Global Access to STD Diagnostics

Global Access to STD Diagnostic is published by the Division of Sexual Health Promotion and STD Prevention and Control, Bureau of HIV/AIDS, STD and TB, Laboratory Centre for Disease Control, Health Canada. It is Health Canada's contribution to the World Health Organization (WHO), to assist in the diagnosis, treatment and prevention of STDs worldwide. The newsletter reports on the activities of the STD Diagnostics Initiative. The Initiative includes leading experts in the field of STD research and control. The content and opinions expressed in Global Access reflect those of the STD Diagnostics Initiative.

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• Return of SDI Secretariat to WHO
• SDI Update
• Diagnostic Monoclonal Antibodies to Hemoglobin-Binding Protein of H. ducreyi
• Rapid Detection for the Diagnosis of Chancroid
• SDI Specimen Collection Panel Progress Report
• Development of a one-step immunochromatographic (ICS) strip test for syphilis

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Return of SDI Secretariat to WHO

The secretariat of the Sexually Transmitted Diseases Diagnostic Initiative (SDI) is returning to the World Health Organization from UNAIDS, where it has been housed since January of 1996. The SDI had previously been located in the WHO Global Programme on AIDS (GPA) in 1994, but with the discontinuation of GPA in 1996, the SDI secretariat moved to the newly operational UNAIDS. SDI is now part of the Communicable Diseases cluster, of which Dr. Heymann was named Executive Director upon the recent reorganization of WHO.

The major reorganization of WHO takes place with the arrival of its new Director General, Dr. Gro Harlem Brundtland, former Prime Minister of Norway. The new structure of WHO has consolidated multiple separate programs into nine clusters, each led by its own executive director. The structure of the Communicable Diseases Cluster, shown below, is organized horizontally by functional area rather than vertically by disease, and is intended to offer more flexibility and enhance collaboration. In addition to the consolidation of existing programs, a number of new activities have been initiated. The Communicable Diseases Cluster merges activities from several programs, including those dealing with leprosy, tuberculosis, and the control of tropical diseases; emerging and other communicable diseases surveillance and control; and research and training in tropical diseases.

The SDI will be managed by Dr. Mark Perkins as part of a diagnostics operation within the Product Research and Development team (circled). The Tuberculosis Diagnostic Initiative and new diagnostics projects for other priority diseases are also managed within that operation. Diagnostics joins Tropical Disease Drug and Vaccine Discovery operations led by Dr. Win Gutteridge in the Product Research and Development team. The SDI expects to benefit from the product discovery and development expertise of that team and from its collaborative relations with industry. The Research and Development Department, in which product research and development is housed, is directed by Dr. Carlos Morel and includes the 25-year-old Special Programme for Research and Training in Tropical Diseases.

SDI Update

There have been some major changes in the SDI organization and administrative activities in the past year. The secretariat of SDI is now incorporated into the Communicable Diseases Cluster of WHO. A search to recruit an administrative officer for the SDI is under way.

In the immediate past, the SDI had two major committees responsible for guiding its activities. The Steering Committee provided leadership and direction, and was responsible for reviewing grant applications and recommending funding. A Committee of Interested Parties (CIP) was open to those involved in the SDI's goals of developing inexpensive, rapid diagnostic tests for use in resource-poor settings. As the move was made from UNAIDS to WHO, the organization of the SDI also changed.

The existing committees were dissolved, and the members were thanked for giving their time and expertise in this endeavour. The previous chairmen, Max Chernesky of the Steering Committee and Paul Delay of the CIP, resigned from those activities. Both are still active within the SDI: Dr. Delay is still a member of the SDI, and Dr. Chernesky chairs the Scientific Advisory Committee.

The SDI is now the remaining structure and is chaired by Dr. Julius Schachter.

There has been a change in the activities of the SDI. Rather than trying to be a mini funding agency, open to grant applications on test development, the SDI is now endeavouring to evaluate existing rapid STD diagnostic tests in resource-poor settings in preparation for what we presume will be the next generation of rapid diagnostic tests. Thus, we are attempting to develop experience in evaluating tests in the conditions under which they will be used. This is obviously more difficult than test evaluation in sophisticated research laboratories and their collaborating clinics.

The SDI will continue funding research activities, within budgetary limitations, and these applications will be reviewed on an ad hoc basis by a Scientific Advisory Committee. Rather than having a standing committee, specific relevant expertise will be sought for each review; Dr. Chernesky will be directing this endeavour. Existing grants will be continued for their committed duration, and if adequate progress is made and appropriate funds are available these projects may receive continued funding.

