Identification of a new human immunodeficiency virus type 1 distinct from group M and group O.

A highly divergent HIV-1 isolate, designated YBF 30, was obtained in 1995 from a 40-year-old Cameroonian woman with AIDS. Depending on the genes studied, phylogenetic analysis showed that YBF30 branched either with SIVcpz-gab or between SIVcpz-gab and HIV-1 group M. The structural genes and tat, vpr, and nef of YBF30 are approximately equidistant from those of HIV-1 group M and SIVcpz-gab. In contrast, vif and rev are closer to HIV-1 group M, and vpu is highly divergent. Using a YBF30 V3 loop peptide enzyme immunoassay, we screened 700 HIV-1-positive sera collected in Cameroon; three reacted strongly with the YBF30 peptides and one was confirmed as being related to YBF30 by genetic analysis of a pol fragment. YBF30 is as distinct from SIVcpz-gab as it is from HIV-1 group M and can thus be considered as the prototype strain of a new human immunodeficiency virus group.

Assessment of adherence to malaria chemoprophylaxis in a French Navy frigate deployed in Southeast Asia.

During a 2-month mission in Southeast Asia, including numerous stopovers in coastal cities, the crew of a frigate of the French Navy received doxycycline for antimalarial prophylaxis. Adherence to this chemoprophylaxis was evaluated with an anonymous questionnaire distributed at the end of the malaria exposure period. The response rate was 74 % (72 crew members). Among them, 67 sailors felt they had received clear information about the risks of malaria. Overall, 19 (27 %) respondents reported adherence (one forgotten pill, by no more than 15 days), 18 (25 %) irregular adherence (one or more pills forgotten weekly or stopped during the mission), and 35 recognized that they had not taken the treatment. These results, in the light of recent international recommendations, suggest that strategies for the prevention of malaria without systematic use of chemoprophylaxis (personal vector protection measures, an "Army posture" strategy), would be more suitable for medicalized ships cruising in this area.

Serological and virological characterization of HIV-1 group O infection in Cameroon.

OBJECTIVES: To study the presence of HIV-1 group O infection among HIV-infected people in Cameroon and to further characterize the HIV-1 group O infections. DESIGN AND METHODS: During a 2-year survey (1994-1995), all samples tested positive in screening methods in the National Reference and Public Health Laboratory, Centre Pasteur, Yaoundé, Cameroon were identified as HIV-1 group M, HIV-1 group O or HIV-2 by using a serological algorithm. HIV-1 group M and HIV-1 group O were distinguished on the basis of competitive enzyme-linked immunosorbent assay (ELISA) reactivity against gp41 group M recombinant protein. HIV-1 group O infections were confirmed by using group O-specific V3 synthetic peptides. HIV-1 group O strains were isolated by lymphocyte cocultures, proviral DNA was amplified with specific primers, and sequencing was performed on the C2V3 and gag regions. RESULTS: Of the 8,331 screened samples, 3,193 were HIV-reactive, 2,376 (74%) of which were considered to belong to group M. The 817 (26%) that had reacted poorly or not at all against group M gp41 were further characterized: 10 were confirmed as HIV-2 and 82 as HIV-1 group O, the others being indeterminate (n = 285) or negative (n = 440). The frequency of group O relative to group M ranged from 1% in Far North province to 6.3% in the capital. There was no difference in sex, age or frequency of clinical manifestations between group M and group O infections. Group O infection was confirmed in a subset of cases by polymerase chain reaction (n = 14), with perfect concordance. Sequencing and phylogenetic analyses confirmed the high variability inside group O. CONCLUSIONS: Group O and group M epidemiological patterns are known to be similar so the reason for the lower prevalence of group O remains to be found. The wide distribution of group O infection in all Cameroonian provinces underlines the importance of further characterizing the epidemic spread and diffusion of this group.

Molecular characterization and phylogenetic analyses of a new simian T cell lymphotropic virus type 1 in a wild-caught african baboon (Papio anubis) with an indeterminate STLV type 2-like serology.

