Background: Controlled Human Infection Models (CHIM) are an important tool in biomedical research in which pathogens are inoculated into human volunteers to study pathogenesis and test vaccines or treatments. Production of CHIM inoculum, however, presents specific challenges in safety, reproducibility and replication of the desired dose. The principles of Good Manufacturing Practice (GMP) developed for production of medications can be applied to the preparation of CHIM inocula, but licensed GMP facilities are scarce in low resource settings. Methods: We applied GMP principles to develop protocols for CHIM inocula production at Liverpool School of Tropical Medicine, UK and subsequently at Malawi-Liverpool Wellcome Programme, Malawi. We used published guidelines to evaluate these protocols and to advise selection, characterisation, manufacture, quality control and storage. We established in-house production of Streptococcus pneumoniae serotypes 3 and 6B for use in Experimental Human Pneumococcal Challenge models. Results: The manufacturing process underwent regulatory review in both the UK and Malawi. CHIM inocula production in Malawi was approved by the National Health Sciences Research Committee after written and oral submission. We successfully implemented our procedure and manufactured batch lots of Streptococcus pneumoniae serotype 3 (n = 2) and serotype 6B (n = 2). We safely, accurately and successfully inoculated participants in CHIM studies and achieved experimental human pneumococcal carriage with both serotype strains. Discussion: CHIM inoculum manufacture of pneumococcus was feasible in Malawi. This allowed the Malawi scientific ecosystem to demonstrate scientific and regulatory autonomy as well as having the potential to improve operational efficiency compared to importation of challenge agents.

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.

Background The aim of this study was to characterize the epidemiology of human seasonal coronaviruses (HCoVs) in southern Malawi.Methods We tested for HCoVs 229E, OC43, NL63, and HKU1 using real-time polymerase chain reaction (PCR) on upper respiratory specimens from asymptomatic controls and individuals of all ages recruited through severe acute respiratory illness (SARI) surveillance at Queen Elizabeth Central Hospital, Blantyre, and a prospective influenza-like illness (ILI) observational study between 2011 and 2017. We modeled the probability of having a positive PCR for each HCoV using negative binomial models, and calculated pathogen-attributable fractions (PAFs).Results Overall, 8.8% (539/6107) of specimens were positive for >= 1 HCoV. OC43 was the most frequently detected HCoV (3.1% [191/6107]). NL63 was more frequently detected in ILI patients (adjusted incidence rate ratio [aIRR], 9.60 [95% confidence interval {CI}, 3.25-28.30]), while 229E (aIRR, 8.99 [95% CI, 1.81-44.70]) was more frequent in SARI patients than asymptomatic controls. In adults, 229E and OC43 were associated with SARI (PAF, 86.5% and 89.4%, respectively), while NL63 was associated with ILI (PAF, 85.1%). The prevalence of HCoVs was similar between children with SARI and controls. All HCoVs had bimodal peaks but distinct seasonality.Conclusions OC43 was the most prevalent HCoV in acute respiratory illness of all ages. Individual HCoVs had distinct seasonality that differed from temperate settings. Human seasonal coronaviruses (HCoVs) were prevalent among children and adults with mild and severe acute respiratory illness (SARI) in Malawi between 2011 and 2017. 229E and OC43 were associated with SARI, and NL63 with influenza-like illness. Individual HCoVs had distinct seasonality.

Background Compared to the abundance of clinical and genomic information available on patients hospitalised with COVID-19 disease from high-income countries, there is a paucity of data from low-income countries. Our aim was to explore the relationship between viral lineage and patient outcome. Methods We enrolled a prospective observational cohort of adult patients hospitalised with PCR-confirmed COVID-19 disease between July 2020 and March 2022 from Blantyre, Malawi, covering four waves of SARS-CoV-2 infections. Clinical and diagnostic data were collected using an adapted ISARIC clinical characterization protocol for COVID-19. SARS-CoV-2 isolates were sequenced using the MinION™ in Blantyre. Results We enrolled 314 patients, good quality sequencing data was available for 55 patients. The sequencing data showed that 8 of 11 participants recruited in wave one had B.1 infections, 6/6 in wave two had Beta, 25/26 in wave three had Delta and 11/12 in wave four had Omicron. Patients infected during the Delta and Omicron waves reported fewer underlying chronic conditions and a shorter time to presentation. Significantly fewer patients required oxygen (22.7% [17/75] vs. 58.6% [140/239], p < 0.001) and steroids (38.7% [29/75] vs. 70.3% [167/239], p < 0.001) in the Omicron wave compared with the other waves. Multivariable logistic-regression demonstrated a trend toward increased mortality in the Delta wave (OR 4.99 [95% CI 1.0–25.0 p = 0.05) compared to the first wave of infection. Conclusions Our data show that each wave of patients hospitalised with SARS-CoV-2 was infected with a distinct viral variant. The clinical data suggests that patients with severe COVID-19 disease were more likely to die during the Delta wave.

