Longitudinal analysis within one hospital in sub-Saharan Africa over 20 years reveals repeated replacements of dominant clones of Klebsiella pneumoniae and stresses the importance to include temporal patterns for vaccine design considerations.

BACKGROUND: Infections caused by multidrug-resistant gram-negative bacteria present a severe threat to global public health. The WHO defines drug-resistant Klebsiella pneumoniae as a priority pathogen for which alternative treatments are needed given the limited treatment options and the rapid acquisition of novel resistance mechanisms by this species. Longitudinal descriptions of genomic epidemiology of Klebsiella pneumoniae can inform management strategies but data from sub-Saharan Africa are lacking. METHODS: We present a longitudinal analysis of all invasive K. pneumoniae isolates from a single hospital in Blantyre, Malawi, southern Africa, from 1998 to 2020, combining clinical data with genome sequence analysis of the isolates. RESULTS: We show that after a dramatic increase in the number of infections from 2016 K. pneumoniae becomes hyperendemic, driven by an increase in neonatal infections. Genomic data show repeated waves of clonal expansion of different, often ward-restricted, lineages, suggestive of hospital-associated transmission. We describe temporal trends in resistance and surface antigens, of relevance for vaccine development. CONCLUSIONS: Our data highlight a clear need for new interventions to prevent rather than treat K. pneumoniae infections in our setting. Whilst one option may be a vaccine, the majority of cases could be avoided by an increased focus on and investment in infection prevention and control measures, which would reduce all healthcare-associated infections and not just one.

Prevalence, risk factors, and antimicrobial resistance of endemic healthcare-associated infections in Africa: a systematic review and meta-analysis.

BACKGROUND: Healthcare-associated infections (HCAI) place a significant burden on healthcare systems globally. This systematic review and meta-analysis aimed to investigate the prevalence, risk factors, and aetiologic agents of endemic HCAI in Africa. METHODS: MEDLINE/PubMed, CINAHL, and Global Health databases (EBSCOhost interface) were searched for studies published in English and French describing HCAI in Africa from 2010 to 2022. We extracted data on prevalence of HCAI, risk factors, aetiologic agents, and associated antimicrobial resistance patterns. We used random-effects models to estimate parameter values with 95% confidence intervals for risk factors associated with HCAI. This study was registered in PROSPERO (CRD42022374559) and followed PRISMA 2020 guidelines. RESULTS: Of 2541 records screened, 92 were included, comprising data from 81,968 patients. Prevalence of HCAI varied between 1.6 and 90.2% with a median of 15% across studies. Heterogeneity (I2) varied from 93 to 99%. Contaminated wound (OR: 1.75, 95% CI: 1.31-2.19), long hospital stay (OR: 1.39, 95% CI: 0.92-1.80), urinary catheter (OR: 1.57, 95% CI: 0.35-2.78), intubation and ventilation (OR: 1.53, 95% CI: 0.85-2.22), vascular catheters (OR: 1.49, 95% CI: 0.52-2.45) were among risk factors associated with HCAI. Bacteria reported from included studies comprised 6463 isolates, with E. coli (18.3%, n = 1182), S. aureus (17.3%, n = 1118), Klebsiella spp. (17.2%, n = 1115), Pseudomonas spp. (10.3%, n = 671), and Acinetobacter spp. (6.8%, n = 438) being most common. Resistance to multiple antibiotics was common; 70.3% (IQR: 50-100) of Enterobacterales were 3rd -generation cephalosporin resistant, 70.5% (IQR: 58.8-80.3) of S. aureus were methicillin resistant and 55% (IQR: 27.3-81.3) Pseudomonas spp. were resistant to all agents tested. CONCLUSIONS: HCAI is a greater problem in Africa than other regions, however, there remains a paucity of data to guide local action. There is a clear need to develop and validate sustainable HCAI definitions in Africa to support the implementation of routine HCAI surveillance and inform implementation of context appropriate infection prevention and control strategies.

Evidence of widespread endemic populations of highly multidrug resistant Klebsiella pneumoniae in hospital settings in Hanoi, Vietnam: a prospective cohort study.

