Multiplex Real-time Polymerase Chain Reaction Research Articles

Quantitative Multiplex Real-Time Reverse Transcriptase–Polymerase Chain Reaction with Fluorescent Probe Detection of Killer Immunoglobulin-Like Receptors, KIR2DL4/3DL3

(1) Background: KIR2DL4/KIR3DL3 are the framework genes present in all KIR haplotypes, with unique expression patterns being present only in women and CD56bright NK cells. KIR genes have a high degree of DNA sequence identity. Consequently, they are one of the most challenging genes for molecular detection—especially regarding expressions; (2) Methods: We developed an effective method to determine KIR3DL3/KIR2DL4 expressions based on a multiplex quantitative real-time Reverse transcription polymerase chain reaction (qRT-PCR )with fluorescent probes using NK92; (3) Results: Standardizations of the singleplex KIR2DL4 and KIR3DL3 were performed to evaluate the sensitivity and specificity for further development of the multiplex assay. The limit of detection was at 500 copies each. There was cross-amplification with the presence of related KIR genes at a level of 5 × 107 copies. This is not biologically significant because this high level of KIR expression has not been found in clinical samples. The multiplex assay was reproducible equivalent to its singleplex (KIR2DL4; R2 = 0.995, KIR3DL3; R2 = 0.996, but lower sensitivity of 103 copies). Furthermore, the validation of the developed method on samples of blood donors showed high sensitivity (100%) and specificity (99.9%); (4) Conclusions: The developed method is reliable and highly specific suitable for evaluation of the KIR2DL4/3DL3 mRNA expressions in further applications.

Simultaneous Characterization of Deletional and Nondeletional Globin Gene Mutations by Multiplex Real-Time-Polymerase Chain Reaction and High-Resolution Melting Curve Analysis

Both deletional and nondeletional globin gene mutations are common in Southeast Asians. Normally, deletional gene mutations are characterized separately from nondeletional gene mutations. Therefore, we developed a new approach of multiplex real-time polymerase chain reaction (qPCR) followed by high-resolution melting (HRM) analysis without a fluorescently-labeled probe for the simultaneous detection of deletional and nondeletional gene mutations in a single tube. Three sets of primer pairs were used to establish the qPCR-HRM method that was used to genotype more than 20 different globin genotypes. Twenty known genotypes were used to optimize the qPCR and HRM conditions. Eight genotypes were used to determine the reproducibility of the method. A total of 351 blinded known DNA samples were used for the validation study in three separate reactions and revealed 16 distinct patterns of fragments and/or HRM. The melting temperatures (Tm) of the 3.5 kb, – –THAI, HBB-FR2 (exon 1 of the HBB gene), – –SEA (Southeast Asian), α2 and 3'-ψζ1 fragments were 79.44, 81.01, 86.47, 87.89, 90.54 and 94.15 °C, respectively. The HRM analysis was performed with the HBB-FR2 fragment to differentiate several alleles. We report a rapid and high-throughput technique that showed 100.0% concordance and low variability for each run. Our developed technique is one of the alternative techniques recommended for screening samples with both deletional and nondeletional globin gene mutations.

Comparison of Real-Time PCR Quantification Methods in the Identification of Poultry Species in Meat Products.

Poultry meat is consumed worldwide and is prone to food fraud because of large price differences among meat from different poultry species. Precise and sensitive analytical methods are necessary to control poultry meat products. We chose species–specific sequences of the cytochrome b gene to develop two multiplex real-time polymerase chain reaction (real-time PCR) systems: one for chicken (Gallus gallus), guinea fowl (Numida meleagris), and pheasant (Phasianus colchicus), and one for quail (Coturnix japonica) and turkey (Meleagris gallopavo). For each species, added meat could be detected down to 0.5 % w/w. No cross reactions were seen. For these two real-time PCR systems, we applied three different quantification methods: (A) with relative standard curves, (B) with matrix-specific multiplication factors, and (C) with an internal DNA reference sequence to normalize and to control inhibition. All three quantification methods had reasonable recovery rates from 43% to 173%. Method B had more accepted recovery rates, i.e., in the range 70–130%, namely 83% compared to 75% for method A or C.

