Establishment and Performance Evaluation of Multiplex PCR-Dipstick DNA Chromatography for Mycoplasma pneumoniae and Chlamydia pneumoniae Rapid Detection
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ABSTRACT:
SUBMITTER: Hu L
PROVIDER: S-EPMC10555492 | biostudies-literature | 2023 Jan
REPOSITORIES: biostudies-literature
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Project description:A multiplex real-time PCR assay for the detection of Mycoplasma pneumoniae (MP181), Chlamydia (Chlamydophila) pneumoniae (CP-Arg), Legionella spp. (Pan-Leg), and the human RNase P (RNase P) gene was developed for rapid testing of atypical bacterial respiratory pathogens in clinical specimens. This method uses 4 distinct hydrolysis probes to detect 3 leading causes of community-acquired pneumonia. The assay was evaluated for specificity and sensitivity by testing against 35 related organisms, a dilution series of each specific target and 197 clinical specimens. Specificity testing demonstrated no cross-reactivity. A comparison to previously validated singleplex real-time PCR assays for each agent was also performed. The analytical sensitivity for specific pathogen targets in both the singleplex and multiplex was identical (50 fg), while efficiencies ranged from 82% to 97% for the singleplex assays and from 90% to 100% for the multiplex assay. The clinical sensitivity of the multiplex assay was improved for the Pan-Leg and CP-Arg targets when compared to the singleplex. The MP181 assay displayed equivalent performance. This multiplex assay provides an overall improvement in the diagnostic capability for these agents by demonstrating a sensitive, high-throughput and rapid method. This procedure may allow for a practical and efficient means to test respiratory clinical specimens for atypical pneumonia agents in health care settings and facilitate an appropriate public health response to outbreaks.
| S-EPMC7126442 | biostudies-literature
Project description:The aim of this study was to improve detection of Mycoplasma pneumoniae and Chlamydia pneumoniae in clinical specimens by developing a multiplex real-time PCR assay that includes identification of macrolide-resistant M. pneumoniae. Novel assays targeting a M. pneumoniae conserved hypothetical protein gene, M. pneumoniae 23S rRNA gene mutations associated with macrolide resistance and human β-globin gene (an endogenous internal control) were designed and combined with a previously published C. pneumoniae PCR targeting ompA gene. The resulting quadraplex PCR was validated with a panel of clinical specimens supplemented with external quality assessment specimens, simulated specimens and various bacterial and viral strains. The obtained results were compared to those obtained by reference PCRs or confirmed by sequencing (typing of macrolide resistance). The novel multiplex PCR assay was in 100 % agreement with reference PCRs. Four M. pneumoniae strains with macrolide resistance-associated mutations were identified among 42 strains, which comprises 9.5 % of the study material. Amplification of an internal control excluded sample-derived inhibition possibly leading to false-negative reporting. In conclusion, we have developed a resources conserving multiplex real-time PCR assay for simultaneous detection of M. pneumoniae, C. pneumoniae and the most common mutations leading to macrolide resistance in M. pneumoniae. The assay is a widely useful tool for detection of these respiratory pathogens and will also shed light on the occurrence of macrolide resistance in M. pneumoniae.
| S-EPMC4641141 | biostudies-literature
Project description:We designed several new primers and modified previously described species- and type-specific primers targeting the Mycoplasma pneumoniae P1 adhesin gene. Optimized thermal profiles allowed one-step or nested PCR to be completed in less than 1 h. In 10 patients with pneumonia, M. pneumoniae type 1 was identified in 3 and type 2 in 7.
