Genomic Characterization of Multidrug-Resistant Pathogenic Enteric Bacteria from Healthy Children in Osun State, Nigeria.

Antimicrobial resistance (AMR) is responsible for the spread and persistence of bacterial infections. Surveillance of AMR in healthy individuals is usually not considered, though these individuals serve as reservoirs for continuous disease transmission. Therefore, it is essential to conduct epidemiological surveillance of AMR in healthy individuals to fully understand the dynamics of AMR transmission in Nigeria. Thirteen multidrug-resistant Citrobacter spp., Enterobacter spp., Klebsiella pneumoniae, and Escherichia coli isolated from stool samples of healthy children were subjected to whole genome sequencing (WGS) using Illumina and Oxford nanopore sequencing platforms. A bioinformatics analysis revealed antimicrobial resistance genes such as the pmrB_Y358N gene responsible for colistin resistance detected in E. coli ST219, virulence genes such as senB, and ybtP&Q, and plasmids in the isolates sequenced. All isolates harbored more than three plasmid replicons of either the Col and/or Inc type. Plasmid reconstruction revealed an integrated tetA gene, a toxin production caa gene in two E. coli isolates, and a cusC gene in K. quasivariicola ST3879, which induces neonatal meningitis. The global spread of AMR pathogenic enteric bacteria is of concern, and surveillance should be extended to healthy individuals, especially children. WGS for epidemiological surveillance will improve the detection of AMR pathogens for management and control.

Seroprevalence of Hepatitis B virus and human immunodeficiency virus co-infection in pregnant women from Osun State, Nigeria.

INTRODUCTION: Hepatitis B virus and human immunodeficiency virus (HBV/HIV) co-infection is a global health concern due to its significant impact on morbidity and mortality. Reports of HBV/HIV co-infections are increasing in Nigeria, but information on the disease burden in pregnant women and its implications on the fetus is scarce. This study aimed to determine the prevalence of HBV/HIV co-infection in pregnant women. In addition, the study identified the risk factors for the disease in pregnant women attending antenatal clinics in Osun State, Nigeria. METHODOLOGY: We collected plasma samples from 303 consenting pregnant women and used enzyme-linked immunosorbent assay (ELISA) to test for HBV (HBsAg) and HIV I/II antigens. We obtained demographic and risk factor data on HBV and HIV transmission using a structured questionnaire. RESULTS: Our analysis revealed a prevalence of 3.96% for HBV/HIV co-infection in pregnant women. Bivariate analysis indicated a history of blood transfusion, oral or anal sex, and multiple sexual partners may be associated with an increased likelihood of HBV/HIV co-infection in pregnant women. After adjusting for other variables in multivariate analysis, none of these risk factors were significant at the 5% level. In contrast, formal education was a potential preventive factor in this population. CONCLUSIONS: Our study provides valuable information on the disease burden of HBV/HIV co-infection in pregnant women in Osun State, Nigeria, highlighting the importance of routine screening for HBV and HIV during antenatal care and emphasizing the importance of implementing preventive measures to reduce the morbidity and mortality associated with HBV/HIV co-infection.

Detection of Immune Escape and Basal Core Promoter/Precore Gene Mutations in Hepatitis B Virus Isolated from Asymptomatic Hospital Attendees in Two Southwestern States in Nigeria.

