Virology

Immunology

HIV/STD research and intervention

Projects

Publications

Objective

The Mycobacteriology Unit aims to optimize the diagnosis and control of tuberculosis and non-tuberculous mycobacteria (NTM). We therefore focus on the rapid detection and worldwide surveillance of multidrug resistant tuberculosis (MDR TB), and the control of Buruli Ulcer (BU) in developing countries.

Research activities

The specific objectives and recent results of the current projects can be summarized as follows:

1. Evaluation of Category II
retreatment in a prison with a high prevalence of drug resistant TB
In collaboration with MSF Belgium, and the Ministry of Justice in Siberia, we started a cohort study on 234 patients receiving the WHO recommended Category II retreatment regimen in the prison hospital of Mariinsk (Siberia). DST and DNA-fingerprinting results from patients’ isolates before, after 3 months of treatment and at the end of 8 months treatment were compared. This study evidenced the shortcomings of this regimen in a prison population with a high prevalence of drug-resistance: (1) exogenous reinfections with multi-or poly-drug resistant strains occurred during treatment, (2) resistance amplification was demonstrated, and (3) MDR disease persisted despite treatment.

Research activities

Assistance and
education activities

Scientific awards

Cooperation

• The problem of drug-resistant tuberculosis. Overcrowding contributes to TB transmission in Russian prisons.
Reproduced with permission from "The global impact of drug-resistant tuberculosis" by Harvard Medical School & the Open Society Institute. Photographer: Sergei Gitman, Public Health Research Institute, 1999.

3. Drug susceptibility (DST) as a factor for evaluation of national treatment regimens
In collaboration with Damian Foundation a project on DST has been running in Bangladesh since a base-line survey was conducted there at the end of 1995. The main aim is to see what is happening in cases that relapse or fail after treatment, and to give clinically useful information. The study has shown that (i) the prevalence of drug resistance in Bangladesh is surprisingly low, (ii) national treatment regimens are appropriate for smear positive cases, (iii) indications for retreament regimens should be extended to include all patients treated for at least one month with any drug, and (iv) NTP regimen for smear-negative cases runs the risk of leading to MDR under present field conditions.

4. Detection of resistance to second-line drugs / Evaluation of different solid media and drug concentrations
Using more than 700 clinical isolates, we evaluated different solid media and drug concentrations to detect resistance to kanamycin (KA), ofloxacin (OFL), capreomycin (CAP), ethionamide (ETH) and cycloserine (CYCL) using the proportion method.
The conclusion is that LJ medium provided better growth for MDR isolates, but appeared not the best medium for performing second-line DST, particularly for ETH, CAP and CYCL. From the agar media tested, 7H9 provided the best growth. Comparison of different agar media for OFL and KA gave only a few discrepant results. Further evaluations are needed to correctly set the critical concentration of CYCL.

5. Rapid detection of drug resistance
We have continued the evaluation of the Mycobacteria Growth Indicator Tube (MGIT) as a rapid method for assessing drug resistance in M. tuberculosis.

In collaboration with Médecins sans Frontières (MSF) and Becton Dick-inson Microbiology Systems, our laboratory is trialing the MGIT system for rapid direct DST in the prison hospital in Mariinsk. Validation of KA critical concentration in MGIT is ongoing. This work is important for the newly-established ‘DOTS-plus’ programs in MDRTB ‘hot spots’ because MDRTB patients with associated kanamycin resistance must be detected promptly and given alter-native treatment.

Our laboratory has also modified the Alamar blue microtiter technique to perform simple susceptibility tests at just one critical concentration for each antibiotic. To reduce discrepancies found with this proportion method for EMB and SM, and to be able to differentiate between high and low level resistance, several concentrations of each antibiotic need to be evaluated. To this end we have started to evaluate this technique for determination of minimal inhibitory concentrations (MIC) of INH, RMP, EMB and SM.

6. Molecular analysis of drug resistance
Sixty-two Mycobacterium tuberculosis isolates from 10 different locations around the world were tested for pyrazinamidase (PZase) activity, and their pyrazinamide (PZA) susceptibility determined by the radiometric method. Interestingly, six PZA resistant isolates have a wild type (wt) pncA sequence (ie. the gene encoding PZase), four isolates lacked pyrazinamidase activity. Four out 17 pncA mutants are presenting a low or moderate level of resistance (R), while 4 out 6 pncA wt isolates present moderate or high level R to PZA. The search for alternative molecular markers of resistance to PZA is required.

In previous experiments with the hybridization assay INNO LiPA Rif TB, no mutations were found in the 509 to 534 codon region of the rpoB for 4 out 203 RMP R isolates. Further sequencing of the rpoB gene revealed a Val146Phe in 3 isolates out of 4. Several molecular and epidemiological arguments support the hypothesis that this substitution is responsible for decreased susceptibility to RMP.

7. Low-oxygen conditions: their effect on the pathogenesis and treatment of tuberculosis and other mycobacterial infections
This new project has already discovered the presence of novel genes encoding alpha-crystallin homologs (a 16kDa heat shock protein playing a central role in the adaptation of M. tuberculosis to reduced oxygen growth conditions) in M. ulcerans, M. genavense, M, marinum and 9 other mycobacterial species.

8. Diagnosis of Buruli Ulcer and detection of Mycobacterium ulcerans
Specimens from patients with Buruli ulcer (BU) were collected during passive case finding in two treatment centers in Benin, one in the department of Zou (Zagnanado) and another in the department of Mono (Lalo). One of the most important findings is that the total number of patients treated in Zagnanado increased exponentially between 1989 and 1997. This increase was also observed in 1998 (357 cases versus 300 in 1997, an increase of 19%). In some endemic departments or subprefectures, the total number of detected cases exceeded those of leprosy and tuberculosis. For example, in 1997, the detection rate in the Department of Zou was 24.2/100.000 inhabitants for BU and 18/100.000 inhabitants for tuberculosis. As in 1998, the detection rate of M. ulcerans was significantly better by PCR (96.9%) and by histopathologic analysis (89.8%) than by Ziehl-Neelsen staining (63.3% positivity) or culture (61.3% positivity when contaminated cultures are excluded).

We have confirmed the first case of M. ulcerans infection in China. Our findings confirm that the disease is not only found in tropical regions and temperate areas of Australia, but that it also occurs above latitude 30° north.

9. Clinical manifestation of M. ulcerans infection
Microbiologic analysis of the cases treated at Zagnanado confirmed that non-ulcerative forms are now more frequently diagnosed than ulcerative forms. During the period 1989-1997, the proportion of non-ulcerative forms to all forms increased from zero to 45.2% in 1997. In 1998, 60.5% of all the patients presented a non-ulcerative form of the disease. The disseminated forms also increased considerably during the years: from 3.8% of all forms seen in 1996 to 7.3% in 1997 (Aguiar et al., 1997) to 10% in 1998. Severe cases of osteomyelitis very infrequently discovered before, were also diagnosed more commonly: 14.6% of all the cases in 1998 compared to 4.5% among 1167 patients reported during the period 1989-1997.

10. Development of effective chemotherapeutic regimens against M. ulcerans
The sensitivity of M. ulcerans to several antimycobacterial drugs was tested in vitro. M. ulcerans was found to be sensitive to rifampicin, clarithromycin and to streptomycin. In collaboration with the St. George's Hospital Medical School (London, UK) and with the University of Science and Technology (Kumasi, Ghana), we found three quinolones (ciprofloxacin, sparfloxacin, ofloxacin) were active in vitro against all tested isolates. Sparfloxacin exhibited the highest ac-tivity. Finally, clarithromycin at a dosage of 100 mg/kg inhibits partially the multiplication of M. ulcerans in the mouse. It is possible that a higher dosage shows a greater efficacy.

