Expansion of pneumococcal serotype 23F and 14 lineages with genotypic changes in capsule polysaccharide locus and virulence gene profiles post introduction of pneumococcal conjugate vaccine in Blantyre, Malawi.

Since the introduction of the 13-valent pneumococcal conjugate vaccine (PCV13) in Malawi in 2011, there has been persistent carriage of vaccine serotype (VT) Streptococcus pneumoniae, despite high vaccine coverage. To determine if there has been a genetic change within the VT capsule polysaccharide (cps) loci since the vaccine's introduction, we compared 1022 whole-genome-sequenced VT isolates from 1998 to 2019. We identified the clonal expansion of a multidrug-resistant, penicillin non-susceptible serotype 23F GPSC14-ST2059 lineage, a serotype 14 GPSC9-ST782 lineage and a novel serotype 14 sequence type GPSC9-ST18728 lineage. Serotype 23F GPSC14-ST2059 had an I253T mutation within the capsule oligosaccharide repeat unit polymerase Wzy protein, which is predicted in silico to alter the protein pocket cavity. Moreover, serotype 23F GPSC14-ST2059 had SNPs in the DNA binding sites for the cps transcriptional repressors CspR and SpxR. Serotype 14 GPSC9-ST782 harbours a non-truncated version of the large repetitive protein (Lrp), containing a Cna protein B-type domain which is also present in proteins associated with infection and colonisation. These emergent lineages also harboured genes associated with antibiotic resistance, and the promotion of colonisation and infection which were absent in other lineages of the same serotype. Together these data suggest that in addition to serotype replacement, modifications of the capsule locus associated with changes in virulence factor expression and antibiotic resistance may promote vaccine escape. In summary, the study highlights that the persistence of vaccine serotype carriage despite high vaccine coverage in Malawi may be partly caused by expansion of VT lineages post-PCV13 rollout.

Clonal Expansion of a Streptococcus pneumoniae Serotype 3 Capsule Variant Sequence Type 700 With Enhanced Vaccine Escape Potential After 13-Valent Pneumococcal Conjugate Vaccine Introduction.

BACKGROUND: Streptococcus pneumoniae serotype 3 remains a problem globally. Malawi introduced 13-valent pneumococcal conjugate vaccine (PCV13) in 2011, but there has been no direct protection against serotype 3 carriage. We explored whether vaccine escape by serotype 3 is due to clonal expansion of a lineage with a competitive advantage. METHODS: The distribution of serotype 3 Global Pneumococcal Sequence Clusters (GPSCs) and sequence types (STs) globally was assessed using sequences from the Global Pneumococcal Sequencing Project. Whole-genome sequences of 135 serotype 3 carriage isolates from Blantyre, Malawi (2015-2019) were analyzed. Comparative analysis of the capsule locus, entire genomes, antimicrobial resistance, and phylogenetic reconstructions were undertaken. Opsonophagocytosis was evaluated using serum samples from vaccinated adults and children. RESULTS: Serotype 3 GPSC10-ST700 isolates were most prominent in Malawi. Compared with the prototypical serotype 3 capsular polysaccharide locus sequence, 6 genes are absent, with retention of capsule polysaccharide biosynthesis. This lineage is characterized by increased antimicrobial resistance and lower susceptibility to opsonophagocytic killing. CONCLUSIONS: A serotype 3 variant in Malawi has genotypic and phenotypic characteristics that could enhance vaccine escape and clonal expansion after post-PCV13 introduction. Genomic surveillance among high-burden populations is essential to improve the effectiveness of next-generation pneumococcal vaccines.

The metabolic, virulence and antimicrobial resistance profiles of colonising Streptococcus pneumoniae shift after PCV13 introduction in urban Malawi.

