Preclinical efficacy and immunogenicity assessment to show that a chimeric Plasmodium falciparum UB05-09 antigen could be a malaria vaccine candidate.

Although it is generally agreed that an effective vaccine would greatly accelerate the control of malaria, the lone registered malaria vaccine Mosquirix™ has an efficacy of 30%-60% that wanes rapidly, indicating a need for improved second-generation malaria vaccines. Previous studies suggested that immune responses to a chimeric Plasmodium falciparum antigen UB05-09 are associated with immune protection against malaria. Herein, the preclinical efficacy and immunogenicity of UB05-09 are tested. Growth inhibition assay was employed to measure the effect of anti-UB05-09 antibodies on P. falciparum growth in vitro. BALB/c mice were immunized with UB05-09 and challenged with the lethal Plasmodium yoelii 17XL infection. ELISA was used to measure antigen-specific antibody production. ELISPOT assays were employed to measure interferon-gamma production ex vivo after stimulation with chimeric UB05-09 and its constituent antigens. Purified immunoglobulins raised in rabbits against UB05-09 significantly inhibited P. falciparum growth in vitro compared to that of its respective constituent antigens. A combination of antibodies to UB05-09 and the apical membrane antigen (AMA1) completely inhibited P. falciparum growth in culture. Immunization of BALB/c mice with recombinant UB05-09 blocked parasitaemia and protected them against lethal P. yoelii 17XL challenge infection. These data suggest that UB05-09 is a malaria vaccine candidate that could be developed further and used in conjunction with AMA1 to create a potent malaria vaccine.

Enhanced acquired antibodies to a chimeric Plasmodium falciparum antigen; UB05-09 is associated with protective immunity against malaria.

It has been shown that covalently linking two antigens could enhance the immunogenicity of the chimeric construct. To prioritize such a chimera for malaria vaccine development, it is necessary to demonstrate that naturally acquired antibodies against the chimera are associated with protection from malaria. Here, we probe the ability of a chimeric construct of UB05 and UB09 antigens (UB05-09) to better differentiate between acquired immune protection and susceptibility to malaria. In a cross-sectional study, recombinant UB05-09 chimera and the constituent antigens were used to probe for specific antibodies in the plasma from children and adults resident in a malaria-endemic zone, using the enzyme-linked immunosorbent assay (ELISA). Anti-UB05-09 antibody levels doubled that of its constituent antigens, UB09 and UB05, and this correlated with protection against malaria. The presence of enhanced UB05-09-specific antibody correlated with the absence of fever and parasitaemia, which are the main symptoms of malaria infection. The chimera is more effective in detecting and distinguishing acquired protective immunity against malaria than any of its constituents taken alone. Online B-cell epitope prediction tools confirmed the presence of B-cell epitopes in the study antigens. UB05-09 chimera is a marker of protective immunity against malaria that needs to be studied further.

Differential T-cell responses to a chimeric Plasmodium falciparum antigen; UB05-09, correlates with acquired immunity to malaria.

The development of a sterilizing and cost-effective vaccine against malaria remains a major problem despite recent advances. In this study, it is demonstrated that two antigens of P. falciparum UB05, UB09 and their chimera UB05-09 can serve as protective immunity markers by eliciting higher T-cell responses in malaria semi-immune subjects (SIS) than in frequently sick subjects (FSS) and could be used to distinguish these two groups. UB05, UB09 and UB05-09 were cloned, expressed in E. coli, purified and used to stimulate PBMCs isolated from 63 subjects in a malaria endemic area, for IFN-γ production, which was measured by the ELISpot assay. The polymorphism of UB09 gene in the malaria infected population was also studied by PCR/sequencing of the gene in P. falciparum field isolates. All three antigens were preferentially recognized by PBMCs from SIS. IFN-γ production induced by these antigens correlated with the absence of fever and parasitaemia. UB09 was shown to be relatively well-conserved in nature. It is concluded that UB05, UB09 and the chimera UB05-09 posses T-cell epitopes that are associated with protection against malaria and could thus be used to distinguish SIS from FSS eventhough acute infection with malaria has been shown to reduce cytokine production in some studies. Further investigations of these antigens as potential diagnostic and/or vaccine candidates for malaria are indicated.

