Bacterial and Candida Colonization of Neonates in a Regional Hospital in South Africa.

BACKGROUND: Neonatal colonization with multidrug-resistant (MDR) Enterobacter spp., Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterococcus faecium (ESKAPE) and Candida spp. often precedes invasive hospital-acquired infections. We investigated the prevalence and dynamics of neonatal ESKAPE and Candida spp. colonization from hospital admission until discharge (or death) and followed up for invasive disease. METHODS: Prospective longitudinal surveillance for neonatal ESKAPE and Candida spp. colonization was conducted over 6 months at a South African regional hospital. Neonates enrolled at birth had swabs (nasal, 2× skin and rectal) collected within 24 hours and every 48-96 hours thereafter, until discharge or death. ESKAPE and Candida spp. were cultured for and antimicrobial susceptibility was performed on bacterial isolates. Whole-genome sequencing was undertaken on paired samples with the same bacterial species from colonizing and invasive disease episodes in the same child. RESULTS: Of 102 enrolled neonates, 79% (n = 81) were colonized by ≥1 ESKAPE organism by time of discharge or death. Forty-four percent (36/81) were colonized within 24 hours of birth. Common colonizers were K. pneumoniae (70%; n = 57) and Enterobacter spp. (43%; n = 35). Almost all MDR organisms (93%) were Gram-negative. Forty-two (45%, 42/93) newborns acquired Candida spp. (skin only) colonization, commonly Candida parapsilosis (69%; n = 29). For 2 children with K. pneumoniae colonization and sepsis, the bloodstream and colonizing isolates were genetically different, whereas the single A. baumannii colonizing and blood isolate pair were genetically identical. CONCLUSIONS: We report a high prevalence of MDR ESKAPE and Candida spp. colonization in a regional neonatal unit. Interventions to reduce the high incidence of hospital-acquired neonatal infections should include reducing high colonization rates.

Bacterial nasopharyngeal colonisation in children in South Africa before and during the COVID-19 pandemic: an observational study.

