Targeted Transcriptomic Screen of Pneumococcal Genes Expressed during Murine and Human Infection.

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.

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.

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.

Prediction of Outcome From Adult Bacterial Meningitis in a High-HIV-Seroprevalence, Resource-Poor Setting Using the Malawi Adult Meningitis Score (MAMS).

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.

Mathematical Modeling to Assess the Drivers of the Recent Emergence of Typhoid Fever in Blantyre, Malawi.

BACKGROUND: Multiyear epidemics of Salmonella enterica serovar Typhi have been reported from countries across eastern and southern Africa in recent years. In Blantyre, Malawi, a dramatic increase in typhoid fever cases has recently occurred, and may be linked to the emergence of the H58 haplotype. Strains belonging to the H58 haplotype often exhibit multidrug resistance and may have a fitness advantage relative to other Salmonella Typhi strains. METHODS: To explore hypotheses for the increased number of typhoid fever cases in Blantyre, we fit a mathematical model to culture-confirmed cases of Salmonella enterica infections at Queen Elizabeth Central Hospital, Blantyre. We explored 4 hypotheses: (1) an increase in the basic reproductive number (R0) in response to increasing population density; (2) a decrease in the incidence of cross-immunizing infection with Salmonella Enteritidis; (3) an increase in the duration of infectiousness due to failure to respond to first-line antibiotics; and (4) an increase in the transmission rate following the emergence of the H58 haplotype. RESULTS: Increasing population density or decreasing cross-immunity could not fully explain the observed pattern of typhoid emergence in Blantyre, whereas models allowing for an increase in the duration of infectiousness and/or the transmission rate of typhoid following the emergence of the H58 haplotype provided a good fit to the data. CONCLUSIONS: Our results suggest that an increase in the transmissibility of typhoid due to the emergence of drug resistance associated with the H58 haplotype may help to explain recent outbreaks of typhoid in Malawi and similar settings in Africa.

Prevalence of Endemic Respiratory Viruses During the COVID-19 Pandemic in Urban and Rural Malawi.

Background: We investigated endemic respiratory virus circulation patterns in Malawi, where no lockdown was imposed, during the COVID-19 pandemic. Methods: Within a prospective household cohort in urban and rural Malawi, adult participants provided upper respiratory tract (URT) samples at 4 time points between February 2021 and April 2022. Polymerase chain reaction (PCR) was performed for SARS-CoV-2, influenza, and other endemic respiratory viruses. Results: 1626 URT samples from 945 participants in 542 households were included. Overall, 7.6% (n = 123) samples were PCR- positive for >1 respiratory virus; SARS-CoV-2 (4.4%) and rhinovirus (2.0%) were most common. No influenza A virus was detected. Influenza B and respiratory syncytial virus (RSV) were rare. Higher virus positivity were detected in the rural setting and at earlier time points. Coinfections were infrequent. Conclusions: Endemic respiratory viruses circulated in the community in Malawi during the pandemic, though influenza and RSV were rarely detected. Distinct differences in virus positivity and demographics were observed between urban and rural cohorts.

Human melioidosis, Malawi, 2011.

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.

Pf7: an open dataset of Plasmodium falciparum genome variation in 20,000 worldwide samples.

We describe the MalariaGEN Pf7 data resource, the seventh release of Plasmodium falciparum genome variation data from the MalariaGEN network.  It comprises over 20,000 samples from 82 partner studies in 33 countries, including several malaria endemic regions that were previously underrepresented.  For the first time we include dried blood spot samples that were sequenced after selective whole genome amplification, necessitating new methods to genotype copy number variations.  We identify a large number of newly emerging crt mutations in parts of Southeast Asia, and show examples of heterogeneities in patterns of drug resistance within Africa and within the Indian subcontinent.  We describe the profile of variations in the C-terminal of the csp gene and relate this to the sequence used in the RTS,S and R21 malaria vaccines.  Pf7 provides high-quality data on genotype calls for 6 million SNPs and short indels, analysis of large deletions that cause failure of rapid diagnostic tests, and systematic characterisation of six major drug resistance loci, all of which can be freely downloaded from the MalariaGEN website.

Incidence of invasive non-typhoidal Salmonella in Blantyre, Malawi between January 2011-December 2019.

