Genetic polymorphisms in malaria vaccine candidate Plasmodium falciparum reticulocyte-binding protein homologue-5 among populations in Lagos, Nigeria.

BACKGROUND: Vaccines are the most reliable alternative to elicit sterile immunity against malaria but their development has been hindered by polymorphisms and strain-specificity in previously studied antigens. New vaccine candidates are therefore urgently needed. Highly conserved Plasmodium falciparum reticulocyte-binding protein homologue-5 (PfRH5) has been identified as a potential candidate for anti-disease vaccine development. PfRH5 is essential for erythrocyte invasion by merozoites and crucial for parasite survival. However, there is paucity of data on the extent of genetic variations on PfRH5 in field isolates of Plasmodium falciparum. This study described genetic polymorphisms at the high affinity binding polypeptides (HABPs) 36718, 36727, 36728 of PfRH5 in Nigerian isolates of P. falciparum. This study tested the hypothesis that only specific conserved B and T cell epitopes on PfRH5 HABPs are crucial for vaccine development. METHODS: One hundred and ninety-five microscopically confirmed P. falciparum samples collected in a prospective cross-sectional study of three different populations in Lagos, Nigeria. Genetic diversity and haplotype construct of Pfrh5 gene were determined using bi-directional sequencing approach. Tajima's D and the ratio of nonsynonymous vs synonymous mutations were utilized to estimate the extent of balancing and directional selection in the pfrh5 gene. RESULTS: Sequence analysis revealed three haplotypes of PfRH5 with negative Tajima's D and dN/dS value of - 1.717 and 0.011 ± 0.020, respectively. A single nucleotide polymorphism, SNP (G → A) at position 608 was observed, which resulted in a change of the amino acid cysteine at position 203 to tyrosine. Haplotype and nucleotide diversities were 0.318 ± 0.016 and 0.0046 ± 0.0001 while inter-population genetic differentiation ranged from 0.007 to 0.037. Five polypeptide variants were identified, the most frequent being KTKYH with a frequency of 51.3%. One B-cell epitope, 151 major histocompatibility complex (MHC) class II T-cell epitopes, four intrinsically unstructured regions (IURs) and six MHC class I T-cell epitopes were observed in the study. Phylogenetic analysis of the sequences showed clustering and evidence of evolutionary relationship with 3D7, PAS-2 and FCB-2 RH5 sequences. CONCLUSIONS: This study has revealed low level of genetic polymorphisms in PfRH5 antigen with B- and T-cell epitopes in intrinsically unstructured regions along the PfRH5 gene in Lagos, Nigeria. A broader investigation is however required in other parts of the country to support the possible inclusion of PfRH5 in a cross-protective multi-component vaccine.

Microsatellite markers reveal low levels of population sub-structuring of Plasmodium falciparum in southwestern Nigeria.

BACKGROUND: Genetic diversity studies provide evidence of Plasmodium falciparum differentiation that could affect fitness and adaptation to drugs and target antigens for vaccine development. This study describes the genetic structure of P. falciparum populations in urban and rural sites from southwestern Nigeria. METHODOLOGY: Ten neutral microsatellite loci were genotyped in 196 P. falciparum infections from three localities: Aramoko-Ekiti, a rural community; Lekki, an urban location and Badagry, a peri-urban border settlement. Analysis was performed on the genetic diversity, linkage disequilibrium, population structure and inter-population differentiation. RESULTS: Allelic diversity values were similar across all populations, with mean expected heterozygosity (HE) values between 0.65 and 0.79. No matching multilocus haplotypes were found and analysis of multilocus LD showed no significant index of association. Genetic differentiation between populations was low (ΦPT = 0.017). CONCLUSION: The absence of detectable population structure of P. falciparum in southwestern Nigeria is evident in the lack of significant differentiation between populations separated by about 200 km. This implies that a fairly uniform malaria control strategy may be effective over a wide geographic range in this highly endemic region. However, more wide-scale survey across the country will be required to inform malaria control in this large and densely populated endemic region.

Mutations in Pfcrt and Pfmdr1 genes of Plasmodium falciparum isolates from two sites in Northcentral and Southwest Nigeria.