The Global Access Newsletter which was published for the past six years as part of the Program for Appropriate Technology in Health (PATH) in Seattle is now being published at Health Canada and coordinated by Dr. Ann Jolly in the Bureau of HIV/AIDS, STD and TB. We are indebted to individuals such as Jaqueline Sherris, Diane Lachman and coworkers at PATH, who provided unyielding leadership in this project as well as the Rockefeller Foundation for financial support.

For the past four years, six projects have been funded by the SDI. This edition of the Newsletter provides summaries of the progress achieved from several of the projects.

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Diagnostic Monoclonal Antibodies to Hemoglobin-Binding Protein of H. ducreyi

The objective of this project was to develop a non-culture, monoclonal antibody-based diagnostic field test for chancroid. The monoclonal antibodies (Mabs) to the hemoglobin-binding protein (HgbA) of Haemophilus ducreyi are used. The rationale for choosing HgbA for detection of H. ducreyi is that HgbA is a major protein found in all tested isolates; it is immunologically and functionally conserved; small amounts can be readily purified in a single step; and it is highly immunogenic in laboratory animals. Isogenic mutants of HgbA are avirulent in the human model of H. ducreyi infection (unpublished data), suggesting that this gene must be expressed to cause disease and that naturally occurring mutants do not exist. Therefore, HgbA would be a good target for immunologic detection.

Native (nHgbA, from H. ducreyi) and recombinant HgbA (rHgbA, over-expressed in E. coli) were purified, mice were immunized, and several thousand hybridoma supernatants were screened for reactivity. Several hybridoma cell lines were cloned by limiting dilution, and clones proving to be stable were used to produce 0.5 L amounts of cell supernatant for further characterization. We have purified analytical amounts of IgG from each. We have developed a simple capture ELISA assay using each purified Mab as the capture antibody and rabbit anti-peptide sera as the detection antibody. Mabs have been tested for cross-reactivity against a panel of geographically diverse strains that contain a variety of ribotypes (P.A. Totten, manuscript in preparation).

Several Mabs were isolated, characterized and purified. We have grown the geographically diverse strains (above) and prepared the cellular extracts for testing. In our format, H. ducreyi are detergent solubilized, and all Mabs recognize this form of HgbA. Among the several tested, we have identified three unique Mabs that are non-competitive and recognize the HgbA protein from all strains of H. ducreyi tested. We estimate that in a simple, non-optimized capture ELISA, we can readily detect 50 ng of purified HgbA mixed with a cell extract from a mutant H. ducreyi unable to make HgbA. This corresponds roughly to 100,000 bacteria (depending on strain, amount of heme starvation and other variables). We have not tried to optimize this ELISA system, but have used it only for strain surveys. Thus, we have three Mabs in hand, suitable for capture and detection.

Christopher Elkins, Ph.D.

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Rapid Detection for the Diagnosis of Chancroid

A feasibility study was undertaken to test the ability of polyclonal and monoclonal (Mab) antibodies to heat shock proteins of H. ducreyi for use in a capture assay to detect H. ducreyi from clinical specimens; it is also hoped to assess their value in a lateral flow format for use in developing countries with no laboratory facilities.

During this study, high-titre polyclonal and monoclonal antibodies were produced and purified. Mab BB11 reacted with all strains of H. ducreyi, whereas Mab CC11 and the polyclonal antibody reacted additionally with some other species of Haemophilus. The detection of the epitope for BB11 but not CC11 was enhanced by prior incubation with 0.1% SDS, but not with Triton X-100, Tween 20 or Tween 80. Mab BB11 has proved to be the most useful of the antibodies, and in combination with the polyclonal antibody has been used to develop a capture assay. In addition, Mab BB11 has been successfully conjugated to colloidal gold, and these conjugates recognize H. ducreyi in the lateral flow format.

In order to establish the sensitivity and specificity of these antibodies for the detection of H. ducreyi antigen in clinical samples, it is proposed to test them in the capture assay before developing the lateral format further. Antibodies with high sensitivities and specificities often transfer well from ELISA to the lateral flow format. As a source of clinical material, swabs from genital ulcers of more than 200 male patients with genital ulcer disease (GUD) presenting to clinics in South Africa have been collected and transferred, frozen, to St. Mary's, in London. The cause of GUD in these patients has been determined at SAIMR, Johannesburg, by means of tests for Treponema pallidum, H. ducreyi, herpes simplex virus and Chlamydia trachomatis. Optimization of the release of antigen from the swabs and subsequent testing of these samples is currently being undertaken.