BACKGROUND: Deployment of non-pharmaceutical interventions such as face masking and physical distancing during the COVID-19 pandemic could have altered the transmission dynamics and carriage of respiratory organisms. We evaluated colonisation with Streptococcus pneumoniae and other upper respiratory tract bacterial colonisers before and during the COVID-19 pandemic. METHODS: We did two cross-sectional surveys in Soweto, South Africa from July 3 to Dec 13, 2018 (pre-COVID-19 period) and from Aug 4, 2021, to March 31, 2022 (COVID-19 period) in healthy children (aged ≤60 months) who had recorded HIV status and had not received antibiotics in the 21 days before enrolment. At enrolment, we collected nasopharyngeal swab samples from child participants. Following nucleic acid extraction, nanofluidic quantitative PCR was used to screen all samples for 92 S pneumoniae serotypes and 14 other bacteria. The primary objective was to compare the prevalence and density of pneumococcal nasopharyngeal colonisation, overall and stratified by 13-valent pneumococcal conjugate vaccine (PCV13) serotypes and non-vaccine serotypes. Secondary study objectives included a comparison of serotype-specific pneumococcal colonisation and density, as well as colonisation by the 14 other bacteria in the COVID-19 versus pre-COVID-19 period. We used an adjusted multiple logistic and linear regression model to compare the colonisation prevalence and density between study periods. FINDINGS: We analysed nasopharyngeal swabs from 1107 children (n=571 in the pre-COVID-19 period; n=536 in the COVID-19 period). We observed no change in overall pneumococcal colonisation between periods (274 [51%] of 536 in the COVID-19 period vs 282 [49%] of 571 in the pre-COVID-19 period; adjusted odds ratio [aOR] 1·03 [95% CI 0·95-1·12]). The prevalence of PCV13 serotypes was lower in the COVID-19 than in the pre-COVID-19 period (72 [13%] vs 106 [19%]; 0·87 [0·78-0·97]), whereas the prevalence of non-typeable S pneumoniae was higher (34 [6%] vs 63 [12%]; 1·30 [1·12-1·50]). The mean log10 density for overall pneumococcal colonisation was lower in the COVID-19 period than in the pre-COVID-19 period (3·96 [95% CI 3·85-4·07] vs 4·72 [4·63-4·80] log10 genome equivalents per mL; p<0·0001). A lower density of non-vaccine serotypes (3·63 [3·51-3·74] vs 4·08 [3·95-4·22] log10 genome equivalents per mL; p<0·0001) and non-typeable S pneumoniae (3·11 [2·94-3·29] vs 4·41 [4·06-4·75] log10 genome equivalents per mL; p<0·00001) was also observed in the COVID-19 period. There was no difference in the density of PCV13 serotypes between the periods. The prevalence of colonisation during the COVID-19 versus pre-COVID-19 period was lower for non-typeable Haemophilus influenzae (280 [49%] vs 165 [31%]; aOR 0·77 [95% CI 0·71-0·84]), Moraxella catarrhalis (328 [57%] vs 242 [45%]; 0·85 [0·79-0·92]), and Neisseria lactamica (51 [9%] vs 13 [2%]; 0·64 [0·52-0·78]), but higher for Acinetobacter baumannii (34 [6%] vs 102 [19%]; 1·55 [1·35-1·77]) and Staphylococcus aureus (29 [5%] vs 52 [10%]; 1·28 [1·10-1·50]). INTERPRETATION: There were variable effects on the colonisation prevalence and density of bacterial organisms during the COVID-19 compared with the pre-COVID-19 period. The lower prevalence of PCV13 serotype together with other respiratory organisms including non-typeable H influenzae and M catarrhalis could have in part contributed to a decrease in all-cause lower respiratory tract infections observed in South Africa during the initial stage of the COVID-19 pandemic. The pathophysiological mechanism for the increase in A baumannii and S aureus colonisation warrants further investigation, as does the clinical relevance of these findings. FUNDING: The Bill & Melinda Gates Foundation.

Nanofluidic qPCR unable to detect and serotype Streptococcus pneumoniae in urine samples of hospitalized South African patients with community-acquired pneumonia.

Pneumonia is a major cause of death among adults living with HIV in South Africa, but the etiology of many cases remains unknown. This study evaluated the utility of a nanofluidic qPCR assay to detect and serotype Streptococcus pneumoniae in urine samples from patients hospitalized with community-acquired pneumonia (CAP). The nanofluidic qPCR assay was optimized to target 13 pneumococcal serotypes and 4 reference genes. Archived urine samples collected from patients > 15 years of age hospitalized with pneumonia between April 2018 and August 2019 were retrospectively tested using the nanofluidic qPCR assay, BinaxNOW urine antigen test, and standard LytA qPCR. Blood culture was undertaken on a subset of the samples at the discretion of the attending physician. Cohens' Kappa statistics were used to determine the concordance between the methods. Of the 828 adults hospitalized for CAP, urine samples were available in 53% (n = 439). Of those, a random subset of 96 (22%) samples underwent testing. Of the participants included in the final analysis, the mean age was 45.8 years (SD 16.2), 49% (n = 47) were female, 98% (n = 94) were black, and 66% (n = 63) were living with HIV infection. The nanofluidic qPCR method was able to detect PCV13 vaccine strains spiked into urine samples; however, the method failed to detect any pneumococcus in clinical samples. In comparison, 19% of the pneumonia cases were attributed to S. pneumoniae using urine antigen testing. Nanofluidic qPCR is unable to detect and serotype Streptococcus pneumoniae in urine samples of South Africans hospitalized with CAP.

Metagenomic sequencing of post-mortem tissue samples for the identification of pathogens associated with neonatal deaths.

