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 < 0.001), however msp2 genetic diversity remained high. A high heterozygosity index (He) (> 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.

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.

Repeated clinical malaria episodes are associated with modification of the immune system in children.

BACKGROUND: There are over 200 million reported cases of malaria each year, and most children living in endemic areas will experience multiple episodes of clinical disease before puberty. We set out to understand how frequent clinical malaria, which elicits a strong inflammatory response, affects the immune system and whether these modifications are observable in the absence of detectable parasitaemia. METHODS: We used a multi-dimensional approach comprising whole blood transcriptomic, cellular and plasma cytokine analyses on a cohort of children living with endemic malaria, but uninfected at sampling, who had been under active surveillance for malaria for 8 years. Children were categorised into two groups depending on the cumulative number of episodes experienced: high (≥ 8) or low (< 5). RESULTS: We observe that multiple episodes of malaria are associated with modification of the immune system. Children who had experienced a large number of episodes demonstrated upregulation of interferon-inducible genes, a clear increase in circulating levels of the immunoregulatory cytokine IL-10 and enhanced activation of neutrophils, B cells and CD8+ T cells. CONCLUSION: Transcriptomic analysis together with cytokine and immune cell profiling of peripheral blood can robustly detect immune differences between children with different numbers of prior malaria episodes. Multiple episodes of malaria are associated with modification of the immune system in children. Such immune modifications may have implications for the initiation of subsequent immune responses and the induction of vaccine-mediated protection.

Gene copy number variation in natural populations of Plasmodium falciparum in Eastern Africa.

BACKGROUND: Gene copy number variants (CNVs), which consist of deletions and amplifications of single or sets of contiguous genes, contribute to the great diversity in the Plasmodium falciparum genome. In vitro studies in the laboratory have revealed their important role in parasite fitness phenotypes such as red cell invasion, transmissibility and cytoadherence. Studies of natural parasite populations indicate that CNVs are also common in the field and thus may facilitate adaptation of the parasite to its local environment. RESULTS: In a survey of 183 fresh field isolates from three populations in Eastern Africa with different malaria transmission intensities, we identified 94 CNV loci using microarrays. All CNVs had low population frequencies (minor allele frequency < 5%) but each parasite isolate carried an average of 8 CNVs. Nine CNVs showed high levels of population differentiation (FST > 0.3) and nine exhibited significant clines in population frequency across a gradient in transmission intensity. The clearest example of this was a large deletion on chromosome 9 previously reported only in laboratory-adapted isolates. This deletion was present in 33% of isolates from a population with low and highly seasonal malaria transmission, and in < 9% of isolates from populations with higher transmission. Subsets of CNVs were strongly correlated in their population frequencies, implying co-selection. CONCLUSIONS: These results support the hypothesis that CNVs are the target of selection in natural populations of P. falciparum. Their environment-specific patterns observed here imply an important role for them in conferring adaptability to the parasite thus enabling it to persist in its highly diverse ecological environment.

Detecting Malaria Hotspots: A Comparison of Rapid Diagnostic Test, Microscopy, and Polymerase Chain Reaction.

Background: Malaria control strategies need to respond to geographical hotspots of transmission. Detection of hotspots depends on the sensitivity of the diagnostic tool used. Methods: We conducted cross-sectional surveys in 3 sites within Kilifi County, Kenya, that had variable transmission intensities. Rapid diagnostic test (RDT), microscopy, and polymerase chain reaction (PCR) were used to detect asymptomatic parasitemia, and hotspots were detected using the spatial scan statistic. Results: Eight thousand five hundred eighty-one study participants were surveyed in 3 sites. There were statistically significant malaria hotspots by RDT, microscopy, and PCR for all sites except by microscopy in 1 low transmission site. Pooled data analysis of hotspots by PCR overlapped with hotspots by microscopy at a moderate setting but not at 2 lower transmission settings. However, variations in degree of overlap were noted when data were analyzed by year. Hotspots by RDT were predictive of PCR/microscopy at the moderate setting, but not at the 2 low transmission settings. We observed long-term stability of hotspots by PCR and microscopy but not RDT. Conclusion: Malaria control programs may consider PCR testing to guide asymptomatic malaria hotspot detection once the prevalence of infection falls.

The effect of declining exposure on T cell-mediated immunity to Plasmodium falciparum - an epidemiological "natural experiment".

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Association of symptomatic upper respiratory tract infections with the alteration of the oropharyngeal microbiome in a cohort of school children in Côte d'Ivoire.