11. Quest for the source of infection of M. ulcerans
A total of 158 environmental specimens were collected (plants, insects, molluscs, mud, fish). They were obtained from marshes and rivers in regions in Benin where BU is endemic: Mono (Tandji), Zou (Kpedekpo, Wedja, Agonita) and the Ouémé (Bonou). M. ulcerans has not been cultured. However, three specimens were positive by PCR: a water bug, a firefly larva and an aquatic beetle. These insects are aggressive predators of some other species of aquatic arthropods and crustacea. These other aquatic organisms may be water-filtering organisms which could concentrate M. ulcerans present in water.

12. Characterisation of the virulence factors of M. ulcerans
Two putative cytolytic protein factors have been analysed: (1)Phospholipase C and D enzymatic activity has been demonstrated for the first time by thin-layer chromatography using a phosphatidylcholine radio-labeled substrate for detecting lipid hydrolysis produced by M. ulcerans. M. ul-cerans DNA sequences homologous to the genes encoding PLC in Pseudo-monas aeruginosa and M. tuberculosis were identified with the help of polymerase chain reaction, partial DNA sequencing and DNA-DNA hybridization. (2) a gene in M. ulcerans homologous to a M. tuberculosis gene coding for a hemolysin has been cloned and fully sequenced.

13. Development of diagnostic tools for the control of paratuberculosis
A new sequence capture based DNA extraction method has been devel-oped It allows PCR detection of M. paratuberculosis in faeces with a sensitivity of 100 acid-fast bacilli (afb) per reaction.

Assistance and education activities

In May 1999 our laboratory was selected as the new worldwide coordinator of the WHO/IUATLD Supranational Reference Laboratory (SRL) Network for Tuberculosis Drug Resistance Surveillance (DRS).

We also became a member of the WHO 'DOTS-Plus' working group, and we will coordinate together with Dr. M. Salfinger (New York State Department of Health, USA), a sub-group on antimycobacterial susceptibility testing for second-line drugs.
As International Reference Laboratory for the Control of Buruli Ulcer, rec-ognized by the WHO, the Unit performed bacteriological analysis for the diagnosis of BU in West-African patients.

Scientific awards

Prof. F. Portaels behaalde de SmithKline-Beecham prijs voor Overzeese Wetenschappen voor haar werk op BU (zie Focus on).

Cooperation

Within the framework of the worldwide surveillance of MDR TB, the Unit collaborated closely with the International Red Cross and with Médecins sans Frontières (Belgium, France and Luxemburg) in some countries of the former Soviet Union.

Within the scope of the control of Buruli Ulcus there was a close coop-eration with several West-African countries (Benin, Togo, Ghana and Ivory Coast) and preliminary work is being done in Peru.

Virology Unit

Objectives

Our main research subject is the phenotypic and genotypic diversity of HIV, and its impact on vaccine development, antiviral therapy and diagnosis of HIV.

Objectives

Research activities

Assistance and
Teaching Activities

Collaboration

• The virology lab, a real “beehive”

Research activities

1. Diagnosis
In the present investigation, we have developed a highly sensitive molecular detection procedure that can detect viral RNA from plasma. It can be used as an early diagnostic tool and might potentially be used for viral load determination for both group M and group O HIV-infections. We have developed a generic primer set based on the conserved immunodominant region of transmembrane protein gp41 that can reliably amplify as few as 10 copies/PCR of viral DNA from near-full length clones representing group M subtypes A to H (subtypes I and J were not available). The assay is highly sensitive in detecting plasma viral RNA from HIV-1 strains of diverse geographic origins representing different subtypes of HIV-1 group M as well as HIV-1 group O. Of the 253 group M plasma specimens (subtypes A, 68 specimens; B, 71; C, 19; D, 27; E, 23; F, 33; and G, 12), 250 (98.8%) were amplified by using the gp41 M/O primer set. More importantly, all 32 (100%) group O plasma samples were also amplified with these primers. In vitro spiking experiments further revealed that the assay could reliably detect as few as 25 copies/ml of viral RNA and gave positive signals in HIV-1 seropositive specimens with plasma copy num-bers below the limits of detection by all commercially available viral load assays. In addition, analysis of five seroconversion panels indicated that the assay is highly sensitive for early detection of plasma viremia during the "window period”. Thus, the highly sensitive assay will be useful for early detection of HIV-1 in clinical specimens from all known HIV-1 infections, regardless of their genotypes and geographic origins.

2. Genetic diversity of HIV-1
Here we report on a study to provide insight in the 10-year follow-up of the intrapatient env V3C3 sequence variability of HIV-1 ANT70. HIV-1 ANT70 is the first HIV-1 group O virus isolate obtained; it is from a 25-year-old Cameroonian woman, who seroconverted in March 1987. This individual has remained asymptomatic and clinically healthy (clinical stage WHO 1, CDC II) even though she did not receive any antiretroviral therapy for HIV-1 before 97 months post-seroconversion. CD4+ T cell counts declined steadily to 200/µl at 70 months post-seroconversion.

RT-PCR, cloning, sequencing and genetic analysis were performed on eight plasma follow-up samples. Extensive increasing intra- and intersample variation was observed. This is the first long-term (> 10 years) follow-up of the genetic variability of an HIV-1 group O-infected individual. As the course of the disease in the HIV-1 ANT70-infected woman was similar in many aspects to that of group M-infected individuals, it remains to be elucidated whether the changes observed in the V3 loop are critical for disease progression.

Here we report the presence of multiple forms of intersubtype recombi-nant HIV-1 strains in Nigeria, using 10 strains collected between 1994 and 1995 in the northeast region of Nigeria near the border with Cameroon. In this study, peripheral blood mononuclear cells obtained in 1994-1995 from 10 patients hospitalized in northeastern Nigeria were evaluated by sequencing of the complete envelope and, from 7 patients, a portion of gag. Four patients harbored subtype G viruses and six patients had recombinant viruses. Two had strains sharing the A/G recombinant structure of IbNG. Two had a previously undescribed recombinant, mostly subtype A, whose carboxyl-terminal gp41 could not be classified. An A/G recombinant different from IbNG but similar to CA1, a Cameroonian strain, was found in one patient. The remaining patient had a strain that was otherwise subtype G but shared an unclassified carboxyl-terminal gp41 segment with the CA1 like strains. Other subtypes and group O were not found.

3. HIV vaccine development
The dichotomy of type-1 and type-2 T-helper (Th) immune responses is thought to be an obstacle to develop human immunodeficiency virus-type-1 (HIV-1) vaccines capable of inducing effective cellular as well as humoral immune responses. Macaca mulatta were immunized using two different HIV-1 SF2 envelope vaccine strategies, based on either immune-stimulating complexes (ISCOM) or chimeric Fowlpox (FP) vaccines. One month following the third immunization all animals were heterologously challenged with a simian/human immunodeficiency virus (SHIV SF13). Vaccinated monkeys, which were protected had the highest levels of both type-1 and type-2 HIV-1 specific T-helper cell (Th) responses in addition to the highest homologous and heterogeneous virus neutralizing antibodies. To determine how long Th responses persisted and if they correlated with protection, animals were re-challenged after waiting for four months without re-boosting. Macaques which maintained the highest gp120-specific type-1 (IFN-g) responses were protected, while there was evidence of viral clearance in two others. These findings demonstrate the importance of both or mixed type-1 and type-2 Th responses in HIV-1 vaccine induced immunity while suggesting a possible role of persistent type-1 responses in main-taining protective immunity over time.

