Characterisation of Staphylococci species from neonatal blood cultures in low- and middle-income countries.

BACKGROUND: In low- and middle-income countries (LMIC) Staphylococcus aureus is regarded as one of the leading bacterial causes of neonatal sepsis, however there is limited knowledge on the species diversity and antimicrobial resistance caused by Gram-positive bacteria (GPB). METHODS: We characterised GPB isolates from neonatal blood cultures from LMICs in Africa (Ethiopia, Nigeria, Rwanda, and South Africa) and South-Asia (Bangladesh and Pakistan) between 2015-2017. We determined minimum inhibitory concentrations and performed whole genome sequencing (WGS) on Staphylococci isolates recovered and clinical data collected related to the onset of sepsis and the outcome of the neonate up to 60 days of age. RESULTS: From the isolates recovered from blood cultures, Staphylococci species were most frequently identified. Out of 100 S. aureus isolates sequenced, 18 different sequence types (ST) were found which unveiled two small epidemiological clusters caused by methicillin resistant S. aureus (MRSA) in Pakistan (ST8) and South Africa (ST5), both with high mortality (n = 6/17). One-third of S. aureus was MRSA, with methicillin resistance also detected in Staphylococcus epidermidis, Staphylococcus haemolyticus and Mammaliicoccus sciuri. Through additional WGS analysis we report a cluster of M. sciuri in Pakistan identified between July-November 2017. CONCLUSIONS: In total we identified 14 different GPB bacterial species, however Staphylococci was dominant. These findings highlight the need of a prospective genomic epidemiology study to comprehensively assess the true burden of GPB neonatal sepsis focusing specifically on mechanisms of resistance and virulence across species and in relation to neonatal outcome.

Effect of deworming on disease progression markers in HIV-1-infected pregnant women on antiretroviral therapy: a longitudinal observational study from Rwanda.

BACKGROUND: Deworming human immunodeficiency virus (HIV)-infected individuals on antiretroviral therapy (ART) may be beneficial, particularly during pregnancy. We determined the efficacy of targeted and nontargeted antihelminth therapy and its effects on Plasmodium falciparum infection status, hemoglobin levels, CD4 counts, and viral load in pregnant, HIV-positive women receiving ART. METHODS: Nine hundred eighty HIV-infected pregnant women receiving ART were examined at 2 visits during pregnancy and 2 postpartum visits within 12 weeks. Women were given antimalarials when malaria-positive whereas albendazole was given in a targeted (n = 467; treatment when helminth stool screening was positive) or nontargeted (n = 513; treatment at all time points, with stool screening) fashion. RESULTS: No significant differences were noted between targeted and nontargeted albendazole treatments for the variables measured at each study visit except for CD4 counts, which were lower (P < .05) in the latter group at the final visit. Albendazole therapy was associated with favorable changes in subjects' hemoglobin levels, CD4 counts, and viral loads, particularly with helminth infections. CONCLUSIONS: Antihelminthic therapy reduces detectable viral load, and increases CD4 counts and hemoglobin levels in pregnant HIV-infected women with helminth coinfections receiving ART.

Colonisation of hospital surfaces from low- and middle-income countries by extended spectrum ß-lactamase- and carbapenemase-producing bacteria.

Hospital surfaces can harbour bacterial pathogens, which may disseminate and cause nosocomial infections, contributing towards mortality in low- and middle-income countries (LMICs). During the BARNARDS study, hospital surfaces from neonatal wards were sampled to assess the degree of environmental surface and patient care equipment colonisation by Gram-negative bacteria (GNB) carrying antibiotic resistance genes (ARGs). Here, we perform PCR screening for extended-spectrum ß-lactamases (blaCTX-M-15) and carbapenemases (blaNDM, blaOXA-48-like and blaKPC), MALDI-TOF MS identification of GNB carrying ARGs, and further analysis by whole genome sequencing of bacterial isolates. We determine presence of consistently dominant clones and their relatedness to strains causing neonatal sepsis. Higher prevalence of carbapenemases is observed in Pakistan, Bangladesh, and Ethiopia, compared to other countries, and are mostly found in surfaces near the sink drain. Klebsiella pneumoniae, Enterobacter hormaechei, Acinetobacter baumannii, Serratia marcescens and Leclercia adecarboxylata are dominant; ST15 K. pneumoniae is identified from the same ward on multiple occasions suggesting clonal persistence within the same environment, and is found to be identical to isolates causing neonatal sepsis in Pakistan over similar time periods. Our data suggests persistence of dominant clones across multiple time points, highlighting the need for assessment of Infection Prevention and Control guidelines.