Genetic diversity of Plasmodium falciparum in Bolifamba, on the slopes of Mount Cameroon: influence of MSP1 allelic variants on symptomatic malaria and anaemia.

The influence of the genetic diversity of Plasmodium falciparum infection on the clinical presentation of human malaria was investigated in rural Bolifamba, Cameroon. Parasite DNA was obtained from the blood of 208 children (aged 1-15 years) with malarial infection. The prevalences of anaemia and symptomatic and asymptomatic malaria among these children were 57.7%, 51.4% and 48.6%, respectively. The frequencies of parasites carrying each of the block-2 allelic variants (MAD20, K1 and RO33) of merozoite surface protein 1 (MSP1) were compared among the symptomatic and asymptomatic cases of malaria, the anaemic and non-anaemic subjects, and in various age groupings. Although all three allelic variants were found in Bolifamba, 32.7% of the children investigated were co-infected with parasites carrying the RO33 and K1 variants. There was a significant difference in the prevalence of each MSP1 allelic variant both between age-groups and between the various categories of anaemia considered (P<0.0001 for each), with the highest number of alleles occurring in the children with severe anaemia. The combination of RO33/K1 co-infection and anaemia was detected in most (57.7%) of the children aged 3-<6 years. The RO33/K1 co-infection was also strongly associated with both fever and high levels of parasitaemia (P<0.0001 for each). Although the children of Bolifamba are exposed to all three allelic variants of MSP1, which occur either singly or in varying combinations in the infected children, RO33/K1 co-infections are particularly associated with fever and this association appears independent of age and parasite density. The preliminary data presented here should facilitate the design of future research towards the development and testing of malaria candidate vaccines in the study area.

Silent circulation of arboviruses in Cameroon.

OBJECTIVES: To investigate the silent circulation and transmission of arthropod-borne viruses (arboviruses) in the Fako Division of Cameroon. DESIGN: This survey was conducted based on clinical observations and laboratory diagnosis; field collections of mosquitoes. SETTING: This study was conducted in the Fako Division of South West Cameroon. SUBJECTS: One hundred and two sera were obtained from febrile patients (with negative laboratory findings for malaria and typhoid fever) at clinics in the Fako Division, and diurnal anthropophilic mosquitoes (4,764) collected. INTERVENTIONS: Virus isolation was attempted from these, and sera were screened for antibodies against 18 African arboviruses by haemagglutination inhibition (HI) and complement fixation (CF) tests. RESULTS: No virus was isolated. Fifty three of 79 (67.1%) sera reacted with one or more viral antigens. Twenty nine sera (36.7%) reacted with members of the genus Alphavirus, with Chikungunya (CHIKV) and O'nyong-nyong (ONNV) viruses as the most frequent (34.2%). Forty six sera (58.2%) reacted with members of the genus Flavivirus: 24 (30.4%) were cross-reactive, but 11.4% reacted monotypically with Zika, 5.1% with yellow fever virus (YFV), 5.1% with dengue virus-2 (DENV-2), 2.5% with DENV-1 and 1.3% with Wesselsbron virus, respectively. The plaque reduction neutralisation test used to specify the agent that elicited the response could not resolve 33.3% of the cross reactions between CHIKV and ONNV. Neutralising antibody titres against ONNV and CHIKV were very high indicating probable re-infection. CONCLUSION: Our results indicate previously undetected circulation of arboviruses in Cameroon, and suggest that they are important, overlooked public health problems.

Applications of biotechnology techniques to the study of medicinal plants.

Medicinal plants are widely used world wide to address a variety of health problems. The major obstacles faced in the study of medicinal plants include inaccurate identification and speciation, low yield of bioactive metabolites prepared by chemical methods, variability of traditional protocols etc. In the present article we review a range of biotechnological methods that can be employed to facilitate medicinal plant studies. DNA-based techniques like PCR, RFLP, AFLP, RAPD and sequencing can be employed to resolve ambiguities in plant identification and speciation. In vitro plant organ and tissue culture methods can be employed to produce bioactive metabolites (alkaloids, flavonoids, terpenoids etc) under defined conditions. Recombinant DNA techniques can be used to manipulate metabolic pathways and produce protein pharmaceuticals such as antibodies, and protein hormones. The new disciplines of Bioinformatics and Genomics can find application in drug discovery from plant-based products. We conclude that biotechnological procedures can enhance and advance the studies of medicinal plants.