Streptococcus pneumoniae causes substantial mortality among children under 5-years-old worldwide. Polysaccharide conjugate vaccines (PCVs) are highly effective at reducing vaccine serotype disease, but emergence of non-vaccine serotypes and persistent nasopharyngeal carriage threaten this success. We investigated the hypothesis that following vaccine, adapted pneumococcal genotypes emerge with the potential for vaccine escape. We genome sequenced 2804 penumococcal isolates, collected 4-8 years after introduction of PCV13 in Blantyre, Malawi. We developed a pipeline to cluster the pneumococcal population based on metabolic core genes into "Metabolic genotypes" (MTs). We show that S. pneumoniae population genetics are characterised by emergence of MTs with distinct virulence and antimicrobial resistance (AMR) profiles. Preliminary in vitro and murine experiments revealed that representative isolates from emerging MTs differed in growth, haemolytic, epithelial infection, and murine colonisation characteristics. Our results suggest that in the context of PCV13 introduction, pneumococcal population dynamics had shifted, a phenomenon that could further undermine vaccine control and promote spread of AMR.

Risk factors for pneumococcal carriage in adults living with HIV on antiretroviral therapy in the infant pneumococcal vaccine era in Malawi.

OBJECTIVE: Adults living with HIV (ALWHIV) on antiretroviral therapy (ART) are at high risk of pneumococcal carriage and disease. To help evaluate carriage risk in African ALWHIV at least 4 years after infant pneumococcal conjugate vaccination introduction in 2011, we assessed association between pneumococcal carriage and potential risk factors. METHODS: Nasopharyngeal swabs were collected from adults aged 18-40 years attending an ART clinic during rolling, cross-sectional surveys in Blantyre, Malawi between 2015 and 2019. We fitted generalized additive models to estimate the risk of sex, social economic status (SES), living with a child less than 5 years, and ART duration on carriage. RESULTS: Of 2067 adults, median age was 33 years (range 28-37), 1427 (69.0%) were women, 1087 (61.4%) were in low-middle socioeconomic-status (SES), 910 (44.0%) were living with a child less than 5 years, and median ART duration was 3 years (range 0.004-17). We estimated 38.2 and 60.6% reductions in overall and vaccine-serotype carriage prevalence. Overall carriage was associated with low SES, living with a child less than 5 years and shorter duration on ART. By contrast, vaccine-type carriage was associated with living without a child less than 5 years and male sex. CONCLUSION: Despite temporal reductions in overall and vaccine-serotype carriage, there is evidence of incomplete vaccine-serotype indirect protection. A targeted-vaccination campaign should be considered for ALWHIV, along with other public health measures to further reduce vaccine-serotype carriage and therefore disease.

Association between household air pollution and nasopharyngeal pneumococcal carriage in Malawian infants (MSCAPE): a nested, prospective, observational study.

BACKGROUND: Household air pollution from solid fuels increases the risk of childhood pneumonia. Nasopharyngeal carriage of Streptococcus pneumoniae is a necessary step in the development of pneumococcal pneumonia. We aimed to assess the association between exposure to household air pollution and the prevalence and density of S pneumoniae carriage among children. METHODS: The Malawi Streptococcus pneumoniae Carriage and Air Pollution Exposure study was a nested, prospective, observational study of children participating in the cluster randomised controlled Cooking and Pneumonia Study (CAPS) in the Karonga Health and Demographic Surveillance System (HDSS) area in northern Malawi. CAPS compared the effects of a cleaner burning biomass-fuelled cookstove (intervention group) with traditional open-fire cooking (control group) on the incidence of pneumonia in children. Eligible children aged 6 weeks or 6 months (those recruited a 6 weeks were also followed up at age 6 months) were identified by the Karonga HDSS centre. Nasopharyngeal swabs were taken to detect S pneumoniae, and infant exposure to particulate matter with a diameter of ≤2·5 µm (PM2·5) exposure was assessed by use of a MicroPEM device. The primary outcome was the prevalence of nasopharyngeal S pneumoniae carriage in all children aged 6 months, assessed in all children with valid data on PM2·5. The effects of the intervention stoves (intention-to-treat analysis) and PM2·5 (adjusted exposure-response analysis) on the prevalence of S pneumoniae carriage were also assessed in the study children. FINDINGS: Between Nov 15, 2015, and Nov 2, 2017, 485 children were recruited (240 from the intervention group and 245 from the control group). Of all 450 children with available data at age 6 months, 387 (86% [95% CI 82-89]) were positive for S pneumoniae. Geometric mean PM2·5 exposure was 60·3 µg/m3 (95% CI 55·8-65·3) in S pneumoniae-positive children and 47·0 µg/m3 (38·3-57·7) in S pneumoniae-negative children (p=0·044). In the intention-to-treat analysis, a non-significant increase in the risk of S pneumoniae carriage was observed in intervention group children compared with control group children (odds ratio 1·36 [95% CI 0·95-1·94]; p=0·093). In the exposure-response analysis, a significant association between PM2·5 exposure and S pneumoniae carriage was observed; a one unit increase in decile of PM2·5 was found to significantly increase the risk of S pneumoniae carriage by 10% (1·10 [1·01-1·20]; p=0·035), after adjustment for age, sex, 13-valent pneumococcal conjugate vaccination status, season, current use of antibiotics, and MicroPEM run-time. INTERPRETATION: Despite the absence of effect from the intervention cookstove, household air pollution exposure was significantly associated with the prevalence of nasopharyngeal S pneumoniae carriage. These results provide empirical evidence for the potential mechanistic association between exposure to household air pollution and childhood pneumonia. FUNDING: Bill & Melinda Gates Foundation.