The ability of malaria parasites to develop resistance to antimalarial drugs has made it necessary to continuously survey malaria parasite populations for resistance markers. Mutations in specific malaria parasite genes confer resistance to antimalarial drugs. The study compared mutations in Pfcrt and Pfmdr1 genes of P. falciparum from two ecologically different areas of Nigeria. Plasmodium falciparum dried blood spots collected from New Bussa (Northcentral Nigeria) and Ijede (Southwest Nigeria) were analysed by PCR-RFLP for Pfcrt, K76 T, Pfmdr1, N86Y and Y184F mutations. Pfmdr1 copy number was determined by quantitative-PCR. A total of 145 blood spots [Ijede = 55; New Bussa = 90 blood spots] were analysed, but Pfcrt gene was successfully amplified in 144 samples while Pfmdr1 was amplified in 132 samples. Overall, prevalence of mutant forms of Pfcrt 76 T,Pfmdr1 86Y and 184F were 74.3% (95% CI: 66.4-81.2%), 18.2% (95% CI: 12.0-25.8%) and 35.6% (95% CI: 27.5-44.4%). The frequency of Pfcrt 76 T was similar in both study sites [Ijede: 81.8% (95%CI: 69.1-90.9%); New Bussa: 69.7% (95%CI: 59.0-79.0), p = 0.105]. However, the frequencies of Pfmdr1 86Y and 184F were significantly higher in Ijede (28.3% and 62.3%) than in New Bussa (11.4% and 17.7%), respectively (P < 0.05). Eight parasite genotypes based on three codons of the two genes were identified. The most frequent genotype was TNY 53(40.5%) while the least was KYF 1 (0.8%). The most frequent genotype in Ijede and New Bussa were TNF 18(34.0%) and TNY 40 (51.3%) respectively. The frequency of wild strain KNF in Ijede and New Bussa were 3 (5.7%) and 18 (23.1%), respectively. The distribution of the genotypes differed significantly by location. The genotypes with more than two or more mutations were more in Ijede 32 (60.4%) than in New Bussa 16 (20.5%) (p < 0.001). Amplification of Pfmdr1 copy number was not observed in the two study sites. The prevalence of Pfcrt 76 T was similar in both locations while Pfmdr1 86Y and 184F differed in both locations. Single nucleotide polymorphisms in the three codons assessed were more in Ijede than in New Bussa.

Impact of training of mothers, drug shop attendants and voluntary health workers on effective diagnosis and treatment of malaria in Lagos, Nigeria.

BACKGROUND: The National Malaria Eradication Program and international agencies are keen on scaling up the use of malaria rapid diagnostic tests (mRDTs) and artemisinin-based combination therapies (ACTs) for effective diagnosis and treatment of the disease. However, poor diagnostic skills and inappropriate treatment are limiting the efforts. In Nigeria, a large proportion of infected patients self-diagnose and treat while many others seek care from informal drug attendants and voluntary health workers. AIMS: This study describes the impact of training voluntary health workers, drug shop attendants, and mothers on effective case detection and treatment of malaria in Lagos, Nigeria. METHODS: We trained mothers accessing antenatal care, drug shop attendants, and voluntary health workers selected from the three districts of Lagos, on the use of histidine-rich protein-2-based mRDTs and ACTs. Pre- and post-training assessments, focus group discussions (FGDs), and in-depth interviews (IDIs) were carried out. RESULTS: The knowledge, attitude, and skill of the participants to achieve the goal of "test, treat, and track" using mRDT and ACTs were low (11%-55%). There was a low awareness of other non-malaria fevers among mothers. Self-medication was widely practiced (31.3%). FGDs and IDIs revealed that health-care providers administered antimalarials without diagnosis. Training significantly improved participants' knowledge and expertise on the use of mRDTs and ACTs (P = 0.02). The participants' field performance on mRDT use was significantly correlated with their category (bivariate r = 0.51, P = 0.001). There was no statistically significant association between the participants' level of education or previous field experience and their field performance on mRDT (r = 0.12, P = 0.9; χ 2= 38, df = 2 and P = 0.49). CONCLUSION: These findings suggest that training of stakeholders in malaria control improves diagnosis and treatment of malaria. However, a broader scope of training in other settings may be required for an effective malaria control in Nigeria.

Pooled-DNA sequencing identifies genomic regions of selection in Nigerian isolates of Plasmodium falciparum.

