Clinical Sequencing Uncovers Origins and Evolution of Lassa Virus.

The 2013-2015 West African epidemic of Ebola virus disease (EVD) reminds us of how little is known about biosafety level 4 viruses. Like Ebola virus, Lassa virus (LASV) can cause hemorrhagic fever with high case fatality rates. We generated a genomic catalog of almost 200 LASV sequences from clinical and rodent reservoir samples. We show that whereas the 2013-2015 EVD epidemic is fueled by human-to-human transmissions, LASV infections mainly result from reservoir-to-human infections. We elucidated the spread of LASV across West Africa and show that this migration was accompanied by changes in LASV genome abundance, fatality rates, codon adaptation, and translational efficiency. By investigating intrahost evolution, we found that mutations accumulate in epitopes of viral surface proteins, suggesting selection for immune escape. This catalog will serve as a foundation for the development of vaccines and diagnostics. VIDEO ABSTRACT.

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.

Genomic Analysis of Lassa Virus during an Increase in Cases in Nigeria in 2018.

During 2018, an unusual increase in Lassa fever cases occurred in Nigeria, raising concern among national and international public health agencies. We analyzed 220 Lassa virus genomes from infected patients, including 129 from the 2017-2018 transmission season, to understand the viral populations underpinning the increase. A total of 14 initial genomes from 2018 samples were generated at Redeemer's University in Nigeria, and the findings were shared with the Nigerian Center for Disease Control in real time. We found that the increase in cases was not attributable to a particular Lassa virus strain or sustained by human-to-human transmission. Instead, the data were consistent with ongoing cross-species transmission from local rodent populations. Phylogenetic analysis also revealed extensive viral diversity that was structured according to geography, with major rivers appearing to act as barriers to migration of the rodent reservoir.

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.

Characterization of Plasmodium falciparum structure in Nigeria with malaria SNPs barcode.

BACKGROUND: Plasmodium falciparum malaria remains a major health challenge in Nigeria despite the global decline of its incidence and mortality rates. Although significant progress has been made in preventing the transmission of P. falciparum and controlling the spread of the infection, there is much to be done in the area of proper monitoring, surveillance of the parasite, investigating the population dynamics and drug resistance profiling of the parasite as these are important to its eventual eradication. Polymorphic loci of msp1, msp2 and/or glurp genes or microsatellites have been traditionally used to characterize P. falciparum population structure in various parts of Nigeria. The lack of standardization in the interpretation of results, as well as the inability of these methods to distinguish closely related parasites, remains a limitation of these techniques. Conversely, the recently developed 24 single nucleotide polymorphism (SNP)-based molecular barcode assay has the possibility of differentiating between closely related parasites and offer additional information in determining the population diversity of P. falciparum within and between parasite populations. This study is therefore aimed at defining the population diversity of P. falciparum in and between two localities in Nigeria using the SNPs barcode technique. METHODS: The 24-SNP high-resolution melt (HRM) barcode assay and msp2 genotyping was used to investigate both intra and inter population diversity of the parasite population in two urban cities of Nigeria. RESULTS: Based on SNP barcode analysis, polygenomic malaria infections were observed in 17.9% and 13.5% of population from Enugu and Ibadan, respectively, while msp2 analyses showed 21% and 19.4% polygenomic infections in Enugu and Ibadan, respectively. Low levels of genetic diversity (π) of 0.328 and 0.318 were observed in Enugu and Ibadan parasite populations, respectively, while the FST value of 0.02 (p = 0.055) was obtained when the genetic divergence of both populations was considered. CONCLUSIONS: The 24-SNP barcode assay was effective in analysing P. falciparum population diversity. This study also showed that P. falciparum populations in Enugu and Ibadan had a degree of intra-population diversity, but very low divergence between the population. A low degree of polygenomic infections were also observed in the two parasite populations unlike previous years. This maybe as a result of the effect of artemisinin-based combination therapy (ACT), long-lasting insecticide-treated nets (LLITNs) and intermittent preventive treatments in the study populations.

Early rising asexual parasitaemia in Nigerian children following a first dose of artemisinin-based combination treatments of falciparum malaria.

