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Background: \ud \ud Invasive pneumococcal disease (IPD) in young infants is uncommon but associated with high morbidity and mortality. Accurate data on the burden of IPD in young infants in low-income countries are lacking. We examined the burden of IPD in infants

The advent of pneumococcal conjugate vaccines led to the near disappearance of most of the included serotypes in high-income settings but also the rise of nonvaccine-type colonization and disease. Alternative strategies, using genetically conserved proteins as antigens, have been evaluated preclinically and clinically for years, so far unsuccessfully. One possible explanation for the failure of these efforts is that the choice of antigens may not have been sufficiently guided by an understanding of the gene expression pattern in the context of infection. Here, we present a targeted transcriptomic analysis of 160 pneumococcal genes encoding bacterial surface-exposed proteins in mouse models, human colonization, and human meningitis. We present the overlap of these different transcriptomic profiles. We identify two bacterial genes that are highly expressed in the context of mouse and human infection: SP_0282, an IID component of a mannose phosphotransferase system (PTS), and SP_1739, encoding RNase Y. We show that these two proteins can confer protection against pneumococcal nasopharyngeal colonization and intraperitoneal challenge in a murine model and generate opsonophagocytic antibodies. This study emphasizes and confirms the importance of studies of pneumococcal gene expression of bacterial surface proteins during human infection and colonization and may pave the way for the selection of a protein-based vaccine candidate.

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.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.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.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.Wellcome Trust and Wellcome Asia and Africa Programme.

BackgroundMortality from bacterial meningitis, predominately caused by Streptococcus pneumoniae, exceeds 50% in sub-Saharan African countries with high HIV prevalence. Underlying causes of high mortality are poorly understood. We examined the host and pathogen proteome in the CSF of adults with proven pneumococcal meningitis (PM), testing if there was an association between differentially expressed proteins and outcome.Materials/MethodsCSF proteomes were analyzed by quantitative Mass-Spectrometry. Spectra were identified using the Swissprot human and TIGR4 pneumococcal protein libraries. Proteins were quantitated and analyzed against mortality. Unique proteins in PM were identified against published normal CSF proteome. Random-Forest models were used to test for protein signatures discriminating outcome. Proteins of interest were tested for their effects on growth and neutrophil opsonophagocytic killing of S. pneumoniae.ResultsCSF proteomes were available for 57 Adults with PM (median age 32 years, 60% male, 70% HIV-1 co-infected, mortality 63%). Three hundred sixty individual human and 23 pneumococcal proteins were identified. Of the human protein hits, 30% were not expressed in normal CSF, and these were strongly associated with inflammation and primarily related to neutrophil activity. No human protein signature predicted outcome. However, expression of the essential S. pneumoniae protein Elongation Factor Tu (EF-Tu) was significantly increased in CSF of non-survivors [False Discovery Rate (q) -correlated against expression of Neutrophil defensin (r 0.4 p p < 0.002), but not against complement proteins C3 or Factor H. In vitro, addition of EF-Tu protein impaired S. pneumoniae neutrophil killing in CSF.ConclusionsExcessive S. pneumoniae EF-Tu protein in CSF was associated with reduced survival in meningitis in a high HIV prevalence population. We show EF-Tu may inhibit neutrophil mediated killing of S. pneumoniae in CSF. Further mechanistic work is required to better understand how S. pneumoniae avoids essential innate immune responses during PM through production of excess EF-Tu.

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.

Typhoid fever, caused by infection with the human-restricted bacterial pathogen Salmonella enterica serovar Typhi, is a major cause of illness and mortality in regions of the world with limited access to improved water and sanitation [1]. Recent estimates have placed the global burden of typhoid fever at 11.9–26.9 million cases and 129 000–270 000 deaths per year [2–5]. Although the endemic burden of typhoid fever in South and Southeast Asia has long been recognized, less is known about the burden of disease in sub-Saharan Africa. In Malawi, as in much of sub-Saharan Africa, nontyphoidal Salmonella (NTS) serovars have been a much more common cause of bloodstream infections over the past 2 decades. Sequential epidemics of Salmonella enterica serovars Typhimurium and Enteritidis have been observed [6]. Salmonella Typhi represented just 2% (105/5061) of Salmonella isolates detected by sentinel surveillance at Queen Elizabeth Central Hospital (QECH) in Blantyre, the largest hospital in Malawi, between 1998 and 2004 [6]. Since 2011, however, there has been a substantial increase in the number of confirmed cases of Salmonella Typhi at QECH. The number of typhoid cases increased from 14 per year during 1998–2010 to 843 cases in 2013. This epidemic of typhoid fever has continued for at least 3 years [7] and coincides with numerous reports of ongoing epidemics of typhoid fever in settings across Africa [7–12]. These epidemics have closely followed the recent global emergence of the H58 haplotype of Salmonella Typhi [13]. The H58 lineage is highly clonal and differentiated from other haplotypes using a simple single nucleotide polymorphism–based typing scheme [14], and is associated with high levels of multidrug resistance [13, 15]. There are a variety of hypotheses that could explain the large increase in typhoid fever cases in Blantyre, including (1) an increase in population density in Blantyre beyond a critical threshold for transmission; (2) waning heterotypic immunity to Salmonella Enteritidis that is cross-protective against Salmonella Typhi; (3) an increase in the prevalence of multidrug resistant (MDR) strains, resulting in prolonged persistence; and (4) the emergence of the MDR H58 haplotype, which has become dominant in many places around the world and may be more transmissible than other strains [13]. The latter 2 hypotheses are tightly coupled, but describe slightly different mechanisms by which the H58 haplotype, and MDR strains more broadly, may have led to the outbreak. Mathematical models provide a platform for assessing the plausibility of these hypotheses to help direct future research efforts. We therefore adapted a mathematical model for the transmission dynamics of typhoid developed previously [16] by fitting to age-specific data on microbiologically confirmed cases of typhoid fever in Blantyre, Malawi. We modified the model to explore the 4 hypotheses outlined above, and examined whether each of them could explain the recent prolonged outbreak of typhoid fever cases in this setting.

