Molecular detection and genotypic characterisation of Streptococcus pneumoniae isolated from children in Malaysia.
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ABSTRACT:
SUBMITTER: Goh SL
PROVIDER: S-EPMC7144269 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundThe 13-valent pneumococcal vaccine (PCV13) was introduced in Cambodia in January 2015. There are limited data concerning the common serotypes causing invasive pneumococcal disease (IPD). Knowledge of the circulating pneumococcal serotypes is important to monitor epidemiological changes before and after vaccine implementation.MethodsAll episodes of IPD defined by the isolation of Streptococcus pneumoniae from blood, cerebrospinal fluid or other sterile site in Cambodian children admitted to the Angkor Hospital for Children in Siem Reap, Northwestern Cambodia, between 1st January 2007 and 1st July 2012 were retrospectively studied. Streptococcus pneumoniae isolates that could be retrieved underwent phenotypic typing and whole genome sequencing.ResultsThere were 90 Cambodian children hospitalized with IPD with a median (IQR) age of 2.3 years (0.9-6.2). The case fatality was 15.6% (95% CI 8-23). Of 50 Streptococcus pneumoniae isolates available for further testing, 46% were penicillin non-susceptible and 8% were ceftriaxone non-susceptible, 78% were cotrimoxazole resistant, 30% were erythromycin resistant and 30% chloramphenicol resistant. There were no significant changes in resistance levels over the five-year period. The most common serotypes were 1 (11/50; 22%), 23F (8/50; 16%), 14 (6/50; 12%), 5 (5/50; 10%) and 19A (3/50; 6%). Coverage by PCV7, PCV10 and PCV13 was 44%, 76% and 92% respectively. We identified novel multilocus sequence types and resistotypes using whole genome sequencing.ConclusionsThis study suggests IPD is an important disease in Cambodian children and can have a significant mortality. PCV13 coverage of the serotypes determined in studied strains was high and consistent with another recent study. The phenotypic resistance patterns observed were similar to other regional studies. The use of whole genome sequencing in the present study provides additional typing and resistance information together with the description of novel sequence types and resistotypes.
| S-EPMC4957771 | biostudies-literature
Project description:Streptococcus pneumoniae is a common pathogenic cause of pediatric infections. This study investigated the serotype distribution, antimicrobial susceptibility, and molecular epidemiology of pneumococci before the introduction of conjugate vaccines in Shanghai, China.A total of 284 clinical pneumococcal isolates (270, 5, 4,3, and 2 of which were isolated from sputum, bronchoalveolar lavage fluid, blood, cerebral spinal fluid, and ear secretions, respectively) from children less than 14 years of age who had not been vaccinated with a conjugate vaccine, were collected between January and December in 2013. All isolates were serotyped by multiplex polymerase chain reaction or quellung reactions and antimicrobial susceptibility testing was performed using the broth microdilution method. The molecular epidemiology of S.pneumoniae was analyzed by multilocus sequence typing (MLST).Among the 284 pneumococcal isolates, 19F (33.5%), 19A (14.1%), 23F (12.0%), and 6A (8.8%) were the most common serotypes and the coverage rates of the 7-, 10-, and 13-valent pneumococcal conjugate vaccines (PCV7, PCV10, and PCV13) were 58.6%, 59.4% and 85.1%, respectively. Antimicrobial susceptibility showed that the prevalence rates of S.pneumoniae resistance to penicillin were 11.3% (32/284). Approximately 88.0% (250/284) of the isolates exhibited multi-drug resistance. MLST analysis revealed a high level of diversity, with 65 sequence types (STs) among 267 isolates. Specifically, the four predominant STs were ST271 (24.3%, 65/267), ST320 (11.2%, 30/267), ST81 (9.7%, 26/267), and ST3173 (5.2%, 14/267), which were mainly associated with serotypes 19F, 19A, 23F, and 6A, respectively.The prevalent serotypes among clinical isolates from children were 19F, 19A, 23F, and 6A and these isolates showed high resistance rates to β-lactams and macrolides. The Taiwan19F-14 clone played a predominant role in the dissemination of pneumococcal isolates in Shanghai, China. Therefore, continued and regional surveillance on pneumococcal isolates may be necessary.
