Sequential Multiplex PCR Research Articles

Development of a PCR algorithm to detect and characterize Neisseria meningitidis carriage isolates in the African meningitis belt.

Improved methods for the detection and characterization of carried Neisseria meningitidis isolates are needed. We evaluated a multiplex PCR algorithm for the detection of a variety of carriage strains in the meningitis belt. To further improve the sensitivity and specificity of the existing PCR assays, primers for gel-based PCR assays (sodC, H, Z) and primers/probe for real-time quantitative PCR (qPCR) assays (porA, cnl, sodC, H, E, Z) were modified or created using Primer Express software. Optimized multiplex PCR assays were tested on 247 well-characterised carriage isolates from six countries of the African meningitis belt. The PCR algorithm developed enabled the detection of N. meningitidis species using gel-based and real-time multiplex PCR targeting porA, sodC, cnl and characterization of capsule genes through sequential multiplex PCR assays for genogroups (A, W, X, then B, C, Y and finally H, E and Z). Targeting both porA and sodC genes together allowed the detection of meningococci with a sensitivity of 96% and 89% and a specificity of 78% and 67%, for qPCR and gel-based PCR respectively. The sensitivity and specificity ranges for capsular genogrouping of N. meningitidis are 67% - 100% and 98%-100% respectively for gel-based PCR and 90%-100% and 99%-100% for qPCR. We developed a PCR algorithm that allows simple, rapid and systematic detection and characterisation of most major and minor N. meningitidis capsular groups, including uncommon capsular groups (H, E, Z).

Nasopharyngeal carriage of Streptococcus pneumoniae in children under 5 years of age before introduction of pneumococcal vaccine (PCV10) in urban and rural districts in Pakistan

BackgroundBenefits of pneumococcal conjugate vaccine programs have been linked to the vaccine’s ability to disrupt nasopharyngeal carriage and transmission. The 10-valent pneumococcal vaccine (PCV10) was included in the Expanded Program on Immunization (EPI) in Sindh, Pakistan in February 2013. This study was carried out immediately before PCV10 introduction to establish baseline pneumococcal carriage and prevalent serotypes in young children and to determine if carriage differed in urban and rural communities.MethodsNasopharyngeal specimens were collected from a random sample of children 3-11 and 12-59 months of age in an urban community (Karachi) and children 3-11 months of age in a rural community (Matiari). Samples were processed in a research laboratory in Karachi. Samples were transported in STGG media, enriched in Todd Hewitt broth, rabbit serum and yeast extract, cultured on 5% sheep blood agar, and serotyped using the CDC standardized sequential multiplex PCR assay. Serotypes were categorized into PCV10-type and non-vaccine types.ResultsWe enrolled 670 children. Pneumococci were detected in 73.6% and 79.5 % of children in the infant group in Karachi and Matiari, respectively, and 78.2% of children 12 to 59 months of age in Karachi. In infants, 38.9% and 33.5% of those carrying pneumococci in Karachi and Matiari, respectively, had PCV10 types. In the older age group in Karachi, the proportion was 30.7%, not significantly different from infants. The most common serotypes were 6A, 23F, 19A, 6B and 19F.ConclusionWe found that about 3 of 4 children carried pneumococci, and this figure did not vary with age group or urban or rural residence. Planned annual surveys in the same communities will inform change in carriage of PCV10 serotype pneumococci after the introduction and uptake of PCV10 in these communities

Streptococcus pneumoniae carriage among healthy and sick paediatric patients before the generalized implementation of the 13-valent pneumococcal vaccine in Morocco, 2010–2011