Background \ud Mortality from acute bacterial meningitis (ABM) in sub-Saharan African adults and adolescents exceeds 50%. We tested if Goal Directed Therapy (GDT) was feasible for adults and adolescents with clinically suspected ABM in Malawi. \ud \ud Materials and methods \ud Sequential patient cohorts of adults and adolescents with clinically suspected ABM were recruited in the emergency department of a teaching hospital in Malawi using a before/after design. Routine care was monitored in year one (P1). In year two (P2), nurses delivered protocolised GDT (rapid antibiotics, airway support, oxygenation, seizure control and fluid resuscitation) to a second cohort. The primary endpoint was composite mean number of clinical goals attained. Secondary endpoints were individual goals attained and death or disability from proven or probable ABM at day 40. \ud \ud Results \ud 563 patients with suspected ABM were enrolled in the study; 273 were monitored in P1; 290 patients with suspected ABM received GDT in P2. 61% were male, median age 33 years and 90% were HIV co-infected. ABM was proven or probable in 132 (23%) patients. GDT attained more clinical goals compared to routine care: composite mean number of goals in P1 was 0·55 vs. 1·57 in P2 GDT (p

BACKGROUNDBacterial 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.FINDINGSBetween 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 (pINTERPRETATIONThe 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.FUNDINGWellcome Trust, H3ABionet, Southern Africa Consortium for Research Excellence (SACORE).

BACKGROUND Acute bacterial meningitis (ABM) causes significant death and disability in children worldwide, with HIV recognized as an established risk factor for infection and negative outcomes. However, additional major risk factors for death and disability in pediatric ABM remain unclear. METHODS We conducted a retrospective analysis of case data from 3 departmental studies of ABM involving 1784 children

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.

Summary The Malawi Adult Meningitis Score prediction tool accurately estimates risk of clinical outcome from bacterial meningitis in sub-Saharan Africa. Clinical trial analysis by risk stratification reveals more severe outcomes in low-risk groups receiving adjunctive glycerol compared to placebo., Background. Acute bacterial meningitis (ABM) in adults residing in resource-poor countries is associated with mortality rates >50%. To improve outcome, interventional trials and standardized clinical algorithms are urgently required. To optimize these processes, we developed and validated an outcome prediction tool to identify ABM patients at greatest risk of death. Methods. We derived a nomogram using mortality predictors derived from a logistic regression model of a discovery database of adult Malawian patients with ABM (n = 523 [65%] cerebrospinal fluid [CSF] culture positive). We validated the nomogram internally using a bootstrap procedure and subsequently used the nomogram scores to further interpret the effects of adjunctive dexamethasone and glycerol using clinical trial data from Malawi. Results. ABM mortality at 6-week follow-up was 54%. Five of 15 variables tested were strongly associated with poor outcome (CSF culture positivity, CSF white blood cell count, hemoglobin, Glasgow Coma Scale, and pulse rate), and were used in the derivation of the Malawi Adult Meningitis Score (MAMS) nomogram. The C-index (area under the curve) was 0.76 (95% confidence interval, .71–.80) and calibration was good (Hosmer-Lemeshow C-statistic = 5.48, df = 8, P = .705). Harmful effects of adjunctive glycerol were observed in groups with relatively low predicted risk of poor outcome (25%–50% risk): Case Fatality Rate of 21% in the placebo group and 52% in the glycerol group (P < .001). This effect was not seen with adjunctive dexamethasone. Conclusions. MAMS provides a novel tool for predicting prognosis and improving interpretation of ABM clinical trials by risk stratification in resource-poor settings. Whether MAMS can be applied to non-HIV-endemic countries requires further evaluation.

A case of human melioidosis caused by a novel sequence type of Burkholderia pseudomallei occurred in a child in Malawi, southern Africa. A literature review showed that human cases reported from the continent have been increasing.