STLV-1 viruses are closely related to HTLV-1 and infect many African monkey species. Seroreactivities of monkeys infected by STLV-1 are nearly identical to those of HTLV-1-infected individuals. In some cases, STLV-1 are, sequence-wise, indistinguishable from HTLV-1, and cannot be separated from them on the basis of phylogenetic analyses. HTLV-2-related simian viruses have been rarely reported. Such STLV-2 viruses, present in African bonobo (Pan paniscus), possess a genomic organization related to but different from all known HTLV-2 subtypes. We report here the molecular characterization and the subtyping of a new STLV-1 in a wild-caught baboon (Papio anubis) whose serum exhibited an indeterminate STLV-2-like serology (p24, GD21, MTA-1 with no p19). In the env and LTR regions, this virus is phylogenetically related to the large African STLV-1 group, but does not cluster with any STLV-1 baboon sequence. The complete p19 sequence reveals amino acid changes at critical positions. This is the first report of an African STLV-1 virus leading to an STLV-2-like serological profile in its host.

An unusual HIV type 1 env sequence embedded in a mosaic virus from Cameroon: identification of a new env clade. European Network on the study of in utero transmission of HIV-1.

An atypical HIV-1 strain (CAM001) was identified in a pregnant Cameroonian woman in 1995. HMA subtyping of the env region was unsuccessful, and sequence analyses were performed. Unique sequence motifs were found at the V3 tip (GAGRALHA and GAGRAWIHA), and phylogenetic studies showed that the env C2-V5 sequence branched within group M but remained distinct from all known HIV-1 subtypes, while p17 gag branched with the subtype F sequences. Four other HIV group M viruses, undetermined by HMA, of African origin were found to cluster with CAM001 in the C2-V5 sequences. With the BLAST method, we found in databases three strains whose V3 sequences also clustered with CAM001. These unusual env sequences from eight HIV-1 strains derived from Cameroon formed a separate cluster in HIV-1 group M, which we designated k.

A new HTLV type II subtype A isolate in an HIV type 1-infected prostitute from Cameroon, Central Africa.

Among 332 female sex workers in Douala, Cameroon, 113 were HIV-1 seropositive, 3 were HTLV-I seropositive, and only 1 had specific anti-HTLV-II antibodies. By cocultivation with BJAB cells, an HTLV-II was isolated from the peripheral blood mononuclear cells of this 32-year-old woman coinfected by HIV-1. This new African HTLV-II isolate (PH230PCAM) belongs to the molecular subtype A, exhibiting, however, a nucleotide variability of 2.4% and 0.8%, vis-à-vis the MO prototype, in the LTR and in the gp21 env gene, respectively. These data, as well as the previous findings of another HTLV-II subtype A in a Ghanean prostitute, suggest that this viral subtype had been imported into Africa, while the HTLV-II subtype B, described in remote areas of Zaire, Gabon, and Cameroon, could be a genuine African HTLV-II, present in this continent for a long period of time.

Evidence of the existence of a new circulating recombinant form of HIV type 1 subtype A/J in Cameroon. The European Network on the Study of In Utero Transmission of HIV-1.

Several genetic subtypes and circulating recombinant forms (CRFs) of HIV-1 have been identified. The greatest degree of genetic diversity is displayed by variants from Central and West Africa. HIV-1 env C2-V5 and protease sequences were obtained from 15 HIV-1-infected pregnant women, who were selected from a larger cohort study in Yaoundé, Cameroon. Fourteen of 15 virus variants were shown to be recombinant, whereas a single variant appeared to be nonrecombinant subtype A. Five viruses were subtype A/J recombinants, with env genes derived from subtype A and protease genes derived from subtype J. Seven viruses clustered with reference sequences for CRF02 AG(IbNG) in both the env and protease gene fragments, and were thus subtype A/G recombinants. Two variants displayed even more complex recombination patterns. Phylogenetic analyses indicated that the five subtype A/J recombinants might be the first representatives of a previously unrecognized CRF.

HIV type 1 diversity and the reliability of the heteroduplex mobility assay.

We investigated HIV-1 diversity by means of heteroduplex mobility assay (HMA) genotyping. We studied 199 samples from patients originating from 26 countries and living in France. The HMA successfully genotyped 182 (91%) of these samples, as follows: 77 (42%) subtype A, 57 (31%) subtype B, 5 (3%) subtype C, 5 (3%) subtype D, 8 (4%) subtype E, 22 (12%) subtype F, 5 (3%) subtype G, and 3 (2%) subtype H. We were not able to genotype 12 samples by means of the HMA. These latter strains were sequenced, and phylogenetic analyses revealed that they were highly divergent subtype A-, D-, or G-related strains. Eight (of 12) subtype D strains were indeterminate by HMA, owing to the broad intrasubtype diversity, suggesting that new reference subtype D plasmids are required, as previously proposed. Thirty-seven strains belonging to the different subtypes were sequenced, and the results showed perfect concordance with the HMA results. Interlaboratory quality controls confirmed the reliability of the HMA for HIV-1 subtyping, despite the extensive viral variability. However, plasmid selection must be continuously revised to cover viral diversification.