BACKGROUND: Early rising asexual parasitaemia (ERAP), initially defined as 'an increase in the parasite count over the baseline pre-treatment level during the first 24 h of treatment' of falciparum malaria with artemisinin derivatives is well documented, but there is no characterization of its risk factors, kinetics, molecular features or relationship to late-appearing anaemia (LAA) in acute falciparum malaria in African children following oral artemisinin-based combination therapies (ACTs). METHODS: ERAP was defined as ≥5% increase in pre-treatment parasitaemia within 8 h of initiating treatment. Parasitaemia was quantified pre-treatment and 1-2 hourly for 8 h, and less frequently thereafter for 6 weeks following randomized treatment of acutely malarious children with artesunate-amodiaquine, artemether-lumefantrine or dihydroartemisinin-piperaquine. Risk factors were determined by stepwise multiple logistic regression model. Kinetics of release into and of elimination of asexual parasites and DNA clones from peripheral blood were evaluated by method of residuals and non-compartment model, respectively. Parasite population changes were evaluated morphologically and by molecular genotyping. RESULTS: ERAP occurred in 205 of 416 children. A parasitaemia <100,000/µL and parasitaemia 1 day post-treatment initiation were independent predictors of ERAP. In children with ERAP: mean and peak time of increase in parasitaemia were 105.6% (95% CI 81-130.1) and 2.5 h (95% CI 2.2-2.7), respectively. Mean lag time, half-time and rate constant of release were 0.2 h (95% CI 0.2-0.3), 1 h (95% CI 0.9-1.1), and 0.9 h-1 (95% CI 0.8-1), respectively. Schizonts and young gametocytes were seen only in peripheral blood of few children with ERAP. In age-, gender-, baseline parasitaemia- and treatment-matched children with and without ERAP, parasite DNA clearance time and area under curve of number of DNA clones versus time were significantly higher in children with ERAP indicating peripheral retention of released parasites followed by elimination. DNA clone elimination was monoexponential. CONCLUSION: ERAP is common, occurs rapidly as first order process and may be due to mobilization of parasites from deep tissue following a first dose of ACTs of acute childhood falciparum malaria. TRIALS REGISTRATION: Pan African Clinical Trial Registry PACTR201508001188143 , 3 July 2015; PACTR201510001189370, 3 July 2015; PACTR201508001191898, 7 July 2015 and PACTR201508001193368, 8 July 2015.

Factors contributing to anaemia after uncomplicated falciparum malaria in under five year-old Nigerian children ten years following adoption of artemisinin-based combination therapies as first-line antimalarials.

BACKGROUND: Artemisinin-based combination therapies (ACTs) have remained efficacious treatments of acute falciparum malaria in many endemic areas but there is little evaluation of factors contributing to the anaemia of acute falciparum malaria following long term adoption of ACTs as first-line antimalarials in African children. METHODS: Malarious <5 year-olds randomized to artemether-lumefantrine, artesunate-amodiaquine or dihydroartemisinin-piperaquine treatments were followed up clinically for 6 weeks. Anaemia was defined as haematocrit <30%; Malaria-attributable fall in haematocrit (MAFH) as the difference between haematocrit 28-42 days post- and pre-treatment; Total MAFH (TMAFH) as the difference between days 28-42 haematocrit and the lowest haematocrit recorded in the first week post-treatment initiation; Drug-attributable fall in haematocrit (DAFH) as the difference between MAFH and TMAFH; Early appearing anaemia (EAA) as haematocrit <30% occurring within 1 week in children with normal haematocrit pre-treatment. Predictors of anaemia pre-treatment, EAA, MAFH or DAFH >4% were evaluated by stepwise multiple logistic regression models. Survival analysis and kinetics of DAFH were evaluated by Kaplan-Meier estimator and non-compartment model, respectively. RESULTS: Pre-treatment, 355 of 959 children were anaemic. Duration of illness >2 days and parasitaemia ≤10,000 µL-1 were independent predictors of anaemia pre-treatment. EAA occurred in 301 of 604 children. Predictors of EAA were age ≤ 15 months, history of fever pre-treatment and enrolment haematocrit ≤35%. The probabilities of progression from normal haematocrit to EAA were similar for all treatments. MAFH >4% occurred in 446 of 694 children; its predictors were anaemia pre-treatment, enrolment parasitaemia ≤50,000 µL-1, parasitaemia one day post-treatment initiation and gametocytaemia. DAFH >4% occurred in 334 of 719 children; its predictors were history of fever pre-and fever 1 day post-treatment initiation, haematocrit ≥37%, and parasitaemia >100,000 µL-1. In 432 children, declines in DAFH deficits were monoexponential with overall estimated half-time of 2.2d (95% CI 1.9-2.6). Area under curve of deficits in DAFH versus time and estimated half-time were significantly higher in non-anaemic children indicating greater loss of haematocrit in these children. CONCLUSION: After ten years of adoption of ACTs, anaemia is common pre-and early post-treatment, falls in haematocrit attributable to a single infection is high, and DAFH >4% is common and significantly lower in anaemic compared to non-anaemic Nigerian children. TRIAL REGISTRATION: Pan African Clinical Trial Registry (PACTR) [ PACTR201709002064150, 1 March 2017 ].

