Oct 25, 2018 rapid detection of macrolide resistanceassociated mutations in mycoplasma pneumoniae is crucial for effective antimicrobial treatment. 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. Detection of streptococcus pneumoniae in whole blood by pcr. 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. The assay was validated in a blinded manner using 30 mycoplasma pneumoniaepositive specimens received from a reference lab and 6 negative specimens. Here, we established a quadruplex quantitative pcr qpcr method to rapidly identify and simultaneously detect a single infection or coinfection. 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. This study used realtime rtpcr targeting the genes encoding autolysin lyta and capsular biosynthesis. The zkir assay, a realtime pcr method for the detection of. 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. Diagnostic assays are needed to rapidly discriminate between causes, because this will guide decisions on appropriate treatment. 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. A rapid and sensitive molecular method for the detection of k.
They are usually found in pairs and do not form spores and are nonmotile. Mycoplasma pneumoniae, molecular detection, pcr, varies. Pneumococcus carriage cocolonization quantitative detection a b s t r a c t detection of pneumococcal carriage by multiple cocolonizing serotypes is important in assessing the bene. 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. Four commercial realtime pcr assays to detect mycoplasma pneumoniae were tested, and the results were compared with the results for an inhouse approach. In the carbapenemases detection studies malditof based assay and realtime pcr the following control strains were used. Rapid diagnosis of streptococcus pneumoniae can play a significant role in decreasing. 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 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. For example, in pneumococcal carriage studies investigating vaccine. Identification of the human pathogen streptococcus pneumoniae or.
We conducted a retrospective analysis of 240 patients with positive cultures for k. 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. Monitoring streptococcus pneumoniae serotype distribution is important to assess the impact and effectiveness of pneumococcal vaccine programs. 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.
Streptococcus pneumoniae and haemophilus influenzae are common cause of. Methods active surveillance for communityacquired pneumonia cap in hospitalised adults was performed. This study describes the diagnostic accuracy of pcrbased detection of s. The spread of carbapenemaseproducing enterobacteriaceae is a global problem. A separate analysis according to site of detection of m. Identification of the human pathogen streptococcus pneumoniae or pneumococcus is an important task that may pose challenges. The m pneumoniae pcr mayo had 100% sensitivity and specificity when compared to the focus diagnostics m pneumoniae primer pair pcr assay. Pdf assessing the diagnostic accuracy of pcrbased detection of.
We identified only 17 cases by culture, but molecular methods identified s. Preliminary evaluation of an lyta pcr assay for detection of. To increase sensitivity and specificity, target dna is amplified in a 2step procedure using 2 different primer pairs. Communityacquired pneumonia may present with similar clinical symptoms, regardless of viral or bacterial cause. Antimicrobial susceptibility and genotyping of mycoplasma pneumoniae isolates in beijing, china, from 2014 to 2016. With the challenges of quellung serotyping, pcrbased serotype prediction is increasingly being used for largescale epidemiological studies. Pfaller ma, yolken rh, eds manual of clinical microbiology. Publication recombinase polymerase amplification assay for. Singlenucleotide polymorphism pcr for the detection of. Pdf a method for the detection of streptococcus pneumoniae in sputum.
For multiplex snp pcr, two outer primers for amplification of the 23s rrna gene and two mutantspecific primers for the. With the challenges of quellung serotyping, pcrbased serotype prediction is. Laboratory diagnosis of pneumonia in the molecular age. 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.
Rapid and combined detection of mycoplasma pneumoniae. Sputum and serum specimens collected from 3146 hospitalized children were tested by gexp assay and pa assay, respectively. Multiplex pcr for detection of seven virulence factors and. The presence of macrolideresistant myocplasma pneumoniae has been frequently reported in recent years, especially in china.
We aimed to evaluate the epidemiology of carbapenemaseproducing escherichia coli and klebsiella pneumoniae in the baltic states and st. International journal of development research 2014. Realtime pcr detection of mycoplasma pneumoniae in the. Detection and analysis of klebsiella pneumoniae causing liver. Assessing the diagnostic accuracy of pcrbased detection of.
Klebsiella pneumoniae is a widespread nosocomial pathogen. Identification of streptococcus pneumoniae by a realtime. Rapid and direct molecular detection of streptococcus pneumoniae. Jan 24, 2019 the presence of macrolideresistant myocplasma pneumoniae has been frequently reported in recent years, especially in china. In this study, we investigated the antimicrobial susceptibility and genotype against m.
Methods active surveillance for communityacquired pneumonia cap in hospitalised. Streptococcus pneumoniae is a major cause of bacteremia in both children and adults. Thus, the rapid and sensitive detection of this pathogen is required if the appropriate therapy is to be administered and outbreaks controlled. Two simulated csf samples were included in the quality assessment panel to assess methods used for the nonculture detection of s. In 2008, clsi changed the interpretative standard for benzyl penicillin and s. Rapid detection of the macrolide sensitivity of pneumonia. Assessing the diagnostic accuracy of pcrbased detection. This assay may find utility as a rapid pointofcare diagnostic test for the detection of s. The lyta, piab, and cpsa genes were used as targets for s. 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.
Pdf detection of streptococcus pneumoniae in sputum samples. Molecular detection of mycoplasma pneumoniae by quantitative realtime pcr in patients with community acquired pneumonia. The presence of macrolideresistant myocplasma pneumoniae has been frequently reported in recent. Pdf detection and prediction of streptococcus pneumoniae. We cultured and performed pcr on 152 pleural fluid samples recovered from pediatric patients in portugal during 20102015 to identify and serotype streptococcus pneumoniae. Streptococcus pneumoniae is a major cause of human disease. Full text detection of mycoplasma pneumoniae in mexican. Acute respiratory infections aris cause substantial morbidity and mortality worldwide. The sensitivity of np swab pcr for the detection of s. Performance of the biomark hd realtime qpcr system fluidigm for the detection of nasopharyngeal bacterial pathogens and streptococcus pneumoniae typing. Laboratory diagnosis of pneumonia in the molecular age european. Mycoplasma pneumoniae is associated with up to 40% of community. We have developed a sensitive assay for the detection of s.