Malaria parasitemia and systemic cytokine bias in pregnancy.

OBJECTIVES: To investigate the effects of age, gravidity, and gestational age on peripheral malaria parasitemia and functional T helper (Th) cell heterogeneity in pregnant women. METHODS: Maternal age, gravidity, and gestational age were recorded and peripheral venous blood collected from 175 women attending antenatal clinics in south western Cameroon between March and September 2002. The blood was checked for malaria parasitemia by light microscopy and plasma levels of interleukin (IL)-4 and human interferon (IFN)-gamma were measured by indirect enzyme-linked immunosorbent assay. RESULTS: Malaria parasites were detected in 45 (25.4%) of 174 women, with rates similar for different age groups, trimesters of pregnancy, and gravidity. The geometric mean parasite density was 565, and parasite density was significantly higher in younger than in older women. For all groups combined, the mean IL-4 level was significantly higher than the mean IFN-gamma (P=0.0004), irrespective of the presence and density of malaria parasites, gravidity except for women in their first trimester of pregnancy and grandmultiparas, who had similar levels of IFN-gamma and IL-4. In general, the cytokine profile was biased toward Th2-type of responses in 112 (84.3%) of 132 women. CONCLUSIONS: In this study the ability to control malaria parasitemia during pregnancy was found to be predominantly age dependent, suggesting naturally acquired immunity. Furthermore, the systemic cytokine profile was found to be biased towards Th2 responses, a prerequisite for a successful pregnancy. This pattern was unaffected by maternal age, gestational age, gravidity, or parasitemia.

Molecular characterisation of Pseudomonas aeruginosa recovered in the Buea health district of Cameroon: implications for nosocomial spread.

BACKGROUND: Pseudomonas aeruginosa is a ubiquitous gram-negative pathogen with a propensity to cause opportunistic infections in humans. Different strains of the organism could colonise patients heralding a wide spectrum of P. aeruginosa infections in the environment. OBJECTIVE: To analyse isolates of P. aeruginosa from clinical and environmental samples using the Polymerase Chain Reaction (PCR) to establish strain relatedness. METHODS: Fifty-two strains of the organism were isolated from wound swabs, urine, sputum of patients and environmental samples from the hospital environment using standard microbiological techniques and ethical consideration. Genomic DNA of the isolates was amplified with primers AF1 (5'-AGA GTT TGA TCC TGG CTCA-3') and 1541R (5'-AAG GAG GTG ATC CAG CC-3'). RESULTS: At least two bands were observed in all isolates typed and band sizes ranged from 0.07 - 1.5kb. The strains were genetically diverse, displaying profiles of 2 - 6 bands between 0.07 - 1.5kb. CONCLUSION: The study demonstrates that strain diversity could be discerned between strains of P. aeruginosa, circulating in the environment of Buea, a finding which has important epidemiological and clinical significance bearing in mind that this pathogen is highly incriminated in nosocomial infections with attendant social implications. This therefore calls for more attention in the diagnosis and management of P. aeruginosa infections in the environment of Buea, Cameroon.

Seasonal variation and prevalence of tuberculosis among health seekers in the South Western Cameroon.

OBJECTIVES: To determine the prevalence of tuberculosis (TB) in Fako health District, to assess the effects of seasonal variation on the incidence of TB in the study area and to use sentinel analysis to predict areas of greatest infection. DESIGN: A prospective cross sectional study based on laboratory investigations. SETTING: Fako health District, South Western Cameroon. RESULTS: The prevalence of TB was 23.3%. Tuberculosis was significantly more prevalent in males (12.6%) as compared with females (10.7%) (P = 0.034). TB prevalence was significantly different between age groups, with the highest number of cases recorded in the age group 21-30 (P = 0.002). When the health areas were compared, TB prevalence varied significantly (P = 0.001), with Limbe Town recording the highest number of TB cases. We recorded more TB cases in the wet season compared with the dry season and the difference was statistically significant (P = 0.000). There was a significant drop in the prevalence of TB over the study period (P = 0.000). CONCLUSION: This study is the first to report on the effects of season on the prevalence of TB in Cameroon. These findings will therefore provide additional useful base line data for setting up TB control strategies in Cameroon.