BACKGROUND: The burden of falciparum malaria is especially high in sub-Saharan Africa. Differences in pressure from host immunity and antimalarial drugs lead to adaptive changes responsible for high level of genetic variations within and between the parasite populations. Population-specific genetic studies to survey for genes under positive or balancing selection resulting from drug pressure or host immunity will allow for refinement of interventions. METHODS: We performed a pooled sequencing (pool-seq) of the genomes of 100 Plasmodium falciparum isolates from Nigeria. We explored allele-frequency based neutrality test (Tajima's D) and integrated haplotype score (iHS) to identify genes under selection. RESULTS: Fourteen shared iHS regions that had at least 2 SNPs with a score > 2.5 were identified. These regions code for genes that were likely to have been under strong directional selection. Two of these genes were the chloroquine resistance transporter (CRT) on chromosome 7 and the multidrug resistance 1 (MDR1) on chromosome 5. There was a weak signature of selection in the dihydrofolate reductase (DHFR) gene on chromosome 4 and MDR5 genes on chromosome 13, with only 2 and 3 SNPs respectively identified within the iHS window. We observed strong selection pressure attributable to continued chloroquine and sulfadoxine-pyrimethamine use despite their official proscription for the treatment of uncomplicated malaria. There was also a major selective sweep on chromosome 6 which had 32 SNPs within the shared iHS region. Tajima's D of circumsporozoite protein (CSP), erythrocyte-binding antigen (EBA-175), merozoite surface proteins - MSP3 and MSP7, merozoite surface protein duffy binding-like (MSPDBL2) and serine repeat antigen (SERA-5) were 1.38, 1.29, 0.73, 0.84 and 0.21, respectively. CONCLUSION: We have demonstrated the use of pool-seq to understand genomic patterns of selection and variability in P. falciparum from Nigeria, which bears the highest burden of infections. This investigation identified known genomic signatures of selection from drug pressure and host immunity. This is evidence that P. falciparum populations explore common adaptive strategies that can be targeted for the development of new interventions.

Study on the susceptibility of Sahel goats to experimental Trypanosoma vivax infection.

Sahel goats, also known as Borno whites are found in the northern semi-arid, tsetse free Sahel region of Nigeria. They are transported alongside cattle from this zone to all other zones in the country, including the tsetse-infested zones, for commercial purposes and are kept for some time in these tsetse-infested zones until they are sold. This study therefore assessed the susceptibility of this breed of goats to trypanosome infection and its response to treatment with Berenil. Six bucks were inoculated intravenously with Trypanosoma vivax through the jugular vein while two served as uninfected control. The mean pre-patent period was 4.5 days and increasing parasitaemia followed the establishment of infection. Onset of parasitaemia was associated with increase in rectal temperature in all the infected goats and the temperature peak coincided with the only parasitaemic peak second week post-infection. The infected goats were treated with Berenil (Hoechst, Germany) 3.5mg/kg body weight at 4 weeks post-infection. The packed cell volume (PCV) continued to fall from a mean 30.73+/-0.01% pre-infection to a mean 13.21+/-0.18% at 1 week post-treatment. Deaths were recorded for 4 of the infected goats 1 week post-treatment while the remaining two died 2 weeks post-treatment, not responding to treatment.

Assessment of competence of participants before and after 7-day intensive malaria microscopy training courses in Nigeria.

Background: The accuracy of malaria diagnosis by microscopy has been a challenge in health facilities in Nigeria due to poor competence of microscopists and inability to report on malaria species other than Plasmodium falciparum. Short microscopy courses were conducted to improve the skills of laboratory personnel to perform malaria microscopy in public health facilities in Nigeria. Materials and Methods: Seven-day malaria microscopy courses were conducted annually between 2011 and 2013 for microscopists in public health facilities. The training courses contained theoretical and practical sessions. Impact of the training was evaluated by practical and theoretical pre- and post-training assessments on malaria slide reading, parasite enumeration and basic malariology. Results: The 102 participants who completed the training consisted of medical laboratory scientists (62; 60.8%), medical laboratory technicians (24; 23.5%) and other healthcare workers (16; 15.7%). The knowledge of basic malariology (theory) at pre- and post-tests were 34% (95% CI 31.7-36.3%) and 74.9% (95% CI 71.8-78.0%), respectively (P<0.001). The mean slide reading detection, species and counting agreements in pre-training assessment were 48.9%, 27.9% and 0%, respectively, and in post-training 56.8%, 39.2% and 25%, respectively. The mean species agreements in picture test pre- and post-training were 21.9% and 55.1%, respectively. There were significant differences (P<0.05) in the median pre-test scores in picture tests and basic malariology of the three categories of participants but not in malaria slide reading and parasite counting tests. However, post-training, a significant difference in test scores of the three categories of participants was recorded only for basic malariology (P=0.0003). Conclusions: The 7-day malaria microscopy courses significantly increased the knowledge and microscopy skills of the trainees and were sufficient to bridge the significant difference in baseline microscopy skills of the different categories of trainees that participated in the training courses.