Introduction Nasopharyngeal carriage studies provide insights into the local prevalence of circulating pneumococcal serotypes. These data are critical to vaccination monitoring, as they allow for the prediction and assessment of impact. Very little data are available on the carriage of pneumococcal serotypes in Morocco. The aim of the study was to describe the prevalence of Streptococcus pneumoniae carriage and serotype distribution among healthy and sick pediatric patients with ages ranging from 2 to 59 months. Methods Were included, patients admitted for severe clinical pneumonia according to WHO definition during a one-year survey from November 2010 to December 2011 and healthy children visiting primary health care centers for routine vaccination in the province of Rabat. These sites were selected randomly and the sample size for the community study (n = 200) was calculated based on the preliminary estimation of carriage rates. Nasopharyngeal samples were obtained from consent children using a mini culturette extra-thin flexible wire swab. Nasal samples were cultured using conventional methods Molecular. Capsular typing of pneumococcal strains was performed using sequential multiplex real-time PCR targeting 24 common serotypes (1, 3, 4, 5, 6A, 6B, 7F/A, 8, 9 V/A/N/l, 14, 15B/C, 18 C/B, 19A, 19F/B/C, 23A and 23F) [8]. Data analysis Study variables were analyzed and summarized in frequency tables. Quantitative variables are expressed as the means with their corresponding standard deviations. Qualitative variables are presented as percentages with 95% confidence intervals. Results The proportion of infants less than one year of age was 30.6% (214/700) for the admitted patients and 70.7% (138/195) for the children from the community. Carriage rates were 40.5% (79/195) for healthy children and 22.8% (159/697) for sick children. Vaccine coverage for the circulating serotypes was 55.9% (66/118) among hospitalized children and 52.1% (24/46) for healthy children. Among admitted patients, the monthly distribution of carriage was significantly different (P Conclusion Monitoring of circulating serotypes remains necessary after vaccine introduction to assess whether serotype replacement is occurring.

Clinical usefulness and accuracy of polymerase chain reaction in the detection of bacterial meningitis agents in pediatric cerebrospinal fluid

Bacterial meningitis poses enormous healthcare challenges due to a high mortality, morbidity and sequelae. Neisseria (N.) meningitidis, Haemophilus (H.) influenzae, Streptococcus (S.) pneumoniae and S. agalactiae remain among the most prevalent infectious agents that cause bacterial meningitis in children. The objective of this study was the simultaneous detection of these pathogens in suspected cerebrospinal fluid (CSF) by using multiplex polymerase chain reaction (mPCR) and compare PCR results with standard diagnostics currently used in clinical practice. CSF specimens were obtained from 515 children (<5 years) clinically suspected of having acute bacterial meningitis. Based on bacterial culture, four isolates of salmonella sp and one Citrobacter freundii isolate were identified. The remaining 510 CSF specimens, having negative culture, were subjected to mPCR. Twenty-three (4.51%) CSF samples yielded a PCR positive signal. The pathogens identified were: S. pneumoniae (n=13), H. influenzae (n=7) and N. meningitidis (n=3). S. agalactiae was not detected. Using sequential multiplex PCR, serogrouping of S. pneumoniae revealed 3 different serotypes: serotype 19A (n=6), 19F (n=4) and serotype 23F (n=3). Only the serotype A was identified for the 3N. meningitidis isolates. Despite vaccination, S. pneumoniae remains a leading cause of pediatric invasive disease. Detecting causative organism remains the most critical aspect for management of children with suspected meningitis. PCR method is more sensitive and rapid than culture for detecting the infectious agents. Institution of PCR diagnostics is recommended for early and appropriate therapy.

Burden of invasive pneumococcal disease among children in rural Mozambique: 2001-2012.

BackgroundInvasive pneumococcal disease (IPD) is a major cause of illness and death among children worldwide. 10-valent pneumococcal conjugate vaccine (PCV10) was introduced as part of the Mozambican routine immunization program in April 2013. We characterized the IPD burden in a rural area of Mozambique before PCV introduction and estimated the potential impact of this intervention.MethodsWe conducted population-based surveillance for IPD, defined as S. pneumoniae isolated from blood or cerebrospinal fluid, among children <5 years old admitted to Manhiça District Hospital, a referral hospital in a rural area with high prevalence of human immunodiciency virus infection. S. pneumoniae was identified using standard microbiologic methods and serotyped using sequential multiplex PCR or Quellung. IPD incidence was calculated among cases from a defined catchment area.ResultsFrom January 2001 through December 2012, we isolated 768 cases of IPD, 498 (65%) of which were bacteraemic pneumonia episodes. A total of 391 (51%) were from the catchment area, yielding IPD incidence rates of 479, 390 and 107 episodes per 100,000 children-years at risk among children <12, 12–23 and 24-<60 months old, respectively. The overall IPD incidence fluctuated and showed a downward trend over time. In these same age groups, in-hospital death occurred in 48 (17%), 26 (12%), and 21 (13%) of all IPD cases, respectively. Overall 90% (543/603) of IPD isolates were available for serotyping; of those, 65% were covered by PCV10 and 83% by PCV13. Among 77 hospital deaths associated with serotyped IPD, 49% and 69% were caused by isolates included in the PCV10 and PCV13, respectively.ConclusionsWe describe very high rates of IPD among children in rural Mozambique that were declining before PCV introduction. Children <1 year old have the greatest incidence and case fatality; although the rates remain high among older groups as well. Most IPD episodes and many deaths among children <5 years old will likely be prevented through PCV10 introduction in Mozambique.

