Biological and molecular variability of human immunodeficiency virus type 2 isolates from The Gambia.

Seven new human immunodeficiency virus type 2 (HIV-2) isolates (CBL-20 to CBL-26) from The Gambia were characterized. Their cytopathogenicity and growth in vitro correlated with the severity of clinical disease. CBL-22 was highly sensitive to neutralization by HIV-2 sera and was cross-neutralized by some HIV-1 sera. These findings, the differing sizes of envelope glycoproteins of individual isolates, and the sequence analysis of amplified regions of the viral DNAs show that these HIV-2 isolates from one geographical region in West Africa exhibit biological and genome variability comparable to that observed for HIV-1.     

A Tuberculin Skin Test Survey and the Annual Risk of Mycobacterium tuberculosis Infection in Gambian School Children

Background

A Tuberculin skin test (TST) survey was conducted to assess the prevalence of latent TB Infection (LTBI) and to estimate the annual risk of M. tuberculosis infection (ARTI) in Gambian school children. The results are expected to contribute to understanding of Tuberculosis epidemiology in The Gambia.

Methods

This was a nationwide, multi-cluster survey in children aged 6–11 years. Districts, 20 of 37, were selected by probability proportional to size and schools by simple random sampling. All TST were performed using the Mantoux method. Height and weight measurements were obtained for all participants. We calculated prevalence of LTBI using cut-off points of 10mm, the mirror and mixture modelling methods.

Results

TST readings were completed 13,386 children with median age of 9 years (interquartile range [IQR] 8–10 years). Mixture analysis yielded a cut-off point of 12 mm, and LTBI prevalence of 6.9% [95%CI 6.47–7.37] and the ARTI was 0.75% [95%CI 0.60–0.91]. LTBI was associated gender and urban residence (p <0.01). Nutritional status was not associated with non-reactive TST or sizes of TST indurations. ARTI did not differ significantly by age, gender, BCG vaccination or residence.

Conclusions

This estimates for LTBI prevalence and ARTI were low but this survey provides updated data. Malnutrition did not affect estimates of LTBI and ARTI. Given the low ARTI in this survey and the overlapping distribution of indurations with mixture modelling, further surveys may require complementary tests such as interferon gamma release assays or novel diagnostic tools.     

Erythropoietin in Cerebrospinal Fluid: Age-related Reference Values and Relevance in Neurological Disease

We aimed to establish age-related reference values for Erythropoietin (EPO) in cerebrospinal fluid (CSF) and to evaluate concentrations in neurological diseases. CSF and serum EPO was measured in controls with tension-type headache (CTTH), in patients with ALS, dementia and depression using ELISA technique. Stability experiments showed CSF EPO to be stable for two and a half months and over two thaw/freeze cycles. A positive correlation of CSF EPO with age was found (P < 0.01). We found a CSF/serum EPO concentration ratio of 0.126, pointing towards an intrathecal synthesis of EPO. The ALS group showed significantly lowered CSF EPO compared to age-matched CTTH (P < 0.012), whereas the dementia and depression group showed no significant differences compared to CTTH. The establishment of age-related reference values in a large cohort of controls will improve the interpretation of future CSF EPO evaluations in neurological diseases. The authors have not reported any conflicts of interest.     

The Chemokine CXCL13 Is a Prognostic Marker in Clinically Isolated Syndrome (CIS)

Background

There is increasing recognition of the importance of B lymphocytes in the immunopathogenesis of multiple sclerosis (MS), encouraging the evaluation of B cell-associated biomarkers in the cerebrospinal fluid (CSF). We aimed to evaluate the relevance of the B cell chemoattractant CXCL13 as a prognostic marker in patients with clinically isolated syndrome (CIS) regarding conversion to MS, and to compare it to Barkhof criteria in magnetic resonance imaging (MRI), oligoclonal bands (OCB) and the polyspecific intrathecal B cell response against measles, rubella and varicella zoster virus (MRZR).

Methodology/Principal Findings

CXCL13 was determined in a prospective study over 2 years including 46 patients that remained CIS over follow-up (CIS-CIS), 45 patients that developed MS (CIS-RRMS), and 30 controls using ELISA. CSF CXCL13 was significantly elevated in CIS-RRMS as compared to CIS-CIS and controls (p<0.001). It was significantly elevated in CIS with OCB (p<0.001), positive MRZR (p = 0.04), and gadolinium enhancement in MRI (p = 0.02) and showed a significant correlation with CSF leukocyte count (p<0.001) and QIgG (p<0.001). CXCL13 showed the best positive predictive value (PPV) of all parameters investigated (70%, 95%-CI: 53–84%), which could be further increased by combination with Barkhof criteria in MRI (80%).

Conclusions/Significance

Our data indicate the relevance of CXCL13 in CIS to predict conversion to MS. It furthermore shows CXCL13 to be an important mediator in the inflammatory cascade associated with the polyspecific intrathecal B cell response that manifests itself in OCB and MRZR.     