Several mutations in the surface (S), basal core promoter (BCP), and precore (PC) genes of the hepatitis B virus have been linked to inaccurate diagnosis and the development of immune escape mutants (IEMs) of the infection, which can lead to chronic infection. Understanding the prevalence and spread of these mutations is critical in the global effort to eliminate HBV. Blood samples were collected from 410 people in Osun and Ekiti states, southwest Nigeria, between 2019 and 2021. Participants were drawn from a group of asymptomatic people who were either blood donors, outpatients, or antenatal patients with no record of HBV infection at the medical outpatients' unit of the hospital. DNA was extracted from plasma using a Qiagen DNEasy kit, followed by nested PCR targeting HBV S and BCP/PC genes. The Sanger sequencing method was used to sequence the positive PCR amplicons, which were further analyzed for IEMs, BCP, and PC mutations. HBV-DNA was detected in 12.4% (51/410) of individuals. After DNA amplification and purification, 47.1% (24) of the S gene and 76.5% (39) of the BCP/PC gene amplicons were successfully sequenced. Phylogenetic analysis showed that all the HBV sequences obtained in this study were classified as HBV genotype E. Mutational analysis of the major hydrophilic region (MHR) and a-determinant domain of S gene sequences revealed the presence of three immune escape mutations: two samples harbored a T116N substitution, six samples had heterogenous D144A/N/S/H substitution, and one sample had a G145E substitution, respectively. The BCP/PC region analysis revealed a preponderance of major BCP mutants, with the prevalence of BCP double substitutions ranging from 38.5% (A1762T) to 43.6% (G1764A). Previously reported classical PC mutant variants were observed in high proportion, including G1896A (33.3%) and G1899A (12.8%) mutations. This study confirms the strong presence of HBV genotype E in Nigeria, the ongoing circulation of HBV IEMs, and a high prevalence of BCP/PC mutants in the cohorts. This has implications for diagnosis and vaccine efficacy for efficient management and control of HBV in the country.

Metagenomic surveillance uncovers diverse and novel viral taxa in febrile patients from Nigeria.

Effective infectious disease surveillance in high-risk regions is critical for clinical care and pandemic preemption; however, few clinical diagnostics are available for the wide range of potential human pathogens. Here, we conduct unbiased metagenomic sequencing of 593 samples from febrile Nigerian patients collected in three settings: i) population-level surveillance of individuals presenting with symptoms consistent with Lassa Fever (LF); ii) real-time investigations of outbreaks with suspected infectious etiologies; and iii) undiagnosed clinically challenging cases. We identify 13 distinct viruses, including the second and third documented cases of human blood-associated dicistrovirus, and a highly divergent, unclassified dicistrovirus that we name human blood-associated dicistrovirus 2. We show that pegivirus C is a common co-infection in individuals with LF and is associated with lower Lassa viral loads and favorable outcomes. We help uncover the causes of three outbreaks as yellow fever virus, monkeypox virus, and a noninfectious cause, the latter ultimately determined to be pesticide poisoning. We demonstrate that a local, Nigerian-driven metagenomics response to complex public health scenarios generates accurate, real-time differential diagnoses, yielding insights that inform policy.

Genomic Characterisation of Multidrug-Resistant Pathogenic Enteric Bacteria from healthy children in Osun State, Nigeria.

Antimicrobial resistance (AMR) has been established to be a significant driver for the persistence and spread of bacterial infections. It is, therefore, essential to conduct epidemiological surveillance of AMR in healthy individuals to understand the actual dynamics of AMR in Nigeria. Multi-drug resistant Klebsiella quasivariicola (n=1), Enterobacter hormaechei (n=1), and Escherichia coli (n=3) from stool samples of healthy children were subjected to whole genome sequencing using Illumina Nextseq1000/2000 and Oxford nanopore. Bioinformatics analysis reveals antimicrobial resistance, virulence genes, and plasmids. This pathogenic enteric bacteria harbored more than three plasmid replicons of either Col and/or Inc type associated with outbreaks and AMR resistant gene pmrB responsible for colistin resistance. Plasmid reconstruction revealed an integrated tetA gene responsible for tetracycline resistance, and caa gene responsible for toxin production in two of the E.coli isolates, and a cusC gene known to induce neonatal meningitis in the K. quasivariicola ST3879. The global spread of MDR pathogenic enteric bacteria is a worrying phenomenon, and close surveillance of healthy individuals, especially children, is strongly recommended to prevent the continuous spread and achieve the elimination and eradication of these infections. Molecular epidemiological surveillance using whole genome sequencing (WGS) will improve the detection of MDR pathogens in Nigeria.