BACKGROUND: Patients with prolonged hospitalisation have a significant risk of carriage of and subsequent infection with extended spectrum ß-lactamase (ESBL)-producing and carbapenemase-producing Klebsiella pneumoniae. However, the distinctive roles of the community and hospital environments in the transmission of ESBL-producing or carbapenemase-producing K pneumoniae remain elusive. We aimed to investigate the prevalence and transmission of K pneumoniae within and between the two tertiary hospitals in Hanoi, Viet Nam, using whole-genome sequencing. METHODS: We did a prospective cohort study of 69 patients in intensive care units (ICUs) from two hospitals in Hanoi, Viet Nam. Patients were included if they were aged 18 years or older, admitted for longer than the mean length of stay in their ICU, and cultured K pneumoniae from their clinical samples. Longitudinally collected samples from patients (collected weekly) and the ICU environment (collected monthly) were cultured on selective media, and whole-genome sequences from K pneumoniae colonies analysed. We did phylogenetic analyses and correlated phenotypic antimicrobial susceptibility testing with genotypic features of K pneumoniae isolates. We constructed transmission networks of patient samples, relating ICU admission times and locations with genetic similarity of infecting K pneumoniae. FINDINGS: Between June 1, 2017, and Jan 31, 2018, 69 patients were in the ICUs and eligible for inclusion, and a total of 357 K pneumoniae isolates were cultured and successfully sequenced. 228 (64%) of K pneumoniae isolates carried between two and four different ESBL-encoding and carbapenemase-encoding genes, with 164 (46%) isolates carrying genes encoding both, with high minimum inhibitory concentrations. We found a novel co-occurrence of blaKPC-2 and blaNDM-1 in 46·6% of samples from the globally successful ST15 lineage. Despite being physically and clinically separated, the two hospitals shared closely related strains carrying the same array of antimicrobial resistance genes. INTERPRETATION: These results highlight the high prevalence of ESBL-positive carbapenem-resistant K pneumoniae in ICUs in Viet Nam. Through studying K pneumoniae ST15 in detail, we showed how important resistance genes are contained within these strains that are carried broadly by patients entering the two hospitals directly or through referral. FUNDING: Medical Research Council Newton Fund, Ministry of Science and Technology, Wellcome Trust, Academy of Medical Sciences, Health Foundation, and National Institute for Health and Care Research Cambridge Biomedical Research Centre.

Effect of resistance to third-generation cephalosporins on morbidity and mortality from bloodstream infections in Blantyre, Malawi: a prospective cohort study.

BACKGROUND: The burden of antimicrobial resistance is a major threat to global health; however, prospective clinical outcome data from Africa are scarce. In Malawi, third-generation cephalosporins are the antibiotics of choice in patients admitted to hospital despite a rapid proliferation of resistance to these drugs. We aimed to quantify the effect of resistance to third-generation cephalosporins on mortality and length of hospital stay among patients with bloodstream infections. METHODS: We did a prospective cohort study of patients admitted to Queen Elizabeth Central Hospital in Blantyre, Malawi. Patients of all ages who had positive blood cultures for Enterobacterales were included, with the exception of those from the genus Salmonella, and were followed up for 180 days. We characterised blood culture isolates using whole-genome sequencing and used Cox regression models to estimate the effect of resistance to third-generation cephalosporins on length of hospital stay, in-hospital mortality, and survival. FINDINGS: Between Jan 31, 2018, and Jan 13, 2020, we recruited 326 patients, from whom 220 (68%) of 326 isolates were resistant to third-generation cephalosporins. The case fatality proportion was 45% (99 of 220) in patients with bloodstream infections that were resistant to third-generation cephalosporins, and 34% (36 of 106) in patients with bloodstream infections that were sensitive to third-generation cephalosporins. Resistance to third-generation cephalosporins was associated with an increased probability of in-hospital mortality (hazard ratio [HR] 1·44, 95% CI 1·02-2·04), longer hospital stays (1·5 days, 1·0-2·0) and decreased probability of discharge alive (HR 0·31, 0·22-0·45). Whole-genome sequencing showed a high diversity of sequence types of both Escherichia coli and Klebsiella pneumoniae. Although isolates associated with death were distributed across clades, we identified three E coli clades (ST410, ST617, and ST648) that were isolated from 14 patients who all died. INTERPRETATION: Resistance to third-generation cephalosporins is associated with increased mortality and longer hospital stays in patients with bloodstream infections in Malawi. These data show the urgent need for allocation of resources towards antimicrobial resistance mitigation strategies in Africa. FUNDING: Wellcome Trust and Wellcome Asia and Africa Programme.