| S-EPMC4646667 | biostudies-other
Project description:Feline morbillivirus (FeMV), a novel virus from the family of Paramyxoviridae, was first identified in stray cat populations. The objectives of the current study were to (i) determine the molecular prevalence of FeMV in Malaysia; (ii) identify risk factors associated with FeMV infection; and (iii) characterise any FeMV isolates by phylogenetic analyses. Molecular analysis utilising nested RT-PCR assay targeting the L gene of FeMV performed on either urine, blood and/or kidney samples collected from 208 cats in this study revealed 82 (39.4%) positive cats. FeMV-positive samples were obtained from 63/124 (50.8%) urine and 20/25 (80.0%) kidneys while all blood samples were negative for FeMV. In addition, from the 35 cats that had more than one type of samples collected (blood and urine; blood and kidney; blood, urine and kidney), only one cat had FeMV RNA in the urine and kidney samples. Risk factors such as gender, presence of kidney-associated symptoms and cat source were also investigated. Male cats had a higher risk (p = 0.031) of FeMV infection than females. In addition, no significant association (p = 0.083) was observed between the presence of kidney-associated symptoms with FeMV status. From the 82 positive samples, FeMV RNA was detected from 48/82 (58.5%) pet cats and 34/126 (27.0%) shelter cats (p < 0.0001). Partial L and N gene sequencing of the RT-PCR-positive samples showed 85-99% identity to the published FeMV sequences and it was significantly different from all other morbilliviruses. A phylogenetic analysis of the identified Malaysian FeMVs was performed with isolates from Japan, Thailand and China. Molecular characterisation revealed high relatedness of the Malaysian isolates with other Asian FeMVs, indicating that the virus had been circulating only within the region. Therefore, this study confirmed the existence of FeMV among domestic cats in Malaysia. The findings suggest further characterisation of the local isolates, including the whole genome sequencing and that studies at determining the direct consequences of FeMV infection in domestic cats are needed.
| S-EPMC7117105 | biostudies-literature
Project description:BackgroundNigeria has one of the highest burdens of pneumococcal disease in the world, but accurate surveillance is lacking. Molecular detection of infectious pathogens in dried blood spots (DBS) is an ideal method for surveillance of infections in resource-limited settings because of its low cost, minimal blood volumes involved, and ease of storage at ambient temperature. Our study aim was to evaluate a Streptococcus pneumoniae real-time polymerase chain reaction (rt-PCR) assay on DBS from febrile Nigerian children on Whatman 903 and FTA filter papers, compared to the gold standard of culture.MethodsBetween September 2011 to May 2015, blood was collected from children 5 years of age or under who presented to six hospital study sites throughout northern and central Nigeria with febrile illness, and inoculated into blood culture bottles or spotted onto Whatman 903 or FTA filter paper. Culture and rt-PCR were performed on all samples.ResultsA total of 537 DBS specimens from 535 children were included in the study, of which 15 were culture-positive for S. pneumoniae. The rt-PCR assay detected S. pneumoniae in 12 DBS specimens (2.2%). One positive rt-PCR result was identified in a culture-negative specimen from a high-risk subject, and two positive rt-PCR results were negative on repeat testing. Six culture-confirmed cases of S. pneumoniae bacteremia were missed. Compared to culture, the overall sensitivities of Whatman 903 and FTA DBS for detection of S. pneumoniae were 57.1% (95% CI 18.4-90.1%) and 62.5% (95% CI 24.5-91.5%), respectively. Nonspecific amplification was noted in an additional 22 DBS (4.1%). Among these, six were positive for a non-S. pneumoniae pathogen on culture.ConclusionsRt-PCR was able to detect S. pneumoniae from clinical DBS specimens, including from a culture-negative specimen. Our findings show promise of this approach as a surveillance diagnostic, but also raise important cautionary questions. Several DBS specimens were detected as S. pneumoniae by rt-PCR despite growth of a non-S. pneumoniae pathogen on culture. A precise definition of what constitutes a positive result is required to avoid falsely over-identifying specimens.
