Evaluation of a multiplex PCR assay for simultaneous detection of bacterial and viral enteropathogens in stool samples of paediatric patients

We evaluated a multiplex PCR assay, the Seeplex Diarrhoea ACE detection, that simultaneously detects 15 enteric pathogens, including Salmonella spp., Shigella spp., Vibrio spp., toxin B producer Clostridium difficile, Campylobacter spp., Clostridium perfringens, Yersinia enterocolitica, Aeromonas spp., Escherichia coli O157:H7, verocytotoxin-producing Escherichia coli, adenovirus, Group A rotavirus, norovirus GI and GII, and astrovirus. We compared this assay with clinical methods routinely used in our laboratory, for detecting enteropathogens in stool samples collected from 245 paediatric patients with suspected infectious gastroenteritis. We recovered 61 bacterial pathogens and 121 enteric viruses with our laboratory assays, while we detected 78 bacteria and 167 viruses with the molecular assay. We calculated specificity and sensitivity for both methods after analysis of discordant results and demonstrated greater sensitivity for multiplex PCR than for our routine methods, with the exception of Salmonella spp. and toxigenic C. difficile detection. The multiplex PCR assay proved to be a reliable tool to directly detect the most common enteropathogens in stool samples but with some limitations.     

Evaluation of the EasyScreen™ Enteric Parasite Detection Kit for the detection of Blastocystis spp., Cryptosporidium spp., Dientamoeba fragilis, Entamoeba complex,and Giardia intestinalis from clinical stool samples

The aim of this study was to evaluate the EasyScreen™ Enteric Parasite Detection Kit (Genetic Signatures, Sydney, Australia) for the detection and identification of 5 common enteric parasites: Blastocystis spp., Cryptosporidium spp., Dientamoeba fragilis, Entamoeba complex, and Giardia intestinalis in human clinical samples. A total of 358 faecal samples were included in the study. When compared to real-time PCR and microscopy, the EasyScreen™ Enteric Parasite Detection Kit exhibited 92–100% sensitivity and 100% specificity and detected all commonly found genotypes and subtypes of clinically important human parasites. No cross reactivity was detected in stool samples containing various other bacterial, viral, and/or protozoan species. The EasyScreen™ PCR assay was able to provide rapid, sensitive, and specific simultaneous detection and identification of the 5 most important diarrhoea-causing enteric parasites that infect humans. It should be noted, however, that the EasyScreen™ Kit does not substitute for microscopy or for additional PCRs as it does not detect the pathogenic Coccidia spp. Cystoisospora belli or Cyclospora cayetanensis and it does not differentiate between pathogenic and nonpathogenic Entamoeba spp. This study also highlights the lack of sensitivity demonstrated by microscopy; as such, molecular methods should be considered the diagnostic method of choice for enteric parasites.     

Laboratory diagnosis of chancroid using species-specific primers from Haemophilus ducreyi groEL and the polymerase chain reaction

To enhance laboratory identification of Haemophilus ducreyi, the causative agent of the genital ulcer disease chancroid, a polymerase chain reaction (PCR) assay was developed using target DNA sequences from the essential H. ducreyi gene, groEL. Positive reactions were obtained in this PCR assay with 139 isolates of H. ducreyi from patients in worldwide locations from the 1940s to the 1990s. In contrast, 24 other bacterial species were negative. When genital ulcer specimens from 162 African patients with clinically diagnosed chancroid were evaluated, 66 were culture positive. The sensitivity of PCR as compared with culture was 89% (59 of 66), and specificity was 79% (76 of 96). However, representative samples of the 20 culture-negative, PCR-positive specimens were confirmed as positive by a second PCR assay using different H. ducreyi-specific primers. Thus, combined results of culture and PCR detected H. ducreyi in 86 specimens, with resolved sensitivities of 92% (79 of 86) for PCR, and 77% (66 of 86) for culture. These results suggest that PCR assays for H. ducreyi have great potential for augmenting or replacing problematic cultural techniques.     

