Multiplex Real-time PCR Research Articles

Metagenomics characterization of respiratory viral RNA pathogens in children under five years with severe acute respiratory infection in the Free State, South Africa.

Prompt detection of viral respiratory pathogens is crucial in managing respiratory infection including severe acute respiratory infection (SARI). Metagenomics next-generation sequencing (mNGS) and bioinformatics analyses remain reliable strategies for diagnostic and surveillance purposes. This study evaluated the diagnostic utility of mNGS using multiple analysis tools compared with multiplex real-time PCR for the detection of viral respiratory pathogens in children under 5 years with SARI. Nasopharyngeal swabs collected in viral transport media from 84 children admitted with SARI as per the World Health Organization definition between December 2020 and August 2021 in the Free State Province, South Africa, were used in this study. The obtained specimens were subjected to mNGS using the Illumina MiSeq system, and bioinformatics analysis was performed using three web-based analysis tools; Genome Detective, One Codex and Twist Respiratory Viral Research Panel. With average reads of 211323, mNGS detected viral pathogens in 82 (97.6%) of the 84 patients. Viral aetiologies were established in nine previously undetected/missed cases with an additional bacterial aetiology (Neisseria meningitidis) detected in one patient. Furthermore, mNGS enabled the much needed viral genotypic and subtype differentiation and provided significant information on bacterial co-infection despite enrichment for RNA viruses. Sequences of nonhuman viruses, bacteriophages, and endogenous retrovirus K113 (constituting the respiratory virome) were also uncovered. Notably, mNGS had lower detectability rate for severe acute respiratory syndrome coronavirus 2 (missing 18/32 cases). This study suggests that mNGS, combined with multiple/improved bioinformatics tools, is practically feasible for increased viral and bacterial pathogen detection in SARI, especially in cases where no aetiological agent could be identified by available traditional methods.

Surveillance of Viral Respiratory Infections in the Neonatal Intensive Care Unit-Evolution in the Last 5 Years.

Viral respiratory infections (VRIs) in very low birthweight infants can be associated with high rates of morbidity. The COVID-19 pandemic has exerted a strong impact on viral circulation. The purpose of this study is to report on VRIs during NICU admission in infants below 32 weeks' gestation and compare data collected between the pre-and post-COVID-19 pandemic periods. A prospective surveillance study was conducted at a tertiary NICU between April 2016 and June 2022. The COVID-19 post-pandemic period was established as being from March 2020 onwards. Respiratory virus detection was performed by real-time multiplex PCR assays in nasopharyngeal aspirates (NPAs). A total of 366 infants were enrolled. There were no statistical differences between periods regarding infants' birth weight, gestational age, gender distribution, or rates of bronchopulmonary dysplasia. Among the 1589 NPA collected during the pre-COVID-19 period, 8.9% were positive, and among the 1147 NPA collected during the post-pandemic period, only 3% were positive (p < 0.005). The type of viruses detected did not differ according to the study period (pre-COVID19 vs. post-COVID-19): rhinovirus (49.5% vs. 37.5%), adenovirus (22.6% vs. 25%), and human coronavirus (12.9% vs. 16.7%). SARS-CoV-2 was only detected in one patient. In conclusion, the viral profile causing VRI during the pre-COVID-19 and post-COVID-19 era was similar. However, the total number of VRI dropped significantly, most probably due to the global increase in infection prevention measures.

Diagnostic Performance and Tolerability of Saliva and Nasopharyngeal Swab Specimens in the Detection of SARS-CoV-2 by RT-PCR.