Environmental Factors Affecting Malaria Parasite Prevalence in Rural Bolifamba; South-West Cameroon

The impact of some environmental factors on malaria parasite prevalence was investigated in rural Bolifamba; Cameroon. The study population comprised 1454 subjects aged 0 - 65 years. Malaria parasite prevalence was higher in the rainy (50.1) than in the dry season (44.2) with a significant difference (P=0.001) in mean parasite density between seasons. Individuals 15 years old; had significantly higher malaria parasite prevalence (55.5) than those 15 years (37.4). Malaria parasite prevalence (P=0.001) and parasite density (P=0.03) were higher in the individuals of wooden plank houses than those of cement brick houses. Inhabitants of houses surrounded by bushes or garbage heaps and swamps or stagnant water showed higher malaria parasite prevalence and densities compared with those from cleaner surroundings. Anopheles gambiae (63.8) and A. funestus (32.8) were associated with perennial transmission of malaria. Our data indicates that poor environmental sanitation and housing conditions may be significant risk factors for malaria parasite burden in Bolifamba

The epidemiology of malaria in Bolifamba, a rural community on the eastern slopes of Mount Cameroon: seasonal variation in the parasitological indices of transmission.

The prevalences of malarial parasitaemia, fever, splenomegaly and anaemia and the levels of parasitaemia were investigated, through part of one wet season (in 2001) and the following dry season (in 2002), in 2157 subjects in the village of Bolifamba, in south-western Cameroon. Overall, 55.9% of the villagers checked in the wet season but only 49.5% of those examined in the dry season were found smear-positive for malaria (P<0.0001). Rainfall was found to be significantly associated with the mean level of parasitaemia (P=0.001). The prevalences of fever (40.3% v. 19.6%), splenomegaly (37.4% v. 4.0%) and marked splenomegaly (i.e. a Hackett's score of 2 or higher; 25.8% v. 2.4%) were all significantly higher in the wet season than in the dry (P<0.0001 for each). No seasonal difference was observed, however, in the prevalence of anaemia. Parasitaemia, fever, splenomegaly and anaemia were all significantly more common in the young children investigated (i.e. those aged < 5 years) than in the older subjects. When the data were subjected to a multiple logistic regression, age-group, anaemia, fever, and month of examination were all found to be significantly associated with the presence of malarial parasitaemia. The results of this large-scale study, the first of its kind in the Buea district of Cameroon, indicate the intense transmission of malarial parasites in rural Bolifamba, with young children at greatest risk. The data collected provide a useful 'base line' for an ongoing study to assess the immune status of the residents of Bolifamba.

Evaluating a malaria intervention strategy using knowledge, practices and coverage surveys in rural Bolifamba, southwest Cameroon.

We evaluated the impact of a malaria intervention in Bolifamba in rural Cameroon. The intervention consisted of educating the community on management of malaria and provision of a dispensary for early diagnosis and treatment. In July 2001, prior to the intervention, a questionnaire was used to obtain information on knowledge of and practices toward childhood malaria of 185 mothers of children aged 0-5 years. The same questionnaire was administered to 120 of the 185 mothers, one-year post-intervention. Clinical and laboratory investigations were carried out on children whose mothers were interviewed. A comparison of pre- and post-intervention data indicated significant changes in (i) the use of appropriate malaria treatment (from 50% to 81.7%); (ii) recognition of splenomegaly as a feature of malaria (from 18.4% to 80.8%); (iii) prevalence of splenomegaly (from 26.5% to 13.3%); (iv) prevalence of fever (from 27.8% to 13.3%); (v) parasite prevalence (from 60.5% to 44.2%) and (vi) severe malaria anaemia (from 2.6% to 0.0%). These findings revealed that proper education of villagers, particularly mothers, on malaria and the presence of health facilities, where treatment is readily available at affordable cost, close to villages, are important strategies that would reduce malaria morbidity and mortality significantly.

Characterisation of a heparan sulfate proteoglycan homologue OvPG-1 from Onchocerca volvulus.

