March 26-31, 2001

Laboratoire Bactériologie et Virologie
Hopital le Dantec
Cheikh Anta Diop University

Dakar, Senegal

Draft Workshop Report

Introduction

The AIDS Prevention Initiative in Nigeria (APIN) held its first workshop on HIV testing and surveillance from March 26-31, in Dakar, Senegal, under the auspices of Professor Souleymane Mboup's Laboratoire Bactériologie et Virologie at Hopital le Dantec, Cheikh Anta Diop Université, which has played an essential role in Senegal's response to the HIV and AIDS epidemic. The workshop was a step towards meeting one of the 18-month milestones of the APIN program, which is to establish accurate and thorough surveillance of HIV infection rates in three Nigerian states, Plateau, Oyo and Lagos.

The APIN workshop had three main objectives. First, to review the most recent survey data, instruments and survey methodologies being implemented in the target Nigerian states. Second, to review laboratory methods and perform additional testing on banked samples used in previous surveys. And last, to develop the design and planning of statewide surveys which are to be conducted in the spring of 2001.

As observed by the Senegalese experience, establishment of clear baseline HIV prevalence rates has been critical in designing and implementing targeted intervention strategies to prevent the spread of HIV. Thus, improved epidemiological surveillance facilitating the inclusion of more populations, better representative sampling, development of field-tested risk determinant questionnaires and appropriate informed consent forms and improved methodology for collection of blood samples that permit detection of virus levels and the host immune response, is a critical part of Nigeria's efforts to prevent the spread of HIV.

Logistics

Twelve participants from Nigeria representing laboratories involved in surveying for HIV infection among various populations in Plateau, Oyo and Lagos states attended the workshop (Annex 1). These participants included laboratory personnel actively involved in screening for and confirming HIV infection, as well as scientists and physicians involved in advising the Nigerian government and the Nigerian Action Committee on AIDS on surveillance methods and HIV prevention and treatment strategies for the entire country. In addition, there were several participants from the Harvard School of Public Health and laboratories at Cheikh Anta Diop University and affiliated labs (Annex 1).

The workshop consisted of overview presentations/seminars from 8h30 to 13h (Annex 2), each day (March 26-30). Seminars covered the goals of the APIN program, principles of epidemiological surveillance, sentinel surveillance, the virology and epidemiology of the Nigerian HIV epidemic, scientific theory and background of various laboratory assays performed in lab sessions, management of the lab and management of research programs. From 14h30 to late evening March 26-29, laboratory protocols to screen and confirm HIV infections were performed on banked blood and plasma samples brought from Nigeria (Annex 2, Annex 4). This was important for evaluating the current testing algorithms in the labs represented.

Key Issues Raised During Seminar Sessions

Mobilizing national political commitment:

Professor Mboup emphasized the need for political commitment at the national and local level for establishing an effective response to HIV and AIDS in any given country. This has proven to be key in Senegal's response to the epidemic. The government as well as moral and social institutions supported AIDS interventions very early on in the epidemic.

National responses to confront AIDS in Senegal were immediate once the first cases were identified for example, a blood bank policy to screen all blood donations for HIV infection was established by 1987. In Senegal, there were existing progressive policies that were able to strengthen the national response for example, since 1970 female commercial sex workers have been registered. Since this group engaged in "high-risk" behavior could be easily identified they could be targeted for interventions. Senegal has also been progressive in the availability of antiretroviral treatment for AIDS patients and was one of the first countries to participate in UNAIDS Accelerating Access Initiative.

Collaboration and communication among the stakeholders necessary for the national and local response to the AIDS epidemic is also critical. The National AIDS Program in Senegal is a multisectoral national program, with the main coordinating person being the Minister of Health. The Ministry of Health works closely with the National AIDS Committee, hospitals, laboratories and other institutions in its efforts to confront the HIV and AIDS epidemic. This local collaboration and support mechanism also strengthens the ability of individual stakeholders to collaborate with other groups in the region and internationally.

Members of the Nigerian delegation acknowledged Senegal's stable political situation over the past few decades, and noted the impact of political stability to the success of the country's HIV and AIDS interventions.

