Multiplex Real-time Polymerase Chain Reaction Assay Research Articles

Utility of multiplex real-time polymerase chain reaction for the detection of bacteria from sputum samples of community acquired pneumonia patients: A study from Dhaka, Bangladesh

Background: This study was carried out to evaluate the utility of multiplex real-time polymerase chain reaction (PCR) to identify the common bacterial agents of community acquired pneumonia (CAP). Methods: Sputum and blood samples were collected from 80 clinically suspected CAP patients in three tertiary-level hospitals in Dhaka city. Multiplex real-time PCR assay was carried out to simultaneously detect five common bacterial agents of CAP; Streptococcus pneumoniae, Haemophilus influenzae, Mycoplasma pneumoniae, Chlamydophila pneumoniae, and Legionella pneumophila. Routine microbiological methods and serology were carried out. The results of PCR were compared with culture, Gram stain and serology. Results: Among the 80 patients, sputum samples of 35 (43.7%) patients were positive by PCR, of which the most commonly detected bacteria were S. pneumoniae (25/35, 71.4%), followed by H. influenzae (9/35, 25.7%) and L. pneumophila (1/35, 2.9%). All 80 sputum samples were negative for both M. pneumoniae and C. pneumoniae by PCR. Out of the 26 culture positive sputum samples, 8 (30.7%) were positive for S. pneumoniae and 1 (3.8%) was positive for H. influenzae. Among the 52 Gram stain valid sputum samples, 24 (46.1%) were S. pneumoniae and 7 (13.5%) were H. influenzae. By serology, out of the 80 cases, M. pneumoniae was detected in 32 (40%) and C. pneumoniae in 24 (30%) of cases. Mixed infections comprised of 38.8% (31/80) cases. Conclusion: Multiplex real-time PCR is useful for the rapid and simultaneous detection of bacterial pathogens of CAP in sputum and can help support traditional laboratory methods for the accurate diagnosis of CAP patients.

Evaluation of the Performance of a Multiplex Real-Time PCR Assay for the Identification of Aspergillus, Cryptococcus neoformans, and Pneumocystis jirovecii Simultaneously from Sputum in Multicenter.

PurposeTo evaluate the performance of a multiplex real-time polymerase chain reaction (PCR) assay for the simultaneous identification of Aspergillus, Cryptococcus neoformans, and Pneumocystis jirovecii from sputum.Patients and MethodsSputum samples (n=537) from patients with suspected invasive fungal infection (IFI) were collected from four centers; they were tested by both multiplex real-time PCR assay and DNA sequencing. DNA sequencing was considered as the reference method, and the performance of the multiplex real-time assay was evaluated by determining the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV). The interference experiment, repeatability, reproducibility, and stability of the multiplex real-time PCR assay were also evaluated.ResultsThe detection performance of the multiplex real-time assay, compared with that of DNA sequencing, for the three pathogens was as follows: sensitivity, specificity, PPV, and NPV for Aspergillus, Cryptococcus neoformans, and Pneumocystis jirovecii were 99.40%, 98.64%, 97.09%, and 99.73%; 100%, 99.59%, 96.36%, and 100.00%; and 99.28%, 98.50%, 95.80%, and 99.75%, respectively. The consistency of the two methods was almost perfect: the kappa value was between 0.97 and 0.98. The minimum detection limit of the multiplex real-time assay for each of the three pathogens was 1250 copies/mL. Interference experiment showed that blood and normally used antifungal drugs had no effect on the results. No cross-reactivity was detected for any bacteria or fungi. In 40 patients, mixed infections by Aspergillus and/or Cryptococcus neoformans and/or Pneumocystis jirovecii were detected by the multiplex real-time assay. Among these patients, those with acquired immune deficiency syndrome (AIDS) ranked first, with Aspergillus and Pneumocystis mixed infection accounting for 75%.ConclusionThe multiplex real-time PCR assay is fast, sensitive, and specific and has good clinical application prospects.

