Complete nucleotide sequence of an African human T-lymphotropic virus type II subtype b isolate (HTLV-II-Gab): molecular and phylogenetic analysis.

We report the first complete nucleotide sequence of an African human T-cell lymphotropic virus type II. This new strain, called HTLV-II-Gab (Gab), was obtained from the uncultured peripheral blood mononuclear cells of a 44-year-old healthy Gabonese male who lived in a remote rural area, with neither history of blood transfusion nor sexual intercourse with non-Africans. Using nested PCR, 25 overlapping fragments, representing the entire proviral genome, were obtained, cloned and sequenced. The overall nucleotide sequence comparison with the four other available complete HTLV-II genomes indicated that Gab was more closely related to the HTLV-II subtype b prototypes (98.9, 99.3 and 98.2% nucleotide similarity with G12, NRA and GU respectively) than to the subtype a prototype (95.1% nucleotide similarity with Mo). Restriction profiles studies and phylogenetic analyses confirmed that Gab was a subtype b strain. However, this strain represents a newly described restriction fragment length polymorphism subtype, closely related to one of the rare partially sequenced African isolates originating from a pygmy living in Cameroon (PYGCAM). Nevertheless, the very low genetic divergence observed between this new African strain and the American strains raises several questions on the origins and level of genetic variability over time of this human retrovirus.

Fluorometric detection of HIV-1 genome through use of an internal control, inosine-substituted primers, and microtiter plate format.

We describe a PCR-based fluorometric assay for the detection of the HIV-1 genome. This technique consists of a reverse hybridization with oligonucleotide probes covalently coated onto a microtiter plate as a solid support. Several improvements to the PCR amplification and detection steps gave greater sensitivity and specificity for HIV-1 screening and resulted in a convenient and rapid technique. False-positive results were avoided by using uracyl DNA glycosylase. False-negative results from the presence of PCR inhibitors were detected by coamplifying an internal control with the viral sequence. False-negative results from viral genome variability were limited by using two pairs of primers and by incorporating inosine at the primer positions corresponding to viral polymorphic nucleotides. Furthermore, the hybridization buffer and enzymatic reaction were optimized to increase the assay's sensitivity. The sensitivity and specificity of the fluorometric detection were similar to those of radioisotopic oligonucleotide solution hybridization; however, hands-on time was reduced, and the use of radioactivity was eliminated. We have used this technique routinely on 115 samples and obtained 100% specificity and high sensitivity (only one false-negative result) according to viral culture and (or) serological status of the patients.

HIV/HTLV-I coinfection and clinical grade at diagnosis.

A total of 963 HIV-infected patients have been identified or followed up in Martinique since 1985. Medical files were used to retrieve information about age, sex, circumstances of diagnosis, HTLV-I status, and HIV clinical grade at first examination according to CDC criteria from 1987. Complete information was available for 774 patients. At the first clinical examination, the clinical grade of 65 coinfected patients was more severe than that of the monoinfected patients (GIV versus GII, OR = 2.60, p < 0.01), but after adjustment for age and sex, this odds ratio was reduced 1.57. Although this study cannot invalidate the hypothesis of a faster progression toward AIDS of coinfected than of monoinfected patients, it shows that one or several other mechanisms contribute to the different grades of severity at the first clinical examination observed between these two categories of patients. We believe that HTLV-I infection acquired during adulthood is a marker of high-risk behavior and that it might be associated with early or multiple HIV infections.

Prospective monitoring and quantitation of residual blasts in childhood acute lymphoblastic leukemia by polymerase chain reaction study of delta and gamma T-cell receptor genes.

We have developed a strategy based on polymerase chain reaction (PCR) for detecting all possible gamma T-cell receptor (gamma TCR) rearrangements and the most common delta TCR rearrangements found in B-lineage and T-acute lymphoblastic leukemia (T-ALL). The segments amplified from blasts are then directly sequenced to derive clonospecific probes. From a series of 45 patients aged 1 to 15 years (42 B-lineage ALL, 3 T-ALL), 35 (83%) could be followed for minimal residual disease with at least one clonospecific probe. Detection of clonal markers using clonospecific probes routinely allowed the detection of 1 to 10 blasts out of 10(5) cells as determined by serial dilutions of the initial samples. Residual disease was quantitated by a competitive PCR assay based on the coamplification of an internal standard. Twenty children were prospectively followed for periods varying from 7 to 30 months. In most children, a progressive decrease of the tumor load was observed, and blasts became undetectable within 6 months after the initiation of treatment. A slower kinetics of decrease in tumor cells was found in three children. These three patients relapsed with blasts that continued to display the initial clonospecific markers. Three other children had a central nervous system relapse despite the absence of detectable medullary residual disease. The use of both delta and gamma TCR genes as clonal markers, as well as simplification in the methods to detect and quantify residual blasts reported here, will allow the study of the large number of patients required to determine the role of the detection of minimal residual disease by PCR in the follow-up of childhood ALL.