Postmortem minimally invasive tissue sampling together with the detailed review of clinical records has been shown to be highly successful in determining the cause of neonatal deaths. However, conventional tests including traditional culture methods and nucleic acid amplification tests have periodically proven to be insufficient to detect the causative agent in the infectious deaths. In this study, metagenomic next generation sequencing was used to explore for putative pathogens associated with neonatal deaths in post-mortem blood and lung tissue samples, in Soweto, South Africa. Here we show that the metagenomic sequencing results corroborate the findings using conventional methods of culture and nucleic acid amplifications tests on post-mortem samples in detecting the pathogens attributed in the causal pathway of death in 90% (18/20) of the decedents. Furthermore, metagenomic sequencing detected a putative pathogen, including Acinetobacter baumannii, Klebsiella pneumoniae, Escherichia coli, and Serratia marcescens, in a further nine of 11 (81%) cases where no causative pathogen was identified. The antimicrobial susceptibility profile was also determined by the metagenomic sequencing for all pathogens with numerous multi drug resistant organism identified. In conclusion, metagenomic sequencing is able to successfully identify pathogens contributing to infection associated deaths on postmortem blood and tissue samples.

Streptococcus pneumoniae and other bacterial nasopharyngeal colonization seven years post-introduction of 13-valent pneumococcal conjugate vaccine in South African children.

OBJECTIVES: Pneumococcal conjugate vaccines (PCVs) reduce pneumococcal-associated disease by reducing vaccine-serotype (VT) acquisition in vaccinated children, thereby interrupting VT transmission. The 7-valent-PCV was introduced in the South African immunization program in 2009 (13-valent-PCV since 2011) using a 2+1 schedule (at 6, 14, and 40 weeks of age). We aimed to evaluate temporal changes in VT and non-vaccine-serotype (NVT) colonization after 9 years of childhood PCV immunization in South Africa. METHODS: Nasopharyngeal swabs were collected from healthy children <60-month-old (n = 571) in 2018 (period-2) and compared with samples (n = 1135) collected during early PCV7-introduction (period-1, 2010-11) in an urban low-income setting (Soweto). Pneumococci were tested for using a multiplex quantitative-polymerase chain reaction serotyping reaction-set. RESULTS: Overall pneumococcal colonization in period-2 (49.4%; 282/571) was 27.5% lower than period-1 (68.1%; 773/1135; adjusted odds ratio [aOR]: 0.66; 95% confidence interval [CI]: 0.54-0.88). Colonization by VT was reduced by 54.5% in period-2 (18.6%; 106/571) compared with period-1 (40.9%; 465/1135; aOR: 0.41; 95% CI: 0.3-0.56). Nevertheless, serotype 19F carriage prevalence was higher (8.1%; 46/571) in period-2 compared with period-1 (6.6%; 75/1135; aOR: 2.0; 95% CI: 1.09-3.56). NVT colonization prevalence was similar in period-2 and period-1 (37.8%; 216/571 and 42.4%; 481/1135). CONCLUSION: There remains a high residual prevalence of VT, particularly 19F, colonization nine years post-introduction of PCV in the South African childhood immunization program.

Single priming and booster dose of ten-valent and 13-valent pneumococcal conjugate vaccines and Streptococcus pneumoniae colonisation in children in South Africa: a single-centre, open-label, randomised trial.