Saliva is a promising alternative for a nasopharyngeal swab (NPS) in specimen collection to detect SARS-CoV-2. We compared the diagnostic performance and tolerability of saliva collection versus NPS in a clinical setting. Paired NPS and saliva specimens were collected sequentially from participants (n = 250) at the Turku University Hospital drive-in coronavirus testing station in the spring of 2022, with Omicron BA.2 as the dominant SARS-CoV-2 variant. Discomfort and preference for the sampling method were assessed. The specimens were analyzed for SARS-CoV-2 using real-time multiplex reverse transcriptase PCR (RT-PCR) with a laboratory-developed test (LDT) and two commercial kits (PerkinElmer SARS-CoV-2 and PerkinElmer SARS-CoV-2 Plus) for several target genes. Among the 250 participants, 246 had respiratory symptoms. With LDT, SARS-CoV-2 was detected in 135 and 134 participants from NPS and saliva, respectively. Of the 250 specimens, 11 gave a discordant outcome, resulting in excellent agreement between the specimen types (Cohen's kappa coefficient of 0.911; P = 0.763). The cycle threshold (CT) values of LDT and commercial kit target genes were significantly lower from NPS than from saliva. A total of 172 (69%) participants assessed saliva sampling as more tolerable than NPS (P < 0.0001). Our findings present saliva as an applicable alternative for SARS-CoV-2 diagnostics. However, the lower CT values obtained from NPS indicate that NPS may be a slightly more sensitive specimen type. Participants preferred saliva sampling, although delivering an adequate volume of saliva was challenging for some participants. IMPORTANCE The extensive testing of SARS-CoV-2 is vital in controlling the spread of COVID-19. The reference standard for specimen collection is a nasopharyngeal swab (NPS). However, the discomfort of NPS sampling, the risk of nosocomial infections, and global material shortages have accelerated the development of alternative testing methods. Our study demonstrates that patients tolerate saliva sampling better than NPS. Of importance, although the RT-PCR qualitative test results seem to correspond between NPS and saliva, we show significantly lower CT values for NPS, compared to saliva, thus contradicting the suggested superiority of the saliva specimen over NPS in the detection of the Omicron variants of SARS-CoV-2. Future research is still required to enable individual planning for specimen collection and to determine the effects of different SARS-CoV-2 variants on the sensitivity of the saliva matrix.

Local Immune Biomarker Expression Depending on the Uterine Microbiota in Patients with Idiopathic Infertility.

The endometrium has traditionally been considered sterile. Nowadays, active studies are performed on the female upper genital tract microbiota. Bacteria and/or viruses colonizing the endometrium are known to alter its functional properties, including receptivity and embryo implantation. Uterine cavity inflammation caused by microorganisms leads to disrupted cytokine expression, which, in turn, is mandatory for the successful implantation of the embryo. The present study assessed the vaginal and endometrial microbiota composition and its relation to the levels of cytokines produced by the endometrium in reproductive-aged women complaining of secondary infertility of unknown origin. The multiplex real-time PCR assay was applied for vaginal and endometrial microbiota analysis. The quantitative measurement of endometrial α-defensin (DEFa1), transforming growth factor (TGFβ1), and basic fibroblast growth factor (bFGF2) was carried out using the ELISA (Cloud-Clone Corporation (Katy, TX, USA; manufactured in Wuhan, China). A reliable decline in endometrial TGFβ1 and bFGF2 and an increase in DEFa1 were demonstrated in women with idiopathic infertility when compared to fertile patients. However, TGFβ1, bFGF2, and DEFa1 expression correlated reliably only with the presence of Peptostreptococcus spp. and HPV in the uterine cavity. The obtained results highlight the importance of local immune biomarker determination in the assessment of certain bacteria and viruses' significance as causative agents of infertility.

Identification of priority pathogens for aetiological diagnosis in adults with community-acquired pneumonia in China: a multicentre prospective study