Development of a multiplex polymerase chain reaction assay for diarrheagenic Escherichia coli and Shigella spp. and its evaluation on colonies, culture broths, and stool

Detection of diarrheagenic Escherichia coli (DEC) typically depends on identification of virulence genes from stool cultures, not on stool itself. We developed a multiplex polymerase chain reaction (PCR) assay that detects key DEC virulence genes (stx1, stx2, eae, bfpA, ipaH, LT, STh, aaiC, aatA). The assay involved a multiplex PCR reaction followed by detection of amplicon(s) using Luminex beads. The assay was evaluated on over 100 colony and broth specimens. We then evaluated the assay using DNA extracted from stool, colony pools, and Gram-negative broths, using stool spiked with known quantities of DEC. Performance of the assay on stool DNA was most quantitative, while stool broth DNA offered the lowest limit of detection. The assay was prospectively evaluated on clinical specimens in Tanzania. Stool DNA yielded higher sensitivity than colony pools compared with broth DNA as the standard. We propose using this assay to screen for DEC directly in stool or stool broths.     

Evaluation of Seeplex® STD6 ACE Detection kit for the diagnosis of six bacterial sexually transmitted infections

Traditionally, the diagnosis of bacterial sexually transmitted infection (STI) has been dependent on the isolation of the causative pathogens by culturing endocervical or urethral swab specimens on selective media. While such procedures typically provide excellent diagnostic accuracy, they are often time-consuming and expensive. A multiplex polymerase chain reaction (PCR) assay, based on a semi-automated detection system, was evaluated for the detection of six STI causative organisms. The Seeplex^® STD6 ACE (auto-capillary electrophoresis) Detection assay employed six pairs of dual priming oligonucleotide (DPO?) primers specifically targeted to unique genes of Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, Ureaplasma urealyticum, Mycoplasma hominis, and Trichomonas vaginalis. A total of 739 specimens (304 cervical swabs and 435 urine samples) collected for 4 months were tested, and results were compared to those obtained with a combined monoplex PCR. The concordance between the multiplex PCR and monoplex PCR assay was 100% for both sensitivity and specificity. We also tested for the presence of two pathogenic bacteria (C. trachomatis and N. gonorrhoeae) and compared the results obtained with the multiplex PCR and BD ProbeTec duplex strand displacement amplification (SDA). The results of the multiplex PCR and duplex SDA were 99.7% concordant for C. trachomatis and 100% concordant for N. gonorrhoeae. The multiplex PCR assay using the Seeplex^® STD6 ACE Detection kit proved to be a novel cost-effective and fast diagnostic tool with high sensitivity and specificity for the simultaneous detection of six STI pathogens.     

A new chromogenic agar medium,chromID VRE,to screen for vancomycin-resistant Enterococcus faecium and Enterococcus faecalis

We compared the performance of a chromogenic agar medium chromID VRE (bioMérieux, Marcy-l'Etoile, France) designed to recover and identify vancomycin-resistant enterococci (VRE) from clinical specimens with bile esculin azide vancomycin (BEAV) agar. For this study, 120 stool specimens were plated on chromID VRE and BEAV and examined after 24 and 48 h. At 24 h, the sensitivity and specificity were as follows: BEAV, 90.2% and 73%, respectively; chromID VRE, 86.3% and 100.0%, respectively. Furthermore, we determined that the sensitivity and specificity of chromID VRE for Enterococcus faecium were 85.4% and 100%, respectively, and for Enterococcus faecalis, 90% and 100%, respectively. We conclude that chromID VRE provides an equivalent sensitivity for the recovery of VRE from stool specimens, with improved specificity, and the added advantage of providing differentiation between vancomycin-resistant E. faecium and E. faecalis.     

Comparison between ImmunoCard STAT! and real-time PCR as screening tools for both O157:H7 and non-O157 Shiga toxin-producing Escherichia coli in Southern Alberta,Canada

