Simultaneous detection of gastrointestinal pathogens with a multiplex Luminex-based molecular assay in stool samples from diarrhoeic patients

We have evaluated the multiplex molecular method xTAG® Gastrointestinal Panel (GPP) for detecting pathogens in stool samples of diarrhoeic patients. We collected 440 samples from 329 patients (male:female ratio of 1.2:1), including 102 immunosuppressed adults, 50 immunosuppressed children, 56 children attending the neonatal unit and 121 children attending the emergency unit. Of these, 176 samples from 162 patients were xTAG® GPP positive (102 viruses, 61 bacteria and 13 parasites) and the assay was more sensitive than the conventional test for detecting rotavirus (p <0.01), noroviruses (p <0.0001), Salmonella spp. (p <0.001), Campylobacter spp. (p <0.001) and toxigenic Clostridium difficile (p 0.005). The predominant pathogens were viruses (23.2%), with rotavirus (15.9%) being the most common. Bacterial agents were detected in 13.9%; the most common was Salmonella spp. (4.8%). Parasites were detected in 2.9%; Cryptosporidium spp. (2%) was the most common. There were 31 co-infections (7% of samples), involving two pathogens in 23 (5.2%) and three pathogens in eight (1.8%) samples. There were 113 (92.6%) positive samples from the children attending the emergency unit, 25 (17%) positive samples from immunosuppressed adults, 22 (25.3%) positive samples from immunosuppressed children and 16 (19%) positive samples from children attending the neonatal unit. The low turnaround time and technical hands-on time make this multiplex technique convenient for routine use. Nevertheless, conventional bacterial culture and parasitological stool examination are still required to detect other pathogens in specific cases and to determine susceptibility to antibiotics.     

Use of molecular beacon probes for real-time PCR detection of Plasmodium falciparum and other plasmodium species in peripheral blood specimens

We describe the development and evaluation of a novel pair of real-time, fluorescence-based PCR assays using molecular beacon probes for rapid, sensitive, and specific detection and quantification of Plasmodium falciparum and other Plasmodium species organisms.     

Molecular beacon real-time PCR detection of swine viruses

Rapid and reliable detection of viral pathogens is critical for the management of the diseases threatening the economic competitiveness of the swine farming industry worldwide. Molecular beacon assays are one type of real-time polymerase chain reaction (PCR) technology capable of fast, specific, sensitive, and reliable viral detection. In this paper, the development of molecular beacon assays as novel tools for the rapid detection of Aujeszky's disease virus, African swine fever virus, porcine circovirus type 2 and porcine parvovirus is described. The assays are capable of rapidly detecting 2 × 10¹ copies of target and are linear between 2 × 10⁹ and 2 × 10² copies. They can detect virus specifically in clinical samples such as whole blood, serum and tissue. In comparison to conventional PCR they are either as sensitive or more sensitive. As such these molecular beacon assays represent a powerful tool for the detection of these viruses in swine.     

Rapid detection of human parechoviruses in clinical samples by real-time PCR.

BACKGROUND: Human parechoviruses (HPeVs) have been associated with severe conditions such as neonatal sepsis and meningitis in young children. Rapid identification of an infectious agent in such serious conditions in these patients is essential for adequate decision making regarding treatment and hospital stay. OBJECTIVES: We have developed an HPeV specific real-time PCR assay based on the conserved 5'untranslated region. STUDY DESIGN: To determine the detection limit of the assay, serial dilutions of HPeV in vitro RNA were tested in a background of HPeV and EV RNA-negative cerebrospinal fluid (CSF). The specificity was tested by analyzing culture isolates of HPeV 1-6, enterovirus (EV) types, human rhinoviruses (HRVs) and hepatitis A virus (HAV). To establish diagnostic relevance, 522 CSF samples from children <5 years were tested. RESULTS: The detection limit of the assay was 75 copies of HPeV cDNA per reaction. The assay was highly specific for HPeV, detecting all HPeV types. We identified HPeV infections in CSF of 20 children (3.8%), all with severe conditions such as sepsis and meningitis. CONCLUSIONS: These results suggest that HPeV screening of paediatric clinical samples should be included in viral diagnostics in suspected cases of neonatal sepsis and meningitis.     