Catherine Ison, Ph.D.
Ron Ballard, Ph.D.

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SDI Specimen Collection Panel Progress Report

The efforts to develop a specimen panel of pathogen-positive clinical materials have continued. The numbers of stored lower genital specimens from males and females are listed in Table 1A. These specimens have been drawn from studies in North America, Africa, and Asia. In addition, a collection of swab specimens from genital ulcers has also been obtained, from S. Morse and R. Ballard. The distribution of these specimens is presented in Table 1B.

Laboratory-grown agents are also available for both sensitivity and specificity panels for tests attempting to diagnose sexually transmitted diseases. These include rabbit testicular material for Treponema pallidum as well as normal flora of the genital tract for the specificity panel.

J. Schachter, Ph.D.

Table 1. SDI Specimen Collection

 

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Development of a one-step immunochromatographic (ICS) strip test for syphilis

PATH and Omega Diagnostics have recently completed the initial product development of a one step  immunochromatographic strip test to detect antibodies specific to T. pallidum. Results can be obtained in 10 minutes or less using patient plasma or serum. The strips, packaged in foil pouches, are suitable for use for months at ambient temperatures.

In the last half of 1998, Omega and PATH successfully completed transfer of the test to Quorum Diagnostics (Vancouver, Canada); a subsidiary of Omega. Standard operating procedures and other manufacturing documents were drafted and commercial production has recently commenced.

An initial prospective field evaluation for the syphilis ICS test on 187 specimens from an STD clinic in Mumbia, India, indicated a sensitivity of 96.6% and a specificity of 96.1% when compared with results of RPR and TPHA tests. This study indicated that the test was positive in several patients with early syphilis that were not yet reactive to RPR test antigen. A retrospective study in Birmingham, Alabama, using 260 STD clinic specimens also demonstrated a high sensitivity and specificity. Similar data have been obtained from panels of sera or plasmas from South Africa and Eastern Europe. Currently, prospective clinical studies are in progress in Mexico, Peru, and the Philippines.  Additional studies are being planned. PATH and Omega are presently working on additional improvements to the tests, including the modification of the strip to use whole blood as a specimen. This would allow the test to be run directly from a sample of venous blood, or preferably a finger stick.

Milton Tam, Ph.D.

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STD Diagnostics Initiative Founding Members

. Seth Berkley, M.D., The Rockefeller Foundation . Claude Betts, M.D., Pan American Health Organization . Richard Frank, Population Services International . Lieve Fransen, M.D., Ph.D., Commission of the European Communities . Jeffrey Harris, M.D., United States Agency for International Development . Subhash Hira, M.D., Ministry of Health/Zambia . Penelope J. Hitchcock, D.V.M., M.S., National Institutes of Health . King K. Holmes, M.D., Ph.D., University of Washington . Franklyn Judson, M.D., Denver Department of Health and Hospitals . Michael Norgard, Ph.D., University of Texas . Sheila Mitchell, Family Health International . Stephen Morse, Ph.D., Centers for Disease Control and Prevention . Peter Piot, M.D., Ph.D., Institute of Tropical Medicine . Lair Guerra de Macedo Rodrigues, Dr. P.H., Ministry of Health/Brazil . Wendy Roseberry, World Bank . Julius Schachter, Ph.D., University of California, San Francisco . Jimmy E. H. Sng, M.D., Ministry of Health/Singapore . Milton Tam, Ph.D., PATH/Seattle . Hiko Tamashiro, Ph.D., World Health Organization . Kathleen Toomey, M.D., M.P.H., Centers for Disease Control and Prevention . Judith N. Wasserheit, M.D., M.P.H., Centers for Disease Control and Prevention

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Global Access to STD Diagnostics is published by the Division of STD Prevention and Control, Bureau of HIV/AIDS, STD & TB, Laboratory Centre for Disease Control, Health Canada. It reports on the activities of the STD Diagnostics Initiative. The Initiative includes leading experts in the field of STD research and control. Content and opinions expressed in Global Access reflect those of the STD Diagnostics Initiative. Questions and comments should be directed to:

Division of EMC WHO 20, avenue Appia CH-1211 Geneva

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