Whole genome analysis of local Kenyan and global sequences unravels the epidemiological and molecular evolutionary dynamics of RSV genotype ON1 strains

The respiratory syncytial virus (RSV) group A variant with the 72-nucleotide duplication in the G gene, genotype ON1, was first detected in Kilifi in 2012 and has almost completely replaced previously circulating genotype GA2 strains. This replacement suggests some fitness advantage of ON1 over the GA2 viruses, and might be accompanied by important genomic substitutions in ON1 viruses. Close observation of such a new virus introduction over time provides an opportunity to better understand the transmission and evolutionary dynamics of the pathogen. We have generated and analyzed 184 RSV-A whole genome sequences (WGS) from Kilifi (Kenya) collected between 2011 and 2016, the first ON1 genomes from Africa and the largest collection globally from a single location. Phylogenetic analysis indicates that RSV-A transmission into this coastal Kenya location is characterized by multiple introductions of viral lineages from diverse origins but with varied success in local transmission. We identify signature amino acid substitutions between ON1 and GA2 viruses within genes encoding the surface proteins (G, F), polymerase (L) and matrix M2-1 proteins, some of which were identified as positively selected, and thereby provide an enhanced picture of RSV-A diversity. Furthermore, five of the eleven RSV open reading frames (ORF) (i.e. G, F, L, N and P), analyzed separately, formed distinct phylogenetic clusters for the two genotypes. This might suggest that coding regions outside of the most frequently studied G ORF play a role in the adaptation of RSV to host populations with the alternative possibility that some of the substitutions are nothing more than genetic hitchhikers. Our analysis provides insight into the epidemiological processes that define RSV spread, highlights the genetic substitutions that characterize emerging strains, and demonstrates the utility of large-scale WGS in molecular epidemiological studies

Examining the human infectious reservoir for Plasmodium falciparum malaria in areas of differing transmission intensity.

A detailed understanding of the human infectious reservoir is essential for improving malaria transmission-reducing interventions. Here we report a multi-regional assessment of population-wide malaria transmission potential based on 1209 mosquito feeding assays in endemic areas of Burkina Faso and Kenya. Across both sites, we identified 39 infectious individuals. In high endemicity settings, infectious individuals were identifiable by research-grade microscopy (92.6%; 25/27), whilst one of three infectious individuals in the lowest endemicity setting was detected by molecular techniques alone. The percentages of infected mosquitoes in the different surveys ranged from 0.05 (4/7716) to 1.6% (121/7749), and correlate positively with transmission intensity. We also estimated exposure to malaria vectors through genetic matching of blood from 1094 wild-caught bloodfed mosquitoes with that of humans resident in the same houses. Although adults transmitted fewer parasites to mosquitoes than children, they received more mosquito bites, thus balancing their contribution to the infectious reservoir

Longitudinal analysis of infant stool bacteria communities before and after acute febrile malaria and artemether-lumefantrine treatment

Background Gut microbiota were recently shown to impact malaria disease progression and outcome, and prior studies have shown thatandnbsp;Plasmodiumandnbsp;infections increase the likelihood of enteric bacteria causing systemic infections. Currently, it is not known whetherandnbsp;Plasmodiumandnbsp;infection impacts human gut microbiota as a prelude to bacteremia or whether antimalarials affect gut microbiota. Our goal was to determine to what degreeandnbsp;Plasmodiumandnbsp;infections and antimalarial treatment affect human gut microbiota. Methods One hundred Kenyan infants underwent active surveillance for malaria from birth to 10 months of age. Each malaria episode was treated with artemether-lumefantrine (AL). Any other treatments, including antibiotics, were recorded. Stool samples were collected on an approximately biweekly basis. Ten children were selected on the basis of stool samples having been collected before (n = 27) or after (n = 17) a malaria episode and without antibiotics having been administered between collections. These samples were subjected to 16S ribosomal ribonucleic acid gene (V3andndash;V4 region) sequencing. Results Bacterial community network analysis revealed no obvious differences in the before and after malaria/AL samples, which was consistent with no difference in alpha and beta diversity and taxonomic analysis at the family and genus level with one exception. At the sequence variant (SV) level, akin to bacterial species, only 1 of the top 100 SVs was significantly different. In addition, predicted metagenome analysis revealed no significant difference in metagenomic capacity between before and after malaria/AL samples. The number of malaria episodes, 1 versus 2, explained significant variation in gut microbiota composition of the infants. Conclusions In-depth bioinformatics analysis of stool bacteria has revealed for the first time that human malaria episode/AL treatment have minimal effects on gut microbiota in Kenyan infants.</p

Longitudinal analysis of infant stool bacteria communities before and after acute febrile malaria and artemether-lumefantrine treatment

