Development and laboratory evaluation of a realtime pcr. The diagnostic value of serological studies in pediatric. Preliminary evaluation of an lyta pcr assay for detection. Standardisation and evaluation of a quantitative multiplex realtime.
Pdf detection of streptococcus pneumoniae in sputum samples. Strategies for the treatment of mrmp are a key focus of research. Singlenucleotide polymorphism pcr for the detection of. They are usually found in pairs and do not form spores and are nonmotile. Macrolideresistant mycoplasma pneumoniae mrmp have been reported worldwide. Resistant genes bla shv, bla tem, bla oxa1, bla kpc, bla oxa48, bla ctxm15, bla vim, bla imp and bla ndm were investigated by polymerase chain reaction pcr. The lyta, piab, and cpsa genes were used as targets for s. Mycoplasma pneumoniae is an important pathogen of communityacquired pneumonia in pediatric patients. The assay was validated in a blinded manner using 30 mycoplasma pneumoniaepositive specimens received from a reference lab and 6 negative specimens. A separate analysis according to site of detection of m.
Detection of pathogens by realtime pcr in adult patients. Specific characteristics for this species are the production of toxins such as pneumolysin ply, as well as the presence of surface antigens such as pneumococcal surface adhesin a psaa and pneumococcal autolysin a lyta 39. Cap capspn was performed by urine antigen detection. We aimed to evaluate the epidemiology of carbapenemaseproducing escherichia coli and klebsiella pneumoniae in the baltic states and st. Development of a rapid recombinase polymerase amplification. Pdf assessing the diagnostic accuracy of pcrbased detection of. Petersburg, russia, and to compare the different methods. Laboratory diagnosis of pneumonia in the molecular age european.
Methods active surveillance for communityacquired pneumonia cap in hospitalised. Evaluation of five realtime pcr assays for detection of. Streptococcus pneumoniae is a major cause of human disease. Oct 25, 2018 rapid detection of macrolide resistanceassociated mutations in mycoplasma pneumoniae is crucial for effective antimicrobial treatment.
Klebsiella pneumoniae is a widespread nosocomial pathogen. Detection of pneumococcus by pcr university of oulu. Rapid detection of the macrolide sensitivity of pneumonia. Pfaller ma, yolken rh, eds manual of clinical microbiology. Pdf study design detection and serotyping of streptococcus pneumoniae are important to assess the impact of pneumococcal vaccines. Acute respiratory infections aris cause substantial morbidity and mortality worldwide. The enterovirus d68 2014 realtime rtpcr assay evd68 2014 rrtpcr is intended for the in vitro qualitative detection of rna from the enterovirus d68 evd68 strains detected in north america. Pcr detection of streptococcus pneumoniae dna in serum.
For multiplex snp pcr, two outer primers for amplification of the 23s rrna gene and two mutantspecific primers for the. The assay is useful for the surveillance of emerging highly virulent strains. In addition to its role as a leading cause of communityacquired pneumonia, s. Rapid diagnosis of streptococcus pneumoniae can play a significant role in decreasing. For instance, the xisco gene was recently described as a biomarker for pcrbased detection of s. Whole organism spiking studies near the limit of detection of the assay were also performed using the following specimens.
Rapid and combined detection of mycoplasma pneumoniae. The aim of this study was to develop a singlenucleotide polymorphism snp pcr assay to be performed directly on respiratory samples for the simultaneous detection of mycoplasma pneumoniae and its 23s rrna gene mutations, which are responsible for macrolide resistance. Identification of the human pathogen streptococcus pneumoniae or pneumococcus is an important task that may pose challenges. Full text detection of mycoplasma pneumoniae in mexican.
We identified only 17 cases by culture, but molecular methods identified s. The sensitivity of np swab pcr for the detection of s. Reliability of nested pcr for the detection of chlamydia. Assessing the diagnostic accuracy of pcrbased detection.
We conducted a retrospective analysis of 240 patients with positive cultures for k. Survey and rapid detection of klebsiella pneumoniae in. In the carbapenemases detection studies malditof based assay and realtime pcr the following control strains were used. This suggests that autolysin pcr is suitable for the detection of s. Two simulated csf samples were included in the quality assessment panel to assess methods used for the nonculture detection of s. Study design detection and serotyping of streptococcus pneumoniae are important to assess the impact of pneumococcal vaccines. With the challenges of quellung serotyping, pcrbased serotype prediction is increasingly being used for largescale epidemiological studies. The goals of this study were to assess the macrolide sensitivity of m.
Development of realtime pcr methods for the detection of. Klebsiella pneumoniae is of growing public health concern due to the emergence of strains that are multidrug resistant, virulent, or both. Evaluation of modified hodge test as a nonmolecular assay. Realtime pcr targeting lyta the major autolysin gene and piab permease. Jan 24, 2019 the presence of macrolideresistant myocplasma pneumoniae has been frequently reported in recent years, especially in china.