BACKGROUND: Pneumococcal conjugate vaccine (PCV) immunisation has reduced vaccine-serotype colonisation and invasive pneumococcal disease in South Africa, providing the opportunity to consider transitioning from a two-dose (2 + 1) to one-dose (1 + 1) primary series and a booster dose. METHODS: In this single-centre, open-label, randomised trial done in South Africa, infants aged 35-49 days without HIV infection, without childhood immunisations except for BCG and polio, and with gestation age at delivery of at least 37 weeks of age, a birthweight of at least 2500 g, and weight of at least 3500 g at the time of enrolment were randomly assigned (1:1:1:1:1:1), through block randomisation (block size of 30), to receive a single priming dose of ten-valent PCV (PCV10) or 13-valent PCV (PCV13) at either 6 weeks (6-week 1 + 1 group) or 14 weeks (14-week 1 + 1 group), compared with two priming doses at 6 weeks and 14 weeks (2 + 1 group), followed by a booster dose at 9 months of age in all groups. The primary objective of the trial has been published previously. We report the secondary objective of the effect of alternative doses of PCV10 and PCV13 on serotype-specific Streptococcus pneumoniae colonisation at 9 months, 15 months, and 18 months of age and a further exploratory analysis in which we assessed non-inferiority of serotype-specific serum IgG geometric mean concentrations 1 month after the booster (10 months of age) and the percentage of participants with serotype-specific IgG titre above the putative thresholds associated with a risk reduction of serotype-specific colonisation between the 1 + 1 and 2 + 1 groups for both vaccines. Non-inferiority was established if the lower limit of the 95% CI for the difference between the proportion of participants (1 + 1 group vs 2 + 1 group) above the putative thresholds was greater than or equal to -10%. All analyses were done in the modified intention-to-treat population, which included all participants who received PCV10 or PCV13 according to assigned randomisation group and for whom laboratory results were available. The trial is registered with ClinicalTrials.gov, NCT02943902. FINDINGS: 1564 nasopharyngeal swabs were available for molecular serotyping from 600 infants who were enrolled (100 were randomly assigned to each of the six study groups) between Jan 9 and Sept 20, 2017. There was no significant difference in the prevalence of overall or non-vaccine serotype colonisation between all PCV13 or PCV10 groups. PCV13 serotype colonisation was lower at 15 months of age in the 14-week 1 + 1 group than in the 2 + 1 group (seven [8%] of 85 vs 17 [20%] of 87; odds ratio 0·61 [95% CI 0·38-0·97], p=0·037), but no difference was seen at 9 months (nine [11%] of 86 vs ten [11%] of 89; 0·92 [0·60-1·55], p=0·87) or 18 months (nine [11%] of 85 vs 11 [14%] of 87; 0·78 [0·45-1·22], p=0·61). Compared with the PCV13 2 + 1 group, both PCV13 1 + 1 groups did not meet the non-inferiority criteria for serotype-specific anti-capsular antibody concentrations above the putative thresholds purportedly associated with risk reduction for colonisation; however, the PCV10 14-week 1 + 1 group was non-inferior to the PCV10 2 + 1 group. INTERPRETATION: The serotype-specific colonisation data reported in this study together with the primary immunogenicity endpoints of the control trial support transitioning to a reduced 1 + 1 schedule in South Africa. Ongoing monitoring of colonisation should, however, be undertaken immediately before and after transitioning to a PCV 1 + 1 schedule to serve as an early indicator of whether PCV 1 + 1 could lead to an increase in vaccine-serotype disease. FUNDING: The Bill & Melinda Gates Foundation.

Optimization of a high-throughput nanofluidic real-time PCR to detect and quantify of 15 bacterial species and 92 Streptococcus pneumoniae serotypes.

Sensitive tools for detecting concurrent colonizing pneumococcal serotypes are needed for detailed evaluation of the direct and indirect impact of routine pneumococcal conjugate vaccine (PCV) immunization. A high-throughput quantitative nanofluidic real-time PCR (Standard BioTools 'Fluidigm') reaction-set was developed to detect and quantify 92 pneumococcal serotypes in archived clinical samples. Nasopharyngeal swabs collected in 2009-2011 from South African children ≤ 5 years-old, previously serotyped with standard culture-based methods were used for comparison. The reaction-set within the 'Fluidigm' effectively amplified all targets with high efficiency (90-110%), reproducibility (R2 ≥ 0.98), and at low limit-of-detection (< 102 CFU/ml). A blind analysis of 1 973 nasopharyngeal swab samples showed diagnostic sensitivity > 80% and specificity > 95% compared with the referent standard, culture based Quellung method. The qPCR method was able to serotype pneumococcal types with good discrimination compared with Quellung (ROC-AUC: > 0.73). The high-throughput nanofluidic real-time PCR method simultaneously detects 57 individual serotypes, and 35 serotypes within 16 serogroups in 96 samples (including controls), within a single qPCR run. This method can be used to evaluate the impact of current PCV formulations on vaccine-serotype and non-vaccine-serotype colonization, including detection of multiple concurrently colonizing serotypes. Our qPCR method can allow for monitoring of serotype-specific bacterial load, as well as emergence or ongoing transmission of minor or co-colonizing serotypes that may have invasive disease potential.