A comparison study between gexpbased multiplexpcr and. Study design detection and serotyping of streptococcus pneumoniae are important to assess the impact of pneumococcal vaccines. The present crosssectional study was conducted from march 2014 to march 2015. Chaudhry r, sharma s, javed s, passi k, dey ab, malhotra p. In this study, we first described a loopmediated isothermal amplification lamp method for the rapid detection of capsular polysaccharide synthesis regulating gene rcsa from k. Community acquired pneumonia cap due to streptococcus pneumoniae still occurs in at risk populations, despite the availability of effective vaccines. Development of realtime pcr methods for the detection of. Pediatric complicated pneumonia caused by streptococcus. The assay is useful for the surveillance of emerging highly virulent strains. We compared the accuracy of our multiplex snp pcr assay with sequencing for the detection of macrolideresistant m. Antimicrobial susceptibility and genotyping of mycoplasma.
The diagnostic value of serological studies in pediatric. The zkir assay, a realtime pcr method for the detection. Mycoplasma pneumoniae is a small bacterium transmitted via organismcontaining droplets. 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. Moreover, antibioticresistant klebsiella pneumoniae emerged in recent years has become a serious problem in clinics ali abdel rahim and ali mohamed, 2014. Petersburg, russia, and to compare the different methods. Detection of pathogens by realtime pcr in adult patients.
Detection and analysis of klebsiella pneumoniae causing. Detection of antimicrobial resistance genes in beta. Identification of streptococcus pneumoniae by a realtime pcr. With the challenges of quellung serotyping, pcr based serotype prediction is increasingly being used for largescale epidemiological studies. Neisseria meningitidis nm, haemophilus influenzae hi, and streptococcus pneumoniae sp are the lead causes of bacterial meningitis. Klebsiella pneumoniae is of growing public health concern due to the emergence of strains that are multidrug resistant, virulent, or both.
The causes of ari are dynamic, and coinfections of mycoplasma pneumoniae, epsteinbarr virus and human cytomegalovirus are recently developed causes of ari. Swedish institute for communicable disease control sweden carbapenemases detection control set including imppositive pseudomonas aeruginosa ps540, vimpositive k. Pcr is a molecular diagnostic technique based on dna detection and offers the advantage of. Standardisation and evaluation of a quantitative multiplex realtime. Detection and serotyping of streptococcus pneumoniae are.
Polymerase chain reaction is a useful technique in microbial diagnostics to detect and quantify dna or rna of low abundance. Kp can be present in environmental sources such as soils and vegetation, which could act as reservoirs of. Detection of cocolonization with streptococcus pneumoniae by. 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. Streptococcus pneumoniae dna by using polymerase chain reaction and microwell hybridization with europiumlabelled probes. 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. Therefore, a quantitative realtime pcr qpcr assay with duplex reactions targeting eight bacteria and six viruses was developed. Realtime pcr targeting lyta the major autolysin gene and piab permease. 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. Mycoplasma pneumoniae is an important pathogen of communityacquired pneumonia in pediatric patients. Pcr detection of streptococcus pneumoniae dna in serum.
We investigated the activities of four antibiotics against 81 m. External quality assurance scheme for streptococcus. Evaluation of modified hodge test as a nonmolecular assay. Development and laboratory evaluation of a realtime pcr. Rapid detection of macrolide resistanceassociated mutations in mycoplasma pneumoniae is crucial for effective antimicrobial treatment. Strategies for the treatment of mrmp are a key focus of research. Detection of cocolonization with streptococcus pneumoniae. This suggests that autolysin pcr is suitable for the detection of s. Macrolideresistant mycoplasma pneumoniae mrmp have been reported worldwide.
Detection of carbapenemaseproducing enterobacteriaceae in. Antimicrobial susceptibility was assessed by vitek 2 system. Reliability of nested pcr for the detection of chlamydia. Preliminary evaluation of an lyta pcr assay for detection. The goals of this study were to assess the macrolide sensitivity of m. Evaluation of five realtime pcr assays for detection of. Currently, the diagnosis of pneumococcal bacteremia relies on the isolation and identification of the bacteria from blood cultures. Whole organism spiking studies near the limit of detection of the. Development of a rapid recombinase polymerase amplification. Multiplex pcr for detection of seven virulence factors and k1.
Despite differences of crossing threshold values of up to 4, assays were able to detect at least 20 cfu5. Future work will focus on the development of an internal amplification control. Streptococcus pneumoniae, or pneumococcus, is a grampositive, alphahemolytic under aerobic conditions or betahemolytic under anaerobic conditions, facultative anaerobic member of the genus streptococcus. We have shown this assay to be as specific and as, sensitive as pcr when applied to the detection of s. Expression of streptococcus pneumoniae virulencerelated genes. Detection of pneumococcus by pcr university of oulu. Detection and prediction of streptococcus pneumoniae.
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. 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. Cap capspn was performed by urine antigen detection. For instance, the xisco gene was recently described as a biomarker for pcrbased detection of s. Evaluation of lightmix mycoplasma macrolide assay for. Survey and rapid detection of klebsiella pneumoniae in. In addition to its role as a leading cause of communityacquired pneumonia, s. Whole organism spiking studies near the limit of detection of the assay were also performed using the following specimens. Pdf study design detection and serotyping of streptococcus pneumoniae are important to assess the impact of pneumococcal vaccines.
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