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)

Lower density and shorter duration of nasopharyngeal carriage by pneumococcal serotype 1 (St217) may explain its increased invasiveness over other serotypes

ABSTRACT Streptococcus pneumoniae is a frequent colonizer of the human nasopharynx and a major cause of life-threating invasive infections such as pneumonia, meningitis and sepsis. Over 1 million people die every year due to invasive pneumococcal disease (IPD), mainly in developing countries. Serotype 1 is a common cause of IPD; however, unlike other serotypes, it is rarely found in the carrier state in the nasopharynx, which is often considered a prerequisite for disease. The aim of this study was to understand this dichotomy. We used murine models of carriage and IPD to characterize the pathogenesis of African serotype 1 (sequence type 217) pneumococcal strains obtained from the Queen Elizabeth Central Hospital in Blantyre, Malawi. We found that ST217 pneumococcal strains were highly virulent in a mouse model of invasive pneumonia, but in contrast to the generally accepted assumption, can also successfully establish nasopharyngeal carriage. Interestingly, we found that cocolonizing serotypes may proliferate in the presence of serotype 1, suggesting that acquisition of serotype 1 carriage could increase the risk of developing IPD by other serotypes. RNA sequencing analysis confirmed that key virulence genes associated with inflammation and tissue invasiveness were upregulated in serotype 1. These data reveal important new insights into serotype 1 pathogenesis, with implications for carriage potential and risk of invasive disease through interactions with other cocolonizing serotypes, an often-overlooked factor in transmission and disease progression. IMPORTANCE The pneumococcus causes serious diseases such as pneumonia, sepsis, and meningitis and is a major cause of morbidity and mortality worldwide. Serotype 1 accounts for the majority of invasive pneumococcal disease cases in sub-Saharan Africa but is rarely found during nasopharyngeal carriage. Understanding the mechanisms leading to nasopharyngeal carriage and invasive disease by this serotype can help reduce its burden on health care systems worldwide. In this study, we also uncovered the potential impact of serotype 1 on disease progression of other coinfecting serotypes, which can have important implications for vaccine efficacy. Understanding the interactions between different serotypes during nasopharyngeal carriage may lead to improved intervention methods and therapies to reduce pneumococcal invasive disease levels

Global distribution of invasive serotype 35D streptococcus pneumoniae isolates following introduction of 13-valent pneumococcal conjugate vaccine

A newly recognized pneumococcal serotype 35D, which differs from the 35B polysaccharide in structure and serology by not binding to factor serum 35a, was recently reported. The genetic basis for this distinctive serology is due to the presence of an inactivating mutation in wciG, which encodes an O-acetyltransferase responsible for O-acetylation of a galactofuranose. Here, we assessed the genomic data of a worldwide pneumococcal collection to identify serotype 35D isolates and understand their geographical distribution, genetic background and invasiveness potential. Of 21,980 pneumococcal isolates, 444 were originally typed as serotype 35B by PneumoCaT. Analysis of wciGrevealed 23 isolates from carriage (n=4) and disease (n=19) with partial or complete loss-of-funtion mutations, including mutations resulting in pre-mature stop codons (n=22) and an in-frame mutation (n=1). These were selected for further analysis. The putative 35D isolates were geographically widespread and 65.2% (15/23) of them was recovered after PCV13 introduction. Compared with serotype 35B, putative serotype 35D isolates have higher invasive disease potentials based on odds ratio (OR) (11.58; 95% CI, 1.42-94.19 vs 0.61; 95% CI, 0.40-0.92) and a higher prevalence of macrolide resistance mediated by mefA (26.1% vs 7.6%, p=0.009). Using Quellung, 50% (10/20) of viable isolates were serotype 35D, 25% (5/20) serotype 35B, and 25% (5/20) a mixture of 35B/35D. The discrepancy between phenotype and genotype requires further investigation. These findings illustrated a global distribution of an invasive serotype 35D among young children post-PCV13 introduction and underlined the invasive potential conferred by the loss of O-acetylation in the pneumococcal capsule