Introduction: The oropharyngeal microbiome plays an important role in protection against infectious agents when in balance. Despite use of vaccines and antibiotic therapy to prevent respiratory tract infections, they remain one of the major causes of mortality and morbidity in Low- and middle-income countries. Hence the need to explore other approaches to prevention by identifying microbial biomarkers that could be leveraged to modify the microbiota in order to enhance protection against pathogenic bacteria. The aim of this study was to analyze the oropharyngeal microbiome (OPM) of schoolchildren in Côte d'Ivoire presenting symptoms of upper respiratory tract infections (URTI) for better prevention strategy. Methods: Primary schools' children in Korhogo (n = 37) and Abidjan (n = 39) were followed for six months with monthly oropharyngeal sampling. Clinical diagnostic of URT infection was performed and nucleic acid extracted from oropharyngeal swabs were used for 16S rRNA metagenomic analysis and RT-PCR. Results: The clinical examination of children's throat in Abidjan and Korhogo identified respectively 17 (43.59%) and 15 (40.54%) participants with visible symptoms of URTIs, with 26 episodes of infection in Abidjan and 24 in Korhogo. Carriage of Haemophilus influenzae (12%), Streptococcus pneumoniae (6%) and SARS-CoV-2 (6%) was confirmed by PCR. A significant difference in alpha diversity was found between children colonized by S. pneumoniae and those that were not (p = 0.022). There was also a significant difference in alpha diversity between children colonised with H. influenzae and those who were not (p = 0.017). No significant difference was found for SARS-CoV-2. Sphingomonas, Ralstonia and Rothia were significantly enriched in non-carriers of S. pneumoniae; Actinobacillus was significantly enriched in non-carriers of H. influenzae; Actinobacillus and Porphyromonas were significantly enriched in non-carriers of SARS-CoV-2 (p < 0.001). Discussion: Nearly 40% of children showed clinical symptoms of infection not related to geographical location. The OPM showed an imbalance during H. influenzae and S. pneumoniae carriage. This study provides a baseline understanding of microbiome markers in URTIs in children for future research, to develop targeted interventions aimed at restoring the microbial balance and reducing the symptoms associated with RTIs.

Radiologic patterns of distant organ metastasis in advanced breast cancer patients: Prospective review of computed tomography images.

BACKGROUND: Breast cancer (BC) metastases to the abdomen and pelvis affect the liver, mesentery, retroperitoneum, peritoneum, bladder, kidney, ovary, and uterus. The study documented the radiological pattern and features of the chest, bone, abdominal and pelvic (AP) metastases among advanced BC patients. AIM: The aim is to document the radiological pattern and features of breast cancer metastasis in the chest, abdomen, pelvis and bones. MATERIALS AND RESULTS: Chest, abdominal, and pelvic computed tomography scan images of 36 patients with advanced BC were collated from Cape Coast Teaching Hospital and RAAJ Diagnostics. The images were prospectively assessed for metastasis to the organs of the chest, AP soft tissues, and bones. Radiologic features of metastasis of the lungs, liver, lymph nodes (LNs), and bones were documented. Patients' demographics, clinical data, and histopathology reports were also collected. The data were captured using UVOSYO and exported to Microsoft Excel templates. The data obtained were descriptively analyzed. Only 2.8% of BCs exhibited metaplastic BC, whereas 97.2% had invasive ductal BC. Triple-negative cases were 55.6%. Of 36 patients, 31 (86.1%), 21 (58.3%), and 14(38.8%) were diagnosed of chest, AP, and bone tissues metastasis, respectively. LN involvement was reported in 26 (72.2%) patients. Majority, 21 (58.3%) were diagnosed of multiple sites metastasis with 15 (41.7%) showing single site. Lungs (77.4%, 24/31) and liver (47.6%, 10/21) were the most affected distant organs. Most bone metastases were lytic lesions (92.9%, 13/14) with the vertebrae (85.7%, 12/14) been the most affected. CONCLUSION: According to the study, advanced BC patients have a higher-than-average radiologic incidence of lung, liver, bone, and LN metastases.

Drug resistance and vaccine target surveillance of Plasmodium falciparum using nanopore sequencing in Ghana.