Cerebrospinal Fluid Immunoglobulin Kappa Light Chain in Clinically Isolated Syndrome and Multiple Sclerosis

Background

Oligoclonal bands (OCB) are the most widely used CSF test to support the diagnosis of MS and to predict conversion of clinically isolated syndrome (CIS) to multiple sclerosis (MS). Since OCB tests are based on non-quantitative and difficult to standardise techniques, measurement of immunoglobulin kappa free light chains (KFLC) may represent an easier to use quantitative test.

Methods

KFLC were measured in CSF and serum of 211 patients using ELISA. These include patients without any inflammatory central nervous system reaction (NIND, n = 77), MS (n = 20), viral CNS infections (V-CNS-I, n = 10), neuroborreliosis (NB, n = 17) and other bacterial CNS infections (B-CNS-I, n = 10). Furthermore a cohort of 77 patients with CIS, including 39 patients that remained CIS over follow-up of two years (CIS-CIS) and 38 patients that developed MS over the same follow-up time (CIS-MS).

Results

CSF-serum ratio of KFLC (Q KFLC) was elevated in all patients with MS, 86.8% of patients with CIS-MS and 61.5% of patients with CIS-CIS. It was significantly elevated in CIS with presence of OCB (p<0.001). Q KFLC significantly correlated with other CSF variables such as CSF leukocyte count (p<0.001, R = 0.46), CSF CXCL13 levels (p<0.001, R = 0.64) and also intrathecal IgG synthesis (p<0.001, R = 0.74) as determined by nephelometry and quotient diagram. OCB were detected in 66.7% of CIS-CIS and in 92.1% of CIS-MS.

Conclusions

Although the measurement of CSF KFLC is a rapid and quantitative easy to standardize tool, it is almost equal but not superior to OCB with regard to diagnostic sensitivity and specificity in patients with early MS.     

Proteome Profiling in Murine Models of Multiple Sclerosis: Identification of Stage Specific Markers and Culprits for Tissue Damage

The identification of new biomarkers is of high interest for the prediction of the disease course and also for the identification of pathomechanisms in multiple sclerosis (MS). To specify markers of the chronic disease phase, we performed proteome profiling during the later phase of myelin oligodendrocyte glycoprotein induced experimental autoimmune encephalomyelitis (MOG-EAE, day 35 after immunization) as a model disease mimicking many aspects of secondary progressive MS. In comparison to healthy controls, high resolution 2 dimensional gel electrophoresis revealed a number of regulated proteins, among them glial fibrilary acidic protein (GFAP). Phase specific up-regulation of GFAP in chronic EAE was confirmed by western blotting and immunohistochemistry. Protein levels of GFAP were also increased in the cerebrospinal fluid of MS patients with specificity for the secondary progressive disease phase. In a next step, proteome profiling of an EAE model with enhanced degenerative mechanisms revealed regulation of alpha-internexin, syntaxin binding protein 1, annexin V and glutamate decarboxylase in the ciliary neurotrophic factor (CNTF) knockout mouse. The identification of these proteins implicate an increased apoptosis and enhanced axonal disintegration and correlate well the described pattern of tissue injury in CNTF −/− mice which involve oligodendrocyte (OL) apoptosis and axonal injury.In summary, our findings underscore the value of proteome analyses as screening method for stage specific biomarkers and for the identification of new culprits for tissue damage in chronic autoimmune demyelination.     

Cytotoxic T Cells from Human Immunodeficiency Virus Type 2-Infected Patients Frequently Cross-React with Different Human Immunodeficiency Virus Type 1 Clades