Support for regional collaboration:

Workshop participants addressed the need for regional collaboration among subxSaharan African nations particularly among those nations facing similar kinds of epidemics. The APIN workshop represented an example of such collaboration and demonstrated how Senegal and Nigeria could work together to confront HIV and AIDS in the region. The IbNg HIV recombinant virus is the major circulating recombinant form of HIV in both countries and throughout Western Africa. Therefore, prevention efforts in Nigeria can be modeled after previous efforts in Senegal.

Regional collaboration can also occur through the exchange and development of technical expertise. Professor Mboup commented on the fact that many students from all over West Africa have been trained in various disciplines at universities in Dakar. These students represent future contacts for collaborative efforts when they return to their home countries.

Support for international collaboration:

Professors Mboup and Kanki discussed their 16 year collaboration in Senegal to confront HIV and AIDS. This collaboration has built capacity in both Professor Mboup's laboratory at Cheikh Anta Diop Université and in Professor Kanki's laboratory at the Harvard School of Public Health. Scientists from each country have had the opportunity to be trained in Senegal and in the United States. The collaboration has mutually benefited both labs and institutions, and has lead to exciting novel scientific discoveries. Through the APIN program, Nigerian labs have the opportunity to be strengthened by this international collaborative effort.

Building capacity:

The importance of building capacity within Nigeria, locally (at the state level) and nationally, to respond to the HIV and AIDS crisis was a consistent theme raised by participants during the workshop. Several suggestions for building capacity were identified and are listed below:

a. Improving infrastructure:

Participants emphasized the need for improving labs and other facilities that form the first line response to the epidemic, particularly with regards to their ability to adequately handle and store samples to be screened and diagnosed with HIV infection.

One key issue raised by participants from Nigeria was the lack of a consistent electric power supply in many of the labs and the impact this has on the ability to store samples. The need for labs to have a constant electric supply or to have "back-up" storage mechanisms for samples was addressed. Several participants from Nigeria indicated a lack of access to liquid nitrogen and/or dry ice in their laboratories in Pre-Workshop Laboratory Evaluation forms distributed prior to the workshop. Access to these alternative means for storing samples must be investigated. Importantly, the costs and availability of liquid nitrogen and dry ice must be considered to ensure the sustainability of supply.

Another issue raised by participants from Nigeria was the need to repair or upgrade much of the general laboratory equipment currently in use in the labs. Many of the labs surveyed reported limited sample storage capacity as demonstrated by the limited number of freezers at their disposal. Many labs had -20°C freezers and of those that had a -70°C freezer, in more than half the cases the freezer was not functional. No labs indicated access to a refrigerated micro-centrifuge. The -70°C freezer and the refrigerated micro-centrifuge are useful for the proper storage and handling of samples to be processed for RNA. Therefore, this is an important need that should be addressed if these labs are to be able to perform viral load tests.

All participants from the labs in Nigeria had access to kits or equipment to perform enzyme immunoassays for HIV-1 and HIV-2. All labs had access to some rapid test kit that could screen for HIV-1 and/or HIV-2. Kits commonly used include the Capillus rapid test kit and Gennie II rapid test kit for HIV-1 and HIV-2. Further discussion by participants revealed that these kits were not consistently available and that labs were subject to whatever the market would provide at any given time point. This of course, significantly affects quality control standards within each lab since testing algorithms would be inconsistent. Another important problem was identified concerning kits--that is--many times the kits available are expired and thus useless. Participants discussed the need for the Nigerian government and other agencies involved in the procurement of screening kits such as DFID, to negotiate mass purchase of kits and a sustainable means of having good quality kits regularly available to labs in Nigeria.

Few labs reported the capacity to perform Western Blots as a confirmatory test for HIV diagnosis and none of the Nigerian labs reported the use of line immunoassays for confirming HIV infection. The use of Western Blots will be discussed later. (Note algorithms currently in use in labs in Nigeria will be addressed later in the report.)

b. Strengthening and supporting technical expertise:

Participants addressed the importance of strengthening local expertise to respond to the epidemic. This includes the training of lab personnel not only to perform HIV diagnostic assays but also to perform "state of the art" virology, immunology and public health research. The need to support individuals and labs currently performing HIV and AIDS research was also addressed. One component of the APIN program is the opportunity for graduate student training in virology, immunology, epidemiology and public health at the Harvard School of Public Health. As mentioned previously, technical expertise and knowledge, at both HSPH and Cheikh Anta Diop Université, have been improved as a result of collaborations between the Kanki and Mboup labs.