Surveillance of impact of PCV-10 vaccine on pneumococcal meningitis in Mozambique, 2013 - 2015.

BackgroundVaccination using the 10-valent conjugate vaccine (PCV-10) was introduced into the Extended Program on Immunization in Mozambique in March 2013, however its impact on pediatric pneumococcal meningitis is unknown. In this study, we assessed for the first time the impact of PCV10 on the burden of pneumococcal meningitis in children less than 5 years of age at the three largest hospitals in Mozambique.MethodBetween March 2013 and December 2015, a total of 744 cerebrospinal fluid (CSF) samples were collected from eligible children, of which 160 (21.5%) were positive for S. pneumoniae. Of these, only 86 samples met the criteria for serotyping and were subsequently serotyped using sequential multiplex PCR (SM-PCR), but 17 samples were non-typable.ResultsThe proportion of cases of pneumococcal meningitis decreased from 33.6% (124 of 369) in 2013 to 1.9% (3 of 160) in 2015 (p < 0.001). The relative frequency of PCV10 serotype cases also decreased from 84.2% (48 of 57) in 2013 to 0% (0 of 3) in 2015 (p = 0.006). Between 2013 and 2015, serotype coverage of PCV-10 and PCV13 vaccine formulations was 66.7% and 81.2%, respectively.ConclusionAltogether, our findings shows that introduction of PCV-10 immunization resulted in rapid decline of pneumococcal meningitis children less than 5 years old in Mozambique. This decline was accompanied by substantial changes in the pattern of circulating pneumococcal serotypes.

Distribution of capsular types and drug resistance patterns of invasive pediatric Streptococcus pneumoniae isolates in Teheran, Iran

To explore the serotype distribution and drug resistance patterns of invasive pneumococcal isolates from children under 5 years of age. During a 32-month period, 585 clinical samples (including blood, cerebrospinal fluid (CSF), and synovial fluid) from children suspected of having meningitis, sepsis, pneumonia, or septic arthritis were analyzed using the BACTEC culture system. Positive cultures were examined using biochemical tests and lytA amplification for the identification of pneumococcal strains. The confirmed pneumococcal isolates were examined to determine capsular types using a modified sequential multiplex PCR and susceptibility to antimicrobial agents. Fifty-three pneumococcal isolates were detected in the 585 clinical samples: 21 (39.6%) blood samples and 32 (60.4%) CSF samples. The most frequent serotype was 23F (24.5%), followed by serotypes 19F (18.9%), 19A (7.5%), and 9V (7.5%). Twenty-one percent of pneumococcal isolates were penicillin-non-susceptible and serotype 19A was significantly associated with resistance to penicillin. This study indicated that the 13-valent pneumococcal conjugate vaccine (PCV13) could cover the majority of the invasive pneumococcal isolates. Drug-resistant and multidrug-resistant Streptococcus pneumoniae strains are circulating in Iran. Therefore, public immunization of infants using PCV13 is recommended to reduce the incidence of pneumococcal disease and pneumococcal-resistant strains in Teheran.

Customized sequential multiplex PCR for accurate and early determination of invasive pneumococcal serotypes found in India

For accurate and earlier detection of invasive pneumococcal serogroup/serotypes from India, we have rearranged the African sequence of multiplex PCR provided by the Centers for Disease Control and Prevention, USA. This modified approach can successfully be adapted for earlier serotype detection of 95% of the pneumococcal strains prevalent in India.

Serotype distribution and antimicrobial resistance of Streptococcus pneumoniae in children with acute bacterial meningitis in Mozambique: implications for a national immunization strategy.