Invasive Streptococcus pneumoniae in Children, Malawi, 2004–2006

Of 176 invasive Streptococcus pneumoniae isolates from children in Malawi, common serotypes were 1 (23%), 6A/B (18%), 14 (6%), and 23F (6%). Coverage with the 7-valent pneumococcal conjugate vaccine (PCV) was 39%; PCV10 and PCV13 increased coverage to 66% and 88%, respectively. We found chloramphenicol resistance in 27% of isolates and penicillin nonsusceptibility in 10% (by using meningitis breakpoints); all were ceftriaxone susceptible

Recombination in Streptococcus pneumoniae Lineages Increase with Carriage Duration and Size of the Polysaccharide Capsule

Streptococcus pneumoniae causes a high burden of invasive pneumococcal disease (IPD) globally, especially in children from resource-poor settings. Like many bacteria, the pneumococcus can import DNA from other strains or even species by transformation and homologous recombination, which has allowed the pneumococcus to evade clinical interventions such as antibiotics and pneumococcal conjugate vaccines (PCVs). Pneumococci are enclosed in a complex polysaccharide capsule that determines the serotype; the capsule varies in size and is associated with properties including carriage prevalence and virulence. We determined and quantified the association between capsule and recombination events using genomic data from a diverse collection of serotypes sampled in Malawi. We determined both the amount of variation introduced by recombination relative to mutation (the relative rate) and how many individual recombination events occur per isolate (the frequency). Using univariate analyses, we found an association between both recombination measures and multiple factors associated with the capsule, including duration and prevalence of carriage. Because many capsular factors are correlated, we used multivariate analysis to correct for collinearity. Capsule size and carriage duration remained positively associated with recombination, although with a reduced P value, and this effect may be mediated through some unassayed additional property associated with larger capsules. This work describes an important impact of serotype on recombination that has been previously overlooked. While the details of how this effect is achieved remain to be determined, it may have important consequences for the serotype-specific response to vaccines and other interventions

Epidemiological and Molecular Characterization of an Invasive Group A Streptococcus32.2 Outbreak

Anemm32.2 invasive group A streptococcus (iGAS) outbreak occurred in Liverpool from January 2010 to September 2012. This genotype had not previously been identified in Liverpool, but was responsible for 32% (14/44) of all iGAS cases reported during this time period. We performed a case-case comparison ofemm32.2 iGAS cases with non-emm32.2 control iGAS cases identified in the Liverpool population over the same time period to assess patient risk factors foremm32.2 iGAS infection. Theemm32.2 iGAS cases were confined to the adult population. We show that homelessness, intravenous drug use, and alcohol abuse predisposed patients toemm32.2 iGAS disease; however, no obvious epidemiological linkage between the patients withemm32.2 iGAS could be identified. Comparative whole-genome sequencing analysis ofemm32.2 iGAS and non-emm32.2 control isolates was also performed to identify pathogen factors which might have driven the outbreak. We identified 19 genes, five of which had previously been implicated in virulence, which were present in all of theemm32.2 iGAS isolates but not present in any of the non-emm32.2 control isolates. We report that a novelemm32.2 genotype emerged in Liverpool in 2010 and identified a specific subset of genes, which could have allowed this novelemm32.2 genotype to persist in a disadvantaged population in the region over a 3-year period.</p

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

Early signals of vaccine driven perturbation seen in pneumococcal carriage population genomic data

