

► Identifying the prevalence of in vitro resistance in this population

1.6. Surveillance Questions

▪ What is the prevalence of trichomoniasis in women attending STD clinics? Does the prevalence differ by race/ethnicity or geographic region?

▪ What is the prevalence of T. vaginalis antibiotic resistance? Does the prevalence differ among different populations (ie regional, racial/ethnic, socioeconomic status, symptomatic)?

▪ Do the susceptibility patterns of isolates from wet-prep negative specimens differ from isolates from wet-prep positive specimens?

▪ Are antibiotic-resistant isolates associated with a greater symptom burden?

▪ Have women with resistant T. vaginalis (or their partners) recently taken antibiotics?

▪ What is the sensitivity of wet-prep at each SSuN site?

▪ What is the prevalence of STD coinfection among women with trichomoniasis?

1.7. General Approach

This surveillance activity is observational and descriptive. Sixty T. vaginalis cultures will be collected from each site over one year and isolates will undergo antibiotic susceptibility testing.

1.8. Case definitions

Trichomoniasis

Isolation of T. vaginalis by InPouch culture of vaginal secretions, after incubation at 37o Celsius for 72 hours.

Antimicrobial (metronidazole or tinidazole) resistant T. vaginalis infection

Minimal resistance: T. vaginalis isolate demonstrating an MLC of 50-100 μg/mL under aerobic conditions

Moderate resistance: T. vaginalis isolate demonstrating an MLC of 200 μg/mL under aerobic conditions

Highly resistant: T. vaginalis isolate demonstrating an MLC of > 400 μm/mL under aerobic conditions

2. PROCEDURES/METHODS: Design

2.1. How surveillance system address hypotheses and meets objectives

By systematically collecting T. vaginalis isolates and wet-preps from women undergoing speculum exams in STD clinics, we will be able to assess the prevalence of trichomoniasis in this population and sensitivity of wet-preps at each site. Conducting ongoing systematic susceptibility testing on T. vaginalis isolates will allow us to evaluate and monitor the level of antibiotic resistance and monitor for emergence of resistance in geographic regions or among sub-populations.

2.2. Audience and Stakeholder Participation

Stakeholders include the patients, providers, SSuN collaborators, and the Centers for Disease Control and Prevention (CDC).

STD clinic clients will provide written informed consent for participation in this surveillance activity. Providers will work with SSuN collaborators in the development of the protocol, implementation, and analysis of the data. CDC will assist with protocol development, implementation, analysis, and translation of the data into public health recommendations.

2.3. Surveillance time line

▪ November- December, 2008: Development of protocol

▪ December 4, 2008: SSuN Collaborators Meeting

▪ January, 2009: Finalization of protocol

▪ March, 2009: Sites begin collection and transport of T. vaginalis culture specimens

▪ March, 2010: First year of data analyzed [program has been funded for one year]

3. PROCEDURES/METHODS: Surveillance Population

3.1. Description and source of sample population and catchment area

Jefferson County Health Department (JCHD) STD Clinic, Birmingham, Alabama

The JCHD STD Clinic is the only specialty public health STD clinic in Jefferson County, Alabama. In 2007, there were 14,356 visits by 10,360 unique patients. African Americans comprised 85 percent of the patient population, Whites comprised 12 percent, and Hispanics comprised 2 percent. Three percent of the clinic population is gay or bisexual.

Denver Metro Health Clinic (DMHC), Denver, Colorado

DMHC is the largest publicly funded STD clinic in the Rocky Mountain Region. In 2007, DMHC had over 18,000 visits, 60 percent of which were by male and 40 percent by female patients. The DMHC serves a diverse population: 36 percent White, 30 percent Latino, 25 percent African American, and 10 percent other races. MSM account for nearly 10 percent of all clinic visits.

Bureau of STD Control (BSTDC) clinics, New York City (NYC), New York

The population of New York City is large and diverse. The BSTDC delivers clinical services in 10 STD clinics located in all five counties of NYC. The distribution of STD clinics in different neighborhoods in five counties ensures access to STD care by persons of varying age, gender, race/ethnicity, and sexual orientation. In 2007, there were over 110,000 clinic visits by approximately 78,000 patients to the BSTDC STD clinics. Over 43 percent of patients were female. Over 50 percent of patients were African-American, 15.8 percent were White, and 25 percent were Hispanic.