Therefore, a quantitative realtime pcr qpcr assay with duplex reactions targeting eight bacteria and six viruses was developed. Community acquired pneumonia cap due to streptococcus pneumoniae still occurs in at risk populations, despite the availability of effective vaccines. Detection of cocolonization with streptococcus pneumoniae by. This assay may find utility as a rapid pointofcare diagnostic test for the detection of s.
The m pneumoniae pcr mayo had 100% sensitivity and specificity when compared to the focus diagnostics m pneumoniae primer pair pcr assay. The zkir assay, a realtime pcr method for the detection of. Pcr is a molecular diagnostic technique based on dna detection and offers the advantage of. External quality assurance scheme for streptococcus. The presence of macrolideresistant myocplasma pneumoniae has been frequently reported in recent years, especially in china. Mycoplasma pneumoniae by pcr new sparrow dna laboratory test november 2016 mycoplasma pneumoniae molecular testing is now performed at sparrow laboratories and significantly enhances your diagnostic options for the detection of mycoplasma pneumoniae from multiple sample types. Despite differences of crossing threshold values of up to 4, assays were able to detect at least 20 cfu5. Detection and serotyping of streptococcus pneumoniae are. Neisseria meningitidis nm, haemophilus influenzae hi, and streptococcus pneumoniae sp are the lead causes of bacterial meningitis.
A single multiplex pcr assay targeting seven virulence factors and the wzi gene specific for the k1 and k2 capsular serotypes of klebsiella pneumoniae was developed and tested on 65 clinical isolates, which included 45 isolates responsible for communityacquired severe human infections. With the challenges of quellung serotyping, pcr based serotype prediction is increasingly being used for largescale epidemiological studies. Detection of these pathogens from clinical specimens using traditional realtime pcr rtpcr requires dna extraction to remove the pcr inhibitors prior to testing, which is time consuming and labor intensive. Future work will focus on the development of an internal amplification control. Bacterial and viral nucleic acid can be amplified by pcr upon clinical sample extraction using specific primers for classical qualitative pcr and primers and probes for realtime pcr. Methods active surveillance for communityacquired pneumonia cap in hospitalised adults was performed. Identification of streptococcus pneumoniae by a realtime pcr. Moreover, antibioticresistant klebsiella pneumoniae emerged in recent years has become a serious problem in clinics ali abdel rahim and ali mohamed, 2014. We cultured and performed pcr on 152 pleural fluid samples recovered from pediatric patients in portugal during 20102015 to identify and serotype streptococcus pneumoniae. Assessing the diagnostic accuracy of pcrbased detection of. The aim of this study was to improve detection of mycoplasma pneumoniae and chlamydia pneumoniae in clinical specimens by developing a multiplex realtime pcr assay that includes identification of macrolideresistant m. Detection of antimicrobial resistance genes in beta. Streptococcus pneumoniae dna by using polymerase chain reaction and microwell hybridization with europiumlabelled probes. Thus, the rapid and sensitive detection of this pathogen is required if the appropriate therapy is to be administered and outbreaks controlled.
Streptococcus pneumoniae and haemophilus influenzae are common cause of. The spread of carbapenemaseproducing enterobacteriaceae is a global problem. Detection and prediction of streptococcus pneumoniae. Whole organism spiking studies near the limit of detection of the. The enterovirus d68 2014 realtime rt pcr assay evd68 2014 rrt pcr is intended for the in vitro qualitative detection of rna from the enterovirus d68 evd68 strains detected in north america. Mycoplasma pneumoniae, molecular detection, pcr, varies. Mycoplasma pneumoniae is a small bacterium transmitted via organismcontaining droplets. Evaluation of lightmix mycoplasma macrolide assay for. Rapid detection of macrolide resistanceassociated mutations in mycoplasma pneumoniae is crucial for effective antimicrobial treatment. Four commercial realtime pcr assays to detect mycoplasma pneumoniae were tested, and the results were compared with the results for an inhouse approach. We compared the accuracy of our multiplex snp pcr assay with sequencing for the detection of macrolideresistant m. Kp can be present in environmental sources such as soils and vegetation, which could act as reservoirs of. Ii saukkoriipi a, palmu a, kilpi t, leinonen m 2002 realtime quantitative pcr for the detection of streptococcus pneumoniae in the middle ear fluid of children with acute otitis media. Currently, the diagnosis of pneumococcal bacteremia relies on the isolation and identification of the bacteria from blood cultures.