Evaluation of Newborns with Non-COVID-19 Pneumonia Hospitalized in the Neonatal Intensive Care Unit during the COVID-19 Pandemic, Turkey, Izmir 2020–2021

Abstract Objective In this study, we aimed to compare the clinical, laboratory, and radiological findings of noncoronavirus disease 2019 (COVID-19) viral agents in newborn infants hospitalized for lower respiratory tract infection during the COVID-19 pandemic. Methods This prospective cross-sectional study conducted between 11 March 2020 and 31 July 2021 included neonates with lower respiratory tract infections admitted to the neonatal intensive care unit of the Dr. Behcet Uz Children's Hospital. Nasopharyngeal swab samples were taken from all hospitalized patients for multiplex respiratory polymerase chain reaction (PCR) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) PCR. The detection of respiratory viral pathogens was performed by multiplex real-time PCR assay (Bosphore Respiratory Pathogens Panel Kit V4, Anatolia Geneworks, Turkey). Infants with SARS-CoV-2 PCR positivity were excluded from the study. Patients' data were obtained from the electronic medical registry system. The non-COVID-19 viruses of the cases were analyzed according to seasonal variation (in/off-season). The pulmonary findings of the cases were classified as normal, infiltration, air bronchogram, and reticulogranular appearance at the time of admission. Results A total of 80 infants were included during the study period. A multiplex PCR test was performed to identify viral agents affecting the lower respiratory tract of infants; it was determined that 31% (25 out of 80) were respiratory syncytial virus (RSV), 41% (33 out of 80) were rhinovirus (Rhino), and the remaining portion (28%, 22 out of 80) were other viral agents (enterovirus, bocavirus, adenovirus, influenza, and parainfluenza). Compared with Rhino and other viral agents, RSV was detected most frequently in seasonal hospitalizations (p < 0.05). When chest radiography and laboratory findings were evaluated, the rate of “infiltration” /“lymphopenia” was significantly associated with infants with RSV lower respiratory tract infections (p < 0.05). Conclusion During the pandemic period, RSV affected the prognosis in intensive care unit admissions due to lower respiratory tract infection in newborns.

A multiplex real-time PCR assay for differential identification of avian Chlamydia

ABSTRACT Avian chlamydiosis is an acute or chronic disease of birds after infection by Chlamydia. Although Chlamydia psittaci is the primary agent of the disease, two additional species, Chlamydia avium and Chlamydia gallinacea, have also been recognized as potential disease agents. Therefore, the diagnosis of avian chlamydiosis requires differential identification of these avian Chlamydia species. The objective of the present study was to develop a multiplex real-time polymerase chain reaction (PCR) assay to rapidly differentiate between these three species of avian Chlamydia (C. psittaci, C. avium, and C. gallinacea) as well as to detect the genus Chlamydia. Specific genetic regions of the three species were identified by comparative analysis of their genome sequences. Also, the genus-specific region was selected based on 23S rRNA sequences. PCR primers and probes specific to the genus and each species were designed and integrated in the multiplex real-time PCR assay. The assay was highly efficient (94.8–100.7%). It could detect fewer than 10 copies of each target sequence of the genus and each species. Twenty-five Chlamydia control and field DNA samples were differentially identified while 20 other bacterial strains comprising 10 bacterial genera were negative in the assay. This assay allows rapid, sensitive, and specific detection of the genus and the three species of avian Chlamydia in a single protocol that is suitable for routine diagnostic purposes in avian diagnostic laboratories.

Development of a One-Step Multiplex Real-Time PCR Assay for the Detection of Viral Pathogens Associated With the Bovine Respiratory Disease Complex.

Bovine respiratory disease complex (BRDC) occurs widely in cattle farms. The main viral pathogens include bovine viral diarrhea virus (BVDV), Bovine herpesvirus 1 (BoHV-1), bovine parainfluenza virus type 3 (BPIV3), and bovine respiratory syncytial virus (BRSV), and the newly emerged influenza D virus (IDV). In this study, we have developed a one-step multiplex real-time Polymerase Chain Reaction (PCR) capable of simultaneously detecting these five viral pathogens causing BRDC. The established assay could specifically detect targeted viruses without cross-reaction with others. The detection limit was ~10 copies/reaction for single real-time PCR and 100 copies/ reaction for multiplex real-time PCR assay. A total of 213 nasal samples from cattle with signs of respiratory tract disease were then collected for performance evaluation of the established platform, proving that the method has good specificity and sensitivity. The surveillance data suggested that BVDV and BoHV-1 infections are the dominant cause of BRDC in the herd, whereas the detection rate of IDV, BIPV3, and BRSV is relatively lower. In summary, the established assay provides technical support for rapid clinical detection of BRDC associated viral pathogens to guide the formulation of BRDC prevention and control measures.

Evaluation of Sensitivity and Cost-Effectiveness of Molecular Methods for the Co-detection of Waterborne Pathogens in India.