Philadelphia Department of Public Health District Health Center # 1 STD Clinic (DHC #1), Philadelphia, Pennsylvania

The DHC #1 is the larger of two Health Department categorical STD clinics in Philadelphia, Pennsylvania. This clinic has a large and varied patient population. In 2006, 14,431 patients attended this clinic generating 23,254 patient visits. In 2007, 21,317 patient-visits by 12,983 unique patients were managed. In 2007, 7639 (35%) of visits were by women. Among women, 79 percent were African-American, 9.5 percent were White, and 4 percent were Hispanic.

The City Clinic, San Francisco, California

Approximately 100 patients per day are seen on a drop-in basis at San Francisco City Clinic, with 21,764 visits in 2006. The proportion of all visits by race/ethnicity was: 13 percent Asian/Pacific Islander, 16 percent African-American, 21 percent Hispanic, 0.5 percent Native American, and 49 percent White. Persons aged 20 years old or older comprised 97 percent of the visits. Women comprised 25 percent of the patient-visits.

Harborview STD Clinic, at the University of Washington’s Harborview Medical Center, Seattle, Washington

Harborview STD Clinic in Seattle, Washington, at the University of Washington’s Harborview Medical Center, is the state’s primary center for excellence in STD treatment and clinical care. The clinic provides STD-related care for high-risk populations and persons with little alternative access to care. In 2007, clinic staff evaluated 7,703 unique patients during 11,471 clinical evaluations, including 2,146 women and 5,557 men (1,711 of whom were MSM).

3.2. Proposed participant inclusion criteria

All women seeking care at the SSuN sentinel clinic sites who undergo a speculum exam are eligible for inclusion. Sites will collect specimens until 60 positive cultures are obtained and transported to CDC.

3.3. Participant exclusion criteria

Men, women who do not undergo a speculum exam and women who refuse to participate will be excluded.

3.4. Justification of exclusion of any sub-segment of the population

The surveillance will focus on vaginal specimens collected from women. T. vaginalis can be difficult to detect by diagnostic testing in men. Women who refuse to participate will not be included on ethical grounds.

3.5. Estimated number of participants

Each site will be asked to send at least 50 viable initial visit culture isolates per year (6 x 50 = 300) and all follow-up culture isolates. Ten to 20% of the isolates are expected to be non-viable upon arrival at CDC, so approximately 60 isolates will be requested from each site.

3.6. Sampling

4. PROCEDURES/METHODS: Surveillance Flow

4.1. Enrollment

► How will this be conducted at each site?

4.3. History and physical exam

► Should data be collected per routine STD clinic surveillance or special questionnaire?

4.4. Specimen Collection, Handling, and Transport

Specimens of vaginal sections will be collected by the health care provider performing the gynecological exam at the initial visit and at a 4-week follow-up visit. The health care provider will inoculate the InPouch TV culture system with the collected specimen.

The InPouch TV culture will be incubated for 72 hours at 37 degrees Celsius. The culture will be considered positive if trichomonads are present and seen by microscopy.

All positive cultures will be packaged and transported to the Division of Parasitic Diseases, CDC. Antimicrobial susceptibility testing will be performed by the standardized CDC method. The Mean Lethal Concentration (MLC) of metronidazole and tinidazole will be reported.

The results of susceptibility testing will be reported to the SSuN site that sent the specimen within approximately 30 days.

5. PROCEDURES/METHODS: Variables

5.1. Variables

5.2. Surveillance instruments

In addition to routinely collected clinic information, each site may develop a supplemental site-specific questionnaire that includes activity-related questions.

5.3. Outcomes

Outcome measures will consist of the presence or absence of T. vaginalis infection, the presence or absence of antibiotic resistance, and the presence or absence of clinical treatment failure.

5.4. Training for all surveillance personnel

Each site will develop and conduct training of surveillance personnel.

6. PROCEDURES/METHODS: Data Handling and Analysis

6.1. Data collection

6.2. Information management and analysis software

6.3. Data entry, editing and management, including handling data collection forms, different versions of data and data storage and disposition

6.4. Quality control/assurance

7. PROCEDURES/METHODS: Dissemination, Notification, and Reporting of Results

7.1. Notifying participants of their individual results

Participating clinics are responsible for notifying patients of their individual results, as per existing clinic protocols.

