Genomic epidemiology of escherichia coli isolates from a tertiary referral center in lilongwe, Malawi

Antimicrobial resistance (AMR) is a global threat, including in sub-Saharan Africa. However, little is known about the genetics of resistant bacteria in the region. In Malawi, there is growing concern about increasing rates of antimicrobial resistance to most empirically used antimicrobials. The highly drug resistant Escherichia coli sequence type (ST) 131, which is associated with the extended spectrum β-lactamase blaCTX-M-15, has been increasing in prevalence globally. Previous data from isolates collected between 2006 and 2013 in southern Malawi have revealed the presence of ST131 and the blaCTX-M-15 gene in the country. We performed whole genome sequencing (WGS) of 58 clinical E. coli isolates at Kamuzu Central Hospital, a tertiary care centre in central Malawi, collected from 2012 to 2018. We used Oxford Nanopore Technologies (ONT) sequencing, which was performed in Malawi. We show that ST131 is observed more often (14.9% increasing to 32.8%) and that the blaCTX-M-15 gene is occurring at a higher frequency (21.3% increasing to 44.8%). Phylogenetics indicates that isolates are highly related between the central and southern geographic regions and confirms that ST131 isolates are contained in a single group. All AMR genes, including blaCTX-M-15, were widely distributed across sequence types. We also identified an increased number of ST410 isolates, which in this study tend to carry a plasmid-located copy of blaCTX-M-15 gene at a higher frequency than blaCTX-M-15 occurs in ST131. This study confirms the expanding nature of ST131 and the wide distribution of the blaCTX-M-15 gene in Malawi. We also highlight the feasibility of conducting longitudinal genomic epidemiology studies of important bacteria with the sequencing done on site using a nanopore platform that requires minimal infrastructure

Genomic Variation across a Clinical Cryptococcus Population Linked to Disease Outcome.

Cryptococcus neoformans is the causative agent of cryptococcosis, a disease with poor patient outcomes that accounts for approximately 180,000 deaths each year. Patient outcomes may be impacted by the underlying genetics of the infecting isolate; however, our current understanding of how genetic diversity contributes to clinical outcomes is limited. Here, we leverage clinical, in vitro growth and genomic data for 284 C. neoformans isolates to identify clinically relevant pathogen variants within a population of clinical isolates from patients with human immunodeficiency virus (HIV)-associated cryptococcosis in Malawi. Through a genome-wide association study (GWAS) approach, we identify variants associated with the fungal burden and the growth rate. We also find both small and large-scale variation, including aneuploidy, associated with alternate growth phenotypes, which may impact the course of infection. Genes impacted by these variants are involved in transcriptional regulation, signal transduction, glycosylation, sugar transport, and glycolysis. We show that growth within the central nervous system (CNS) is reliant upon glycolysis in an animal model and likely impacts patient mortality, as the CNS yeast burden likely modulates patient outcome. Additionally, we find that genes with roles in sugar transport are enriched in regions under selection in specific lineages of this clinical population. Further, we demonstrate that genomic variants in two genes identified by GWAS impact virulence in animal models. Our approach identifies links between the genetic variation in C. neoformans and clinically relevant phenotypes and animal model pathogenesis, thereby shedding light on specific survival mechanisms within the CNS and identifying the pathways involved in yeast persistence. IMPORTANCE Infection outcomes for cryptococcosis, most commonly caused by C. neoformans, are influenced by host immune responses as well as by host and pathogen genetics. Infecting yeast isolates are genetically diverse; however, we lack a deep understanding of how this diversity impacts patient outcomes. To better understand both clinical isolate diversity and how diversity contributes to infection outcomes, we utilize a large collection of clinical C. neoformans samples that were isolated from patients enrolled in a clinical trial across 3 hospitals in Malawi. By combining whole-genome sequence data, clinical data, and in vitro growth data, we utilize genome-wide association approaches to examine the genetic basis of virulence. Genes with significant associations display virulence attributes in both murine and rabbit models, demonstrating that our approach can identify potential links between genetic variants and patho-biologically significant phenotypes

Gentamicin Susceptibility in Neisseria gonorrhoeae and Treatment Outcomes for Urogenital Gonorrhea After 25 Years of Sustained Gentamicin Use in Malawi