BackgroundCommunity-acquired pneumonia (CAP) is a major public health challenge worldwide. However, the aetiological and disease severity-related pathogens associated with CAP in adults in China are not well established based on the detection of both viral and bacterial agents.MethodsA multicentre, prospective study was conducted involving 10 hospitals located in nine geographical regions in China from 2014 to 2019. Sputum or bronchoalveolar lavage fluid (BALF) samples were collected from each recruited CAP patient. Multiplex real-time PCR and bacteria culture methods were used to detect respiratory pathogens. The association between detected pathogens and CAP severity was evaluated.ResultsAmong the 3,403 recruited eligible patients, 462 (13.58%) had severe CAP, and the in-hospital mortality rate was 1.94% (66/3,403). At least one pathogen was detected in 2,054 (60.36%) patients, with two or more pathogens were co-detected in 725 patients. The ten major pathogens detected were Mycoplasma pneumoniae (11.05%), Haemophilus influenzae (10.67%), Klebsiella pneumoniae (10.43%), influenza A virus (9.49%), human rhinovirus (9.02%), Streptococcus pneumoniae (7.43%), Staphylococcus aureus (4.50%), adenovirus (2.94%), respiratory syncytial viruses (2.35%), and Legionella pneumophila (1.03%), which accounted for 76.06–92.52% of all positive detection results across sampling sites. Klebsiella pneumoniae (p < 0.001) and influenza viruses (p = 0.005) were more frequently detected in older patients, whereas Mycoplasma pneumoniae was more frequently detected in younger patients (p < 0.001). Infections with Klebsiella pneumoniae, Staphylococcus aureus, influenza viruses and respiratory syncytial viruses were risk factors for severe CAP.ConclusionsThe major respiratory pathogens causing CAP in adults in China were different from those in USA and European countries, which were consistent across different geographical regions over study years. Given the detection rate of pathogens and their association with severe CAP, we propose to include the ten major pathogens as priorities for clinical pathogen screening in China.

Multiplex RT Real-Time PCR Based on Target Failure to Detect and Identify Different Variants of SARS-CoV-2: A Feasible Method That Can Be Applied in Clinical Laboratories.

Shortly after its emergence, Omicron and its sub-variants have quickly replaced the Delta variant during the current COVID-19 outbreaks in Vietnam and around the world. To enable the rapid and timely detection of existing and future variants for epidemiological surveillance and diagnostic applications, a robust, economical real-time PCR method that can specifically and sensitively detect and identify multiple different circulating variants is needed. The principle of target- failure (TF) real-time PCR is simple. If a target contains a deletion mutation, then there is a mismatch with the primer or probe, and the real-time PCR will fail to amplify the target. In this study, we designed and evaluated a novel multiplex RT real-time PCR (MPL RT-rPCR) based on the principle of target failure to detect and identify different variants of SARS-CoV-2 directly from the nasopharyngeal swabs collected from COVID-19 suspected cases. The primers and probes were designed based on the specific deletion mutations of current circulating variants. To evaluate the results from the MPL RT-rPCR, this study also designed nine pairs of primers for amplifying and sequencing of nine fragments from the S gene containing mutations of known variants. We demonstrated that (i) our MPL RT-rPCR was able to accurately detect multiple variants that existed in a single sample; (ii) the limit of detection of the MPL RT-rPCR in the detection of the variants ranged from 1 to 10 copies for Omicron BA.2 and BA.5, and from 10 to 100 copies for Delta, Omicron BA.1, recombination of BA.1 and BA.2, and BA.4; (iii) between January and September 2022, Omicron BA.1 emerged and co-existed with the Delta variant during the early period, both of which were rapidly replaced by Omicron BA.2, and this was followed by Omicron BA.5 as the dominant variant toward the later period. Our results showed that SARS-CoV-2 variants rapidly evolved within a short period of time, proving the importance of a robust, economical, and easy-to-access method not just for epidemiological surveillance but also for diagnoses around the world where SARS-CoV-2 variants remain the WHO's highest health concern. Our highly sensitive and specific MPL RT-rPCR is considered suitable for further implementation in many laboratories, especially in developing countries.

Evaluating the prognostic significance of p53 and TP53 mutations in HPV-negative hypopharyngeal carcinoma patients: a 5-year follow-up retrospective study

PurposeTo evaluate prognostic significance of human papillomavirus (HPV) in hypopharyngeal squamous cell carcinoma patients, and to investigate the effect of p53 and TP53 mutations on the prognosis of patients.MethodsA total of 111 patients were enrolled in our retrospective study. HPV infection status was detected in formalin-fixed paraffin-embedded tissue by real-time multiplex PCR test. p53 expression was evaluate by immunohistochemical staining. TP53 exon mutations were analyzed by PCR amplification and Sanger sequencing. HPV infection status, p53 expression and TP53 mutation were compared with clinical outcome including overall survival and recurrence-free survival by Kaplan-Meier method and Log-rank test.ResultsOf the 111 investigated patients, 18 (16.22%) were positive for HPV infection. HPV(-) patients have a worse clinical outcome than HPV(+) patients. TP53 mutations have similar mutation rates in patients with and without HPV (55.56% vs. 41.94%). p53 and TP53 mutation were not associated with prognosis of patients in HPV(-) patients. TP53 disruptive mutations were found both in patients with or without HPV infection. Furthermore, TP53 non-disruptive mutation had a significantly better clinical outcome than those with disruptive mutation in HPV(-) patients.ConclusionOur results showed that HPV infection status is a strong prognostic indicator of survival. p53 and TP53 mutations do not appear to significantly impact survival in HPV(-) patients. TP53 disruptive mutation is associated with reduced survival in HPV(-)/TP53 mutation patients.