Salmonella Typhi whole genome sequencing in Rwanda shows a diverse historical population with recent introduction of haplotype H58.

Salmonella enterica serovar Typhi (S. Typhi) is the cause of typhoid fever, presenting high rates of morbidity and mortality in low- and middle-income countries. The H58 haplotype shows high levels of antimicrobial resistance (AMR) and is the dominant S. Typhi haplotype in endemic areas of Asia and East sub-Saharan Africa. The situation in Rwanda is currently unknown and therefore to reveal the genetic diversity and AMR of S. Typhi in Rwanda, 25 historical (1984-1985) and 26 recent (2010-2018) isolates from Rwanda were analysed using whole genome sequencing (WGS). WGS was locally implemented using Illumina MiniSeq and web-based analysis tools, thereafter complemented with bioinformatic approaches for more in-depth analyses. Whereas historical S. Typhi isolates were found to be fully susceptible to antimicrobials and show a diversity of genotypes, i.e 2.2.2, 2.5, 3.3.1 and 4.1; the recent isolates showed high AMR rates and were predominantly associated with genotype 4.3.1.2 (H58, 22/26; 84,6%), possibly resulting from a single introduction in Rwanda from South Asia before 2010. We identified practical challenges for the use of WGS in endemic regions, including a high cost for shipment of molecular reagents and lack of high-end computational infrastructure for the analyses, but also identified WGS to be feasible in the studied setting and giving opportunity for synergy with other programs.

Multidrug-resistant tuberculosis control in Rwanda overcomes a successful clone that causes most disease over a quarter century.

SUMMARY BACKGROUND: Multidrug-resistant (MDR) tuberculosis (TB) poses an important challenge in TB management and control. Rifampicin resistance (RR) is a solid surrogate marker of MDR-TB. We investigated the RR-TB clustering rates, bacterial population dynamics to infer transmission dynamics, and the impact of changes to patient management on these dynamics over 27 years in Rwanda. METHODS: We analysed whole genome sequences of a longitudinal collection of nationwide RR-TB isolates. The collection covered three important periods: before programmatic management of MDR-TB (PMDT; 1991-2005), the early PMDT phase (2006-2013), in which rifampicin drug-susceptibility testing (DST) was offered to retreatment patients only, and the consolidated phase (2014-2018), in which all bacteriologically confirmed TB patients had rifampicin DST done mostly via Xpert MTB/RIF assay. We constructed clusters based on a 5 SNP cut-off and resistance conferring SNPs. We used Bayesian modelling for dating and population size estimations, TransPhylo to estimate the number of secondary cases infected by each patient, and multivariable logistic regression to assess predictors of being infected by the dominant clone. RESULTS: Of 308 baseline RR-TB isolates considered for transmission analysis, the clustering analysis grouped 259 (84.1%) isolates into 13 clusters. Within these clusters, a single dominant clone was discovered containing 213 isolates (82.2% of clustered and 69.1% of all RR-TB), which we named the "Rwanda Rifampicin-Resistant clone" (R3clone). R3clone isolates belonged to Ugandan sub-lineage 4.6.1.2 and its rifampicin and isoniazid resistance were conferred by the Ser450Leu mutation in rpoB and Ser315Thr in katG genes, respectively. All R3clone isolates had Pro481Thr, a putative compensatory mutation in the rpoC gene that likely restored its fitness. The R3clone was estimated to first arise in 1987 and its population size increased exponentially through the 1990s', reaching maximum size (∼84%) in early 2000 s', with a declining trend since 2014. Indeed, the highest proportion of R3clone (129/157; 82·2%, 95%CI: 75·3-87·8%) occurred between 2000 and 13, declining to 64·4% (95%CI: 55·1-73·0%) from 2014 onward. We showed that patients with R3clone detected after an unsuccessful category 2 treatment were more likely to generate secondary cases than patients with R3clone detected after an unsuccessful category 1 treatment regimen. CONCLUSIONS: RR-TB in Rwanda is largely transmitted. Xpert MTB/RIF assay as first diagnostic test avoids unnecessary rounds of rifampicin-based TB treatment, thus preventing ongoing transmission of the dominant R3clone. As PMDT was intensified and all TB patients accessed rifampicin-resistance testing, the nationwide R3clone burden declined. To our knowledge, our findings provide the first evidence supporting the impact of universal DST on the transmission of RR-TB.