Waterborne microbial diseases are regarded as a major public health concern, particularly in nations with poor sanitation, a lack of social awareness, and problems linked with low socioeconomic status. Waterborne pathogen identification using traditional culture methods is time-consuming and labor-intensive. As a result, there is a growing demand for quick pathogen detection technologies. High sensitivity, specificity, and rapidity are all advantages of using molecular techniques like polymerase chain reaction (PCR) in such instances. In this study, we designed multiplex PCR and quantitative real-time PCR (qPCR) assays for the co-detection and enumeration of waterborne pathogens such as Aeromonas hydrophila, Pseudomonas aeruginosa, Salmonella enterica, Yersinia enterocolitica, Escherichia coli, Vibrio cholerae, and Shigella spp. Specific primers were selected against the virulence and species-specific genes of the seven target pathogens. For all seven target organisms, the detection limits for conventional culture methods were in the range of 103-104 cells/ml. While employing multiplex PCR method in this study, Pseudomonas aeruginosa and Shigella spp. have a detection sensitivity of 101 cells/ml, Vibrio cholerae and Aeromonas hydrophila have a detection sensitivity of 102 cells/ml, whereas Salmonella enterica, E. coli, and Yersinia enterocolitica have a detection sensitivity of only 103 cells/ml. According to our cost-benefit analysis, these molecular technologies are less expensive, with unit analysis costs of ₹52 and ₹173 for qPCR and multiplex PCR, respectively. Furthermore, all of the target genes had a detection limit of 1 cell/ml in qPCR. Because of their speed, sensitivity, specificity, and cost-effectiveness, these multiplex and qPCR assays could be employed for successful co-detection of aquatic pathogens.

The identification and semi-quantitative assessment of gastrointestinal nematodes in faecal samples using multiplex real-time PCR assays

BackgroundThe diagnosis of gastrointestinal nematode (GIN) infections in ruminants is routinely based on morphological/morphometric analysis of parasite specimens recovered by coprological methods, followed by larval culture (LC) techniques. Such an approach is laborious, time-consuming, requires a skilled expert, and moreover suffers from certain limitations. Molecular tools are able to overcome the majority of these issues, providing accurate identification of nematode species and, therefore, may be valuable in sustainable parasite control strategies.MethodsTwo multiplex real-time polymerase chain reaction (PCR) assays for specific detection of five main and one invasive GIN species, including an internal amplification control to avoid false-negative results, were designed targeting SSU rRNA and COI genetic markers, as well as established ITS1/2 sequences. The assays were optimized for analysis of DNA extracted directly from sheep faeces and verified for Haemonchus contortus, Teladorsagia circumcincta, Trichostrongylus colubriformis, Nematodirus battus, Chabertia ovina, and Ashworthius sidemi. Semi-quantitative evaluation of infection intensity was enabled using a plasmid construct and a dilution series of sheep faeces with a known number of nematode eggs. Assays were tested on 44 individually collected faecal samples from three farms, and results were compared to those from faecal egg counts (FEC) using the concentration McMaster technique and LC.ResultsMultiplex real-time PCR assays showed great specificity to target nematodes. During the analysis of faecal samples, the assays proved to have higher sensitivity in strongylid-type egg detection over FEC by revealing three false-negative samples, while showing moderate agreement in evaluation of infection intensity. The multiplex assays further clarified GIN species identification compared to LC, which had confused determination of Teladorsagia spp. for Trichostrongylus spp.ConclusionsOur multiplex assays proved to be a rapid and accurate approach enabling simultaneous and reliable GIN species identification from faeces and semi-quantitative estimation of the number of eggs present. This approach increases diagnostic value and may add a high degree of precision to evaluation of anthelmintic efficacy, where it is important to identify species surviving after treatment.Graphical

Epidemiology and clinical features of intestinal protozoan infections detected by Real-time PCR in non-native children within an Italian tertiary care children's hospital: A cross-sectional study

BackgroundEnteric parasite infections are underestimated due to the limited sensitivity and specificity of microscopy, which remains the diagnostic gold standard in routine clinical practice. This could be a major problem in high-income countries, where the burden of parasitic diseases is low. In recent years, Multiplex Real-Time polymerase chain reaction (RT-PCR) based methods have been implemented. Therefore, the aim of this study was to evaluate the prevalence of four enteric protozoan species detected by RT-PCR in non-native children in Italy, and to describe their clinical characteristics. MethodsAdopted and immigrant children, evaluated for migration health assessment between 2017 and 2020 in a tertiary care children's hospital in Italy, were enrolled. Molecular analysis for Giardia lamblia, Dientamoeba fragilis, Blastocystis hominis, and Entamoeba histolytica, was conducted by in-house RT-PCR. ResultsOverall, 209 children were enrolled and 70% of them resulted positive by RT-PCR for at least one enteric parasite. B. hominis (47.8%) was the most commonly identified protozoa, followed by D. fragilis (44.5%). Co-infections with multiple pathogens were detected in 35.4% of the samples. Almost 80% of parasite-positive children were asymptomatic and the most common symptom was flatulence (60.7% of symptomatic children). Eosinophils were significantly increased in RT-PCR positive children compared to the negative ones and children with D. fragilis presented the highest eosinophils count. ConclusionsThe In-house Multiplex RT-PCR assay provides a valid molecular detection system for selected enteric parasites. This novel and accurate diagnostic method can help in increasing the detection rate of parasite infection, especially in high-risk population.