Drivers of Resistance in Uganda and Malawi (DRUM): a protocol for the evaluation of One-Health drivers of Extended Spectrum Beta Lactamase (ESBL) resistance in Low-Middle Income Countries (LMICs).

In sub-Saharan Africa (sSA), there is high morbidity and mortality from severe bacterial infection and this is compounded by antimicrobial resistance, in particular, resistance to 3rd-generation cephalosporins. This resistance is typically mediated by extended-spectrum beta lactamases (ESBLs). To interrupt ESBL transmission it will be important to investigate how human behaviour, water, sanitation, and hygiene (WASH) practices, environmental contamination, and antibiotic usage in both urban and rural settings interact to contribute to transmission of ESBL E. coli and ESBL K. pneumoniae between humans, animals, and the environment. Here we present the protocol for the Drivers of Resistance in Uganda and Malawi (DRUM) Consortium, in which we will collect demographic, geospatial, clinical, animal husbandry and WASH data from a total of 400 households in Uganda and Malawi. Longitudinal human, animal and environmental sampling at each household will be used to isolate ESBL E. coli and ESBL K. pneumoniae. This will be complimented by a Risks, Attitudes, Norms, Abilities and Self-Regulation (RANAS) survey and structured observations to understand the contextual and psychosocial drivers of regional WASH practices. Bacterial isolates and plate sweeps will be further characterised using a mixture of short-,long-read and metagenomic whole-genome sequencing. These datasets will be integrated into agent-based models to describe the transmission of EBSL resistance in Uganda and Malawi and allow us to inform the design of interventions for interrupting transmission of ESBL-bacteria.

Genomic investigation of a suspected Klebsiella pneumoniae outbreak in a neonatal care unit in sub-Saharan Africa.

A special-care neonatal unit from a large public hospital in Malawi was noted as having more frequent, difficult-to-treat infections, and a suspected outbreak of multi-drug-resistant Klebsiella pneumoniae was investigated using genomic characterisation. All K. pneumoniae bloodstream infections (BSIs) from patients in the neonatal ward (n=62), and a subset of K. pneumoniae BSI isolates (n=38) from other paediatric wards in the hospital, collected over a 4 year period were studied. After whole genome sequencing, the strain sequence types (STs), plasmid types, virulence and resistance genes were identified. One ST340 clone, part of clonal complex 258 (CC258) and an ST that drives hospital outbreaks worldwide, harbouring numerous resistance genes and plasmids, was implicated as the likely cause of the outbreak. This study contributes molecular information necessary for tracking and characterizing this important hospital pathogen in sub-Saharan Africa.

A novel air-dried multiplex high-resolution melt assay for the detection of extended-spectrum ß-lactamase and carbapenemase genes.