Implementation of population-based newborn screening reveals low incidence of spinal muscular atrophy

PurposeSpinal muscular atrophy (SMA) was added to the Recommended Uniform Screening Panel (RUSP) in July 2018, following FDA approval of the first effective SMA treatment, and demonstration of feasibility of high-throughput newborn screening using a primary molecular assay. SMA newborn screening was implemented in New York State (NYS) on 1 October 2018. MethodsScreening was conducted using DNA extracted from dried blood spots with a multiplex real-time quantitative polymerase chain reaction (qPCR) assay targeting the recurrent SMN1 exon 7 gene deletion. ResultsDuring the first year, 225,093 infants were tested. Eight screened positive, were referred for follow-up, and confirmed to be homozygous for the deletion. Infants with two or three copies of the SMN2 gene, predicting more severe, earlier-onset SMA, were treated with antisense oligonucleotide and/or gene therapy. One infant with ≥4 copies SMN2 also received gene therapy. ConclusionNewborn screening permits presymptomatic SMA diagnosis, when treatment initiation is most beneficial. At 1 in 28,137 (95% confidence interval [CI]: 1 in 14,259 to 55,525), the NYS SMA incidence is 2.6- to 4.7-fold lower than expected. The low SMA incidence is likely attributable to imprecise and biased estimates, coupled with increased awareness, access to and uptake of carrier screening, genetic counseling, cascade testing, prenatal diagnosis, and advanced reproductive technologies.

Clinical features and prognostic factors of parainfluenza virus infections in adult patients

Objective: To explore the clinical features and prognostic factors of parainfluenza viral lower respiratory tract infections in adults. Methods: A total of 70 patients withpositive nucleic acid of parainfluenza virus (PIV) admitted to China-Japan Friendship Hospital between August 2016 and November 2019 were enrolled. Multiplex real-time polymerase chain reaction (RT-PCR) assays for viral detection were implemented to specimens (nasopharynx swab, sputum or bronchoalveolar lavage) obtained from all the patients, which was consistent with the diagnosis of lower respiratory tract infection. Patients were divided into two groups depending on the status of immune function (immunocompromised group, n=26; immunocompetent group, n=44), and patients were divided into PIV infection group (n=43) and mixed infection group (n=27) according to whether there was mixed infection. Characteristics including age, gender, underlying diseases, symptoms, disease severity, imaging manifestations, etiology, respiratory failure, mechanical ventilation, vasoactive drug, antimicrobial drug and 30-day mortality between the groups were compared, and the prognostic factors of PIV infections were investigated using Cox regression. Results: The peak incidence of PIV infection time was in May, August, September and December, accounting for 58.6% of all cases. The enrolled 70 cases included 43 cases with pneumonia, 16 cases with interstitial lung disease and infection, 7 cases with bronchiectasis and infection, and 4 cases with acute exacerbation of chronic obstructive pulmonary disease. Eight patients (11.4%) had no underlying diseases, 21 patients (30.0%) had respiratory failure, 18 patients (25.7%) were treated with mechanical ventilation, 15 patients (21.4%) died within 30 days after admission. The pneumonia severity index score, percentage of patients with ground-glass opacity according CT scan, with honeycomb or reticular pattern, with mechanical ventilation, with respiratory failure, with ICU admission, and 30-day mortality in immunocompromised group were higher than those of immunocompetent group [(91.5 vs 84.0), (60.0% vs 34.1%), (44.0% vs 11.4%), (42.3% vs 15.9%), (50.0% vs 18.2%), (38.5% vs 22.7%), (34.6% vs 13.6%)] (all P<0.05). There were 27 cases (38.6%) with mixed infection, including 17 viruses (24.3%), 19 bacteria (27.1%), 14 (20.0%) fungi (PCP, aspergillus) and 1 (1.4%) Mycobacterium intracellulare. Sixteen patients (59.3%) in the mixed infection group were immunocompromised patients and 21 patients (77.8%) had chronic lung disease. Cox regression analysis showed that mechanical ventilation and interstitial lung disease were independent predictors of prognosis in all patients, and mechanical ventilation was an independent predictor of prognosis in PIV infection group. Conclusions: Most of the patients with PIV lower respiratory tract infection in adults are complicated with underlying diseases and mixed infection, with a high 30-day mortality. Interstitial lung disease and mechanical ventilation indicate poor prognosis in these patients.