The importance of expertly trained laboratory personnel for HIV surveillance cannot be overemphasized. This becomes especially critical for labs performing Western Blot as a confirmatory test. This "gold standard" for confirming HIV infection is an extremely delicate technique and requires a well-trained individual to interpret the results. Criteria for positive results vary by institution and some organizations such as WHO have sought to standardize the interpretation criteria for Western Blots. Professor Kanki's lab at Harvard School of Public Health has long-time experience with performing this assay, particularly for confirming HIV-2 infection and can help labs through the APIN program in building technical expertise to perform this assay.

c. Improving mechanisms for technology transfer:

Participants from Nigeria expressed the hope that the first APIN workshop and similar workshops be a catalyst for creating the continuous exchange of information among the labs present. They expressed the need for access to better diagnostic and surveillance tools, more reading materials, updates on laboratory assays and practices, and other kinds of information. An email discussion group was cited as one possible means for ensuring the constant flow of information between labs. Most individuals present had access to computers and could obtain information electronically via the Internet. Again technology transfer could also be facilitated by training individuals in "state of the art techniques" both at HSPH and Cheikh Anta Diop Université.

Improving biological and behavioral surveillance:

As discussed previously, accurate epidemiological surveillance in Nigeria is one of the main goals of the APIN program and can be achieved once there is political commitment, financial support, and technical capacity and infrastructure in place to do so. Typically, surveillance is achieved through the establishment of sentinel sites or "watchposts," that provide access to various populations. The advantages of these sites were discussed by workshop participants and were identified as the opportunity to generate a public response to HIV, target prevention activities, monitor the success of the national response and help with plans to reduce the impact of HIV and AIDS. The weaknesses of sentinel surveillance sites were also covered including problems with tracking risk behaviors that contribute to the spread of HIV, the lack of coordination with other useful sources of information and lastly, the overemphasis of surveillance in the general population where little infection exists, as opposed to at risk sub-populations.

The importance of second-generation surveillance was emphasized throughout the workshop. Professor Olaleye from Ibadan (Oyo State), addressed the use of second-generation surveillance as defined by WHO and UNAIDS, and the use of groups that can be followed and counseled in prevention strategies. In principle, the approaches of second-generation surveillance should be more flexible and appropriate to the changing epidemic state over time. Second generation surveillance advocates comparison of biological and behavioral data for maximum explanatory power and the integration of information from other sources to improve the national response. By considering this information, better use of data to plan prevention and care programs can be ensured.

The concept of "bridge groups " which connect individuals with "high-risk" behaviors to the general population and the need to target prevention interventions to these groups was also discussed. Professor Mboup identified two groups in Senegal as examples of these "bridge groups": migrant workers and fishermen. Professor Kanki emphasized the need to identify similar kinds of "bridge groups" in Nigeria in order to adequately target prevention interventions.

Data collection methods for HIV surveillance were covered, including methods for collecting biological and behavioral data, and other sources of information such as HIV and AIDS cases, pediatric AIDS cases, registrations of deaths, sexually transmitted infection (STI) surveillance and tuberculosis (TB) surveillance.

Confidentiality and informed consent were also important topics covered. Anonymous and semi-anonymous approaches to HIV surveillance were discussed. Professor Shokunbi from Ibadan (Oyo State) commented on the fact that few blood donors in Nigeria are truly voluntary and that many of the blood donors screened in her lab were paid donors, and when needed to be contacted, often times the information they provided was proven false.

a. Monitoring Trends in HIV infection in Nigeria:

Professor Olaleye gave an overview of trends in HIV infection and the molecular epidemiology of HIV in Nigeria. The first AIDS case was reported in Nigeria in 1986. Subsequent sero-surveys have demonstrated a 10-fold increase in HIV prevalence among different categories of people in Nigeria between 1987 and 1991. Molecular epidemiology of HIV in Nigeria:

