Characterization of occult hepatitis B virus infection among HIV positive patients in Cameroon.

PURPOSE: Occult hepatitis B infection (OBI) among HIV positive patients varies widely in different geographic regions. We undertook a study to determine the prevalence of occult hepatitis B infection among HIV infected individuals visiting a health facility in South West Cameroon and characterized occult HBV strains based on sequence analyses. METHODS: Plasma samples (n = 337), which previously tested negative for hepatitis B surface antigen (HBsAg), were screened for antibodies against hepatitis B core (anti-HBc) and surface (anti-HBs) antigens followed by DNA extraction. A 366 bp region covering the overlapping surface/polymerase gene of HBV was then amplified in a nested PCR and the amplicons sequenced using Sanger sequencing. The resulting sequences were then analyzed for genotypes and for escape and drug resistance mutations. RESULTS: Twenty samples were HBV DNA positive and were classified as OBI giving a prevalence of 5.9%. Out of these, 9 (45%) were anti-HBs positive, while 10 (52.6%) were anti-HBc positive. Additionally, 2 had dual anti-HBs and anti-HBc reactivity, while 6 had no detectable HBV antibodies. Out of the ten samples that were successfully sequenced, nine were classified as genotype E and one as genotype A. Three sequences possessed mutations associated with lamivudine resistance. We detected a number of mutations within the major hydrophilic region of the surface gene where most immune escape mutations occur. CONCLUSIONS: Findings from this study show the presence of hepatitis B in patients without any of the HBV serological markers. Further prospective studies are required to determine the risk factors and markers of OBI.

Serologic and genotypic characterization of hepatitis B virus in HIV-1 infected patients from South West and Littoral Regions of Cameroon.

BACKGROUND: HBV and HIV share similar transmission routes. Concurrent infection with the two viruses usually results in more severe and progressive liver disease, and a higher incidence of cirrhosis, liver cancer and mortality. Further, this co-infection may lead to cross-resistance between HIV and HBV drugs and increased liver injury, either due to direct hepatotoxicity or drug-related immune-reconstitution hepatitis. These challenges necessitate continuous surveillance for HBV among HIV infected individuals to guide patient management. We conducted this study to understand the serologic and genotypic characteristics of HBV among HIV/HBV infected patients in South West and Littoral Regions of Cameroon. METHODS: Plasma samples were screened for HBsAg, HBeAg, Anti-HBs and anti-HBc using ELISA followed by DNA extraction from all HBsAg positive samples. A 366 bp region covering the overlapping surface/polymerase gene was amplified by a nested PCR and the product sequenced using Big Dye sequencing chemistry. The resulting sequences were then analyzed for genotypes and both escape and drug resistance mutations. RESULTS: Of the 455 samples in this study, 25.5 % (n = 116) were HBsAg positive and 46 of these had their DNA successfully amplified. Genotype E was found in 32 samples (69.6 %) and genotype A in the rest of the samples. Escape mutations associated with failure of diagnosis (Y100C, R122K and Q129H) and with vaccine escape (Q129R and T131N) were detected in varying frequencies in the population. Polymerase mutations implicated in resistance to lamivudine and other ʟ-nucleoside analogues were detected in seven patients (15.2 %), while all the samples lacked mutations associated with resistance to adefovir and tenofovir. CONCLUSIONS: These findings suggest the endemicity of HBV and the predominance of genotypes A and E in the study population. Also, drug resistance findings support the use of tenofovir based ART regimens among HIV/HBV co-infected persons. There is need for continuous HBV screening and monitoring in HIV infected individuals in these regions.

Malaria infection and anaemia in HIV-infected children in Mutengene, Southwest Cameroon: a cross sectional study.