Evaluation of a molecular beacon real-time PCR assay for detection of Baylisascaris procyonis in different soil types and water samples

Baylisascaris procyonis is a helminth parasite commonly found in North American raccoons (Procyon lotor) that is a cause of clinical neural, ocular, and visceral larva migrans in humans when infective eggs are ingested. Rapid detection of B. procyonis eggs in contaminated soil and water would assist public health analysts in evaluating risks associated with public exposure to areas of known raccoon activity. In this study, a molecular beacon probe-based real-time polymerase chain reaction (PCR) assay was developed to enable rapid and specific detection of eggs of Baylisascaris spp. The molecular beacon assay targeted the cytochrome oxidase subunit 2 (cox-2) gene of B. procyonis. To determine method sensitivity, experiments testing various egg levels (250, 25, and five eggs) were performed by seeding into 0.5-g soil samples or 0.5-mL water samples. Different soil sample types were extracted using a commercial nucleic acid extraction kit. Specificity testing using previously characterized helminth tissue specimens indicated that the assay was specific to Baylisascaris spp. Little real-time PCR inhibition was observed for most of the soil and water samples. A seed level of 250 eggs was detected for all soil types, and two seed levels (25 and five eggs) were detected for surface water samples. These results demonstrate that the reported real-time PCR assay was effective for the sensitive detection of B. procyonis in a wide range of soil types, and should be a useful tool for investigations of soil or water potentially contaminated with eggs of this parasite.     

Successful nanolitre real-time PCR detection of respiratory pathogens in clinical specimens

We performed a proof-of-concept study to determine if human pathogens could be detected in clinical specimens using nanolitre-volume real-time PCR. Nanolitre PCR for Bordetella pertussis/ B. parapertussis and respiratory syncytial virus (RSV) was performed on nasopharyngeal specimens and results compared with conventional methods. B. pertussis/B. parapertussis nanolitre PCR detection was 100% sensitive (20/20; 95% CI, 84–100%) and 100% specific (26/26; 95% CI, 87–100%). RSV nanolitre PCR was also 100% sensitive (21/21; 95% CI, 85–100%) and specific (25/25; 95%, CI 87–100%). Respiratory pathogens can be successfully detected in clinical specimens using nanolitre-volume PCR.     

A novel real-time PCR system for simultaneous detection of human viruses in clinical samples from patients with uncertain diagnoses

A novel simultaneous detection system for human viruses was developed using a real-time polymerase chain reaction (PCR) system to identify causes of infection in clinical samples from patients with uncertain diagnoses. This system, designated as the "multivirus real-time PCR," has the potential to detect 163 human viruses (47 DNA viruses and 116 RNA viruses) in a 96-well plate simultaneously. The specificity and sensitivity of each probe-primer set were confirmed with cells or tissues infected with specific viruses. The multivirus real-time PCR system showed profiles of virus infection in 20 autopsies of acquired immunodeficiency syndrome patients, and detected frequently TT virus, cytomegalovirus, human herpesvirus 6, and Epstein-Barr virus in various organs; however, RNA viruses were detected rarely except for human immunodeficiency virus-1. Pathology samples from 40 patients with uncertain diagnoses were examined, including cases of encephalitis, hepatitis, and myocarditis. Herpes simplex virus 1, human herpesvirus 6, and parechovirus 3 were identified as causes of diseases in four cases of encephalitis, while no viruses were identified in other cases as causing disease. This multivirus real-time PCR system can be useful for detecting virus in specimens from patients with uncertain diagnoses.     