BackgroundS. pneumoniae is the leading cause of acute bacterial meningitis (ABM) in children. Vaccination using the 10-valent conjugate vaccine (PCV-10) was recently introduced into the National Immunization Program in Mozambique, but data on serotype coverage of this vaccine formulation are scarce. In this study, we investigated the serotype distribution and antimicrobial resistance of isolates of S. pneumoniae causing ABM in children < 5 years at the two largest hospitals in Mozambique.MethodsBetween March 2013 and March 2014, a total of 352 cerebrospinal fluid (CSF) samples were collected from eligible children, of which 119 (33.8 %) were positive for S. pneumoniae. Of these, only 50 samples met the criteria for serotyping and were subsequently serotyped using sequential multiplex PCR (SM-PCR), but 15 samples were non-typable.ResultsThe most common serotypes of S. pneumoniae were 1 (18.2 %), 5 (15.2 %), 14 (12.1 %), 9 V (12.1 %), 23 F (9.1 %), 6A (9.1 %), 4 (9.1 %) and 6B (6.1 %). Serotypes 1, 5, 9 V, 6A and 12 were mostly prevalent in Northern Mozambique, while serotypes 23 F, 4, 6B, 3 and 15B were predominant in Southern. Serotype coverage of PCV-10 and PCV-13 vaccine formulations were 81.8 % and 93.9 %, respectively. Serotypes 1, 3, 4, 6B, 14, 23 F were resistant to penicillin and sensitive to ceftriaxone.ConclusionsOur findings shows that changing the current in use PCV-10 vaccine formulation to PCV-13 formulation might increase substantially the protection against invasive strains of S. pneumoniae as the PCV-10 vaccine formulation does not cover the serotypes 3 and 6A, which are prevalent in Mozambique.

Serotypes and Antibiotic Susceptibility of Streptococcus pneumoniae Isolates from Invasive Pneumococcal Disease and Asymptomatic Carriage in a Pre-vaccination Period, in Algeria.

In Algeria, few data is available concerning the distribution of pneumococcal serotypes and respective antibiotic resistance for the current pre-vaccination period, which is a public health concern. We identified the most frequent Streptococcus pneumoniae serogroup/types implicated in invasive pneumococcal disease (IPD; n = 80) and carriage (n = 138) in Algerian children younger than 5 years old. Serogroup/types of 78 IPD isolates were identified by capsular typing using a sequential multiplex PCR. Overall, serotypes 14, 19F, 6B, 23F, 18C, 1, 5, 7F, 19A, and 3 (55% of PCV7 serotypes, 71.3% of PCV10, and 90% of PCV13) were identified. Additionally, 7.5% of the non-vaccine serotypes 6C, 9N/L, 20, 24F, 35B, and 35F, were observed. In the case of S. pneumoniae asymptomatic children carriers, the most common serogroup/types were 6B, 14, 19F, 23F, 4, 9V/A, 1, 19A, 6A, and 3 (42.7% of PCV7 serotypes, 44.2% of PCV10, and 58% of PCV13). For 6.1% of the cases co-colonization was detected. Serotypes 14, 1, 5, and 19A were more implicated in IPD (p < 0.01), whereas serotype 6A was exclusively isolated from carriers (p < 0.01). Deaths associated with IPD were related to serotypes 19A, 14, 18C, and one non-typeable isolate. Among IPD related to vaccine serotypes, the rates of penicillin non-susceptible isolates were higher in no meningitis cases (80%) than in meningitis (66.7%), with serotypes 14, 19A, 19F, and 23F presenting the highest MIC levels (>2μg/ml). Resistance to cefotaxime was higher in isolates from meningitis (40.5%); however, resistance to erythromycin and co-trimoxazole (>40%) was more pronounced in no-meningeal forms. Overall, our results showed that PCV13 conjugate vaccine would cover up to 90% of the circulating isolates associated with IPD in Algeria, highlighting the importance of monitoring the frequency of S. pneumoniae serogroups/types during pre- and post-vaccination periods.

Using a practical molecular capsular serotype prediction strategy to investigate Streptococcus pneumoniae serotype distribution and antimicrobial resistance in Chinese local hospitalized children.