A comparison study between gexpbased multiplexpcr and. We evaluated the lightmix mycoplasma macrolide assay for the detection of point mutations at nucleotide positions 2063 and 2064 in the 23s ribosomal rna rrna gene of m. Detection of carbapenemaseproducing enterobacteriaceae in. This study used realtime rtpcr targeting the genes encoding autolysin lyta and capsular biosynthesis. Direct detection of c pneumoniae requires an exceptional level of knowhow and expertise, considering not only cell culture and quantification but also detection of this pathogen by pcr, especially nested methods. With the challenges of quellung serotyping, pcrbased serotype prediction is. Here, we established a quadruplex quantitative pcr qpcr method to rapidly identify and simultaneously detect a single infection or coinfection.
To compare the detection limit of lamp using either realtime turbidity measurements or visual color change with traditional pcr, pure genomic dna was extracted from k. Streptococcus pneumoniae, or pneumococcus, is a grampositive, alphahemolytic under aerobic conditions or betahemolytic under anaerobic conditions, facultative anaerobic member of the genus streptococcus. We investigated the activities of four antibiotics against 81 m. Swedish institute for communicable disease control sweden carbapenemases detection control set including imppositive pseudomonas aeruginosa ps540, vimpositive k. Communityacquired pneumonia may present with similar clinical symptoms, regardless of viral or bacterial cause. Sputum and serum specimens collected from 3146 hospitalized children were tested by gexp assay and pa assay, respectively.
Pediatric complicated pneumonia caused by streptococcus. Mycoplasma pneumoniae is associated with up to 40% of community. Rapid and direct molecular detection of streptococcus pneumoniae. Molecular detection of mycoplasma pneumoniae by quantitative realtime pcr in patients with community acquired pneumonia. Detection and analysis of klebsiella pneumoniae causing liver. Streptococcus pneumoniae is a major cause of bacteremia in both children and adults. Realtime pcr detection of mycoplasma pneumoniae in the. In 2008, clsi changed the interpretative standard for benzyl penicillin and s. A rapid and sensitive molecular method for the detection of k. Antimicrobial susceptibility and genotyping of mycoplasma pneumoniae isolates in beijing, china, from 2014 to 2016. Antimicrobial susceptibility and genotyping of mycoplasma. Identification of streptococcus pneumoniae by a realtime. Multiplex pcr for detection of seven virulence factors and k1.
The causes of ari are dynamic, and coinfections of mycoplasma pneumoniae, epsteinbarr virus and human cytomegalovirus are recently developed causes of ari. Pdf detection and prediction of streptococcus pneumoniae. Multiplex pcr for detection of seven virulence factors and. The zkir assay, a realtime pcr method for the detection. Chaudhry r, sharma s, javed s, passi k, dey ab, malhotra p.
Detection of cocolonization with streptococcus pneumoniae. The presence of macrolideresistant myocplasma pneumoniae has been frequently reported in recent. To increase sensitivity and specificity, target dna is amplified in a 2step procedure using 2 different primer pairs. Preliminary evaluation of an lyta pcr assay for detection of. The present crosssectional study was conducted from march 2014 to march 2015. For example, in pneumococcal carriage studies investigating vaccine.
Publication recombinase polymerase amplification assay for. Laboratory diagnosis of pneumonia in the molecular age. This study describes the diagnostic accuracy of pcrbased detection of s. Antimicrobial susceptibility was assessed by vitek 2 system. Rapid and combined detection of mycoplasma pneumoniae, epsteinbarr virus and human cytomegalovirus using allglo quadruplex quantitative pcr yi chen 1,2, hui he 1,2, ping pan 1,2, songzhe he 3, xueyan dong 2, yueming chen 2, shuying wang 2, daojun yu 1,2. Diagnostic assays are needed to rapidly discriminate between causes, because this will guide decisions on appropriate treatment. Monitoring streptococcus pneumoniae serotype distribution is important to assess the impact and effectiveness of pneumococcal vaccine programs. In this study, we investigated the antimicrobial susceptibility and genotype against m. Expression of streptococcus pneumoniae virulencerelated genes. We have developed a sensitive assay for the detection of s.
Klebsiella pneumoniae causes serious hospitalacquired infections of the urinary tract, respiratory tract, surgical sites, and the bloodstream and can cause severe diseases, such as pneumonia, sepsis, and bacteremia maroncle et al. Antimicrobial susceptibility and genotyping of mycoplasma pneumoniae isolates in beijing, china, from 2014 to 2016 fei zhao1,2, jinrong liu3, weixian shi4, fang huang4, liyong liu1,2, shunying zhao3 and jianzhong zhang1,2 abstract background. Detection and analysis of klebsiella pneumoniae causing. It is a cause of upper respiratory infection, pharyngitis, and tracheobronchitis, particularly in children, and has been associated with approximately 20% of cases of community acquired pneumonia.
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