Synthetic peptide ELISAs for detection of and discrimination between group M and group O HIV type 1 infection.

We developed and evaluated two peptide-based immunoassays to confirm and discriminate between group M and group O HIV-1 infection. These assays are based on in vitro competition for antibody binding between M and O peptides. The first EIA is based on competition between group M and group O gp41 immunodominant domains and the second on competition between group O and group M V3 regions of gp120. Two panels of sera were used: the first consisted of 109 sera collected from 27 group O- and 92 group M-infected patients in whom the HIV isolates had been genotyped by sequencing or heteroduplex mobility assay. In this panel, the combination of the two assays correctly discriminated 106 samples (100% group O and 96.7% group M samples). The second panel, used for the field evaluation of the two assays, consisted of 157 samples from HIV-1-infected Cameroonian patients, 33 strains having been genotyped. The combination of the two techniques in a serogrouping algorithm discriminated 147 of these samples, 74 being HIV-1 group O and 73 group M. These results always correlated with genotyping results. The 10 sera that were not successfully classified by these assays were from early seroconverters. Altogether, the two assays clearly differentiated 263 of 276 (94.9%) samples in the two panels. On the basis of the genotyping results, the positive predictive value for group discrimination in the two panels was 100% for both GSEIA assays. Our peptide-blocking group-specific EIAs for differentiation and confirmation of HIV-1 group M and group O infection are complementary tools for epidemiological studies and surveillance of HIV-1 group O strain trafficking.

Antenatal HIV prevalence in Yaounde, Cameroon.

From June 1994 to July 1996, 4100 pregnant women living in Yaounde, Cameroon, were tested for human immunodeficiency virus type 1 (HIV-1) and syphilis. The HIV seroprevalence was 4.2% (95% confidence interval (CI): 3.6%-4.8%), and that of antibodies to Treponema pallidum was 17.4% (95% CI: 16.3%-18.6%) (HIV infection was twice as common in women with positive syphilis serology) (7.2% vs 3.6%). Over the study period, the antenatal seroprevalence of syphilis remained stable, while there was an increase in the HIV seroprevalence rate. There was an increase in HIV seropositivity in women uninfected with syphilis between 1994/1995 and 1995/1996 from 2.9% to 4.3%. By the end of the study, HIV infection was no commoner in women with negative compared with positive syphilis serology. It is therefore postulated that HIV infection in Yaounde has entered the general, sexually active female population. We suggest that management of pregnant women in Cameroon should include routine screening for both HIV infection and other sexually transmitted diseases (STDs).

Multicenter study of street foods in 13 towns on four continents by the food and environmental hygiene study group of the international network of pasteur and associated institutes.

An international multicenter study of ready-to-eat foods, sandwiches, and ice creams or sorbets sold in the streets and their vendors was carried out to assess the microbiological quality of these foods and to identify characteristics of the vendors possibly associated with pathogens. Thirteen towns in Africa, America, Asia, and Oceania were involved in the study. A single protocol was used in all 13 centers: representative sampling was by random selection of vendors and a sample of foods bought from each of these vendors at a time and date selected at random. Microbiological analyses were carried out using standardized Association Française de Normalisation methods, and the use of a standardized questionnaire to collect data concerning the characteristics of the vendors. Fifteen surveys were carried out, with 3,003 food samples from 1,268 vendors. The proportion of unsatisfactory food samples was between 12.7 and 82.9% for ice creams and sorbets and between 11.3 and 92% for sandwiches. For ice creams and sorbets, the sale of a large number of units (>80 per day) increased the risk of unsatisfactory food by a factor of 2.8 (95% confidence interval [CI]: 1.5 to 5.1), lack of training in food hygiene by 6.6 (95% CI: 1.1 to 50). and by a factor of 2.8 (95% CI: 1.4 to 5.4) for mobile vendors. These risk factors were not identified for sandwiches, this difference may be due to the presence of a cooking step in their preparation. These results show that the poor microbiological quality of these street foods constitutes a potential hazard to public health, that the extent of this hazard varies between the cities studied, and that vendors' health education in food safety is a crucial factor in the prevention of foodborne infections.