Prevalence of SARS-COV-2 and other respiratory pathogens among a Jordanian subpopulation during Delta-to-Omicron transition: Winter 2021/2022.

Acute respiratory tract infections (ARTIs) during the winter months are associated with higher morbidity and mortality compared to other seasons of the year, with children below five, elderly, and immunocompromised patients being the most susceptible. Influenza A and B viruses, rhinovirus, coronaviruses, respiratory syncytial virus, adenovirus, and parainfluenza viruses, are the most frequently identified causes of viral ARTIs. In addition, the emergence of SARS-CoV-2 in 2019 provided an additional viral cause of ARTIs. The aim of this study was to provide an overview of the epidemiological status of upper respiratory infections, their main causative agents, and reported clinical presentation in the winter months of 2021, during two important surges of COVID-19 in Jordan. Nasopharyngeal samples were collected from 339 symptomatic patients during the period from December 2021 to March 2022, followed by nucleic acid isolation using a Viral RNA/DNA extraction Kit. The causative virus species associated with the patient's respiratory symptoms was determined utilizing a multiplex real-time PCR targeting 21 viruses, 11 bacteria, and a single fungus. SARS-CoV-2 was identified in 39.2% of the patients (n = 133/339). A total of 15 different pathogens were also identified as co-infections among these 133 patients (n = 67/133). SARS-CoV-2-Bacterial coinfections (37.6%, n = 50/133) were the most frequent, with Bordetella species being the most common, followed by Staphylococcus aureus, and H.influenzae type B. Viral coinfection rate was 27.8% (n = 37/133), with Influenza B virus and Human bocavirus being the most common. In Conclusion, Both SARS-CoV-2, influenza B virus, and Bordetella accounted for the majority of infections in patients with URTI during the winter months of 2021-2022. Interestingly, more than 50% of the patients with symptoms of URTIs were confirmed to have a coinfection with two or more respiratory pathogens, with SARS-CoV-2 and Bordetella coinfection being most predominant.

Development of TaqMan Real-Time PCR Protocols for Simultaneous Detection and Quantification of the Bacterial Pathogen Ralstonia solanacearum and Their Specific Lytic Bacteriophages.

Ralstonia solanacearum is the causal agent of bacterial wilt, one of the most destructive diseases of solanaceous plants, affecting staple crops worldwide. The bacterium survives in water, soil, and other reservoirs, and is difficult to control. In this sense, the use of three specific lytic R. solanacearum bacteriophages was recently patented for bacterial wilt biocontrol in environmental water and in plants. To optimize their applications, the phages and the bacterium need to be accurately monitored and quantified, which is laborious and time-consuming with biological methods. In this work, primers and TaqMan probes were designed, and duplex and multiplex real-time quantitative PCR (qPCR) protocols were developed and optimized for the simultaneous quantification of R. solanacearum and their phages. The quantification range was established from 108 to 10 PFU/mL for the phages and from 108 to 102 CFU/mL for R. solanacearum. Additionally, the multiplex qPCR protocol was validated for the detection and quantification of the phages with a limit ranging from 102 targets/mL in water and plant extracts to 103 targets/g in soil, and the target bacterium with a limit ranging from 103 targets/mL in water and plant extracts to 104 targets/g in soil, using direct methods of sample preparation.