Standardization of an in-house multiplex real-time polymerase chain reaction for the simultaneous detection of Toxoplasma gondii, Rubella virus, cytomegalovirus, herpes simplex Virus 1 and 2, and Treponema pallidum infection among pregnant women.

An in-house multiplex real-time polymerase chain reaction (PCR) was developed in two cocktails for the identification of six Toxoplasma gondii, Rubella virus, cytomegalovirus, herpes simplex virus (1 and 2), and Treponema pallidum (syphilis) (TORCH-S) agents, which causes congenital infection among pregnant women. Standardization and validation of an in-house multiplex real-time PCR assay for the detection of TORCH-S infection. This study was conducted from February 2017 to February 2019. Primers specific for T. gondii, Rubella virus, cytomegalovirus, herpes simplex virus (1 and 2), and T. pallidum were designed using Primer3 software (https://bioinfo.ut.ee/primer3-0.4.0/). The primer sequences obtained were subjected to BLAST analysis using BLAST database. Synthetic DNA was obtained to use as positive control templates for all the six TORCH-S agents. The lower limit of the detection was performed using plasmid construct for each virus serially diluted from 10-1 to 10-9. An in-house multiplex real-time PCR was standardized and validated in two cocktails for TORCH-S agents, cocktail-1 (HSV1, rubella, and T. gondii), and cocktail-2 (HSV2, CMV, and T. pallidum). The lower limit of the detection for HSV1, rubella, and Toxoplasma were 60.7 copies/10 μl input, 76.4 copies/10 μl input, and 34.4 copies/10 μl input and for HSV2, CMV, and T. pallidum were 80.8 copies/10 μl input, 166 copies/10 μl input, and 43.7 copies/10 μl input, respectively. TORCH-S infection is one of the significant reasons for irregular pregnant outcomes. It is absolutely important to screen TORCH-S infection for women who had the histories of abnormal pregnancies to prevent birth defects and perinatal complications. This multiplex real-time PCR assay provides a rapid, sensitive, and specific technique to detect these six TORCH-S agents.

Detection of Giardia lamblia by Microscopic Examination, Rapid Chromatographic Immunoassay Test, and Molecular Technique.

BackgroundGiardia lamblia is a pathogenic intestinal flagellate transmitted by the ingestion of contaminated water or food with the cyst stage of the parasite. Giardiasis can cause severe acute diarrhea and malabsorption or may persist as a chronic infection. Effective treatment and control measures depend on proper laboratory diagnosis using diagnostic methods with high sensitivity and specificity.ObjectiveTo compare the sensitivity and specificity of direct smear, Ritchie sedimentation technique, two brands of rapid chromatographic immunoassay test, and real-time polymerase chain reaction (PCR) for the detection of G. lamblia in clinical human fecal samples.Materials and methodsUnpreserved 100 stool specimens were collected in clean plastic containers and labeled with the patient’s information and examined through light microscopy, immunochromatographic test (ICTs), and real-time PCR.ResultsOut of 100 fresh stool samples obtained from workers analyzed, real-time PCR targeting the SSU rRNA gene was able to detect Giardia deoxyribonucleic acid (DNA) in (42) samples followed by ImmunoCard STAT! (31) samples (Meridian Bioscience, Germany), direct smear (23) samples, CerTest (19) samples (Biotec, Zaragoza, Spain), and Ritchie technique (17) samples. Real-time PCR was the most sensitive for the diagnosis of G. lamblia in comparison to the other techniques.ConclusionsAll the techniques investigated were sensitive for the detection of G. lamblia in stool samples. Further studies are recommended using multiplex real-time PCR assay in order to increase the possibility of the presence or absence of the infection.