Neonatal sepsis and mortality in low-income and middle-income countries from a facility-based birth cohort: an international multisite prospective observational study.

BACKGROUND: Neonatal sepsis is a primary cause of neonatal mortality and is an urgent global health concern, especially within low-income and middle-income countries (LMICs), where 99% of global neonatal mortality occurs. The aims of this study were to determine the incidence and associations with neonatal sepsis and all-cause mortality in facility-born neonates in LMICs. METHODS: The Burden of Antibiotic Resistance in Neonates from Developing Societies (BARNARDS) study recruited mothers and their neonates into a prospective observational cohort study across 12 clinical sites from Bangladesh, Ethiopia, India, Pakistan, Nigeria, Rwanda, and South Africa. Data for sepsis-associated factors in the four domains of health care, maternal, birth and neonatal, and living environment were collected for all mothers and neonates enrolled. Primary outcomes were clinically suspected sepsis, laboratory-confirmed sepsis, and all-cause mortality in neonates during the first 60 days of life. Incidence proportion of livebirths for clinically suspected sepsis and laboratory-confirmed sepsis and incidence rate per 1000 neonate-days for all-cause mortality were calculated. Modified Poisson regression was used to investigate factors associated with neonatal sepsis and parametric survival models for factors associated with all-cause mortality. FINDINGS: Between Nov 12, 2015 and Feb 1, 2018, 29 483 mothers and 30 557 neonates were enrolled. The incidence of clinically suspected sepsis was 166·0 (95% CI 97·69-234·24) per 1000 livebirths, laboratory-confirmed sepsis was 46·9 (19·04-74·79) per 1000 livebirths, and all-cause mortality was 0·83 (0·37-2·00) per 1000 neonate-days. Maternal hypertension, previous maternal hospitalisation within 12 months, average or higher monthly household income, ward size (>11 beds), ward type (neonatal), living in a rural environment, preterm birth, perinatal asphyxia, and multiple births were associated with an increased risk of clinically suspected sepsis, laboratory-confirmed sepsis, and all-cause mortality. The majority (881 [72·5%] of 1215) of laboratory-confirmed sepsis cases occurred within the first 3 days of life. INTERPRETATION: Findings from this study highlight the substantial proportion of neonates who develop neonatal sepsis, and the high mortality rates among neonates with sepsis in LMICs. More efficient and effective identification of neonatal sepsis is needed to target interventions to reduce its incidence and subsequent mortality in LMICs. FUNDING: Bill & Melinda Gates Foundation.

Strengthening Antimicrobial Resistance Diagnostic Capacity in Rural Rwanda: A Feasibility Assessment.