7.2. Anticipated products or interventions resulting from surveillance and their use

Results will be distributed to surveillance collaborators through periodic reports, and may be disseminated by presentations and publications in the peer-reviewed literature

.

APPENDIX 1. Summary Tables of Evidence

1. Prevalence of T. vaginalis in the general population

|Author |Year published |Year data collected |Type of Site |Specimen/ |# isolates |

| | | | |Test | |

|Birmingham, AL |Est. annual |Culture |~1700 |25% |Estimation of # cases based on research data of 25% |

| | | | | |prevalence; ~ 6 cases/day (=1700/260); |

| | | | | |~30 cases/week |

|DMHC, Denver, CO |2007 |? |224 | |~0.9 cases/day (=224/260); |

| | | | | |~4.5 cases/week |

|NYC |2006 |Wet mount |~600 | |All BSTDC clinics, ~2.3 cases/day for all clinics, |

| | | | | |~11.5 cases/week |

|NYC |2008 |Culture |17/110 |15% |One clinic, January 1 – April 16, 2008, ~0.2 cases/days |

| | | | | |(=17/75 business days); |

| | | | | |~ 1 case/week |

|San Francisco City Clinic, SF, CA |2006 |? |88 | |82 (93%) diagnosed among women; 82 cases /260 days = 0.3 |

| | | | | |cases/day; |

| | | | | |~1.5 cases/week |

|DHC # 1 STD Clinic, Philadelphia, PA |2006 |Wet mount |630 | |630/260 = 2.4 cases/day = |

| | | | | |~12 cases/week |

|DHC # 1 STD Clinic, Philadelphia, PA |2007 |Wet mount |774 | | |

|Harborview STD Clinic, WA |2007 |? |116 | |116/260 = 0.45 cases/day = |

| | | | | |~2 cases/week |

4. Prevalence of antibiotic resistance among T. vaginalis isolates

|Author |Year published |

|Edward W. Hook,, III, MD |Consultant, UAB |

|ehook@uab.edu |Medical Director for JCHD STD Program |

| |Professor of Medicine, Microbiology, and Epidemiology |

|Elizabeth Turnipseed, MD, MSPH |Principal STD Clinic collaborator |

|elizabeth.turnipseed@ |Director of Communicable Diseases for JCHD |

|Shuying Yu, MPH |Senior analyst |

| |Epidemiology Analyst |

|Virginia Bozeman |Disease Intervention Specialist |

|Jada Harper |Clerical Staff |

Part B.

|Jane R. Schwebke, MD* |Overall supervisor for project, Responsible for collaborating with CDC on the |

| |development of protocols, instituting protocols in the STD clinic for collection of |

| |specimens and data, overseeing microbiology work in her lab. |

| |Professor of Medicine and Epidemiology, UAB, |

| |Medical Director, Jefferson County Health Department (JCHD) |

|Charles Rivers, PhD. |Laboratory Manager |

| |Performs culture and susceptibility testing. Collaborating with CDC on technical |

| |aspects of common protocols. Responsible for archiving and shipping specimens to CDC.|

|Marga Jones |Database Manager |

| |Responsible for data entry, helping with generating data collection forms |

|Paula Dixon, MLT |Laboratory Technician |

| |Responsible for performing any Chlamydia or GC work |

2. Lab Capacity

This site has performed trichomoniasis susceptibility testing for the past 5 years and has processed over 300 specimens to date and have published the results of 168 isolates. The staff was trained at CDC by Dr. Evan Secor and utilizes the CDC standardized testing method. The site has the capacity to perform testing on 50 women per month.

3. Methods

The clinic nurses will begin to collect cultures for trichomonas and these will be processed by the UAB lab. Specimens are routinely archived in freezers or liquid nitrogen and shipped to various sites as required for multicenters protocols.

All females are screened by wet prep for candidiasis, trichomoniasis and BV.

We propose to culture women attending the JCDH STD Clinic at the beginning of each month, collecting each month until 5 positive cultures are obtained. This will result in 60 isolates over the course of 12 months. We propose the additional 10% due to the fact that viability until susceptibility testing is not 100%. Nurses at the STD Clinic will perform InPouch TV cultures on all women at the beginning of each month until instructed to stop. This should be accomplished in the first few days, allowing for susceptibility testing prior to banking in liquid nitrogen.