Rapid detection of OXA-23-like, OXA-24-like, and OXA-58-like carbapenemases from Acinetobacter species by real-time PCR

Carbapenemase-producing Acinetobacter species, especially A.baumannii, are frequently associated with treatment failures and hospital outbreaks; thus, rapid and reliable detection of specific resistance markers is paramount. The most common carbapenemases found in A.baumannii, namely OXA-23-like, OXA-24-like, and OXA-58-like, belong to the oxacillinase group (class D β-lactamases) which is notoriously difficult to identify phenotypically due to the lack of specific inhibitors. To design and validate a multiplex real-time polymerase chain reaction (PCR) assay to detect and differentiate the above three oxacillinases. All available variants of the above three oxacillinase subfamilies were downloaded (as of November 2019) from the Beta-Lactamase DataBase (http://bldb.eu/) aligned with Clustal Omega and oligonucleotides designed using Primer-BLAST. A multiplex real-time PCR assay that included an internal control to discount inhibition was optimized on the Rotor-Gene Q (Qiagen) using the Rotor-Gene Multiplex PCR Kit (Qiagen) and validated using a panel of 122 previously characterized strains carrying a wide range of β-lactamases, often in combination. The in-silico approach enabled the design of oligonucleotides in conserved regions of the OXA-24-like and OXA-58-like alignments. Among the 42 described OXA-23-like variants, a single nucleotide polymorphism (SNP) was present in one of the oligonucleotide binding sites of OXA-27, OXA-166, OXA-811, OXA-812, and OXA-816. The assay was 100% sensitive and highly specific. Inhibition was not observed. The assay is easy to perform with results available in about 70 min. It enables unequivocal detection and differentiation of OXA-23-like, OXA-24-like, and OXA-58-like carbapenemases even when more than one is simultaneously present.

Comparison of the performance of an amplicon sequencing assay based on Oxford Nanopore technology to real-time PCR assays for detecting bacterial biodefense pathogens

BackgroundThe state-of-the-art in nucleic acid based biodetection continues to be polymerase chain reaction (PCR), and many real-time PCR assays targeting biodefense pathogens for biosurveillance are in widespread use. These assays are predominantly singleplex; i.e. one assay tests for the presence of one target, found in a single organism, one sample at a time. Due to the intrinsic limitations of such tests, there exists a critical need for high-throughput multiplex assays to reduce the time and cost incurred when screening multiple targets, in multiple pathogens, and in multiple samples. Such assays allow users to make an actionable call while maximizing the utility of the small volumes of test samples. Unfortunately, current multiplex real-time PCR assays are limited in the number of targets that can be probed simultaneously due to the availability of fluorescence channels in real-time PCR instruments.ResultsTo address this gap, we developed a pipeline in which the amplicons produced by a 14-plex end-point PCR assay using spiked samples were subsequently sequenced using Nanopore technology. We used bar codes to sequence multiple samples simultaneously, leading to the generation and subsequent analysis of sequence data resulting from a short sequencing run time (< 10 min). We compared the limits of detection (LoD) of real-time PCR assays to Oxford Nanopore Technologies (ONT)-based amplicon sequencing and estimated the sample-to-answer time needed for this approach. Overall, LoDs determined from the first 10 min of sequencing data were at least one to two orders of magnitude lower than real-time PCR. Given enough time, the amplicon sequencing approach is approximately 100 times more sensitive than real-time PCR, with detection of amplicon specific reads even at the lowest tested spiking concentration (around 2.5–50 Colony Forming Units (CFU)/ml).ConclusionsBased on these results, we propose amplicon sequencing assay as a viable alternative to replace the current real-time PCR based singleplex assays for higher throughput biodefense applications. We note, however, that targeted amplicon specific reads were not detectable even at the highest tested spike concentrations (2.5 X 104–5.0 X105 CFU/ml) without an initial amplification step, indicating that PCR is still necessary when utilizing this protocol.

Giardiasis: An Overview.