Introduction: Antimicrobial resistance (AMR) is a global public health threat. Worse still, there is a paucity of data from low- and middle-income countries to inform rational antibiotic use. Objective: Assess the feasibility of setting up microbiology capacity for AMR testing and estimate the cost of setting up microbiology testing capacity at rural district hospitals in Rwanda. Methods: Laboratory needs assessments were conducted, and based on identified equipment gaps, appropriate requisitions were processed. Laboratory technicians were trained on microbiology testing processes and open wound samples were collected and cultured at the district hospital (DH) laboratories before being transported to the National Reference Laboratory (NRL) for bacterial identification and antibiotic susceptibility testing. Quality control (QC) assessments were performed at the DHs and NRL. We then estimated the cost of three scenarios for implementing a decentralized microbiology diagnostic testing system. Results: There was an eight-month delay from the completion of the laboratory needs assessments to the initiation of sample collection due to the regional unavailability of appropriate supplies and equipment. When comparing study samples processed by study laboratory technicians and QC samples processed by other laboratory staff, there was 85.0% test result concordance for samples testing at the DHs and 90.0% concordance at the NRL. The cost for essential equipment and supplies for the three DHs was $245,871. The estimated costs for processing 600 samples ranged from $29,500 to $92,590. Conclusion: There are major gaps in equipment and supply availability needed to conduct basic microbiology assays at rural DHs. Despite these challenges, we demonstrated that it is feasible to establish microbiological testing capacity in Rwandan DHs. Building microbiological testing capacity is essential for improving clinical care, informing rational antibiotics use, and ultimately, contributing to the establishment of robust national antimicrobial stewardship programs in rural Rwanda and comparable settings.

Effects of antibiotic resistance, drug target attainment, bacterial pathogenicity and virulence, and antibiotic access and affordability on outcomes in neonatal sepsis: an international microbiology and drug evaluation prospective substudy (BARNARDS).

BACKGROUND: Sepsis is a major contributor to neonatal mortality, particularly in low-income and middle-income countries (LMICs). WHO advocates ampicillin-gentamicin as first-line therapy for the management of neonatal sepsis. In the BARNARDS observational cohort study of neonatal sepsis and antimicrobial resistance in LMICs, common sepsis pathogens were characterised via whole genome sequencing (WGS) and antimicrobial resistance profiles. In this substudy of BARNARDS, we aimed to assess the use and efficacy of empirical antibiotic therapies commonly used in LMICs for neonatal sepsis. METHODS: In BARNARDS, consenting mother-neonates aged 0-60 days dyads were enrolled on delivery or neonatal presentation with suspected sepsis at 12 BARNARDS clinical sites in Bangladesh, Ethiopia, India, Pakistan, Nigeria, Rwanda, and South Africa. Stillborn babies were excluded from the study. Blood samples were collected from neonates presenting with clinical signs of sepsis, and WGS and minimum inhibitory concentrations for antibiotic treatment were determined for bacterial isolates from culture-confirmed sepsis. Neonatal outcome data were collected following enrolment until 60 days of life. Antibiotic usage and neonatal outcome data were assessed. Survival analyses were adjusted to take into account potential clinical confounding variables related to the birth and pathogen. Additionally, resistance profiles, pharmacokinetic-pharmacodynamic probability of target attainment, and frequency of resistance (ie, resistance defined by in-vitro growth of isolates when challenged by antibiotics) were assessed. Questionnaires on health structures and antibiotic costs evaluated accessibility and affordability. FINDINGS: Between Nov 12, 2015, and Feb 1, 2018, 36 285 neonates were enrolled into the main BARNARDS study, of whom 9874 had clinically diagnosed sepsis and 5749 had available antibiotic data. The four most commonly prescribed antibiotic combinations given to 4451 neonates (77·42%) of 5749 were ampicillin-gentamicin, ceftazidime-amikacin, piperacillin-tazobactam-amikacin, and amoxicillin clavulanate-amikacin. This dataset assessed 476 prescriptions for 442 neonates treated with one of these antibiotic combinations with WGS data (all BARNARDS countries were represented in this subset except India). Multiple pathogens were isolated, totalling 457 isolates. Reported mortality was lower for neonates treated with ceftazidime-amikacin than for neonates treated with ampicillin-gentamicin (hazard ratio [adjusted for clinical variables considered potential confounders to outcomes] 0·32, 95% CI 0·14-0·72; p=0·0060). Of 390 Gram-negative isolates, 379 (97·2%) were resistant to ampicillin and 274 (70·3%) were resistant to gentamicin. Susceptibility of Gram-negative isolates to at least one antibiotic in a treatment combination was noted in 111 (28·5%) to ampicillin-gentamicin; 286 (73·3%) to amoxicillin clavulanate-amikacin; 301 (77·2%) to ceftazidime-amikacin; and 312 (80·0%) to piperacillin-tazobactam-amikacin. A probability of target attainment of 80% or more was noted in 26 neonates (33·7% [SD 0·59]) of 78 with ampicillin-gentamicin; 15 (68·0% [3·84]) of 27 with amoxicillin clavulanate-amikacin; 93 (92·7% [0·24]) of 109 with ceftazidime-amikacin; and 70 (85·3% [0·47]) of 76 with piperacillin-tazobactam-amikacin. However, antibiotic and country effects could not be distinguished. Frequency of resistance was recorded most frequently with fosfomycin (in 78 isolates [68·4%] of 114), followed by colistin (55 isolates [57·3%] of 96), and gentamicin (62 isolates [53·0%] of 117). Sites in six of the seven countries (excluding South Africa) stated that the cost of antibiotics would influence treatment of neonatal sepsis. INTERPRETATION: Our data raise questions about the empirical use of combined ampicillin-gentamicin for neonatal sepsis in LMICs because of its high resistance and high rates of frequency of resistance and low probability of target attainment. Accessibility and affordability need to be considered when advocating antibiotic treatments with variance in economic health structures across LMICs. FUNDING: The Bill & Melinda Gates Foundation.