BackgroundChina is one of ten countries with the highest prevalence rate of pneumococcal infections. However, there is limited serotype surveillance data for Streptococcus pneumoniae, 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 to develop a simple, practical and economic pneumococcal serotype prediction strategy suitable for future serotype surveillance in China.MethodsIn this study, 193 S. pneumoniae isolates were collected from hospitalized children, 96.9 % of whom were < 5 years old. The cpsB sequetyping, complemented by selective and modified USA CDC sequential multiplex-PCR, was performed on all the isolates, and serotypes 6A-6D specific PCRs were done on all serogroup 6 isolates. Based on systematic analysis of available GenBank cpsB sequences, we established a more comprehensive cpsB sequence database than originally published for cpsB sequetyping. Antibiotic susceptibility of all isolates was determined using the disk diffusion or E-test assays.ResultsWe built up a comprehensive S. pneumoniae serotype cpsB sequetyping database for all the 95 described serotypes first, and then developed a simple strategy for serotype prediction based on the improved cpsB sequetyping and selective multiplex-PCR. Using the developed serotype prediction strategy, 191 of 193 isolates were successfully “serotyped”, and only two isolates were “non-serotypeable”. Sixteen serotypes were identified among the 191 “serotypeable” isolates. The serotype distribution of the isolates from high to low was: 19 F (34.7 %), 23 F (17.1 %), 19A (11.9 %), 14 (7.3 %), 15B/15C (6.7 %), 6B (6.7 %), 6A (6.2 %), 9 V/9A (1.6 %); serotypes 6C, 3, 15 F/15A, 23A and 20 (each 1.1 %); serotypes 10B, 28 F/28A and 34 (each 0.5 %). The prevalence of parenteral penicillin resistance was 1.0 % in the non-meningitis isolates and 88.6 % in meningitis isolates. The total rate of multidrug resistance was 86.8 %.ConclusionsThe integrated cpsB sequetyping supplemented with selective mPCR and serotypes 6A-6D specific PCRs “cocktail” strategy is practical, simple and cost-effective for use in pneumococcal infection serotype surveillance in China. For hospitalized children with non-meningitis penicillin-susceptible pneumococcal infections, clinicians still can use narrow-spectrum and cheaper penicillin, using the parenteral route, rather than using broader-spectrum and more expensive antimicrobials.Electronic supplementary materialThe online version of this article (doi:10.1186/s12887-016-0589-7) contains supplementary material, which is available to authorized users.

Nasopharyngeal carriage of Streptococcus pneumoniae in adults infected with human immunodeficiency virus in Jakarta, Indonesia.

This study investigated the distribution of serotype and antimicrobial susceptibility of Streptococcus pneumoniae carried by adults infected with human immunodeficiency virus (HIV) in Jakarta, Indonesia. Specimens of nasopharyngeal swab were collected from 200 HIV infected adults aged 21 to 63 years. Identification of S. pneumoniae was done by optochin susceptibility test and PCR for the presence of psaA and lytA genes. Serotyping was performed with sequential multiplex PCR and antibiotic susceptibility with the disk diffusion method. S. pneumoniae strains were carried by 10% adults with serotype 6A/B 20% was common serotype among cultured strains in 20 adults. Most of isolates were susceptible to chloramphenicol (80%) followed by clindamycin (75%), erythromycin (75%), penicillin (55%), and tetracycline (50%). This study found resistance to sulphamethoxazole/trimethoprim was most common with only 15% of strains being susceptible. High non-susceptibility to sulphamethoxazole/trimethoprim was observed in S. pneumoniae strains carried by HIV infected adults in Jakarta, Indonesia.

Emerging non-PCV13 serotypes of noninvasive Streptococcus pneumoniae with macrolide resistance genes in northern Japan.

In Japan, the 7-valent pneumococcal conjugate vaccine (PCV7) was introduced to the nation's routine immunization program in April 2013 and was replaced by the 13-valent pneumococcal conjugate vaccine (PCV13) in November 2013. Distribution of serotypes and macrolide resistance genotypes was investigated for a total of 1097 (975 children, 122 adults) and 960 (873 children, 87 adults) clinical isolates of Streptococcus pneumoniae from noninvasive infections in Hokkaido (northern main island of Japan) in the routine immunization periods for PCV7 and PCV13 (April–October 2013 and November 2013–November 2014, respectively). Serotype was determined by sequential multiplex PCR and additional genetic analyses. Macrolide resistance genes erm(B) and mef(A/E) were detected by multiplex PCR. Although the most prevalent serotypes in children were 23A and 6C in the PCV7 period, after replacement with PCV13, 19A became the most common, followed by 6C, 15A and 23A. Among adults, serotype 3 was consistently the most frequent throughout the study periods. Compared with values from the pre-PCV7 routine immunization period, PCV7 serotypes decreased from 48.3 to 3.3% in the PCV13 period among children, while the rates of non-PCV13 serotypes (particularly 15A, 23A, 11A, 10A and 35B) increased from 39.7 to 75.1% (p < 0.001). In the PCV13 period, erm(B), mef(A/E) and both of these genes were detected in 75.8, 31.6 and 11.3% of all isolates, respectively. Serotype 19A accounted for 76.9% of the isolates with both the macrolide resistance genes, and emerging non-PCV13 serotypes 15A, 15C and 23A mostly harboured erm(B).