Evaluation of a PCR assay for detection of Streptococcus pneumoniae in respiratory and nonrespiratory samples from adults with community-acquired pneumonia

Streptococcus pneumoniae is the most common cause of community-acquired pneumonia, but it is undoubtedly underdiagnosed. We used a nested PCR assay (targeting the pneumolysin gene) to detect S. pneumoniae DNA in multiple sample types from 474 adults with community-acquired pneumonia and 183 control patients who did not have pneumonia. Plasma or buffy coat samples were PCR positive in only 6 of the 21 patients with positive blood cultures for S. pneumoniae and in 12 other patients (4 of whom had no other laboratory evidence of S. pneumoniae infection). Buffy coat samples from two control patients (neither having evidence of S. pneumoniae infection), but no control plasma samples, were PCR positive. Although pneumococcal antigen was detected in the urine from 120 of 420 (29%) patients, only 4 of 227 (2%) urine samples tested were PCR positive. Overall, 256 of 318 (81%) patients had PCR-positive sputum samples, including 58 of 59 samples from which S. pneumoniae was cultured. Throat swab samples from 229 of 417 (55%) patients were PCR positive and, in those who produced sputum, 96% also had positive PCR results from sputum. Throat swabs from 73 of 126 (58%) control patients were also PCR positive. We conclude that the pneumolysin PCR assay adds little to existing diagnostic tests for S. pneumoniae and is unable to distinguish colonization from infection when respiratory samples are tested.     

Molecular detection of respiratory pathogens in community-acquired pneumonia involving adults

BackgroundCommunity-acquired pneumonia (CAP) causes substantial morbidity and mortality in adults worldwide. The etiology of CAP often remains uncertain, and therapy is empirical. Thus, there is still room for improvement in the diagnosis of pneumonia.MethodsAdults aged >20 years who presented at the outpatient or emergency departments of Linkou and Keelung Chang Gung Memorial Hospital with CAP were prospectively included between November 2016 and December 2018. We collected respiratory specimens for culture and molecular testing and calculated the incidence rates of CAP according to pathogens.ResultsOf 212 hospitalized adult patients with CAP, 69.3% were male, and the median age of the patients was 67.8 years. Bacterial pathogens were detected in 106 (50%) patients, viruses in 77 (36.3%), and fungal pathogens in 1 patient (0.5%). The overall detection rate (culture and molecular testing method) was 70.7% (n = 150). Traditional microbial culture yielded positive results in 36.7% (n = 78), molecular testing in 61.3% (n = 130). The most common pathogens were influenza (16.1%), followed by Klebsiella pneumoniae (14.1%), Pseudomonas aeruginosa (13.6%), human rhinovirus (11.8%), and Streptococcus pneumoniae (9.9%). Multiple pathogen co-infections accounted for 28.7% (n = 61), of which co-infection with K. pneumoniae and human rhinovirus comprised the largest proportion.ConclusionsMolecular diagnostic testing could detect 23.6% more pathogens than traditional culture techniques. However, despite the current diagnostic tests, there is still the possibility that no pathogen was detected.     

Rapid detection of enterohemorrhagic Escherichia coli by real-time PCR with fluorescent hybridization probes

In this report, we present a PCR protocol for rapid identification of enterohemorrhagic Escherichia coli on a LightCycler instrument. In a multiplex assay, the genes encoding Shiga toxin 1 and Shiga toxin 2 are detected in a single reaction capillary. A complete analysis of up to 32 samples takes about 45 min.     

Quantitative detection of hepatitis B virus DNA by real-time nucleic acid sequence-based amplification with molecular beacon detection

We have developed a hepatitis B virus (HBV) DNA detection and quantification system based on amplification with nucleic acid sequence-based amplification (NASBA) technology and real-time detection with molecular beacon technology. NASBA is normally applied to amplify single-stranded target RNA, producing RNA amplicons. In this work we show that with modifications like primer design, sample extraction method, and template denaturation, the NASBA technique can be made suitable for DNA target amplification resulting in RNA amplicons. A major advantage of our assay is the one-tube, isothermal nature of the method, which allows high-throughput applications for nucleic acid detection. The homogeneous real-time detection allows a closed-tube format of the assay, avoiding any postamplification handling of amplified material and therefore minimizing the risk of contamination of subsequent reactions. The assay has a detection range of 10(3) to 10(9) HBV DNA copies/ml of plasma or serum (6 logs), with good reproducibility and precision. Compared with other HBV DNA assays, our assay provides good sensitivity, a wide dynamic range, and high-throughput applicability, making it a viable alternative to those based on other amplification or detection methods.     

