Tonate virus infection in French Guiana: clinical aspects and seroepidemiologic study.

Two recent cases of human infection with Tonate virus, one of which was a fatal case of encephalitis, have renewed interest in these viruses in French Guiana. The clinical aspects of confirmed and probable cases of infection with this virus indicate that it has pathogenic properties in humans similar to those of other viruses of the Venezuelan equine encephalitis complex. To determine the prevalence of antibodies to Tonate virus in the various ethnic groups and areas of French Guiana, 3,516 human sera were tested with a hemagglutination inhibition test. Of these, 11.9% were positive for the virus, but significant differences in seroprevalence were found by age, with an increase with age. After adjustment for age, significant differences were found between places of residence. The prevalence of antibody to Tonate virus was higher in savannah areas, especially in the Bas Maroni (odds ratio [OR] = 22.2, 95% confidence interval [CI] = 15.2-32.4) and Bas Oyapock areas (OR = 13.4; 95% CI = 9.8-18.4). The ethnic differences observed in this study were due mainly to differences in place of residence, except that whites were significantly less frequently infected than other ethnic groups. This study indicates that Tonate virus infection is highly prevalent in French Guiana, especially in savannah areas.

Association of severe malaria with a specific Plasmodium falciparum genotype in French Guiana.

Why severe Plasmodium falciparum malaria occurs in only a small percentage of patients is unclear. The possibility that specific parasite characteristics contribute to severity has been investigated in French Guiana, a hypoendemic area, where parasite diversity is low and all patients with severe cases are referred to a single intensive care unit. Parasite genotyping in geographically and temporally matched patients with mild and severe disease showed that the association of a specific msp-1 allele (B-K1) with a specific var gene (var-D) was overrepresented among patients with severe versus mild disease (47% vs. 3%, respectively; P<.001). Moreover, this genotype combination was consistently observed in the most severe clinical cases. Reverse-transcription polymerase chain reaction demonstrated programmed expression of var-D in vivo, which is consistent with its potential implication in severe disease. These results provide field evidence of an association of severe malaria with specific genetic characteristics of parasites and open the way for intervention strategies targeting key virulence factors of parasites.

Surveillance of dengue fever in French Guiana by monitoring the results of negative malaria diagnoses.

Surveillance of dengue fever is mainly based on specific laboratory tests. However non-specific systems, such as clinical surveillance, are also required. In French Guiana, we have tested a non-specific laboratory surveillance system where different biological examinations performed for other reasons than the diagnosis of dengue fever were analysed as methods for dengue fever surveillance. The number of negative malaria diagnoses in Cayenne and Kourou was found to be the best indicator of dengue fever infections in these towns. This surveillance system appears to be very simple and reliable, and a test which could serve as an indicator that is likely to be found everywhere.

Reduced immune activation and T cell apoptosis in human immunodeficiency virus type 2 compared with type 1: correlation of T cell apoptosis with beta2 microglobulin concentration and disease evolution.

This study analyzes the degree of immune activation and characterizes apoptosis in lymphocytes from healthy West African donors or patients infected with human immunodeficiency virus (HIV)-1 or -2. The lower decline of CD4 T cells in HIV-2- compared with HIV-1-infected donors is associated with lower levels of immune activation, evaluated by HLA-DR expression on lymphocytes and sera concentrations of IgG and beta2 microglobulin (beta2m). Ex vivo apoptosis was found in both infections in all lymphocyte subsets, including CD4 and CD8 T cells, as well as B cells, but was lower in HIV-2 than in HIV-1 infection. Interestingly, high correlations were found in HIV-2- and HIV-1-infected donors between the level of CD4 T cell apoptosis and beta2m concentration and progression of the disease. These observations support the hypothesis that long-term activation of the immune system, weaker in HIV-2 infection, significantly contributes to T cell deletion and disease evolution.

Human immune responses to the highly repetitive Plasmodium falciparum antigen Pf332.

The B and T cell responses to EB200, a repetitive part of the Plasmodium falciparum antigen Pf332, were examined in malaria-exposed Senegalese adults. Most donors had high levels of antibodies to recombinant EB200 and 17 overlapping peptides spanning EB200. Taking proliferation and/or cytokine (interferon-gamma and interleukin-4) production as a measure of T cell activation, eight of the EB200-derived peptides induced responses in > 40% of the donors tested. There was no general association between the different types of T cell responses measured, emphasizing the importance of including multiple parameters when analyzing T cell responses and suggesting that EB200 induces functionally distinct T cell responses. The most efficient peptide for induction of proliferative responses was one previously shown to induce T cell responses in five different H-2 congenic mouse strains primed with EB200, suggesting that this is a universal T cell epitope. The presence of multiple B and T cell epitopes in EB200, widely recognized by humans, is important since EB200 has been shown to elicit protective antibody responses in monkeys and may be considered for inclusion in malaria subunit vaccines.