Background Gentamicin has been used for the treatment of gonorrhea in Malawi since 1993. However, declining clinical cure rates have been suspected. We evaluated current Neisseria gonorrhoeae susceptibility to gentamicin in vitro and clinically. Methods Men with acute urethritis were recruited at the Bwaila District Hospital STI Clinic in Lilongwe, Malawi, between January 2017 and August 2019. All men provided urethral swabs for etiological testing at enrollment and test of cure (TOC), 1 week later, using Gram-stained microscopy and culture. We used Etest to determine minimum inhibitory concentrations (MICs) of gentamicin, azithromycin, cefixime, ceftriaxone, ciprofloxacin, and spectinomycin; disc diffusion for tetracycline susceptibility; and whole-genome sequencing (WGS) to verify/refute treatment failure. Results Among 183 N. gonorrhoeae culture-positive men enrolled, 151 (82.5%) had a swab taken for TOC. Of these 151 men, 16 (10.6%) had a positive culture at TOC. One hundred forty-one baseline isolates were tested for gentamicin susceptibility using Etest: 2 (1.4%), MIC = 2 μg/mL; 111 (78.7%), MIC = 4 μg/mL; and 28 (19.9%), MIC = 8 μg/mL. All isolates were susceptible to azithromycin, cefixime, ceftriaxone, and spectinomycin, whereas 63.1% had intermediate susceptibility or resistance to ciprofloxacin. Almost all (96.1%) isolates were resistant to tetracycline. All examined isolates cultured at TOC (n = 13) had gentamicin MICs ≤8 μg/mL. Ten men had pretreatment and posttreatment isolates examined by whole-genome sequencing; 2 (20%) were verified new infections (4119 and 1272 single-nucleotide polymorphisms), whereas 8 (80%) were confirmed treatment failures (0-1 single-nucleotide polymorphism). Conclusions Gentamicin MICs poorly predict gonorrhea treatment outcome with gentamicin, and treatment failures are verified with gonococcal strains with in vitro susceptibility to gentamicin. The first-line treatment of gonorrhea in Malawi should be reassessed

Poor association between 13-valent pneumococcal conjugate vaccine-induced serum and mucosal antibody responses with experimental Streptococcus pneumoniae serotype 6B colonisation

Background Pneumococcal carriage is the primary reservoir for transmission and a prerequisite for invasive pneumococcal disease. Pneumococcal Conjugate Vaccine 13 (PCV13) showed a 62% efficacy in protection against experimental Streptococcus pneumoniae serotype 6B (Spn6B) carriage in a controlled human infection model (CHIM) of healthy Malawian adults. We, therefore, measured humoral responses to experimental challenge and PCV-13 vaccination and determined the association with protection against pneumococcal carriage. Methods We vaccinated 204 young, healthy Malawian adults with PCV13 or placebo and nasally inoculated them with Spn6B at least four weeks post-vaccination to establish carriage. We collected peripheral blood and nasal lining fluid at baseline, 4 weeks post-vaccination (7 days pre-inoculation), 2, 7, 14 and > 1 year post-inoculation. We measured the concentration of anti-serotype 6B Capsular Polysaccharide (CPS) Immunoglobulin G (IgG) and IgA antibodies in serum and nasal lining fluid using the World Health Organization (WHO) standardised enzyme-linked immunosorbent assay (ELISA). Results PCV13-vaccinated adults had higher serum IgG and nasal IgG/IgA anti-Spn6B CPS-specific binding antibodies than placebo recipients 4 to 6 weeks post-vaccination, which persisted for at least a year after vaccination. Nasal challenge with Spn6B did not significantly alter serum or nasal anti-CPS IgG binding antibody titers with or without experimental pneumococcal carriage. Pre-challenge titers of PCV13-induced serum IgG and nasal IgG/IgA anti-Spn6B CPS binding antibodies did not significantly differ between those that got experimentally colonised by Spn6B compared to those that did not. Conclusion This study demonstrates that despite high PCV13 efficacy against experimental Spn6B carriage in young, healthy Malawian adults, robust vaccine-induced systemic and mucosal anti-Spn6B CPS binding antibodies did not directly relate to protection

Development and validation of quantitative PCR assays for HIV-associated cryptococcal meningitis in sub-Saharan Africa: a diagnostic accuracy study