Real-time polymerase chain reaction assay for detection of Streptococcus pneumoniae in sputum samples from patients with community-acquired pneumonia.

BACKGROUND AND PURPOSE: A quantitative real-time polymerase chain reaction (PCR) method targeting the pneumolysin gene of Streptococcus pneumoniae in sputum specimens from patients with community-acquired pneumonia (CAP) was evaluated for the identification of pneumococci. METHODS: The applicability of the assay to clinical samples was evaluated by studying 140 sputum specimens from patients with CAP. Of the specimens, 96 (68.6%) were found to be positive by real-time PCR. The results were compared to culture findings. RESULTS: The sensitivity and specificity of the real-time PCR assay developed in this study as compared to those of the culture method were 97.2% and 60.9%, respectively. CONCLUSION: Real-time PCR assay was found to be a rapid and sensitive method for the detection of pneumococci.     

DNA microarray-based detection and identification of fungal pathogens in clinical samples from neutropenic patients

The increasing incidence of invasive fungal infections (IFI) in immunocompromised patients emphasizes the need to improve diagnostic tools. We established a DNA microarray to detect and identify DNA from 14 fungal pathogens (Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus, Candida albicans, Candida dubliniensis, Candida glabrata, Candida lusitaniae, Candida tropicalis, Fusarium oxysporum, Fusarium solani, Mucor racemosus, Rhizopus microsporus, Scedosporium prolificans, and Trichosporon asahii) in blood, bronchoalveolar lavage, and tissue samples from high-risk patients. The assay combines multiplex PCR and consecutive DNA microarray hybridization. PCR primers and capture probes were derived from unique sequences of the 18S, 5.8S, and internal transcribed spacer 1 regions of the fungal rRNA genes. Hybridization with genomic DNA of fungal species resulted in species-specific hybridization patterns. By testing clinical samples from 46 neutropenic patients with proven, probable, or possible IFI or without IFI, we detected A. flavus, A. fumigatus, C. albicans, C. dubliniensis, C. glabrata, F. oxysporum, F. solani, R. microsporus, S. prolificans, and T. asahii. For 22 of 22 patients (5 without IFI and 17 with possible IFI), negative diagnostic results corresponded with negative microarray data. For 11 patients with proven (n = 4), probable (n = 2), and possible IFI (n = 5), data for results positive by microarray were validated by other diagnostic findings. For 11 of 11 patients with possible IFI, the microarray results provided additional information. For two patients with proven and probable invasive aspergillosis, respectively, microarray results were negative. The assay detected genomic DNA from 14 fungal pathogens from the clinical samples, pointing to a high significance for improving the diagnosis of IFI.     

Molecular detection and identification of Candida and Aspergillus spp. from clinical samples using real-time PCR

This report describes the development of a real-time LightCycler assay for the detection and identification of Candida and Aspergillus spp., using the MagNa Pure LC Instrument for automated extraction of fungal DNA. The assay takes 5-6 h to perform. The oligonucleotide primers and probes used for species identification were derived from the DNA sequences of the 18S rRNA genes of various fungal pathogens. All samples were screened for Aspergillus and Candida to the genus level in the real-time PCR assay. If a sample was Candida-positive, typing to species level was performed using five species-specific probes. The assay detected and identified most of the clinically relevant Aspergillus and Candida spp. with a sensitivity of 2 CFU/mL blood. Amplification was 100% specific for all Aspergillus and Candida spp. tested. To assess clinical applicability, 1,650 consecutive samples (1,330 blood samples, 295 samples from other body fluids and 25 biopsy samples) from patients with suspected invasive fungal infections were analysed. In total, 114 (6.9%) samples were PCR-positive, 5.3% for Candida and 1.7% for Aspergillus spp. In patients with positive PCR results for Candida and Aspergillus, verification with conventional methods was possible in 83% and 50% of cases, respectively. In conclusion, the real-time PCR assay allows sensitive and specific detection and identification of fungal pathogens in vitro and in vivo.     