An increasing number of non-O157 Shiga toxin-producing Escherichia coli (STEC) infections and outbreaks have been reported. In this study, we evaluated the performance of ImmunoCard STAT!^® (Meridian Bioscience, Inc., Cincinnati, OH, USA) as a method to screen stool specimens for STEC (O157 and non-O157). An in-house real-time PCR method was used as the “gold standard”. We also evaluated the prevalence and clinical characteristics of STEC infections in the Alberta South West Zone. From July to November 2011, 819 stool specimens submitted for routine stool culture were tested. With our in-house real-time PCR, 7 O157:H7 and 10 non-O157 STEC isolates were identified for a total of 17 STECs. In comparison, ImmunoCard STAT!^® identified a total of 6, resulting in a sensitivity and specificity of 35% and 99%, respectively (P < 0.05). Because of the low sensitivity, ImmunoCard STAT!^® cannot be recommended as a routine screening test for STEC from enriched stool specimens. The rate of STEC positivity as detected by PCR was 2.08%, of which 0.86% was O157:H7 and 1.22% non-O157 STEC. Five of the 7 cases of STEC O157 infection experienced bloody diarrhea, and 1 developed hemolytic uremic syndrome.     

Diagnosis of Legionella pneumophila,Mycoplasma pneumoniae,or Chlamydia pneumoniae lower respiratory infection using the polymerase chain reaction on a single throat swab specimen

Diagnosis of Mycoplasma pneumoniae and Chlamydia pneumoniae lower respiratory infections using DNA amplification by polymerase chain reaction (PCR) on throat swab specimens has been reported. In this study we determined the sensitivity of the detection of Legionella pneumophila in simulated throat swab specimens by PCR. Next, we compared the sensitivity and specificity of a single throat swab PCR with the current tests for diagnosis of Legionella spp., M. pneumoniae, and C. pneumoniae in patients with lower respiratory tract infections. Patients' work-up included: (a) throat swab speciment for Legionella spp., M. pneumoniae, and C. pneumoniae PCR; (b) throat swab specimen for C. pneumoniae, culture; (c) sputum specimen for L. pneumophila direct fluorescent antibody and culture; (d) urine specimen for L. pneumophila serogroup 1 antigen detection; and (e) serum specimen for L. pneumophila, M. pneumoniae, and C. pneumoniae acute and convalescent antibody titers. A total of 155 patients with lower respiratory infection were enrolled in this prospective study. Throat swab PCR was positive for Legionella spp. in five of the six patients with legionellosis, indicating the presence of this organism in the oropharynx of patients with Legionnaires disease. Mycoplasma pneumoniae PCR was positive in eight of the nine patients with mycoplasma infection. Chlamydia pneumoniae PCR was positive in the two patients with C. pneumoniae infection. None of the other 138 patients with negative PCR had other positive confirmatory tests for respiratory infection by these three organisms (100% specificity). PCR was able to detect 15 of the 17 infected (88.2%). Results of this investigation indicate that PCR on a single throat swab specimen is a rapid, sensitive, and specific test that may greatly simplify the diagnosis of lower respiratory infection caused by Legionella spp., Mycoplasma pneumoniae, or C. pneumoniae.     

利用PCR快速检测粪便标本中的鸭沙门菌

目的 建立应用PCR技术鉴定鸭沙门菌血清型的方法,并评价其特异度、灵敏度和检测下限。方法 从GenBank下载截至2015年8月的所有沙门菌基因组序列进行比对,得到鸭沙门菌血清型特异的基因序列,设计引物,建立普通PCR检测体系并验证引物的特异度和灵敏度。同时利用模拟粪便样本确定其检测下限。结果 利用鸭沙门菌特异基因AW58_15605建立的PCR检测方法,对全部鸭沙门菌纯菌及其临床样本的扩增结果均为阳性,而对鸭沙门菌血清型以外的沙门菌和肠道菌的扩增结果均为阴性,检测的特异度和灵敏度均为100%。以粪便模拟样本提取DNA为模板进行检测,增菌后该方法检测下限的灵敏度明显提高,样本经过夜选择性增菌后,检测下限可达59.1 cfu/g,较增菌前提高105倍。结论 建立了一种特异度、灵敏度高的筛查鸭沙门菌血清型方法,为快速、准确鉴定鸭沙门菌和及时处置其造成的公共卫生问题有重要作用。     

Evaluation of iNtRON VRE vanA/vanB real-time PCR for follow-up surveillance of VRE-infected or colonized patients