Genetic diversity and complexity of Plasmodium falciparum infections in Lagos, Nigeria.

OBJECTIVE: To analyse the genetic diversity of Plasmodium falciparum (P. falciparum) using msp-1 and msp-2 as antigenic markers. METHODS: Parasite DNA was extracted from 100 blood samples collected from P. falciparum-positive patients confirmed by microscopy, and followed by PCR-genotyping targeting the msp-1 (block2) and msp-2 (block 3) allelic families. RESULTS: All the families of msp-1 (K1, MAD20 and R033) and msp-2 (FC27 and 3D7) locus were observed. Results revealed that K1 (60/100) was the most predominant genotype of msp-1 allelic family followed by the genotypes of MAD20 (50/100) and R033 (45/100). In the msp-2 locus, FC27 genotype (62/100) showed higher frequency than 3D7 genotype (55/100). The allelic families were detected either alone or in combination with other families. However, no R033/MAD20 combination was observed. Multiplicity of infection (MOI) with msp-1 was higher in the locality of Ikorodu (1.50) than in Lekki (1.39). However, MOI with msp-2 was lower in the locality of Ikorodu (1.14) than in Lekki (1.76). There was no significant difference in the mean MOI between the two study areas (P=0.427). CONCLUSIONS: The observation of limited diversity of malaria parasites may imply that the use of antigenic markers as genotyping tools for distinguishing recrudescence and re-infections with P. falciparum during drug trials is subjective.

Persistence of markers of chloroquine resistance among P. falciparum isolates recovered from two Nigerian communities.

Background: A recovery in chloroquine efficacy following a period of cessation has raised the possibility of its reintroduction for malaria chemotherapy. We investigated the prevalence of the major markers of chloroquine resistance years after the withdrawal of the drug in Nigeria. Materials and Methods: Finger prick blood samples were collected from participants presenting with symptoms of malaria in two selected health centres each representing Lekki and Ijede communities of Lagos, Nigeria. Thick and thin blood smears were prepared for microscopy and dry blood spots made from malaria-positive participants for parasite DNA extraction. The detection of mutations in the Plasmodium falciparum chloroquine resistance transporter (pfcrt) and P. falciparum multidrug resistance (pfmdr1) genes was performed by nested polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP). Results: Of the 1527 blood samples that were confirmed by PCR to be P. falciparum positive, 412 and 344 were typed for the molecular detection of pfcrt and pfmdr1 gene mutations, respectively. The mutant alleles of pfcrt were present among 290 (70%) parasite carriers while the pfmdr1 mutant allele was found in 117 (34%) of the total population. There were higher distributions of the mutant alleles for the two loci in Ijede than in Lekki. The observed frequencies of pfcrt mutant alleles in the two parasite populations were in agreement with the expected frequencies predicted by Hardy-Weinberg. In comparing data with studies conducted between 2000 and 2002 in Ijede, we observed an increase in the prevalence of mutant type pfcrt against a marginal decline in the pfmdr1 mutant type. Conclusion: The high frequencies of pfcrt mutation are suggestive of a persistent drug pressure and continuing inefficacy of chloroquine as an antimalarial drug.

Malariometric survey of ibeshe community in ikorodu, lagos state: dry season.

Malariometric surveys generate data on malaria epidemiology and dynamics of transmission necessary for planning and monitoring of control activities. This study determined the prevalence of malaria and the knowledge, attitude, and practice (KAP) towards malaria infection in Ibeshe, a coastal community. The study took place during the dry season in 10 villages of Ibeshe. All the participants were screened for malaria. A semistructured questionnaire was used to capture sociodemographic data and KAP towards malaria. A total of 1489 participants with a mean age of 26.7 ± 20.0 years took part in the study. Malaria prevalence was 14.7% (95% CI 13.0-16.6%) with geometric mean density of 285 parasites/µL. Over 97% of participants were asymptomatic. Only 40 (2.7%) of the participants were febrile, while 227 (18.1%) were anemic. Almost all the participants (95.8%) identified mosquito bite as a cause of malaria, although multiple agents were associated with the cause of malaria. The commonest symptoms associated with malaria were hot body (89.9%) and headache (84.9%). Window nets (77.0%) were preferred to LLIN (29.6%). Malaria is mesoendemic in Ibeshe during the dry season. The participants had good knowledge of symptoms of malaria; however, there were a lot of misconceptions on the cause of malaria.