The specific immune mechanisms necessary and/or sufficient to elicit HIV-vaccine protection remain undefined. Utilizing the SHIV rhesus macaque model the immunogenicity as well as the efficacy of ten different HIV-1 vaccine candidates was evaluated in close collaboration with Dr. J. Heeney and his colleagues (BioMedical Primate Research Centre Rijswijck, The Netherlands). Comparison of the immune responses induced, with the ability of the vaccine to protect from SHIV infection provided a means to determine which type of immune responses were necessary for protection. Vaccine candidates included VLPs, DNA, subunit protein with novel adjuvant formulations, ISCOMs and pox-virus vectors. Protection from SHIV infection was achieved in approximately half of the animals which received a primary intravenous cell-free challenge. The presence of CTL in the absence of other effector responses did not correlate with protection from this route and type of challenge. Virus neutralizing antibodies (Nab) appeared to be necessary but alone were insufficient for protection. If Ag-specific IFN-g and/or IL-4 as well as lymphoproliferative (LP) responses were found with the lack of a detectable IL-2 response, then protection was not observed. Immunity correlated with the magnitude of Nab responses, b-chemokines and as well as balanced, qualitative T-helper responses.

Assistance and Teaching Activities

1. AIDS Reference Laboratory
The virology laboratory is one of the eight AIDS Reference laboratories in Belgium, that were recognized and supported by law (Royal Decree of 8 October 1996).

Their main tasks are:

1. to confirm initial HIV-antibody reactive screening results
2. to develop, adapt and apply techniques which allow determination of prognostic markers and monitoring of treatment failure of HIV and opportunistic infections
3. to evaluate and control pre-commercial and commercially available diagnostic HIV assays; to make research data related to the above mentioned activities, available for epidemiological and scientific research.

2. Evaluation of HIV diagnostic assays
In the period 1 January - 31 December 1999 the operational characteris-tics of 13 commercially available HIV-1/HIV-2 antibody assays were determined on request of WHO. For each test ± 80 operational parameters were monitored. The results of this evaluation were published in WHO/UNAIDS Technical Report n°11 and distributed in 140 countries. On request from industrial companies, 2 pre-commercial HIV tests have been evaluated.

3. Other activities

• Reviews of manuscripts for International Journals (AIDS, Journal of Acquired Immune Deficiency Syndromes and Human Retrovirology, Vaccine, Journal of Clinical Microbiology, Clinical Microbiology, Clinical & Experimental Immunology)
• Courses in virology and practical exercises as part of the International course “Master of Science in Disease Control”.
• Training in HIV molecular biological techniques of numerous national and international thesis students and trainees.

4. Logistical activities
The administrative unit continued its excellent work:

• Administrative coaching of trainees/visitors.
• Organization of meetings and SOA/HIV seminars.
• Organization of international meetings (The HIV/AIDS conference in Lusaka, Zambia)
• Logistical support of overseas projects.

Collaboration

The Virology Unit collaborates intensively with the other units in the Department.
Close ties exert also with the Clinical Research Department:

• Coordination of AIDS Reference Centre activities.
• Follow-up of clinical studies.
• Collaboration with the clinical biology.
• European quality laboratory programme.

The unit collaborates, within Belgium, with the seven other AIDS Reference Laboratories; with the Universities of Liège, Antwerp and Ghent and with the Red Cross Blood Transfusion Centre (Antwerp). Basic scientific work is also performed with and for the companies Innogenetics, Tibotec, Virco and the Janssen Research Foundation. The prime international collaborations are with partners in The Netherlands (Biomedical Primate Research Centre, Rijswijck, Organon Teknika); Ivory Coast (Projet Retro-Ci, Institut Pasteur); Cameroon (CUSS); Ghana (Noguchi Institute); China (Institute of Biological Products of Shanghai); Benin (PNLS); Argentina (Bios Rosario S.A.); USA (Henry M. Jackson Foundation, Los Alamos National Laboratory); France (ORSTOM, INSERM); U.K. (St. Mary’s Hospital Medical School, London); Germany (Max von Pettenkofer Institute, München); Japan (N.I.H., Tokyo); Canada (Hôpital du St. Sacrément, Quebec). The unit is also partner in the European AIDS Virus Surveillance Programme (EASP) and works closely with UNAIDS programmes.

Immunology Unit

Research activities

The research of the Immunology unit is focused on the cellular immunology of HIV in humans and SIVcpz in chimpanzees, and on the immune interaction between HIV and Mycobacterium tuberculosi (MTB).

Research activities

Assistance and educational activities

Collaboration

• HIV-1, transfected with enhanced green fluorescent protein (EGFP) as a tool to study viral transfer between dendritic cells (DC) and CD4 T-cells

An HIV-1 clone, transfected with EGFP, was used to infect normal DC in vitro. The infected DC were co-cultured with purified resting CD4 T-cells from the same healthy donor. From day 7 on, the fluorescent virus was visible in both the originally infected DC and in the co-cultured CD4 T-cells, indicating transfer from the former to the latter. The figure shows the fluorescence picture of the T-cells after 17 days of co-culture. The T-cells were marked with an antibody, specific for CD4 that was conjugated the red fluorochrome phycoerythrin (anti-CD4-PE in the y-axis). From the green fluorescence ( EGFP in the x-axis) it is evident that a significant proportion of the T-cells expresses the virus.

2. Co-pathogenesis of HIV and pulmonary tuberculosis (PTB)
Aims: Uncover the immune mechanisms, which are responsible for the bi-directional unfavourable effects, HIV and PTB have on each other's course.
Irrespective of the HIV serostatus, PTB was associated with granulo-monocytosis in vivo, increased production of the immune suppressive cytokine transforming growth factor beta and diminished interferon-gamma production in vitro. All these alterations were corrected by anti-TB treatment and therefore rather appear a consequence of the disease than a pathogenic mechanism. Typically HIV-associated changes (CD4 T cell depletion, viral load, and abnormal T cell activation) were not improved by anti-TB treatment in dually infected subjects.

3. Natural resistance against infection with HIV in Abidjan
Aim: to investigate the underlying (immunological) mechanism of resis-tance to infection with HIV in seronegative female commercial sexworkers in Abidjan.

This project is being realized in collaboration with Dr. J. Nkengasong from the ‘Projet RETRO-CI’ in Abidjan and, the Virology and HIV/STD Research and Intervention units of ITM. The past year, special attention has been paid to the detection of HIV suppressive chemokines and immunoregulatory cytokines, and their relation to HIV-1 coreceptor expression and parameters of immune activation. Preliminary results do not indicate that there is an increased production of virus suppressive chemokines in peripheral blood in HIV seronegative prostitutes. Currently we are studying whether or not HIV-specific systemic immune responses, as possible indicators of protection against HIV, can be detected in these individuals.

4. HIV and SIVcpz infection in chimpansees
Aim: To evaluate cellular immune responses directed against HIV and SIVcpz in infected chimpanzees.