Diagnosis of Pneumocystis pneumonia by real-time PCR in patients with various underlying diseases

BackgroundPneumocystis pneumonia (PCP) is a disease caused by the opportunistic infection of the fungus Pneumocystis jirovecii. Several PCR methods have been developed to aid in the diagnosis of PCP. In this study, we evaluated the performance of a real-time PCR in the diagnosis of PCP, in patients with various underlying diseases.MethodsNinety-seven BAL samples and 94 sputum samples from 191 patients were used in the study. Patients were classified as PCP (121 patients) or non-PCP (70 patients) based on their clinical and radiological presentations.ResultsReal time PCR amplified the P. jirovecii mitochondrial large-subunit rRNA gene with a detection limit of 68 copies of DNA per reaction. Non-PCP pathogens including 32 different fungi and bacteria were also evaluated. Overall, 71.9% of the samples from PCP patients and 14.5% of those from non-PCP patients were positive for the PCR test with a C_T value of the real-time PCR below 45. The main underlying diseases of the patients were hematological or solid malignancies (47.1%) and HIV infection (8.9%). The C_T values of the test were significantly lower in BAL samples from PCP patients than those from non-PCP patients (p = 0.024). No non-PCP patient had a C_T value below 30, whereas samples from 24.8% of PCP patients with underlying diseases had a C_T value below 30.ConclusionSince false positive PCR results were obtained, perhaps due to colonization, we suggest that the diagnosis of PCP should be based on a combination of clinical symptoms, underlying diseases, and PCR results.     

New diagnostic real-time PCR for specific detection of Parachlamydia acanthamoebae DNA in clinical samples

Given the low sensitivity of amoebal coculture, we developed a specific real-time PCR for the detection of Parachlamydia. The analytical sensitivity was high, and the inter- and intrarun variabilities were low. When the PCR was applied to nasopharyngeal aspirates, it was positive for six patients with bronchiolitis. Future studies should assess the role of Parachlamydia in bronchiolitis.     

Simultaneous detection of Chlamydophila pneumoniae and Mycoplasma pneumoniae by use of molecular beacons in a duplex real-time PCR

A real-time PCR was designed for detection of Chlamydophila pneumoniae and Mycoplasma pneumoniae such that each pathogen could be detected in a single tube and differentiated using molecular beacons marked with different fluorochromes. This duplex PCR, targeting the P1 adhesion gene for M. pneumoniae and the ompA gene for C. pneumoniae, was compared with two conventional PCR assays targeting the 16S rRNA gene and the ompA gene. A total of 120 clinical throat and nasopharyngeal swab samples were tested. DNA extraction was performed using an alkali denaturation/neutralization method, and real-time amplification, detection, and data analysis were performed using a Rotor-Gene 2000 real-time rotary analyzer (Corbett Life Science, Sydney, Australia). Using conventional PCR as a reference in an analysis of 120 samples, 13 of 14 samples positive for C. pneumoniae were detected by the novel real-time PCR. In an analysis of M. pneumoniae, 22 samples were positive in the conventional PCR and the novel assay detected 24 positive samples. When using the conventional PCR as a reference, sensitivity and specificity were 93% and 100%, respectively, for C. pneumoniae and 100% and 98%, respectively, for M. pneumoniae. With an overall agreement of 98.8%, this suggests that performance of the new duplex real-time PCR is comparable to that of conventional PCR.     

Rapid detection of pneumococcal antigens in sputa in patients with community-acquired pneumonia by coagglutination

This paper introduces the clinical use of coagglutination as a method for the rapid detection of pneumococcal antigens in patients with community-acquired pneumonia and compares it with the sputum Gram stain and culture methods. Among 105 patients, 50 (48%) were diagnosed as having pneumococcal pneumonia by at least one of the three methods. Of 95 sputa tested, 44 (46%) were found to be positive by the coaggulation test, 26 (27%) by Gram staining and only 16 (17%) by the bacterial culture method. The rate of detection of pneumococcal antigens was thus greater with coaggulation than with either of the other two methods. The differences were very significant (bothP<0.01). Our study indicates that the advantages of coagglutination over the traditional bacteriological methods are its speed, sensitivity, convenience and also its relative independence of antibiotic therapy. It thus provides a new dimension in the aetiological diagnosis of pneumococcal pneumonia.