Background Compared to the abundance of clinical, molecular, and genomic information available on patients hospitalised with COVID-19 disease from high-income countries, there is a paucity of data from low-income countries. Methods We enrolled a cohort of patients with PCR confirmed COVID-19 disease at Queen Elizabeth Central Hospital, the main hospital for southern Malawi, between July 2020 and September 2021. The recruitment period covered three waves of SARS-CoV-2 infections in Malawi. Clinical and diagnostic data were collected using the ISARIC clinical characterization protocol for COVID-19. The viral material from PCR-positive swabs was amplified with a tiling PCR scheme and sequenced using the MinION sequencer in Malawi. Consensus genomes were generated using the ARTIC pipeline and lineage assignment was performed using Pangolin. Results Sequencing data showed that wave one was predominantly B.1 (8/11 samples), wave two consisted entirely of Beta variant of concern (VOC) (6/6), and wave three was predominantly Delta VOC (25/26). Patients presenting in the second and third waves had progressively fewer underlying chronic conditions, and patients in the third wave had a shorter time to presentation (2 days vs 5 in the original wave). Multivariable logistic regression demonstrated increased mortality in wave three, dominated by the Delta VOC, compared to previous waves (OR 6.6 [CI 1.1-38.8]). Conclusions Patients hospitalised with COVID-19 in Blantyre during the Delta wave had more acute symptom onset; fewer underlying conditions; and were more likely to die. Whilst we demonstrate the value of linking virus sequence data with clinical outcome data in a low-income setting, this study also highlights the considerable barriers to establishing sequencing capacity in a setting heavily affected by disruptions in supply chain and inequity of resource distribution.

Background:The impact of human immunodeficiency virus (HIV) infection on influenza incidence and severity in adults in sub-Saharan Africa is unclear. Seasonal influenza vaccination is recommended for HIV-infected persons in developed settings but is rarely implemented in Africa. Methods:We conducted a prospective cohort study to compare the incidence of laboratory-confirmed influenza illness between HIV-infected and HIV-uninfected adults in Blantyre, Malawi. In a parallel case-control study, we explored risk factors for severe influenza presentation of severe (hospitalized) lower respiratory tract infection, and mild influenza (influenza-like illness [ILI]). Results:The cohort study enrolled 608 adults, of whom 360 (59%) were HIV infected. Between April 2013 and March 2015, 24 of 229 ILI episodes (10.5%) in HIV-infected and 5 of 119 (4.2%) in HIV-uninfected adults were positive for influenza by means of polymerase chain reaction (incidence rate, 46.0 vs 14.5 per 1000 person-years; incidence rate ratio, 2.75; 95% confidence interval, 1.02-7.44; P = .03; adjusted for age, sex, household crowding, and food security). In the case-control study, influenza was identified in 56 of 518 patients (10.8%) with hospitalized lower respiratory tract infection, and 88 or 642 (13.7%) with ILI. The HIV prevalence was 69.6% and 29.6%, respectively, among influenza-positive case patients and controls. HIV was a significant risk factor for severe influenza (odds ratio, 4.98; 95% confidence interval, 2.09-11.88; P < .001; population-attributable fraction, 57%; adjusted for season, sanitation facility, and food security). Conclusions:HIV is an important risk factor for influenza-associated ILI and severe presentation in this high-HIV prevalence African setting. Targeted influenza vaccination of HIV-infected African adults should be reevaluated, and the optimal mechanism for vaccine introduction in overstretched health systems needs to be determined.

