Modelling pathogen load dynamics to elucidate mechanistic determinants of host-Plasmodium falciparum interactions

During infection, increasing pathogen load stimulates both protective and harmful aspects of the host response. The dynamics of this interaction are hard to quantify in humans, but doing so could improve understanding of mechanisms of disease and protection. We sought to model the contributions of parasite multiplication rate and host response to observed parasite load in individual subjects with Plasmodium falciparum malaria, using only data obtained at the time of clinical presentation, and then to identify their mechanistic correlates. We predicted higher parasite multiplication rates and lower host responsiveness in severe malaria cases, with severe anemia being more insidious than cerebral malaria. We predicted that parasite growth-inhibition was associated with platelet consumption, lower expression of CXCL10 and type-1 interferon-associated genes, but increased cathepsin G and matrix metallopeptidase 9 expression. We found that cathepsin G and matrix metallopeptidase 9 directly inhibit parasite invasion into erythrocytes. Parasite multiplication rate was associated with host iron availability and higher complement factor H levels, lower expression of gametocyte-associated genes but higher expression of translation-associated genes in the parasite. Our findings demonstrate the potential of using explicit modelling of pathogen load dynamics to deepen understanding of host-pathogen interactions and identify mechanistic correlates of protection

Investigation of sequential outbreaks of Burkholderia cepacia and multidrug-resistant extended spectrum β-lactamase producing Klebsiella species in a West African tertiary hospital neonatal unit: a retrospective genomic analysis

Background Sick newborns admitted to neonatal units in low-resource settings are at an increased risk of developing hospital-acquired infections due to poor clinical care practices. Clusters of infection, due to the same species, with a consistent antibiotic resistance profile, and in the same ward over a short period of time might be indicative of an outbreak. We used whole-genome sequencing (WGS) to define the transmission pathways and characterise two distinct outbreaks of neonatal bacteraemia in a west African neonatal unit. Methods We studied two outbreaks of Burkholderia cepacia and multidrug-resistant extended spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae in a neonatal unit that provides non-intensive care on the neonatal ward in the Edward Francis Small Teaching Hospital, Banjul, The Gambia. We used WGS to validate and expand findings from the outbreak investigation. We retrospectively sequenced all clinical isolates associated with each outbreak, including isolates obtained from swabs of ward surfaces, environmental fluid cultures, intravenous fluids, and antibiotics administered to newborns. We also sequenced historical B cepacia isolates associated with neonatal sepsis in the same ward. Results Between March 1 and Dec 31, 2016, 321 blood cultures were done, of which 178 (55%) were positive with a clinically significant isolate. 49 episodes of neonatal B cepacia bacteraemia and 45 episodes of bacteraemia due to ESBL-producing K pneumoniae were reported. WGS revealed the suspected K pneumoniae outbreak to be contemporaneous outbreaks of K pneumoniae (ST39) and previously unreported Klebsiella quasipneumoniae subspecies similipneumoniae (ST1535). Genomic analysis showed near-identical strain clusters for each of the three outbreak pathogens, consistent with transmission within the neonatal ward from extrinsically contaminated in-use intravenous fluids and antibiotics. Time-dated phylogeny, including retrospective analysis of archived bacterial strains, suggest B cepacia has been endemic in the neonatal ward over several years, with the Klebsiella species a more recent introduction. Interpretation Our study highlights the emerging threat of previously unreported strains of multidrug-resistant Klebsiella species in this neonatal unit. Genome-based surveillance studies can improve identification of circulating pathogen strains, characterisation of antimicrobial resistance, and help understand probable infection acquisition routes during outbreaks in newborn units in low-resource settings. Our data provide evidence for the need to regularly monitor endemic transmission of bacteria within the hospital setting, identify the introduction of resistant strains from the community, and improve clinical practices to reduce or prevent the spread of infection and resistance

A report on preparation, expansion and future outlook of COVID-19 testing in Gambia