Ebola Virus Epidemiology and Evolution in Nigeria.

Containment limited the 2014 Nigerian Ebola virus (EBOV) disease outbreak to 20 reported cases and 8 fatalities. We present here clinical data and contact information for at least 19 case patients, and full-length EBOV genome sequences for 12 of the 20. The detailed contact data permits nearly complete reconstruction of the transmission tree for the outbreak. The EBOV genomic data are consistent with that tree. It confirms that there was a single source for the Nigerian infections, shows that the Nigerian EBOV lineage nests within a lineage previously seen in Liberia but is genetically distinct from it, and supports the conclusion that transmission from Nigeria to elsewhere did not occur.

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.

In vitro-reduced susceptibility to artemether in P. falciparum and its association with polymorphisms on transporter genes.

Plasmodium falciparum with reduced sensitivity to artemisinin derivatives has been observed in endemic areas, but the molecular mechanisms for this reduced sensitivity remain unclear. We evaluated the association between in vitro susceptibility of P. falciparum isolates obtained from southwest Nigeria and polymorphisms in selected putative transporter genes (PFE0775C, PF13_0271, pfmrp1, pfcrt, and pfmdr1). Modified schizont inhibition assay was used to determine the in vitro parasite susceptibility to artemether (ATH). Polymorphisms in selected genes were detected by polymerase chain reaction followed by direct DNA sequencing. The half-maximal inhibitory concentration (IC(50)) geometric mean (GM) for all P. falciparum isolates was 1.78 nM (range, 0.03-10.43 nM). Polymorphisms at codons 241, 86, and 76 of PFE0775C, pfmdr1, and pfcrt genes, respectively, were associated with reduced susceptibility to ATH. A new S263P single-nucleotide polymorphism on the PFE0775C gene was also detected in 27% of the isolates. Patient isolates harboring V241L or S263P polymorphisms on the PFE0775C gene showed increased IC(50) (GM: 3.08 nM and 1.79 nM, respectively). Plasmodium falciparum isolates harboring mutant Y86 pfmdr1 and P263 PFE0775C alleles showed a 2.5-5.5-fold increase in ATH IC(50.) This study shows that polymorphisms on the PFE0775C and pfmdr1 genes are associated with reduced sensitivity to ATH in fresh isolates of P. falciparum from Nigeria.

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.

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.

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.

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.

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.

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.

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.

Early variations in plasmodium falciparum dynamics in Nigerian children after treatment with two artemisinin-based combinations: implications on delayed parasite clearance.

BACKGROUND: Combination treatments, preferably containing an artemisinin derivative, are recommended to improve efficacy and prevent Plasmodium falciparum drug resistance. Artemether-lumefantrine (AL) and artesunate-amodiaquine (AA) are efficacious regimens that have been widely adopted in sub-Saharan Africa. However, most study designs ignore the effects of these regimens on peripheral parasitaemia in the first 24 hours of therapy. The study protocol was designed to evaluate more closely the early effects and the standard measures of efficacies of these two regimens. METHODS: In an open label, randomized controlled clinical trial, children aged 12 months to 132 months were randomized to receive AL (5-14 kg, one tablet; 15-24 kg, two tablets and 25-34 kg, three tablets twice daily) or artesunate (4 mg/kg daily) plus amodiaquine (10 mg/kg daily) for three days. Peripheral blood smears were made hourly in the first 4 hours, 8 h, 16 h, 24 h, and daily on days 2-7, and on days 7, 14, 21, 28, 35, and 42 for microscopic identification and quantification of Plasmodium falciparum. RESULTS: A total of 193 children were randomized to receive either AL (97) or AA (96). In children that received both medications, early response of peripheral parasitaemia showed that 42% of children who received AL and 36.7% of those who received AA had an immediate rise in peripheral parasitaemia (0-4 h after treatment) followed by a rapid fall. The rise in parasitaemia was significant and seems to suggest a mobilization of asexual parasites from the deep tissues to the periphery. Days 3, 7, 14, 28, and 42 cure rates in the per protocol (PP) population were > 90% in both groups of children. Both drug combinations were well tolerated with minimal side effects. CONCLUSION: The study showed the high efficacy of AL and AA in Nigerian children. In addition the study demonstrated the mobilisation of asexual parasites from the deep to the periphery in the early hours of commencing ACT treatment in a subset of patients in both study groups. It is unclear whether the early parasite dynamics discovered in this study play any role in the development of drug resistance and thus it is important to further evaluate this discovery. It may be useful for studies investigating delay in parasite clearance of artemisinin derivatives as a way of monitoring the development of resistance to artemisinin to assess the early effects of the drugs on the parasites.