Background: The Malawi-Liverpool Wellcome Trust Clinical Research Programme (MLW) has undertaken sentinel surveillance of bloodstream infection and meningitis at Queen Elizabeth Central Hospital (QECH), Blantyre, Malawi for 20 years. Previously, three epidemics of Salmonella bloodstream infection have been identified. Here we provide updated surveillance data on invasive non-typhoidal Salmonella disease from 2011 - 2019. Methods: Surveillance data describing trends in invasive non-typhoidal Salmonella disease and associated antimicrobial susceptibility profiles are presented for the period January 2011 - December 2019. Results: Between January 2011-December 2019, 128,588 blood cultures and 40,769 cerebrospinal fluid cultures were processed at MLW. Overall, 1.00% of these were positive for S. Typhimurium, 0.10% for S. Enteritidis, and 0.05% positive for other Salmonella species. Estimated minimum incidence of invasive non-typhoidal Salmonella (iNTS) disease decreased from 21/100,000 per year in 2011 to 7/100,000 per year in 2019. Over this period, 26 confirmed cases of Salmonella meningitis were recorded (88.5% S. Typhimurium). Between 2011-2019 there was a substantial decrease in proportion of S. Typhimurium (78.5% to 27.7%) and S. Enteritidis (31.8% in 2011 to 0%) that were multidrug-resistant. Resistance to fluoroquinolones and third-generation generation cephalosporins (3GC) remained uncommon, however 3GC increased amongst Salmonella spp. and S. Typhimurium in the latter part of the period. Conclusions: The total number of iNTS bloodstream infections decreased between 2011-2019. Although the number multidrug resistance (MDR) S. Typhimurium and S. Enteritidis isolates has fallen, the number of MDR isolates of other Salmonella spp. has increased, including 3GC isolates.

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.

Changing Incidence of Invasive Pneumococcal Disease in Infants Less Than 90 Days of Age Before and After Introduction of the 13-Valent Pneumococcal Conjugate Vaccine in Blantyre, Malawi: A 14-Year Hospital Based Surveillance Study.

BACKGROUND: 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 <90 days old in Blantyre, Malawi over a 14-year period and evaluated the indirect impact of the 13-valent pneumococcal conjugate vaccine (PCV13) on vaccine-serotype IPD (VT-IPD) in this population. METHODS: We conducted laboratory-based prospective IPD surveillance in infants <90 days of age admitted to Queen Elizabeth Central Hospital in Blantyre between 2005 and 2018, including 7 years pre-PCV13 and 7 years post-PCV13 introduction. IPD was defined as Streptococcus pneumoniae identified by culture from blood or cerebrospinal fluid. Serotypes were determined by multiplex polymerase chain reaction and latex agglutination testing. RESULTS: We identified 130 cases of culture-confirmed IPD in infants <90 days old between 2005 and 2018. Total IPD incidence was declining before PCV13 introduction. The mean incidence of IPD was significantly lower in the post-PCV13 era. Serotypes 5 (27.8%) and 1 (15.6%) were most prevalent. Even after PCV13 introduction, VTs remained the primary cause of IPD, with serotype 5 accounting for 17.4% and serotype 1 for 13.0% of cases in young infants. CONCLUSION: Vaccine serotypes 1 and 5 were the main cause of IPD in neonates and young infants, both before and after PCV13 introduction. This suggests incomplete indirect protection with persisting VT carriage across the population despite vaccination in this setting. Alternative vaccine schedules and other vaccine introduction approaches need to be considered to protect this vulnerable population.

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.

An open dataset of Plasmodium falciparum genome variation in 7,000 worldwide samples.

MalariaGEN is a data-sharing network that enables groups around the world to work together on the genomic epidemiology of malaria. Here we describe a new release of curated genome variation data on 7,000 Plasmodium falciparum samples from MalariaGEN partner studies in 28 malaria-endemic countries. High-quality genotype calls on 3 million single nucleotide polymorphisms (SNPs) and short indels were produced using a standardised analysis pipeline. Copy number variants associated with drug resistance and structural variants that cause failure of rapid diagnostic tests were also analysed.  Almost all samples showed genetic evidence of resistance to at least one antimalarial drug, and some samples from Southeast Asia carried markers of resistance to six commonly-used drugs. Genes expressed during the mosquito stage of the parasite life-cycle are prominent among loci that show strong geographic differentiation. By continuing to enlarge this open data resource we aim to facilitate research into the evolutionary processes affecting malaria control and to accelerate development of the surveillance toolkit required for malaria elimination.

CSF Levels of Elongation Factor Tu Is Associated With Increased Mortality in Malawian Adults With Streptococcus pneumoniae Meningitis.

Background: Mortality 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/Methods: CSF 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. Results: CSF 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) <0.001]. Expression of EF-Tu was negatively co-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. Conclusions: Excessive 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.

Goal directed therapy for suspected acute bacterial meningitis in adults and adolescents in sub-Saharan Africa.

BACKGROUND: 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. MATERIALS AND METHODS: 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. RESULTS: 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<0·001); Death or disability by day 40 from proven or probable ABM occurred in 29/57 (51%) in P1 and 38/60 (63%) in P2 (p = 0·19). CONCLUSION: Nurse-led GDT in a resource-constrained setting was associated with improved delivery of protocolised care. Outcome was unaffected. TRIAL REGISTRATION: www.isrctn.com ISRCTN96218197.

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).

Modelling the Contributions of Malaria, HIV, Malnutrition and Rainfall to the Decline in Paediatric Invasive Non-typhoidal Salmonella Disease in Malawi.

