Genomic transmission analysis of multidrug-resistant Gram-negative bacteria within a newborn unit of a Kenyan tertiary hospital: A four-month prospective colonization study

Objective: Multidrug-resistant organisms (MDRO), especially carbapenem-resistant organisms (CRO), represent a threat for newborns. This study investigates the colonization prevalence of these pathogens in a newborn unit at a Kenyan tertiary hospital in an integrated approach combining routine microbiology, whole genome sequencing (WGS) and hospital surveillance data. Methods: The study was performed in the Kenyatta National Hospital (KNH) in 2019 over a four-month period and included 300 mother-baby pairs. A total of 1,097 swabs from newborns (weekly), mothers (once) and the hospital environment were taken. Routine clinical microbiology methods were applied for surveillance. Of the 288 detected MDRO, 160 isolates were analyzed for antimicrobial resistance genes and phylogenetic relatedness using whole genome sequencing (WGS) and bioinformatic analysis. Results: In maternal vaginal swabs, MDRO detection rate was 15% (n=45/300), including 2% CRO (n=7/300). At admission, MDRO detection rate for neonates was 16% (n=48/300), including 3% CRO (n=8/300) with a threefold increase for MDRO (44%, n=97/218) and a fivefold increase for CRO (14%, n=29/218) until discharge. Among CRO, K. pneumoniae harboring blaNDM-1 (n=20) or blaNDM-5 (n=16) were most frequent. WGS analysis revealed 20 phylogenetically related transmission clusters (including five CRO clusters). In environmental samples, the MDRO detection rate was 11% (n=18/164), including 2% CRO (n=3/164). Conclusion: Our study provides a snapshot of MDRO and CRO in a Kenyan NBU. Rather than a large outbreak scenario, data indicate several independent transmission events. The CRO rate among newborns attributed to the spread of NDM-type carbapenemases is worrisome

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

INTRODUCTION Investment in Africa over the past year with regard to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequencing has led to a massive increase in the number of sequences, which, to date, exceeds 100,000 sequences generated to track the pandemic on the continent. These sequences have profoundly affected how public health officials in Africa have navigated the COVID-19 pandemic. RATIONALE We demonstrate how the first 100,000 SARS-CoV-2 sequences from Africa have helped monitor the epidemic on the continent, how genomic surveillance expanded over the course of the pandemic, and how we adapted our sequencing methods to deal with an evolving virus. Finally, we also examine how viral lineages have spread across the continent in a phylogeographic framework to gain insights into the underlying temporal and spatial transmission dynamics for several variants of concern (VOCs). RESULTS Our results indicate that the number of countries in Africa that can sequence the virus within their own borders is growing and that this is coupled with a shorter turnaround time from the time of sampling to sequence submission. Ongoing evolution necessitated the continual updating of primer sets, and, as a result, eight primer sets were designed in tandem with viral evolution and used to ensure effective sequencing of the virus. The pandemic unfolded through multiple waves of infection that were each driven by distinct genetic lineages, with B.1-like ancestral strains associated with the first pandemic wave of infections in 2020. Successive waves on the continent were fueled by different VOCs, with Alpha and Beta cocirculating in distinct spatial patterns during the second wave and Delta and Omicron affecting the whole continent during the third and fourth waves, respectively. Phylogeographic reconstruction points toward distinct differences in viral importation and exportation patterns associated with the Alpha, Beta, Delta, and Omicron variants and subvariants, when considering both Africa versus the rest of the world and viral dissemination within the continent. Our epidemiological and phylogenetic inferences therefore underscore the heterogeneous nature of the pandemic on the continent and highlight key insights and challenges, for instance, recognizing the limitations of low testing proportions. We also highlight the early warning capacity that genomic surveillance in Africa has had for the rest of the world with the detection of new lineages and variants, the most recent being the characterization of various Omicron subvariants. CONCLUSION Sustained investment for diagnostics and genomic surveillance in Africa is needed as the virus continues to evolve. This is important not only to help combat SARS-CoV-2 on the continent but also because it can be used as a platform to help address the many emerging and reemerging infectious disease threats in Africa. In particular, capacity building for local sequencing within countries or within the continent should be prioritized because this is generally associated with shorter turnaround times, providing the most benefit to local public health authorities tasked with pandemic response and mitigation and allowing for the fastest reaction to localized outbreaks. These investments are crucial for pandemic preparedness and response and will serve the health of the continent well into the 21st century