This project is being realized in collaboration with Dr. J. Heeney from the Biomedical Primate Research Center in Rijswijk, The Netherlands, and with the virology unit of ITM. The most remarkable finding in this study the past year was that the plasma RNA viral load in SIVcpz infected chimpanzees is comparable to that seen in HIV-1 infected humans. Importantly however, the infection in chimpanzees is not associated with non-specific chronic activation of the immune system. This observation suggests that not the viral load but another, yet unknown factor, probably associated with HIV, is the driving force behind this chronic hyperactivation in infected humans.

Assistance and educational activities

The total number of routine CD4 assays performed for the ITM outpatient clinic in 1999 was 2569. These assays are being performed in the context of the immunological follow-up of HIV seropositive patients.

The members of the academic staff acted as referees for international scientific journals (AIDS, Vaccine, Clinical and Experimental Immunology, Tropical Medicine & International Health).

L. Kestens and G. Vanham have given special seminars on the immunology of schistosomiasis and HIV in the Masters course of Disease Control (MDC) at ITM. L. Boel has been engaged in the practical laboratory courses on microbiology in the national course of Tropical Medicine at ITM.

• Wim Jennes and colleagues in the virology laboratory of the ‘projet RETRO-CI’ during the study on HIV-exposed uninfected commercial sex workers in Abidjan, Côte d’Ivoire.

Collaboration

The past year we have collaborated intensively with the Virology Unit and the STD/HIV Unit of our department, with the AIDS unit from the department of clinical sciences and the schistosomiasis unit (J. Scott, B. Gryseels).

Within Belgium, we have collaborated with the experimental hematology unit of the University Hospital of Antwerp (Prof. Dr. Van Bockstaele), with the department of microbiology of the University Hospital of Gent (Prof. Dr. Plum) and with the laboratory of virology and chemotherapy of the Rega Institute of Leuven (Prof. Jan Balzarini).

Ongoing international collaboration has been continued with the Bio-medical Primate Research Center, The Netherlands; with RETRO-CI, Abidjan, Ivory Coast; the Case Western University, Cleveland, USA; the Ugandan Joint Clinical Research Center of the Makerere University, and the American Tuber-culosis Research Unit (NIH) of Kampala, Uganda.

HIV/STD Research
and Intervention Unit

The overall aim of the unit is to identify and develop effective strategies for the prevention and control of STDs and HIV/AIDS in developing countries.

Research activities

Technical assistance/ Service delivery

Scientific collaborations

• From left to right :
Lut Van Damme, Ann Buvé, Thérèse Delvaux, Marie Laga, Pieter Remes, Isolde De Schampheleire, Vicky Jespers, Yves Lafort, Yvette Jacob

Research activities

1. Multicentre study on factors determining the differential spread of HIV in African towns
The spread of HIV has been explosive in some regions of Africa while in other regions the prevalence of HIV infection has remained relatively low and stable. The HIV/STD Research and Intervention Unit is co-ordinating a multicentre study on factors that determine the differences in rate of spread of HIV in different regions of Africa.

The study has been conducted in Cotonou (Benin), Yaoundé (Cameroon), Kisumu (Kenya) and Ndola (Zambia). The prevalence of HIV infection among adults in the general population was 3% in Cotonou, 6% in Yaoundé, 26% in Kisumu and 28% in Ndola. These differences in prevalence of HIV infection could not be explained by differences in sexual behaviour. However there is evidence that the efficiency of the transmission of HIV during sexual intercourse is higher in Kisumu and Ndola than in Cotonou and Yaoundé.

Further qualitative research is planned on sexual behaviour of young people in the four cities. This research should ultimately aid in improving interventions aimed at young people and their partners. The work will be co-ordinated by an anthropologist who recently joined the team. The HIV/STD Research and Intervention Unit has also been asked to co-ordinate another multicentre study on the sexual behaviour of youth in 5 African countries, including Senegal, Burkina Faso, Ivory Coast, Uganda and South Africa.

• The multicentre study on factors determining the differential spread of HIV in four African cities was a collaborative effort of 15 research teams

3. Assessing biologic risk factors for the transmission of HIV
A study on HIV shedding in the genital tract among sex workers in Abidjan has been completed and has shown a significant association between the presence of HIV in vaginal secretions and other concurrent STD, as well as a decrease of HIV shedding after treatment of STD. Currently, a study is ongoing to examine the association between genital herpes and HIV shedding.

4. Research on the delivery of comprehensive sexual health services for female sex workers and other preventive measures
In collaboration with the "Institut National de Sante Publique (INSP)" and the project Rétro-CI, the unit is conducting a research project in Abidjan, Ivory Coast, with the overall aim of designing a feasible and acceptable package of comprehensive sexual health services for female sex workers for the prevention and control of sexually transmitted diseases (STD) and HIV.

Specific aims are: 1) to define optimal STD case management strategies for female sex workers; 2) to assess the health needs and the present health care seeking behaviour of female sex workers in Abidjan with the focus on sexual/reproductive health issues; 3) to assess the quality of sexual health care for female sex workers in existing health services; 4) to translate results of research into recommendations for policy makers with regard to STD/HIV interventions among high-risk women.

During 1999 a validation study of different diagnostic approaches was being conducted in the specialized clinic for sex workers “Clinique de Confiance”, in Abidjan; a study on knowledge, attitudes and HIV prevalence among clients of female sex workers in Abidjan was finalized; and preparatory work has been done for a study, which is planned for 2000, on the quality of sexual health care in Abidjan.

5. Integration of different components of Reproductive Health Programmes
As part of the Health Policy Studies of the BADC-programme the different issues of "Integrated Reproductive Health Care" are being examined. The main goal is to explore the remaining questions with regard to the feasibility of integrating STD/HIV prevention and control into Family Planning services as well as Mother and Child Clinics and primary health care services. The acceptability and the feasibility of integrated reproductive health services for at-risk groups, such as female sex workers, is also studied (Case study of Cambodia).

6. Care and prevention of STD in the Kingdom of Cambodia
At the demand of, and with funding from the EU, a large STD/HIV intervention project has been initiated in Cambodia with the following objectives: 1) to strengthen the public and private sector services providing care and preventive services for STDs and HIV among high and low risk women; 2) to strengthen the management and co-ordination capacities of the National AIDS Control Programme in Cambodia. Dr. F. Crabbé is resident in Cambodia as technical advisor to the project.

7. Technical assistance to IMPACT projects in developing countries
The unit is a partner in the Implementing AIDS Prevention and Care Project (IMPACT), the U.S. Agency for International Development’s (USAID) mechanism for HIV/AIDS interventions co-ordinated by the U.S. based NGO Family Health International (FHI). Dr. Y. Lafort was contracted for this purpose to provide technical assistance to selected projects in issues of STI control and clinical management.

In Zambia, a project targeting truck drivers and female sex workers was started. A baseline survey will measure STI prevalence rates and behavioral indicators. The intervention will focus on peer education, condom promotion and improved STI case management services.

In Rwanda and Tanzania, projects of training health care providers from respectively the government and the private sector in an improved STD case management have successfully been concluded. Evaluations showed a high level of STI case management by the trained providers. In Rwanda, the quality of the STI case management will continue to be ensured through refresher training and improved supervision. New interventions will target the strengthening of partner notification, improved STI case management in the informal sector, including pharmacies, drug stores and traditional healers, and improved STI health care seeking behavior by high-risk groups. The gonococcal antimicrobial susceptibility study that was started last year will be concluded.

