Detection and subtyping of Herpes simplex virus in clinical samples by LightCycler PCR, enzyme immunoassay and cell culture

Background  

Prompt laboratory diagnosis of Herpes simplex virus (HSV) infection facilitates patient management and possible initiation of antiviral therapy. In our laboratory, which receives various specimen types for detection of HSV, we use enzyme immunoassay (EIA) for rapid detection and culture of this virus. The culture of HSV has traditionally been accepted as the diagnostic 'gold standard'. In this study, we compared the use of real time PCR (LightCycler) for amplification, detection and subtyping of specific DNA with our in-house developed rapid and culture tests for HSV.     

Development of real-time PCR for the detection of Clostridium perfringens in meats and vegetables

A real-time PCR assay was developed and validated inhouse specifically for the detection of Clostridium perfringens (Cl. perfringens) in meats and vegetables by comparing with the culture method. The detection limit of the real-time PCR assay in phosphate-buffered saline was 102 CFU/ml. When the two methods were compared in food samples inoculated with Cl. perfringens, the culture method detected 52 positives, whereas real-time PCR detected 51 positives out of 160 samples. The difference was without statistical significance (p>0.05). Real-time PCR assay is an option for quality assurance laboratories to perform standard diagnostic tests, considering its detection ability and time-saving efficiency.     

Comparison of Dermatophyte PCR Kit with Conventional Methods for Detection of Dermatophytes in Skin Specimens

The laboratory diagnosis of dermatophytosis is usually based on direct microscopic examination and culturing of clinical specimens. A commercial polymerase chain reaction kit (Dermatophyte PCR) has had favorable results when used for detection of dermatophytes and identification of Trichophyton rubrum in nail specimens. This study investigated the efficacy of the Dermatophyte PCR kit for detecting dermatophytosis in 191 hair or skin specimens from patients with suspected dermatophytosis. PCR was positive for 37 % of samples, whereas 31 and 39 % of the specimens were positive by culturing and direct microscopy, respectively. The sensitivity, specificity, positive predictive value, and negative predictive value for PCR analysis were 83, 84, 71, and 91 %, respectively. The sensitivity of the PCR test was higher in specimens obtained from skin (88 %) than in those obtained from hair (58 %), while the specificity remained almost the same (84 and 86 % for skin and hair, respectively). Our results show that the Dermatophyte PCR kit is a promising diagnostic tool for detection of dermatophytosis in skin samples, providing clinicians with a rapid diagnosis.     

Detection of Mycoplasma pneumoniae in respiratory samples by real-time PCR using an inhibition control

Polymerase chain reaction (PCR) with real-time detection using two adjacent fluorescent probes in a Lightcycler instrument was applied for detection of the Mycoplasma pneumoniae P1 protein gene. To monitor inhibition in each sample an internal control was constructed that can be amplified by the same primers but detected by different probes and dual color detection. The real-time PCR was applied on 115 respiratory samples from 82 patients and compared to a conventional PCR. There was 100% agreement between the assays, but the real-time PCR proved to be highly superior in speed with a much lower risk of false positives by laboratory contamination.     

Diagnosis of Dermatophytosis Using Molecular Biology

Identification of fungi in dermatological samples using PCR is reliable and provides significantly improved results in comparison with cultures. It is possible to identify the infectious agent when negative results are obtained from cultures. In addition, identification of the infectious agent can be obtained in 1 day. Conventional and real-time PCR methods used for direct fungus identification in collected samples vary by DNA extraction methods, targeted DNA and primers, and the way of analysing the PCR products. The choice of a unique method in a laboratory is complicated because the results expected from skin and hair sample analysis are different from those expected in cases of onychomycosis. In skin and hair samples, one dermatophyte among about a dozen possible species has to be identified. In onychomycosis, the infectious agents are mainly Trichophyton rubrum and, to a lesser extent, Trichophyton interdigitale, but also moulds insensitive to oral treatments used for dermatophytes, which renders fungal identification mandatory. The benefits obtained with the use of PCR methods for routine analysis of dermatological samples have to be put in balance with the relative importance of getting a result in a short time, the price of molecular biology reagents and equipment, and especially the time spent conducting laboratory manipulations.     