The iNtRON vancomycin-resistant enterococci (VRE) vanA/vanB real-time PCR assay was directly applied to stool surveillance (direct-PCR). direct-PCR was compared to 2 culture-based methods using Enterococcosel broth (enrichment-culture) and ChromID VRE media. The positive broth of the enrichment-culture was submitted to phenotypic confirmation of subcultured colonies and genotyping by Seeplex VRE PCR. From September 2011 to May 2012, 208 stool specimens from 188 patients previously positive for VRE were enrolled. Enrichment-culture and direct-PCR detected 178 and 158 positives, respectively. Among 129 specimens cultured with ChromID, direct-PCR and ChromID yielded 105 and 104 positives, respectively. Compared to the enrichment-culture, sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of direct-PCR and ChromID were 86.0%, 83.3%, 96.8%, 50.0%, and 89.5%, 86.7%, 98.1%, 52.0%, respectively. Considering the excellent PPV and low NPV, direct-PCR would be useful to monitor VRE-colonized or infected patients on the day, but enrichment-culture is required for direct-PCR-negative specimens.     

A commercially available multiplex real-time PCR for detection of pathogens in cardiac valves from patients with infective endocarditis

Infective endocarditis (IE) is a life-threatening condition, burdened by high mortality. Current guidelines recommend that, in case of negative culture result, tissues from excised heart valves or vegetations from patients with suspected IE should be referred for broad-range bacterial PCR and sequencing. In this proof-of-concept study, the diagnostic utility of the commercially available multiplex real-time PCR system SeptiFast (SF), performed on cardiac valves, was evaluated in a selected population of 20 patients with definite IE of known origin, in comparison with culture. A significant difference was found between SF and culture in the rate of pathogen detection (19 versus 3 respectively; chi-square 14.06; P = 0.0002). SF sensitivity was 95%; specificity, 100%; positive predictive value (PPV), 100%; and negative predictive value (NPV), 83.3%. Culture sensitivity was 15%; specificity, 100%; PPV, 100%; and NPV, 22.7%. SF assay, performed on culture-negative excised heart valves, can be useful for the etiological diagnosis of IE.     

Detection of pathogenic Leptospira spp. through TaqMan polymerase chain reaction targeting the LipL32 gene

Rapid diagnosis of leptospirosis, through culture and/or serology, can be difficult without proper expertise and is often delayed because of the length of time required to obtain results. In this study, we developed a real-time polymerase chain reaction (PCR) assay using a TaqMan probe targeting lipL32, which is present only in pathogenic Leptospira spp. Using Leptospira interrogans serovar Icterohaemorrhagiae DNA, the lower limit of detection was found to be 20 genomic equivalents/reaction with a 95% cutoff value. The assay detected pathogenic Leptospira strains, but not intermediately pathogenic or nonpathogenic strains. When testing the assay on spiked clinical specimens, whole blood and plasma were better specimens for detecting the same initial number of leptospires compared with serum from clotted and centrifuged blood. Leptospira spiked at the same concentration was better detected in centrifuged urine. This real-time PCR assay with high specificity and sensitivity may prove to be a rapid method for diagnosing acute leptospirosis.     

Apyrase-based colorimetric test for detection of Shigella and enteroinvasive Escherichia coli in stool

For lack of simple inexpensive early detection methods for Shigella spp. and enteroinvasive Escherichia coli (EIEC), bacillary dysentery remains a major cause of childhood mortality and morbidity in India and other developing countries. Rapid stool testing for apyrase, a specific periplasmic enzyme essential for the pathogen's intracellular spread, may provide a solution. We have developed a whole-cell colorimetric pyrophosphate hydrolysis assay based on cheap, stable, and locally available reagents. An innovative filtration–cum–inoculation step eliminates interfering stool solids and ensures sufficient bacterial growth and apyrase expression in 6 to 7 h at 37 °C. In a limited double-blind study of 57 clinical isolates of common enterobacteria, the test showed 100% sensitivity and 80% specificity for Shigella spp. and EIEC. Requiring only widely available equipment and inexpensive consumables, this affordable test is readily adaptable for determining antibiograms and for surveillance of food and water samples for the presence of Shigella and EIEC.     

