Challenges of using molecular serotyping for surveillance of pneumococcal disease.
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ABSTRACT:
SUBMITTER: Magomani V
PROVIDER: S-EPMC4313149 | biostudies-literature | 2014 Sep
REPOSITORIES: biostudies-literature
Publications
Challenges of using molecular serotyping for surveillance of pneumococcal disease.
Magomani Victoria V Wolter Nicole N Tempia Stefano S du Plessis Mignon M de Gouveia Linda L von Gottberg Anne A
Journal of clinical microbiology 20140623 9
Recent advances in the molecular identification and serotyping of Streptococcus pneumoniae are useful for culture-negative samples; however, there are limitations associated with these methods. We aimed to assess the value of molecular assays for invasive pneumococcal disease (IPD) surveillance in South Africa from 2010 through 2012. Nonviable isolates and culture-negative clinical specimens were tested for the lytA gene and, if positive, were serotyped, using real-time PCRs. Multinomial regress ...[more]
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Project description:Surveillance of new cases of invasive pneumococcal disease (IPD) in Italy was started in 2007 by the Ministry of Health (MoH). In 2012, pneumococcal childhood vaccination was introduced at the national level and, in 2017, for citizens aged 65 years and over. We describe here IPD epidemiology in Italy over the past 10 years investigating the impact of the vaccine programme on disease burden. Reports of IPD cases, data on serotype and vaccination coverage (VC) data were obtained from MoH annual reports, for the period 2007-2017. IPD notification rate and proportion by year, region, age and serotype were calculated. In 2007, 525 cases were reported (rate 0.88/100 000), rising to 1703 cases (rate 2.82/100 000) in 2017. The distribution of IPD cases by age group over time registered the largest share among individuals aged 65 years and over. A decreasing trend in notification rate was observed among those aged 0-4 years. During the same period, the 24-month VC increased, ranging from 80.9% to 96.7% in 2017. Molecular data indicated re-emergence of PPSV23-specific serotypes and non-vaccine serotypes. We observed an increase in IPD notifications during 2007-2017, likely due to an improved surveillance system, at least in some regions, with the relative quota of IPD notifications decreasing among vaccinated children cohorts. Further strengthening of IPD surveillance system, including molecular and vaccine coverage data, would be needed to assess and inform pneumococcal vaccination strategies in Italy.
| S-EPMC7482190 | biostudies-literature
Project description:The use of molecular diagnostic techniques for the evaluation of the impact of pneumococcal conjugate vaccines (PCVs) has not been documented. We aimed to evaluate the impact of PCVs on invasive pneumococcal disease (IPD) using polymerase chain reaction (PCR)-based techniques and compare with results obtained from culture-based methods.We implemented two independent surveillance programs for IPD among individuals hospitalized at one large surveillance site in Soweto, South Africa during 2009-2012: (i) PCR-based (targeting the lytA gene) syndromic pneumonia surveillance; and (ii) culture-based laboratory surveillance. Positive samples were serotyped. The molecular serotyping assay included targets for 42 serotypes including all serotypes/serogroups included in the 7-valent (PCV-7) and 13-valent (PCV-13) PCV. The Quellung reaction was used for serotyping of culture-positive cases. We calculated the change in rates of IPD (lytA- or culture-positive) among HIV-uninfected children aged <2 years from the year of PCV-7 introduction (2009) to the post-vaccine years (2011 or 2012).During the study period there were 607 lytA-positive and 1,197 culture-positive cases that were serotyped. Samples with lytA cycle threshold (Ct)-values ?35 (30.2 %; 123/407) were significantly less likely to have a serotype/serogroup detected for serotypes included in the molecular serotyping assay than those with Ct-values <35 (78.0 %; 156/200) (p < 0.001). From 2009 to 2012 rates of PCV-7 serotypes/serogroups decreased -63.8 % (95 % CI: -79.3 % to -39.1 %) among lytA-positive cases and -91.7 % (95 % CI: -98.8 % to -73.6 %) among culture-positive cases. Rates of lytA-positive non-vaccine serotypes/serogroups also significantly decreased (-71.7 %; 95 % CI: -81.1 % to -58.5 %) over the same period. Such decline was not observed among the culture-positive non-vaccine serotypes (1.2 %; 95 % CI: -96.7 % to 58.4 %).Significant downward trends in IPD PCV-7 serotype-associated rates were observed among patients tested by PCR or culture methods; however trends of non-vaccine serotypes/serogroups differed between the two groups. Misclassifications of serotypes/serogroups, affecting the use of non-vaccine serotypes as a control group, may have occurred due to the low performance of the serotyping assay among lytA-positive cases with high Ct-values. Until PCR methods improve further, culture methods should continue to be used to monitor the effects of PCV vaccination programs on IPD incidence.