| S-EPMC4805257 | biostudies-literature
Project description:OBJECTIVES:The objective of this study was to determine the occurrence of chromosomal and plasmid-mediated ?-lactamases (AmpC) genes in a collection of Malaysian isolates of Enterobacter species. Several phenotypic tests for detection of AmpC production of Enterobacter spp. were evaluated and the agreements between tests were determined. METHODS:Antimicrobial susceptibility profiles for 117 Enterobacter clinical isolates obtained from the Medical Microbiology Diagnostic Laboratory, University Malaya Medical Centre, Malaysia, from November 2012-February 2014 were determined in accordance to CLSI guidelines. AmpC genes were detected using a multiplex PCR assay targeting the MIR/ACT gene (closely related to chromosomal EBC family gene) and other plasmid-mediated genes, including DHA, MOX, CMY, ACC, and FOX. The AmpC ?-lactamase production of the isolates was assessed using cefoxitin disk screening test, D69C AmpC detection set, cefoxitin-cloxacillin double disk synergy test (CC-DDS) and AmpC induction test. RESULTS:Among the Enterobacter isolates in this study, 39.3% were resistant to cefotaxime and ceftriaxone and 23.9% were resistant to ceftazidime. Ten (8.5%) of the isolates were resistant to cefepime, and one isolate was resistant to meropenem. Chromosomal EBC family gene was amplified from 36 (47.4%) E. cloacae and three (25%) E. asburiae. A novel blaDHA type plasmid-mediated AmpC gene was identified for the first time from an E. cloacae isolate. AmpC ?-lactamase production was detected in 99 (89.2%) of 111 potential AmpC ?-lactamase producers (positive in cefoxitin disk screening) using D69C AmpC detection set. The detection rates were lower with CC-DDS (80.2%) and AmpC induction tests (50.5%). There was low agreement between the D69C AmpC detection set and the other two phenotypic tests. Of the 40 isolates with AmpC genes detected in this study, 87.5%, 77.5% and 50.0% of these isolates were positive by the D69C AmpC detection set, CC-DDS and AmpC induction tests, respectively. CONCLUSIONS:Besides MIR/ACT gene, a novel plasmid-mediated AmpC gene belonging to the DHA-type was identified in this study. Low agreement was noted between the D69C AmpC detection set and two other phenotypic tests for detection of AmpC production in Enterobacter spp. As plasmid-mediated genes may serve as the reservoir for the emergence of antibiotic resistance in a clinical setting, surveillance and infection control measures are necessary to limit the spread of these genes in the hospital.
| S-EPMC4786217 | biostudies-literature
Project description:Although Streptococcus pneumoniae is a leading cause of childhood disease in South East Asia, little has previously been reported regarding the epidemiology of invasive pneumococcal disease in Malaysia and very few studies have explored pneumococcal epidemiology using multilocus sequence typing (MLST). Here we describe serotype, multilocus sequence type (ST), and penicillin susceptibility of thirty pneumococcal invasive disease isolates received by the University of Malaya Medical Centre between February 2000 and January 2007 and relate this to the serotypes included in current pneumococcal conjugate vaccines. A high level of diversity was observed; fourteen serotypes and 26 sequence types (ST), (11 of which were not previously described) were detected from 30 isolates. Penicillin non-susceptible pneumococci accounted for 33% of isolates. The extent of molecular heterogeneity within carried and disease-causing Malaysian pneumococci remains unknown. Larger surveillance and epidemiological studies are now required in this region to provide robust evidence on which to base future vaccine policy.
| S-EPMC4062404 | biostudies-literature
Project description:BackgroundStreptococcus pneumoniae is a major causative agent of severe infections, including sepsis, pneumonia, meningitis, and otitis media, that has since become a major public health concern. In this study, the serotypes distribution of pneumococcal isolates was investigated to predict the efficacy of the 7-valent pneumococcal conjugate vaccine (PCV7) among the Malaysian populations.Methodology/principal findingsA total of 151 clinical isolates were serotyped using multiplex PCR assays. Out of them, there were 21.2% penicillin-resistant, 29.1% penicillin-intermediate, and 49.7% penicillin-susceptible S. pneumoniae strains. Serotypes detected among the Malaysian isolates were 1, 3, 10A, 11A/11D, 12F/12A, 14, 15A, 15B/15C, 16F, 18C/18B/18A/18F, 19A, 19F, 23F, 35B, 35F/47F, 6A/6B, 7C/7B/40, 7F/7A, 9V/9A, and 34. Serotype 19F and 23F were the two most prevalent serotypes detected. Serotypes are highly associated with invasiveness of isolates (p?=?0.001) and penicillin susceptibility (p<0.001). Serotype 19F was observed to have increased resistance against penicillin while serotype 19A has high invasive tendency. Age of patients was an important factor underlying the pneumococcal serotypes (p?=?0.03) and clinical sites of infections (p<0.001). High prevalence of pneumococcal isolates were detected among children <5 years old at nasopharyngeal sites while elderly adults ?60 years old were at increased risk for pneumococcal bacteremia.Conclusion/significanceCurrent study revealed that a number of serotypes, especially those associated with high penicillin resistance, have been formulated in the PCV7. Therefore, the protections expected from the routine use of PCV7 would be encouraging for the Malaysian. However, it is not possible to predict serotypes that might become predominant in the future and hence continued surveillance of circulating serotypes will be needed.
| S-EPMC3095606 | biostudies-literature
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.