OBJECTIVES: This study aimed to develop and evaluate a novel air-dried high-resolution melt (HRM) assay to detect eight major extended-spectrum ß-lactamase (ESBL) (blaSHV and blaCTX-M groups 1 and 9) and carbapenemase (blaNDM, blaIMP, blaKPC, blaVIM and blaOXA-48-like) genes that confer resistance to cephalosporins and carbapenems. METHODS: The assay was evaluated using 439 DNA samples extracted from bacterial isolates from Nepal, Malawi and the UK and 390 clinical isolates from Nepal with known antimicrobial susceptibility. Assay reproducibility was evaluated across five different real-time quantitative PCR (qPCR) instruments [Rotor-Gene® Q, QuantStudioTM 5, CFX96, LightCycler® 480 and Magnetic Induction Cycler (Mic)]. Assay stability was also assessed under different storage temperatures (6.2 ± 0.9°C, 20.4 ± 0.7°C and 29.7 ± 1.4°C) at six time points over 8 months. RESULTS: The sensitivity and specificity (with 95% confidence intervals) for detecting ESBL and carbapenemase genes was 94.7% (92.5-96.5%) and 99.2% (98.8-99.5%) compared with the reference gel-based PCR and sequencing and 98.3% (97.0-99.3%) and 98.5% (98.0-98.9%) compared with the original HRM wet PCR mix format. Overall agreement was 91.1% (90.0-92.9%) when predicting phenotypic resistance to cefotaxime and meropenem among Enterobacteriaceae isolates. We observed almost perfect inter-machine reproducibility of the air-dried HRM assay, and no loss of sensitivity occurred under all storage conditions and time points. CONCLUSION: We present a ready-to-use air-dried HRM PCR assay that offers an easy, thermostable, fast and accurate tool for the detection of ESBL and carbapenemase genes in DNA samples to improve antimicrobial resistance detection.

Pervasive transmission of a carbapenem resistance plasmid in the gut microbiota of hospitalized patients.

Infections caused by carbapenemase-producing enterobacteria (CPE) are a major concern in clinical settings worldwide. Two fundamentally different processes shape the epidemiology of CPE in hospitals: the dissemination of CPE clones from patient to patient (between-patient transfer), and the transfer of carbapenemase-encoding plasmids between enterobacteria in the gut microbiota of individual patients (within-patient transfer). The relative contribution of each process to the overall dissemination of carbapenem resistance in hospitals remains poorly understood. Here, we used mechanistic models combining epidemiological data from more than 9,000 patients with whole genome sequence information from 250 enterobacteria clones to characterize the dissemination routes of a pOXA-48-like carbapenemase-encoding plasmid in a hospital setting over a 2-yr period. Our results revealed frequent between-patient transmission of high-risk pOXA-48-carrying clones, mostly of Klebsiella pneumoniae and sporadically Escherichia coli. The results also identified pOXA-48 dissemination hotspots within the hospital, such as specific wards and individual rooms within wards. Using high-resolution plasmid sequence analysis, we uncovered the pervasive within-patient transfer of pOXA-48, suggesting that horizontal plasmid transfer occurs in the gut of virtually every colonized patient. The complex and multifaceted epidemiological scenario exposed by this study provides insights for the development of intervention strategies to control the in-hospital spread of CPE.

Duration of Carbapenemase-Producing Enterobacteriaceae Carriage in Hospital Patients.

To determine the duration of carbapenemase-producing Enterobacteriaceae (CPE) carriage, we studied 21 CPE carriers for ¼1 year. Mean carriage duration was 86 days; probability of decolonization in 1 year was 98.5%, suggesting that CPE-carriers' status can be reviewed yearly. Prolonged carriage was associated with use of antimicrobial drugs.

Early Signals of Vaccine-driven Perturbation Seen in Pneumococcal Carriage Population Genomic Data.

BACKGROUND: Pneumococcal conjugate vaccines (PCVs) have reduced pneumococcal diseases globally. Pneumococcal genomic surveys elucidate PCV effects on population structure but are rarely conducted in low-income settings despite the high disease burden. METHODS: We undertook whole-genome sequencing (WGS) of 660 pneumococcal isolates collected through surveys from healthy carriers 2 years from 13-valent PCV (PCV13) introduction and 1 year after rollout in northern Malawi. We investigated changes in population structure, within-lineage serotype dynamics, serotype diversity, and frequency of antibiotic resistance (ABR) and accessory genes. RESULTS: In children <5 years of age, frequency and diversity of vaccine serotypes (VTs) decreased significantly post-PCV, but no significant changes occurred in persons ≥5 years of age. Clearance of VT serotypes was consistent across different genetic backgrounds (lineages). There was an increase of nonvaccine serotypes (NVTs)-namely 7C, 15B/C, and 23A-in children <5 years of age, but 28F increased in both age groups. While carriage rates have been recently shown to remain stable post-PCV due to replacement serotypes, there was no change in diversity of NVTs. Additionally, frequency of intermediate-penicillin-resistant lineages decreased post-PCV. Although frequency of ABR genes remained stable, other accessory genes, especially those associated with mobile genetic element and bacteriocins, showed changes in frequency post-PCV. CONCLUSIONS: We demonstrate evidence of significant population restructuring post-PCV driven by decreasing frequency of vaccine serotypes and increasing frequency of few NVTs mainly in children under 5. Continued surveillance with WGS remains crucial to fully understand dynamics of the residual VTs and replacement NVT serotypes post-PCV.