Simultaneous detection of food pathogens, Staphylococcus aureus, Salmonella Enterica, Bacillus cereus and Vibrio parahaemolyticus by multiplex real-time polymerase chain reaction

The study established a multiplex real-time polymerase chain reaction (PCR) for simultaneous detection and quantitation of Staphylococcus aureus, Salmonella Enterica, Bacillus cereus and Vibrio parahaemolyticus in the same reaction at one time, thus reducing the amounts of reagents and cutting down on the labor and time. Four sets of genetic marker specific primers corresponding to SAOUHSC-02297, invA, hbl and tlhA gene, and probes were labeled with FAM, HEX, TEXAS RED and Cy5 respectively. Multiplex real-time PCR was carried out with an iQ™ Multiplex Powermix Kit (BIO-RAD) Sequence Detection System. The result showed that the correlation coefficient between multiplex real-time PCR estimates and plate counts of 10-serial dilutions in specific bacteria was above 0.98, independent of 101-4-fold numbers of three other pathogenic strains. With optimized conditions, the lowest detection concentrations of four pathogens were 102.5 CFU/mL for Sta. aureus, S. Enterica and V. parahaemolyticus and 103.5 CFU/mL for B. cereus. The duration of the entire experiment from DNA isolation and purification to PCR amplification was less than 12 h.

Utility of a Multiplex Real-Time Polymerase Chain Reaction for Combined Detection and Serotyping of Dengue Virus in Paediatric Patients Hospitalised with Severe Dengue: A Report from Chennai

AbstractObjective: Molecular detection and serotyping are rapid, sensitive and accurate techniques for early diagnosis of paediatric dengue. The present study evaluates multiplex real-time polymerase chain reaction (PCR) for diagnosis of dengue virus in children hospitalised with severe dengue (SD) and attempts to establish an association of clinical severity with specific serotypes. Methods: Four hundred and eighty-five samples were received from hospitalised paediatric patients with suspected dengue from March 2019 to February 2020. Multiplex real time PCR was employed for diagnosis. An in-house real-time PCR that combined diagnosis and serotyping was established. Non-structural protein 1 (NS1) assay and real-time PCR were assessed for their accuracy in diagnosing severe paediatric dengue. Results: Three hundred and twenty-five (67%) patients were positive for dengue RNA by real-time PCR. All four serotypes were identified throughout the year; dengue serotype 2 (DEN-2) was predominant (61%) followed by DEN-3, 20%. Compared to the commonly used NS1 testing, multiplex real-time PCR showed greater sensitivity in diagnosing SD. Conclusions: Compared to NS1, multiplex real-time PCR is a rapid and accurate diagnostic test for children hospitalised with SD. DEN-2 was the predominant serotype in severe cases. Continued surveillance of serotypes should be carried out year-round in endemic areas.

Multi-assay investigation of viral etiology in pediatric central nervous system infections.

In an attempt to identify a wide spectrum of viral infections, cerebrospinal fluid (CSF) specimens were collected from pediatric cases with the preliminary diagnosis of viral encephalitis/meningoencephalitis in two reference hospitals, from October 2011 to December 2015. A combination of nucleic acid-based assays, including in house generic polymerase chain reaction (PCR) assays for enteroviruses, flaviviruses and phleboviruses, a commercial real-time PCR assay for herpesviruses and a commercial real time multiplex PCR, enabling detection of frequently-observed viral, bacterial and fungal agents were employed for screening. The microbial agent could be characterized in 10 (10%) of the 100 specimens. Viral etiology could be demonstrated in 7 (70%) specimens, which comprises Human Herpesvirus 6 (4/7), Herpes Simplex virus type1 (2/7) and Enteroviruses (1/7). In 3 specimens (30%), Streptococcus pneumoniae, Listeria monocytogenes and Staphylococcus aureus were detected via the multiplex PCR, which were also isolated in bacteriological media. All specimens with detectable viral nucleic acids, as well as unreactive specimens via nucleic acid testing remained negative in bacteriological cultures. Herpes and enteroviruses were identified as the primary causative agents of central nervous system infections in children. Enterovirus testing must be included in the diagnostic work-up of relevant cases.