Background: The outbreak of COVID-19 disease and rapid spread of the virus outside China led to its declaration as a Public Health Emer-gency of International Concern (PHEIC) in January 2020. Key elements of the early intervention strategy focused on laboratory diagnosis and screening at points of entry and imposition of restrictions in cross-border activities. Objective: We report the role the Medical Research Council Unit, The Gambia (MRCG) played in the early implementation of molecular testing for COVID-19 in The Gambia as part of the national outbreak response. Methods: Laboratory staff members, with experience in molecular biology assays, were identified and trained on COVID-19 testing at the Africa CDC training workshop in Dakar, Senegal. Thereafter risks assessments, drafting of standard operating procedures (SOPs) and in-house training enabled commencement of testing using commercial RT-PCR kits. Subsequently, testing was expanded to the National Public Health Laboratroy and also implemented across field sites for rapid response across the country. Results: Capacity for COVID-19 testing at MRCG was developed and can process aproximately 350 tests per day, which can be further scaled up as the demand for testing increases. Conclusion: The long presence of the Unit in The Gambia and strong collaborative relationship with the National Health Ministry, allowed for a synergistc approach in mounting an effective response that con-tributed in delaying the establishment of community transmission in the country

A real-time, quantitative PCR method using hydrolysis probes for the monitoring of Plasmodium falciparum load in experimentally infected human volunteers

Background: The accurate quantification of Plasmodium falciparum parasite numbers by PCR is an important tool for monitoring growth kinetics in subjects infected and subsequently treated with anti-malarial agents

West Africa International Centers of Excellence for Malaria Research: Drug Resistance Patterns to Artemether-Lumefantrine in Senegal, Mali, and The Gambia.

In 2006, artemether-lumefantrine (AL) became the first-line treatment of uncomplicated malaria in Senegal, Mali, and the Gambia. To monitor its efficacy, between August 2011 and November 2014, children with uncomplicated Plasmodium falciparum malaria were treated with AL and followed up for 42 days. A total of 463 subjects were enrolled in three sites (246 in Senegal, 97 in Mali, and 120 in Gambia). No early treatment failure was observed and malaria infection cleared in all patients by day 3. Polymerase chain reaction (PCR)-adjusted adequate clinical and parasitological response (ACPR) was 100% in Mali, and the Gambia, and 98.8% in Senegal. However, without PCR adjustment, ACPR was 89.4% overall; 91.5% in Mali, 98.8% in Senegal, and 64.3% in the Gambia (the lower value in the Gambia attributed to poor compliance of the full antimalarial course). However, pfmdr1 mutations were prevalent in Senegal and a decrease in parasite sensitivity to artesunate and lumefantrine (as measured by ex vivo drug assay) was observed at all sites. Recrudescent parasites did not show Kelch 13 (K13) mutations and AL remains highly efficacious in these west African sites

Alternative splicing of the Anopheles gambiae Dscam gene in diverse Plasmodium falciparum infections

Background: In insects, including Anopheles mosquitoes, Dscam (Down syndrome cell adhesion molecule) appears to be involved in phagocytosis of pathogens, and shows pathogen-specific splice-form expression between divergent pathogen (or parasite) types (e.g. between bacteria and Plasmodium or between Plasmodium berghei and Plasmodium falciparum). Here, data are presented from the first study of Dscam expression in response to genetic diversity within a parasite species. Methods: In independent field and laboratory studies, a measure of Dscam splice-form diversity was compared between mosquitoes fed on blood that was free of P. falciparum to mosquitoes exposed to either single or mixed genotype infections of P. falciparum. Results: Significant increases in Anopheles gambiae Dscam (AgDscam) receptor diversity were observed in parasite-exposed mosquitoes, but only weak evidence that AgDscam diversity rises further upon exposure to mixed genotype parasite infections was found. Finally, a cluster of AgDscam exon 4 variants that become especially common during Plasmodium invasion was identified. Conclusions: While the data clearly indicate that AgDscam diversity increases with P. falciparum exposure, they do not suggest that AgDscam diversity rises further in response to increased parasite diversit

Community-acquired invasive bacterial disease in urban Gambia, 2005–2015: A hospital-based surveillance