OBJECTIVE: To determine the role of an immunodominant antigen OvPG-1 in human onchocerciasis. DESIGN: Cross-sectional study of subjects living in three onchocerciasis endemic areas. SETTING: Mbonge and Tubah divisions of Western Cameroon and in Esmeralda Province of Ecuador. SUBJECTS: There were 94 and 99 subjects from the Cameroon rain forest and savannah respectively, and 83 endemic residents from Ecuador. RESULTS: The IgG2 anti-OvPG-1 responses of visually impaired and microfiladermic patients were significantly higher than for their age and sex matched counterparts with normal vision and no microfiladermia (p=0.024). Furthermore, the isotype specificity of anti-OvPG-I responses varied for the various onchocerciasis endemic zones. IgG1, IgG2, IgG3 and IgE levels correlated with the presence of microfilariae in Cameroon, but not in Ecuador. CONCLUSION: Increased IgG and IgE levels to the antigen OvPG-1 seem to correlate with the development of onchocercal eye pathology. The present results suggest that the OvPG1 is a dominant antigen of Onchocerca volvulus with a significant role in the pathogenesis of onchocerciasis.

Helicobacter pylori antigens in the faeces of asymptomatic children in the Buea and Limbe health districts of Cameroon: a pilot study.

OBJECTIVE: To determine the prevalence and identify intra-familial risk factors associated with Helicobacter pylori infection in a paediatric population. METHODS: Cross-sectional study in the Buea and Limbe health districts, South West Cameroon. Stool samples were collected from 176 randomly selected apparently healthy children from two communities with different socioeconomic status. They comprised 86 males and 90 females aged 0-10 years with a mean age of 4.29. Helicobacter pylori status was determined using an enzyme-linked immunosorbent assay, the H. pylori stool antigen (HpSA) test. The test uses polyclonal anti-H. pylori capture antibody to detect H. pylori antigens in human stool. Epidemiological data were analysed using the Fisher test and odds ratio (OR) at 95% confidence intervals (CI). RESULTS: The overall prevalence of H. pylori was 52.27% (92 of 176). Univariate analysis showed that H. pylori prevalence was significantly higher in children of the low socioeconomic class, 62.50% (55 of 88) than in those of the high socioeconomic class, 42.05% (37 of 88) (P < 0.05; OR = 2.41, 95% CI: 1.26-4.64). Helicobacter pylori prevalence increased with age from 37.50% (12 of 32) for children aged <3 years, 50.00% (53 of 106) aged 3-6 years and 71.05% (27 of 38) aged 7-10 years (P > 0.05; OR = 0.81, 95% CI: 0.34-1.91). The frequency of infection was significantly higher in males, 64.00% (55 of 86) than in females, 41.11% (37 of 90), (P < 0.05; OR = 2.67, 95% CI: 1.39-5.17). CONCLUSIONS: This study highlights the continuing importance of age, sex and socioeconomic status in the acquisition of H. pylori infection.

Acute spotted fever rickettsiosis among febrile patients, Cameroon.

Although potential arthropod vectors are abundant in Cameroon, acute febrile illnesses are rarely evaluated for arboviral or rickettsial infections. Serum samples from 234 acutely febrile patients at clinics in Tiko and Buea, Cameroon, were examined for antibodies to Rickettsia africae and African alphaviruses and flaviviruses. These serum samples did not contain antibodies against typhoid, and blood malarial parasites were not detected. Serum samples of 32% contained immunoglobulin M antibodies reactive with R. africae by immunofluorescence assay and were reactive with outer membrane proteins A and B of R. africae by immunoblotting. These findings established a diagnosis of acute rickettsiosis, most likely African tick-bite fever. Hemagglutination inhibition testing of the serum samples also detected antibodies to Chikungunya virus (47%) and flaviviruses (47%). High prevalence of antibodies to arboviruses may represent a major, previously unrecognized public health problem in an area where endemic malaria and typhoid fever have been the principal diagnostic considerations.

Antibiogram and plasmid profiles of Neisseria gonorrhoeae isolates from Cameroon: useful tools for epidemiological survey.