Genomic characterization of Alphacoronavirus from Mops condylurus bats in Nigeria.

Coronaviruses (CoVs) are responsible for sporadic, epidemic and pandemic respiratory diseases worldwide. Bats have been identified as the reservoir for CoVs. To increase the number of complete coronavirus genomes in Africa and to comprehend the molecular epidemiology of bat Alphacoronaviruses (AlphaCoVs), we used deep metagenomics shotgun sequencing to obtain three (3) near-complete genomes of AlphaCoVs from Mops condylurus (Angolan free-tailed) bat in Nigeria. Phylogenetic and pairwise identity analysis of open reading frame 1ab (ORF1ab), spike (S), envelope (E), membrane (M) and nucleocapsid (N) genes of AlphaCoV in this study to previously described AlphaCoVs subgenera showed that the Nigerian AlphaCoVs may be members of potentially unique AlphaCoV subgenera circulating exclusively in bats in the Molossidae bat family. Recombination events were detected, suggesting the evolution of AlphaCoVs within the Molossidae family. The pairwise identity of the S gene in this study and previously published S gene sequences of other AlphaCoVs indicate that the Nigerian strains may have a genetically unique spike protein that is distantly related to other AlphaCoVs. Variations involving non-polar to polar amino acid substitution in both the Heptad Repeat (HR) regions 1 and 2 were observed. Further monitoring of bats to understand the host receptor use requirements of CoVs and interspecies CoV transmission in Africa is necessary to identify and prevent the potential danger that bat CoVs pose to public health.

Patterns of hepatitis b virus immune escape and pol/rt mutations across clinical cohorts of patients with genotypes a, e and occult hepatitis b infection in Nigeria: A multi-centre study.

Hepatitis B virus (HBV) immune escape and Pol/RT mutations account for HBV immunoprophylactic, therapeutic, and diagnostic failure globally. Little is known about circulating HBV immune escape and Pol/RT mutants in Nigeria. This study focused on narrowing the knowledge gap of the pattern and prevalence of the HBV mutants across clinical cohorts of infected patients in southwestern Nigeria. Ninety-five enrollees were purposively recruited across clinical cohorts of HBV-infected patients with HBsAg or anti-HBc positive serological outcome and occult HBV infection. Total DNA was extracted from patients' sera. HBV S and Pol gene-specific nested PCR amplification was carried out. The amplicons were further sequenced for serotypic, genotypic, phylogenetic, and mutational analysis. HBV S and Pol genes were amplified in 60 (63.2%) and 19 (20%) of HBV isolates, respectively. All the sixty HBV S gene and 14 of 19 Pol gene sequences were exploitable. The ayw4 serotype was predominant (95%) while ayw1 serotype was identified in 5% of isolates. Genotype E predominates in 95% of sequences, while genotype A, sub-genotype A3 was observed in 5%. Prevalence of HBV IEMs in the "a" determinant region was 29%. Commonest HBV IEM was S113T followed by G145A and D144E. The Pol/RT mutations rtV214A and rtI163V among others were identified in this study. This study provided data on the occurrence of existing and new HBV IEMs and Pol gene mutations in Nigeria.

The Prevalence of Undiagnosed Salmonella enterica Serovar Typhi in Healthy School-Aged Children in Osun State, Nigeria.

Typhoid fever remains a significant public health concern due to cases of mis-/overdiagnosis. Asymptomatic carriers play a role in the transmission and persistence of typhoid fever, especially among children, where limited data exist in Nigeria and other endemic countries. We aim to elucidate the burden of typhoid fever among healthy school-aged children using the best surveillance tool(s). In a semi-urban/urban state (Osun), 120 healthy school-aged children under 15 years were enrolled. Whole blood and fecal samples were obtained from consenting children. ELISA targeting the antigen lipopolysaccharide (LPS) and anti-LPS antibodies of Salmonella Typhi, culture, polymerase chain reaction (PCR), and next-generation sequencing (NGS) were used to analyze the samples. At least one of the immunological markers was detected in 65.8% of children, with 40.8%, 37.5%, and 39% of children testing positive for IgM, IgG, and antigen, respectively. Culture, PCR, and NGS assays did not detect the presence of Salmonella Typhi in the isolates. This study demonstrates a high seroprevalence of Salmonella Typhi in these healthy children but no carriage, indicating the inability to sustain transmission. We also demonstrate that using a single technique is insufficient for typhoid fever surveillance in healthy children living in endemic areas.