| S-EPMC4460616 | biostudies-literature
Project description:Streptococcus pneumoniae serotype 24F is one of the most prevalent non-vaccine serotypes that causes invasive pneumococcal disease (IPD) in many countries, including Japan. This study aimed to analyze the bacteriological and molecular characteristics of serotype 24F sequence type (ST) 162, which has been increasingly isolated from pediatric patients with IPD in Japan recently. The examination of growth conditions, sequencing of genes associated with the CO2 dependence, and antimicrobial susceptibility testing at 35°C under 5% CO2 were performed for 10 isolates obtained from Japanese children with IPD caused by serotype 24F ST162. All isolates failed to grow at 35°C in ambient air; however, they showed growth at 30°C in ambient air and at 35°C under 5% CO2. Sequencing of murF involved in cell wall synthesis indicated that all isolates had a single amino acid substitution, MurFA179V. Additionally, all isolates were sensitive to penicillin G and resistant to trimethoprim-sulfamethoxazole. Furthermore, 25 non-capnophilic strains were obtained from all CO2-dependent isolates, and their murF sequences were compared. Thirteen of the 25 non-capnophilic strains displayed a different amino acid substitution, MurFV179A, whereas the other 12 presented the previously described MurFA179V. This suggests that a proportion of the CO2-dependent phenotype of serotype 24F ST162 may have been conferred by MurFA179V; however, the mechanism for the CO2 dependence of these isolates warrants further investigation. The CO2-dependent serotype 24F ST162 pneumococcal isolates shared common characteristics in terms of growth patterns, molecular basis, and antimicrobial susceptibility; therefore, future epidemiological trends of this clone must be closely monitored. IMPORTANCE We characterized Streptococcus pneumoniae serotype 24F sequence type (ST) 162 isolated from Japanese children with invasive pneumococcal disease (IPD). Owing to its highly invasive nature, serotype 24F is expected to be isolated from clinically significant cases. Serotype 24F ST162 isolates tested in the present study did not grow at 35°C in ambient air. Therefore, antimicrobial susceptibility testing using the broth microdilution method, which is usually conducted in ambient air, cannot be performed, posing a clinical challenge. Clinical practitioners and laboratory personnel should be aware of the epidemiological, bacteriological, and molecular characteristics of serotype 24F ST162. We believe that our findings can help diagnose and treat IPD caused by serotype 24F ST162, a serotype expected to become problematic in the post-13 valent pneumococcal conjugate vaccine era.
| S-EPMC10714769 | biostudies-literature
Project description:A total of 125 isolates were recovered from adenoids and/or nasopharynx of 170 children aged 2 to 5 from south-east Poland; they had undergone adenoidectomy for recurrent and/or persistent symptoms of upper respiratory tract infections. Pneumococcal isolates were analyzed by phenotyping (serotyping and antimicrobial resistance tests) and genotyping together with the clonality of the pneumococcal isolates based on resistance determinants, transposon distribution and multilocus sequence typing (MLST). Serotypes 19F, 6B and 23F constituted 44.8% of the isolates. Among all of the strains, 44.8% showed decreased susceptibility to penicillin and resistance to co-trimoxazole (52.8%), tetracycline (38.4%), erythromycin (53.6%), clindamycin (52.8%) and chloramphenicol (27.2%) was observed. Tn6002 was found in 34.8% of erythromycin-resistant isolates while composite Tn2010-in 16.7% of erm(B)-carrying isolates that harboured also mef(E) gene. Tn3872-related elements were detected in 27.3% of erythromycin-resistant strains. In the majority of chloramphenicol-resistant catpC194-carrying isolates (79.4%), ICESp23FST81-family elements were detected. The genotyping showed that pneumococcal population was very heterogeneous; 82 sequence types (STs) were identified, and the most frequent contributed to not more than 8% of the isolates. Nearly 44% STs were novel, each of them was recovered only from one child. Four STs belonged to one of the 43 worldwide spread resistant pneumococcal clones currently accepted by Pneumococcal Molecular Epidemiology Network (PMEN), i.e. Spain 9V-3, Spain 23F-1, Norway NT-42 and Poland 6B-20, accounting for 12 (16.7%) of the 75 nonususceptible isolates, and five STs were single-locus variants of PMEN resistant clones (England 14-9, Spain 9V-3, Spain 23F-1, Greece 21-30, Denmark 14-32), accounting 9 (12%) of nonsusceptible isolates. A few MDR clones belonging to 6B and 19F serotypes found among preschool children emphasizes rather the role of clonal dissemination of local strains in the community than international clones spreading in the increase of resistance among pneumococcal strains.
| S-EPMC4945090 | biostudies-literature