In Ivory Coast, STI case management was integrated in Family Planning (FP) clinics through the training of the national FP trainers and supervisors, the training of the FP providers in the non governmental sector, and through introducing STI case management supervision and STI drug supply into the existing FP systems. The feasibility and acceptability of the integration will be thoroughly documented and evaluated. Additionally, the cost-efficiency will be evaluated by measuring the STI prevalence and the validity of the current treatment guidelines in the FP population.

8. Activities of the STD diagnostics laboratory
All the sera that were collected in the multicentre study on factors determining the differential spread of HIV in four African cities were tested for specific herpes simplex type 2 IgG antibodies. In addition, a representative sample of sera was tested for antibodies against herpes simplex type 1.

Diagnostic tests for Neisseria gonorrhoeae, Chlamydia trachomatis, Treponema pallidum and HIV were performed for the study on the use of vaginal microbicides for the prevention of HIV and STI in high-risk women in Africa and Asia. The unit was also responsible for the internal and external quality control, the field supervision and capacity building.

PCR tests for the detection of Trichomonas vaginalis (TV) were developed using different DNA sequences. The evaluation of these techniques on urine specimens and vaginal swabs from African women showed sub-optimal results for PCR on urine, but a significantly higher sensitivity of the PCR on vaginal swabs compared to culture.

Three different PCR tests for the detection, confirmation and typing of herpes simplex virus (HSV) type 1 and 2 in clinical specimen of different origin were optimised. These techniques proved to be as specific and significantly more sensitive than culture, and to be very accurate in the detection of HSV DNA in genital samples of asymptomatic patients.

A comparative study between PCR, Ligase Chain Reaction and Strand Displacement Amplification for the detection of Neisseria gonorrhoeae and Chlamydia trachomatis DNA was performed on 740 consecutive, endocervical swabs taken from high-risk women in the multicentre trial of vaginal microbicides

A study on antimicrobial susceptibility and plasmid resistance typing of Neisseria gonorrhoeae isolates from Benin was concluded.

More than 100 swabs of genital ulcers from patients from Dhaka, Bangladesh were tested for Haemophilus ducreyi en herpes simplex virus using inhouse DNA amplification techniques.

The unit started providing laboratory support to the IMPACT STI/HIV pre-vention project in Cambodia. This support consists of field training, quality control and the testing for Neisseria gonorrhoeae and Chlamydia trachomatis using DNA amplification techniques.

The storage life of diagnostic tests for syphilis serology was evaluated by testing reagents that were kept at 37°C for 12 months after the expiry date.

The diagnosis of Chlamydia trachomatis and herpes simplex virus in patients attending the outpatient department of the institute, used to be made by culture and is now done by DNA amplification tests.

9. STD/HIV prevention and AIDS care for migrants from sub-Saharan Africa and asylum seekers living in Flanders
As part of a global strategic plan and action plan of AIDS control in Flanders which is supervised by the Interprovincial AIDS Co-ordinating Centre (IPAC), an action-programme aimed to provide care and prevention of STDs and HIV to migrants from Africa living in the cities of Antwerp and Gent and the asylum seekers living in the twelve centres in Flanders was initiated.

This action-programme which covers 3 years (1999-2001) is based on the results of research and pilot projects which were conducted in the commu-nities of African migrants and in the centres for asylum seekers, between 1996 and 1998.

According to the epidemiological data from the Pasteur Institute of Bel-gium, more than 50% of the persons living with HIV in Flanders don't have the Belgian nationality.

The HIV/STD Research and Intervention Unit of the Institute of Tropical Medicine is responsible for the management of the action-programme.

The objectives of the programme are:

• To increase the level of basic knowledge of the target groups and their intermediaries (including to increase awareness) about STDs and HIV/AIDS
• To provide training to the target groups and their intermediaries on the prevention of STDs and HIV and on culturally adapted support for persons living with HIV
• To set up a network of health professionals who:
  = will organise the prevention activities and provide support that is culturally adapted to the migrants
  = will be able to make contact with hard to reach migrant groups
  = will ensure the continuity of HIV prevention activities of HIV and care
• In collaboration with the migrants, to develop a new strategic plan and action programme for AIDS control to be implemented after 2001.
  
  

• Conference 23.11.99 and workshop on basic knowledge, education and setting up networks with African migrants and health care professionals

Technical assistance / Service delivery

• At present, staff members of the unit are regular consultants for the Belgian Cooperation, Family Health International (IMPACT), UNAIDS, The World Bank, International Family Health.
• Several staff of the Unit are regularly asked to review papers submitted to international journals (AIDS, Genitourinary Medicine, The Lancet, Health Policy and Planning, Sexually Transmitted Diseases, European Journal of Microbiology and Infectious Diseases, Tropical Medicine & International Health…). M. Laga is editor of AIDS.
• The Unit is represented in scientific committees of international confer-ences (international AIDS conferences, conferences on AIDS and STD in Africa, International Conference on Home and Commuity Care for persons living with HIV/AIDS). Several staff of the Unit are asked to review abstracts submitted to these international conferences.
• Staff members of the unit are part of review committees for funding agencies for research (World AIDS Foundation, European Union, HIVNET, Agence Nationale de Recherches sur le SIDA, Wellcome Trust, UNAIDS/SDI).
• The unit is also asked by African organisations to provide technical advise and support to the organisation of conferences and meetings. Most of this support is provided on behalf of UNAIDS.

Scientific collaborations

Within the Institute
• The Unit was responsible for the new Master Course "Master of Science in Disease Control / Contrôle des Maladies", co-ordinated by Marie Laga.
• There is intense collaboration with the virology unit on several research projects. The mycobacteriology unit and the STD laboratory collaborate on studies on the mechanisms of drug resistance. Further collaboration with the mycobacteriology unit has not yet materialised though the unit has a keen interest in tuberculosis as well.
• Collaboration with the Public Health Department expresses itself in joint teaching, collaborative scientific meetings, joint research projects and publications. The unit has undoubtedly gained from this collaboration in terms of developing methodologies for research and control in existing health systems.
• The unit is occasionally involved in studies of the Department of Clinical Sciences.

Outside the Institute
• The research institutions with which the unit has a long standing collaboration include: London School of Hygiene and Tropical Medicine; INSERM U88 (Paris);ORSTOM (Montpellier); Projet Rétro-CI, Abidjan; Karolinska Institute, Sweden; South African Institute of Medical Research.
• New collaborations were initiated with Rega Institute Leuven and St. Mary’s London on Microbicides.

Bastian I, Colebunders R. Treatment and prevention of multidrug-resistant tuberculosis. Drugs 1999; 58: 633-661.

Breman JG, Johnson KM, van der Groen G, Robbins CB, Szczeniowski MV, Ruti K, Webb PA, Meier F, Heymann DL. A search for Ebola virus in animals in the Democratic Republic of the Congo and Cameroon: ecologic, virologic, and serologic surveys, 1979-1980. J Infect Dis 1999; 179(Suppl.1): S139-S147.

Buvé A. Genital ulcer disease in Africa: many pieces are still missing from the puzzle [editorial]. Sex Transm Infect 1999; 75: 85-86.

Crabbé F, Laga M, Piot P. Human immunodeficiency virus and AIDS; epidemiology and prevention. In: Root RK, editor. Clinical infectious diseases; a practical approach. New York: Oxford University Press, 1999: 905-913.

Glynn JR, Buvé A, Caraël M, Zaba B. Adjustment of antenatal clinic HIV surveillance data for HIV-associated differences in fertility. AIDS 1999; 13: 1598-1599.