Giardiasis is an important cause of waterborne and foodborne diarrhea, daycare center outbreaks, and traveler's diarrhea. The study aimed to provide an update on the evaluation, diagnosis, and treatment of giardiasis. A PubMed search was completed in Clinical Queries using the key terms "giardiasis", "Giardia lamblia", "Giardia duodenalis" and "Giardia intestinalis". The search strategy included metaanalyses, randomized controlled trials, clinical trials, observational studies, and reviews. The search was restricted to the English literature. Patents were searched using the key term "giardiasis" from www.freepatentsonline.com. Giardiasis is caused by the protozoan parasite Giardia lamblia. The parasite is transmitted by the fecal-oral route, frequently through ingestion of contaminated water and food or person-to person transmission. Risk factors for infection include children in day-care settings, child-care workers, institutionalized individuals, travelers in endemic areas, ingestion of contaminated or recreational water, immunodeficiency, cystic fibrosis, and oral-anal sex. Approximately 50 to 75% of infected children are asymptomatic. Other children present acute or chronic diarrhea. Direct fluorescent antibody tests that detect intact organisms, enzyme immunoassays that detect soluble antigens, and multiplex real-time polymerase chain reaction assays that detect specific genes of the parasite in stool samples have improved sensitivity and specificity compared with microscopic examination of stool specimens for the detection of Giardia trophozoites or cysts. Drugs used in the treatment of symptomatic giardiasis are reviewed in this study. Moreover, recent patents related to the management of giardiasis are also discussed. Metronidazole, tinidazole, and nitazoxanide are drugs of choice. Resistance to common antigiardial drugs has increased in recent years, therefore, the search for new molecular targets for antigiardial drugs is urgently needed. In general, treatment of asymptomatic carriers is not recommended. Purification of water supply is an important preventive measure.

A fast multiplex real-time PCR assay for simultaneous detection of pork, chicken, and beef in commercial processed meat products

As rapid and efficient methods for species identification were required, we developed a fast multiplex real-time polymerase chain reaction (PCR) assay based on TaqMan® probes to simultaneously detect pork, chicken, and beef in processed meat samples within 40 min. For multiplex PCR reactions, the 5’ ends of the pork-, chicken-, and beef-specific probes were labeled with FAM, VIC, and NED, respectively. The specificity of this assay was evaluated using 20 animal species and no cross-contamination was observed. The absolute detection limit was 0.1 pg of DNA extracted for all three target species, and the limit of detection (LOD) of reference meat mixtures was determined to be 0.5%. The optimized method was applied to 66 commercial processed products and specific targets were successfully amplified in under 40 min. Therefore, the fast multiple real-time PCR method can be utilized for easily and rapidly monitoring the presence of three meat species in various processed meat products.

Fast diagnosis of sporotrichosis caused by Sporothrix globosa, Sporothrix schenckii, and Sporothrix brasiliensis based on multiplex real-time PCR.

The accurate diagnosis of sporotrichosis and identification at the species level are critical for public health and appropriate patient management. Compared with morphological identification methods, molecular diagnostic tests are rapid and have high sensitivity and standardized operating processes. Therefore, we designed a novel multiplex real-time polymerase chain reaction (PCR) method based on the calmodulin (CAL) gene for the identification of clinically relevant Sporothrix species: S. globosa, S. schenckii s. str., and S. brasiliensis. We evaluated the assay with clinical and spiked samples and assessed its diagnostic performance by comparing the results to those of culture and species-specific PCR. Thirty-three DNA templates were used to detect assay specificity, and three plasmids were constructed to create a standard curve and determine the limits of detection (LODs). For nucleic acid detection, the sensitivity and specificity reached 100%. The LODs were 10 copies, 10 copies and 100 copies for S. globosa, S. schenckii s. str and S. brasiliensis, respectively. For the clinical samples, the positive detection rates by culture, species-specific PCR and the multiplex real-time PCR assay were 87.9% (29/33), 39.4% (13/33), and 93.9% (31/33), respectively. For the spiked samples, the positive detection rates were both 100% for S. schenckii s. str and S. brasiliensis. Based on the above results, compared with culture and other molecular diagnosis methods, the novel multiplex real-time PCR assay is effective, fast, accurate, and highly sensitive. It has a lower reaction cost and lower sample volume requirements, can detect co-infections, and allows for standardized operation and easier interpretation of results. In the future, this assay could be developed into a commercial kit for the diagnosis and identification of S. globosa, S. schenckii s. str, and S. brasiliensis.

Viral respiratory infections diagnosed by multiplex polymerase chain reaction in pediatric patients.