When a culture is observed to be positive, 500 uL are directly removed from the pouch, centrifuged to pellet the trichomonads, and resuspended in 1 mL of fresh Diamond’s TYM media containing 1.15% agar and 5% DMSO. The sample is then cryopreserved in liquid nitrogen as a backup sample. The remainder is recultured in 3 successive passes of fresh media supplemented with amphotericin B and tetracycline. After the final passage, cultures are centrifuged to pellet the trichomonads. Pellets are resuspended in 2 mL of fresh Diamond’s TYM media containing 1.15% agar and 5% DMSO, split into two 1 ml aliquots, and cryopreserved in liquid nitrogen for future use. Susceptibility testing in performed in triplicate on two trays, one aerobic and one anaerobic. Each section contains one row of control wells that contain trichomonads only and no drug in Diamond’s medium. The remaining 3 rows are plated in triplicate and contain either metronidazole or tinidazole in a 2-fold drug dilutions. Trichomonads are seeded into the plates at a concentration of approximately 10,000 cells per well. Plates are covered. Aerobic plates are incubated at 37oC directly in the incubator. Anaerobic plates are placed in Mitsubishi boxes with anaerobic environment generators and then incubated at 37oC. Plates are incubated 46-50 hours before scoring under an inverted microscope at 100X. Each ‘run’ of tests includes two known controls: a sensitive strain (CDC 520) and a resistant strain (CDC 955). The MLC endpoint is the lowest dilution concordant across the three replica well with 0% movement.

Colorado Department of Public Health and Environment (CPDPHE)

Denver, Colorado

1. Personnel

Part A.

|Kees Rietmeijer, MD |Principal Collaborator |

|Cornelis.Rietmeijer@ | |

|Christie Mettenbrink |Data Analyst |

|Christie.mettenbrink@ | |

|Isabel Mendez |RN Program Manager |

Part B

|Melanie Mattson |Program Manager |

|Jennifer Donnelly |Primary contact for CDC, |

|jennifer.donnelly@state.co.us |Program Coordinator |

|Gary Lilly |Fiscal Coordinator |

|Jean Finn |Administration Manager |

2. Lab Capacity

The DMHC onsite STD stat laboratory, which is part of the Denver Health laboratory system, is staffed full time and conducts the following stat tests: HIV rapid test, RPR, urine pregnancy test, dipstick and microscopic evaluations, gram stained smear, and vaginal wet preps (potassium hydroxide and saline preps for yeast, BV and TV). With the exception of routine RPR testing and HIV pooled testing (performed at the CDPHE lab), all tests are conducted at the STD/Denver Health lab.

The CDPHE laboratory is a fully functional public health laboratory with the capacity to perform cultures for GC and TV, perform NAAT testing for both GC and CT, conducts treponemal and non-treponemal assays, and conducts confirmatory testing for HIV infection.

Question: What is the culture and storage capacity?

3. Methods

DMHC is the largest STD clinic in the Rocky Mountain Region, with over 18,000 patient visits annually. The Part B activities will involve a collaborative effort between DMHC, the DMHC laboratory and the CDPHE laboratory.

With 20-25 women with T. vaginalis infection diagnosed by wet prep at DMHC every month, there will be ample prevalence of TV infection to select 50 women for the project annually. DPH and CDPHE will engage in discussions with other SSuN sites to determine the best sampling methodology for obtaining a representative sample of women with TV.

Vaginal specimens of selected cases will be collected and transferred to the InPouch culture/transport media during vaginal examination by DMHC. Specimens will be labeled with bar-coded labels to allow for electronic linkage to patient information in the clinic’s EMR. Specimens will be stored temporarily in the onsite DMHC laboratory incubator at 37oC and then transported to the CDPHE laboratory using routine courier services. Specimens will be further tested via culture, if necessary, and stored by the CPPHE laboratory until transported in batches to the CDC laboratory or as outlined in SSuN project guidelines once they are developed.