Prevalence and outcome of bloodstream infections due to third-generation cephalosporin-resistant Enterobacteriaceae in sub-Saharan Africa: a systematic review.

BACKGROUND: The prevalence of bacterial bloodstream infections (BSIs) in sub-Saharan Africa (sSA) is high and antimicrobial resistance is likely to increase mortality from these infections. Third-generation cephalosporin-resistant (3GC-R) Enterobacteriaceae are of particular concern, given the widespread reliance on ceftriaxone for management of sepsis in Africa. OBJECTIVES: Reviewing studies from sSA, we aimed to describe the prevalence of 3GC resistance in Escherichia coli, Klebsiella and Salmonella BSIs and the in-hospital mortality from 3GC-R BSIs. METHODS: We systematically reviewed studies reporting 3GC susceptibility testing of E. coli, Klebsiella and Salmonella BSI. We searched PubMed and Scopus from January 1990 to September 2019 for primary data reporting 3GC susceptibility testing of Enterobacteriaceae associated with BSI in sSA and studies reporting mortality from 3GC-R BSI. 3GC-R was defined as phenotypic resistance to ceftriaxone, cefotaxime or ceftazidime. Outcomes were reported as median prevalence of 3GC resistance for each pathogen. RESULTS: We identified 40 articles, including 7 reporting mortality. Median prevalence of 3GC resistance in E. coli was 18.4% (IQR 10.5 to 35.2) from 20 studies and in Klebsiella spp. was 54.4% (IQR 24.3 to 81.2) from 28 studies. Amongst non-typhoidal salmonellae, 3GC resistance was 1.9% (IQR 0 to 6.1) from 12 studies. A pooled mortality estimate was prohibited by heterogeneity. CONCLUSIONS: Levels of 3GC resistance amongst bloodstream Enterobacteriaceae in sSA are high, yet the mortality burden is unknown. The lack of clinical outcome data from drug-resistant infections in Africa represents a major knowledge gap and future work must link laboratory surveillance to clinical data.

Parallel evolution leading to impaired biofilm formation in invasive Salmonella strains.

Pathogenic Salmonella strains that cause gastroenteritis are able to colonize and replicate within the intestines of multiple host species. In general, these strains have retained an ability to form the rdar morphotype, a resistant biofilm physiology hypothesized to be important for Salmonella transmission. In contrast, Salmonella strains that are host-adapted or even host-restricted like Salmonella enterica serovar Typhi, tend to cause systemic infections and have lost the ability to form the rdar morphotype. Here, we investigated the rdar morphotype and CsgD-regulated biofilm formation in two non-typhoidal Salmonella (NTS) strains that caused invasive disease in Malawian children, S. Typhimurium D23580 and S. Enteritidis D7795, and compared them to a panel of NTS strains associated with gastroenteritis, as well as S. Typhi strains. Sequence comparisons combined with luciferase reporter technology identified key SNPs in the promoter region of csgD that either shut off biofilm formation completely (D7795) or reduced transcription of this key biofilm regulator (D23580). Phylogenetic analysis showed that these SNPs are conserved throughout the African clades of invasive isolates, dating as far back as 80 years ago. S. Typhi isolates were negative for the rdar morphotype due to truncation of eight amino acids from the C-terminus of CsgD. We present new evidence in support of parallel evolution between lineages of nontyphoidal Salmonella associated with invasive disease in Africa and the archetypal host-restricted invasive serovar; S. Typhi. We hypothesize that the African invasive isolates are becoming human-adapted and 'niche specialized' with less reliance on environmental survival, as compared to gastroenteritis-causing isolates.