HTLV-I maternal transmission in Martinique, using serology and polymerase chain reaction.

We have investigated HTLV-I and HTLV-II infection in children born to HTLV-I-seropositive or indeterminate Western blot mothers in Martinique by using the polymerase chain reaction (PCR). Only HTLV-I and no HTLV-II-positive samples were found in this study. All the samples from HTLV-I-seropositive children and adults were PCR positive, whereas the four HIV-I-seropositive and Western blot HTLV-I-negative mothers and their eight children were all PCR negative. Therefore, PCR and serology were in complete agreement in these patients. However, two of the six mothers who were first indeterminate by Western blot, and who later became seronegative, were found positive by PCR. Of the 27 children (ages 2-12 years), born to HTLV-I-seropositive and PCR-positive mothers, 2 were seropositive and PCR positive, 5 were seronegative and PCR positive with 2 primer pairs in gag and pol, and 4 were seronegative and PCR positive with only 1 of the primer pairs. In contrast to an initial rate of transmission of 7% estimated by serology we found a rate of transmission of 28 to 41% (whether or not children who were positive with only one of the primer pairs were included). Thus, our study confirms that PCR is useful in detecting HTLV-I infection in children before seroconversion and underlines the potential lack of sensitivity of serology to detect contaminating HTLV-I blood units in endemic areas.

[Epidemiological characteristics of HIV infection in Martinique]. / Caractéristiques épidémiologiques de l'infection à VIH en Martinique.

The epidemiologic study of HIV infected patients in Martinique from 1985 to 1992 allowed to point out a stability of new cases by year, and confirm the heterosexual transmission in this area. The sexual comportment of Martinicans seems to be different of the continental French people and different between men and women. This constation is important to know for the preventive strategy of the infection.

Prevalence of HTLV-I and HTLV-II infection in Gabon, Africa: comparison of the serological and PCR results.

A cluster sampling survey was performed in 1989 in Libreville, Gabon, to determine HTLV-I and HTLV-II prevalence and to compare the efficacy of polymerase chain reaction (PCR) and serology in detecting HTLV-I and HTLV-II infections. A total of 322 sera from adults were tested by ELISA and by Western blot (WB). The WB patterns were interpreted according to WHO criteria and those of the manufacturer. PCR analysis using primer pairs in the gag and pol region, with a specific probe for HTLV-I and HTLV-II, was performed on the lymphocytes of the 322 adults. In addition, 134/322 samples were re-tested with tax primers, in a second laboratory. Using WHO criteria, 8/322 (2.5%) samples were positive on WB and 25 were indeterminate; with the criteria of the kit, 26/322 (8.1%) were positive and 7 were indeterminate by WB. By PCR, 13 (4%) samples were positive, including 12 for HTLV-I (3.7%) and one for HTLV-II (0.3%). All 8 seropositive samples (by the WHO criteria) were positive by PCR, as were 4 out of 25 indeterminate samples. Only one out of 289 seronegative samples was positive by PCR. In contrast, only 12/26 positive samples by the kit criteria were confirmed by PCR. These results confirm the relatively high HTLV-I/II seroprevalence observed in Gabon. HTLV-I infection is preponderant, but HTLV-II is also present. The WHO criteria for WB give a better fit with PCR results than the kit criteria for WB. In the absence of a specific confirmatory test and based on the uncommon "seronegative" HTLV-I/II infection, the indication for PCR appears limited to the positive WB samples (to differentiate HTLV-I and II infection) and to the indeterminate WB samples.

HTLV-I infection in French West Indies: a case-control study.

A case-control study was performed in Martinique, French West Indies, comparing 66 anti-p24 antibody carriers to 91 seronegative subjects for HTLV-I, matched for age and place of residence. The aim of our study was to identify factors associated with HTLV-I infection and to observe whether clinical examination and biological measurements would reveal any abnormalities among the seropositive subjects. We observed a predominance of females among seropositive subjects (74% compared to 59%, p less than 0.05), and a greater risk due to earlier blood transfusions (p less than 0.001). This survey revealed important differences between cases and controls regarding socioeconomic factors: cases had fewer luxuries or advantages (i.e. bathroom, toilets, refrigerator, telephone, p less than 0.01), were more corpulent (p less than 0.05), and more often widowed, divorced or separated (p less than 0.01) than the controls. Although the differences were not significant, the seropositive donors seemed to be less educated, and were from a lower socioprofessional class than the seronegative donors. With regard to clinical symptoms (infections, adenopathies, splenomegaly, hepatomegaly) and biological parameters (blood count; T-cell subsets, electrophoresis of protids, immunoglobulins, calcemia, antischistosomal antibody), seropositive subjects appeared to be healthy; no parameters, except for alpha 1 globulin (p less than 0.05) and monocytes (p less than 0.05), were found to be correlated with seropositivity; but these two parameters remained within their normal ranges. This study confirms blood transfusion as a risk factor. It underscored the importance of socioeconomic factors for seropositivity.