New York City Department of Health and Mental Health (NYC DOHMH)

New York City, New York

1. Personnel

Part A

|Preeti Pathela, DrPH |Principal project collaborator; |

|ppathela@health. | |

2. Lab Capacity

The PHL performs clinical and environmental testing on almost half a million specimens annually. A high volume of STD clinical specimens are received from DOHMH Bureau of STD Control (BSTDC) clinics and community based and juvenile detention center for the laboratory diagnosis of CT and GC. The PHL conducts CT/GC NAAT testing on approximately 90,000 specimens submitted by the 10 BSTDC clinics annually. The NYC PHL also performs cultures for gonorrhea on oral, rectal, cervical, and urethral specimens sent from BSTDC clinics for such testing. Wet mount is performed to detect T. vaginalis. T. vaginalis culture using InPouch TV was piloted between January and April, 2008, and BSTDC is conducting a project by which positive InPouch TV media from female STD clinic clients are tested by PCR to describe the genetic diversity of T. vaginalis (Principal investigator: Jane Carlton, PhD).

Question: Any limitations to culture or storage capacity?

3. Methods

Currently wet mounts being performed. The specimens tested at the Public Health Laboratory (PHL) would come from BSTDC clinic patients through the route by which STD clinic specimens are currently delivered to PHL, on a daily basis. Specimens could be collected from patients attending one or more of the 10 STD Clinics. Laboratory results would be made available through either existing electronic data systems.

Philadelphia Department of Public Health, Division of Disease Control,

Sexually Transmitted Disease Control Program

Philadelphia, Pennsylvania

1. Personnel

Part A.

|Lenore Asbel, MD |Medical Director, STD Clinic |

|Lenore.Asbell@ | |

Part B.

|Lenore Asbel, MD |Principal STD Clinic Collaborator; |

| |Responsible for modifying clinic protocols to assure collection of T. vaginalis |

| |specimens using InPouch TV, training staff, maintaining quality assurance, and |

| |coordinating clinic activities with PHL and SsuN; |

| |Medical Director, STD Clinic |

|Anne DeMeis |PHL point of contact with other SsuN Lab staff; |

| |Will maintain QA of TV culturing, supervise medical technician; |

| |Immunology Lab Supervisor |

2. Lab Capacity

DHC # 1 has an on-site laboratory capable of performing rapid RPR, rapid HIV, Gram staining, darkfield microscopy, and wet preps for TV, yeast, and BV. All other tests (GC culture, FTA-ABS, TPPA, NAAT for GC/CT, herpes direct fluorescence antibody (DFA) and hepatitis serologies) are performed at PHL.

The Philadelphia Bureau of Laboratory Services Public Health Laboratory (PHL) is a full-service public health laboratory located in the same building as DHC #1. PHL conducts wet mounts for BV and TV, NAAT testing of genital specimens for GC/CT, treponemal and non-treponemal tests for syphilis, including rapid RPR, GC culture, and rapid HIV testing, in addition to other tests. PHL is flexible and able to implement novel and collaborative standard protocols to adapt to public health needs. Current NAAT volume exceeds 100,000 tests/year.

At existing staffing levels, 10 specimens/week can be processed during 6 hours/week of lab time.

3. Methods

The Philadelphia Bureau of Laboratory Services Public Health Laboratory (PHL) in conjunction with PDPH District Health Center #1 STD Clinic (DHC #1), will implement T. vaginalis culture. Staff will inoculate the InPouch TV system from female specimens for culture in PHL. PHL and its stat DHC #1 laboratory will continue to perform wet-prep for T. vaginalis. DHC #1 will perform cultures on those women patients seen by staff clinicians each Monday who have a pelvic exam, regardless of wet-prep results or symptomatic status (~25 women). This will ensure that representative specimens are obtained. The initial wet-prep procedure will be performed by clinicians in DHC#1 and cultures inoculated there. The culture will be transported upstairs to PHL, incubated and read as recommended. ~ 3 positive cultures are expected per Monday of testing. PHL expects to meet the objective of obtaining 50 positive TV cultures within 17 weeks of initiating the procedure. Specimens will be packaged and shipped to CDC for antimicrobial testing on a scheduled basis.