Comparison of different PCR methods for detection of Brucella spp. in human blood samples

For detection of Brucella species by PCR four DNA extraction methods and four targets were compared using pure culture of Brucella melitensis and the best conditions were applied in clinical samples. It was found that the MagNA Pure LC method was the most efficient and sensitive method showing a positive PCR reaction with DNA extracted from as low as 25 and 100 CFU suspended in one ml blood and one ml water, respectively. Detection of Brucella spp. by conventional PCR was investigated using four different targets. The results indicated that The B4-B5 amplification method was the most sensitive one as it could amplify DNA extracted from as a low as 25 and 100 CFU/ml suspended in one ml water and blood, respectively. Furthermore real-time PCR was able to detect Brucella using DNA extracted from as low as 50 CFU/ml blood and 15 CFU/ml water, respectively. The best and optimum detection conditions were applied to the clinical samples. Evaluation of conventional PCR assays on blood specimens confirmed 72% of the results obtained by conventional blood culture methods with a specificity of 95%, while serum samples had a sensitivity of 54% and specificity of 100%. Real-time PCR was generally found to be more sensitive and specific for detecting Brucella spp. in blood and serum samples compared to conventional PCR. The real-time PCR done on blood specimens confirmed 77.5% of the results obtained by conventional blood culture methods with specificity of 100%, while 60% of serum samples were found to be positive with specificity of 100%. These results suggest that serum and blood analysis by conventional and real time PCR is a convenient and safe method for rapid and accurate diagnosis of brucellosis.     

Detection of Mycobacterium avium subspecies paratuberculosis in Swiss dairy cattle by real-time PCR and culture: a comparison of the two assays

AIMS: To compare the two different diagnostic assays for the detection of Mycobacterium avium ssp. paratuberculosis, the aetiological agent of paratuberculosis. METHODS AND RESULTS: Faecal samples were derived from 310 cows, representing 13 commercial dairy herds in various locations in Switzerland with expected increased risk because of a past history of disease. Detection assays for M. avium ssp. paratuberculosis were culture (gold standard) and a newly designed real-time PCR. Real-time PCR identified 31 of 310 animals as positive within this risk population whereas culture identified 20 positive animals. The specificity of real-time PCR was confirmed by DNA sequencing of the PCR product. Depending on the test used, the paratuberculosis prevalence in our tested risk population ranged from 6.5 to 10%. CONCLUSIONS: Real-time PCR and culture data were in good agreement, and real-time PCR generates data in a short time in contrast to culture. SIGNIFICANCE AND IMPACT OF THE STUDY: We consider real-time PCR as a suitable alternative method to culture for the detection of M. avium ssp. paratuberculosis in a national surveillance programme.     

Quantification of enterococci and human adenoviruses in environmental samples by real-time PCR

Pathogenic bacteria and enteric viruses can be introduced into the environment via human waste discharge. Methods for rapid detection and quantification of human viruses and fecal indicator bacteria in water are urgently needed to prevent human exposure to pathogens through drinking and recreational waters. Here we describe the development of two real-time PCR methods to detect and quantify human adenoviruses and enterococci in environmental waters. For real-time quantification of enterococci, a set of primers and a probe targeting the 23S rRNA gene were used. The standard curve generated using Enterococcus faecalis genomic DNA was linear over a 7-log-dilution series. Serial dilutions of E. faecalis suspensions resulted in a lower limit of detection (LLD) of 5 CFU/reaction. To develop real-time PCR for adenoviruses, degenerate primers and a Taqman probe targeting a 163-bp region of the adenovirus hexon gene were designed to specifically amplify 14 different serotypes of human adenoviruses, including enteric adenovirus serotype 40 and 41. The standard curve generated was linear over a 5-log-dilution series, and the LLD was 100 PFU/reaction using serial dilutions of purified adenoviral particles of serotype 40. Both methods were optimized to be applicable to environmental samples. The real-time PCR methods showed a greater sensitivity in detection of adenoviruses in sewage samples than the viral plaque assay and in detection of enterococci in coastal waters than the bacterial culture method. However, enterococcus real-time PCR overestimated the number of bacteria in chlorinated sewage in comparison with the bacterial culture method. Overall, the ability via real-time PCR to detect enterococci and adenoviruses rapidly and quantitatively in the various environmental samples represents a considerable advancement and a great potential for environmental applications.     