Guimaraes-Peres A, Portaels F, de Rijk P, Fisette K, Pattyn SR, Van Vooren JP, Fonteyne PA. Comparison of two PCRs for detection of Mycobacterium ulcerans. J Clin Microbiol 1999; 37: 206-208.

Heeney J, Akerblom L, Barnett S, Bogers W, Davis D, Fuller D, Koopman G, Lehner T, Mooij P, Morein B, De Giuli-Morghen C, Rosenwirth B, Verschoor E, Wagner R, Wolf H. HIV-1 vaccine-induced immune responses which correlate with protection from SHIV infection: compiled preclinical efficacy data from trials with ten different HIV-1 vaccine candidates. Immunol Lett 1999; 66: 189-195.

Hirsch CS, Toossi Z, Vanham G,
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Hu DJ, Buvé A, Baggs J, van der Groen G, Dondero TJ. What role does HIV-1 subtype play in transmission and pathogenesis? An epidemiological perspective. AIDS 1999; 13: 873-881.

Janssens W, Heyndrickx L, Van der Auwera G, Nkengasong J, Beirnaert E, Vereecken K, Coppens S, Willems B, Fransen K, Peeters M, Ndumbe P, Delaporte E, van der Groen G. Interpatient genetic variability of HIV-1 group O. AIDS 1999; 13: 41-48.

Janssens W, Nkengasong J, Heyndrickx L, Van der Auwera G, Vereecken K, Coppens S, Willems B, Beirnaert E, Fransen K, Peeters M, van der Groen G. Intrapatient variability of HIV type 1 group O ANT70 during a 10-year follow-up. AIDS Res Hum Retroviruses 1999; 15: 1325-1332.

Kamali A, Nunn AJ, Mulder DW, Van Dyck E, Dobbins JG, Whitworth JAG. Seroprevalence and incidence of genital ulcer infections in a rural Ugandan population. Sex Transm Infect 1999; 75: 98-102.

Kimerling ME, Kluge H, Vezhnina N, Iacovazzi T, Demeulenaere T, Portaels F, Matthys F. Inadequacy of the current WHO re-treatment regimen in a central Siberian prison: treatment failure and MDR-TB. Int J Tuberc Lung Dis 1999; 3: 451-453.

McCutchan FE, Carr JK, Bajani M, Sanders-Buell E, Harry TO, Stoeckli TC, Robbins KE, Gashau W, Nasidi A, Janssens W, Kalish ML. Subtype G and multiple forms of A/G intersubtype recombinant human immunodeficiency virus type 1 in Nigeria. Virology 1999; 254: 226-234.

Mestdagh M, Fonteyne PA, Realini L, Rossau R, Jannes G, Mijs W, De Smet KAL, Portaels F, Van den Eeckhout E. Relationship between pyrazinamide resistance, loss of pyrazinamidase activity, and mutations in the pncA locus in multidrug-resistant clinical isolates of Mycobacterium tuberculosis. Antimicrob Agents Chemother 1999; 43: 2317-2319.

Mve-Obiang A, Remacle J, Palomino JC, Houbion A, Portaels F. Growth and cytotoxic activity by Mycobacterium ulcerans in protein-free media. FEMS Microbiol Lett 1999; 181: 153-157.

Nicoll A, Johnson AM, Adler MW, Laga M. Preventing HIV-1: lessons from Mwanza and Rakai [letter]. Lancet 1999; 353: 1522.

Padian N, Buvé A. Epidemiology [editorial]. AIDS 1999; 13(Suppl.A): S59-S60.

Palomino JC, Portaels F. Simple procedure for drug susceptibility testing of Mycobacterium tuberculosis using a commercial colorimetric assay. Eur J Clin Microbiol Infect Dis 1999; 18: 380-383.

Palomino JC, Traoré H, Fissette K, Portaels F. Evaluation of Mycobacteria Growth Indicator Tube (MGIT) for drug susceptibility testing of Mycobacterium tuberculosis. Int J Tuberc Lung Dis 1999; 3: 344-348.

Pattyn SR. Rechutes très tardives après traitement des maladies multibacillaires. Bull ALLF 1999; (5): 28.

Pattyn SR. Yersinia pestis, bacteriologie. Verh K Acad Geneeskd België 1999; 61: 103-107.

Ping Z, Guizheng L, Wei Z, Fransen K, Heyndrickx L, Janssens W, van der Groen G. Genotyping of HIV-1 in anti-HIV-1 sero-positive plasma. Virol Sin 1999; 14: 17-22.

Piper MA, Severin ST, Wiktor SZ, Unger ER, Ghys PD, Miller DL, Horowitz IR, Greenberg AE, Reeves WC, Vernon SD. Association of human papillomavirus with HIV and CD4 cell count in women with high or low numbers of sex partners. Sex Transm Infect 1999; 75: 253-257.

Portaels F, Elsen P, Guimaraes-Peres A, Fonteyne PA, Meyers WM. Insects in the transmission of Mycobacterium ulcerans infection [letter]. Lancet 1999; 353: 986.

Portaels F, Rigouts L, Bastian I. Addressing multidrug-resistant tuberculosis in penitentiary hospitals and in the general population of the former Soviet Union. Int J Tuberc Lung Dis 1999; 3: 582-588.

Realini L, De Ridder R, Hirschel B, Portaels F. Blood and charcoal added to acidified agar media promote the growth of Mycobacterium genavense. Diagn Microbiol Infect Dis 1999; 34: 45-50.

Rudnicka W, Brzychcy M, KLink M, Lopez AG, Fonteyne PA, Rusch-Gerdes S, Rozalska B. The production of nitric oxide and tumor necrosis factor by murine macrophages infected with mycobacterial strains differing by hemolytic activity. Microbiol Immunol 1999; 43: 637-644.

Rustomjee R, Abdool Karim Q, Abdool Karim SS, Laga M, Stein Z. Phase 1 trial of nonoxynol-9 film among sex workers in South Africa. AIDS 1999; 13: 1511-1515.

Tsegaye TM. Potentials of a Haemophilus ducreyi-specific antigen and a monoclonal antibody for the laboratory diagnosis of chancroid (Dissertation). [Brussel]: Vrije Universiteit Brussel, Faculty of Science, Institute of Molecular Biology and Biotechnology, 1999: 131 pp.

Van Bockstaele DR, Deneys V, Philippé J, Bernier M, Kestens L, Chatelain B, De Waele M, Demanet C. Belgian consensus recommendations for flow cytometric immuno-phenotyping. Acta Clin Belg 1999; 54: 88-98.

Van Damme L, Laga M. Pointer: ethical and technical aspects of microbicide effectiveness trials. Int Antivir News 1999; 7(3).

Van Damme L, Rosenberg ZF. Microbicides and barrier methods in HIV prevention. AIDS 1999; 13(Suppl.A): S85-S92.

van der Groen G. Ebola en Marburg; dodelijk maar weinig gevaarlijk. Natuur & Techniek 1999; 67(12): 62-69.

Van Deun A, Aung KJM, Chowdhury S, Saha S, Pankaj A, Ashraf A, Rigouts L, Fissette K, Portaels F. Drug susceptibility of Mycobacterium tuberculosis in a rural area of Bangladesh and its relevance to the national treatment regimens. Int J Tuberc Lung Dis 1999; 3: 143-148.