| S-EPMC87576 | biostudies-literature
Project description:Influenza A, influenza B, severe acute respiratory syndrome coronavirus 2, adenovirus, respiratory syncytial virus, Mycoplasma pneumoniae, and Chlamydophila pneumoniae are common pathogens that can cause severe pneumonia and other symptoms, resulting in acute lower respiratory tract infections. The objective of this study was to design and evaluate a sensitive and specific multiplex one-step reverse transcription PCR (RT-PCR)-dipstick chromatography method for simultaneous rapid detection of these seven pathogens. Streptavidin-coated blue latex particles were used to read out a positive signal. Based on the DNA-DNA hybridization of oligonucleotide sequences (Tag) for forward primer with the complementary oligonucleotide sequence (cTag) on the dipstick and biotin-streptavidin interactions, PCR products were able to be illuminated visually on the dipstick. The specificity and the limit of detection (LOD) were also evaluated. Moreover, the clinical performance of this method was compared with Sanger sequencing for 896 samples. No cross reaction with other pathogens was found, confirming the high specificity of this method. The LOD was 10 copies/µL for each of the tested pathogens, and the whole procedure took less than 40 min. Using 896 samples, the sensitivity and specificity were shown to be no lower than 94.5%. The positive predictive value was higher than 82.1%, and the negative predictive value was higher than 99.5%. The kappa value between the PCR-dipstick chromatography method and Sanger sequencing ranged from 0.869 to 0.940. In summary, our one-step RT-PCR-dipstick chromatography method is a sensitive and specific tool for rapidly detecting multiplex respiratory pathogens.
| S-EPMC8326646 | biostudies-literature
Project description:The capsule is a major virulence factor of pneumococci, and it was shown that some capsular variants are associated with antimicrobial resistance and certain types of disease. Moreover, pneumococcal capsular typing has received renewed interest since the availability of conjugate vaccines, which include serotypes frequently associated with pediatric disease. Our aim was to develop a simple, reliable, and economical method for detecting epidemiologically important serotypes present in the proposed 11-valent conjugate vaccine. We designed primers based on the sequences available for the capsular types 1, 3, 4, 6B, 14, 18C, 19F, 19A, and 23F and combined them into seven multiplex PCRs. The method involves streamlined DNA template preparation and agarose gel electrophoresis to analyze the amplification products. A total of 446 pneumococci selected from among isolates colonizing the nasopharynx of children attending day care centers in Lisbon, Portugal, were typed both by conventional immunological techniques and by multiplex PCR. Capsular types identified by the PCR method invariably produced results concordant with the conventional serotyping technique. Even when the method presented does not fully type an isolate, the PCR data can guide the experimenter when using immunological serotyping. Multiplex PCR for the analysis of pneumococci provides an accurate, expeditious, and cost-effective way of reducing the number of strains that have to be serotyped by conventional immunological techniques.
| S-EPMC156574 | biostudies-literature
Project description:Mycoplasma and Chlamydia multiplex test
| PRJEB2281 | ENA
Project description:Community-acquired pneumonia caused by Mycoplasma pneumoniae or Chlamydia pneumoniae is usually mild. Mycoplasma pneumoniae-related and C. pneumoniae-related acute respiratory distress syndromes (ARDSs) are rare. Moreover, to our knowledge, there are no published reports on ARDS caused by M. pneumoniae and C. pneumoniae coinfection. Here, we report a case of an immunocompetent young woman who was co-infected with M. pneumoniae and C. pneumoniae and was started on treatment with piperacillin and clarithromycin. Two days later, she developed ARDS. She recovered rapidly following a change of antibiotic treatment to levofloxacin and was discharged on day 12. We conducted exome sequencing followed by alternative filtering to search for candidate ARDS-related genes. We identified an intronic variant of unknown significance within leucine-rich repeat-containing 16A (LRRC16A), a gene previously identified as a significant locus for platelet count with a possible role in ARDS. This is a rare case of ARDS in a young adult caused by M. pneumoniae and C. pneumoniae coinfection. This case suggests that ARDS in young adults may be correlated with variants in LRRC16A. This requires confirmation by further case reports.