Background. Invasive bacterial diseases cause significant disease and death in sub-Saharan Africa. Several are vaccine preventable, although the impact of new vaccines and vaccine policies on disease patterns in these communities is poorly understood owing to limited surveillance data. Methods. We conducted a hospital-based surveillance of invasive bacterial diseases in The Gambia where blood and cerebrospinal fluid (CSF) samples of hospitalized participants were processed. Three surveillance periods were defined in relation to the introduction of pneumococcal conjugate vaccines (PCVs), before (2005- 2009), during (2010–2011) and after (2012–2015) PCV introduction. We determined the prevalences of commonly isolated bacteria and compared them between the different surveillance periods. Results. A total of 14 715 blood and 1103 CSF samples were collected over 11 years; overall, 1045 clinically significant organisms were isolated from 957 patients (972 organisms [6.6%] from blood and 73 [6.6%] from CSF). The most common blood culture isolates were Streptococcus pneumoniae (24.9%), Staphylococcus aureus (22.0%), Escherichia coli (10.9%), and nontyphoidal Salmonella (10.0%). Between the pre-PCV and post-PCV eras, the prevalence of S. pneumoniae bacteremia dropped across all age groups (from 32.4% to 16.5%; odds ratio, 0.41; 95% confidence interval, .29–.58) while S. aureus increased in prevalence, becoming the most prevalent bacteria (from 16.9% to 27.2%; 1.75; 1.26–2.44). Overall, S. pneumoniae (53.4%), Neisseria meningitidis (13.7%), and Haemophilus influenzae (12.3%) were the predominant isolates from CSF. Antimicrobial resistance to common antibiotics was low. Conclusions. Our findings demonstrate that surveillance data on the predominant pathogens associated with invasive disease is necessary to inform vaccine priorities and appropriate management of patients

Genome variation and population structure among 1142 mosquitoes of the African malaria vector species Anopheles gambiae and Anopheles coluzzii

Mosquito control remains a central pillar of efforts to reduce malaria burden in sub-Saharan Africa. However, insecticide resistance is entrenched in malaria vector populations, and countries with a high malaria burden face a daunting challenge to sustain malaria control with a limited set of surveillance and intervention tools. Here we report on the second phase of a project to build an open resource of high-quality data on genome variation among natural populations of the major African malaria vector species Anopheles gambiae and Anopheles coluzzii. We analyzed whole genomes of 1142 individual mosquitoes sampled from the wild in 13 African countries, as well as a further 234 individuals comprising parents and progeny of 11 laboratory crosses. The data resource includes high-confidence single-nucleotide polymorphism (SNP) calls at 57 million variable sites, genome-wide copy number variation (CNV) calls, and haplotypes phased at biallelic SNPs. We use these data to analyze genetic population structure and characterize genetic diversity within and between populations. We illustrate the utility of these data by investigating species differences in isolation by distance, genetic variation within proposed gene drive target sequences, and patterns of resistance to pyrethroid insecticides. This data resource provides a foundation for developing new operational systems for molecular surveillance and for accelerating research and development of new vector control tools. It also provides a unique resource for the study of population genomics and evolutionary biology in eukaryotic species with high levels of genetic diversity under strong anthropogenic evolutionary pressures

Ex vivo susceptibility and genotyping of Plasmodium falciparum isolates from Pikine, Senegal

Background: The monitoring of Plasmodium falciparum sensitivity to anti-malarial drugs is a necessity for effective case management of malaria. This species is characterized by a strong resistance to anti-malarial drugs. In Senegal, the first cases of chloroquine resistance were reported in the Dakar region in 1988 with nearly 7% population prevalence, reaching 47% by 1990. It is in this context that sulfadoxine–pyrimethamine temporarily replaced chloroquine as first line treatment in 2003, pending the introduction of artemisinin-based combination therapy in 2006. The purpose of this study is to assess the ex vivo sensitivity to different anti-malarial drugs of the P. falciparum population from Pikine. Methods: Fifty-four samples were collected from patients with non-complicated malaria and aged between 2 and 20 years in the Deggo health centre in Pikine in 2014. An assay in which parasites are stained with 4′, 6-di-amidino-2-phenylindole (DAPI), was used to study the ex vivo sensitivity of isolates to chloroquine, amodiaquine, piperaquine, pyrimethamine, and dihydroartemisinin. High resolution melting was used for genotyping of pfdhps, pfdhfr, pfmdr1, and pfcrt genes. Results: The mean IC50s of chloroquine, amodiaquine, piperaquine, dihydroartemisinin, and pyrimethamine were, respectively, 39.44, 54.02, 15.28, 2.23, and 64.70 nM. Resistance mutations in pfdhfr gene, in codon 437 of pfdhps gene, and an absence of mutation at position 540 of pfdhps were observed. Mutations in codons K76T of pfcrt and N86Y of pfmdr1 were observed at 51 and 11% population prevalence, respectively. A relationship was found between the K76T and N86Y mutations and ex vivo resistance to chloroquine. Conclusion: An increase in sensitivity of isolates to chloroquine was observed. A high sensitivity to dihydroartemisinin was observed; whereas, a decrease in sensitivity to pyrimethamine was observed in the parasite population from Pikine