INTRODUCTION: Nontyphoidal Salmonellae (NTS) are responsible for a huge burden of bloodstream infection in Sub-Saharan African children. Recent reports of a decline in invasive NTS (iNTS) disease from Kenya and The Gambia have emphasised an association with malaria control. Following a similar decline in iNTS disease in Malawi, we have used 9 years of continuous longitudinal data to model the interrelationships between iNTS disease, malaria, HIV and malnutrition. METHODS: Trends in monthly numbers of childhood iNTS disease presenting at Queen's Hospital, Blantyre, Malawi from 2002 to 2010 were reviewed in the context of longitudinal monthly data describing malaria slide-positivity among paediatric febrile admissions, paediatric HIV prevalence, nutritional rehabilitation unit admissions and monthly rainfall over the same 9 years, using structural equation models (SEM). RESULTS: Analysis of 3,105 iNTS episodes identified from 49,093 blood cultures, showed an 11.8% annual decline in iNTS (p < 0.001). SEM analysis produced a stable model with good fit, revealing direct and statistically significant seasonal effects of malaria and malnutrition on the prevalence of iNTS disease. When these data were smoothed to eliminate seasonal cyclic changes, these associations remained strong and there were additional significant effects of HIV prevalence. CONCLUSIONS: These data suggest that the overall decline in iNTS disease observed in Malawi is attributable to multiple public health interventions leading to reductions in malaria, HIV and acute malnutrition. Understanding the impacts of public health programmes on iNTS disease is essential to plan and evaluate interventions.

Risk factors for death and severe sequelae in Malawian children with bacterial meningitis, 1997-2010.

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 <15 years old who attended Queen Elizabeth Central Hospital in Blantyre, Malawi during 1997 to 2010. Univariate and multivariate logistic regression models were used to estimate the effects of HIV seropositivity, impaired consciousness and causative organism on death and severe sequelae. RESULTS: Impaired consciousness or coma at the time of admission was strongly associated with death (coma: odds ratio [OR] = 14.4, 95% confidence interval [CI]: 9.42, 22.1) and severe sequelae (Coma: OR = 3.27, 95% CI: 2.02, 5.29) in multivariate logistic regression models. HIV seropositivity was significantly associated with increased odds of death (OR = 1.65, 95% CI: 1.20, 2.26) but not with developing severe sequelae (OR = 0.88, 95% CI: 0.56, 1.38). After adjustment, infection with Salmonella spp. was associated with increased odds of death (OR = 2.11, 95% CI: 1.06, 4.08) and pneumococcal meningitis was associated with increased odds of severe sequelae (OR = 1.84, 95% CI: 1.03, 3.29). CONCLUSIONS: Impaired consciousness and HIV infection increased the odds of death from ABM in Malawian children. Use of pneumococcal conjugate vaccine could greatly reduce the burden of ABM in Malawi.

A prospective longitudinal study of the clinical outcomes from cryptococcal meningitis following treatment induction with 800 mg oral fluconazole in Blantyre, Malawi.

INTRODUCTION: 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. METHODS: 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. RESULTS: 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 <14 of 15), moderate/severe neurological disability (modified Rankin Score >3 of 5) and confusion (Abbreviated Mental Test Score <8 of 10) were all common at baseline and associated with death or treatment failure. ART prior to recruitment was not associated with better outcomes. CONCLUSIONS: Mortality and treatment failure from cryptococcal meningitis following initiation of treatment with 800 mg oral fluconazole is unacceptably high. To improve outcomes, there is an urgent need for better therapeutic strategies and point-of-care diagnostics, allowing earlier diagnosis before development of neurological deficit.

Novel biomarker combination improves the diagnosis of serious bacterial infections in Malawian children.

BACKGROUND: High throughput technologies offer insight into disease processes and heightens opportunities for improved diagnostics. Using transcriptomic analyses, we aimed to discover and to evaluate the clinical validity of a combination of reliable and functionally important biomarkers of serious bacterial infection (SBI). METHODS: We identified three previously reported biomarkers of infection (neutrophil gelatinase-associated lipocalin (NGAL), granulysin and resistin) and measured gene expression using quantitative real-time PCR. Protein products related to the three transcripts were measured by immunoassays. RESULTS: Relative gene expression values of NGAL and resistin were significantly increased, and expression of granulysin significantly decreased in cases compared to controls. Plasma concentrations of NGAL and resistin were significantly increased in children with confirmed SBI compared to children with no detectable bacterial infection (NBI), and to controls (287 versus 128 versus 62 ng/ml and 195 versus 90 versus 18 ng/ml, respectively, p < 0.05). Plasma protein concentrations of NGAL and resistin were significantly increased in non-survivors compared to survivors (306 versus 211 and 214 versus 150 ng/ml, p = 0.02). The respective areas under the curve (AUC) for NGAL, resistin and procalcitonin in predicting SBI were 0.79, 0.80 and 0.86, whilst a combination of NGAL, resistin and procalcitonin achieved an AUC of 0.90. CONCLUSIONS: We have demonstrated a unique combination of diagnostic biomarkers of SBI using transcriptomics, and demonstrated translational concordance with the corresponding protein. The addition of NGAL and resistin protein measurement to procalcitonin significantly improved the diagnosis of SBI.