BACKGROUND: Malaria is one of the leading causes of morbidity and mortality in children and HIV infection as well as other factors may worsen the situation. This study was aimed at determining the factors influencing malaria parasite prevalence and density as well as anaemia in HIV-infected children in Mutengene, Cameroon from November, 2012 to April, 2013. METHODS: A semi-structured questionnaire was used to record information on socio-demographic factors and use of preventive measures by caregivers of HIV-infected children aged 1-15 years and of both sexes. Venous blood was collected; blood films were prepared and Giemsa-stained for parasite detection and speciation. Haemoglobin concentration was measured and the anaemic status determined. Data was analysed using Epi Info 7 software. RESULTS: A total of 234 children were studied. The overall malaria parasite prevalence was 24.8 % (58) and was significantly higher (31.9 %, P = 0 .004) in females, those who did not implement any preventive measure at all (66.7 %, P = 0.03) and children who used antiretroviral therapy (ART) (28.6 %, P = 0.02) when compared with their respective counterparts. Geometric mean parasite density (GMPD) was significantly higher (3098.4, P = 0.02) in children who presented with fever, had CD4 T cells ≥500 cells/µL (491.3, P = 0.003) and those with moderate anaemia (1658.8, P = 0.03) than their respective counterparts. Although there was no significant difference, GMPD was however higher in males (549.0); those not on ART (635.0) and highest in children <5 years old (633.0) than their respective counterparts. The overall prevalence of anaemia was 49.6 % (116). The value was significantly highest (58.3 %, P = 0.01) in the 11-15 years age group; those with CD4 T cell level 200-499 (72.7 %, P = 0.001) and children with fever (85.7 %, P = 0.01). CONCLUSION: Implementation of proper and integrated malaria preventive measures as well as frequent monitoring of anaemia on prescription of ART could likely improve the health conditions of HIV-infected children thus avoiding malaria-related morbidity and mortality.

An evolutionary approach to identify potentially protective B cell epitopes involved in naturally acquired immunity to malaria and the role of EBA-175 in protection amongst denizens of Bolifamba, Cameroon.

BACKGROUND: The search for a vaccine against malaria caused by Plasmodium falciparum has lasted for more than 100 years, with considerable progress in the identification of a number of vaccine candidates. The post-genomic era offers new opportunities for an expedited search using rational vaccine design and prioritization of key B-cell epitopes involved in natural acquired immunity. METHODS: Malaria vaccine candidate genes that have reached clinical trial were searched on an evolutionary relationship tree, to determine their level of lineage-specificity. Ten other genes with similar protein features and level of lineage specificity to the vaccine candidates were randomly selected, and computationally evaluated for the presence of B-cell epitopes. The protein fragment with maximum probability of putative epitopes were synthesized and used in an ELISA experiment to determine the presence of antibodies to these peptides, in the serum of malaria patients and healthy malaria uninfected inhabitants from a malaria endemic region (Bolifamba), alongside with a vaccine candidate EBA-175. RESULTS: Two peptide fragments of 25 and 30 amino acid length from PF3D7_1233400 and PF3D7_1437500 respectively, coded as PF4-123 and PF4-143 were shown to contain B-cell epitope(s). Total IgG antibodies to these peptides were not significantly different between sick and healthy participants, but cytophilic antibodies to these peptides were significantly higher in healthy participants (p < 0.03). Total IgG to the vaccine candidate EBA-175 was significantly higher in sick participants than in healthy participants, likewise cytophilic antibodies (p < 0.04). Antibodies to the peptides PF4-123 and PF4-143 correlated negatively (p = 0.025 and 0.008 and r = -0.291 and -0.345, respectively) to parasite load. Total IgG antibodies to EBA-175 showed a negative correlation to parasite load (r = -0.144), which was not significant (p = 0.276). Duration of stay in Bolifamba also negatively correlated with parasite load (p = 0.026, r = -0.419) and total IgG to PF4-143 was significantly associated with prolonged duration of stay in the locality of Bolifamba, Cameroon (p = 0.006, r = 0.361). CONCLUSIONS: The present study has identified two genes PF3D7_1233400 and PF3D7_1437500 containing peptide fragment (PF4-123 and PF4-143) with B-cell epitopes that are correlated with naturally acquired immunity to malaria. A pipeline has been developed for rapid identification of other B-cell epitopes involved in naturally acquired immunity.