Genomic analysis of Klebsiella pneumoniae isolates from Malawi reveals acquisition of multiple ESBL determinants across diverse lineages.

OBJECTIVES: ESBL-producing Klebsiella pneumoniae (KPN) pose a major threat to human health globally. We carried out a WGS study to understand the genetic background of ESBL-producing KPN in Malawi and place them in the context of other global isolates. METHODS: We sequenced genomes of 72 invasive and carriage KPN isolates collected from patients admitted to Queen Elizabeth Central Hospital, Blantyre, Malawi. We performed phylogenetic and population structure analyses on these and previously published genomes from Kenya (n = 66) and from outside sub-Saharan Africa (n = 67). We screened for presence of antimicrobial resistance (AMR) genetic determinants and carried out association analyses by genomic sequence cluster, AMR phenotype and time. RESULTS: Malawian isolates fit within the global population structure of KPN, clustering into the major lineages of KpI, KpII and KpIII. KpI isolates from Malawi were more related to those from Kenya, with both collections exhibiting more clonality than isolates from the rest of the world. We identified multiple ESBL genes, including blaCTX-M-15, several blaSHV, blaTEM-63 and blaOXA-10, and other AMR genes, across diverse lineages of the KPN isolates from Malawi. No carbapenem resistance genes were detected; however, we detected IncFII and IncFIB plasmids that were similar to the carbapenem resistance-associated plasmid pNDM-mar. CONCLUSIONS: There are multiple ESBL genes across diverse KPN lineages in Malawi and plasmids in circulation that are capable of carrying carbapenem resistance. Unless appropriate interventions are rapidly put in place, these may lead to a high burden of locally untreatable infection in vulnerable populations.

ChloS-HRM, a novel assay to identify chloramphenicol-susceptible Escherichia coli and Klebsiella pneumoniae in Malawi.

OBJECTIVES: Chloramphenicol is a broad-spectrum antimicrobial widely available in sub-Saharan Africa. With susceptibility re-emerging among Enterobacteriaceae in Blantyre, Malawi, we designed and evaluated a new high-resolution melt (HRM) RT-PCR assay, ChloS-HRM, to identify chloramphenicol-susceptible infections in a hospital setting. METHODS: Seventy-two previously whole-genome sequenced isolates of Escherichia coli and Klebsiella pneumoniae from the Queen Elizabeth Central Hospital, Malawi, were subjected to determination of chloramphenicol MICs. Primers were designed to detect 18 chloramphenicol resistance genes that produce seven distinct peaks correlating with different gene groups (catA1, catA2, catA3, catB2, catB group 3, cmlA and floR) following HRM analysis. ChloS-HRM results were compared with MIC and WGS results. RESULTS: ChloS-HRM correctly identified 15 of 17 phenotypically susceptible isolates and 54 of 55 resistant isolates, giving an accuracy of 88% in identifying susceptibility and 98% in identifying resistance. WGS identified 16 of 17 susceptible and 54 of 55 resistant isolates, giving an accuracy of 94% in identifying susceptibility and 98% in identifying resistance. The single false-susceptible result had no detectable gene by ChloS-HRM or WGS. Compared with WGS, ChloS-HRM had 100% sensitivity and specificity for catA (catA1-3), cmlA and floR, and 96% specificity for catB; sensitivity could not be estimated due to the lack of catB in the clinical sample collection. The overall agreement between MIC and HRM was 96% and between MIC and WGS it was 97%. CONCLUSIONS: ChloS-HRM could support antimicrobial stewardship in enabling de-escalation from third-generation cephalosporins by identifying chloramphenicol-susceptible infections. This would be valuable in areas with chloramphenicol-susceptible MDR and XDR Enterobacteriaceae.

Emerging Resistance to Empiric Antimicrobial Regimens for Pediatric Bloodstream Infections in Malawi (1998-2017).