Development of a single-tube polymerase chain reaction assay for the simultaneous detection of Haemophilus influenzae, Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus spp. directly in clinical samples

This study describes the development and evaluation of a multiplex single-tube polymerase chain reaction assay for the simultaneous detection of Haemophilus influenzae, Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus spp. used as target species-specific or genus-specific genes. The assay enables the detection of 5 to 50 pg of bacterial DNA. The sensitivity of the assay was evaluated as 100% for P. aeruginosa, S. aureus, and Streptococcus spp., and 94.3% for H. influenzae; the specificity was 100% for all 4 microorganisms (positive predictive value, 100%; negative predictive value, 98.2%). The assay permits rapid and accurate detection of these 4 microorganisms in a wide range of clinical samples such as whole blood, cerebrospinal, ear, pleural and ophthalmic fluids, as well as bronchoalveolar lavage and bronchial secretions.     

Comparison of staining techniques and multiplex nested PCR for diagnosis of intestinal microsporidiosis

Microsporidiosis is increasingly being recognized as the cause for diarrhea in immunocompromised patients. The 2 most common microsporidia causing gastrointestinal infection worldwide are Enterocytozoon bieneusi and Encephalitozoon intestinalis. The aim of present study was to evaluate different techniques for detection of intestinal microsporidia in human stool samples. The fecal samples of 395 individuals including 125 HIV-seropositive patients with diarrhoea, 158 HIV-seropositive patients without diarrhoea, 55 HIV-seronegative patients with diarrhoea, and 57 healthy controls were used for detection of microsporidia by modified trichrome staining, calcofluor staining, and multiplex polymerase chain reaction (PCR). PCR had the highest sensitivity of 100%, while its specificity was 97.9%. Trichrome staining had highest specificity of 100% but a sensitivity of 63.8% only, and calcofluor white had a sensitivity and specificity of 79.7% and 82.2%, respectively. Thus, for diagnosis of intestinal microsporidiosis, it is important to perform PCR as staining techniques are not good enough to detect microsporidia in stool samples and for their species identification.     

Real-time Leishmania genus master mix: a platform compatibility and stability study

Performing diagnostics and vector-pathogen surveillance in austere environments is challenging. On-site diagnostic/detection mitigates vector-borne disease complications during military or humanitarian deployments to disease endemic locals. The mobile molecular diagnostic platform, Joint Biological Agent Identification and Diagnostic System (JBAIDS; BioFire Diagnostics Inc., Salt Lake City, UT, USA), rapidly identifies biothreat pathogens. Although ideal for remote diagnostics, the platform was validated for specific pathogens of insignificant epidemiological consequence. Recognizing the JBAIDS's remote diagnostic/detection versatility, we tested a Leishmania genus real-time PCR master mix validated for use on the SmartCycler® (Cepheid, Sunnyvale, CA, USA) for concomitant use on the JBAIDS. We evaluated assay sensitivity, precision, and specificity of one or more Leishmania spp. on the JBAIDS and found that the JBAIDS produces superior detection sensitivity and specificity compared to the SmartCycler®. We also examined the storage stability of a bulk lot preparation of the Leishmania genus real-time PCR master mix on the SmartCycler® to ensure that long periods of frozen storage that would translate to a field environment with the JBAIDS were not detrimental to the reagent. We found that the bulk master mix maintains its stability over a 13-month time period. Overall, these studies confirm JBAIDS's versatility and demonstrate a streamlined assay development approach where reagents are compatible with both platforms.     

Detection of Haemophilus influenzae in respiratory secretions from pneumonia patients by quantitative real-time polymerase chain reaction

A quantitative real-time polymerase chain reaction (PCR) based on the omp P6 gene was developed to detect Haemophilus influenzae. Its specificity was determined by analysis of 29 strains of 11 different Haemophilus spp. and was compared with PCR assays having other target genes: rnpB, 16S rRNA, and bexA. The method was evaluated on nasopharyngeal aspirates from 166 adult patients with community-acquired pneumonia. When 10⁴ DNA copies/mL was used as cutoff limit for the method, P6 PCR had a sensitivity of 97.5% and a specificity of 96.0% compared with the culture. Of 20 culture-negative but P6 PCR-positive cases, 18 were confirmed by fucK PCR as H. influenzae. Five (5.9%) of 84 nasopharyngeal aspirates from adult controls tested PCR positive. We conclude that the P6 real-time PCR is both sensitive and specific for identification of H. influenzae in respiratory secretions. Quantification facilitates discrimination between disease-causing H. influenzae strains and commensal colonization.     