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 two self-sampling methods for human papillomavirus (HPV) DNA testing among women with high prevalence rates.

One major advantage of molecular assays for human papillomavirus (HPV) DNA detection is that these assays can be performed on self-collected samples unlike cytology or visual inspection with acetic acid (VIA). This cross-sectional study was carried out between March 2017 and April 2019 to compare the diagnostic performance in self-collected urine and vaginal samples for HPV DNA detection. Viral DNA was extracted from processed samples using aQiagen viral DNA extraction Kit (QIAamp DNA Mini Kit). To detect four common high-risk HPV types (16, 18, 31, 45), multiplex real-time polymerase chain reaction (PCR) targeting the LCR/E6/E7 region of the HPV genome was performed in ABI 7500 cycler (Applied Biosystems). The negative samples were screened by conventional PCR targeting the L1 capsid region to exclude other HPV types. The overall agreement between the two self-collecting sampling methods was 64.04% with a κ value of 0.29 pointing towards a fair agreement (P < .01). The sensitivity of HPV DNA detection in urine samples was 57.95% (47.52%, 67.72), and specificity was 84.6% (66.47%, 93.85%) when compared with vaginal samples. The study concludes that self-collected vaginal HPV DNA testing is more sensitive than unpreserved-urine samples for HPV DNA detection in a hospital-based setting.

Molecular evidence of bovine herpesvirus 1 and 5 in cattle with suspected rabies in Rio Grande do Sul state, Brazil

Rabies and herpetic encephalitis are the main viral infections in bovines with neurological symptoms. Bovine rabies has a high prevalence in Central and South America, while bovine encephalitis associated with herpesvirus is especially important in South America. Viral isolation is the classical way to confirm herpesvirus infection, but molecular evidence of the presence of the virus in affected animals is gaining importance in the diagnosis of the disease in the laboratory. This study investigated the presence of herpesvirus type 1 and 5 (BoHV-1 and BoHV-5) in 182 encephalon of rabies-suspected cattle in Rio Grande do Sul state (RS), Brazil using multiplex real-time polymerase chain reaction (mRT-PCR). The rabies virus was investigated by direct fluorescent antibody assay and intracerebral suckling mouse inoculation. The genomes of BoHV-1 and BoHV-5 were detected in 17% of samples. BoHV-5 and BoHV-1 were detected in 100% and 19% of BoHV positive samples, respectively, indicating the circulation of the pathogens in cattle herds in RS. The high Ct values and the absence of isolation suggest viral latency. Coinfection of herpesvirus and the rabies virus was detected in 28% of samples, although no significant association between pathogens was observed. Rabies was detected in 57.7% of suspected samples, confirming the importance of the disease in the state. Concerning the method by which samples were conserved, no significant difference was observed between the number of positive results in frozen and refrigerated samples.

High-Frequency Direct Detection of Triazole Resistance in Aspergillus fumigatus from Patients with Chronic Pulmonary Fungal Diseases in India.