A prospective laboratory-based investigative study was carried out on clinical isolates of N. gonorrhoea to determine their antibiotic susceptibility patterns and plasmid profile using standard microbiological and molecular techniques. All the 32 isolates studied showed total resistance to penicillin, spectinomycin and amoxyclinE. On the other hand, susceptibilities of 100%, 98.6% and 98.6% were noted for ciprofloxacin, ofloxacin and norfloxacin respectively. Thirty (93.8%) of the 32 isolates were found to harbour plasmids of molecular weights ranging from 9.2 to 25.2 Mdal. Three distinct groups of N. gonorrhoea isolates were identified based on the molecular weights of the plasmids, namely, group one (9.2 Mdal), group two (12.6 Mdal) and group three (25.2 Mdal). These results suggest that different strains of N. gonorrhoea may be circulating in Fako Division of Cameroon, a finding that is of clinical and epidemiological significance.

Genetic variability and linkage disequilibrium within the HLA-DP region: analysis of 15 different populations.

In order to understand the forces governing the evolution of the genetic diversity in the HLA-DP molecule, polymerase chain reaction (PCR)-based methods were used to characterize genetic variation at the DPA1 and DPB1 loci encoding this heterodimer on 2,807 chromosomes from 15 different populations including individuals of African, Asian, Amerindian, Indian and European origin. These ethnically diverse samples represent a variety of population substructures and include small, isolated populations as well as larger, presumably admixed populations. Ten DPA1 and 39 DPB1 alleles were identified and observed on 87 distinct DP haplotypes, 34 of which were found to be in significant positive linkage disequilibrium in at least one population. Some haplotypes were found in all ethnic groups while others were confined to a single ethnic group or population. Strong positive global linkage disequilibrium (Wn) between DPA1 and DPB1 was present in all 15 populations. The African populations displayed the lowest values of Wn whereas the Amerindian populations displayed near absolute disequilibrium. Analysis of the distribution of haplotypes using the normalized deviate of the Ewens-Watterson homozygosity statistic, F, suggests that DP haplotypes encoding the functional heterodimer are subject to much lower degrees of balancing selection than other loci within the HLA region. Finally, neighbor joining tree analyses demonstrate the power of haplotype diversity for inferring the relationships between the different populations.

Reduced levels of chloroquine resistant Plasmodium falciparum in selected foci of the South West Province, Cameroon.

OBJECTIVES: To evaluate the state of chloroquine resistant malaria in the South West Province of Cameroon. To estimate the prevalence of different species of Plasmodium in the area. DESIGN: A cross sectional study. SETTING: South West Province of Cameroon. SUBJECTS: 326 febrile patients. MAIN OUTCOME MEASURES: Chloroquine resistance studies using the WHO seven-day standard in vivo test. Speciation of malaria by polymerase chain reaction. Prevalence of falciparum malaria by light microscopy of thick blood smears. RESULTS: Prevalence of chloroquine resistance ranged from 4.3 to 13.2%. A total of six cases showed RIII resistance, and three cases showed RII resistance. Plasmodium falciparum was the predominant species, 96.8% of those testing positive for P. falciparum, 6.3% of cases showed mixed infections of P. falciparum and P. malariae. P. ovale accounted for 3% of cases. CONCLUSION: P. falciparum resistance to chloroquine may not be on the increase compared to earlier reports from the South West Province. The results have important implications in treatment of malaria in view of the low cost of chloroquine.

Antibiogram of Klebsiella pneumoniae isolates from Buea, Cameroon.

OBJECTIVE: To determine the antibiotic susceptibility of K. pneumoniae isolates from Buea, Cameroon. DESIGN: A prospective study of K. pneumoniae isolates from clinical samples of nosocomial origin. SETTING: A laboratory based investigative study at the Biotechnology Centres of the Universities of Buea and Yaounde 1, Cameroon, and three Buea based hospitals. K. pneumoniae isolates were obtained from sputum, wound swabs and urine and screened for their antibiogram using standard procedures. RESULTS: Results on the antibiogram showed seven distinct antibiotypes distinguished by different susceptibilities to aminoglycosides (Spectinomycin and Gentamicin), Chloramphenicol and Augmentin. All the isolates shared multi-resistance to Amoxicillin and Trimethoprim. However, the isolates showed marked susceptibilities to Norfloxacin (90.01%), Cefuroxime (95.45%) and Ciprofloxacin (86.36%). CONCLUSION: The study has revealed that K. pneumoniae isolates in the environment of Buea, Cameroon are multi-drug resistant. This finding is of clinical and epidemiological significance.