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Project description:IntroductionElectronic influenza surveillance systems aid in health surveillance and clinical decision-making within the emergency department (ED). While major advances have been made in integrating clinical decision-making tools within the electronic health record (EHR), tools for sharing surveillance data are often piecemeal, with the need for data downloads and manual uploads to shared servers, delaying time from data acquisition to end-user. Real-time surveillance can help both clinicians and public health professionals recognize circulating influenza earlier in the season and provide ongoing situational awareness.MethodsWe created a prototype, cloud-based, real-time reporting system in two large, academically affiliated EDs that streamed continuous data on a web-based dashboard within hours of specimen collection during the influenza season. Data included influenza test results (positive or negative) coupled with test date, test instrument geolocation, and basic patient demographics. The system provided immediate reporting to frontline clinicians and to local, state, and federal health department partners.ResultsWe describe the process, infrastructure requirements, and challenges of developing and implementing the prototype system. Key process-related requirements for system development included merging data from the molecular test (GeneXpert) with the hospitals' EHRs, securing data, authorizing/authenticating users, and providing permissions for data access refining visualizations for end-users.ConclusionIn this case study, we effectively integrated multiple data systems at four distinct hospital EDs, relaying data in near real time to hospital-based staff and local and national public health entities, to provide laboratory-confirmed influenza test results during the 2014-2015 influenza season. Future innovations need to focus on integrating the dashboard within the EHR and clinical decision tools.
| S-EPMC8967469 | biostudies-literature
Project description:All currently available vaccines against Streptococcus pneumoniae are based on selections of the over 90 different serotypes, which underlines the importance of serotyping for surveillance and vaccine efficacy monitoring. In this study, we modified and validated a PCR-based scheme for deducing the serotypes of the invasive pneumococci isolated in Finland. For validation, 170 isolates were serotyped using the new protocol with six sequential multiplex PCRs for the deduction of serotypes, supplemented with Quellung testing when needed. The results were compared with those obtained by traditional serotyping methods. We found that 98.8% (168/170) of the isolates were correctly serotyped by the new protocol. Subsequently, the scheme was taken into regular use for serotyping the invasive pneumococci isolated in Finland for serotype-specific surveillance purposes and has been applied in the serotyping of more than 1,500 invasive isolates so far. The sequential multiplex PCRs (mPCRs) have given a result for over 99% of the isolates and allowed us to both handle samples in bulk and noticeably reduce the cost of reagents. While serotyping primarily by PCR is precise and effective, Quellung testing remains the most reliable way to discover possible discrepancies between the DNA deduced and the phenotypic serotype of an isolate. Since implementing the protocol for regular use, two serotype 19F PCR-positive isolates were found to be serotype 19A by the Quellung reaction. While a rare occurrence, this is an important observation, which prompted a revision of our serotyping protocol to prevent possible underreporting of serotype 19A, a potential replacement serotype following large-scale vaccination.