Evaluation of the impact of HIV-1 infection and density of common nasopharyngeal bacterial colonizers in South African children immunized with 7-valent pneumococcal conjugate vaccine.

BACKGROUND: Due to limitations in standard culture methods, the impact of pneumococcal conjugate vaccine (PCV) immunization on nasopharyngeal bacterial carriage density is unclear, including among HIV-infected children. METHODS: The prevalence and density of serotype/serogroup-specific pneumococcal and other nasopharyngeal colonizing bacteria were investigated in archived swabs of HIV-infected and HIV-uninfected, PCV-7 immunized (at 6, 10 and 14 weeks of age) South African children collected at 9 and 16 months of age. During the course of the study, PCV-immunization of children in Soweto was limited to study-participants, as the vaccine had not been introduced into the public immunization program. RESULTS: At 9 months of age, the prevalence of overall pneumococcal colonization was lower in HIV-infected (58.6%) than HIV-uninfected children (69.9%, p = 0.02), mainly due to lower prevalence of non-vaccine-serotype colonization (27.8% vs. 40%, respectively; p = 0.047). The mean-log10 density of pneumococcal colonization was, however, higher in HIV-infected (4.81 CFU/ml) than HIV-uninfected pneumococcal colonized children (4.44 CFU/ml; p = 0.014); mainly due to higher mean-log10 density of PCV7-serotype colonization (4.21 vs. 3.72 CFU/ml; p = 0.014). No difference in the prevalence or density of overall pneumococci was found at 16 months of age. The prevalence of non-vaccine serotype colonization remained 1.7 fold higher in HIV-uninfected (60.4%) than HIV-infected children (50.9%, p = 0.049). Other differences included a lower prevalence of H. influenzae colonization in HIV-infected (42.3% and 56%) than HIV-uninfected children (64.2% and 73.4%) at both 9 and 16 months of age respectively; however, the density of colonization was similar. CONCLUSION: Increased carriage density of residual PCV7-serotypes might cause HIV-infected children to have a higher risk of pneumococcal disease. The higher carriage density observed in HIV-infected children could be attributed to a combination of factors, including HIV treatment and impaired host immunity. Additional studies are needed.

Performance of the Biomark HD real-time qPCR System (Fluidigm) for the detection of nasopharyngeal bacterial pathogens and Streptococcus pneumoniae typing.

Traditional qPCR assays for pneumococcal detection and serotype characterization require large sample volume, is expensive and labor intensive. We aimed to develop a quantitative nanofluidic Fluidigm assay to overcome some of these shortcomings. A quantitative Fluidigm assay was established to detect 11 bacterial pathogens, 55 pneumococcal serotypes and 6 serotypes of H. influenzae. The Fluidigm assay results were compared to conventional qPCR and culture. All reactions in the Fluidigm assay effectively amplified their respective targets with high sensitivity and specificity compared to qPCR. There was excellent concordance between qPCR and Fluidigm for detection of carriage prevalence (kappa > 0.75) and density (Rho > 0.95). Fluidigm identified an additional 7 (4.2%) serotypes over those detected by qPCR. There was a modest concordance between culture and Fluidigm for the majority of reactions detecting S. pneumoniae serotypes/serogroups (kappa > 0.6), with Fluidigm identifying an additional 113 (39.1%) serotypes. Discordant results between the three methods were associated with a low carriage density. The Fluidigm assay was able to detect common pneumococcal serotypes, H. influenzae serotypes, and other common nasopharyngeal bacterial organisms simultaneously. Deployment of this assay in epidemiological studies could provide better insight into the effect of PCV immunization on the nasopharyngeal microbiota in the community.