HLA Gm systems and susceptibility to alcoholic cirrhosis: a study of mixed-race subjects.

The fact that only a small percentage of excessive drinkers develop cirrhosis may be due to a genetic susceptibility to the disease. In order to identify possible genetic risk factors for cirrhosis, we studied mixed-race (Negroid-Caucasian) inhabitants of the French West Indies and compared: (1) the frequency of 51 HLA-A, -B, -C and -DR antigens in 41 subjects with alcoholic cirrhosis and in two control groups consisting of 41 excessive drinkers free of liver disease and 51 healthy non-drinkers; and (2) the frequency of Gm and Km haplotypes in the same groups. Analysis of the Gm system also determined the patients' ethnic origins. The frequency of the HLA-A2 antigen was significantly higher in the cirrhotic patients than in the control group of excessive drinkers (chi 2 = 4.47; P less than 0.05), while that of the HLA-B15 antigen was significantly lower (chi 2 = 5.14; P less than 0.05). The frequency of the Cw4 antigen was significantly higher in the cirrhotics than in the non-drinkers (chi 2 = 5.59; P less than 0.05). However, these differences did not persist when the number of comparisons was taken into account. The frequency of Gm and Km haplotypes was not significantly different in the three groups. In conclusion, complementary studies are required to determine the value of the Gm-Km system as a marker of susceptibility to alcoholic cirrhosis. Our results do not identify an association between HLA antigens and cirrhosis specific to a negroid ethnic group and support the notion that such an association is weak.

[Epidemiology of HTLV-I infection in its hyperendemic foci (Japan, tropical Africa, Caribbean)]. / Epidémiologie de l'infection par HTLV-I dans ses foyers d'hyperendémie (Japon, Afrique intertropicale, Caraïbes).

HTLV-1 infection is endemic in Japan, black Africa, the Caribbean and several regions of South America. In these foci, the infections is very heterogeneously distributed (variations from village to village, intrafamilial clustering). The virus is transmitted from mother to child, and breast feedings seems to play a major role. Sexual transmission is usually from man to woman. The frequency of transmission by blood transfusion must not be underestimated. It justifies the systematic detection of HTLV-1 infection in areas where it is economically feasible.

Sex ratio of human T-lymphotropic virus type I infection and blood transfusion.

The prevalence of human T-lymphotropic virus type I (HTLV-I) infection is higher for females than for males. Blood transfusion is a potential confounding factor which might contribute to this high female:male ratio. Two studies were performed in Martinique (French West Indies) to clarify this issue: a case-control survey comparing the experience of previous blood transfusion among 62 HTLV-I-seropositive and 88 HTLV-I-seronegative blood donors, and a retrospective study of the sex of recipients of blood. Blood transfusion was strongly associated with HTLV-I infection (odds ratio = 6.4, p less than 0.001). Females were more often given blood transfusions (57.9 percent, p less than 0.001) and received a higher percentage of blood units (53.5 percent, p less than 0.05) than could be expected from their proportion in the general population (51.6 percent). Thus, the high female:male sex ratio of HTLV-I-infected subjects might be due partially to a sex difference for blood transfusion.

Diagnosis of HTLV-I infected seronegative neurological patients by polymerase chain reaction amplification in Martinique.

HTLV-I is a type C human retrovirus, endemic in Japan, central Africa, South America and the Caribbean, which causes adult T cell leukemia/lymphoma and chronic progressive paraparesis. Some neurological patients with chronic progressive myelopathies, but seronegative for HTLV-I have been reported in Martinique. We performed polymerase chain reaction (PCR), to look for the presence of HTLV-I genomic sequences in those patients. Two primers were chosen for gag and tax genes. Liquid hybridization was performed on the amplified products using an internal 32p labelled oligonucleotide as a probe. The hybridized material was run on a 6% polyacrylamide gel. Forty-three of forty-four seropositive subjects analysed as "positive controls" were positive for gag, but only 16/29 for tax. Twenty HTLV-I seronegative neurological patients with a symptomatology suggesting HAM/TSP were tested: 18 were found positive for at least one of the 2 oligonucleotide pairs. Two "negative controls": Moya-Moya and vascular brain failure were found negative. 8/9 subjects with uninterpretable WB were also studied by PCR, and 8 were found positive for at least one oligo pair. Our conclusion is that PCR methodology is able to detect genetic information related to HTLV-I in a number of cases where classical serology is negative.