Atypical serologic profiles of hepatitis B virus infection across clinical cohorts of patients in Southwestern Nigeria.

Hepatitis B virus (HBV) infection follows a natural course of events predicted by a dynamic interaction between viral antigen and the host immune system, which forms the basis for HBV serological diagnosis. These interactions may deviate from the typical serologic patterns. This study investigates the types of atypical HBV serologic profiles (AHBSP) across clinical cohorts of patients with HBV infection in southwestern Nigeria. This is a cross-sectional, hospital-based, multi-centered study. Patients' sera were analyzed for HBsAg, anti-HBs, HBeAg, anti-HBe, anti-HBc IgM, and anti-HBc IgG by ELISA from 279 study participants attending selected gastroenterology clinics between August 2019 and December 2020. The prevalence of atypical HBV serologic profiles was 27% (n = 76). The mean age of patients was 35.7 ± 11.2 years. The gender distribution involved 183 females (65.6%) and 96 males (34.4%). Across clinical cohorts of patients with atypical serologic profiles, HBeAg Negative, anti-HBe positive with detectable HBV DNA had the highest prevalence of 21% followed by isolated anti-HBc antibody positive, HBsAg negative and detectable HBV DNA, 5%. The atypical serologic profiles, HBeAg positive, HBsAg negative with detectable HBV DNA and concurrent anti-HBs with HBsAg, had the lowest prevalence, 0.4%, respectively. This study identified the considerable presence of atypical HBV serologic profiles across clinical cohorts of HBV infection in southwestern Nigeria.

Seroprevalence, co-infection and risk of transmission of Hepatitis B and D virus among hospital attendees in two South-western states in Nigeria.

Infection with both Hepatitis B (HBV) and D (HDV) virus causes more severe liver damage than HBV alone. Superinfections among chronic HBV infected cohorts often lead to HDV persistence with rapid progression to cirrhosis, necessitating continuous surveillance to determine their prevalence and relative contribution to liver pathology. A cross-sectional study among hospital outpatients in Ekiti and Osunstates was conducted using random sampling technique. Blood samples were collected from 410 participants and tested for HBV serological markers. All samples positive for HBsAg samples were tested for Hepatitis D virus antigen (HDAg), serum anti-HDV IgM, and serum anti-HDV IgG using enzyme-linked immunosorbent assay kits. The prevalence of HBV infection among the 410 samples was 12.4% (CI 9.5-15.9). Past HBV exposure was detected in 120 (29.2%), while 147(35.8%) were susceptible to HBV infection. Among the HBsAg positive individuals, 9.8% were hepatitis D antigen (HDAg) positive, while 3.9% and 1.9% were positive for IgG anti-HDV and IgM anti-HDV, respectively. Risk factors associated with HBV infections in this study were multiple sexual partners and sharing of sharp objects. Our investigation has verified the endemicity of HBV in Nigeria and revealed that HBV- HDV co-infection is highly prevalent in south-west Nigeria.

Detection of Alpha- and Betacoronaviruses in Frugivorous and Insectivorous Bats in Nigeria.