First case of yellow fever in French Guiana since 1902.

The first case of yellow fever in French Guiana since 1902 was reported in March 1998. The yellow fever virus genome was detected in postmortem liver biopsies by seminested polymerase chain reaction. Sequence analysis showed that this strain was most closely related to strains from Brazil and Ecuador.

Plasmodium falciparum parasites in French Guiana: limited genetic diversity and high selfing rate.

The genetic characteristics of Plasmodium falciparum isolates collected in French Guiana, where malaria transmission is low and occurs in isolated foci, were studied. Blood samples were collected from 142 patients with symptomatic malaria and typed using a polymerase chain reaction-based strategy for merozoite surface protein-(MSP-1) block 2, the MSP-2 central domain, and glutamate-rich protein (GLURP) repeat domain polymorphism. This showed that the parasite population circulating in French Guiana presented a limited number of allelic forms (4, 2, and 3 for MSP-1 block 2, MSP-1, and GLURP, respectively) and a small number of mixed infections, contrasting with the large genetic diversity of parasite populations and infection complexity reported for Africa, Asia, and other parts of South America. Two groups of isolates displaying identical 3 loci allele combinations were further studied for the Pf332 antigen, histidine-rich protein-1, thrombospondin-related anonymous protein, and Pf60 multigene family polymorphism. Within each group, most isolates were identical for all markers tested. This suggests a high rate of self-fertilization of P. falciparum parasites in French Guiana, resulting in homogenization of the population. The implications of these findings for malaria control in areas of low endemicity are discussed.

[Susceptibility to neuro-malaria and HLA-DR alleles in Senegal]. / Susceptibilité au neuropaludisme et allèles HLA-DR au Sénégal.

A prospective study was carried out in 46 patients suffering from severe malaria. The control group included 220 persons of which the HLA-DR distribution was known. The HLA-DRB1 alleles were typed by PCR-SSP (Sequence Specific Primers). The most frequent HLA-DR alleles found in patients group were: DR52 (82.8%), DR13 (57.1%), DR10 (28.6%), DR53 (25.7%), DR3 (20%), DR18 (20%). A significant difference was observed between patients with severe malaria and control group for the following alleles: DR3, DR10, DR13 (p < 0.001; Chi square with Yates' correction) and their relative risk were respectively 14.67; 6.29; 2.84. HLA-DR3 was considered as the major marker associated to severe malaria.

Dengue encephalitis in French Guiana.

Thousands of cases of dengue fever (DF) and several cases of dengue haemorrhagic fever were recorded in French Guiana during the recent outbreak of dengue-2 virus (1991-1992) and in subsequent years. One case with clinical signs typical of classical DF with neurological complications is reported in this study. The neurological features (encephalitis) appeared during the acute phase, 2 days after the onset of fever. Dengue-2 virus was detected in both the cerebrospinal fluid and blood sample. This case was fatal. This first reported case of classical DF with encephalitis in French Guiana is a new demonstration of the potential neurovirulence of dengue viruses.

Mayaro virus fever in French Guiana: isolation, identification, and seroprevalence.

This paper reports the first isolation of Mayaro (MAY) virus from a patient infected in French Guiana. The identification was initially performed using immunofluorescent antibody testing with specific mouse antibody, and confirmed by plaque-reduction neutralization testing and reverse transcription-polymerase chain reaction. To determine if MAY virus infection is widespread in French Guiana, a serosurvey was performed to determine the prevalence of antibody to this virus in various ethnic groups and areas of French Guiana. Human sera (n = 1,962) were screened using the hemagglutination inhibition (HI) test. To determine whether MAY virus circulates in the rain forest, a serosurvey in monkey populations was performed. Monkey sera (n = 150) were also screened for antibody to MAY virus using HI testing. Of the human sera tested, 6.3% were positive for anti-MAY virus antibodies. Significant differences in MAY virus seroprevalence between different age groups were observed. Seroprevalence rates increased with age, with a large increase in people 10-19 years of age in comparison with those less than 10 years of age. After adjustment for age, significant differences were also found between places of residence. The prevalence of anti-MAY virus antibody was higher in people living in contact with the forest, especially in the Haut Oyapock area (odds ratio [OR] = 97.7, 95% confidence interval [CI] = 48.2-197.9) and along the Maroni River (OR = 39.7, 95% CI = 20.6-76.6). The ethnic differences observed in this study were probably due to differences in residence. Among monkeys, higher seroprevalence rates were found in Alouatta seniculus (66.0%) than in Saguinus midas (18.2%). Among Alouatta, the seroprevalence increased significantly with weight (and therefore with age). This study indicates that MAY virus is present in French Guiana, and human infections occur in areas where people live near the tropical rain forest.