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Project description:Streptococcus pneumoniae (the pneumococcus) carriage is commonly used to measure effects of pneumococcal vaccines. Based on findings from culture-based studies, the World Health Organization recommends both nasopharyngeal (NP) and oropharyngeal (OP) sampling for detecting adult carriage. Given evidence of potential confounding by other streptococci, we evaluated molecular methods for pneumococcal identification and serotyping from 250 OP samples collected from adults in Fiji, using paired NP samples for comparison. Samples were screened using lytA quantitative PCR (qPCR), as well as pneumococcal identification and serotyping conducted by DNA microarray. A subset of OP samples were characterized by latex sweep agglutination and multiplex PCR. Alternate qPCR assays (piaB and bguR) for pneumococcal identification were evaluated. The lytA qPCR was less specific and had poor positive predictive value (PPV) in OP samples (88% and 26%, respectively) compared with NP samples (95% and 64%, respectively). Using additional targets piaB and/or bguR improved qPCR specificity in OP, although the PPV (42 to 53%) was still poor. Using microarray, we found that 102/107 (95%) of OP samples contained nonpneumococcal streptococci with partial or divergent complements of pneumococcal capsule genes. We explored 91 colonies isolated from 11 OP samples using various techniques, including multiplex PCR, latex agglutination, and microarray. We found that nonpneumococcal streptococci contribute to false positives in pneumococcal serotyping and may also contribute to spurious identification by qPCR. Our results highlight that molecular approaches should include multiple loci to minimize false-positive results when testing OP samples. Regardless of method, pneumococcal identification and serotyping results from OP samples should be interpreted with caution.IMPORTANCE Streptococcus pneumoniae (the pneumococcus) is a significant global pathogen. Accurate identification and serotyping are vital. In contrast with World Health Organization recommendations based on culture methods, we demonstrate that pneumococcal identification and serotyping with molecular methods are affected by sample type. Results from oropharyngeal samples from adults were often inaccurate. This is particularly important for assessment of vaccine impact using carriage studies, particularly in low- and middle-income countries where there are significant barriers for disease surveillance.
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Project description:BackgroundPneumococcal serotype identification is essential to monitor pneumococcal vaccine effectiveness and serotype replacement. Serotyping by conventional serological methods are costly, labour-intensive, and require significant technical expertise. We compared two different molecular methods to serotype pneumococci isolated from the nasopharynx of South African infants participating in a birth cohort study, the Drakenstein Child Health Study, in an area with high 13-valent pneumococcal conjugate vaccine (PCV13) coverage.MethodsA real-time multiplex PCR (rmPCR) assay detecting 21 different serotypes/-groups and a sequetyping assay, based on the sequence of the wzh gene within the pneumococcal capsular locus, were compared. Forty pneumococcal control isolates, with serotypes determined by the Quellung reaction, were tested. In addition, 135 pneumococcal isolates obtained from the nasopharynx of healthy children were tested by both serotyping assays and confirmed by Quellung testing. Discordant results were further investigated by whole genome sequencing of four isolates.ResultsOf the 40 control isolates tested, 25 had a serotype covered by the rmPCR assay. These were all correctly serotyped/-grouped. Sequetyping PCR failed in 7/40 (18%) isolates. For the remaining isolates, sequetyping assigned the correct serotype/-group to 29/33 (88%) control isolates. Of the 132/135 (98%) nasopharyngeal pneumococcal isolates that could be typed, 69/132 (52%) and 112/132 (85%) were assigned the correct serotype/-group by rmPCR and sequetyping respectively. The serotypes of 63/132 (48%) isolates were not included in the rmPCR panel. All except three isolates (serotype 25A and 38) were theoretically amplified and differentiated into the correct serotype/-group with some strains giving ambigous results (serotype 13/20, 17F/33C, and 11A/D/1818F). Of the pneumococcal serotypes detected in this study, 69/91 (76%) were not included in the current PCV13. The most frequently identified serotypes were 11A, 13, 15B/15C, 16F and 10A.ConclusionThe rmPCR assay performed well for the 21 serotypes/-groups included in the assay. However, in our study setting, a large proportion of serotypes were not detected by rmPCR. The sequetyping assay performed well, but did misassign specific serotypes. It may be useful for regions where vaccine serotypes are less common, however confirmatory testing is advisable.