The rise of bat-associated zoonotic viruses necessitates a close monitoring of their natural hosts. Since the detection of severe acute respiratory syndrome coronavirus (SARS-CoV), it is evident that bats are vital reservoirs of coronaviruses (CoVs). In this study, we investigated the presence of CoVs in multiple bat species in Nigeria to identify viruses in bats at high-risk human contact interfaces. Four hundred and nine bats comprising four bat species close to human habitats were individually sampled from five states in Nigeria between 2019 and 2021. Coronavirus detection was done using broadly reactive consensus PCR primers targeting the RNA-dependent RNA polymerase (RdRp) gene of CoVs. Coronavirus RNA was detected in 39 samples (9.5%, CI 95%: [7.0, 12.8]), of which 29 were successfully sequenced. The identified CoVs in Nigerian bats were from the unclassified African alphacoronavirus lineage and betacoronavirus lineage D (Nobecovirus), with one sample from Hipposideros ruber coinfected with alphacoronavirus and betacoronavirus. Different bat species roosting in similar or other places had CoVs from the same genetic lineage. The phylogenetic and evolutionary dynamics data indicated a high CoV diversity in Nigeria, while host switching may have contributed to CoV evolution. Robust sentinel surveillance is recommended to enhance our knowledge of emerging and re-emerging coronaviruses.

Metagenomic sequencing characterizes a wide diversity of viruses in field mosquito samples in Nigeria.

Mosquito vectors are a tremendous public health threat. One in six diseases worldwide is vector-borne transmitted mainly by mosquitoes. In the last couple of years, there have been active Yellow fever virus (YFV) outbreaks in many settings in Nigeria, and nationwide, entomological surveillance has been a significant effort geared towards understanding these outbreaks. In this study, we used a metagenomic sequencing approach to characterize viruses present in vector samples collected during various outbreaks of Yellow fever (YF) in Nigeria between 2017 and 2020. Mosquito samples were grouped into pools of 1 to 50 mosquitoes, each based on species, sex and location. Twenty-five pools of Aedes spp and one pool of Anopheles spp collected from nine states were sequenced and metagenomic analysis was carried out. We identified a wide diversity of viruses belonging to various families in this sample set. Seven different viruses detected included: Fako virus, Phasi Charoen-like virus, Verdadero virus, Chaq like-virus, Aedes aegypti totivirus, cell fusing agent virus and Tesano Aedes virus. Although there are no reports of these viruses being pathogenic, they are an understudied group in the same families and closely related to known pathogenic arboviruses. Our study highlights the power of next generation sequencing in identifying Insect specific viruses (ISVs), and provide insight into mosquito vectors virome in Nigeria.

The evolving SARS-CoV-2 epidemic in Africa: Insights from rapidly expanding genomic surveillance.

Investment in Africa over the past year with regards to SARS-CoV-2 genotyping has led to a massive increase in the number of sequences, exceeding 100,000 genomes generated to track the pandemic on the continent. Our results show an increase in the number of African countries able to sequence within their own borders, coupled with a decrease in sequencing turnaround time. Findings from this genomic surveillance underscores the heterogeneous nature of the pandemic but we observe repeated dissemination of SARS-CoV-2 variants within the continent. Sustained investment for genomic surveillance in Africa is needed as the virus continues to evolve, particularly in the low vaccination landscape. These investments are very crucial for preparedness and response for future pathogen outbreaks. One-Sentence SummaryExpanding Africa SARS-CoV-2 sequencing capacity in a fast evolving pandemic.

Molecular profiling of the artemisinin resistance Kelch 13 gene in Plasmodium falciparum from Nigeria.