Data on the epidemiology of severe acute respiratory illness (SARI) in adults from low-income, high human immunodeficiency virus (HIV) prevalence African settings are scarce. We conducted adult SARI surveillance in Blantyre, Malawi. From January 2011 to December 2013, individuals aged ≥ 15 years with SARI (both inpatients and outpatients) were enrolled at a large teaching hospital in Blantyre, Malawi. Nasopharyngeal aspirates were tested for influenza and other respiratory viruses by polymerase chain reaction. We estimated hospital-attended influenza-positive SARI incidence rates and assessed factors associated with influenza positivity and clinical severity (Modified Early Warning Score > 4). We enrolled 1,126 SARI cases; 163 (14.5%) were positive for influenza. Human immunodeficiency virus prevalence was 50.3%. Annual incidence of hospital-attended influenza-associated SARI was 9.7–16.8 cases per 100,000 population. Human immunodeficiency virus was associated with a 5-fold greater incidence (incidence rate ratio 4.91, 95% confidence interval [CI]: 3.83–6.32). On multivariable analysis, female gender, as well as recruitment in hot, rainy season (December to March; adjusted odds ratios (aOR): 2.82, 95% CI: 1.57–5.06) and cool, dry season (April to August; aOR: 2.47, 95% CI: 1.35–4.15), was associated with influenza positivity, whereas influenza-positive patients were less likely to be HIV-infected (aOR: 0.59, 95% CI: 0.43–0.80) or have viral coinfection (aOR: 0.51, 95% CI: 0.36–0.73). Human immunodeficiency virus infection (aOR: 1.86; 95% CI: 1.35–2.56) and recruitment in hot, rainy season (aOR: 4.98, 95% CI: 3.17–7.81) were independently associated with clinical severity. In this high HIV prevalence population, influenza was associated with nearly 15% of hospital-attended SARI. Human immunodeficiency virus infection is an important risk factor for clinical severity in all-cause and influenza-associated SARI. Expanded access to HIV testing and antiretroviral treatment, as well as targeted influenza vaccination, may reduce the burden of SARI in Malawi and other high HIV prevalence settings.

BACKGROUND:We used data from 4 years of pediatric severe acute respiratory illness (SARI) sentinel surveillance in Blantyre, Malawi, to identify factors associated with clinical severity and coviral clustering. METHODS:From January 2011 to December 2014, 2363 children aged 3 months to 14 years presenting to the hospital with SARI were enrolled. Nasopharyngeal aspirates were tested for influenza virus and other respiratory viruses. We assessed risk factors for clinical severity and conducted clustering analysis to identify viral clusters in children with viral codetection. RESULTS:Hospital-attended influenza virus-positive SARI incidence was 2.0 cases per 10 000 children annually; it was highest among children aged <1 year (6.3 cases per 10 000), and human immunodeficiency virus (HIV)-infected children aged 5-9 years (6.0 cases per 10 000). A total of 605 SARI cases (26.8%) had warning signs, which were positively associated with HIV infection (adjusted risk ratio [aRR], 2.4; 95% confidence interval [CI], 1.4-3.9), respiratory syncytial virus infection (aRR, 1.9; 95% CI, 1.3-3.0) and rainy season (aRR, 2.4; 95% CI, 1.6-3.8). We identified 6 coviral clusters; 1 cluster was associated with SARI with warning signs. CONCLUSIONS:Influenza vaccination may benefit young children and HIV-infected children in this setting. Viral clustering may be associated with SARI severity; its assessment should be included in routine SARI surveillance.

BackgroundAlthough annual vaccination of at-risk groups is the cornerstone of influenza prevention in many developed settings, it is unavailable in most sub-Saharan African countries. Since 70% of the estimated total population of HIV-infected persons reside in these countries, they might be an important group for whom policy makers could target influenza control strategies. This study aimed to determine the impact of HIV on the burden and severity of influenza illness in adults in a setting with high HIV prevalence.MethodsAt the Queen Elizabeth Central Hospital, Blantyre, Malawi, we conducted a prospective cohort study between April 1, 2013, and March 31, 2015, to compare influenza incidence between HIV-infected and HIV-uninfected adults. We also did a case-control study of adults with mild influenza (influenza-like illness) and severe influenza (admission to hospital with lower respiratory tract infection) to explore risk factors for severe influenza presentation. Poisson and unconditional logistic regression models, respectively, were performed.Findings608 adults were enrolled in the cohort study, of whom 360 (59%) were HIV-reactive (median CD4 count 390 cells per μL [IQR 244–547]). 24 (11%) of 229 episodes of influenza-like illness in HIV-infected and five (4%) of 119 in HIV-uninfected adults were influenza PCR positive (incidence rates 46 vs 15 per 1000 person-years, incidence rate ratio 2·75 [95% CI 1·02–7·44]). In the case-control study, 56 (11%) of 518 patients admitted with lower respiratory tract infection and 88 (14%) of 642 patients with influenza-like illness were influenza PCR positive. HIV prevalence in the influenza-positive cases and controls were 70% (39/56) and 30% (26/88), respectively. HIV was a significant risk factor for severe influenza presentation (odds ratio 4·98 [95% CI 2·09–11·88], population attributable fraction 57%). There was a tendency towards increased incidence and severity in individuals with CD4 count less than 200 cells per μL.InterpretationIn a setting with high HIV prevalence, HIV infection is an important risk factor for acquiring influenza infection and for severe presentation. Individuals with advanced immunosuppression can be particularly vulnerable. These results present a strong case to policy makers to consider targeted vaccination policy in HIV-infected adults in sub-Saharan Africa. However, the optimum mechanism for vaccine introduction and evaluation in overstretched health systems will need to be determined.FundingWellcome Trust.