Background: HIV-associated cryptococcal meningitis is the second leading cause of AIDS-related deaths, with a 10-week mortality rate of 25–30%. Fungal load assessed by colony-forming unit (CFU) counts is used as a prognostic marker and to monitor response to treatment in research studies. PCR-based assessment of fungal load could be quicker and less labour-intensive. We sought to design, optimise, and validate quantitative PCR (qPCR) assays for the detection, identification, and quantification of Cryptococcus infections in patients with cryptococcal meningitis in sub-Saharan Africa. Methods: We developed and validated species-specific qPCR assays based on DNA amplification of QSP1 (QSP1A specific to Cryptococcus neoformans, QSP1B/C specific to Cryptococcus deneoformans, and QSP1D specific to Cryptococcus gattii species) and a pan-Cryptococcus assay based on a multicopy 28S rRNA gene. This was a longitudinal study that validated the designed assays on cerebrospinal fluid (CSF) of 209 patients with cryptococcal meningitis at baseline (day 0) and during anti-fungal therapy (day 7 and day 14), from the AMBITION-cm trial in Botswana and Malawi (2018–21). Eligible patients were aged 18 years or older and presenting with a first case of cryptococcal meningitis. Findings: When compared with quantitative cryptococcal culture as the reference, the sensitivity of the 28S rRNA was 98·2% (95% CI 95·1–99·5) and of the QSP1 assay was 90·4% (85·2–94·0) in CSF at day 0. Quantification of the fungal load with QSP1 and 28S rRNA qPCR correlated with quantitative cryptococcal culture (R2=0·73 and R2=0·78, respectively). Both Botswana and Malawi had a predominant C neoformans prevalence of 67% (95% CI 55–75) and 68% (57–73), respectively, and lower C gattii rates of 21% (14–31) and 8% (4–14), respectively. We identified ten patients that, after 14 days of treatment, harboured viable but non-culturable yeasts based on QSP1 RNA detection (without any positive CFU in CSF culture). Interpretation: QSP1 and 28S rRNA assays are useful in identifying Cryptococcus species. qPCR results correlate well with baseline quantitative cryptococcal culture and show a similar decline in fungal load during induction therapy. These assays could be a faster alternative to quantitative cryptococcal culture to determine fungal load clearance. The clinical implications of the possible detection of viable but non-culturable cells in CSF during induction therapy remain unclear

Development and validation of quantitative PCR assays for HIV-associated cryptococcal meningitis in sub-Saharan Africa: a diagnostic accuracy study

Background: HIV-associated cryptococcal meningitis is the second leading cause of AIDS-related deaths, with a 10-week mortality rate of 25–30%. Fungal load assessed by colony-forming unit (CFU) counts is used as a prognostic marker and to monitor response to treatment in research studies. PCR-based assessment of fungal load could be quicker and less labour-intensive. We sought to design, optimise, and validate quantitative PCR (qPCR) assays for the detection, identification, and quantification of Cryptococcus infections in patients with cryptococcal meningitis in sub-Saharan Africa. Methods: We developed and validated species-specific qPCR assays based on DNA amplification of QSP1 (QSP1A specific to Cryptococcus neoformans, QSP1B/C specific to Cryptococcus deneoformans, and QSP1D specific to Cryptococcus gattii species) and a pan-Cryptococcus assay based on a multicopy 28S rRNA gene. This was a longitudinal study that validated the designed assays on cerebrospinal fluid (CSF) of 209 patients with cryptococcal meningitis at baseline (day 0) and during anti-fungal therapy (day 7 and day 14), from the AMBITION-cm trial in Botswana and Malawi (2018–21). Eligible patients were aged 18 years or older and presenting with a first case of cryptococcal meningitis. Findings: When compared with quantitative cryptococcal culture as the reference, the sensitivity of the 28S rRNA was 98·2% (95% CI 95·1–99·5) and of the QSP1 assay was 90·4% (85·2–94·0) in CSF at day 0. Quantification of the fungal load with QSP1 and 28S rRNA qPCR correlated with quantitative cryptococcal culture (R2=0·73 and R2=0·78, respectively). Both Botswana and Malawi had a predominant C neoformans prevalence of 67% (95% CI 55–75) and 68% (57–73), respectively, and lower C gattii rates of 21% (14–31) and 8% (4–14), respectively. We identified ten patients that, after 14 days of treatment, harboured viable but non-culturable yeasts based on QSP1 RNA detection (without any positive CFU in CSF culture). Interpretation: QSP1 and 28S rRNA assays are useful in identifying Cryptococcus species. qPCR results correlate well with baseline quantitative cryptococcal culture and show a similar decline in fungal load during induction therapy. These assays could be a faster alternative to quantitative cryptococcal culture to determine fungal load clearance. The clinical implications of the possible detection of viable but non-culturable cells in CSF during induction therapy remain unclear. Funding: European and Developing Countries Clinical Trials Partnership; Swedish International Development Cooperation Agency; Wellcome Trust/UK Medical Research Council/UKAID Joint Global Health Trials; and UK National Institute for Health Research

Simple measures are as effective as invasive techniques in the diagnosis of pulmonary tuberculosis in Malawi.