Introduction\ud Cryptococcal meningitis is the most common neurological infection in HIV infected patients in Sub Saharan Africa, where gold standard treatment with intravenous amphotericin B and 5 flucytosine is often unavailable or difficult to administer. Fluconazole monotherapy is frequently recommended in national guidelines but is a fungistatic drug compromised by uncertainty over optimal dosing and a paucity of clinical end-point outcome data.\ud \ud Methods\ud From July 2010 until March 2011, HIV infected adults with a first episode of cryptococcal meningitis were recruited at Queen Elizabeth Central Hospital, Blantyre, Malawi. Patients were treated with oral fluconazole monotherapy 800 mg daily, as per national guidelines. ART was started at 4 weeks. Outcomes and factors associated with treatment failure were assessed 4, 10 and 52 weeks after fluconazole initiation.\ud \ud Results\ud Sixty patients were recruited. 26/60 (43%) died by 4 weeks. 35/60 (58.0%) and 43/56 (77%) died or failed treatment by 10 or 52 weeks respectively. Reduced consciousness (Glasgow Coma Score 3 of 5) and confusion (Abbreviated Mental Test Score

Background\ud \ud Malawi commenced the introduction of the 13-valent pneumococcal conjugate vaccine (PCV13) into the routine infant immunisation schedule in November 2011. Here we have tested the utility of high throughput whole genome sequencing to provide a high-resolution view of pre-vaccine pneumococcal epidemiology and population evolutionary trends to predict potential future change in population structure post introduction.\ud \ud Methods\ud \ud One hundred and twenty seven (127) archived pneumococcal isolates from randomly selected adults and children presenting to the Queen Elizabeth Central Hospital, Blantyre, Malawi underwent whole genome sequencing.\ud \ud Results\ud \ud The pneumococcal population was dominated by serotype 1 (20.5% of invasive isolates) prior to vaccine introduction. PCV13 is likely to protect against 62.9% of all circulating invasive pneumococci (78.3% in under-5-year-olds). Several Pneumococcal Molecular Epidemiology Network (PMEN) clones are now in circulation in Malawi which were previously undetected but the pandemic multidrug resistant PMEN1 lineage was not identified. Genome analysis identified a number of novel sequence types and serotype switching.\ud \ud Conclusions\ud \ud High throughput genome sequencing is now feasible and has the capacity to simultaneously elucidate serotype, sequence type and as well as detailed genetic information. It enables population level characterization, providing a detailed picture of population structure and genome evolution relevant to disease control. Post-vaccine introduction surveillance supported by genome sequencing is essential to providing a comprehensive picture of the impact of PCV13 on pneumococcal population structure and informing future public health interventions.

Objective\ud To document trends in invasive pneumococcal disease (IPD) in a central hospital in Malawi during the period of national scale-up of antiretroviral therapy (ART) and cotrimoxazole prophylaxis.\ud \ud Methods\ud Between 1 January 2000 and 31 December 2009 almost 100,000 blood cultures and 40,000 cerebrospinal fluid (CSF) cultures were obtained from adults and children admitted to the Queen Elizabeth Central Hospital, Blantyre, Malawi with suspected severe bacterial infection.\ud \ud Results\ud 4,445 pneumococcal isolates were obtained over the 10 year period. 1,837 were from children: 885 (19.9%) from blood and 952 (21.4%) from CSF. 2,608 were from adults: 1,813 (40.8%) from blood and 795 (17.9%) from CSF. At the start of the surveillance period cotrimoxazole resistance was 73.8% and at the end was 92.6%. Multidrug resistance (MDR) was present in almost one third of isolates and was constant over time. Free ART was introduced in Malawi in 2004. From 2005 onwards there was a decline in invasive pneumococcal infections with a negative correlation between ART scale-up and the decline in IPD (Pearson's correlation r = −0.91; p

Background Accurate data on childhood pneumonia aetiology are essential especially from regions where mortality is high, in order to inform case-management guidelines and the potential of prevention strategies such as bacterial conjugate vaccines. Yield from blood culture is low, but lung aspirate culture provides a higher diagnostic yield. We aimed to determine if diagnostic yield could be increased further by polymerase chain reaction (PCR) detection of bacteria (Streptococcus pneumoniae and Haemophilus influenzae b) and viruses in lung aspirate fluid. Methods A total of 95 children with radiological focal, lobar or segmental consolidation had lung aspirate performed and sent for bacterial culture and for PCR for detection of bacteria, viruses and Pneumocystis jirovecii. In children with a pneumococcal aetiology, pneumococcal bacterial loads were calculated in blood and lung aspirate fluid. Results Blood culture identified a bacterial pathogen in only 8 patients (8%). With the addition of PCR on lung aspirate samples, causative pathogens (bacterial, viral, pneumocystis) were identified singly or as co-infections in 59 children (62%). The commonest bacterial organism was S.pneumoniae (41%), followed by H. influenzae b (6%), and the commonest virus identified was adenovirus (16%), followed by human bocavirus (HBoV) (4%), either as single or co-infection. Conclusions In a select group of African children, lung aspirate PCR significantly improves diagnostic yield. Our study confirms a major role of S.pneumoniae and viruses in the aetiology of childhood pneumonia in Africa.