Knowledge and perceptions towards malaria prevention among vulnerable groups in the Buea Health District, Cameroon.

BACKGROUND: Malaria is a public health problem especially in vulnerable groups such as pregnant women and children under five years in Cameroon including the Buea Health District (BHD). Misconceptions concerning it exist. This study assessed the level of knowledge and perceptions towards malaria control among pregnant women and mothers/caretakers of under-fives in the BHD. METHODS: A community-based cross-sectional study was conducted in the BHD in August, 2011 in five health areas. A questionnaire was used to collect data on demographic variables, knowledge and perceptions towards malaria control from 443 respondents aged 15-73 years. RESULTS: Of the 443 respondents interviewed, 99% had heard about malaria. Awareness of malaria was similar in rural (98.04%) and urban (98.97%) areas. The health facility was the most popular source of information (74%). The radio, television, tracts/posters and the community relay agents (CRAs) all informed significantly higher proportions of respondents in the urban than rural communities (P < 0.05). Overall, 92% of respondents had the right perception of malaria and 88% knew at least one correct sign/symptom of malaria. The most recognised sign of malaria was fever. When all aspects of malaria were considered, majority (88%) of respondents had good levels of knowledge on malaria. The level of good knowledge in respondents with ≥ secondary school education (91%) was significantly higher (P = 0.01) than in those with ≤ primary school level (83%). Overall, 99% had heard about insecticide treated nets (ITNs); 99% perceived ITNs as a good means to prevent malaria; most respondents (57%) used ITNs mainly for protection against mosquito bites while 48% used them for protection against malaria. CONCLUSION: Respondents with no formal education had a poor level of knowledge on malaria. Hence, new strategies for sensitization messages involving their active participation need to be developed.

Malaria and haematologic parameters of pupils at different altitudes along the slope of Mount Cameroon: a cross-sectional study.

BACKGROUND: Haematologic abnormalities are features in Plasmodium falciparum infection, and anaemia is an inevitable outcome. This study examines the influence of malaria status and altitude on haematologic parameters in school-aged pupils. METHODS: A cross-sectional study was conducted among 728 school pupils aged between four and 15 years at three different altitudinal ranges along the slope of the Mount Cameroon region. The investigative methods included the use of questionnaire, clinical evaluation and laboratory investigations. Blood sample collected from each child was used for the preparation of blood films for detection of malaria parasites and assessment of malaria parasite density as well as full blood count determination using an automated haematology analyzer. RESULTS: The prevalence of malaria in the study population was 33.8% and 64.2% (158/246) of these were asymptomatic (AM). Pupils in lowlands had a significantly (P <0.05) prevalence (95% confidence interval, CI) of malaria (60.6%, CI = 54.6-65.9%) than those in middle belt (29.1%, CI = 23.9-34.8%) and highlands (7.7%, CI = 6.1-9.8%), while those in middle belt had significantly higher geometric mean parasite density (475) than those in lowlands (233) and highlands (388). The prevalence of malaria was significantly higher in children that presented with fever (40.4%, CI = 33.8-47.2%) when compared with afebrile subjects (31%, CI = 27-35.2%). Pupils with AM had a higher prevalence of leucopaenia (43.7%, CI = 35.8-51.8%), microcytosis (27.2%, CI = 20.5-34.9%), hypochromasia (27.8%, CI = 21-35.5%) and thrombocytopaenia (14.9%, CI = 8.9-22.8%) when compared with those with clinical malaria (CM). All mean haematological parameters were comparable in pupils with CM and AM, except for the mean white blood cell (WBC) counts. Pupils with AM had significantly lower (P = 0.02) mean WBC counts (5.1 ± 2.5 × 109/L) than those with CM (5.9 ± 2.3 × 109/L). Age, altitude and malaria parasitaemia was of significant influence on several haematological parameters. CONCLUSION: Altitude influenced the distribution and density of malaria parasites and was of confounding influence on the haematologic profiles. These results highlight the insidious effects of AM on the haematologic components.