BACKGROUND: The adequacy of the World Health Organization's Integrated Management of Childhood Illness (IMCI) antimicrobial guidelines for the treatment of suspected severe bacterial infections is dependent on a low prevalence of antimicrobial resistance (AMR). We describe trends in etiologies and susceptibility patterns of bloodstream infections (BSI) in hospitalized children in Malawi. METHODS: We determined the change in the population-based incidence of BSI in children admitted to Queen Elizabeth Central Hospital, Blantyre, Malawi (1998-2017). AMR profiles were assessed by the disc diffusion method, and trends over time were evaluated. RESULTS: A total 89643 pediatric blood cultures were performed, and 10621 pathogens were included in the analysis. Estimated minimum incidence rates of BSI for those ≤5 years of age fell from a peak of 11.4 per 1000 persons in 2002 to 3.4 per 1000 persons in 2017. Over 2 decades, the resistance of Gram-negative pathogens to all empiric, first-line antimicrobials (ampicillin/penicillin, gentamicin, ceftriaxone) among children ≤5 years increased from 3.4% to 30.2% (P < .001). Among those ≤60 days, AMR to all first-line antimicrobials increased from 7.0% to 67.7% (P < .001). Among children ≤5 years, Klebsiella spp. resistance to all first-line antimicrobial regimens increased from 5.9% to 93.7% (P < .001). CONCLUSIONS: The incidence of BSI among hospitalized children has decreased substantially over the last 20 years, although gains have been offset by increases in Gram-negative pathogens' resistance to all empiric first-line antimicrobials. There is an urgent need to address the broader challenge of adapting IMCI guidelines to the local setting in the face of rapidly-expanding AMR in childhood BSI.

HIV-associated disruption of lung cytokine networks is incompletely restored in asymptomatic HIV-infected Malawian adults on antiretroviral therapy.

Disruption of lung cytokine networks during chronic HIV infection is incompletely restored in individuals on antiretroviral therapy.

Trends in antimicrobial resistance in bloodstream infection isolates at a large urban hospital in Malawi (1998-2016): a surveillance study.

BACKGROUND: Bacterial bloodstream infection is a common cause of morbidity and mortality in sub-Saharan Africa, yet few facilities are able to maintain long-term surveillance. The Malawi-Liverpool-Wellcome Trust Clinical Research Programme has done sentinel surveillance of bacteraemia since 1998. We report long-term trends in bloodstream infection and antimicrobial resistance from this surveillance. METHODS: In this surveillance study, we analysed blood cultures that were routinely taken from adult and paediatric patients with fever or suspicion of sepsis admitted to Queen Elizabeth Central Hospital, Blantyre, Malawi from 1998 to 2016. The hospital served an urban population of 920 000 in 2016, with 1000 beds, although occupancy often exceeds capacity. The hospital admits about 10 000 adults and 30 000 children each year. Antimicrobial susceptibility tests were done by the disc diffusion method according to British Society of Antimicrobial Chemotherapy guidelines. We used the Cochran-Armitage test for trend to examine trends in rates of antimicrobial resistance, and negative binomial regression to examine trends in icidence of bloodstream infection over time. FINDINGS: Between Jan 1, 1998, and Dec 31, 2016, we isolated 29 183 pathogens from 194 539 blood cultures. Pathogen detection decreased significantly from 327·1/100 000 in 1998 to 120·2/100 000 in 2016 (p<0·0001). 13 366 (51·1%) of 26 174 bacterial isolates were resistant to the Malawian first-line antibiotics amoxicillin or penicillin, chloramphenicol, and co-trimoxazole; 68·3% of Gram-negative and 6·6% of Gram-positive pathogens. The proportions of non-Salmonella Enterobacteriaceae with extended spectrum beta-lactamase (ESBL) or fluoroquinolone resistance rose significantly after 2003 to 61·9% in 2016 (p<0·0001). Between 2003 and 2016, ESBL resistance rose from 0·7% to 30·3% in Escherichia coli, from 11·8% to 90·5% in Klebsiella spp and from 30·4% to 71·9% in other Enterobacteriaceae. Similarly, resistance to ciprofloxacin rose from 2·5% to 31·1% in E coli, from 1·7% to 70·2% in Klebsiella spp and from 5·9% to 68·8% in other Enterobacteriaceae. By contrast, more than 92·0% of common Gram-positive pathogens remain susceptible to either penicillin or chloramphenicol. Meticillin-resistant Staphylococcus aureus (MRSA) was first reported in 1998 at 7·7% and represented 18·4% of S aureus isolates in 2016. INTERPRETATION: The rapid expansion of ESBL and fluoroquinolone resistance among common Gram-negative pathogens, and the emergence of MRSA, highlight the growing challenge of bloodstream infections that are effectively impossible to treat in this resource-limited setting. FUNDING: Wellcome Trust, H3ABionet, Southern Africa Consortium for Research Excellence (SACORE).