Serotype distribution and antibiotic susceptibility of Streptococcus pneumoniae strains carried by children infected with human immunodeficiency virus.

BackgroundWe studied the serotype distribution and antibiotic susceptibility of Streptococcus pneumoniae isolates carried by children infected with HIV in Jakarta, Indonesia.MethodsNasopharyngeal swabs were collected from 90 HIV infected children aged 4 to 144 months. S. pneumoniae was identified by conventional and molecular methods. Serotyping was performed with sequential multiplex PCR and antibiotic susceptibility with the disk diffusion method.ResultsWe identified S. pneumoniae carriage in 41 children (46%). Serotype 19F was most common among 42 cultured strains (19%) followed by 19A and 6A/B (10% each), and 23F (7%). Most isolates were susceptible to chloramphenicol (86%), followed by clindamycin (79%), erythromycin (76%), tetracycline (43%), and sulphamethoxazole/trimethoprim (41%). Resistance to penicillin was most common with only 33% of strains being susceptible. Strains of serotypes targeted by the 13-valent pneumococcal conjugate polysaccharide vaccine (PCV13) were more likely to be multidrug resistant (13 of 25 or 52%) compared to non-PCV13 serotype isolates (3 of 17 or 18%; Fisher exact test p = 0.05).ConclusionOur study provides insight into the epidemiology of pneumococcal carriage in young HIV patients in Indonesia. These findings may facilitate potential preventive strategies that target invasive pneumococcal disease in Indonesia.

Distribution of serotypes and antibiotic susceptibility patterns among invasive pneumococcal diseases in Saudi Arabia.

BackgroundStreptococcus pneumoniae causes life-threatening infections such as meningitis, pneumonia, and febrile bacteremia, particularly in young children. The increasing number of drug-resistant isolates has highlighted the necessity for intervening and controlling disease. To achieve this, information is needed on serotype distribution and patterns of antibiotic resistance in children.MethodsAll cases of invasive pneumococcal disease (IPD) in children aged less than 15 yr recorded at King Khalid University Hospital, King Saud University, Riyadh, Saudi Arabia, were reviewed for serotyping and antibiotic susceptibility. Isolates were collected from 78 consecutive patients with IPD between 2009 and 2012. All collected isolates were subjected to serotyping by co-agglutination, sequential multiplex PCR, and single PCR sequetyping as previously described.ResultsThe most frequently isolated IPD serotypes were 23F, 6B, 19F, 18C, 4, 14, and 19A, which are listed in decreasing order and cover 77% of total isolates. The serotype coverage for the pneumococcal conjugate vaccine (PCV)7, PCV10, and PCV13 was 77%, 81%, and 90%, respectively. Results from sequential multiplex PCR agreed with co-agglutination results. All serotypes could not be correctly identified using single PCR sequetyping. Minimum inhibitory concentration showed that 50 (64%) isolates were susceptible to penicillin, whereas 70 (90%) isolates were susceptible to cefotaxime.ConclusionsThe most common pneumococcal serotypes occur with frequencies similar to those found in countries where the PCV has been introduced. The most common serotypes in this study are included in the PCVs. Addition of 23A and 15 to the vaccine would improve the PCV performance in IPD prevention.

Historical development of typing methods for Streptococcus pneumoniae

Streptococcus pneumoniae is a major cause of morbidity and mortality in developing countries, especially in children, even though this is a vaccine-preventable disease. Several vaccines are available for which programmes are in regular immunization, but the success of this depends upon their efficacy and vaccine coverage of the serotypes prevalent in the region. A so-called replacement phenomenon has made it mandatory that there should be a regular and continuous survey of serotype prevalence whenever the vaccine is used. Many typing methods have been described including serology as well as molecular-based typing. We have analysed most of the described typing methods for pneumococcal isolates and discussed their prospects for future surveillance programmes. In brief, molecular typing such as sequential multiplex PCR is more reliable for typing, but it has limitations as it types the isolates only up to serogroup level in few cases, and therefore is still dependent on conventional serotyping methods like Quellung and co-agglutination. However, conventional typing methods are limited because of cross-reactivity with other serotypes within serogroups. Conventional serological methods are costly, labour-intensive, and prone to misidentification, whereas current DNA-based methods have limited serotype coverage requiring multiple PCR primers. Implementation of pneumococcal conjugate vaccines necessitates continued monitoring of circulating strains to consider vaccine effectiveness and subsequent substitution of serotypes.