Genetic diversity of Plasmodium falciparum merozoite surface protein-1 block 2 in sites of contrasting altitudes and malaria endemicities in the Mount Cameroon region.

The present study analyzed the relationship between the genetic diversity of Plasmodium falciparum and parasitologic/entomologic indices in the Mount Cameroon region by using merozoite surface protein 1 as a genetic marker. Blood samples were collected from asymptomatic children from three altitude zones (high, intermediate, and low). Parasitologic and entomologic indices were determined by microscopy and landing catch mosquito collection/circumsporozoite protein-enzyme-linked immunosorbent assay, respectively. A total of 142 randomly selected P. falciparum-positive blood samples were genotyped by using a nested polymerase chain reaction-based technique. K-1 polymerase chain reaction products were also sequenced. As opposed to high altitude, the highest malaria prevalence (70.65%) and entomologic inoculation rate (2.43 infective/bites/night) were recorded at a low altitude site. Seven (18.91%), 22 (36.66%), and 19 (42.22%) samples from high, intermediate, and low altitudes, respectively, contained multiclonal infections. A new K-1 polymorphism was identified. This study shows a positive non-linear association between low/intermediate altitude (high malaria transmission) and an increase in P. falciparum merozoite surface protein 1 block 2 polymorphisms.

Performance and usefulness of the Hexagon rapid diagnostic test in children with asymptomatic malaria living in the Mount Cameroon region.

BACKGROUND: Rapid and correct diagnosis of malaria is considered an important strategy in the control of the disease. However, it remains to be determined how well these tests can perform in those who harbour the parasite, but are asymptomatic, so that rapid diagnostic tests (RDTs) could be used in rapid mass surveillance in malaria control programmes. METHODS: Microscopic and immunochromatographic diagnosis of malaria were performed on blood samples from the hyperendemic Mount Cameroon region. Thin and thick blood films were stained with Giemsa and examined under light microscopy for malaria parasites. The RDT was performed on the blood samples for the detection of Plasmodium species. In addition, the performance characteristics of the test were determined using microscopy as gold standard. RESULTS: Results revealed 40.32% to be positive for microscopy and 34.41% to be positive for the RDT. Parasites were detected in a greater proportion of samples as the parasite density increase. Plasmodium falciparum was the predominant Plasmodium species detected in the study population either by microscopy or by the RDT. Overall, the test recorded a sensitivity and specificity of 85.33% and 95.05% respectively, and an accuracy of 91.40%. The sensitivity and specificity of the RDT increased as parasite densities increased. CONCLUSION: The Hexagon Malaria Combi test showed a high sensitivity and specificity in diagnosing malaria in asymptomatic subjects and so could be suitable for use in mass surveillance programmes for the management and control of malaria.

Cross-sectional study of specific antibodies to a polymorphic Plasmodium falciparum antigen and of parasite antigen genotypes in school children on the slope of Mount Cameroon.

To investigate relationships between Plasmodium falciparum parasitaemia, parasite genotypes, and specific anti-parasite antibodies, 244 school children (aged 4 to 16 years) were studied in April/May 2002, the peak malaria transmission season in Buea, Cameroon. Antibody reactivities were analysed by ELISA using an array of recombinant antigens representing different sequences from the polymorphic block 2 region of the merozoite surface protein 1 (MSP1), and the blood samples that were slide-positive for P. falciparum were genotyped for msp1 block 2 alleles. The prevalence of antibodies to the specific MSP1 block 2 antigens was significantly higher in children at one particular school (situated at the lowest altitude) compared to the others, although the prevalence of infection or particular parasite genotypes did not differ. Thus, at a population level, the prevalence of these antibodies does not simply reflect prevalence of parasites, but rather may be due to differences in the incidence of past infections. However, there were weak positive associations between specific antibody reactivity and the presence of the corresponding allele in the blood of individuals (statistically significant for the MAD20-type allele of block 2), indicating that antibody specificities are to some extent determined by current parasite infections.