Accurate assessment and monitoring of the Plasmodium falciparum Kelch 13 (pfk13) gene associated with artemisinin resistance is critical to understand the emergence and spread of drug-resistant parasites in malaria-endemic regions. In this study, we evaluated the genomic profile of the pfk13 gene associated with artemisinin resistance in P. falciparum in Nigerian children by targeted sequencing of the pfk13 gene. Genomic DNA was extracted from 332 dried blood (DBS) spot filter paper samples from three Nigerian States. The pfk13 gene was amplified by nested polymerase chain reaction (PCR), and amplicons were sequenced to detect known and novel polymorphisms across the gene. Consensus sequences of samples were mapped to the reference gene sequence obtained from the National Center for Biotechnology Information (NCBI). Out of the 13 single nucleotide polymorphisms (SNPs) detected in the pfk13 gene, five (F451L, N664I, V487E, V692G and Q661H) have not been reported in other endemic countries to the best of our knowledge. Three of these SNPs (V692G, N664I and Q661H) and a non-novel SNP, C469C, were consistent with late parasitological failure (LPF) in two States (Enugu and Plateau States). There was no validated mutation associated with artemisinin resistance in this study. However, a correlation of our study with in vivo and in vitro phenotypes is needed to establish the functional role of detected mutations as markers of artemisinin resistance in Nigeria. This baseline information will be essential in tracking and monitoring P. falciparum resistance to artemisinin in Nigeria.

VGEA: an RNA viral assembly toolkit.

Next generation sequencing (NGS)-based studies have vastly increased our understanding of viral diversity. Viral sequence data obtained from NGS experiments are a rich source of information, these data can be used to study their epidemiology, evolution, transmission patterns, and can also inform drug and vaccine design. Viral genomes, however, represent a great challenge to bioinformatics due to their high mutation rate and forming quasispecies in the same infected host, bringing about the need to implement advanced bioinformatics tools to assemble consensus genomes well-representative of the viral population circulating in individual patients. Many tools have been developed to preprocess sequencing reads, carry-out de novo or reference-assisted assembly of viral genomes and assess the quality of the genomes obtained. Most of these tools however exist as standalone workflows and usually require huge computational resources. Here we present (Viral Genomes Easily Analyzed), a Snakemake workflow for analyzing RNA viral genomes. VGEA enables users to map sequencing reads to the human genome to remove human contaminants, split bam files into forward and reverse reads, carry out de novo assembly of forward and reverse reads to generate contigs, pre-process reads for quality and contamination, map reads to a reference tailored to the sample using corrected contigs supplemented by the user's choice of reference sequences and evaluate/compare genome assemblies. We designed a project with the aim of creating a flexible, easy-to-use and all-in-one pipeline from existing/stand-alone bioinformatics tools for viral genome analysis that can be deployed on a personal computer. VGEA was built on the Snakemake workflow management system and utilizes existing tools for each step: fastp (Chen et al., 2018) for read trimming and read-level quality control, BWA (Li & Durbin, 2009) for mapping sequencing reads to the human reference genome, SAMtools (Li et al., 2009) for extracting unmapped reads and also for splitting bam files into fastq files, IVA (Hunt et al., 2015) for de novo assembly to generate contigs, shiver (Wymant et al., 2018) to pre-process reads for quality and contamination, then map to a reference tailored to the sample using corrected contigs supplemented with the user's choice of existing reference sequences, SeqKit (Shen et al., 2016) for cleaning shiver assembly for QUAST, QUAST (Gurevich et al., 2013) to evaluate/assess the quality of genome assemblies and MultiQC (Ewels et al., 2016) for aggregation of the results from fastp, BWA and QUAST. Our pipeline was successfully tested and validated with SARS-CoV-2 (n = 20), HIV-1 (n = 20) and Lassa Virus (n = 20) datasets all of which have been made publicly available. VGEA is freely available on GitHub at: https://github.com/pauloluniyi/VGEA under the GNU General Public License.

The Origins and Future of Sentinel: An Early-Warning System for Pandemic Preemption and Response.