Direct simultaneous detection of 6 sexually transmitted pathogens from clinical specimens by multiplex polymerase chain reaction and auto-capillary electrophoresis

The availability of a reliable and user-friendly method to identify pathogens causing sexually transmitted diseases (STDs) is essential to reduce the complications and spread of infection. In this study, genital/urinary specimens from 113 patients with STDs were simultaneously tested for 6 pathogens using the automated Seeplex® (Seegene, Seoul, Korea) multiplex polymerase chain reaction (PCR)-based STD6B auto-capillary electrophoresis (ACE) system. The results were compared with conventional reference methods, including culture and PCR tests. The sensitivity of STD6B ACE was found to be 100% for Chlamydia trachomatis, Mycoplasma hominis, Mycoplasma genitalium, Neisseria gonorrhoeae, and Trichomonas vaginalis, and 98% for genital Ureoplasma (U. urealyticum and U. parvum). Specificity ranged from 97% to 100%. One pathogen was detected in 51 specimens, and 2 or more pathogens were detected in 24. In conclusion, the multiplex PCR and ACE system is highly sensitive and specific for the rapid, simultaneous detection of STD pathogens directly from a single specimen.     

Evaluation of a new multiplex polymerase chain reaction assay STDFinder for the simultaneous detection of 7 sexually transmitted disease pathogens

We evaluated a new multiplex polymerase chain reaction (mPCR), “STDFinder assay”, a novel multiplex ligation-dependent probe amplification (MLPA) assay for the simultaneous detection of 7 clinically relevant pathogens of STDs, i.e., Neisseria gonorrhoeae, Chlamydia trachomatis, Trichomonas vaginalis, Mycoplasma genitalium, Treponema pallidum, and herpes simplex virus type 1 and 2 (HSV-1 and HSV-2). An internal amplification control was included in the mPCR reaction. The limits of detection for the STDFinder assay varied among the 7 target organisms from 1 to 20 copies per MLPA assay. There were no cross-reactions among any of the probes. Two hundred and forty-two vaginal swabs and an additional 80 specimens with known results for N. gonorrhoeae and C. trachomatis, obtained from infertile women seen at an infertility research clinic at the Kigali Teaching Hospital in Rwanda, were tested by STDFinder assay and the results were confirmed by single real-time PCR using different species-specific targets. Compared to the reference standard, the STDFinder assay showed specificities and sensitivities of 100% and 100%, respectively, for N. gonorrhoeae, C. trachomatis, and M. genitalium; 90.2% and 100%, respectively, for Trichomonas vaginalis; and 96.1% and 100%, respectively, for HSV-2. No specimen was found to be positive for HSV-1 by either the STDFinder assay or the comparator method. Similarly, the sensitivity for Treponema pallidum could not be calculated due to the absence of any Treponema pallidum-positive samples. In conclusion, the STDFinder assays have comparable clinical sensitivity to the conventional mono and duplex real-time PCR assay and are suitable for the routine detection of a broad spectrum of these STDs at relatively low cost due to multiplexing.     

Rapid and accurate detection of Pseudomonas aeruginosa by real-time polymerase chain reaction with melting curve analysis targeting gyrB gene

Laboratory detection of Pseudomonas spp., particularly Pseudomonas aeruginosa, is an important assay in the nosocomial control. The study was designed firstly to establish a new assay-applied LightCycler polymerase chain reaction (PCR) technology with melting curve analysis (MCA). A total of 224 Gram-negative isolates were used to verify the assay system. The PCR with MCA method using the P. aeruginosa-specific gyrase B gene primers was rapid and accurate; the total run is approximately 3 h, and the sensitivity and specificity relative to the Vitek (bioMerieux, Hazelwood, MO) results were 98.1% and 100%, respectively. Vitek identification system was not able to identify the isolates from the new Pseudomonas otitidis spp. opposite to the real-time PCR. This assay was validated to be accurate with an overall sensitivity and specificity of 98.7% and 98.9%, respectively. Conclusively, this rapid and accurate PCR assay with MCA will help to manage and control infections with P. aeruginosa.