Risk factors for HIV-associated neurocognitive disorders (HAND) in sub-Saharan Africa: the case of Yaoundé-Cameroon.

BACKGROUND: The prevalence of HIV-associated neurocognitive disorders (HAND), especially HIV-associated dementia (HAD) is influenced by several risk factors. The prevalence as well as risk factors for HAD are not well known in sub-Saharan Africa (SSA). We have shown that the International HIV Dementia Scale (IHDS) is a useful screening tool for HAND in Yaoundé [Njamnshi AK, Djientcheu VdP, Fonsah JY, Yepnjio FN, Njamnshi DM, Muna WFT. The IHDS is a useful screening tool for HAD/Cognitive Impairment in HIV-infected adults in Yaoundé-Cameroon. Journal of Acquired Immune Deficiency Syndromes 2008;49(4):393-397], but no study in Cameroon has yet investigated the risk factors for HAND or HAD. PATIENTS AND METHODS: A cross-sectional study was conducted in Yaoundé, the capital of Cameroon from September to December 2006. One hundred and eighty-five HIV-positive subjects were included. Diagnosis of HAND was done using the IHDS with a score < or = 10 considered as abnormal. Age, sex, level of education, IV drug use, body mass index (BMI), CDC clinical stage, CD4 counts, hemoglobin levels, administration of highly active antiretroviral therapy (HAART) and type of regimen used, were considered in univariate analysis, with level of significance set at P < or = 0.05. A binary logistic regression was used to determine independent risk factors. RESULTS: The following factors were independent predictors of HAND: advanced clinical stage (OR=7.43, P=0.001), low CD4 count especially CD4 < or = 200 cells/microL (OR=4.88, P=0.045) and low hemoglobin concentration (OR=1.16, P=0.048). CONCLUSION: This first study of the risk factors for HAND in Yaoundé-Cameroon shows findings similar to those described in other studies. These results call for rapid action by policy makers to include HAND prevention strategies such as providing early universal access to HAART based on these risk factors, in the management of HIV patients at risk of HAND in resource-limited settings of SSA like ours.

Analysis of human lung endothelial cells for susceptibility to HIV type 1 infection, coreceptor expression, and cytotoxicity of gp120 protein.

The lung represents a potential target during HIV infection, and the onset of AIDS is associated with severe pulmonary complications in many patients. T-lymphocytes and alveolar macrophages form the majority of HIV-infected cells in the lung. However, other cell types in the lung could participate in HIV-mediated lung pathology and their role has not been investigated. The aims of this study were to determine if human lung microvascular endothelial cells (HLMEC) express HIV receptor and coreceptors, and if HIV can directly infect HLMEC. Specifically, we wished to determine if these cells constitute a viral reservoir in the lung, and if HIV-1 envelope proteins induce cytotoxic effects on HLMEC. Our results showed that by flow cytometry, HLMEC failed to express any CXCR4 or CCR5 on their surface. In contrast, RT-PCR revealed the presence of CXCR4 and CCR5 mRNA, but not CD4 in HLMEC. Two dual-tropic HIV-1 isolates failed to infect HLMEC in vitro, as determined by (1) p24 antigen capture ELISA, (2) reverse transcriptase assay, RT-PCR, and (3) DNA PCR. However, a recombinant HIV-1 gp120 preparation induced apoptotic cell death of HLMEC. These data support the hypothesis that no productive HIV-1 infection of HLMEC occurs in vitro. This suggests that in vivo, HLMEC may not be a major reservoir of HIV in the lung and the primary route for HIV invasion of the lung. Thus, while other mechanisms must play a role in HIV invasion and subsequent dissemination in the lung, lung endothelial cells do represent potential targets for the lethal effects of HIV viral proteins.

Analysis of human endothelial cells and cortical neurons for susceptibility to HIV-1 infection and co-receptor expression.

Neuronal cell death is believed to be the underlying cause of neurological diseases and AIDS dementia often seen in human immunodeficiency virus (HIV) infected patients. The means by which HIV invades the brain is still unknown and the mechanism of neuronal cell death remains to be elucidated. The aim of this study was to determine if direct infection of human brain endothelial cells and neurons play a role in viral invasion of the brain and neuronal cell death, respectively. To this effect, we evaluated human brain microvascular endothelial cells (HBMEC) and human cortical neurons (HCN) for the expression of HIV co-receptors and their susceptibility to HIV-1 infection. While both HBMEC and HCN failed to express any CXCR4 and CCR5 on their cell surface, as assessed by flow cytometry, RT - PCR revealed the presence of CXCR4 and CCR5 mRNA in HBMEC but not in HCN. Two dual tropic HIV-1 primary isolates failed to infect both cell types as determined by p24 antigen capture ELISA, RT - PCR and DNA PCR. These data support the hypothesis that no productive infection of HBMEC and HCN occurs in vitro and suggest that other cell types are the primary focus of HIV-1 infection in the brain.