Aspergillosis due to azole-resistant Aspergillus fumigatus is a worldwide problem with major therapeutic implications. In patients with invasive aspergillosis, a low yield of fungal cultures results in underestimation of azole resistance. To detect azole resistance in A. fumigatus, we applied the AsperGenius® Resistance multiplex real-time polymerase chain reaction (PCR) assay to detect TR34/L98H, and TR46/T289A/Y121F mutations and the AsperGenius® G54/M220 RUO PCR assay to detect G54/M220 mutations directly in bronchoalveolar lavage (BAL) samples of 160 patients with chronic respiratory diseases in Delhi, India. Only 23% of samples were culture-positive compared to 83% positivity by A. fumigatus species PCR highlighting concerns about the low yield of cultures. Notably, 25% of BAL samples (33/160 patients) had azole resistance-associated mutation by direct detection using PCR assay. Detection of resistance-associated mutations was found mainly in 59% and 43% patients with chronic pulmonary aspergillosis (CPA) and allergic bronchopulmonary aspergillosis (ABPA), respectively. Overall, a G54 mutation, conferring itraconazole resistance, was the predominant finding in 87.5% and 67% of patients with CPA and ABPA, respectively. In culture-negative, PCR-positive samples, we detected azole-resistant mutations in 34% of BAL samples. Azole resistance in chronic Aspergillus diseases remains undiagnosed, warranting standardization of respiratory culture and inclusion of rapid techniques to detect resistance markers directly in respiratory samples.

Detection of bovine pestiviruses by a multiplex real-time polymerase chain reaction.

Pestiviruses are the cause of reproductive problems, diseases of the gastrointestinal and respiratory tracts of animals. Three species are important for cattle: Pestivirus A, B, and H. Fast and reliable methods of differentiation of these pathogens are currently needed. Aims and objectives of the study: the development of multiplex real time PCR for the simultaneous detection and differentiation of three viruses. The nucleotide sequences of the conserved regions of the 5´-UTR genes of pes tivirusesA, B, and H served as a target. The reaction showed high specificity, sensitivity, reproducibility and was able to detect virus RNA at a concentration of not less than 0.6-1.2 lg TCID50/cm3. Cross-reactions with other pestiviruses wer e not observed. Real time PCR confirmed the results obtained previously in RT-PCR with gel electrophoresis detection. In a parallel study of 1823 biological samples, the results of the two reactions were completely consistent. Pestivirus spp. was detectedin 76 samples, Pestivirus A was present in 73 samples, Pestivirus B - in 3 samples, and Pestivirus H was not detected. A two-step real time PCR was developed for the simultaneous detection and differentiation of three pestiviruses. Modified pan primers of S. Vilcek et al. were used for the first reaction, and primers and probes of our own design were used for virus typing, which resulted in high reaction efficiency. On the big dairy farms for livestock maintenance, there are favorable conditions for the circulation of pathogenic viruses. In this situation, rapid diagnostic methods are needed to quickly identify of several viruses. Real-time triplex analysis can be recommended as the rapid method for mass epidemiological studies, as well as for screening fetal calf serum used for virus cultivation in medicine and veterinary practice.

Pattern of Enteropathogens during Diarrhoeal Episode in Children with Haematological Malignancy

Background: Haematological malignancies comprise 82% of all malignancies in children at the department of Paediatric Haematology and Oncology, Bangabandhu Sheikh Mujib Medical University (BSMMU), Bangladesh; Gastro-intestinal infections were the leading causes of infection in paediatric oncology patients. Objectives: This study was conducted to see the type of enteropathogens in stool samples during diarrhoeal episode in children with haematological malignancy. Methods: This observational study was conducted from April 2012 through March 2013 at BSMMU, Bangladesh. A total of 58 diarrhoeal episodes experienced by 51 children of various types of haematological malignancies were included in the study. Faecal samples from studied children were sent to Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR, B), laboratory to do aerobic culture for common bacteria; Enzyme Immunoassay (EIA) for Clostridium difficile and multiplex real-time polymerase chain reaction (PCR) for parasitic infection. Results: Among the total 58 diarrhoeal episodes, potential pathogenic bacteria were isolated from only 5.17% sample. Faecal samples from 22.41% episodes were found positive for GDH antigen for Clostridium difficile; none of the focal samples were positive for toxin A and/ or B. Several different parasites were identified from 70.37% samples and most frequently identified protozoa was Giardia lamblia in 68.52%. Conclusion: The study found 22.41% colonization rate with Clostridium difficile but none was toxigenic. Parasitic infections were seen more frequently in children with haematological malignancy. The study also found significant association of severe neutropenia with GDH positive diarrhoeal episodes.

Influenza species and subtypes circulation among hospitalized patients in Laleh hospital during two influenza seasonal (2016-2017 and 2017-2018) using a multiplex Real Time-Polymerase Chain Reaction.