San Francisco Department of Public Health

San Francisco, California

1. Personnel

Part A.

|Kyle Bernstein, PhD, ScM |Principal Collaborator |

|kyle.bernstein@ |Chief of the Epidemiology, Research, and Surveillance Unit of the STD Section, SF |

| |Department of Public Health (SFDPH) |

|Susan Philip, MD, MPH |Principal STD Clinic Collaborator |

| |Medical Director of the City Clinic |

|Robert Kohn |Surveillance Coordinator |

| |Responsible for managing and analyzing the surveillance data and transmitting to |

| |CDC |

|Julia Marcus |Project manager for SsuN |

| |Epidemiologist |

|Armanda Litiatco |Data entry |

Part B.

|Mark Pandori, PhD. |Principal Collaborator |

| |Chief Microbiologist (SFDPH PHL) |

|Lily Lew |Laboratory technician |

2. Lab Capacity

The PHL provides over 100,000 lab analyses annually to assist in the diagnosis and screening of STDs. This includes NAAT methods for GC, CT, and herpes, as well as serological tests for syphilis, herpes, and HIV. A variety of specimen types are tested including genital, rectal and oropharyngeal. Service is provided to over 30 clinic sites and the results are transmitted electronically to the majority of these providers.

Question: culture and storage capacity?

3. Methods

We plan to collaborate with the San Francisco Public Health Laboratory to … collect culture positive Trichomonas vaginalis specimens from a representative sample of 50 females seen at the SF City Clinic, the City’s only municipal STD Clinic.

Currently T. vaginalis is diagnosed through examining wet mount slides collected during clinical exam and read at the City Clinic stat-lab. As part of SsuN, all female patients with a positive wet mount will have specimens collected for culture. After inoculation, culture pouches will be labeled and transferred to the PHL for incubation, analysis, storage, and transport to CDC for resistance testing. Per protocols, demographic, clinical and behavioral data from patients with T. vaginalis culture specimens will be electronically sent to CDC.

Washington State Department of Health, Infectious Disease & Reproductive Health Assessment Unit and STD/TB Services Section, and the

Public Health – Seattle & King County Laboratory (PHSKCL)

Olympia, Washington

1. Personnel

Part A

|Mark Stenger |Epidemiologist III |

|mark.stenger@doh. | |

|Todd Rime |Research Investigator III |

|Todd.Rime@doh. | |

|Roxanne Pieper Kerani |Epidemiologist |

|rkerani@u.washington.edu | |

Part B

|Paul Swenson, PhD | |

2. Lab Capacity

PHSKCL is a modern 5,000 square foot laboratory located at Harborview Medical Center in Seattle. It is staffed by 14.5 FTEs including clerical staff, laboratory technicians, and microbiologists and performs over 100,000 tests a year, including tests for HIV, syphilis, hepatitis, Chlamydia, gonorrhea, and other pathogens of interest, for the Harborview STD Clinic and other Public Health clinics in Seattle and King County.

Question: Culture and storage capacity?

3. Methods

Cultures will be collected from the first 1000 females presenting for exam at Harborview STD Clinic with a preference for screening those with vaginal discharge. Vaginal swabs from the posterior fornix or equivalent sites will be used to inoculate InPouch TV culture pouches in the clinic. Inoculated media will be transported to the laboratory for incubation. Isolates found to be positive after three days of incubation at 37oC will be prepared and shipped to CDC for resistance testing.

Reference List

(1) World Health Organization. Global Prevalence and Incidence of Selected Curable Sexually Transmitted Infections: Overview and Estimates. 1-50. 2001. Geneva.

Ref Type: Report

(2) Centers for Disease Control and Prevention. Trichomoniasis. 2008.

Ref Type: Internet Communication

(3) Miller WC, Swygard H, Hobbs MM et al. The Prevalence of Trichomoniasis in Young Adults in the United States. Sexually Transmitted Diseases. 2005;32:593-598.

(4) Weinstock H, Berman S, Cates W, Jr. Sexually transmitted diseases among American youth: incidence and prevalence estimates, 2000. Perspect Sex Reprod Health. 2004;36:6-10.

(5) Sutton M, Sternberg M, Koumans E, McQuillan G, Berman S, Markowitz L. The Prevalence of Trichomonas vaginalis Infection among Reproductive-Age Women in the United States, 2001-2004. Clin Infect Dis. 2007;45:1319-1326.

(6) Helms DJ, Mosure DJ, Metcalf CA et al. Risk factors for prevalent and incident Trichomonas vaginalis among women attending three sexually transmitted disease clinics. Sex Transm Dis. 2008;35:484-488.