Distribution of CD4+ T-lymphocytes levels in patients with clinical symptoms of AIDS in three west African countries.

OBJECTIVES: To study the CD4 T-lymphocyte distribution in patients with clinical signs suggestive of AIDS in West Africa. DESIGN AND METHODS: Selected patients had clinical AIDS, according to the WHO clinical definition of AIDS in Africa. Serum samples were tested for the presence of HIV antibodies with two different enzyme immunoassays (EIA), and whole blood was used to determine the CD4 lymphocyte levels of each patient, using the TRAx CD4 Test Kit. RESULTS: In patients with AIDS, the mean CD4+ cell level was 466/microliter; 34% of patients had less than 200/microliter and 62.1% less than 400/microliter. In patients with clinical AIDS but without HIV antibodies, the mean CD4+ cell level was 807/microliter; with 4% below 200/microliter and 14.7% below 400/microliter. The optimal CD4+ cell cut-off between the two groups of patients (with and without antibody to HIV) was 400/microliter. CONCLUSIONS: The mean CD4 cell levels of AIDS patients was more than twice the 200 CD4+ cells/microliter which, alone or associated with clinical criteria is used to differentiate HIV seropositive patients with and without AIDS. A cut-off of 400 T-lymphocyte equivalents per microlitre (TLE/microliter) will be more appropriate. Only 4% of the anti-HIV negative patients had < 200 CD4 TLE/microliter, and could be infected with unknown immunodeficiency viruses.

Wide variation in DNA content among isolates of Trypanosoma brucei ssp.

The DNA contents of 18 Trypanosoma brucei ssp. stocks were compared using flow cytometry, karyotype analysis and quantitation of repetitive DNA by Southern blotting and hybridisation. The DNA contents of Type 1 T. b. gambiense stocks were lower than those of non-gambiense stocks, but both groups showed a wide range of variation in DNA content. Amongst T. b. gambiense stocks. Mabia at the lower end of the range had 14% less DNA than Dal 972 at the top of the range. Similarly, amongst non-gambiense stocks. 117R at the lower end of the range had 14% less DNA than LM55 at the top of the range. The T. b. gambiense stock Mabia had 29% less DNA than non-gambiense stock LM55. The DNA content of Type II T. b. gambiense stocks had minichromosomes albeit fewer than non-gambiense stocks. This result was verified by hybridisation with probes for satellite DNA and a telomere-specific repeat. Hybridisation with the probe for the beta-tubulin genes also revealed an apparent reduction of gene copy number T. b. gambiense relative to non-gambiense stocks. In conclusion, there is a wide range of variation in genome size in T. brucei ssp., with T. b. gambiense stocks at the lower end of the range. The reduction in genome size correlates with loss of repeated genes and non-coding sequences in T. b. gambiense stocks, and is not continued to chromosomes of a particular size.

Detection of Trypanosoma brucei gambiense, in serologically positive but aparasitaemic sleeping-sickness suspects in Cameroon, by PCR.

Diagnosis of Gambian sleeping sickness is problematic because of the very low levels of parasitaemia encountered in the field. A PCR method developed for the sensitive detection of Trypanosoma brucei was used to diagnose parasitologically negative suspects in a recent survey in Cameroon. Individuals were screened in two foci (Mbam and Fontem), firstly with the card agglutination test for trypanosomiasis (CATT) as a primary serological test, together with palpation and puncture of enlarged cervical lymph glands. Any suspects found positive by CATT (CATT+) and any clinical suspects were then subjected to several parasitological tests (examination of thick blood films and use of hematocrit centrifugation, mini-anion-exchange chromatography and a commercial kit for in-vitro isolation). Overall, 43 of the 1703 subjects screened in the Mbam focus were CATT+ and three (two of whom were CATT+) had enlarged glands. In Fontem, 56 of the 1210 subjects screened were CATT+, 78 (24 of whom were CATT+) had enlarged glands and two (both CATT+) had trypanosomes in their gland juice. However, all the suspected cases of sleeping sickness, including the two gland-positives, gave negative results in the secondary, parasitological tests. Blood samples from 28 suspects from Mbam and 30 from Fontem were selected for PCR analysis on the basis of high CATT response or clinical grounds. For each suspect, DNA was prepared from 0.5 ml blood by phenol extraction or differential lysis and then amplified by PCR using specific primers for T. brucei ssp. Four samples from Mbam and nine from Fontem, including the two gland-positives, were found positive by PCR. Compared with the other parasitological techniques, therefore, PCR was the most sensitive diagnostic method in this study, with an estimated sensitivity of 25 trypanosomes/ml blood. Although PCR analysis is too expensive for routine diagnosis, it could be very useful in determining which sleeping-suspects should be closely followed up.