Panel of primers for evaluation of antibiotic resistance genes with real time detection of results

Introduction. The problem of the spread of antibiotic resistance among pathogens of nosocomial infections is becoming increasingly important. In order to improve microbiological monitoring, it is advisable to use methods that allow you to quickly determine the largest number of antibiotic resistance determinants. In this regard, it seems relevant to develop PCR test systems for detecting antibiotic resistance genes, in particular, for screening hospitalized patients and identifying cases of nosocomial infection.Purpose. Develop a panel of primers for detection of antibiotic resistance genes using PCR with real-time visualization of results. Materials and methods. For the design of specific primers and probes, reference sequences from the databases of the National Center for Biotechnology Information (NCBI) were used. Primer selection and specificity assessment were performed using the NCBI PrimerBlast and Primer3 programs. Primers for PCR amplification of a number of antibiotic resistance genes were tested on strains of Klebsiella pneumoniae. These strains were isolated from the clinical material of intensive care patients with COVID-19 and whole genome sequencing of the strains was carried out with a detailed assessment of the resistome, virulome. The results of whole genome sequencing and multiplex real-time PCR were compared.Results. We have developed a set of primers for the detection of antibiotic resistance genes, including carbapenemase, using real-time multiplex PCR.Conclusion. The developed panel of primers can be used to screen Klebsiella pneumoniae isolates for the presence of resistance genes; further expansion of the spectrum of detected genes and testing of the panel on clinical material is required.

Prevalence of Chlamydia trachomatis, Neisseria gonorrhoeae, and Mycoplasma genitalium among Patients with Urogenital Symptoms in Istanbul

Chlamydia trachomatis, Neisseria gonorrhoeae, and Mycoplasma genitalium are the three most commonly reported sexually transmitted bacteria. The present study aimed to investigate the presence of C. trachomatis, N. gonorrhoeae, and M. genitalium in urogenital samples collected from 18-68-year-old Turkish patients who were admitted to the hospital with various urogenital symptoms. A total of 360 patients with symptoms of STD were included in the study. Following DNA extraction by QIAamp Mini Kit, the presence of C. trachomatis, N. gonorrhoeae, and M. genitalium were investigated using multiplex real-time PCR. Causative organisms were identified in 68 (18.9%) of 360 patients. C. trachomatis, N. gonorrhoeae, and M. genitalium were detected in 40 (11.1%), 14 (3.9%), and 28 (7.8%) of the patients, respectively. Patients 21-30 years of age represented more than one-third (37.8%) of positive patients. Of all patients, dual infections of C. trachomatis-M. genitalium, N. gonorrhoeae-C. trachomatis, N. gonorrhoeae-M. genitalium, and triple infection of C. trachomatis-N. gonorrhoeae-M. genitalium were determined in 1.6% (6/360), 1.3% (5/360), 0.2% (1/360), and 0.2% (1/360) of the patients, respectively. In CT-, NG-, and MG-positive patients, different STI agents were also found such as HIV, HBV, HPV, HSV2, T. pallidum, and T. vaginalis. In conclusion, among C. trachomatis, N. gonorrhoeae, and M. genitalium, CT was the most frequently detected bacterial cause of STDs in our hospital at Istanbul. Co-infections, which comprise more than one-fifth of the cases, should not be underestimated. Regular screening and following up of STD agents using multiplex real-time PCR-based diagnostic methods enabling the immediate detection of co-infections are essential for the treatment and primary prevention of STDs.

Development of multiplex real-time PCR for detection of clarithromycin resistance genes for the Mycobacterium abscessus group.

Introduction. The M. abscessus molecular identification and its drug-resistance profile are important to choose the correct therapy.Aim. This work developed a multiplex real-time PCR (mqPCR) for detection of clarithromycin resistance genes for the Mycobacterium abscessus group.Methodology. Isolates received by Adolfo Lutz Institute from 2010 to 2012, identified by PCR restriction enzyme analysis of a fragment of the hsp65 gene (PRA-hsp65) as M. abscessus type 1 (n=135) and 2 (n=71) were used. Drug susceptibility test (DST) for CLA were performed with reading on days 3 and 14. Subespecies identification by hsp65 and rpoB genes sequencing and erm(41) and rrl genes for mutation detection and primer design were performed. erm(41) gene deletion was detected by conventional PCR. Primers and probes were designed for five detections: erm(41) gene full size and with deletion; erm(41) gene T28 and C28; rrl gene A2058.Results. In total, 191/206 (92.7 %) isolates were concordant by all methods and 13/206 (6.3 %) were concordant only between molecular methods. Two isolates (1.0 %) were discordant by mqPCR compared to rrl gene sequencing. The mqPCR obtained 204/206 (99.0 %) isolates in agreement with the gold standard, with sensitivity and specificity of 98 and 100 %, respectively, considering the gold standard method and 92 and 93 % regarding DST.Conclusion. The mqPCR developed by us proved to be an easy-to-apply tool, minimizing time, errors and contamination.