(7) Cotch MF, Pastorek JG, Nugent RP et al. Trichomonas vaginalis associated with low birth weight and preterm delivery. The Vaginal Infections and Prematurity Study Group. Sex Transm Dis. 1997;24:353-360.

(8) Minkoff H, Grunebaum AN, Schwarz RH et al. Risk factors for prematurity and premature rupture of membranes: a prospective study of the vaginal flora in pregnancy. Am J Obstet Gynecol. 1984;150:965-972.

(9) Moodley P, Wilkinson D, Connolly C, Moodley J, Sturm AW. Trichomonas vaginalis is associated with pelvic inflammatory disease in women infected with human immunodeficiency virus. Clin Infect Dis. 2002;34:519-522.

(10) Laga M, Manoka A, Kivuvu M et al. Non-ulcerative sexually transmitted diseases as risk factors for HIV-1 transmission in women: results from a cohort study. AIDS. 1993;7:95-102.

(11) McClelland SA, Sangare L, Hassan WM et al. Infection with Trichomonas vaginalis Increases the Risk of HIV-1 Acquisition. Journal of Infectious Diseases. 2007;195:698-702.

(12) Workowski KA, Berman SM. Sexually transmitted diseases treatment guidelines, 2006. MMWR Recomm Rep. 2006;55:1-94.

(13) Hager WD, Brown ST, Kraus SJ, Kleris GS, Perkins GJ, Henderson M. Metronidazole for vaginal trichomoniasis. Seven-day vs single-dose regimens. JAMA. 1980;244:1219-1220.

(14) Manorama HT, Shenoy DR. Single-dose oral treatment of vaginal trichomoniasis with tinidazole and metronidazole. J Int Med Res. 1978;6:46-49.

(15) Forna F, Gulmezoglu AM. Interventions for treating trichomoniasis in women. Cochrane Database Syst Rev. 2003;CD000218.

(16) Wendel KA, Workowski KA. Trichomoniasis: Challenges to Appropriate Management. Clin Infect Dis. 2007;44:S123-S129.

(17) Schmid G, Narcisi E, Mosure D, Secor WE, Higgins J, Moreno H. Prevalence of metronidazole-resistant Trichomonas vaginalis in a gynecology clinic. J Reprod Med. 2001;46:545-549.

(18) Schwebke JR, Barrientes FJ. Prevalence of Trichomonas vaginalis Isolates with Resistance to Metronidazole and Tinidazole. Antimicrobial Agents and Chemotherapy. 2006;50:4209-4210.

(19) Lossick JG, Muller M, Gorrell TE. In vitro drug susceptibility and doses of metronidazole required for cure in cases of refractory vaginal trichomoniasis. J Infect Dis. 1986;153:948-955.

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SSuN

STD Clinic

Results

Negative

Positive

InPouch TV incubated at 37oC for 72 hours

Sent to CDC for susceptibility testing

Routine clinical management, no further participation

Culture Result

Clinician performs wet-prep and InPouch TV culture

Clinical history and physical/speculum exam

Possible Sampling Methods

See Excel Spreadsheets to illustrate examples

* Rate of NYC collection will depend on whether sampling from one site or all sites – some examples use one site, some use all sites

▪ All sites start collecting at same time and collect specimens until 60 obtained (Example 1)

▪ All sites start collecting at the same time, but are restricted to only at most 5 specimens per week to reduce burden on CDC lab (Example 2)

▪ On the first of each month, each SSuN site takes T. vaginalis cultures from every woman undergoing a pelvic exam until 5 positive T. vaginalis cultures obtained (Example 3)

▪ Three sites start collecting at the start on the first month, three sites start collecting at the second month. All sites collect at the start of every other month (Examples 4 [1 NYC site] and 5 [all NYC sites])

▪ Staggered participation:

o Some sites start March 1, and as the end of collection of some sites is projected, other sites start collecting (July 1, Sept 1). (Examples 6 and 7)

Inclusion Criteria to be determined:

▪ Symptom status: Propose to include symptomatic and asymptomatic, thus can evaluate association with symptomatic status

▪ Presence of vaginal discharge: Propose to include those without a discharge

▪ Wet-prep positive: Propose to include wet-prep positives and negatives, performing cultures at the time of wet-prep, until requested number of samples obtained by chosen sampling strategy.

Women agrees to participate

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