Characterization of Trypanosoma brucei gambiense isolates using restriction fragment length polymorphisms in 5 variant surface glycoprotein genes.

Fifty-eight Type I Trypanosoma brucei gambiense (G) stocks, including 16 from 3 sleeping sickness foci in Cameroon, were compared by Restriction Fragment Length Polymorphism (RFLP) analysis with 14 T.b. brucei and T.b. rhodesiense stocks from various endemic areas of Africa. Loci examined were for 5 variant surface glycoprotein (VSG) genes: the LiTat 1.3, AnTat 11.17 and 2K genes were present as single copy genes, while the VSG 117 and U2 gene probes hybridised with a family of related genes. The RFLP data were subjected to cluster analysis to produce a dendrogram constructed from similarity coefficients. The LiTat 1.3 and AnTat 11.17 genes are considered to be characteristic of G stocks, and neither gene was found in the non-G stocks; however, the LiTat 1.3 gene was absent from 6 of the 58 G stocks, while the AnTat 11.17 gene was absent from 8. Supplementation of the LiTat 1.3 antigen in the Card Agglutination Test for Trypanosomiasis with the AnTat 11.17 antigen might thus improve performance of the test, particularly in Cameroon. The U2 VSG gene probe gave a characteristic RFLP pattern for G stocks, as did the VSG 117 gene; the latter is an isogene of AnTat 1.8 previously used extensively to characterise G stocks by other workers. The 2K gene was absent in some G stocks, while present in some non-G stocks, and was not therefore useful for characterisation of G stocks. In cluster analysis, the T.b. gambiense stocks formed a large homogeneous group, subdivided into 5 subgroups, with the non-gambiense stocks as a heterogeneous outgroup.

Genetic heterogeneity in the Trypanosoma brucei gambiense genome analysed by random amplification of polymorphic DNA.

The random amplification of polymorphic DNA (RAPD) technique has the potential to produce large amounts of characterisation data very quickly and simply, using far less DNA than conventional restriction-fragment-length polymorphism (RFLP) analysis. In the present study we assessed genetic heterogeneity among 34 Trypanosoma brucei gambiense isolates from various endemic areas in Africa by the RAPD technique using 8 arbitrary primers and compared the results with those obtained previously from RFLP analysis of polymorphisms in 5 variant surface glycoprotein (VSG) genes. The isolates were compared both among themselves and with 3 T. b. non-gambiense isolates. Most of the primers produced RAPD profiles specific for T. b. gambiense, with 4 primers showing marked polymorphisms between T. b. gambiense and non-gambiense stocks. These primers also showed minor variations between the T. b. gambiense stocks, and 2 revealed differences between Cameroonian stocks. These results were comparable with those produced by RFLP analysis, where certain polymorphisms are characteristic of T. b. gambiense. Numerical analysis showed a high correlation between the RAPD and RFLP data, with genetic variation being detected at a finer level by RAPD analysis. We conclude that RAPD analysis provides a simple and accurate method for the characterisation of T. b. gambiense.

The isolation and genetic heterogeneity of Trypanosoma brucei gambiense from north-west Uganda.

Fifty-two samples of blood were taken from sleeping sickness patients in north-west Uganda. All samples failed to infect immunosuppressed mice. Ten cryopreserved blood samples were fed to laboratory bred Glossina morsitans morsitans; eight flies developed midgut infections from which procyclic cultures were established in vitro. Isoenzyme electrophoretic analysis of 9 enzymes revealed that 7 of the 8 trypanosome isolates had a combination of enzyme patterns already described for Trypanosoma brucei gambiense. The eighth isolate had a different aspartate aminotransferase polymorphism which placed it in a new zymodeme. Analysis of polymorphisms in genes for 3 variant surface glycoproteins (VSGs) confirmed that the 8 Ugandan trypanosome isolates were T.b.gambiense and revealed further heterogeneity. The VSG 117 gene was present in all the isolates in a pattern of fragments (equivalent to AnTat 1.8) characteristic for T.b.gambiense. For two other VSG genes characteristic of T.b.gambiense, the LiTat 1.3 gene was present in all the isolates, while the AnTat 11.17 gene was present in only 2 of the 8 isolates.