Background Gut microbiota were recently shown to impact malaria disease progression and outcome, and prior studies have shown that Plasmodium infections increase the likelihood of enteric bacteria causing systemic infections. Currently, it is not known whether Plasmodium infection impacts human gut microbiota as a prelude to bacteremia or whether antimalarials affect gut microbiota. Our goal was to determine to what degree Plasmodium infections and antimalarial treatment affect human gut microbiota. Methods One hundred Kenyan infants underwent active surveillance for malaria from birth to 10 months of age. Each malaria episode was treated with artemether-lumefantrine (AL). Any other treatments, including antibiotics, were recorded. Stool samples were collected on an approximately biweekly basis. Ten children were selected on the basis of stool samples having been collected before (n = 27) or after (n = 17) a malaria episode and without antibiotics having been administered between collections. These samples were subjected to 16S ribosomal ribonucleic acid gene (V3–V4 region) sequencing. Results Bacterial community network analysis revealed no obvious differences in the before and after malaria/AL samples, which was consistent with no difference in alpha and beta diversity and taxonomic analysis at the family and genus level with one exception. At the sequence variant (SV) level, akin to bacterial species, only 1 of the top 100 SVs was significantly different. In addition, predicted metagenome analysis revealed no significant difference in metagenomic capacity between before and after malaria/AL samples. The number of malaria episodes, 1 versus 2, explained significant variation in gut microbiota composition of the infants. Conclusions In-depth bioinformatics analysis of stool bacteria has revealed for the first time that human malaria episode/AL treatment have minimal effects on gut microbiota in Kenyan infants.</p

Comparative analysis of the vaginal microbiome of pregnant women with either Trichomonas vaginalis or Chlamydia trachomatis

Background Although the significance of the human vaginal microbiome for health and disease is increasingly acknowledged, there is paucity of data on the differences in the composition of the vaginal microbiome upon infection with different sexually transmitted pathogens. Method The composition of the vaginal bacterial community of women with Trichomonas vaginalis (TV, N = 18) was compared to that of women with Chlamydia trachomatis (CT, N = 14), and to that of controls (N = 21) (women negative for TV, CT and bacterial vaginosis). The vaginal bacterial composition was determined using high throughput sequencing with the Ion 16S metagenomics kit of the variable regions 2, 4 and 8 of the bacterial 16S ribosomal RNA gene from the vaginal swab DNA extract of the women. QIIME and R package “Phyloseq” were used to assess the α- and β-diversity and absolute abundance of the 16S rRNA gene per sample in the three groups. Differences in taxa at various levels were determined using the independent T-test. Results A total of 545 operational taxonomic units (OTUs) were identified in all the three groups of which 488 occurred in all three groups (core OTUs). Bacterial α-diversity, by both Simpson’s and Shannon’s indices, was significantly higher, (p = 0.056) and (p = 0.001) respectively, among women with either TV or CT than among controls (mean α-diversity TV-infected &gt; CT-infected &gt; Controls). At the genus level, women infected with TV had a significantly (p &lt; 0.01) higher abundance of Parvimonas and Prevotella species compared to both controls and CT-infected women, whereas women infected with CT had a significantly (p &lt; 0.05) higher abundance of Anaerococcus, Collinsella, Corynebacterium and Dialister. Conclusion The vaginal microbiomes of TV and CT-infected women were markedly different from each other and from women without TV and CT. Future studies should determine whether the altered microbiomes are merely markers of disease, or whether they actively contribute to the pathology of the two genital infections

Safety and PCR monitoring in 161 semi-immune Kenyan adults following controlled human malaria infection

BACKGROUND. Naturally acquired immunity to malaria is incompletely understood. We used controlled human malaria infection (CHMI) to study the impact of past exposure on malaria in Kenyan adults in relation to infection with a non-Kenyan parasite strain. METHODS. We administered 3.2 × 103 aseptic, purified, cryopreserved Plasmodium falciparum sporozoites (Sanaria PfSPZ Challenge, NF54 West African strain) by direct venous inoculation and undertook clinical monitoring and serial quantitative PCR (qPCR) of the 18S ribosomal RNA gene. The study endpoint was met when parasitemia reached 500 or more parasites per μL blood, clinically important symptoms were seen, or at 21 days after inoculation. All volunteers received antimalarial drug treatment upon meeting the endpoint. RESULTS. One hundred and sixty-one volunteers underwent CHMI between August 4, 2016, and February 14, 2018. CHMI was well tolerated, with no severe or serious adverse events. Nineteen volunteers (11.8%) were excluded from the analysis based on detection of antimalarial drugs above the minimal inhibitory concentration or parasites genotyped as non-NF54. Of the 142 volunteers who were eligible for analysis, 26 (18.3%) had febrile symptoms and were treated; 30 (21.1%) reached 500 or more parasites per μL and were treated; 53 (37.3%) had parasitemia without meeting thresholds for treatment; and 33 (23.2%) remained qPCR negative. CONCLUSION. We found that past exposure to malaria, as evidenced by location of residence, in some Kenyan adults can completely suppress in vivo growth of a parasite strain originating from outside Kenya. TRIAL REGISTRATION. ClinicalTrials.gov NCT02739763. FUNDING. Wellcome Trust

The effect of declining exposure on T cell-mediated immunity to Plasmodium falciparum – an epidemiological “natural experiment”