Susceptibility profiles and correlation with pneumococcal serotypes soon after implementation of the 10-valent pneumococcal conjugate vaccine in Brazil

To evaluate the susceptibility patterns among Streptococcus pneumoniae recovered during the years 2010-2012 and to correlate these with serotypes. Pneumococci from invasive sites were serotyped by sequential multiplex PCR and/or Quellung reaction. Etest strips were used to determine the minimal inhibitory concentrations, and the Clinical and Laboratory Standards Institute (CLSI) guidelines were used for interpretation. Genetic determinants of macrolide resistance were assessed by PCR, and the occurrence of the D phenotype was analyzed following the recommendations of the CLSI. One hundred fifty-nine S. pneumoniae were studied; most were recovered from blood and were associated with serotypes 14, 3, 4, 23F, 20, 7F, 12F, 19A, and 19F. Pneumococcal conjugate vaccine PCV7, PCV10, and PCV13 and 23-valent polysaccharide vaccine serotypes represented 38.2%, 48.7%, 64.5%, and 85.5%, respectively. β-Lactam non-susceptibility (non-meningitis) was basically related to serotype 19A. For meningitis, it was observed in 21.4% (serotypes 14, 3, 9V, 23F, and 24F). Resistance to erythromycin occurred in 8.2% and mefA was the most common macrolide genetic determinant. One isolate was resistant to levofloxacin. Non-susceptibility to trimethoprim-sulfamethoxazole was 37.7% and to tetracycline was 22.0%. Our population of pneumococci represents a transition era, soon after the introduction of PCV10. Non-susceptible patterns were found to be associated with classical PCV serotypes (especially serotype 14), which is still highly prevalent, and non-PCV10 ones (19A), which may disseminate, occupying the biological niche left by the vaccine serotypes.

Nasopharyngeal carriage, serotype distribution and antimicrobial resistance of Streptococcus pneumoniae among children from Brazil before the introduction of the 10-valent conjugate vaccine

BackgroundStreptococcus pneumoniae remains a major cause of childhood morbidity and mortality worldwide. Nasopharyngeal colonization plays an important role in the development and transmission of pneumococcal diseases, and infants and young children are considered to be the main reservoir of this pathogen. The aim of this study was to evaluate the rates and characteristics associated with nasopharyngeal carriage, the distribution of serotypes and the antimicrobial resistance profiles of Streptococcus pneumoniae among children in a large metropolitan area in Brazil before the introduction of the 10-valent pneumococcal conjugate vaccine.MethodsBetween March and June 2010, nasopharyngeal swabs were collected from 242 children aged <6 years attending one day care center and the emergency room of a pediatric hospital. Pneumococcal isolates were identified by conventional methods and serotypes were determined by a sequential multiplex PCR assay and/or the Quellung reaction. The antimicrobial susceptibilities of the pneumococci were assessed by the disk diffusion method. MICs for erythromycin and penicillin were also performed. Erythromycin resistance genes were investigated by PCR.ResultsThe overall colonization rate was 49.2% and it was considerably higher among children in the day care center. Pneumococcal carriage was more common among day care attenders and cohabitants with young siblings. The most prevalent serotypes were 6B, 19F, 6A, 14, 15C and 23F, which accounted for 61.2% of the isolates. All isolates were susceptible to clindamycin, levofloxacin, rifampicin and vancomycin. The highest rate of non-susceptibility was observed for sulphamethoxazole-trimethoprim (51.2%). Penicillin non-susceptible pneumococci (PNSP) accounted for 27.3% of the isolates (MICs of 0.12-4 μg/ml). Penicillin non-susceptibility was strongly associated with serotypes 14 and 23F. Hospital attendance and the presence of respiratory or general symptoms were frequently associated with PNSP carriage. The two erythromycin-resistant isolates (MICs of 2 and 4 μg/ml) belonged to serotype 6A, presented the M phenotype and harbored the mef(A/E) gene.ConclusionsCorrelations between serotypes, settings and penicillin non-susceptibility were observed. Serotypes coverage projected for the 10-valent pneumococcal conjugate vaccine was low (45.5%), but pointed out the potential reduction of PNSP nasopharyngeal colonization by nearly 20%.