Van Dyck E, Meheus AZ, Piot P. Laboratory diagnosis of sexually transmitted diseases. Geneva: World Health Organization (WHO), 1999:
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Vanham G, Penne L, Devalck J, Kestens L, Colebunders R, Bosmans E, Thielemans K, Ceuppens JL. Decreased CD40 ligand induction in CD4 T cells and dysregulated IL-12 production during HIV infection. Clin Exp Immunol 1999; 117: 335-342.

Vernon SD, Unger ER, Piper MA, Severin ST, Wiktor SZ, Ghys PD, Miller DL, Horowitz IR, Greenberg AE, Reeves WC. HIV and human papillomavirus as independent risk factors for cervical neoplasia in women with high or low numbers of sex partners. Sex Transm Infect 1999; 75: 258-260.

Verschoor EJ, Davis D, van Gils M, Koopman G, Mooij P, Oostermeijer H, Haaft PT, Verstrepen B, Rosenwirth B, Morein B, Barnett SW, Heeney JL. Efforts to broaden HIV-1-specific immunity by boosting with heterologous peptides or envelope protein and the influence of prior exposure to virus. J Med Primatol 1999; 28: 224-232.

Vuylsteke B, Laga M. Approach to management of STDs in developing countries. In: Holmes KK, Sparling PF, Mardh PA, Lemon SM, Stamm WF, Piot P, Wasserheit JN, editors. Sexually transmitted diseases; 3rd ed. New York: McGraw-Hill, 1999: 1399-1408.

Vuylsteke B, Vandenbruaene M, Vandenbulcke P, Van Dyck E, Laga M. Chlamydia trachomatis prevalence and sexual behaviour among female adolescents in Belgium. Sex Transm Infect 1999; 75: 152-155.

Yang C, Pieniazek D, Owen SM, Fridlund C, Nkengasong J, Mastro TD, Rayfield MA, Downing R, Biryawaho B, Tanuri A, Zekeng L, van der Groen G, Gao F, Lal RB. Detection of phylogenetically diverse human immunodeficiency virus type 1 groups M and O from plasma by using highly sensitive and specific generic primers. J Clin Microbiol 1999; 37: 2581-2586.

Abstracts

Allemeersch H, Penne L, Willems B, Rich E, Vanham G. Suivi par cytométrie en flux de l'infection à VIH dans les macrophages, les cellules dendritiques et les cellules T4 in vitro [résumé]. In: Club Francophone des Cellules Dendritiques, Paris, 14-15 décembre 1999. [s.l.]: [s.n.], 1999.

Anagonou S, Lafia YE, Laourou M, Lydie L, Guedou F, Buvé A. Epidémiologie de l'infection à VIH dans la population générale de Cotonou en République du Bénin [résumé]. In: XIème Conférence Internationale sur le SIDA et les MST en Afrique: "Regard vers l'Avenir; priorités pour le VIH/SIDA en Afrique"; 12-16 septembre, 1999, Lusaka, Zambie; livre des résumés. [Lusaka]: [s.n.], 1999: 30, Abstract 13PT31-33.

Atibu L, Denolf D, Edidi , Nzila N, Beelaert G, Watelle S, Colebunders R. Quality control of HIV testing in Kinshasa [abstract]. In: XIth International Conference on AIDS and STD in Africa: "Looking into the Future; setting priorities for HIV/AIDS in

Africa"; 12-16 September, 1999, Lusaka, Zambia; abstract book. [Lusaka]: [s.n.], 1999: 157, Abstract 15PT12-5.

Beelaert G, Vercauteren G, van der Wal B, Fransen K, van der Groen G. An evaluation of 15 enzyme immunoassays and 20 simple assays for detection of antibodies to HIV [abstract]. In: XIth International Conference on AIDS and STD in Africa: "Looking into the Future; setting priorities for HIV/AIDS in Africa"; 12-16 September, 1999, Lusaka, Zambia; abstract book. [Lusaka]: [s.n.], 1999: 158, Abstract 15PT12-9.

Beirnaert E, Janssens W, Davis D, van der Groen G. Potent broad cross-neutralizing sera inhibit binding of HIV-1 (group M and O) to host cells [abstract]. In: 1999 UCLA Symposium on Molecular & Cellular Biology, Keystone, January 7-13: HIV Vaccine Development: Opportunities and Challenges; AIDS Pathogenesis. [s.l.]: [s.n.], 1999: 103, Abstract 215. (UCLA Symposia on Molecular and Cellular Biology)

Bennani MA, Metahri M, Guermaz M, Hassaine M, Portaels F. Evaluation de la technique LiPA dans le diagnostic de la tuberculose et de la résistance à la rifampicine [résumé]. Int J Tuberc Lung Dis 1999; 3(Suppl.1): S113, abstract 457-PD.

Buvé A, Morison L, Zekeng L, Musonda R, Anagonou S, Heyndrickx L, Janssens W, Study Group on Heterogenerity of HIV Epidemics in African Cities. HIV-1 subtypes and recombinants in the general population and in commercial sex workers in four African cities [abstract]. In: XIth International Conference on AIDS and STD in Africa: "Looking into the Future; setting priorities for HIV/AIDS in Africa"; 12-16 September, 1999, Lusaka, Zambia; abstract book. [Lusaka]: [s.n.], 1999: 7, Abstract 13BT5-4.

Cham F, Heyndrickx L, Janssens W, Vereecken K, Coppens S, Van der Auwera G, van der Groen G. Development of a multiplex RT-PCR for the simultaneous amplifica-tion of HIV-1 group M gag and env fragments to be used in a Heteroduplex Mobility Assay (HMA) [abstract]. In: 1999 Meeting of the Institute of Human Virology, August 28-September 2 1999. [s.l.]: [s.n.], 1999: 180, Abstract 22.

Clement J, Colson P, Vervoort T, van der Groen G, van Ranst M. Hantavirussen [abstract]. Ned Tijdschr Geneeskd 1999; 143: 2642.

de Béthune MP, Hertogs K, Heyndrickx L, Vingerhoets J, Fransen K, Azijn H, Michiels L, Janssens W, Scholliers A, Larder , Bloor S, Pauwels R, van der Groen G. Does natural or acquired resistance to RT- and Pls, observed in HIV-1 group M (subtypes A-H) and group O, differ from subtype? [abstract]. In: 3rd International Workshop on HIV Drug Resistance and Treatment Strategies, 23-26 June, 1999, San Diego, USA. [s.l.]: [s.n.], 1999: 33.

Dosso M, Josse R, Bouzid AM, Migliani R, Portaels F, Rey JL. Une nouvelle endémie tropicale: l'ulcère de Buruli, pourquoi? [résumé]. Méd Trop 1999; 59(Suppl): 79S, Abstract CB 18.

Ettiègne-Traoré V, Ghys PD, Diallo MO, Maurice C, Kadjio JC, Kalé K, Tawil O, Carael M, Traoré M, Coulibaly IM, Greenberg AE, Laga M, Wiktor SZ. Seven year trends in HIV infection and sexually transmitted diseases (STD), and sociodemographic and behavioural characteristics in female sex workers in Abidjan, Côte d'Ivoire, 1991-1998 [abstract]. In: XIth International Conference on AIDS and STD in Africa: "Looking into the Future; setting priorities for HIV/AIDS in Africa"; 12-16 September, 1999, Lusaka, Zambia; abstract book. [Lusaka]: [s.n.], 1999: 90, Abstract 14PT37-33.

Gomez A, Realini L, Portaels F, Obiang A, Remacle J, Vray B, Fonteyne PA. Hemolysis and hemolytic activity in pathogenic and non-pathogenic mycobacteria [abstract]. Clin Microbiol Infect 1999; 5(Suppl.3): 326, Abstract P900.