Introduction: The impact of HIV infection on influenza incidence and presentation in adults in sub-Saharan Africa is unclear. Although seasonal influenza vaccination is recommended for HIV-infected persons in developed settings, it is not part of routine HIV care in Africa.

Background: A substantial proportion of Influenza morbidity and mortality is due to secondary pneumococcal infection. Although the synergistic interaction between influenza and pneumococcal disease is well studied, the interplay between influenza and nasopharyngeal carriage of Streptococcus pneumoniae remains unclear. We test the hypothesis that influenza infection increases susceptibility of pneumococcal carriage in a high HIV prevalence setting. Methods & Materials: From January to December 2011, patients with severe acute respiratory infection (SARI), defined as fever (>38 °c), cough or sore throat, requiring hospitalization, and symptoms <7 days were enrolled in the sentinel influenza surveillance at Queen Elizabeth Central Hospital in Blantyre, Malawi. Demographic and clinical information were recorded; nasopharyngeal aspirates were obtained to detect influenza, S. pneumoniae, and 19 additional respiratory pathogens by real-time reverse transcriptase polymerase chain reaction. We explored the relationship between influenza infection and pneumococcal carriage using logistic regression analysis. Results: Of 511 SARI cases enrolled, 255 (50%) were male, and 155 (57%) were children (aged <15 years). 11% children and 39% adults were HIV-positive. Influenza was identified in 72 (14%) (children 9%, adults 19%) and S. pneumoniae in342 (69.6%) individuals (children 78%, adults 60%). Influenza had a bimodal peak (April and July). In contrast, pneumococcal carriage prevalence was highest from September to January. Individuals with a positive influenza PCR had a lower risk of pneumococcal carriage (odds ratio (OR) 0.51, 95%CI 0.30-0.86). A strong negative association was also demonstrated between presence of Coronavirus OC43 and S. pneumoniae. Independent risk factors for carriage were young age and hot season. The effect of influenza infection on carriage disappeared after adjusting for age, sex, HIV status, seasonality and the presence of coronavirus OC43 (adjusted OR 0.71, 95% CI 0.40-1.26, p = 0.25). Conclusion: Contrary to our hypothesis, there was a trend towards lower pneumococcal carriage in subjects with influenza infection. This observation was most likely confounded by age and season. We plan to further characterize this relationship by incorporating surveillance data from 2012-2013, ascertaining pneumococcal colonization density using quantitative PCR for lytA gene, and examining the novel finding of the inverse association between Coronavirus OC43 and pneumococcal colonization.

Mortality from adult bacterial meningitis exceeds 50% in sub-Saharan Africa. We postulated that-particularly in individuals infected with human immunodeficiency virus (HIV)-herpes simplex virus, varicella zoster virus, Epstein-Barr virus (EBV), and cytomegalovirus (CMV) in the cerebrospinal fluid (CSF) contribute to poor outcome. CSF from 149 Malawian adults with bacterial meningitis and 39 controls were analyzed using polymerase chain reaction. EBV was detected in 79 of 149 bacterial meningitis patients. Mortality (54%) was associated with higher CSF EBV load when adjusted for HIV (P = .01). CMV was detected in 11 of 115 HIV-infected patients, 8 of whom died. The mechanisms by which EBV and CMV contribute to poor outcome require further investigation.