While investigating a signal of adaptive evolution in humans at the gene LARGE, we encountered an intriguing finding by Dr. Stefan Kunz that the gene plays a critical role in Lassa virus binding and entry. This led us to pursue field work to test our hypothesis that natural selection acting on LARGE-detected in the Yoruba population of Nigeria-conferred resistance to Lassa Fever in some West African populations. As we delved further, we conjectured that the "emerging" nature of recently discovered diseases like Lassa fever is related to a newfound capacity for detection, rather than a novel viral presence, and that humans have in fact been exposed to the viruses that cause such diseases for much longer than previously suspected. Dr. Stefan Kunz's critical efforts not only laid the groundwork for this discovery, but also inspired and catalyzed a series of events that birthed Sentinel, an ambitious and large-scale pandemic prevention effort in West Africa. Sentinel aims to detect and characterize deadly pathogens before they spread across the globe, through implementation of its three fundamental pillars: Detect, Connect, and Empower. More specifically, Sentinel is designed to detect known and novel infections rapidly, connect and share information in real time to identify emerging threats, and empower the public health community to improve pandemic preparedness and response anywhere in the world. We are proud to dedicate this work to Stefan Kunz, and eagerly invite new collaborators, experts, and others to join us in our efforts.

Molecular surveillance of shiga toxigenic Escherichia coli in selected beef abattoirs in Osun State Nigeria.

Shiga toxigenic strains of E. coli (STEC) known to be etiological agents for diarrhea were screened for their incidence/occurrence in selected abattoirs sources in Osogbo metropolis of Osun State, Nigeria using a randomized block design. Samples were plated directly on selective and differential media and E. coli isolates. Multiplex PCR analysis was used to screen for the presence of specific virulence factors. These were confirmed serologically as non-O157 STEC using latex agglutination serotyping kit. Sequence analysis of PCR products was performed on a representative isolate showing the highest combination of virulence genes using the 16S gene for identification purposes only. Results showed that the average cfu/cm2 was significantly lower in the samples collected at Sekona-2 slaughter slab compared with those collected at Al-maleek batch abattoir and Sekona-1 slaughter slab in ascending order at P = 0.03. Moreover, the average cfu/cm2 E. coli in samples collected from butchering knife was significantly lower when compared with that of the workers' hand (P = 0.047) and slaughtering floor (P = 0.047) but not with the slaughter table (P = 0.98) and effluent water from the abattoir house (P = 0.39). These data suggest that the abattoir type may not be as important in the prevalence and spread of STEC as the hygiene practices of the workers. Sequence analysis of a representative isolate showed 100% coverage and 96.46% percentage identity with Escherichia coli O113:H21 (GenBank Accession number: CP031892.1) strain from Canada. This sequence was subsequently submitted to GenBank with accession number MW463885. From evolutionary analyses, the strain from Nigeria, sequenced in this study, is evolutionarily distant when compared with the publicly available sequences from Nigeria. Although no case of E. coli O157 was found within the study area, percent occurrence of non-O157 STEC as high as 46.3% at some of the sampled sites is worrisome and requires regulatory interventions in ensuring hygienic practices at the abattoirs within the study area.

Genetic diversity and population structure of Plasmodium falciparum in Nigeria: insights from microsatellite loci analysis.

BACKGROUND: Malaria remains a public health burden especially in Nigeria. To develop new malaria control and elimination strategies or refine existing ones, understanding parasite population diversity and transmission patterns is crucial. METHODS: In this study, characterization of the parasite diversity and structure of Plasmodium falciparum isolates from 633 dried blood spot samples in Nigeria was carried out using 12 microsatellite loci of P. falciparum. These microsatellite loci were amplified via semi-nested polymerase chain reaction (PCR) and fragments were analysed using population genetic tools. RESULTS: Estimates of parasite genetic diversity, such as mean number of different alleles (13.52), effective alleles (7.13), allelic richness (11.15) and expected heterozygosity (0.804), were high. Overall linkage disequilibrium was weak (0.006, P < 0.001). Parasite population structure was low (Fst: 0.008-0.105, AMOVA: 0.039). CONCLUSION: The high level of parasite genetic diversity and low population structuring in this study suggests that parasite populations circulating in Nigeria are homogenous. However, higher resolution methods, such as the 24 SNP barcode and whole genome sequencing, may capture more specific parasite genetic signatures circulating in the country. The results obtained can be used as a baseline for parasite genetic diversity and structure, aiding in the formulation of appropriate therapeutic and control strategies in Nigeria.

