| Pneumococcal diseases (PDs) are a significant public health problem globally. Streptococcus pneumonia (SP) is a major cause of serious invasive diseases, including meningitis, bacteraemia, and pneumonia in infants and children. It is also a common cause of noninvasive diseases such as sinusitis and acute otitis media.A study conducted by the Pneumococcal Global Burden of Disease Study Team conducted that six of the 10 countries with the highest numbers and proportions of pneumococcal cases caused by SP in the year 2000 in children aged<5 years were located in Asia (India 27%, China 12%). In the same study, it also reported that approximately 1.74 million children cases suffered from PDs and thirty thousand deaths each year in China. Invasive pneumococcal disease (IPDs) is an important cause of severe diseases and death, especially in small children, the elderly and immunocompromised individuals. A study on IPDs burden among children in the Asia-Pacific region suggested that except for those in Australia and New Zealand, many of the surveillance studies that have been conducted can be assumed to underestimate the incidence of pneumococcal disease. For example, many of the surveillance studies included investigated disease only in hospitalized patients. The data from the United States showed approximately 5 million cases of S. pneumonia otitis media occurred each year before 2007, as well as bacteremia and meningitis. The burden of pneumococcal disease is immense. The World Health Organization (WHO) recommended use of pneumococcal conjugate vaccines (PCVs) in developed and developing countries.Capsular polysaccharide (CPS) is the most important and critical virulence factors in SP, which is associated with the serotype-specific and can serve as targets of the host antibody, which is also the basis for vaccine applications. To date, depending on the structure of CPS, using immunological methods have identified 46 serogroups and 95 serotypes. Pneumococcal vaccines, which induce the development of anticapsular antibodies to type-specific capsular polysaccharide antigens, confer immunity against the disease caused by vaccine serotypes. Seven-valent pneumococcal conjugate vaccine (PCV-7) includes serotypes 4,6B,9 V,14,18C,19F, and 23F. It was first licensed in 2000 in the United States for rountine use in children aged<5 years. To date, over 40 countries or regions have included pneumococcal vaccination in their national immunization programs. The incidence of IPDs decreased significantly in those countries or regions.After the introduction of the PCV-7 into childhood vaccination calendars, rates of IPDs caused by the seven serotypes covered by the vaccine (4,6B,9V,14,18C, 19F, and 23F) have decreased substantially in all aged. However, since the introduction of PCV-7, "serotype replacement" has been observed, with an increased in the proportion of invasive and non-invasive disease caused by serotypes not represented in the vaccine. The changing of disease type and emerging of replacement disease following the introduction of PCV-7 has been noted.PCV-10 and PCV-13, which newer conjugate vaccines with additional serotypes, are currently licensed in children aged<5 years. The long-term impacts of the vaccine include some replacement of vaccine serotypes (VT) by non-vaccine types (NVT), both among the nasopharyngeal colonizers and among invasive isolates should be concerned. Such changes in the seroepidemiology of the organism need to be constantly monitored to evaluate the effect and appropriateness of newer vaccines. Increases in disease caused by previously uncommon NVT could necessitate changes in vaccine composition, emphasizing the need for continued surveillance.Conventional serotyping by the Quellung reaction is complex, costly and require highly skilled personnel. Quellung serotyping is available in only a couple of big city centers such as Beijing, Shanghai, and Guangzhou in China. In recent years, a variety of DNA-based methods that rely on the capsular polysaccharide synthesis (cps) locus for the deduction of pneumococcal serotypes have been described, including approaches based on amplicon sequencing, multilocus sequence typing (MLST), real time PCR, multiplex PCR, reverse line blot hybridization (RLB), restriction fragment length polymorphism (RFLP), and micrroarry methods. Though these molecular approaches to serotyping appear promising, but they have different advantages and disadvantages. For example, RLB is an objective way to identify up to 43 targets in 43 individual specimens simultaneously (using a 45-lane membrane format). The number of targets is adequate for epidemiological and most clinical diagnostic applications; based on the same target (43) and specimen numbers (43). It takes about one and half working days to complete from DNA extraction to the final results of the RLB. However, it need skilled person to design primer and probe which are critical to success of the assay, trained personnel to operation and special equipments. Micrroarry methods for serotyping can provide coverage of all known serotypes, but its high cost would limit its introduction in resource-poor regions with high burdens of disease. While specificity and sensitivity are increased by these PCR-based serotyping methods, a large number of serotype-specific primers pairs are required to provide broad serotype coverage. Most DNA-based methods are multiplex or multistep, increasing the complexity of the assays.Leung and collaborators used a single PCR sequencing method targeting cpsB gene (sequence type) to identify S. pneumonia serotypes. They identified 46 serotypes from 138 pneumococcal stains covering 48 serotypes. They also developed a cpsB gene sequetyping database. The database designed by Leung et al didn’t include all the 95 serotype cpsB seuquences. S. pneumoniae is characterized by high-frequency recombination within the cps gene cluster, leading to serotype’switching’ among isolates within genetic lineages defined by relationships between the more conserved housekeeping genes. This make difficult to identify cross-reactive serotypes, new sequence type, and mixed serotypes by sequencing method.The USA Centers for Disease Control and Prevention (CDC) has published a sequential multiplex PCR (mPCR) protocol, which was proven precise and effective for serotyping the majority of invasive pneumococci for national surveillance. But it is complicated by the need to perform eight sets of multiplex PCRs. The CDC serotyping schemes have been modified in different ways for use in several countries as well, which indicates that the oligonucleotide primers used for amplification can be combined according to the serotype distribution of different geographical regions of the world. This flexibility is important.The serotypes distribution of 5. pneumoniae reported varied with age, geography, diseases, and time. Continuous surveillance is crucial to monitor vaccine efficacy and serotype replacement in different geographical regions. Although China is among the ten countries with the highest prevalence of pneumococcal cases, there is limited serotype surveillance data for S. pneumonia, especially from the community or rural regions, partly due to limited serotyping capacity because Quellung serotyping is only available in few centers in China. The aim of this study was, as a showcase, to investigate the best combination of the aforementioned methods for use as an initial serotype screening method especially for developing countries. Furthermore, in order to provide some local epidemiological data for current and future planning purposes, we studied the serotype distribution, antibiotic susceptibility and clinical presentation, amongst the 193 S. pneumoniae isolates.Part 1 cpsB sequetyping for determining serotypes of Streptococcus pneumoniaePurpose:Using the previous cpsB sequetyping method to identify serotypes of S.pneumoniae isolates and improved this work by developed a new cpsB gene sequetyping database including all the 95 serotypes described to date.Methods:All the S. pneumoniae sequences in the GenBank that contained the full-length of cpsB (as of Jan 1,2015) were downloaded, and after sequence alignment using ClustalW and/or Blastn, each GenBank sequence was given a unique cpsB sequetype name, ensuring that all GenBank sequences with the same cpsB sequence, were given the same sequetype name. To building up a comprehensive 95 serotypes cpsB sequetyping database based on GenBank sequences.193 S. pneumoniae isolates were collected from hospitalized children whom were< 5 years old, during the period January 2009 to December 2013. The cpsB gene was amplified by single PCR using specific primers (cpsl/cps2), the amplicon purified, and the nucleotide sequence determined by double strand sequencing. The amplicon nucleotide sequences were then used to Blastn GenBank database, and if it was identical to any one or more of the GenBank sequences, the serotype (s)/cpsB sequetype was decided according to our cpsB sequetyping database.Results:We developed a comprehensive S. pneumoniae serotype cpsB sequence (732-bp) sequetyping reference database for all the 95 described serotypes, including all 390 available GenBank sequences with full length of the cpsB sequence name. All the 193 isolates included in the study could be amplified by cpsB PCR and yielded satisfactory sequencing results. Based on cpsB sequence heterogeneity at one or more sites for all isolates,21 different sequetypes were identified:eight serotype-specific sequetypes could predict isolates to serotype level (3,9V,6B,10B,14,19A,23F, 23A); five sequetypes shared by different serotypes but in the same serogroups (6C-6D-1,6B-6E-6X-1,15B-15C-1,19F-19A-1, and 28F-28A-1) could predict isolates to the serogroup level; and three sequetypes shared by different serotypes serotypes 13 and 20,15A and 33B,17A and 34, could not be differentiated from each other. Of the 193 isolates tested,66 (34.2%) were sequetyped to the serotype level and 107 (55.4%) to the serogroup level.Conclusion:The comprehensive S. pneumoniae 95 serotype cpsB sequetyping database is a supplement for molecular approaches to serotyping. Single-primer cpsB sequetyping method is simple to perform, and can be a first screening for serotype identification.Part 2 Serotyping of Streptococcus pneumoniae by modified sequential multiplex PCR approachPurpose:Based on the serotype distribution from children in China, to modify USA CDC sequential multiplex-PCR to identify the most common serotypes in the first three sets of mPCR.Methods:Using modified sequential multiplex PCR to identify serotypes of 193 5. pneumonia isolates. Sequential mPCR, as per CDC scheme, was employed to predict the isolate serotype(s) by targeting serotype-specific cps regions. The primer sequences, PCR reactions and product detection are as published by the CDC. We further designed combinations of primer sets in the first three reactions to identify the six most predominant serotypes (19F,19A,14,23F,15,6) in China based on previous studies, three PCV-7 vaccine serotypes (4,9V/9A,18C), and serogroup15 (serotype 15A/15F,15B/15C) which are currently among the most prevalent NVT globally besides serotype 19A. In these modified reactions, reaction 1 contained primers for serotypes 9V/9A,14,23F and 15B/15C; reaction 2 contained primers for serotypes 4, 6, and 19A; and reaction 3 contained primers for serotypes 15 A/15F,19F and 18C. If a sample was negative in the first three reactions, eight sequential mPCR reactions were performed as previously described in CDC (USA) Streptococcus website. An internal positive control targeting all known pneumococcal cps operons was included in each reaction.Results:Employed modified sequential mPCR method,12 serogroup/serotypes were identified from 193 S.pneumoniae isolates. They included 3,6,9V/9A,14, 15F/15A,15B/15C,19F,19A,20,23A,23F and 34. There were not serotype 4 and 18C isolates in this study, so they were negative. Furthermore, using the mPCR, most serotypes were identified in the first three sets of mPCR reactions, and only 11 (5.7%) isolates required further testing of up to eight sequential sets of multiplex reactions. Five isolates, for which sequencing gave ambiguous result as serotypes 15A-33B-1 (2 isolates),13-20A-20B-1 (2 isolates), and 17A-34-1 (1 isolate), were confirmed by mPCR that they were serotypes 15F/15A,20 and 34, respectively. Thirteen serotype 23F isolates belonging to four new cpsB sequetypes, were assigned new sequetype names after mPCR confirmed them as serotype 23F. Two isolates identified by cpsB sequetyping as 10B,28F/28A, were non-typeable by mPCR. Two isolates showed unknown by the both methods. The level of agreement between cpsB sequetyping and multiplex PCR results was 92.2%.Conclusion:Our modified sequential mPCR is an accurate and reliable method which can be used when needed after cpsB sequencing in pneumococcal serotyping.Part 3 Serotype 6A-6D specific PCR assay to differentiate Streptococcus pneumoniae serogroup 6 isolatesPurpose:Using we developed serotype-specific PCR to identify serotypes 6A, 6B,6C and 6D among which cpsB sequetyping and mPCR identified as serogroup 6 isolates.Methods:27 serogroup 6 isolates were collected after cpsB sequetyping and mPCR identified. Specific primers wciP584aS/wciP-r(6B) and wciP584gS/wciP-r(6A) were used by single PCR to differentiates serotypes 6A/6C and 6B/6D. Then we used single PCR that target wciNβ gene by specific primers wciNβS1/wciNβA2 to differentiates serotype 6C and 6D from 6A/6C and 6B/6D isolates respectively.Results:The distribution of serotypes 6A-6D among the 27 serogroup 6 isolates were 6A (12/27,44.4%),6B (13/27,48.1%),6C (2/27,7.4%) and serotype 6D was not detected.Conclusion:In this study, we confirmed that serotype 6A-6D specific PCR was a simple, reliable method to differentiate S. pneumoniae serogroup 6.Part 4 cpsB sequetyping supplemented with mPCR and serotypes 6A-6D specific PCR "cocktail" strategy to investigate Streptococcus pneumoniae serotype distribution in Chinese local hospitalized childrenPurpose:The aim of this study was to develop an integrated cpsB sequetyping supplemented with modified mPCR and serotype 6A-6D specific PCRs "cocktail" strategy suitable for future serotype surveillance in China.193 S. pneumonia isolates from hospitalized children were serotyped using this strategy and antibiotics tested.Methods:In this study,193 S. pneumoniae isolates were collected from hospitalized children whom were< 5 years old suffered from PDs, during the period 2009 to 2013. cpsB sequetyping was performed on all the isolates to identify serotypes or sequence type. After cpsB sequetyping results were known, isolates which with discrepant results, shared the same cpsB sequetype, non-typeable by cpsB sequetyping, and new cpsB sequetypes were further tested by modified sequential mPCR to confirm the results. Serotypes 6A-6D specific PCRs were done on all serogroup 6 isolates to differentiate serotype 6A,6B,6C and 6D. The GenBank accession numbers for the new cpsB sequences from this study submitted to GenBank. In vitro susceptibility tests were performed using disk diffusion on all the isolates. All tests were performed following the United States Clinical and Laboratory Standards Institute (CLSI) recommendations, and CLSI 2013 version of the antibiotic susceptibility breakpoints for S. pneumoniae was adopted as criteria for determining drug resistance.Results:Using the sequence-based method selectively supplemented with sequential mPCR and serotypes 6A-6D specific PCRs strategy, sixteen serotypes were identified from 193 S. pneumoniae isolates. They included 19F (34.7%),23F (17.1%), 19A (11.9%),14 (7.3%),15B/15C (6.7%),6B (6.7%),6A (6.2%),9V/9A (1.6%); serotypes 6C,3,15F/15A,23A and 20 (each,1.1%); serotypes 10B,28F/28A and 34 (each,0.5%). PCV-7 and PCV-10 vaccines cover 67.4% of the serotypes identified, whilst PCV-13 covers 86.5%. Among patients less than 2 years of age, PCV-7 and PCV-10 covers 70.6% of these strains whilst PCV-13 covers 92%. Using cpsB sequetyping as first screening of the 193 isolates,34.2% were sequetyped to the serotype level and 55.4% to the serogroup level.59.5%of sequetyping results needed confirmation by modified mPCR to give a definite serotype, with most of them identified in the first three mPCR reactions. Only 3.6% isolates required further testing of up to eight sequential mPCR reactions. The percentage of MDR isolates was 86.8%, and the most common pattern was resistance to erythromycin+ clindamycin+sulfamethoxazole-trimethoprim. According to the revised CLSI breakpoints, there was no penicillin resistant S.pneumoniae found among non-meningitis isolates. Based on standard for parenteral penicillin, the prevalence rates for penicillin resistance were 1.0% and 88.6% in the non-meningitis and meningitis isolates, respectively.Conclusion:(1) This study provides a cost-effective alternative S. pneumoniae serotype prediction strategy to conventional serotyping. We showcased the utility of this new serotyping strategy by identifying serotypes of 193 S. pneumoniae isolates from children. This strategy enables most routine laboratories equipped with PCR to predict the majority of pneumococcal serotypes without the need for an expensive set of serological reagents in China. (2) This study confirms that serotype 19A is common in south China, and that PCV-13 vaccine would be important for future vaccination in areas such as Shenzhen. Proportion of serogroup 15 (especially serotype 15B/15C) isolates over than the previous report from other regions in China. (3) Considering the low resistance rate in non-meningitis isolates to parenteral penicillin, clinicians should be encouraged to increase the use of penicillin to treat penicillin-susceptible non-meningitis pneumococcal infections, instead of using broader-spectrum antimicrobials. Being high rate of MDR among children<5years, it should be avoid to choose erythromycin, clindamycin and sulfamethoxazole-trimethoprim to treat PDs. Continued surveillance of the serotype distribution and antimicrobial susceptibility of S. pneumoniae isolates in China is warranted. |
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