Evaluation of Pneumococcal Serotyping of Nasopharyngeal-Carriage Isolates by Latex Agglutination, Whole-Genome Sequencing (PneumoCaT), and DNA Microarray in a High-Pneumococcal-Carriage-Prevalence Population in Malawi.

Accurate assessment of the serotype distribution associated with pneumococcal colonization and disease is essential for evaluating and formulating pneumococcal vaccines and for informing vaccine policy. For this reason, we evaluated the concordance between pneumococcal serotyping results by latex agglutination, whole-genome sequencing (WGS) with PneumoCaT, and DNA microarray for samples from community carriage surveillance in Blantyre, Malawi. Nasopharyngeal swabs were collected according to WHO recommendations between 2015 and 2017 by using stratified random sampling among study populations. Participants included healthy children 3 to 6 years old (vaccinated with the 13-valent pneumococcal conjugate vaccine [PCV13] as part of the Expanded Program on Immunization [EPI]), healthy children 5 to 10 years old (age-ineligible for PCV13), and HIV-infected adults (18 to 40 years old) on antiretroviral therapy (ART). For phenotypic serotyping, we used a 13-valent latex kit (Statens Serum Institut [SSI], Denmark). For genomic serotyping, we applied the PneumoCaT pipeline to whole-genome sequence libraries. For molecular serotyping by microarray, we used the BUGS Bioscience Senti-SP microarray. A total of 1,347 samples were analyzed. Concordance was 90.7% (95% confidence interval [CI], 89.0 to 92.2%) between latex agglutination and PneumoCaT, 95.2% (95% CI, 93.9 to 96.3%) between latex agglutination and the microarray, and 96.6% (95% CI, 95.5 to 97.5%) between the microarray and PneumoCaT. By detecting additional vaccine serotype (VT) pneumococci carried at low relative abundances (median, 8%), the microarray increased VT detection by 31.5% over that by latex serotyping. To conclude, all three serotyping methods were highly concordant in identifying dominant serotypes. Latex serotyping is accurate in identifying vaccine serotypes and requires the least expertise and resources for field implementation and analysis. However, WGS, which adds population structure, and microarray, which adds multiple-serotype carriage, should be considered at regional reference laboratories for investigating the importance of vaccine serotypes at low relative abundances in transmission and disease.

High residual carriage of vaccine-serotype Streptococcus pneumoniae after introduction of pneumococcal conjugate vaccine in Malawi.

There are concerns that pneumococcal conjugate vaccines (PCVs) in sub-Saharan Africa sub-optimally interrupt Streptococcus pneumoniae vaccine-serotype (VT) carriage and transmission. Here we assess PCV carriage using rolling, prospective nasopharyngeal carriage surveys between 2015 and 2018, 3.6-7.1 years after Malawi's 2011 PCV13 introduction. Carriage decay rate is analysed using non-linear regression. Despite evidence of reduction in VT carriage over the study period, there is high persistent residual carriage. This includes among PCV-vaccinated children 3-5-year-old (16.1% relative reduction from 19.9% to 16.7%); PCV-unvaccinated children 6-8-year-old (40.5% reduction from 26.4% to 15.7%); HIV-infected adults 18-40-years-old on antiretroviral therapy (41.4% reduction from 15.2% to 8.9%). VT carriage prevalence half-life is similar among PCV-vaccinated and PCV-unvaccinated children (3.26 and 3.34 years, respectively). Compared with high-income settings, there is high residual VT carriage 3.6-7.1 years after PCV introduction. Rigorous evaluation of strategies to augment vaccine-induced control of carriage, including alternative schedules and catch-up campaigns, is required.