Polymorphisms in Plasmodium falciparum dihydropteroate synthetase and dihydrofolate reductase genes in Nigerian children with uncomplicated malaria using high-resolution melting technique.

In 2005, the Nigerian Federal Ministry of Health revised the treatment policy for uncomplicated malaria with the introduction of artemisinin-based combination therapies (ACTs). This policy change discouraged the use of Sulphadoxine-pyrimethamine (SP) as the second-line treatment of uncomplicated falciparum malaria. However, SP is used as an intermittent preventive treatment of malaria in pregnancy (IPTp) and seasonal malaria chemoprevention (SMC) in children aged 3-59 months. There have been increasing reports of SP resistance especially in the non-pregnant population in Nigeria, thus, the need to continually monitor the efficacy of SP as IPTp and SMC by estimating polymorphisms in dihydropteroate synthetase (dhps) and dihydrofolate reductase (dhfr) genes associated with SP resistance. The high resolution-melting (HRM) assay was used to investigate polymorphisms in codons 51, 59, 108 and 164 of the dhfr gene and codons 437, 540, 581 and 613 of the dhps gene. DNA was extracted from 271 dried bloodspot filter paper samples obtained from children (< 5 years old) with uncomplicated malaria. The dhfr triple mutant I51R59N108, dhps double mutant G437G581 and quadruple dhfr I51R59N108 + dhps G437 mutant haplotypes were observed in 80.8%, 13.7% and 52.8% parasites, respectively. Although the quintuple dhfr I51R59N108 + dhps G437E540 and sextuple dhfr I51R59N108 + dhps G437E540G581 mutant haplotypes linked with in-vivo and in-vitro SP resistance were not detected, constant surveillance of these haplotypes should be done in the country to detect any change in prevalence.

Polymorphisms in Plasmodium falciparum chloroquine resistance transporter (Pfcrt) and multidrug-resistant gene 1 (Pfmdr-1) in Nigerian children 10 years post-adoption of artemisinin-based combination treatments.

The emergence and spread of Plasmodium falciparum parasites resistant to artemisinin derivatives and their partners in southeastern Asia threatens malaria control and elimination efforts, and heightens the need for an alternative therapy. We have explored the distribution of P. falciparum chloroquine resistance transporter (Pfcrt) and multidrug-resistant gene 1 (Pfmdr-1) haplotypes 10 years following adoption of artemisinin-based combination therapies in a bid to investigate the possible re-emergence of Chloroquine-sensitive parasites in Nigeria, and investigated the effect of these P. falciparum haplotypes on treatment outcomes of patients treated with artemisinin-based combination therapies. A total of 271 children aged <5 years with uncomplicated falciparum malaria were included in this study. Polymorphisms on codons 72-76 of the Pfcrt gene and codon 86 and 184 of Pfmdr-1 were determined using the high resolution melting assay. Of 240 (88.6%) samples successfully genotyped with HRM for Pfcrt, wildtype C72M74N75K76 (42.9%) and mutant C72I74E75T76 (53.8%) were observed. Also, wildtype N86Y184 (62.9%) and mutant N86F184 (21.1%), Y86Y184 (6.4%), and Y86F184 (0.4%) haplotypes of Pfmdr-1 were observed. Measures of responsiveness to ACTs were similar in children infected with P. falciparum crt haplotypes (C72I74E75T76 and C72M74N75K76) and major mdr-1 haplotypes (N86Y184, N86F184 and Y86Y184). Despite a 10 year gap since the malaria treatment policy changed to ACTs, over 50% of the P. falciparum parasites investigated in this study harboured the Chloroquine-resistant C72I74E75T76 haplotype, however this did not compromise the efficacy of artemisinin-based combination therapies. Should complete artemisinin resistance emerge from or spread to Nigeria, chloroquine might not be a good alternative therapy.