Population genetic structure, antibiotic resistance, capsule switching and evolution of invasive pneumococci before conjugate vaccination in Malawi.

INTRODUCTION: Pneumococcal infections cause a high death toll in Sub Saharan Africa (SSA) but the recently rolled out pneumococcal conjugate vaccines (PCV) will reduce the disease burden. To better understand the population impact of these vaccines, comprehensive analysis of large collections of pneumococcal isolates sampled prior to vaccination is required. Here we present a population genomic study of the invasive pneumococcal isolates sampled before the implementation of PCV13 in Malawi. MATERIALS AND METHODS: We retrospectively sampled and whole genome sequenced 585 invasive isolates from 2004 to 2010. We determine the pneumococcal population genetic structure and assessed serotype prevalence, antibiotic resistance rates, and the occurrence of serotype switching. RESULTS: Population structure analysis revealed 22 genetically distinct sequence clusters (SCs), which consisted of closely related isolates. Serotype 1 (ST217), a vaccine-associated serotype in clade SC2, showed highest prevalence (19.3%), and was associated with the highest MDR rate (81.9%) followed by serotype 12F, a non-vaccine serotype in clade SC10 with an MDR rate of 57.9%. Prevalence of serotypes was stable prior to vaccination although there was an increase in the PMEN19 clone, serotype 5 ST289, in clade SC1 in 2010 suggesting a potential undetected local outbreak. Coalescent analysis revealed recent emergence of the SCs and there was evidence of natural capsule switching in the absence of vaccine induced selection pressure. Furthermore, majority of the highly prevalent capsule-switched isolates were associated with acquisition of vaccine-targeted capsules. CONCLUSIONS: This study provides descriptions of capsule-switched serotypes and serotypes with potential to cause serotype replacement post-vaccination such as 12F. Continued surveillance is critical to monitor these serotypes and antibiotic resistance in order to design better infection prevention and control measures such as inclusion of emerging replacement serotypes in future conjugate vaccines.

Genomic landscape of extended-spectrum ß-lactamase resistance in Escherichia coli from an urban African setting.

Objectives: Efforts to treat Escherichia coli infections are increasingly being compromised by the rapid, global spread of antimicrobial resistance (AMR). Whilst AMR in E. coli has been extensively investigated in resource-rich settings, in sub-Saharan Africa molecular patterns of AMR are not well described. In this study, we have begun to explore the population structure and molecular determinants of AMR amongst E. coli isolates from Malawi. Methods: Ninety-four E. coli isolates from patients admitted to Queen's Hospital, Malawi, were whole-genome sequenced. The isolates were selected on the basis of diversity of phenotypic resistance profiles and clinical source of isolation (blood, CSF and rectal swab). Sequence data were analysed using comparative genomics and phylogenetics. Results: Our results revealed the presence of five clades, which were strongly associated with E. coli phylogroups A, B1, B2, D and F. We identified 43 multilocus STs, of which ST131 (14.9%) and ST12 (9.6%) were the most common. We identified 25 AMR genes. The most common ESBL gene was bla CTX-M-15 and it was present in all five phylogroups and 11 STs, and most commonly detected in ST391 (4/4 isolates), ST648 (3/3 isolates) and ST131 [3/14 (21.4%) isolates]. Conclusions: This study has revealed a high diversity of lineages associated with AMR, including ESBL and fluoroquinolone resistance, in Malawi. The data highlight the value of longitudinal bacteraemia surveillance coupled with detailed molecular epidemiology in all settings, including low-income settings, in describing the global epidemiology of ESBL resistance.