Malaria results in over 600,000 deaths annually, with the highest burden of deaths in young children living in sub-Saharan Africa. Molecular surveillance can provide important information for malaria control policies, including detection of antimalarial drug resistance. However, genome sequencing capacity in malaria-endemic countries is limited. We designed and implemented an end-to-end workflow to detect Plasmodium falciparum antimalarial resistance markers and diversity in the vaccine target circumsporozoite protein (csp) using nanopore sequencing in Ghana. We analysed 196 clinical samples and showed that our method is rapid, robust, accurate and straightforward to implement. Importantly, our method could be applied to dried blood spot samples, which are readily collected in endemic settings. We report that P. falciparum parasites in Ghana are mostly susceptible to chloroquine, with persistent sulfadoxine-pyrimethamine resistance and no evidence of artemisinin resistance. Multiple single nucleotide polymorphisms were identified in csp, but their significance is uncertain. Our study demonstrates the feasibility of nanopore sequencing for malaria genomic surveillance in endemic countries.

Anti-merozoite antibodies induce natural killer cell effector function and are associated with immunity against malaria.

Natural killer (NK) cells are potent immune effectors that can be activated via antibody-mediated Fc receptor engagement. Using multiparameter flow cytometry, we found that NK cells degranulate and release IFN-γ upon stimulation with antibody-opsonized Plasmodium falciparum merozoites. Antibody-dependent NK (Ab-NK) activity was largely strain transcending and enhanced invasion inhibition into erythrocytes. Ab-NK was associated with the successful control of parasitemia after experimental malaria challenge in African adults. In an independent cohort study in children, Ab-NK increased with age, was boosted by concurrent P. falciparum infections, and was associated with a lower risk of clinical episodes of malaria. Nine of the 14 vaccine candidates tested induced Ab-NK, including some less well-characterized antigens: P41, P113, MSP11, RHOPH3, and Pf_11363200. These data highlight an important role of Ab-NK activity in immunity against malaria and provide a potential mechanism for evaluating vaccine candidates.

Cessation of exclusive breastfeeding and seasonality, but not small intestinal bacterial overgrowth, are associated with environmental enteric dysfunction: A birth cohort study amongst infants in rural Kenya.

Background: Environmental Enteric Dysfunction (EED) is a chronic intestinal inflammatory disorder of unclear aetiology prevalent amongst children in low-income settings and associated with stunting. We aimed to characterise development of EED and its putative risk factors amongst rural Kenyan infants. Methods: In a birth cohort study in Junju, rural coastal Kenya, between August 2015 and January 2017, 100 infants were each followed for nine months. Breastfeeding status was recorded weekly and anthropometry monthly. Acute illnesses and antibiotics were captured by active and passive surveillance. Intestinal function and small intestinal bacterial overgrowth (SIBO) were assessed by monthly urinary lactulose mannitol (LM) and breath hydrogen tests. Faecal alpha-1-antitrypsin, myeloperoxidase and neopterin were measured as EED biomarkers, and microbiota composition assessed by 16S sequencing. Findings: Twenty nine of the 88 participants (33%) that underwent length measurement at nine months of age were stunted (length-for-age Z score <-2). During the rainy season, linear growth was slower and LM ratio was higher. In multivariable models, LM ratio, myeloperoxidase and neopterin increased after cessation of continuous-since-birth exclusive breastfeeding. For LM ratio this only occurred during the rainy season. EED markers were not associated with antibiotics, acute illnesses, SIBO, or gut microbiota diversity. Microbiota diversified with age and was not strongly associated with complementary food introduction or linear growth impairment. Interpretation: Our data suggest that intensified promotion of uninterrupted exclusive breastfeeding amongst infants under six months during the rainy season, where rainfall is seasonal, may help prevent EED. Our findings also suggest that therapeutic strategies directed towards SIBO are unlikely to impact on EED in this setting. However, further development of non-invasive diagnostic methods for SIBO is required. Funding: This research was funded in part by the Wellcome Trust (Research Training Fellowship to RJC (103376/Z/13/Z)). EPKP was supported by the MRC/DfID Newton Fund (MR/N006259/1). JAB was supported by the MRC/DFiD/Wellcome Trust Joint Global Health Trials scheme (MR/M007367/1) and the Bill & Melinda Gates Foundation (OPP1131320). HHU was supported by the NIHR Oxford Biomedical Research Centre (IS-BRC-1215-20008).

Genetic diversity of SARS-CoV-2 infections in Ghana from 2020-2021.