Characterization of antimicrobial-resistant Gram-negative bacteria that cause neonatal sepsis in seven low- and middle-income countries.

Antimicrobial resistance in neonatal sepsis is rising, yet mechanisms of resistance that often spread between species via mobile genetic elements, ultimately limiting treatments in low- and middle-income countries (LMICs), are poorly characterized. The Burden of Antibiotic Resistance in Neonates from Developing Societies (BARNARDS) network was initiated to characterize the cause and burden of antimicrobial resistance in neonatal sepsis for seven LMICs in Africa and South Asia. A total of 36,285 neonates were enrolled in the BARNARDS study between November 2015 and December 2017, of whom 2,483 were diagnosed with culture-confirmed sepsis. Klebsiella pneumoniae (n = 258) was the main cause of neonatal sepsis, with Serratia marcescens (n = 151), Klebsiella michiganensis (n = 117), Escherichia coli (n = 75) and Enterobacter cloacae complex (n = 57) also detected. We present whole-genome sequencing, antimicrobial susceptibility and clinical data for 916 out of 1,038 neonatal sepsis isolates (97 isolates were not recovered from initial isolation at local sites). Enterobacterales (K. pneumoniae, E. coli and E. cloacae) harboured multiple cephalosporin and carbapenem resistance genes. All isolated pathogens were resistant to multiple antibiotic classes, including those used to treat neonatal sepsis. Intraspecies diversity of K. pneumoniae and E. coli indicated that multiple antibiotic-resistant lineages cause neonatal sepsis. Our results will underpin research towards better treatments for neonatal sepsis in LMICs.

Geographical distribution and prevalence of podoconiosis in Rwanda: a cross-sectional country-wide survey.