Knowledge of immune mechanisms responsible for the cross-protection between highly divergent viruses such as human immunodeficiency virus type 1 (HIV-1) and HIV-2 may contribute to an understanding of whether virus variability may be overcome in the design of vaccine candidates which are broadly protective across the HIV subtypes. We demonstrate that despite the significant difference in virus amino acid sequence, the majority of HIV-2-infected individuals with different HLA molecules possess a dominant cytotoxic T-cell response which is able to recognize HIV-1 Gag protein. Furthermore, HLA-B5801-positive subjects show broad cross-recognition of HIV-1 subtypes since they mounted a T-cell response that tolerated extensive amino acid substitutions within HLA-B5801-restricted HIV-1 and HIV-2 epitopes. These results suggests that HLA-B5801-positive HIV-2-infected individuals have an enhanced ability to react with HIV-1 that could play a role in cross-protection.Human immunodeficiency virus type 1 (HIV-1) and HIV-2 are related human retroviruses that show various biological and structural differences. HIV-2 is found mainly in West Africa, whereas HIV-1 is spreading throughout the world. HIV-2 is less transmissible, and HIV-2-positive patients exhibit longer clinical latency periods than individuals infected with HIV-1 (23). A recent report has also shown that the mortality in HIV-2-infected individuals is only twice as high as in the uninfected population and, in the majority of adults, survival is not affected by HIV-2 status (31).Although the two viruses are similar in genomic organization, various genetic and enzymatic differences have been found at many stages of the retroviral life cycle. They differ significantly in terms of amino acid sequence, the more conserved being the Pol and Gag sequences, which exhibit less than 60% homology (17).Despite these differences, epidemiological data and animal studies have shown some evidence of cross-protection between the two viral infections. Travers et al. reported that HIV-2-infected women had a lower incidence of HIV-1 infection than did HIV-seronegative women in a cohort of commercial sexual workers in Dakar (37), and rhesus macaques immunized with a recombinant HIV-1 poxvirus vaccine are protected against HIV-2 challenge (2). These studies, though not conclusive (1, 6), suggest that differences in the virus may not necessarily preclude the development of defensive immunity to a subsequent pathogenic infection, an old-fashioned concept pioneered by Jenner, who used cowpox to vaccinate against human smallpox.The immunological basis of cross-protection is largely unknown, and a clear understanding of the role played by the humoral or cell-mediated immune response in HIV protection is still lacking. However, mounting evidence suggests that cytotoxic T-lymphocyte (CTL) response could be the key element. Indeed, the protection afforded in animal models against simian (13) and feline (12) immunodeficiency virus infections is closely correlated with the induction of specific CTL response, and HIV-1 and HIV-2 HLA-B35-restricted cross-reactive CTLs have been postulated to confer protection against repeated HIV exposure (33).CTLs recognize short viral peptides, 8 to 11 amino acids long, that are generated by the intracellular processing of endogenously synthesized viral antigens within the infected cells, which are expressed at the cell surface in the binding groove of HLA class I molecules. The specificity of the T-cell response is determined by the interaction of the antigen-specific T-cell receptor (TCR) with the peptide-HLA complex, and this interaction, together with non-antigen-specific signals, activates the CTLs (15).The presence of cross-reactive CTLs able to lyse HIV-1- or HIV-2-infected cells should be dependent on the extent of conservation between the two viruses within the epitopes selected by particular HLA class I molecules. It is well known that amino acid substitutions within the epitopes can abrogate the CTL response by inhibiting either HLA binding or TCR recognition (32). However, a number of recent studies have shown that T cells can recognize apparently unrelated peptides (10, 41), and crystallographic data have shown physical limits to the TCR epitope specificity due to the limited size of contact between the TCR and the peptide (14), suggesting a flexibility in T-cell recognition of antigen (19).Some individuals with a particular HLA profile which is responsible for presentation of the viral antigen and for selection of the T-cell repertoire may possess a CTL response not affected by mutations within the epitope, as has been demonstrated in subjects with HLA alleles B27 (28) and B35 (33). In these cases, amino acid substitutions within the HIV-1 and -2 epitopes were tolerated by the CTLs.In this study, we have investigated the extent of cross-reacting CTLs between HIV-2 and HIV-1 in a group of HIV-2-infected subjects with different HLA class I types. We have shown that despite differences in amino acid sequence between the two viruses, the majority of HIV-2-positive subjects possess CTLs which are able to recognize HIV-1 Gag protein.Furthermore, analysis of HLA profiles and the fine specificity of the cytotoxic response demonstrated that HLA-B5801-positive subjects show broad cross-recognition of HIV-1 isolates. These subjects mounted a CTL response that tolerated extensive amino acid substitutions within an HLA-B5801-restricted HIV-1 epitope.     

Health centre surveys as a potential tool for monitoring malaria epidemiology by area and over time

Background

Presently, many malaria control programmes use health facility data to evaluate the impact of their interventions. Facility-based malaria data, although useful, have problems with completeness, validity and representativeness and reliance on routinely collected health facility data might undermine demonstration of the magnitude of the impact of the recent scaleups of malaria interventions. To determine whether carefully conducted health centre surveys can be reliable means of monitoring area specific malaria epidemiology, we have compared malaria specific indices obtained from surveys in health centres with indices obtained from cross-sectional surveys conducted in their catchment communities.

Methods

A series of age stratified, seasonal, cross-sectional surveys were conducted during the peak malaria transmission season in 2008 and during the following dry season in 2009 in six ecologically diverse areas in The Gambia. Participants were patients who attended the health centres plus a representative sample from the catchment villages of these health facilities. Parasitaemia, anaemia, attributable proportion of fever and anti-MSP1-₁₉ antibody seroprevalence were compared in the health facility attendees and community participants.

Results

A total of 16,230 subjects completed the study; approximately half participated in the health centre surveys and half in the wet season surveys. Data from both the health centre and community surveys showed that malaria endemicity in The Gambia is now low, heterogeneous and seasonal. In the wet season, parasitaemia, seroprevalence and fever prevalence were higher in subjects seen in the health centres than in the community surveys. Age patterns of parasitaemia, attributable proportions of fever and seroprevalence rates were similar in subjects who participated in the community and health centre surveys.

Conclusion

Health centre surveys have potential as a surveillance tool for evaluating area specific malaria control activities and for monitoring changes in local malaria epidemiology over time.