• Isolation of HIV-1 IbNg recombinant from a donor in 1991, identified as a variant of subtype A. This strain is now the predominant circulating recombinant form in the West African sub-region;
• HIV-1 subtype G was detected in Jos in 1994;
• HIV subtype O was detected in 1995;5. HIV-1 subtype B was detected among samples collected after 1994 (peptide ELISA and sequencing);
• Serologic and genomic identification of multiple HIV-1 subtypes has been reported in samples collected from 1993 to 1997;5. Reports of regional distribution of two main HIV-1 subtypes A and G and the A/G IbNg recombinant in Nigeria in 1995;
• More subtype A has been reported in the south, more subtype G in the north5. Subtypes A, B, G, A/G (IbNg) also detected in samples isolated from the southern regions of the country and analyzed in the U.S.;
• Subtyping by HMA of randomly collected HIV-1 positive samples from the middle belt (26) and SW (21) show more subtype A in the SW and more G and IbNg (A/G) in the middle belt (2000 samples);
• Both HIV-1 and HIV-2 and dual infections have been found in Nigeria (Table 1).

Table 1: HIV Types in Nigeria

	HIV-1	HIV-2	Dual
1985-1990 (urban)	60.0%	30.0%	10.0%
1992-1994 (rural)	37.5%	50.0%	12.5%
1999 (urban)	87.0%	4.0%	7.0%
2000 (urban PTB)	99.6%	0.4%	0.0%

PTB= Primary TB patients
STD = sexually transmitted diseases

b. Specific Surveillance Issues identified by participants from Plateau, Oyo and Lagos State

• Reports suggest that there is a high prevalence of HIV infection among blood donors in Nigeria. Further, surveys among HIV infected individuals reveal that up to 10% of those infected cite contaminated blood/blood transfusion as a source of infection.¹ Therefore, current algorithms for screening blood for transfusion have become quite strict, with labs performing ELISA and confirmatory test regardless of a rapid test result. The high prevalence of HIV infection in donated blood suggests that the practice of paying blood donors may create a potential bias among individuals who donate blood and possibly targets individuals with risky behavior. Laboratory personnel participating in the workshop report difficulty in recruiting low-risk volunteer donors. Discussions at the APIN workshop addressed the need for Nigeria to revise its blood donor policy to attract individuals with low risk behavior as volunteers. Some examples of how other sub--Saharan African countries have chosen to address high prevalence among blood donors were given. Professor Kanki cited the case of Botswana where HIV prevalence is high (30-40%) among adults in the general population. In Botswana, children in the third and fourth grade donate blood in order to ensure that the supply remains uncontaminated.

  ¹Note: at the workshop this was described as self-reported data on a questionnaire and therefore is subject to recall bias.

• Technical expertise in laboratories (as described previously) is critical for successful surveillance efforts. Nigerian participants stated the need for increased manpower and better training of all lab personnel.
• The individual characteristics of the HIV epidemic in Plateau, Oyo and Lagos were addressed. Second generation surveillance mandates that interventions be based on the characteristics of the epidemic in each region. For example, individuals falling into "high-risk" groups and "bridge-groups" may differ in each state based on other variables such as economic situation, main industry, and access to overall health services.
• Some laboratory personnel addressed the issue of high rates of indeterminate screening results seen in some labs. This could be due to many different factors including lab personnel error, lack of familiarity with performing certain assays, defective/inadequate equipment, defective kits and poor testing algorithms. All of these factors must be carefully considered moving forward in the labs that reported high frequency of indeterminate results.
• The need to build capacity in labs performing STD surveillance was covered. Studies from Jos showed that HIV-1 infection was higher among patients with Genital Urinary Disease than those without Genital Urinary Disease (Annex 3). HIV-1 infection was also higher among patients with Non-Ulcerative STDs than those without Non-Ulcerative STDs (Annex 3). HIV and other STD prevalence were found to be highest in the 20-29 and 30-39 age groups. HIV and STD prevalence were found to be highest among university students, where prevalence was found to be even higher than among commercial sex workers. Unpublished reports suggest that many female university students earn money by being companions to older men and therefore may be an important group for targeting interventions. These students represent the so-called "bridge-population" previously defined. High STD prevalence with HIV prevalence represents an opportunity for public health interventions when patients report to clinics for treatment with other STIs, which will most likely occur prior to these individuals seeking voluntary counseling and testing for HIV. Thus, pre and post-test counseling for HIV can be targeted to these individuals. Syndromic management of STDs may also have an impact on HIV transmission as observed in trials in Mwanza, Tanzania.
• Lack of adequate funds remains an issue for many labs in Nigeria. There is currently a lack of funds for HIV research and diagnosis. For example, at Jos University Teaching Hospital, HIV confirmatory assays must be partially subsidized by patients.