Immunoglobulin A-specific capture enzyme-linked immunosorbent assay for diagnosis of dengue fever.

Dengue fever (DF) is usually diagnosed by testing for dengue virus immunoglobulin M (IgM) by a capture enzyme-linked immunosorbent assay (ELISA) (MAC-ELISA). However, IgM can last for months, and its presence might reflect a previous infection. We have tested the use of anti-dengue virus IgA capture ELISA (AAC-ELISA) for the diagnosis of DF by comparing the results of MAC-ELISAs and AAC-ELISAs for 178 serum samples taken from patients with confirmed cases of DF. IgM appears more rapidly (mean delay of positivity, 3.8 days after the onset of DF) than IgA (4.6 days) but lasts longer; the peak IgA titer is obtained on day 8. The specificity and the positive predictive value of AAC-ELISA are 100%; its sensitivity and negative predictive value (NPV) are also 100% between days 6 and 25 after the onset of DF, but they decrease drastically when data for tests conducted with specimens from the first days of infection are included, because the IgA titers, like the IgM titers, have not yet risen. AAC-ELISA is a simple method that can be performed together with MAC-ELISA and that can help in interpreting DF serology.

[Prognostic value of cytokines and HLA markers in patients with cerebral malaria in Senegal]. / Valeurs pronostiques des cytokines et marqueurs HLA chez des patients atteints de neuropaludisme au Sénégal.

41 patients senegalese patients suffering from clinically defined severe malaria were studied in the intensive medical care unit of the Hôpital Principal in Dakar, Senegal. All of these individuals lived in Dakar, an area of low and seasonal Plasmodium falciparum transmission. In this study, we aim to determine in one hand, the cytokine levels of TNF-alpha, TNF-alpha sRI, TNF-alpha sRII, IL-2 sR, IL-6, IL-6 sR, and IL-10 to evaluate their prognostic value in the course of the disease; in the other hand, the influence of the HLA-DR alleles in the susceptibility to get severe malaria. At the day of admission (day 0) and 3 days later, one or two blood samples were collected for each patient to assess different biological parameters. Plasma samples were tested for cytokines cited above by ELISA (Medegenix EASIA kits) and DNA samples for HLA-DR by PCR-SSP genotyping. The concentrations of all the cytokines and/or their receptors were significantly increased at day 0 in the patients who died (TNF-alpha = 455 +/- 480 pg/ml, IL6 = 511 +/- 396 pg/ml) and decreased rapidly in the patients who survived from the disease (TNF-alpha = 354 +/- 629 pg/ml, IL6 = 453 +/- 706 pg/ml). A fatal issue seems likely related to the age of patients (20 +/- 12 years for surviving patients and 31 +/- 16 years for patients who died) and the kinetic of the cytokines. Significant differences were observed (pc < 0.001) between patients with severe malaria and a control group for the following HLA alleles: DR3, DR10 and DR13. The HLA-DR13 allele was found positively and highly associated with severe malaria.

Prognostic value of anti-Plasmodium falciparum-specific immunoglobulin G3, cytokines, and their soluble receptors in West African patients with severe malaria.