Naturally acquired immunity to malaria may be lost with lack of exposure. Recent heterogeneous reductions in transmission in parts of Africa mean that large populations of previously protected people may lose their immunity while remaining at risk of infection.Using two ethnically similar long-term cohorts of children with historically similar levels of exposure to Plasmodium falciparum who now experience very different levels of exposure, we assessed the effect of decreased parasite exposure on antimalarial immunity. Peripheral blood mononuclear cells (PBMCs) from children in each cohort were stimulated with P. falciparum and their P. falciparum-specific proliferative and cytokine responses were compared.We demonstrate that, while P. falciparum-specific CD4+ T cells are maintained in the absence of exposure, the proliferative capacity of these cells is altered considerably. P. falciparum-specific CD4+ T cells isolated from children previously exposed, but now living in an area of minimal exposure ("historically exposed") proliferate significantly more upon stimulation than cells isolated from children continually exposed to the parasite. Similarly, PBMCs from historically exposed children expressed higher levels of pro-inflammatory cytokines and lower levels of anti-inflammatory cytokines after stimulation with P. falciparum. Notably, we found a significant positive association between duration since last febrile episode and P. falciparum-specific CD4+ T cell proliferation, with more recent febrile episodes associated with lower proliferation.Considered in the context of existing knowledge, these data suggest a model explaining how immunity is lost in absence of continuing exposure to P. falciparum

Epidemiological and evolutionary dynamics of influenza B virus in coastal Kenya as revealed by genomic analysis of strains sampled over a single season

The genomic epidemiology of influenza B virus (IBV) remains understudied in Africa despite significance to design of effective local and global control strategies. We undertook surveillance throughout 2016 in coastal Kenya, recruiting individuals presenting with acute respiratory illness at nine outpatient health facilities (any age) or admitted to the Kilifi County Hospital (<5 years old). Whole genomes were sequenced for a selected 111 positives; 94 (84.7%) of B/Victoria lineage and 17 (15.3%) of B/Yamagata lineage. Inter-lineage reassortment was detected in ten viruses; nine with B/Yamagata backbone but B/Victoria NA and NP segments and one with a B/Victoria backbone but B/Yamagata PB2, PB1, PA, and MP segments. Five phylogenomic clusters were identified among the sequenced viruses; (i), pure B/Victoria clade 1A (n = 93, 83.8%), (ii), reassortant B/Victoria clade 1A (n = 1, 0.9%), (iii), pure B/Yamagata clade 2 (n = 2, 1.8%), (iv), pure B/Yamagata clade 3 (n = 6, 5.4%), and (v), reassortant B/Yamagata clade 3 (n = 9, 8.1%). Using divergence dates and clustering patterns in the presence of global background sequences, we counted up to twenty-nine independent IBV strain introductions into the study area (∼900 km2) in 2016. Local viruses, including the reassortant B/Yamagata strains, clustered closely with viruses from neighbouring Tanzania and Uganda. Our study demonstrated that genomic analysis provides a clearer picture of locally circulating IBV diversity. The high number of IBV introductions highlights the challenge in controlling local influenza epidemics by targeted approaches, for example, sub-population vaccination or patient quarantine. The finding of divergent IBV strains co-circulating within a single season emphasises why broad immunity vaccines are the most ideal for influenza control in Kenya

Maintenance of high temporal Plasmodium falciparum genetic diversity and complexity of infection in asymptomatic and symptomatic infections in Kilifi, Kenya from 2007 to 2018

Background High levels of genetic diversity are common characteristics of Plasmodium falciparum parasite populations in high malaria transmission regions. There has been a decline in malaria transmission intensity over 12 years of surveillance in the community in Kilifi, Kenya. This study sought to investigate whether there was a corresponding reduction in P. falciparum genetic diversity, using msp2 as a genetic marker. Methods Blood samples were obtained from children (< 15 years) enrolled into a cohort with active weekly surveillance between 2007 and 2018 in Kilifi, Kenya. Asymptomatic infections were defined during the annual cross-sectional blood survey and the first-febrile malaria episode was detected during the weekly follow-up. Parasite DNA was extracted and successfully genotyped using allele-specific nested polymerase chain reactions for msp2 and capillary electrophoresis fragment analysis. Results Based on cross-sectional surveys conducted in 2007–2018, there was a significant reduction in malaria prevalence (16.2–5.5%: P-value  0.95) was observed in both asymptomatic infections and febrile malaria over time. About 281 (68.5%) asymptomatic infections were polyclonal (> 2 variants per infection) compared to 46 (56%) polyclonal first-febrile infections. There was significant difference in complexity of infection (COI) between asymptomatic 2.3 [95% confidence interval (CI) 2.2–2.5] and febrile infections 2.0 (95% CI 1.7–2.3) (P = 0.016). Majority of asymptomatic infections (44.2%) carried mixed alleles (i.e., both FC27 and IC/3D7), while FC27 alleles were more frequent (53.3%) among the first-febrile infections. Conclusions Plasmodium falciparum infections in Kilifi are still highly diverse and polyclonal, despite the reduction in malaria transmission in the community