In vitro sensitivity of Plasmodium falciparum to amodiaquine compared with other major antimalarials in Madagascar.

Chloroquine has been used in Madagascar since 1945 and remains the first-line treatment for uncomplicated cases of malaria. Low-grades of resistance type R1 and R2 have been reported. Thus, in vitro tests were performed in order to monitor the drug sensitivity of Plasmodium falciparum from different study sites, with the aim of identifying alternatives to chloroquine. Chloroquine IC50 values ranged from 0.2 nM to 283.4 nM (n = 190, mean IC50 = 52.6 nM; 95% CI = 46.1-59.1 nM). Fifteen isolates (7.9%) were chloroquine-resistant. One mefloquine-resistant isolate was detected (1/139). The test isolates were sensitive to amodiaquine (n = 118), quinine (n = 212), pyrimethamine (n = 86) and cycloguanil (n = 79). The median IC50 for amodiaquine was 12.3 nM (mean IC50 = 15.3 nM, 95% CI = 13.3-17.3 nM). Amodiaquine was 3.4 times as active as chloroquine in vitro and 7 times as active as quinine against P. falciparum. These results indicate that amodiaquine may be a potent alternative to chloroquine in Madagascar. There was positive correlation between tested quinoline-containing drugs activities, which suggests in vitro cross-susceptibility.

[HIV-1 group N in Cameroon and apparent viruses in the chimpanzee]. / VIH-1 groupe N au Cameroun et virus apparentés chez le chimpanzé.

The hypothesis of the recent origin of AIDS by way of one of several events of inter-species transmission has been widely accepted. Whilst the primate HIV-2 reservoir has been clearly identified, the origin of HIV-2 is still uncertain. Since 1994, collaborative studies on the variability of HIV-1 conducted at the Yaoundé Pasteur Centre have confirmed the very great diversity of circulating HIV-1 group M subtypes. This research has led to the identification of a further branch of HIV-1 phylogenesis, by the YBF30 strain, prototype for a new N group. A study conducted on chimpanzees isolated the retrovirus in three of them. Complete sequencing for one of the primates showed an important similarity in the env gene with the YBF30 strain, whereas the pol gene is closer to the HIV-1 group M. These findings support the hypothesis that HIV-1 group N resulted from a recombinant event. The origin of the HIV-1 group N pol fragment and more generally the origins of group M and O human viruses are still unclear. In order to complete the phylogenesis of human and non-human primate retrovirus as well as to identify two circumstances of more epidemiogenic variants, it is important to follow the variability of HIV-1 in central Africa and search for intermediary forms amongst simian species.

[Influenza epidemiologic and virologic surveillance in Antananarivo from 1995 to 2002]. / Surveillance épidémiologique et virologique de la grippe à Antananarivo de 1995 à 2002.

The "Institut Pasteur de Madagascar" virology laboratory is the National WHO Centre for Influenza surveillance in Madagascar. On this surveillance collaborate the Ministry of Health with 9 sentinel centres. In the present article, the authors relate the results of influenza surveillance in Antananarivo between 1995 and 2002. Among 6341 patients with nasal and/or pharyngeal swabs, influenza virus were isolated from 427 patients (6.7%): 307 (68.4%) influenza virus A (H3N2), 124 (27.1%) influenza virus B, 8 (4.0%) influenza virus A (H1N1). The virus had been continually spreading all year long. The weak and the strong points of the influenza sentinel surveillance are also discussed in order to ameliorate the collection processes of influenzal and respiratory morbidity data.

[Vibrio cholerae in Madagascar: study of a multiresistant strain]. / Vibrio cholerae à Madagascar: étude d'une souche multirésistante.