Locus assignment of two alpha-globin structural mutants from the Caribbean basin: alpha Fort de France (alpha 45 Arg) and alpha Spanish Town (alpha 27 Val).

Two alpha-globin structural mutants were mapped to their encoding loci by in vitro translation of hybrid-selected alpha 1- and alpha 2-globin mRNA. The more highly expressed mutant, alpha Spanish Town (alpha 27Val), is encoded at the alpha 2 locus and the less expressed mutant, alpha Fort de France (alpha 45Arg), is encoded at the alpha 1 locus. These results further define the distribution of alpha-globin structural mutations within the alpha-globin gene cluster and substantiate the dominant role of the alpha 2-globin locus in alpha-globin expression.

Anti HTLV-I antibodies and HLA phenotypes in the West Indies.

Sera of supposedly healthy blood donors were screened for the presence of anti HTLV-I p24 antibodies, and HLA typing for A, B, C and DR antigens was performed for 68 seropositive subjects and 92 seronegative controls. HLA phenotypes of the two groups were not significantly different but the level of the antibody response was related to the antigens of the HLA-B (P = 0.02) and -C loci (P = 0.003). Subjects with HLA-B12 or -B21 antigens had lower titres than the others. Subjects with HLA-Cw2 or -Cw7 antigens had higher titres than the others, but only the difference between HLA-B12 positive and negative subjects (P = 0.002) remained significant at the alpha = 0.10 level if the classical, although conservative, Bonferroni procedure was used to correct for the number of comparisons performed.

HLA A,B,C and DR association with insulin-dependent diabetes in Martinique.

HLA-A,B,C, and DR frequencies have been determined in 34 Coloured Martinican IDDM patients to establish the HLA and IDDM associations. HLA A3, B15, B18, Cw3 and DR4 antigens associations with IDDM are confirmed by this study. We found an increase of B21 similar to that found in Asiatic Indians. As in some African Black populations and in Cape coloured people, A1, B8, and DR3 are not increased in our population. We should point out that our patients' ages of onset were low, and that some studies have found DR4 association in young patients and DR3 in older ones. The protective role of DR2 is confirmed here. B35 and Cw4 negative associations have been found. We have observed that the antigens associated with IDDM are decreased in our control population, except DR4, and that the negative associated DR/ and Cw4 antigens are increased compared to the Continental French population. This corresponds with the low IDDM incidence in Blacks and Coloured people.

[Sero-epidemiology of viral hepatitis A, B and delta in Martinique]. / Séro-épidémiologie des hépatites virales A, B et delta en Martinique.

In view of the ethnic and geographical peculiarities of the French department of Martinique and of the endemic character of hepatitis in tropical countries, we studied the prevalence of infections with hepatitis A, B and delta viruses in that region. A group of 10,109 blood donors and a group of about 100 patients were selected on account of their liver symptoms. As regards hepatitis A, the study of the 2 groups was completed by a sero-epidemiological survey of 509 children and teenagers aged from 1 to 18 years. The prevalence of the HB antigen among blood donors was 1.3 per cent, i.e. about 10 times higher than in Europe and 7 times lower than in hyperendemic tropical areas. It was 2.5 times higher in the male than in the female population; 84 per cent of HBs-positive donors had anti-HBe antibodies, 9 per cent had HBe antigen and 7 per cent had neither one nor the other. This distribution is coherent with a population of symptomless carriers. The prevalence of anti-HBs-positive sera was 34 per cent as against 70-95 per cent in highly endemic countries and 4-20 per cent in Europe; 1.9 per cent of the HBs donors studied and 8.8 per cent of the patients had anti-delta antibodies; 11 of the 13 anti-delta-positive subjects had anti-HBe antibodies and 2 had neither HBe nor anti-HBe. Between the ages of 1 and 3 years very few anti-HAV-positive subjects were observed. From 3 to 10 years, the percentage of seroconversions increased moderately. Between 10 and 20 years, the number of positive cases increased considerably reaching 67 per cent at 20 years and 100 per cent at and above 45 years. Among the patients, 97 were positive for IgG (96 per cent) and only 5 for IgM (4 per cent).

Study of HLA antigens in systemic lupus erythematosus in the French West Indies.

As incidence of SLE is high in Blacks, we studied HLA and SLE associations in the French West Indies, whose population is racially mixed. Forty-seven coloured SLE patients have been typed in HLA A,B,C and DR. We observed B8 association in nearly all of the studies. B15 association, more frequent in Caucasians, was found, also B53 association, a Black variant of B5 more frequent in Blacks. We did not find any class II association.