P189 Extrapulmonary tuberculosis in Pereira, Colombia, 2011

Introduction: Streptococcus pneumoniae is an important human pathogen in community-acquired respiratory infections. S. pneumoniae with resistance to penicillin and other antibiotics is becoming prevalent. In particular, macrolide resistance among S. pneumoniae isolates has risen in recent years worldwide. The increasing incidence of macrolideand penicillin-resistant S. pneumoniae has been a serious problem in Japan. Objectives: The objective of this study was to investigate the prevalence of serotypes, and macrolideand penicillin-resistance among pneumococcal isolates from outpatients with non-invasive infections. Methods: A total of 1,061 non-invasive S. pneumoniae strains isolated from outpatients who visited various hospitals and clinics in Hokkaido, northern main island of Japan in 2011 were analyzed. The presence of macrolide-resistance genes erm (B ) and mef (A/E ), and 3 unaltered pbp genes (pbp1a, pbp2x, and pbp2b) were detected by multiplex PCR. MICs of all isolates against erythromycin and penicillin were measured by E-test. Serotyping of these isolates was performed genetically by sequential multiplex PCR and direct sequencing. Additionally, we developed a novel mutagenic PCR-RFLP assay for discrimination of serotypes 6A, 6B, 6C, and 6D. Results: Among the 1,061 strains, 95.8% and 7.7% isolates were resistant to erythromycin and penicillin, respectively. The erm (B ) and mef (A/E ) genes were detected in 66.4% and 40%, respectively (including 10.7% of the strains carrying both genes). The rate of strains with a single altered pbp gene was 38.8%, with two and three altered pbp genes being 18.4% and 37.9%, respectively. The most frequent serotypes were 6B (17.0%), 19F (15.6%), 23F (12.1%). While most of strainswith these three serotypes harbored erm (B ) and/ormef (A/E ) (>98%), rate of PRSP with three altered pbp genes was highest in 23F (83.6%), followed by 6B (70.3%) and 19F (62.4%). All the serotype15B/C isolates were resistant to erythromycin (MIC: >256 mg/ml). Serotypes included in the 7and 13-valent conjugate vaccines covered 47.7% and 69.3% of isolates, respectively. Conclusion:The present study indicated the spreadofmacrolideand penicillin-resistant strains among non-invasive S. pneumoniae isolates in Hokkaido.

P187 Withdrawn P188 Serotype distribution and analysis of macrolide- and penicillin-resistance among non-invasive S. pneumoniae isolates in northern Japan

Introduction: Streptococcus pneumoniae is an important human pathogen in community-acquired respiratory infections. S. pneumoniae with resistance to penicillin and other antibiotics is becoming prevalent. In particular, macrolide resistance among S. pneumoniae isolates has risen in recent years worldwide. The increasing incidence of macrolideand penicillin-resistant S. pneumoniae has been a serious problem in Japan. Objectives: The objective of this study was to investigate the prevalence of serotypes, and macrolideand penicillin-resistance among pneumococcal isolates from outpatients with non-invasive infections. Methods: A total of 1,061 non-invasive S. pneumoniae strains isolated from outpatients who visited various hospitals and clinics in Hokkaido, northern main island of Japan in 2011 were analyzed. The presence of macrolide-resistance genes erm (B ) and mef (A/E ), and 3 unaltered pbp genes (pbp1a, pbp2x, and pbp2b) were detected by multiplex PCR. MICs of all isolates against erythromycin and penicillin were measured by E-test. Serotyping of these isolates was performed genetically by sequential multiplex PCR and direct sequencing. Additionally, we developed a novel mutagenic PCR-RFLP assay for discrimination of serotypes 6A, 6B, 6C, and 6D. Results: Among the 1,061 strains, 95.8% and 7.7% isolates were resistant to erythromycin and penicillin, respectively. The erm (B ) and mef (A/E ) genes were detected in 66.4% and 40%, respectively (including 10.7% of the strains carrying both genes). The rate of strains with a single altered pbp gene was 38.8%, with two and three altered pbp genes being 18.4% and 37.9%, respectively. The most frequent serotypes were 6B (17.0%), 19F (15.6%), 23F (12.1%). While most of strainswith these three serotypes harbored erm (B ) and/ormef (A/E ) (>98%), rate of PRSP with three altered pbp genes was highest in 23F (83.6%), followed by 6B (70.3%) and 19F (62.4%). All the serotype15B/C isolates were resistant to erythromycin (MIC: >256 mg/ml). Serotypes included in the 7and 13-valent conjugate vaccines covered 47.7% and 69.3% of isolates, respectively. Conclusion:The present study indicated the spreadofmacrolideand penicillin-resistant strains among non-invasive S. pneumoniae isolates in Hokkaido.