The introduction of polymerase chain reaction (PCR) techniques has improved the detection of respiratory viruses, particularly with the use of multiplex real-time technique with the capability of simultaneous detection of various pathogens in a single reaction. The aim of this study was to apply the above technology for the diagnosis of influenza infections and at the same time to differentiate between common flu species between hospitalized patients in Laleh hospital (Iran) between two flu seasons (2016-2017 and 2017-2018). Different respiratory specimens were collected from 540 patients from a period of December 2016 to May 2018 and were sent to the laboratory for molecular diagnosis. RNAs were extracted and subsequently, a multiplex real time PCR identifying flu A, flu B and typing flu A (H1N1) was carried out. The mean age of patients was 47.54±23.96. 216 (40%) and 321 (60%) of subjects were male and female, respectively. 219 out of 540 (40.5%) were positive for influenza infection including flu A (n=97, 44.3%), flu A (H1N1) (n=45, 20.7%) and flu B (n=77, 35%). Flu A was the dominant species on 2016-2017 and flu B was the major species on 2017-2018. Flu A (H1N1) was comparable in both time periods. Flu infections were most frequently diagnosed in age groups 21-40. Flu-positive patients suffered more from body pain and sore throat than flunegative patients with significant statistical difference (P values <0.001). The mean duration of hospitalization was shorter for flu-positive patients (P value = 0.016). Application of multiplex real time PCR could facilitate the influenza diagnosis in a short period of time, benefiting patients from exclusion of bacterial infections and avoiding unnecessary antibiotic therapy. Influenza diagnosis was not achieved in up to 60% of flu-like respiratory infections, suggesting the potential benefit of adopting the same methodology for assessing the involvement of other viral or/and bacterial pathogens in those patients.

Use of real-time multiplex PCR, malaria rapid diagnostic test and microscopy to investigate the prevalence of Plasmodium species among febrile hospital patients in Sierra Leone

BackgroundMalaria continues to affect over 200 million individuals every year, especially children in Africa. Rapid and sensitive detection and identification of Plasmodium parasites is crucial for treating patients and monitoring of control efforts. Compared to traditional diagnostic methods such as microscopy and rapid diagnostic tests (RDTs), DNA based methods, such as polymerase chain reaction (PCR) offer significantly higher sensitivity, definitive discrimination of Plasmodium species, and detection of mixed infections. While PCR is not currently optimized for routine diagnostics, its role in epidemiological studies is increasing as the world moves closer toward regional and eventually global malaria elimination. This study demonstrates the field use of a novel, ambient temperature-stabilized, multiplexed PCR assay in a small hospital setting in Sierra Leone.MethodsBlood samples from 534 febrile individuals reporting to a hospital in Bo, Sierra Leone, were tested using three methods: a commercial RDT, microscopy, and a Multiplex Malaria Sample Ready (MMSR) PCR designed to detect a universal malaria marker and species-specific markers for Plasmodium falciparum and Plasmodium vivax. A separate PCR assay was used to identify species of Plasmodium in samples in which MMSR detected malaria, but was unable to identify the species.ResultsMMSR detected the presence of any malaria marker in 50.2% of all tested samples with P. falciparum identified in 48.7% of the samples. Plasmodium vivax was not detected. Testing of MMSR P. falciparum-negative/universal malaria-positive specimens with a panel of species-specific PCRs revealed the presence of Plasmodium malariae (n = 2) and Plasmodium ovale (n = 2). The commercial RDT detected P. falciparum in 24.6% of all samples while microscopy was able to detect malaria in 12.8% of tested specimens.ConclusionsWider application of PCR for detection of malaria parasites may help to fill gaps existing as a result of use of microscopy and RDTs. Due to its high sensitivity and specificity, species coverage, room temperature stability and relative low complexity, the MMSR assay may be useful for detection of malaria and epidemiological studies especially in low-resource settings.

Molecular Diagnosis of Pneumonia Using Multiplex Real-Time PCR Assay RespiFinder® SMART 22 FAST in a Group of Moroccan Infants.