Targeted amplicon deep sequencing of ama1 and mdr1 to track within-host P. falciparum diversity throughout treatment in a clinical drug trial

Introduction: Antimalarial therapeutic efficacy studies are routinely conducted in malaria-endemic countries to assess the effectiveness of antimalarial treatment strategies. Targeted amplicon sequencing (AmpSeq) uniquely identifies and quantifies genetically distinct parasites within an infection. In this study, AmpSeq of Plasmodium falciparum apical membrane antigen 1 (ama1), and multidrug resistance gene 1 (mdr1), were used to characterise the complexity of infection (COI) and drug-resistance genotypes, respectively. Methods: P. falciparum-positive samples were obtained from a triple artemisinin combination therapy clinical trial conducted in 30 children under 13 years of age between 2018 and 2019 in Kilifi, Kenya. Nine of the 30 participants presented with recurrent parasitemia from day 26 (624h) onwards. The ama1 and mdr1 genes were amplified and sequenced, while msp1, msp2 and glurp data were obtained from the original clinical study. Results: The COI was comparable between ama1 and msp1, msp2 and glurp; overall, ama1 detected more microhaplotypes. Based on ama1, a stable number of microhaplotypes were detected throughout treatment until day 3. Additionally, a recrudescent infection was identified with an ama1 microhaplotype initially observed at 30h and later in an unscheduled follow-up visit. Using the relative frequencies of ama1 microhaplotypes and parasitemia, we identified a fast (<1h) and slow (>5h) clearing microhaplotype. As expected, only two mdr1 microhaplotypes (NF and NY) were identified based on the combination of amino acid polymorphisms at codons 86 and 184. Conclusions: This study highlights AmpSeq as a tool for highly-resolution tracking of parasite microhaplotypes throughout treatment and can detect variation in microhaplotype clearance estimates. AmpSeq can also identify slow-clearing microhaplotypes, a potential early sign of selection during treatment. Consequently, AmpSeq has the capability of improving the discriminatory power to distinguish recrudescences from reinfections accurately

Targeted amplicon deep sequencing of ama1 and mdr1 to track within-host P. falciparum diversity throughout treatment in a clinical drug trial [version 4; peer review: 2 approved, 2 approved with reservations]

Introduction Antimalarial therapeutic efficacy studies are routinely conducted in malaria-endemic countries to assess the effectiveness of antimalarial treatment strategies. Targeted amplicon sequencing (AmpSeq) uniquely identifies and quantifies genetically distinct parasites within an infection. In this study, AmpSeq of Plasmodium falciparum apical membrane antigen 1 (ama1), and multidrug resistance gene 1 (mdr1), were used to characterise the complexity of infection (COI) and drug-resistance genotypes, respectively. Methods P. falciparum-positive samples were obtained from a triple artemisinin combination therapy clinical trial conducted in 30 children under 13 years of age between 2018 and 2019 in Kilifi, Kenya. Nine of the 30 participants presented with recurrent parasitemia from day 26 (624h) onwards. The ama1 and mdr1 genes were amplified and sequenced, while msp1, msp2 and glurp data were obtained from the original clinical study. Results The COI was comparable between ama1 and msp1, msp2 and glurp; overall, ama1 detected more microhaplotypes. Based on ama1, a stable number of microhaplotypes were detected throughout treatment until day 3. Additionally, a recrudescent infection was identified with an ama1 microhaplotype initially observed at 30h and later in an unscheduled follow-up visit. Using the relative frequencies of ama1 microhaplotypes and parasitemia, we identified a fast (5h) clearing microhaplotype. As expected, only two mdr1 microhaplotypes (NF and NY) were identified based on the combination of amino acid polymorphisms at codons 86 and 184. Conclusions This study highlights AmpSeq as a tool for highly-resolution tracking of parasite microhaplotypes throughout treatment and can detect variation in microhaplotype clearance estimates. AmpSeq can also identify slow-clearing microhaplotypes, a potential early sign of selection during treatment. Consequently, AmpSeq has the capability of improving the discriminatory power to distinguish recrudescences from reinfections accurately