| S-EPMC8775183 | biostudies-literature
Project description:A PCR-dipstick chromatography technique was designed and evaluated for differential identification of blaNDM, blaKPC, blaIMP, and blaOXA-48 carbapenemase genes directly in stool specimens within 2 h. It is a DNA-DNA hybridization-based detection system where PCR products can be easily interpreted by visual observation without electrophoresis. The PCR-dipstick showed high sensitivity (93.3%) and specificity (99.1%) in directly detecting carbapenemase genes in stool specimens compared with multiplex PCR for genomic DNA of the isolates from those stool specimens.
| S-EPMC5444138 | biostudies-literature
Project description:The contribution of intracellular and fastidious bacteria in Cystic fibrosis (CF) pulmonary exacerbations, and progressive lung function decline remains unknown. This project aimed to explore their impact on bacterial microbiota diversity over time in CF children. Sixty-one children enrolled in the MUCOVIB multicentre prospective cohort provided 746 samples, mostly nasopharyngeal swabs, throat swabs and sputa which were analysed using culture, specific real-time qPCRs and 16S rRNA amplicon metagenomics. Chlamydia pneumoniae (n = 3) and Mycoplasma pneumoniae (n = 1) were prospectively documented in 6.6% of CF children. Microbiota alpha-diversity in children with a documented C. pneumoniae was highly variable, similarly to children infected with Staphylococcus aureus or Pseudomonas aeruginosa. The transition from routine follow-up visits to pulmonary exacerbation (n = 17) yielded variable changes in diversity indexes with some extreme loss of diversity. The high rate of C. pneumoniae detection supports the need for regular screenings in CF patients. A minor impact of C. pneumoniae on the microbial community structure was documented. Although detected in a single patient, M. pneumoniae should also be considered as a possible aetiology of lung infection in CF subjects.
| S-EPMC7787906 | biostudies-literature
Project description:BackgroundDiagnosis of community-acquired pneumonia (CAP) caused by Mycoplasma pneumoniae (Mp) in children has been hampered by difficulty in obtaining convalescent serum and time constraints. In this study, the two diagnostic assays that targeted respectively on Mp-antibody and Mp-DNA were retrospectively investigated.MethodsA total of 3146 children were clinically diagnosed to have CAP and were confirmed by chest X-ray during March 2015 to February 2016 in Children's hospital of Hebei Province (China). Both of the sera and sputum samples were collected in 24 h after their admission. The Mp-antibody was examined by the passive particle agglutination assay and a fourfold or greater increase of antibody titers of paired sera or≧1:160 titer of single serum was set as the serology positive. Mp-DNA in the sputum samples was tested by a multiplex-PCR method named GeXP assay (multiplex PCR combined with automated capillary electrophoresis). In order to eliminate the false positive results caused by the asymptomatic carriage after infected by M. pneumoniae, the inconsistent samples were tested by the real-time isothermal transcription-mediated RNA amplification assay (SAT).ResultsThe inter-rated agreement test was performed in 3146 CAP patients, with a highest kappa value in the school-age children as 0.783. There were 6.29% (198/3146) cases showed inconsistent results determined by GeXP and serology assay. All of the 19 GeXP(+)/Serology (-) samples and a randomly chosen 27 from 179 GeXP(-)/Serology (+) samples were tested by SAT assay, and a 97.8% diagnosis agreement was observed between SAT and GeXP assay, but not with the serology assay. In addition, patients who were detected only by serology or only by multiplex-PCR were significantly younger than those with both methods positive (3.0 and 1.5 years vs. 5.0 years, p < 0.01). The Viral-Mp coinfection accounted for 37.0% (97/262), which was more common in winter and spring (p < 0.05) and in the infantile group (p < 0.01), compared to the pure Mp positive ones.ConclusionIn some children CAP cases, the Mp laboratory diagnosis was inconsistent between serology and multiplex-PCR assay. Verified by the SAT assay, the GeXP showed a more sensitive and reliable performance compared with the serology assay. Furthermore, employing the multiplex-PCR could provide more information on the associated pathogens for clinical assessment of CAP.
| S-EPMC5527399 | biostudies-literature