A trial of a 7-valent pneumococcal conjugate vaccine in HIV-infected adults.

BACKGROUND: Streptococcus pneumoniae is a leading and serious coinfection in adults with human immunodeficiency virus (HIV) infection, particularly in Africa. Prevention of this disease by vaccination with the current 23-valent polysaccharide vaccine is suboptimal. Protein conjugate vaccines offer a further option for protection, but data on their clinical efficacy in adults are needed. METHODS: In this double-blind, randomized, placebo-controlled clinical efficacy trial, we studied the efficacy of a 7-valent conjugate pneumococcal vaccine in predominantly HIV-infected Malawian adolescents and adults who had recovered from documented invasive pneumococcal disease. Two doses of vaccine were given 4 weeks apart. The primary end point was a further episode of pneumococcal infection caused by vaccine serotypes or serotype 6A. RESULTS: From February 2003 through October 2007, we followed 496 patients (of whom 44% were male and 88% were HIV-seropositive) for 798 person-years of observation. There were 67 episodes of pneumococcal disease in 52 patients, all in the HIV-infected subgroup. In 24 patients, there were 19 episodes that were caused by vaccine serotypes and 5 episodes that were caused by the 6A serotype. Of these episodes, 5 occurred in the vaccine group and 19 in the placebo group, for a vaccine efficacy of 74% (95% confidence interval [CI], 30 to 90). There were 73 deaths from any cause in the vaccine group and 63 in the placebo group (hazard ratio in the vaccine group, 1.18; 95% CI, 0.84 to 1.66). The number of serious adverse events within 14 days after vaccination was significantly lower in the vaccine group than in the placebo group (3 vs. 17, P = 0.002), and the number of minor adverse events was significantly higher in the vaccine group (41 vs. 13, P = 0.003). CONCLUSIONS: The 7-valent pneumococcal conjugate vaccine protected HIV-infected adults from recurrent pneumococcal infection caused by vaccine serotypes or serotype 6A. (Current Controlled Trials number, ISRCTN54494731.).

Clinical Characteristics and Lung Function in Older Children Vertically Infected With Human Immunodeficiency Virus in Malawi.

BACKGROUND: Antiretroviral therapy (ART) has led to increased survival of children with vertically acquired human immunodeficiency virus infection. Significant morbidity arises from respiratory symptoms, but aetiology and pulmonary function abnormalities have not been systematically studied. METHODS: Human immunodeficiency virus-positive children aged 8-16 years were systematically recruited within clinics in Blantyre, Malawi. Clinical review, quality of life assessment, spirometry, and chest radiography were performed. RESULTS: One hundred sixty participants had a mean of age 11.1 (range, 8-16) years and 50.0% were female. Cough was present in 60 (37.5%) participants, and 55 (34.4%) had moderate or severe dyspnoea. Thirty-four (22.1%) participants had digital clubbing. Thirty-three (20.6%) participants were hypoxic at rest. One hundred eighteen (73.8%) of the children were receiving ART; median CD4 count was 698 cells/µL in these compared with 406 cells/µL in ART-naive individuals (P < .001). From 145 spirometry traces (90.6%), mean forced expiratory volume in 1 second (FEV1) and forced vital capacity (FVC) were 1.06 and 0.89 standard deviations below predicted mean, respectively. Twenty-one (14.5%) traces demonstrated obstructive defects and 26 (17.9%) reduced FVC. Lung function abnormality was not associated with any clinical findings. Of the 51 individuals with abnormal lung function, the mean increase in FEV1 after salbutamol was 3.8% (95% confidence interval, 0.02-7.53). "Tramlines" and ring shadows were seen on chest radiographs in over half of cases. CONCLUSIONS: Symptoms of chronic lung disease were highly prevalent with 2 main clinical phenotypes: "cough" and "hypoxia". Lung function abnormalities are common, poorly responsive to bronchodilators, and apparent throughout the age range of our cohort. Pathological causes remain to be elucidated. Cough and hypoxic phenotypes could be a useful part of diagnostic algorithms if further validated.