Syndromic diagnosis by multiplex nucleic acid amplification tests is the most practical approach to respiratory tract infections since the symptoms are rarely agent‐specific. The aim of this study was to investigate the respiratory viruses in children admitted to a university hospital with acute respiratory tract infection during the last 8 years by a multiplex polymerase chain reaction (PCR) assay. A total of 3162 respiratory samples collected from children between April 2011 and April 2018 tested by a multiplex real‐time PCR assay. Two different commercial assays were used during the study period, "AusDiagnostics/Respiratory Pathogens 12 (AusDiagnostics)" used between April 2011 and December 2015, which changed to "Fast Track Diagnostics/Respiratory Pathogens 21 (Fast Track Diagnostics)" after January 2016 to cover more viruses. Nucleic acid extraction was done by EZ1 Advanced XL platform (QIAGEN). Respiratory pathogens detected in 1857 of the 3162 (58.7%) samples. The most prevalent viruses during the 8‐year period were rhinovirus/enterovirus (RV/EV; 36.2%), respiratory syncytial virus (RSV; 19%), and influenza virus A/B (14.7%). Rhinovirus was the main contributor to the RV/EV group as shown by the assay used during the 2016‐2018 period. RV/EV and adenoviruses detected throughout the year. Influenza virus was most frequently detected during January to March when both RSV and metapneumovirus were also in circulation. The coinfection percentage was 10.2%. Rhinovirus was the most common virus in coinfections while RSV plus rhinovirus/enterovirus were the most frequent combination. RSV and metapneumovirus showed a similar seasonal distribution to the influenza virus, which made it necessary to use a virological diagnostic assay during the influenza season.

Early and simultaneous detection ofNosema bombycis(Microsporidia: Nosematidae), nucleopolyhedrovirus (Baculoviridae), and densovirus (Parvoviridae) by multiplex real-time polymerase chain reaction inBombyx mori(Lepidoptera: Bombycidae)

AbstractAn effective multiplex real-time polymerase chain reaction (PCR) assay for the simultaneous detection of three major pathogens,Nosema bombycisNägeli (Microsporidia: Nosematidae),Bombyx morinucleopolyhedrovirus (Baculoviridae: genusAlphabaculovirus) (NPV), andBombyx moridensovirus (Parvoviridae: genusIteravirus) (DNV), in silkworms (Bombyx mori(Linnaeus); Lepidoptera: Bombycidae) was developed in this study. Polymerase chain reaction and real-time PCR tests and basic local alignment search tool searches revealed that the primers and probes used in this study had high specificities for their target species. The ability of each primer/probe set to detect pure pathogen DNA was determined using a plasmid dilution panel, in which under optimal conditions the multiplex real-time PCR assay showed high efficiency in the detection of three mixed target plasmids with a detection limit of 8.5×103copies forN. bombycisandBombyx moriNPV (BmNPV) and 8.5×104copies forBombyx moriDNV (BmDNV). When the ability to detect these three pathogens was examined in artificially inoculated silkworms, our method presented a number of advantages over traditional microscopy, including specificity, sensitivity, and high-throughput capabilities. Under the optimal volume ratio for the three primer/probe sets (3:2:2=N. bombycis:BmNPV:BmDNV), the multiplex real-time PCR assay showed early detection of BmNPV and BmDNV by day 1 post inoculation using DNA templates of the three pathogens in various combinations from individually infected silkworms; the early detection ofN. bombyciswas possible by day 3 post inoculation using the DNA isolated from the midgut ofN. bombycis-infected silkworms.

Real-time PCR Detection of the Most Common Bacteria and Viruses Causing Meningitis

Central nervous system (CNS) infections require prompt diagnosis, as the clinical condition progresses rapidly and may lead to severe permanent sequelae or death. The causative agents include viruses, bacteria, fungi, and parasites. In this study, samples with the diagnosis of CNS infection based on cerebrospinal fluid (CSF) sent to us from other hospitals/labs, were studied by multiplex real-time Polymerase Chain Reaction (PCR) method. The purpose of this study is to demonstrate, retrospectively, the most common bacteria and viruses causing meningitis and seasonal distribution of these agents using the multiplex real-time PCR method in CSF samples. This study retrospectively evaluated the results of 470 CSF specimens that had been sent to the Molecular Unit of our hospital with a pre-diagnosis of CNS infection and had been tested with the PCR method between January 2014 and December 2015. Specimens were tested using multiplex real-time PCR assay for Adenovirus (AdV), Cytomegalovirus (CMV), Enteroviruses (EV) (Polioviruses, Coxackieviruses, Echoviruses, and other enteroviruses), Epstein- Barr virus (EBV), Herpes simplex virus 1 and 2, Human Herpes virus 6 and 7, Varicella-zoster virus (VZV), Human Parechoviruses and Parvovirus B19, Hemophilus influenzae, Streptococcus pneumoniae or Neisseria meningitidis. (FTD NEURO9 and FTD Bacterial meningitis, multiplex real-time PCR Kit). A bacterial or viral agent was identified in 98 (21%) of the 470 CSF samples. Of the patients, 85% were children and 15% were adults. Of the 98 positive samples, 22 (22.5%) patients were 15 years or older, and the remaining 76 (77.5%) were younger than 15 years. While Enterovirus (25%) was the most frequently identified agent, Adenovirus ranked second (22%) and Streptococcus pneumoniae ranked third (15%) in total. Positivity was highest in the 0 - 5-year age range. Bacteria were detected with the PCR method in 22 patients: S. pneumonia in 14, and N. meningitidis in 8. In cultures, S. pneumonia grew only in 7 and N. meningitidis in one. EV and AdV were seen in the summer months. The two coexisted in 3 (3%) patients. Early diagnosis and treatment of meningitis are very important for reducing its mortality and morbidity. In patients with suspected meningitis, early detection of the responsible agents may be possible with molecular methods, such as PCR. Significant economic benefits may be obtained by preventing unnecessary antibiotic use and hospitalizations through the early detection of the microbial agents.