Hertoghe T, Wajja A, Ntambi L, Okwera A, Abdel Aziz M, Hirsch C, Johnson J, Toossi Z, Mugerwa R, Mugyenyi P, Colebunders R, Ellner J, Vanham G. T cell activation, apoptosis and regulatory cytokine production in the pathogenesis of HIV and pulmonary tuberculosis [abstract]. In: Tuberculosis, the real millenium bug; International Colloquium, December 14-17, 1999, Antwerp. [s.l.]: [s.n.], 1999.

Heyndrickx L, Van der Auwera G, Van Rampelbergh R, De Witte K, Vereecken K, Coppens S, McCutchan F, van der Groen G. Development of an HIV-1 group M heteroduplex mobility assay for gag region [abstract]. In: 1999 UCLA Symposium on Molecular & Cellular Biology, Keystone, January 7-13: HIV Vaccine Development: Opportunities and Challenges; AIDS Pathogenesis. [s.l.]: [s.n.], 1999: 116, Abstract 309. (UCLA Symposia on Molecular and Cellular Biology).

Jennes W, Vereecken C, Ondoa P, van der Groen G, Heeney J, Kestens L. Beta-chemokine expression in HIV-1 infected subjects [abstract]. Anal Cell Pathol 1999; 18(3).

Jimborean G, Nemes M, Portaels F. Observations sur les mycobactérioses atypiques dans le département Mures, Roumanie [résumé]. Int J Tuberc Lung Dis 1999; 3(Suppl.1): S187, abstract 540-PD.

Josse R, Dosso M, Migliani R, Guedenon A, Rey JL, Portaels F. L'ulcère de Buruli, une pathologie endémique émergente: actualité du diagnostic [résumé]. Méd Trop 1999; 59(Suppl): 68S, Abstract CJ 13.

Lungu M. Marketing season increases rises of STDs among peasant farmers [abstract]. In: XIth International Conference on AIDS and STD in Africa: "Looking into the Future; setting priorities for HIV/AIDS in Africa"; 12-16 September, 1999, Lusaka, Zambia; abstract book. [Lusaka]: [s.n.], 1999: 90, Abstract 14PT37-31.

Malele F, Séguy N, Tuliza C, Philips M, Vuylsteke B. Do female sex workers (FSW) need special health care services? A report from Kinshasa, DRC [abstract]. In: XIth International Conference on AIDS and STD in Africa: "Looking into the Future; setting
priorities for HIV/AIDS in Africa"; 12-16 September, 1999, Lusaka, Zambia; abstract book. [Lusaka]: [s.n.], 1999: 83, Abstract 14PT37-4.

Morein B, Vanham G, Akerblom L, Lövgren-Bengtsson K, Davis D, Willems B, Penne L, Barnett S, van der Groen G. A recombinant prime, peptide boost vaccination strategy to induce antibodies able to neutralize primary isolates of HIV-1 [abstract]. In: New concepts in HIV vaccine development, Bethesda, USA, May, 3-5, 1999. [s.l.]: [s.n.], 1999.

Morein B, Vanham G, Akerblom L, Lövgren-Bengtson K, Davis D, Willems B, Penne L, van der Groen G. Unity amongst HIV diversity; impact on vaccine development [abstract]. In: XIth International Congress of Virology, 1999, Sydney, Australia. [s.l.]: [s.n.], 1999: 41, Abstract VW37A.05.

Musonda RM, Tembo F, Weiss HA, Buvé A, Van Dyck E, Sukwa TY, Robinson NJ, Kaona FAD, Carael M, Kaetano L, Mwanakasale V, Manyando C. Prevalence of STDs in the general population of Ndola, Zambia [abstract]. In: XIth International Conference on AIDS and STD in Africa: "Looking into the Future; setting priorities for HIV/AIDS in Africa"; 12-16 September, 1999, Lusaka, Zambia; abstract book. [Lusaka]: [s.n.], 1999: 168, Abstract 16BT3-1.

Ondoa P, Kestens L, Heeney JL, Willems B, Davis D, Heyndrickx L, Janssens W, Vingerhoets J, Peeters M, van der Groen G. Variable region polymorphism cycle while putative neutralizing epitopes accumulate mutations in the env gene during natural infection of a chimpanzee with SIVcpz [abstract]. In: 1999 UCLA Symposium on Molecular & Cellular Biology, Keystone, January 7-13: HIV Vaccine Development: Opportunities and Challenges; AIDS Pathogenesis. [s.l.]: [s.n.], 1999: 109, Abstract 239. (UCLA Symposia on Molecular and Cellular Biology)

Realini L, De Ridder K, Hirschel B, Portaels F. Optimal in vitro growth conditions for Mycobacterium genavense [abstract]. Clin Microbiol Infect 1999; 5(Suppl.3): 63, Abstract 040.

Spicer JT, Diakhaté M, Vereecken K, Fall A, De Clercq D, Diop A, Ly A, Kestens L, Gryseels B. Resistance to Schistosoma mansoni in epidemic foci in northern Senegal [abstract]. In: VII International Symposium on Schistosomiasis, Rio de Janeiro, Brazil, 5-9 December 1999. [s.l.]: [s.n.], 1999: 78, Abstract ST04.36.

Spicer JT, Diakhaté M, Vereecken K, Fall A, Diop A, Ly A, Kestens L, Gryseels B. Intensity of Schistosoma mansoni infection in epidemic foci in northern Senegal suggests that maturity is an important factor in determining resistance to infection [abstract]. In: Parasitic helminths: from genomes to vaccines, II, Edinburgh, 8-11th July 1999. [s.l.]: [s.n.], 1999.

Van den Ende J, Van Gompel A, Van den Enden E, Taelman H, Vanham G, Vervoort T. Hyperreactive malaria in expatriates returning from sub-Saharan Africa: a comparison between 17 cases with the hyperreactive malarial splenomegaly syndrome (HMS) and 32 cases with an incomplete syndrome [abstract]. In: 6th Conference of the International Society of Travel Medicine, Montréal, Québéc, Canada, June 6-10, 1999; final program; book of abstracts. Stone Mountain: International Society of Travel Medicine (ISTM), 1999: 63, Abstract FC8.6.

Van der Auwera G, Heyndrickx L, Van Rampelbergh R, van der Groen G. HMA for fast gag subtyping of HIV-1 group M isolates [abstract]. In: XIth International Conference on AIDS and STD in Africa: "Looking into the Future; setting priorities for HIV/AIDS in Africa"; 12-16 September, 1999, Lusaka, Zambia; abstract book. [Lusaka]: [s.n.], 1999: 43, Abstract 13PT51-1.

van der Groen G. Filovirussen [abstract]. Ned Tijdschr Geneeskd 1999; 143: 2643.

van der Groen G, Heyndrickx L, Janssens W, Zekeng L, Musonda R, Anagonou S, Van der Auwera G, Coppens S, Vereecken K, De Witte K, Van Rampelbergh R, Kahindo M, Morison L, McCutchan FE, Carr JK, Albert J, Essex M, Goudsmit J, Asjö B, Salminen M, Buvé A, Study Group on Heterogeneity of HIV Epidemics in African Cities. Genetic and phenotypic variation of HIV-1: relevance to vaccine development [abstract]. In: International AIDS Vaccine Workshop, November 18-20, 1999, Beijing, China. [s.l.]: [s.n.], 1999.