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Project description:Ninety-two Streptococcus pneumoniae serotypes have been described so far, but the pneumococcal conjugate vaccine introduced in the Brazilian basic vaccination schedule in 2010 covers only the ten most prevalent in the country. Pneumococcal serotype-shifting after massive immunization is a major concern and monitoring this phenomenon requires efficient and accessible serotyping methods. Pneumococcal serotyping based on antisera produced in animals is laborious and restricted to a few reference laboratories. Alternatively, molecular serotyping methods assess polymorphisms in the cps gene cluster, which encodes key enzymes for capsular polysaccharides synthesis in pneumococci. In one such approach, cps-RFLP, the PCR amplified cps loci are digested with an endonuclease, generating serotype-specific fingerprints on agarose gel electrophoresis.In this work, in silico and in vitro approaches were combined to demonstrate that XhoII is the most discriminating endonuclease for cps-RFLP, and to build a database of serotype-specific fingerprints that accommodates the genetic diversity within the cps locus of 92 known pneumococci serotypes.The expected specificity of cps-RFLP using XhoII was 76% for serotyping and 100% for serogrouping. The database of cps-RFLP fingerprints was integrated to Molecular Serotyping Tool (MST), a previously published web-based software for molecular serotyping. In addition, 43 isolates representing 29 serotypes prevalent in the state of Minas Gerais, Brazil, from 2007 to 2013, were examined in vitro; 11 serotypes (nine serogroups) matched the respective in silico patterns calculated for reference strains. The remaining experimental patterns, despite their resemblance to their expected in silico patterns, did not reach the threshold of similarity score to be considered a match and were then added to the database.The cps-RFLP method with XhoII outperformed the antisera-based and other molecular serotyping methods in regard of the expected specificity. In order to accommodate the genetic variability of the pneumococci cps loci, the database of cps-RFLP patterns will be progressively expanded to include new variant in vitro patterns. The cps-RFLP method with endonuclease XhoII coupled with MST for computer-assisted interpretation of results may represent a relevant contribution to the real time detection of changes in regional pneumococci population diversity in response to mass immunization programs.
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Project description:BackgroundVaccine-serotype (VT) invasive pneumococcal disease (IPD) rates declined substantially following introduction of 7-valent pneumococcal conjugate vaccine (PCV7) into national immunization programs. Increases in non-vaccine-serotype (NVT) IPD rates occurred in some sites, presumably representing serotype replacement. We used a standardized approach to describe serotype-specific IPD changes among multiple sites after PCV7 introduction.Methods and findingsOf 32 IPD surveillance datasets received, we identified 21 eligible databases with rate data ? 2 years before and ? 1 year after PCV7 introduction. Expected annual rates of IPD absent PCV7 introduction were estimated by extrapolation using either Poisson regression modeling of pre-PCV7 rates or averaging pre-PCV7 rates. To estimate whether changes in rates had occurred following PCV7 introduction, we calculated site specific rate ratios by dividing observed by expected IPD rates for each post-PCV7 year. We calculated summary rate ratios (RRs) using random effects meta-analysis. For children <5 years old, overall IPD decreased by year 1 post-PCV7 (RR 0.55, 95% CI 0.46-0.65) and remained relatively stable through year 7 (RR 0.49, 95% CI 0.35-0.68). Point estimates for VT IPD decreased annually through year 7 (RR 0.03, 95% CI 0.01-0.10), while NVT IPD increased (year 7 RR 2.81, 95% CI 2.12-3.71). Among adults, decreases in overall IPD also occurred but were smaller and more variable by site than among children. At year 7 after introduction, significant reductions were observed (18-49 year-olds [RR 0.52, 95% CI 0.29-0.91], 50-64 year-olds [RR 0.84, 95% CI 0.77-0.93], and ? 65 year-olds [RR 0.74, 95% CI 0.58-0.95]).ConclusionsConsistent and significant decreases in both overall and VT IPD in children occurred quickly and were sustained for 7 years after PCV7 introduction, supporting use of PCVs. Increases in NVT IPD occurred in most sites, with variable magnitude. These findings may not represent the experience in low-income countries or the effects after introduction of higher valency PCVs. High-quality, population-based surveillance of serotype-specific IPD rates is needed to monitor vaccine impact as more countries, including low-income countries, introduce PCVs and as higher valency PCVs are used. Please see later in the article for the Editors' Summary.
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Project description:Novel data streams (NDS), such as web search data or social media updates, hold promise for enhancing the capabilities of public health surveillance. In this paper, we outline a conceptual framework for integrating NDS into current public health surveillance. Our approach focuses on two key questions: What are the opportunities for using NDS and what are the minimal tests of validity and utility that must be applied when using NDS? Identifying these opportunities will necessitate the involvement of public health authorities and an appreciation of the diversity of objectives and scales across agencies at different levels (local, state, national, international). We present the case that clearly articulating surveillance objectives and systematically evaluating NDS and comparing the performance of NDS to existing surveillance data and alternative NDS data is critical and has not sufficiently been addressed in many applications of NDS currently in the literature.
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