BACKGROUND: Podoconiosis is a type of tropical lymphoedema that causes massive swelling of the lower limbs. The disease is associated with both economic insecurity, due to long-term morbidity-related loss of productivity, and intense social stigma. Reliable and detailed data on the prevalence and distribution of podoconiosis are scarce. We aimed to fill this data gap by doing a nationwide community-based study to estimate the number of cases throughout Rwanda. METHODS: We did a population-based cross-sectional survey to determine the national prevalence of podoconiosis. A podoconiosis case was defined as a person with bilateral, asymmetrical lymphoedema of the lower limb present for more than 1 year, who tested negative for Wuchereria bancrofti antigen (determined by Filariasis Test Strip) and specific IgG4 (determined by Wb123 test), and had a history of any of the associated clinical signs and symptoms. All adults (aged ≥15 years) who resided in any of the 30 districts of Rwanda for 10 or more years were invited at the household level to participate. Participants were interviewed and given a physical examination before Filariasis Test Strip and Wb123 testing. We fitted a binomial mixed model combining the site-level podoconiosis prevalence with continuous environmental covariates to estimate prevalence at unsampled locations. We report estimates of cases by district combining our mean predicted prevalence and a contemporary gridded map of estimated population density. FINDINGS: Between June 12, and July 28, 2017, 1 360 612 individuals-719 730 (53%) women and 640 882 (47%) men-were screened from 80 clusters in 30 districts across Rwanda. 1143 individuals with lymphoedema were identified, of whom 914 (80%) had confirmed podoconiosis, based on the standardised diagnostic algorithm. The overall prevalence of podoconiosis was 68·5 per 100 000 people (95% CI 41·0-109·7). Podoconiosis was found to be widespread in Rwanda. District-level prevalence ranged from 28·3 per 100 000 people (16·8-45·5, Nyarugenge, Kigali province) to 119·2 per 100 000 people (59·9-216·2, Nyamasheke, West province). Prevalence was highest in districts in the North and West provinces: Nyamasheke, Rusizi, Musanze, Nyabihu, Nyaruguru, Burera, and Rubavu. We estimate that 6429 (95% CI 3938-10 088) people live with podoconiosis across Rwanda. INTERPRETATION: Despite relatively low prevalence, podoconiosis is widely distributed geographically throughout Rwanda. Many patients are likely to be undiagnosed and morbidity management is scarce. Targeted interventions through a well coordinated health system response are needed to manage those affected. Our findings should inform national level planning, monitoring, and implementation of interventions. FUNDING: Wellcome Trust.

Malaria and helminthic co-infection among HIV-positive pregnant women: prevalence and effects of antiretroviral therapy.

The impact of malaria on anemia and the interplay with helminths underline the importance of addressing the interactions between HIV/AIDS, malaria and intestinal helminth infections in pregnancy. The aim of this study was to determine the prevalence of malaria-helminth dual infections among HIV positive pregnant mothers after 12 months of ART. A cross sectional study was conducted on intestinal helminths and malaria dual infections among HIV-positive pregnant women attending antenatal health centers in Rwanda. Stool and malaria blood slide examinations were performed on 328 women residing in rural (n=166) and peri-urban locations (n=162). BMI, CD4 cell count, hemoglobin levels, type of ART and viral load of participants were assessed. Within the study group, 38% of individuals harbored helminths, 21% had malaria and 10% were infected with both. The most prevalent helminth species were Ascaris lumbricoides (20.7%), followed by Trichuris trichiura (9.2%), and Ancylostoma duodenale and Necator americanus (1.2%). Helminth infections were characterized by low hemoglobin and CD4 counts. Subjects treated with a d4T, 3TC, NVP regimen had a reduced risk of T. trichiura infection (OR, 0.27; 95% CIs, 0.10-0.76; p<0.05) and malaria-helminth dual infection (OR, 0.29; 95% CI, 0.11-0.75; p<0.05) compared to those receiving AZT, 3TC, NVP. This study shows a high prevalence of malaria and helminth infection among HIV-positive pregnant women in Rwanda. The differential effect of ARTs on the risk of helminth infection is of interest and should be examined prospectively in larger patient groups.