The COVID-19 pandemic is one of the fastest evolving pandemics in recent history. As such, the SARS-CoV-2 viral evolution needs to be continuously tracked. This study sequenced 1123 SARS-CoV-2 genomes from patient isolates (121 from arriving travellers and 1002 from communities) to track the molecular evolution and spatio-temporal dynamics of the SARS-CoV-2 variants in Ghana. The data show that initial local transmission was dominated by B.1.1 lineage, but the second wave was overwhelmingly driven by the Alpha variant. Subsequently, an unheralded variant under monitoring, B.1.1.318, dominated transmission from April to June 2021 before being displaced by Delta variants, which were introduced into community transmission in May 2021. Mutational analysis indicated that variants that took hold in Ghana harboured transmission enhancing and immune escape spike substitutions. The observed rapid viral evolution demonstrates the potential for emergence of novel variants with greater mutational fitness as observed in other parts of the world.

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

BACKGROUNDNaturally 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.METHODSWe 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.RESULTSOne 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.CONCLUSIONWe 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 REGISTRATIONClinicalTrials.gov NCT02739763.FUNDINGWellcome Trust.

Individual-level variations in malaria susceptibility and acquisition of clinical protection.

After decades of research, our understanding of when and why individuals infected with Plasmodium falciparum develop clinical malaria is still limited. Correlates of immune protection are often sought through prospective cohort studies, where measured host factors are correlated against the incidence of clinical disease over a set period of time. However, robustly inferring individual-level protection from these population-level findings has proved difficult due to small effect sizes and high levels of variance underlying such data. In order to better understand the nature of these inter-individual variations, we analysed the long-term malaria epidemiology of children ≤12 years old growing up under seasonal exposure to the parasite in the sub-location of Junju, Kenya. Despite the cohort's limited geographic expanse (ca. 3km x 10km), our data reveal a high degree of spatial and temporal variability in malaria prevalence and incidence rates, causing individuals to experience varying levels of exposure to the parasite at different times during their life. Analysing individual-level infection histories further reveal an unexpectedly high variability in the rate at which children experience clinical malaria episodes. Besides exposure to the parasite, measured as disease prevalence in the surrounding area, we find that the birth time of year has an independent effect on the individual's risk of experiencing a clinical episode. Furthermore, our analyses reveal that those children with a history of an above average number of episodes are more likely to experience further episodes during the upcoming transmission season. These findings are indicative of phenotypic differences in the rates by which children acquire clinical protection to malaria and offer important insights into the natural variability underlying malaria epidemiology.

10-year longitudinal study of malaria in children: Insights into acquisition and maintenance of naturally acquired immunity.

Background: Studies of long-term malaria cohorts have provided essential insights into how Plasmodium falciparum interacts with humans, and influences the development of antimalarial immunity. Immunity to malaria is acquired gradually after multiple infections, some of which present with clinical symptoms. However, there is considerable variation in the number of clinical episodes experienced by children of the same age within the same cohort. Understanding this variation in clinical symptoms and how it relates to the development of naturally acquired immunity is crucial in identifying how and when some children stop experiencing further malaria episodes. Where variability in clinical episodes may result from different rates of acquisition of immunity, or from variable exposure to the parasite. Methods: Using data from a longitudinal cohort of children residing in an area of moderate P. falciparum transmission in Kilifi district, Kenya, we fitted cumulative episode curves as monotonic-increasing splines, to 56 children under surveillance for malaria from the age of 5 to 15. Results: There was large variability in the accumulation of numbers of clinical malaria episodes experienced by the children, despite being of similar age and living in the same general location. One group of children from a particular sub-region of the cohort stopped accumulating clinical malaria episodes earlier than other children in the study. Despite lack of further clinical episodes of malaria, these children had higher asymptomatic parasite densities and higher antibody titres to a panel of P. falciparum blood-stage antigens. Conclusions: This suggests development of clinical immunity rather than lack of exposure to the parasite, and supports the view that this immunity to malaria disease is maintained by a greater exposure to P. falciparum, and thus higher parasite burdens. Our study illustrates the complexity of anti-malaria immunity and underscores the need for analyses which can sufficiently reflect the heterogeneity within endemic populations.

Genomic analysis of SARS-CoV-2 reveals local viral evolution in Ghana.