Forty-one African patients suffering from clinically defined severe malaria were studied in the intensive medical care unit of the main hospital in Dakar, Senegal, West Africa. All of these individuals lived in Greater Dakar, an area of low and seasonal Plasmodium falciparum endemicity. Twenty-seven patients (mean age +/- 1 standard deviation, 19.2 +/- 12.7 years) survived this life-threatening episode, but 14 (30.8 +/- 16.2 years old) died despite initiation of adequate treatment. On the day of admission (day 0) and 3 days later, one to two blood samples (i.e., approximately 10 to 15 ml) were obtained from each subject, and different biological parameters were evaluated in the two groups. Plasma samples were tested for their content in tumor necrosis factor alpha (TNF-alpha), soluble receptors I and II for TNF-alpha (TNF-alpha sRI and TNF-alpha sRII), interleukin-6 (IL-6), IL-6 sR, IL-10, and IL-2 sR. The concentrations of all these cytokines and/or their receptors was significantly elevated in patient plasma samples on day 0, and it rapidly decreased in the group of individuals who survived. By comparison, the mean concentration of the same parameters decreased slowly in the group of patients who died (except for IL-10, which dramatically fell in all patient plasma samples soon after initiation of antimalarial treatment). The TNF-alpha sRI level remained significantly elevated among the patients who died, and the highest levels of soluble TNF-alpha sRI receptor were found among the older patients. Parasite-specific immunoglobulin M (IgM), total IgG, IgG1, IgG2, IgG3, and IgG4 were evaluated by enzyme-linked immunosorbent assay using a crude extract of a local P. falciparum isolate as antigen and human class- and subclass-specific monoclonal antibodies. Parasite-specific IgM, total IgG, and IgG1 were detectable in the plasma samples of most of these African patients, whereas IgG2 and IgG4 mean values were low. The mean level of parasite-specific IgG3 was different (P = 0.024) at day 0, i.e., before initiation of intensive medical care, between the group of the 27 surviving subjects and the group of 14 patients dying of severe malaria. As a consequence, most of the African patients who died had only trace amounts or almost no detectable level of parasite-specific IgG3 at the time of admission. In contrast, the presence of even limited IgG3 activity at day 0 was found to be associated with a significantly increased probability of recovering from severe malaria. Therefore, in our study, both an elevated level of TNF-alpha sRI and absence of IgG3 activity were of bleak prognostic significance, whereas a favorable outcome was usually observed when parasite-specific IgG3 activity was detectable. This finding was strongly suggestive of a prime role for these parasite-specific immunoglobulins in the capacity to help recovery from severe malaria.

Prevalence of antibodies to hepatitis A, C, and E viruses in different ethnic groups in French Guiana.

In order to determine the prevalence of antibodies to hepatitis A, C, and E viruses (HAV, HCV, and HEV) in the various ethnic groups and areas of French Guiana, sera (996 for HCV and HEV, 941 for HAV) were tested for antibodies to these viruses using ELISAs. Differences in HAV seroprevalence were found for different age groups, with a large increase in people aged 20-30 years in comparison with those under 20. After logistic analysis, significant differences were found between places of residence; the prevalence of anti-HAV was higher along the Maroni and Oyapock rivers than in the littoral area. The ethnic differences that were observed were generally due to differences in residence. Of all sera, 5.3% were positive for anti-HCV in preliminary tests, but only 1.5% remained positive after confirmation. Brazilians were significantly more frequently infected by HCV than other ethnic groups (4.7%). Sixty-four sera (6.4%) had antibodies to HEV, and differences were found between ethnic groups. Persons of ethnic groups who had emigrated recently to French Guiana had significantly higher seroprevalence rates: 14.6% for Chinese and Hmongs [odds ratio (OR), 4.4; 95% confidence interval (CI), 1.8-10.7], 13.5% for Brazilians (OR, 4.1; CI, 1.8-9.4), and 10.6% for Haitians (OR, 3.1; CI, 1.1-8.7).

[Identification of HLA-DR alleles for susceptibility to rheumatoid polyarthritis in Senegal]. / Identification des allèles HLA-DR de susceptibilité a la polyarthrite rhumatoïde au Sénégal.

Rheumatoid arthritis is the most frequent inflammatory rheumatism disease. Several studies were aimed to understand its physiopathogenesis in particular the association between some HLA-DR alleles and rheumatoid arthritis. A prospective study was carried out in 34 patients suffering from RA (30 Women and 4 men). The diagnosis was clinically and radiologically made. The control group included 220 persons of which the HLA-DR distribution was known. The HLA-DRB1 alleles were typed by PCR-SSP (Sequence Specific Primers). The most frequent HLA-DR alleles found in patients group were: DR10 (85.3%), DR52 (53%), DR14 (38.2%), DR11 (26.5%), and DR13 (20.3%). A significant difference was observed between RA patients and control group for the following alleles: DR3, DR10, DR18, and DR52 (p < 0.001; Chi square with Yates' correction). HLA-DR3 and DR10 were positively associated with RA. The relative risk was up 30 for DR10.

Extensive genetic diversity of Plasmodium falciparum isolates collected from patients with severe malaria in Dakar, Senegal.