Laboratory Sessions

Table 2: Current Algorithms from Nigerian Labs

LAB	Current Testing Algorithm	Average number of specimens tested
		Per week	Per year
Department of Virology, UCH, Ibadan	Double ELISA and WB when discordant	100	5000
Department of Hematology, UCH, Ibadan	Screening?, Confirmatory WB	200	6000
Department of Medicine, JUTH	EIA then WB
Department of Hematology & Blood Transfusion, UCH, Ibadan	Double EIA and Combfirm	192	9984
Oyo State Blood Transfusion Services, Ibadan	Double EIA, or EIA/Rapid Test	200	9000

UCH: University College Hospital
JUTH: Jos University Teaching Hospital

Conventional HIV algorithms include a screening assay and supplementary or confirmatory assay. Since screening assays should detect all infected individuals they are highly sensitive. Confirmatory assays on the other hand, should be highly specific and ensure that uninfected individuals are not falsely labeled as HIV positive. Typically, for screening, an ELISA/ rapid /simple test is performed and as a confirmatory test western blot is considered the "gold-standard". Other less frequently used confirmatory test strategies include radio-immunoprecipitation assays and line-immunoassays. As discussed previously, western blots require well-trained personnel who can adequately interpret the results. However, the main problem with this assay is that many times it gives indeterminate results. When this happens, WHO recommends that for asymptomatic individuals, a second blood sample is tested in two weeks. For uninfected individuals who give indeterminate results, recommendations are that these individuals be followed for at least 1 year. Since not all labs have the technical capacity to perform western blots, alternative confirmatory strategies have been recommended which may include the use of two or more of the screening tests described above. In this case care should be taken to screen with the most sensitive test initially, and to ensure that the first and second tests use different principles.

Table 2 indicates current testing algorithms for some of the labs participating in the workshop. Labs also estimated the number of samples being tested each year. In Dakar, the following tests and assays were performed on banked samples from the Nigerian labs: Capillus Rapid test, Genescreen ELISA (Indirect), confirmatory immunoblot (home-made Western Blot), Line Immunoassay, nucleic acid extraction methods and DNA and RNA based PCR assays. Results from the lab sessions and the interpretation and recommendations for each lab are contained in Annex 4.

Evaluation of laboratory sessions

The majority of participants indicated that the concepts and protocols presented in the laboratory sessions were clear and that the techniques covered were useful and comprehensive (Annex 5). However, about half of the participants felt that the techniques covered were not realistic and adaptable for their existing lab conditions and infrastructure. Most participants felt that attention was paid to their specific questions concerning laboratory practices and protocols. Participants also felt that future workshops should be more catered to lab work.

Recommendations for the future

The first APIN workshop met its objectives. The most recent survey data, instruments and survey methodologies being implemented in Plateau, Lagos and Oyo states was reviewed. Additional testing on banked samples used in previous surveys was performed. Finally, the design and planning of statewide surveys to be conducted in the spring of 2001 was covered.At the conclusion of the meeting participants made the following recommendations:

1. The need for continuous exchange of information and developing effective communication networks. For example: improve access to email and access to journal articles.

2. All samples that gave indeterminate results in Dakar should be confirmed by western blot (details in Annex 4).

3. Future APIN Lab Workshops should emphasize the following:

• More intense lab work and more time in lab;
• Reference panel- Mboup;
• Increase space, decrease number of people in each lab;
• Emphasize quality control and the proficiency of results;
• Focus on some more difficult techniques for example PCR;
• Emphasize on-site technology transfer-after

4. Develop common SOP

• develop national Quality Control Program

5. A similar workshop on STI diagnosis and surveillance would be extremely useful.

6. The maintenance of equipment in Nigerian labs is a critical need that the APIN program must address.

7. The next workshop could include a component on study design.