Limitation of Simultaneous Analysis of T-Cell Receptor and κ-Deleting Recombination Excision Circles Based on Multiplex Real-Time Polymerase Chain Reaction in Common Variable Immunodeficiency Patients

Aim of Study: We used a triplex real-time polymerase chain reaction (PCR) to classify our common variable immunodeficiency (CVID) patients into distinct groups according to the amount of their T-cell receptor excision circles (TRECs) and κ-deleting recombination excision circles (KRECs). Materials and Methods: TREC and KREC analysis was performed using a multiplex real-time PCR assay. The T- and B-lymphocyte subsets were measured by flow cytometry. Results: The copy number of TRECs and KRECs was significantly reduced in CVID patients compared to healthy controls. The TREC copy number was inversely correlated with age in both healthy subjects and patients; however, the KREC copy number was inversely correlated with age only in CVID patients. Moreover, no association was seen between TREC/KREC copy number and clinical manifestations such as bronchiectasis, splenomegaly, granulomata, autoimmune cytopenias, organ-specific autoimmunity, enteropathy and lymphoid hyperplasia. Conclusion: TREC and KREC quantification might be a useful tool to differentiate between CVID and combined immunodeficiency, but considering the results of this study a classification of CVID patients in certain groups is hardly possible.

Real time PCR for the rapid identification and drug susceptibility of Mycobacteria present in Bronchial washings.

BackgroundMycobacteria have a spectrum of virulence and different susceptibilities to antibiotics. Distinguishing mycobacterial species is vital as patients with non-tuberculous mycobacterial (NTM) infections present clinical features that are similar to those of patients with tuberculosis. Thus, rapid differentiation of Mycobacterium tuberculosis complex from NTM is critical to administer appropriate treatment. Hence the aim of the study was to rapid identification of mycobacterial species present in bronchial washings using multiplex real time Polymerase Chain Reaction (PCR) and to determine the drug susceptibility in identified mycobacterial species.MethodsSputum smear negative bronchoscopy specimens (n = 150) were collected for a period of one year, from patients attending the General Hospital Kandy, Sri Lanka. The specimens were processed with modified Petroff’s method and were cultured on Löwenstein– Jensen medium. DNA, extracted from the mycobacterial isolates were subjected to a SYBR green mediated real time multiplex, PCR assay with primers specific for the M. tuberculosis complex, M. avium complex, M. chelonae-M.abscessus group and M. fortuitum group. DNA sequencing was performed for the species confirmation, by targeting the 16S rRNA gene and the drug susceptibility testing was performed for the molecularly identified isolates of M. tuberculosis and NTM.ResultsThe optimized SYBR Green mediated multiplex real-time PCR assay was able to identify the presence of genus Mycobacterium in 25 out of 26 AFB positive isolates, two M. tuberculosis complex, three M. avium complex and two isolates belonging to M. chelonae-M. abscessus group. DNA sequencing confirmed the presence of M. tuberculosis, M. chelonae-M. abscessus, M. intracellulare, M. avium, Rhodococcus sp. and M. celatum. Remaining isolates were identified as Mycobacterium sp. All the NTM isolates were sensitive to amikacin and seven were resistant to ciproflaxacin. Twenty two of the NTM isolates and the isolate Rhodococcus was resistant to clarithromycin. The two isolates of M. tuberculosis were sensitive to all first line anti tuberculosis drugs.ConclusionThe optimized SYBR Green mediated multiplex real time PCR assay could be an effective tool for the rapid differentiation of pathogenic M. tuberculosis complex from the opportunistic nontuberculous mycobacteria and also it confirmed the presence of NTM in 15.3 % of the study population.