A reduction in adult blood stream infection and case fatality at a large African hospital following antiretroviral therapy roll-out.

INTRODUCTION: Blood-stream infection (BSI) is one of the principle determinants of the morbidity and mortality associated with advanced HIV infection, especially in sub-Saharan Africa. Over the last 10 years, there has been rapid roll-out of anti-retroviral therapy (ART) and cotrimoxazole prophylactic therapy (CPT) in many high HIV prevalence African countries. METHODS: A prospective cohort of adults with suspected BSI presenting to Queen's Hospital, Malawi was recruited between 2009 and 2010 to describe causes of and outcomes from BSI. Comparison was made with a cohort pre-dating ART roll-out to investigate whether and how ART and CPT have affected BSI. Malawian census and Ministry of Health ART data were used to estimate minimum incidence of BSI in Blantyre district. RESULTS: 2,007 patients were recruited, 90% were HIV infected. Since 1997/8, culture-confirmed BSI has fallen from 16% of suspected cases to 10% (p<0.001) and case fatality rate from confirmed BSI has fallen from 40% to 14% (p<0.001). Minimum incidence of BSI was estimated at 0.03/1000 years in HIV uninfected vs. 2.16/1000 years in HIV infected adults. Compared to HIV seronegative patients, the estimated incidence rate-ratio for BSI was 80 (95% CI:46-139) in HIV-infected/untreated adults, 568 (95% CI:302-1069) during the first 3 months of ART and 30 (95% CI:16-59) after 3 months of ART. CONCLUSIONS: Following ART roll-out, the incidence of BSI has fallen and clinical outcomes have improved markedly. Nonetheless, BSI incidence remains high in the first 3 months of ART despite CPT. Further interventions to reduce BSI-associated mortality in the first 3 months of ART require urgent evaluation.

A trial of a 7-valent pneumococcal conjugate vaccine in HIV-infected adults.

BACKGROUND: Streptococcus pneumoniae is a leading and serious coinfection in adults with human immunodeficiency virus (HIV) infection, particularly in Africa. Prevention of this disease by vaccination with the current 23-valent polysaccharide vaccine is suboptimal. Protein conjugate vaccines offer a further option for protection, but data on their clinical efficacy in adults are needed. METHODS: In this double-blind, randomized, placebo-controlled clinical efficacy trial, we studied the efficacy of a 7-valent conjugate pneumococcal vaccine in predominantly HIV-infected Malawian adolescents and adults who had recovered from documented invasive pneumococcal disease. Two doses of vaccine were given 4 weeks apart. The primary end point was a further episode of pneumococcal infection caused by vaccine serotypes or serotype 6A. RESULTS: From February 2003 through October 2007, we followed 496 patients (of whom 44% were male and 88% were HIV-seropositive) for 798 person-years of observation. There were 67 episodes of pneumococcal disease in 52 patients, all in the HIV-infected subgroup. In 24 patients, there were 19 episodes that were caused by vaccine serotypes and 5 episodes that were caused by the 6A serotype. Of these episodes, 5 occurred in the vaccine group and 19 in the placebo group, for a vaccine efficacy of 74% (95% confidence interval [CI], 30 to 90). There were 73 deaths from any cause in the vaccine group and 63 in the placebo group (hazard ratio in the vaccine group, 1.18; 95% CI, 0.84 to 1.66). The number of serious adverse events within 14 days after vaccination was significantly lower in the vaccine group than in the placebo group (3 vs. 17, P=0.002), and the number of minor adverse events was significantly higher in the vaccine group (41 vs. 13, P=0.003). CONCLUSIONS: The 7-valent pneumococcal conjugate vaccine protected HIV-infected adults from recurrent pneumococcal infection caused by vaccine serotypes or serotype 6A. (Current Controlled Trials number, ISRCTN54494731.)