Background In Morocco, pediatric pneumonia remains a serious public health problem, as it constitutes the first cause of mortality due to infectious diseases. The etiological diagnosis of acute respiratory tract infections is difficult. Therefore, it is necessary to use Multiplex real-time polymerase chain reaction assay tests in a routine setting for exact and fast identification. Objectives In this paper, we present the clinical results of pediatric pneumonia and describe their etiology by using molecular diagnosis. Study design: Tracheal secretion was collected from infants presenting respiratory distress isolated or associated with systemic signs, attending the unit of Neonatology between December 1, 2016, and Mai 31, 2018. Samples were tested with the multiplex RespiFinder® SMART 22 FAST which potentially detects 18 viruses and 4 bacteria. Results Of the 86 infants considered in this study (mean age 31 ± 19 days) suspected of acute respiratory tract infections, 71 (83%) were positive for one or multiple viruses or/and bacteria. The majority of acute respiratory tract infections had a viral origin (95%): respiratory syncytial viruses (A and B) (49%), rhinovirus (21%), coronaviruses 229E (11%), humain metapneumovirus (5%), influenza A (3%), influenza H1N1 (1%), adenovirus (2%), and parainfluenza virus type 4 (2%). Among our patients, 6% had Mycoplasma pneumoniae. Coinfections were not associated with severe respiratory symptoms. Conclusion The clinical spectrum of respiratory infections is complex and often nonspecific. Thus, the early and fast detection of related causative agents is crucial. The use of multiplex real time polymerase chain reaction may help choose an accurate treatment, reduce the overall use of unnecessary antibiotics, preserve intestinal flora, and decrease nosocomial infection by reducing the length of hospitalization.

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.

Development and evaluation of a panel of multiplex one-tube nested real time PCR assay for simultaneous detection of 14 respiratory viruses in five reactions.

Multiplex real‐time quantitative polymerase chain reaction (mRT‐qPCR) assay is commonly used to detect respiratory viruses, however, the sensitivity is limited for most reports. A panel of locked nucleic acid based multiplex closed one‐tube nested real‐time PCR (mOTNRT‐PCR) assay consisting of five separate internally controlled RT‐qPCR assays was developed for detection of 14 respiratory viruses. The sensitivity and reproducibility of mOTNRT‐PCR panel were evaluated using plasmid standards and the specificity was evaluated using clinical samples. The clinical performance of mOTNRT‐PCR panel was further evaluated with 468 samples collected from patients with an acute respiratory infection and compared with individual real‐time PCR (RT‐qPCR) assay. The analytical sensitivities of mOTNRT‐PCR panel ranged from 2 to 20 copies/reaction, and no cross‐reaction with common respiratory viruses was observed. The coefficients of variation of intra‐assay and inter‐assay were between 0.35% and 8.29%. Totally 35 clinical samples detected by mOTNRT‐PCR assay panel were missed by RT‐qPCR and confirmed true positive by sequencing of nested PCR products. The mOTNRT‐PCR assay panel provides a more sensitive and high‐throughput method for the detection of 14 respiratory viruses.

Improving the etiological diagnosis of osteoarticular infections with the commercial multiplex real-time polymerase chain reaction SeptiFast®

Detection of etiological agents is pivotal for adequate therapy of osteoarticular bacterial infections. Culture often lacks sensitivity, especially in patients under antibiotic therapy. The present study investigates the potential clinical utility of the commercial multiplex real-time polymerase chain reaction SeptiFast® (SF) in the etiological diagnosis of osteoarticular infections. Results obtained from conventional culture and SF were compared in 86 osteoarticular specimens collected from patients with suspected infection. The number of specimens positive by SF (38/86, 44.18%) was significantly greater (P = 0.001) than that of specimens positive by culture (20/86, 23.25%). The sensitivity of SF was 48.71%, significantly higher than culture sensitivity (25.64%). Specificity was 100% for both tests. The overall diagnostic accuracy for SF was 53.48%, and that of culture was 32.55%. Even with the limitation of the low number of specimens, this study supports the usefulness of SF in the diagnosis of osteoarticular infections.