Review on Clinical and Molecular Epidemiology of Human Rhinovirus-Associated Lower Respiratory Tract Infections in African and Southeast Asian Children.

BACKGROUND: The detection of human rhinoviruses (HRV) is highly prevalent in children with pneumonia, bronchiolitis, acute asthma and croup; however, there is also evidence that HRV is common in asymptomatic individuals. The majority of studies on the role of different HRV serotypes during acute respiratory tract infections episodes have limited sample size to fully characterize the epidemiology of HRV infection, including those from low-middle income countries, where the burden of childhood respiratory disease is greatest. METHODS: We systematically reviewed HRV clinical and molecular epidemiology in low- and middle-income countries in Africa and Southeast Asia before November 2015. RESULTS: We identified 31 studies, which included data from 13 African and 6 Southeast Asian countries, emphasizing the gaps in knowledge surrounding HRV infections. HRV was one of the most prevalent respiratory viruses detected during childhood respiratory disease (13%-59%); however, many studies could not determine the attributable role of HRV in the pathogenesis of acute respiratory infections due to high prevalence of detection among asymptomatic individuals (6%-50%). A meta-analysis showed no significant difference in the prevalence of HRV identification between children of different age groups; or between children with severe disease compared with asymptomatic children. CONCLUSIONS: These data highlight the need for large-scale surveillance projects to determine the attributable etiologic role of HRV in respiratory disease.

Evaluation of the association of pneumococcal conjugate vaccine immunization and density of nasopharyngeal bacterial colonization using a multiplex quantitative polymerase chain reaction assay.

BACKGROUND: Nasopharyngeal bacterial colonization is a pre-requisite for developing bacterial mucosal and invasive disease. Pneumococcal conjugate vaccine (PCV) immunization of children reduces their risk of colonization by vaccine-serotypes, which could affect the biome of the nasopharynx in relation to colonization by other bacteria. This study evaluated the association of PCV immunization on the prevalence density of nasopharyngeal colonization by common, potentially pathogenic bacteria. METHODS: A multiplex qPCR assay was used to evaluate bacterial nasopharyngeal colonization by 7-valent PCV (PCV7) serotypes, non-vaccine serotypes (NVT), Haemophilus influenzae, Staphylococcus aureus, Moraxella catarrhalis, and Neisseria meningitidis in PCV7-vaccinated and PCV-unvaccinated African children at two time points. RESULTS: PCV7 vaccination was associated with a higher prevalence of NVT and H. influenzae at 9 and 16 months, respectively. While the prevalence of S. aureus was higher in PCV7-vaccinated children at 9 months, no difference was found at 16 months. The density of PCV7 serotypes (3.8 vs. 3.4 log10; p = 0.048), NVT (3.6 vs. 3.1 log10; p = 0.018), H. influenzae (4.34 vs. 3.86 log10; p = 0.008), M. catarrhalis (3.52 vs. 2.98 log10; p < 0.001) and S. aureus (4.02 vs. 3.06 log10; p = 0.02) was higher among PCV-vaccinated compared to PCV-unvaccinated children at 9 months, although, this difference diminished at 16 months of age. CONCLUSION: The reduction in PCV7-serotype colonization impacted on colonization prevalence and density of other bacterial species of the nasopharynx. The clinical relevance of this needs further exploration in relation to mucosal and invasive disease outcomes, as well as for higher valency PCV vaccines.