BACKGROUND: Pneumococcal conjugate vaccines (PCV) 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 of 660 pneumococcal isolates collected through surveys from healthy carriers two years from PCV14 introduction and one-year post-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 the under-fives, frequency and diversity of vaccine serotypes (VT) decreased significantly post-PCV but no significant changes occurred in over-fives. Clearance of VT serotypes was consistent across different genetic backgrounds (lineages). There was an increase of non-vaccine serotypes (NVT) namely 7C, 15B/C, 23A in under-fives but 28F increased in both age groups. While carriage rates have been recently shown to remain stable post-PCV due replacement serotypes, there was no change in diversity of NVTs. Additionally, frequency of intermediate-penicillin-resistant lineages decreased post-PCV. While frequency of ABR genes remained stable, other accessory genes especially those associated with MGEs 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 under-fives. Continued surveillance with WGS remains crucial to fully understand dynamics of the residual VTs and replacement NVT serotypes post-PCV

Genomic identification of a novel co-trimoxazole resistance genotype and its prevalence amongst Streptococcus pneumoniae in Malawi.

OBJECTIVES: This study aimed to define the molecular basis of co-trimoxazole resistance in Malawian pneumococci under the dual selective pressure of widespread co-trimoxazole and sulfadoxine/pyrimethamine use. METHODS: We measured the trimethoprim and sulfamethoxazole MICs and analysed folA and folP nucleotide and translated amino acid sequences for 143 pneumococci isolated from carriage and invasive disease in Malawi (2002-08). RESULTS: Pneumococci were highly resistant to both trimethoprim and sulfamethoxazole (96%, 137/143). Sulfamethoxazole-resistant isolates showed a 3 or 6 bp insertion in the sulphonamide-binding site of folP. The trimethoprim-resistant isolates fell into three genotypic groups based on dihydrofolate reductase (encoded by folA) mutations: Ile-100-Leu (10%), the Ile-100-Leu substitution together with a residue 92 substitution (56%) and those with a novel uncharacterized resistance genotype (34%). The nucleotide sequence divergence and dN/dS of folA and folP remained stable from 2004 onwards. CONCLUSIONS: S. pneumoniae exhibit almost universal co-trimoxazole resistance in vitro and in silico that we believe is driven by extensive co-trimoxazole and sulfadoxine/pyrimethamine use. More than one-third of pneumococci employ a novel mechanism of co-trimoxazole resistance. Resistance has now reached a point of stabilizing evolution. The use of co-trimoxazole to prevent pneumococcal infection in HIV/AIDS patients in sub-Saharan Africa should be re-evaluated

The global distribution and diversity of protein vaccine candidate antigens in the highly virulent Streptococcus pnuemoniae serotype 1

Serotype 1 is one of the most common causes of pneumococcal disease worldwide. Pneumococcal protein vaccines are currently being developed as an alternate intervention strategy to pneumococcal conjugate vaccines. Pre-requisites for an efficacious pneumococcal protein vaccine are universal presence and minimal variation of the target antigen in the pneumococcal population, and the capability to induce a robust human immune response. We used in silico analysis to assess the prevalence of seven protein vaccine candidates (CbpA, PcpA, PhtD, PspA, SP0148, SP1912, SP2108) among 445 serotype 1 pneumococci from 26 different countries, across four continents. CbpA (76%), PspA (68%), PhtD (28%), PcpA (11%) were not universally encoded in the study population, and would not provide full coverage against serotype 1. PcpA was widely present in the European (82%), but not in the African (2%) population. A multi-valent vaccine incorporating CbpA, PcpA, PhtD and PspA was predicted to provide coverage against 86% of the global population. SP0148, SP1912 and SP2108 were universally encoded and we further assessed their predicted amino acid, antigenic and structural variation. Multiple allelic variants of these proteins were identified, different allelic variants dominated in different continents; the observed variation was predicted to impact the antigenicity and structure of two SP0148 variants, one SP1912 variant and four SP2108 variants, however these variants were each only present in a small fraction of the global population (<2%). The vast majority of the observed variation was predicted to have no impact on the efficaciousness of a protein vaccine incorporating a single variant of SP0148, SP1912 and/or SP2108 from S. pneumoniae TIGR4. Our findings emphasise the importance of taking geographic differences into account when designing global vaccine interventions and support the continued development of SP0148, SP1912 and SP2108 as protein vaccine candidates against this important pneumococcal serotype