Quantitative Detection of Late Blowing Agents C. tyrobutyricum, C. butyricum, and C. sporogenes in Traditional Turkish Cheese by Multiplex Real-Time PCR

Quantitative Detection of Late Blowing Agents C. tyrobutyricum, C. butyricum, and C. sporogenes in Traditional Turkish Cheese by Multiplex Real-Time PCR

DNA Methylation of JAK2 Gene in Intestinal Biopsy and Peripheral Blood Samples of Patients with Ulcerative Colitis

Background: Comprised of two main subtypes (Ulcerative Colitis (UC) and Crohn), inflammatory bowel disease is caused by an interaction between genetic and environmental factors. As of the important role of innate immunity and JAK/STAT signaling pathway, the current study was designed to investigate the methylation status JAK2 gene in blood and tissue samples of patients with UC.&#x0D; Methods: Genomic DNA was extracted from blood and intestinal biopsy samples of 28 UC patients and 28 controls. After bisulfite DNA conversion, real-time quantitative multiplex methylation specific PCR (QM-MSP) method was applied in order to assess JAK2 promotor methylation status.&#x0D; Results: The JAK2 promotor in the intestinal biopsy samples was significantly hypermethylated in UC as the mean of unmethylated DNA was 1.255±1.865 in the patients group, while it was 1.292±4.726 in control group.&#x0D; Conclusion: Hypermathylation of JAK2 gene may play a part in pathophysiology of UC which could result in gene silencing.

Development and validation of a multiplex real-time PCR assay for accurate authentication of common buckwheat (Fagopyrum esculentum) and tartary buckwheat (Fagopyrum tataricum) in food

The increased demand for accurate information on ingredients listed on food product labels requires identifying a plant's species of origin. To efficiently and rapidly discriminate between common buckwheat (Fagopyrum esculentum) and tartary buckwheat (Fagopyrum tataricum), here we developed a multiplex real-time PCR assay. This assay included target species-specific and shared primer/probe sets representing the two buckwheat species. Multiplex real-time PCR analyses specifically amplified the target species without detectable cross-reactivity with nontarget plant samples. The detection limit was 1% for common buckwheat in tartary buckwheat and 2% for tartary buckwheat in common buckwheat. The method was verified through application to processed products and was confirmed through comparison with the food label information. The species identified were identical to those declared on the product label. Therefore, this multiplex real-time PCR assay will serve as a sensitive and specific diagnostic tool for detection of buckwheat species in foods.

DNA concentration by solid phase reversible immobilization improves its yield and purity, and detection time of E. coli O157:H7 in foods by high resolution melt curve qPCR

For molecular detection of foodborne pathogens to be effective, an efficient technique for bacterial DNA concentration is required. Solid phase reversible immobilization (SPRI) paramagnetic beads can concentrate DNA from foods containing low amounts of bacteria because of their highly specific DNA-binding capacity. This study aimed to develop an upstream bacterial DNA concentration step using SPRI beads and compare its efficacy with immunomagnetic separation (IMS) for the detection of Shiga toxin producing E. coli (STEC) O157:H7 in food samples via a multiplex PCR assay. Food samples were inoculated with STEC O157:H7 at concentrations of 10, 100 and 1000 CFU/mL. The two different concentration methods were compared based on 1) DNA yield 2) DNA purity and 3) detection limit and time of the target pathogen using a high-resolution melt cure multiplex real-time PCR assay for the detection of the major virulence genes, eaeA, stx1 and stx2, in E. coli O157:H7. DNA samples isolated and concentrated by SPRI showed better purity and yield compared to IMS cell concentration. A detection limit of 10 CFU was attained for the majority of inoculated food samples utilizing DNA samples concentrated using SPRI in just 4 h as opposed to 6 or more hours using IMS. SPRI paramagnetic beads in combination with multiplex HRM real-time PCR can be used to rapidly concentrate and detect low concentrations of E. coli O157:H7 in food samples that cannot be detected by conventional culture techniques.