While some genetic host factors are known to protect against severe Plasmodium falciparum malaria, little is known about parasite virulence factors. We have compared the genetic characteristics of P. falciparum isolates collected from 56 severe malaria patients and from 30 mild malaria patients recruited in Hôpital Principal, Dakar, Senegal. All isolates were typed using polymerase chain reaction amplification of polymorphic genetic loci (MSP-1, MSP-2, HRP1, GLURP, CSP, RESA, and the multigene family Pf60). The complexity of infections was lower in severe than in mild malaria and the parasite genetic diversity in both groups was very large. No specific genetic make-up was associated with severity; there were, however, marked differences in allele frequencies in both groups, with a prevalence up to 60% of MSP-2 alleles specifically observed in the severe malaria isolates. In addition, the presence of MSP-1/RO33 alleles was significantly associated with a higher plasma level of tumour necrosis factor alpha receptor 1 (P < 0.05), a reported indicator of severity in human malaria. These results point to potential differences in the genetic characteristics of parasites inducing severe versus mild pathology.

Pattern of immunoglobulin isotype response to Plasmodium falciparum blood-stage antigens in individuals living in a holoendemic area of Senegal (Dielmo, west Africa).

Three cross-sectional studies were conducted in a representative cohort of individuals living continuously in an area holoendemic for malaria in Senegal. Plasma from 145 children and adults were tested. The pattern of antimalarial immunoglobulin class (IgM and IgG) and subclass (IgG1 to IgG4) antibody distribution was determined by enzyme-linked immunosorbent assay using a crude blood-stage antigen of Plasmodium falciparum-infected red blood cells. Adults had higher levels of specific antibodies than children, and IgM, IgG2, and IgG3 accounted for the highest difference (2.9, 6.5, and 4.5 times, respectively). Differences in antibody levels were significant for IgG1 to IgG4 between the lowest and the highest transmission season. No particular isotype distribution pattern could be found associated with any given parasitemia level. The relationship between the optical density (OD) values of each isotype and the risk of clinical malaria attack was tested using a Poisson regression model. Only the IgG3 OD increases were found associated with a significantly reduced risk of malaria attack. These seroepidemiologic data suggest that whereas the total IgG-specific activity is not indicative of any given level of protection against malaria, the level of IgG3 was significantly associated with the relative susceptibility to clinical P. falciparum malaria attacks.

Plasmodium falciparum induces apoptosis in human mononuclear cells.

The level of spontaneous apoptosis in short-term lymphocyte cultures was evaluated in different human immunodeficiency virus-negative groups of either healthy control individuals or patients with clinical malaria. The mean percentage of spontaneous apoptosis found in patients during a malaria attack was significantly higher than in sex- and age-matched healthy controls. The healthy asymptomatic controls were individuals with different degrees of exposure to Plasmodium falciparum as reflected by their various mean levels of specific anti-P. falciparum (immunoglobulin G and M) antibodies. The percentages of apoptotic nuclei were found to be significantly higher in lymphocytes from subjects living in an area where malaria is holoendemic than in lymphocytes from subjects less exposed. Concentrations of soluble plasma interleukin-2 receptor were also higher in subjects from areas where malaria is endemic than in other groups, revealing different levels of lymphocyte activation. Of particular relevance to the in vivo situation, a P. falciparum schizont-rich extract induced a systematic and significant elevation of apoptotic nuclei at day 6 in 87.5% (35 of 40) of the subjects tested. In additional studies with different concentrations of extract, [3H]thymidine incorporation was concomitant with a low or limited level of apoptosis. Taken together, our results strongly suggest that acute as well as chronic asymptomatic P. falciparum infections were consistently associated with a marked increase in the level of mononuclear cell apoptosis. This process could be implicated in some of the alterations reported for the proliferative T-cell responses in areas where malaria is endemic.

HLA-A, -B, -C, -DR, -DQ typing in a population group of Senegal: distribution of HLA antigens and HLA-DRB1*13 and DRB1*11 subtyping by PCR using sequence-specific primers (PCR-SSP).

One-hundred-and sixteen Senegalese Serere were typed for HLA antigens and compared with other ethnic groups in Gambia. We did not find significant differences (Fisher's exact test; P < 0.01) in the HLA antigens distribution between the Serere and Mandinka groups in Senegal and the Serere, Mandinka and Wolof in The Gambia. The most common HLA haplotypes found (P < 0.01; Chi square with Yates' correction) were: A1, B8; A2, B51; A32, B44; A33, B58; A2, Cw2; A2, Cw4; A33, Cw3; A2, DR17; A10, DR10; B35, Cw4; B53, Cw6; B57, Cw3; B65, Cw8; B50, DR15; B52, DR4; Cw2, DR17; DR7, DQ2; DR18, DQ4. The HLA-DRB1*13 and DRB1*11 alleles were subtyped by PCR-SSP and the frequencies of these alleles in the studied population given. HLA-DRB1*1304 and DRB1102 were the most common alleles found respectively 15.0 and 18.5%.