SETTING: Detection of smear-positive pulmonary tuberculosis (PTB) cases is vital for tuberculosis (TB) control. Methods to augment sputum collection are available, but their additional benefit is uncertain in resource-limited settings. OBJECTIVE: To compare the diagnostic yields using five methods to obtain sputum from adults diagnosed with smear-negative PTB in Malawi. DESIGN: Self-expectorated sputum was collected under supervision for microscopy and mycobacterial culture in the study laboratory. Confirmed smear-negative patients provided physiotherapy-assisted sputum and induced sputum, followed the next morning by gastric washing and bronchoalveolar lavage (BAL) samples. RESULTS: A total of 150 patients diagnosed with smear-negative PTB by the hospital service were screened; 39 (26%) were smear-positive from supervised self-expectorated sputum examined in the study laboratory. The remaining 111 confirmed smear-negative patients were enrolled in the study; 89% were human immunodeficiency virus positive. Seven additional smear-positive cases were diagnosed using the augmented sputum collection techniques. No differences were observed in the numbers of cases detected using the different methods. Of the 46 smear-positive cases, 44 (95.6%) could be detected from self-expectorated and physiotherapy-assisted samples. CONCLUSIONS: For countries such as Malawi, the best use of limited resources to detect smear-positive PTB cases would be to improve the quality of self-expectorated sputum collection and microscopy. The additional diagnostic yield using BAL after induced sputum is limited

Development and validation of quantitative PCR assays for HIV-associated cryptococcal meningitis in sub-Saharan Africa: a diagnostic accuracy study

Background: HIV-associated cryptococcal meningitis is the second leading cause of AIDS-related deaths, with a 10-week mortality rate of 25–30%. Fungal load assessed by colony-forming unit (CFU) counts is used as a prognostic marker and to monitor response to treatment in research studies. PCR-based assessment of fungal load could be quicker and less labour-intensive. We sought to design, optimise, and validate quantitative PCR (qPCR) assays for the detection, identification, and quantification of Cryptococcus infections in patients with cryptococcal meningitis in sub-Saharan Africa. Methods: We developed and validated species-specific qPCR assays based on DNA amplification of QSP1 (QSP1A specific to Cryptococcus neoformans, QSP1B/C specific to Cryptococcus deneoformans, and QSP1D specific to Cryptococcus gattii species) and a pan-Cryptococcus assay based on a multicopy 28S rRNA gene. This was a longitudinal study that validated the designed assays on cerebrospinal fluid (CSF) of 209 patients with cryptococcal meningitis at baseline (day 0) and during anti-fungal therapy (day 7 and day 14), from the AMBITION-cm trial in Botswana and Malawi (2018–21). Eligible patients were aged 18 years or older and presenting with a first case of cryptococcal meningitis. Findings: When compared with quantitative cryptococcal culture as the reference, the sensitivity of the 28S rRNA was 98·2% (95% CI 95·1–99·5) and of the QSP1 assay was 90·4% (85·2–94·0) in CSF at day 0. Quantification of the fungal load with QSP1 and 28S rRNA qPCR correlated with quantitative cryptococcal culture (R2=0·73 and R2=0·78, respectively). Both Botswana and Malawi had a predominant C neoformans prevalence of 67% (95% CI 55–75) and 68% (57–73), respectively, and lower C gattii rates of 21% (14–31) and 8% (4–14), respectively. We identified ten patients that, after 14 days of treatment, harboured viable but non-culturable yeasts based on QSP1 RNA detection (without any positive CFU in CSF culture). Interpretation: QSP1 and 28S rRNA assays are useful in identifying Cryptococcus species. qPCR results correlate well with baseline quantitative cryptococcal culture and show a similar decline in fungal load during induction therapy. These assays could be a faster alternative to quantitative cryptococcal culture to determine fungal load clearance. The clinical implications of the possible detection of viable but non-culturable cells in CSF during induction therapy remain unclear. Funding: European and Developing Countries Clinical Trials Partnership; Swedish International Development Cooperation Agency; Wellcome Trust/UK Medical Research Council/UKAID Joint Global Health Trials; and UK National Institute for Health Research