Targeted Amplicon deep sequencing of ama1 and mdr1 to track within-host P. falciparum diversity in Kilifi, KENYA

These data were generated from targeted amplicon sequencing of Plasmodium falciparum ama1 and mdr1 genes in samples collected from Kilifi, at the coast of Kenya. Two objectives were explored: [1] To determine temporal changes in the genetic diversity of malaria parasites in asymptomatic and febrile infections. [2] To track within-host parasite diversity, throughout treatment in a clinical drug trial

Pooled testing conserves SARS-CoV-2 laboratory resources and improves test turn-around time: Experience on the Kenyan Coast

Background: International recommendations for the control of the coronavirus disease 2019 (COVID-19) pandemic emphasize the central role of laboratory testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent, at scale. The availability of testing reagents, laboratory equipment and qualified staff are important bottlenecks to achieving this. Elsewhere, pooled testing (i.e. combining multiple samples in the same reaction) has been suggested to increase testing capacities in the pandemic period. Methods: We discuss our experience with SARS-CoV-2 pooled testing using real-time reverse transcription polymerase chain reaction (RT-PCR) on the Kenyan Coast. Results: In mid-May, 2020, our RT-PCR testing capacity for SARS-CoV-2 was improved by ~100% as a result of adoption of a six-sample pooled testing strategy. This was accompanied with a concomitant saving of ~50% of SARS-CoV-2 laboratory test kits at both the RNA extraction and RT-PCR stages. However, pooled testing came with a slight decline of test sensitivity. The RT-PCR cycle threshold value (ΔCt) was ~1.59 higher for samples tested in pools compared to samples tested singly. Conclusions: Pooled testing is a useful strategy to increase SARS-CoV-2 laboratory testing capacity especially in low-income settings

An optimization of four SARS-CoV-2 qRT-PCR assays in a Kenyan laboratory to support the national COVID-19 rapid response teams

Background: The COVID-19 pandemic relies on real-time polymerase chain reaction (qRT-PCR) for the detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), to facilitate roll-out of patient care and infection control measures. There are several qRT-PCR assays with little evidence on their comparability. We report alterations to the developers’ recommendations to sustain the testing capability in a resource-limited setting. Methods: We used a SARS-CoV-2 positive control RNA sample to generate several 10-fold dilution series that were used for optimization and comparison of the performance of the four qRT-PCR assays: i) Charité Berlin primer-probe set, ii) European Virus Archive – GLOBAL (EVAg) primer-probe set, iii) DAAN premixed commercial kit and iv) Beijing Genomics Institute (BGI) premixed commercial kit. We adjusted the manufacturer- and protocol-recommended reaction component volumes for these assays and assessed the impact on cycle threshold (Ct) values. Results: The Berlin and EVAg E gene and RdRp assays reported mean Ct values within range of each other across the different titrations and with less than 5% difference. The DAAN premixed kit produced comparable Ct values across the titrations, while the BGI kit improved in performance following a reduction of the reaction components. Conclusion: We achieved a 2.6-fold and 4-fold increase in the number of tests per kit for the commercial kits and the primer-probe sets, respectively. All the assays had optimal performance when the primers and probes were used at 0.375X, except for the Berlin N gene assay. The DAAN kit was a reliable assay for primary screening of SARS-CoV-2 whereas the BGI kit’s performance was dependent on the volumes and concentrations of both the reaction buffer and enzyme mix. Our recommendation for SARS-CoV-2 diagnostic testing in resource-limited settings is to optimize the assays available to establish the lowest volume and suitable concentration of reagents required to produce valid results