Use of Multiplex Quantitative PCR To Evaluate the Impact of Pneumococcal Conjugate Vaccine on Nasopharyngeal Pneumococcal Colonization in African Children.

Pneumococcal conjugate vaccine (PCV) immunization of children induces shifts in colonizing pneumococcal serotypes. This study evaluated the effect of infant vaccination with 7-valent PCV (PCV7) on vaccine serotype (VT) colonization and whether the increase in nonvaccine serotype (NVT) was due to either unmasking of previously low-density-colonizing serotypes or increase in acquisition of NVT. A multiplex quantitative PCR (qPCR) was used to evaluate VT and NVT nasopharyngeal colonization in archived swabs of PCV-vaccinated and PCV-unvaccinated African children at 9 and 15 to 16 months of age. Molecular qPCR clearly identified the vaccine effect typified by a decrease in VT colonization and an increase in NVT colonization. Serotype 19A was primarily responsible for the higher NVT carriage among PCV vaccinees at 9 months of age (53.4% difference; P = 0.021) and 16 months of age (70.7% difference; P < 0.001). Furthermore, the density of serotype 19A colonization was higher in PCV-vaccinated groups than in PCV-unvaccinated groups (3.76 versus 2.83 CFU/ml [P = 0.046], respectively, and 4.15 versus 3.04 CFU/ml [P = 0.013], respectively) at 9 and 16 months of age, respectively. Furthermore, serotype 19A was also more commonly reported as a primary isolate (by having the highest density among other cocolonizing serotypes identified in the sample) in PCV7-vaccinated children, while being equally a primary (46.2%) or nonprimary (53.8%) isolate in PCV-unvaccinated children. Molecular qPCR showed both serotype replacement and unmasking to be the cause for the increase in NVT colonization in PCV7-vaccinated children, as some serotypes were associated with an absolute increase in colonization (replacement), while others were associated with an increase in detection (unmasking). IMPORTANCE This study focused on evaluating the effect of infant vaccination with 7-valent pneumococcal conjugate vaccine (PCV7), using a multiplex qPCR method, on the density of serotype-specific nasopharyngeal colonization in order to delineate the relative role of serotype replacement versus unmasking as the cause for the increase in nonvaccine serotype colonization in PCV7-vaccinated children. This is pertinent in the context of the ongoing deployment of PCV immunization in children, with surveillance of colonization considered an early proxy for disease that might arise from nonvaccine serotypes, as well as the success of childhood vaccination on indirect effect in the community through the interruption of pneumococcal transmission from vaccinated young children.

Comparison of traditional culture and molecular qPCR for detection of simultaneous carriage of multiple pneumococcal serotypes in African children.

S. pneumoniae is a common colonizer of the human nasopharynx in high income and low-middle income countries. Due to limitations of standard culture methods, the prevalence of concurrent colonization with multiple serotypes is unclear. We evaluated the use of multiplex quantitative PCR (qPCR) to detect multiple pneumococcal serotypes/group colonization in archived nasopharyngeal swabs of pneumococcal conjugate vaccine naive children who had previously been investigated by traditional culture methods. Overall the detection of pneumococcal colonization was higher by qPCR (82%) compared to standard culture (71%; p < 0.001), with a high concordance (kappa = 0.73) of serotypes/groups identified by culture also being identified by qPCR. Also, qPCR was more sensitive in detecting multiple serotype/groups among colonized cases (28.7%) compared to culture (4.5%; p < 0.001). Of the additional serotypes detected only by qPCR, the majority were of lower density (<104 CFU/ml) than the dominant colonizing serotype, with serotype/group 6A/B, 19B/F and 23F being the highest density colonizers, followed by serotype 5 and serogroup 9A/L/N/V being the most common second and third colonizers respectively. The ability of qPCR to detect multiple pneumococcal serotypes at a low carriage density might provide better insight into underlying mechanism for changes in serotype colonization in PCV vaccinated children.