O-174 Serogroups of Dichelobacter nodosus detected in footrot lesions in sheep using a new multiplex real-time PCR

O-174 Serogroups of Dichelobacter nodosus detected in footrot lesions in sheep using a new multiplex real-time PCR

Correction to: Differentiation of the Flavescence dorée phytoplasma genetic clusters by multiplex real-time PCR assay targeting the map gene

Correction to: Differentiation of the Flavescence dorée phytoplasma genetic clusters by multiplex real-time PCR assay targeting the map gene

Multiplex Real-time PCR Assay for the Detection of all Chlamydia Species and Simultaneous Differentiation of C. psittaci and C. pneumoniae in Human Clinical Specimens.

We developed and assessed the performance of a new multiplex real-time PCR assay for the detection of all Chlamydia species and simultaneous differentiation of Chlamydia psittaci and Chlamydia pneumoniae-two important human respiratory pathogens-in human clinical specimens. Next-generation sequencing was used to identify unique targets to design real-time PCR assays targeting all Chlamydia species, C. psittaci, and C. pneumoniae. To validate the assay, we used a panel of 49 culture isolates comprising seven C. psittaci genotypes, eight C. pneumoniae isolates, seven other Chlamydia species, and 22 near-neighbor bacterial and viral isolates, along with 22 specimens from external quality assessment (EQA) panels and 34 nasopharyngeal and oropharyngeal swabs and cerebrospinal fluid, stool, and sputum specimens previously identified as positive or negative for C. psittaci or C. pneumoniae. The assays were 100% specific, with limits of detection of 7.64- 9.02 fg/μL. The assay results matched with historical assay results for all specimens, except for one owing to the increased sensitivity of the new C. psittaci assay; the results of the EQA specimens were 100% accurate. This assay may improve the timely and accurate clinical diagnosis of Chlamydia infections and provide a greater understanding of the burden of disease caused by these agents.

Development of a new multiplex real-time PCR assay for rapid screening of hospital-acquired infection agents

AimsA new multiplex real-time PCR (qPCR) assay was developed to detect antibiotic-resistant hospital-acquired infectious agents in nasal and rectal swab samples in 1.5 h without the need for nucleic acid extraction. MethodsSpiked negative clinical specimens were used for the analytical performance evaluation. Double-blind samples were collected from 1788 patients to assess the relative clinical performance of the qPCR assay to the conventional culture-based methods. Bio-Speedy® Fast Lysis Buffer (FLB) and 2× qPCR-Mix for hydrolysis probes (Bioeksen R&D Technologies, Istanbul, Turkey) and LightCycler® 96 Instrument (Roche Inc., Branchburg, NJ, USA) were used for all molecular analyses. The samples were transferred into 400 L FLB, homogenized and immediately used in qPCRs. The target DNA regions are vanA and vanB genes for vancomycin-resistant Enterococcus (VRE); blaKPC, blaNDM, blaVIM, blaIMP, blaOXA-23, blaOXA-48, blaOXA-58 genes for carbapenem-resistant Enterobacteriaceae (CRE); and mecA, mecC and spa for methicillin-resistant Staphylococcus aureus (MRSA). ResultsNo qPCR tests produced positive results for the samples spiked with the potential cross-reacting organisms. The limit of detection (LOD) of the assay for all targets was 100 colony-forming unit (cfu)/swab-sample. Results of the repeatability studies in two different centers were in 96%–100% (69/72–72/72) agreement. The relative specificity and sensitivity of the qPCR assay were respectively 96.8% and 98.8% for VRE; 94.9% and 95.1% for CRE; 99.9% and 97.1% for MRSA. ConclusionsThe developed qPCR assay can screen antibiotic-resistant hospital-acquired infectious agents in infected/colonized patients with an equal clinical performance to the culture-based methods.