The confirmed case fatality rate for the coronavirus disease 2019 (COVID-19) in Ghana has dropped from a peak of 2% in March to be consistently below 1% since May 2020. Globally, case fatality rates have been linked to the strains/clades of circulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) within a specific country. Here we present 46 whole genomes of SARS-CoV-2 circulating in Ghana, from two separate sequencing batches: 15 isolates from the early epidemic (March 12-April 1 2020) and 31 from later time-points ( 25-27 May 2020). Sequencing was carried out on an Illumina MiSeq system following an amplicon-based enrichment for SARS-CoV-2 cDNA. After genome assembly and quality control processes, phylogenetic analysis showed that the first batch of 15 genomes clustered into five clades: 19A, 19B, 20A, 20B, and 20C, whereas the second batch of 31 genomes clustered to only three clades 19B, 20A, and 20B. The imported cases (6/46) mapped to circulating viruses in their countries of origin, namely, India, Hungary, Norway, the United Kingdom, and the United States of America. All genomes mapped to the original Wuhan strain with high similarity (99.5-99.8%). All imported strains mapped to the European superclade A, whereas 5/9 locally infected individuals harbored the B4 clade, from the East Asian superclade B. Ghana appears to have 19B and 20B as the two largest circulating clades based on our sequence analyses. In line with global reports, the D614G linked viruses seem to be predominating. Comparison of Ghanaian SARS-CoV-2 genomes with global genomes indicates that Ghanaian strains have not diverged significantly from circulating strains commonly imported into Africa. The low level of diversity in our genomes may indicate lower levels of transmission, even for D614G viruses, which is consistent with the relatively low levels of infection reported in Ghana.

Genomic epidemiology and evolutionary dynamics of respiratory syncytial virus group B in Kilifi, Kenya, 2015-17.

Respiratory syncytial virus (RSV) circulates worldwide, occurring seasonally in communities, and is a leading cause of acute respiratory illness in young children. There is paucity of genomic data from purposively sampled populations by which to investigate evolutionary dynamics and transmission patterns of RSV. Here we present an analysis of 295 RSV group B (RSVB) genomes from Kilifi, coastal Kenya, sampled from individuals seeking outpatient care in nine health facilities across a defined geographical area (∼890 km2), over two RSV epidemics between 2015 and 2017. RSVB diversity was characterized by multiple virus introductions into the area and co-circulation of distinct genetic clusters, which transmitted and diversified locally with varying frequency. Increase in relative genetic diversity paralleled seasonal virus incidence. Importantly, we identified a cluster of viruses that emerged in the 2016/17 epidemic, carrying distinct amino-acid signatures including a novel nonsynonymous change (K68Q) in antigenic site ∅ in the Fusion protein. RSVB diversity was additionally marked by signature nonsynonymous substitutions that were unique to particular genomic clusters, some under diversifying selection. Our findings provide insights into recent evolutionary and epidemiological behaviors of RSVB, and highlight possible emergence of a novel antigenic variant, which has implications on current prophylactic strategies in development.

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.

Genetic characterization of influenza A(H3N2) viruses circulating in coastal Kenya, 2009-2017.

BACKGROUND: Influenza viruses evolve rapidly and undergo immune driven selection, especially in the hemagglutinin (HA) protein. We report amino acid changes affecting antigenic epitopes and receptor-binding sites of A(H3N2) viruses circulating in Kilifi, Kenya, from 2009 to 2017. METHODS: Next-generation sequencing (NGS) was used to generate A(H3N2) virus genomic data from influenza-positive specimens collected from hospital admissions and health facility outpatients presenting with acute respiratory illness to health facilities within the Kilifi Health and Demographic Surveillance System. Full-length HA sequences were utilized to characterize A(H3N2) virus genetic and antigenic changes. RESULTS: From 186 (90 inpatient and 96 outpatient) influenza A virus-positive specimens processed, 101 A(H3N2) virus whole genomes were obtained. Among viruses identified in inpatient specimens from 2009 to 2015, divergence of circulating A(H3N2) viruses from the vaccine strains A/Perth/16/2009, A/Texas/50/2012, and A/Switzerland/9715293/2013 formed 6 genetic clades (A/Victoria/208/2009-like, 3B, 3C, 3C.2a, 4, and 7). Among viruses identified in outpatient specimens from 2015 to 2017, divergence of circulating A(H3N2) viruses from vaccine strain A/Hong Kong/4801/2014 formed clade 3C.2a, subclades 3C.2a2 and 3C.2a3, and subgroup 3C.2a1b. Several amino acid substitutions were associated with the continued genetic evolution of A(H3N2) strains in circulation. CONCLUSIONS: Our results suggest continuing evolution of currently circulating A(H3N2) viruses in Kilifi, coastal Kenya and suggest the need for continuous genetic and antigenic viral surveillance of circulating seasonal influenza viruses with broad geographic representation to facilitate prompt and efficient selection of influenza strains for inclusion in future influenza vaccines.

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 ß-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 > CT-infected > Controls). At the genus level, women infected with TV had a significantly (p < 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 < 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.