Madagascar was cholera free until March 1999. The first case was reported in Mahajanga, a north west coast harbor. Ten months later and despite a massive use of tetracycline as prophylactic drug, cholera had reached every region of the island. All suspected cholera samples were analysed at the Pasteur Institute of Madagascar where susceptibility to tetracycline was systematically performed. On February 2000, a multidrug resistant strain of V. cholerae was isolated. We studied this strain by performing Minimal Inhibitory Concentration (MIC) and by plasmidic and conjugative assay. As the original strain, this multiresistant V. cholerae showed a resistance to cotrimoxazole, to streptomycin and chloramphenicol but, in addition to, appeared strongly resistant to ampicillin and tetracycline. This strain harboured a 26 kb self-transmissible plasmid. Conjugation tests showed the possibility of plasmidic segregates or acquisition of two different plasmids. The weak transfer rate could explain why we have isolated only one multiresistant strain. The emergence of a such multiresistant strain should encourage the medical authorities to reinforce the epidemic survey in every medical Malagasy district and to carry out new antimicrobial surveys to describe the mechanisms of the spread of these resistances.

A novel gamma 2-herpesvirus of the Rhadinovirus 2 lineage in chimpanzees.

Old World monkeys and, recently, African great apes have been shown, by serology and polymerase chain reaction (PCR), to harbor different gamma2-herpesviruses closely related to Kaposi's sarcoma-associated Herpesvirus (KSHV). Although the presence of two distinct lineages of KSHV-like rhadinoviruses, RV1 and RV2, has been revealed in Old World primates (including African green monkeys, macaques, and, recently, mandrills), viruses belonging to the RV2 genogroup have not yet been identified from great apes. Indeed, the three yet known gamma2-herpesviruses in chimpanzees (PanRHV1a/PtRV1, PanRHV1b) and gorillas (GorRHV1) belong to the RV1 group. To investigate the putative existence of a new RV2 Rhadinovirus in chimpanzees and gorillas we have used the degenerate consensus primer PCR strategy for the Herpesviral DNA polymerase gene on 40 wild-caught animals. This study led to the discovery, in common chimpanzees, of a novel gamma2-herpesvirus belonging to the RV2 genogroup, termed Pan Rhadino-herpesvirus 2 (PanRHV2). Use of specific primers and internal oligonucleotide probes demonstrated the presence of this novel gamma2-herpesvirus in three wild-caught animals. Comparison of a 1092-bp fragment of the DNA polymerase obtained from these three animals of the Pan troglodytes troglodytes subspecies, one from Gabon and the two others from Cameroon, revealed <1% of nucleotide divergence. The geographic colocalization as well as the phylogenetic "relationship" of the human and simian gamma2-herpesviruses support the model according to which herpesviruses have diversified from a common ancestor in a manner mediating cospeciation of herpesviruses with their host species. By demonstrating the existence of two distinct Rhadinovirus lineages in common chimpanzees, our finding indicates the possible existence of a novel human gamma2-herpesvirus belonging to the RV2 genogroup.

[National Network study to perpetuate the surveillance of Plasmodium falciparum sensitivity to antimalarials in Madagascar]. / Réseau d'Etude de la Résistance (RER) pour pérenniser la surveillance de la sensibilité de Plasmodium falciparum aux antipaludiques à Madagascar.

To redefine strategy and policy to cure or to prevent malaria, there is a need to get relevant and updated data on Plasmodium sp sensitivity level to antimalarial drugs. Thus, in September 1999, the Madagascan Ministry of Health and the Institut Pasteur de Madagascar (IPM) formed a network named RER for malaria resistance surveillance. To alleviate the lack of experienced medical teams within the health centres, and due to technical and logistic matters, as part of the network activities, it was decided to give a start with the in vitro studies which are carried out at IPM. In vitro sensitivity testing is done by use of the isotopic method. Results from the study done in 2001 demonstrate that the Madagascan P. falciparum isolates are susceptible to amodiaquine (n = 215), to cycloguanil (n = 56), to pyrimethamine (n = 98) and to quinine (n = 214). One isolate (1/110 i.e. 0.9%) of mefloquine-resistant phenotype is detected from the Eastern region. P. falciparum susceptibility to chloroquine is satisfactory with 95.4% (206/216) of in vitro sensitive isolates. RER arises from the partnership and collaboration between the Madagascan Ministry of Health and the IPM. The network set-up is presented. The usefulness of the in vivo approach, and the in vitro investigations (chemosusceptibility test and screening of mutations accounting for resistance to chloroquine) to monitor the emergence and the dissemination of drug-resistant parasites in Madagascar as well as in the subregion of the Indian Ocean is discussed.