Evaluation of a rapid and inexpensive liquid culture system for the detection of Mycobacterium avium subsp. paratuberculosis in bovine faeces

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of paratuberculosis, a chronic granulomatous enteric disease of ruminants. MAP detection by faecal culture provides the definitive diagnosis of the infection. Automated liquid systems for MAP culture are more sensitive and rapid than culture on solid media, but they are expensive and require specialised equipment. In this study, a non-automated culture method using a modified Middlebrook 7H9 liquid medium (7H9+) was compared with Herrold's solid medium (HEYM) and direct real-time PCR on dairy cattle faeces. MAP growth in 7H9+ was monitored weekly by real-time PCR until the 12th week post-inoculation. The analytical sensitivity of the three methods was evaluated using faecal samples from a healthy cow spiked with ten-fold dilutions of MAP organisms (10⁴-10^− 1) and naturally MAP-infected faeces serially diluted 1 to 10 in negative faecal samples. The limits of detection of the solid culture and direct real-time PCR were 10² and 10³ MAP/g, respectively. In comparison, the 7H9+ culture revealed as few as 1 MAP/g. A marked reduction in time to detection of the pathogen, compared with HEYM culture, was obtained. In addition, the three methods were applied to environmental faecal samples collected from a high- and a low-prevalence herd. The culture in 7H9+ showed to be the most sensitive test in the low-prevalence herd and provided faster results than HEYM. In the high-prevalence herd the three methods showed the same sensitivity and the real-time PCR had the shortest turnaround time. In conclusion, the use of 7H9+ for MAP-detection from cattle faeces maximizes diagnostic sensitivity and reduces turnaround time and, therefore, could replace culture in solid medium. Hence, we propose a two-step protocol for the assessment of MAP faecal excretion based on: 1) direct real-time PCR on all samples; and 2) inoculation of negative samples into 7H9+ and analysis after 3 and, if necessary, 6 weeks by real-time PCR.     

Comparison of polymerase chain reaction and conventional culture for the detection of legionellas in hospital water samples

A detection system for Legionella spp. based on the polymerase chain reaction (PCR) was used to assess the diagnostic value of PCR for the surveillance of contamination of man-made water systems by legionellas. A previously-published primer system was chosen to amplify a fragment of the 5S-ribosomal gene of Legionella spp. A total of 78 water samples from various sources were examined by PCR and culture on MWY Legionella selective agar. Fifty-seven of 78 water samples were positive by both test systems (73%), nine were positive by PCR only (11.5%), another nine were positive by culture but negative by PCR (11.5%), and three were negative by both techniques (3.8%). The PCR was inhibited when large amounts of rust were present in the samples. Culture failed to detect legionellas in samples that contained large numbers of other bacteria capable of overgrowing the legionellas. These results show that PCR is a rapid and sensitive technique for the detection of legionella contamination in water samples and that PCR and culture complement each other in monitoring of environmental water samples.     

A real-time PCR antibiogram for drug-resistant sepsis

Current molecular diagnostic techniques for susceptibility testing of septicemia rely on genotyping for the presence of known resistance cassettes. This technique is intrinsically vulnerable due to the inability to detect newly emergent resistance genes. Traditional phenotypic susceptibility testing has always been a superior method to assay for resistance; however, relying on the multi-day growth period to determine which antimicrobial to administer jeopardizes patient survival. These factors have resulted in the widespread and deleterious use of broad-spectrum antimicrobials. The real-time PCR antibiogram, described herein, combines universal phenotypic susceptibility testing with the rapid diagnostic capabilities of PCR. We have developed a procedure that determines susceptibility by monitoring pathogenic load with the highly conserved 16S rRNA gene in blood samples exposed to different antimicrobial drugs. The optimized protocol removes heme and human background DNA from blood, which allows standard real-time PCR detection systems to be employed with high sensitivity (<100 CFU/mL). Three strains of E. coli, two of which were antimicrobial resistant, were spiked into whole blood and exposed to three different antibiotics. After real-time PCR-based determination of pathogenic load, a ΔC(t)<3.0 between untreated and treated samples was found to indicate antimicrobial resistance (P<0.01). Minimum inhibitory concentration was determined for susceptible bacteria and pan-bacterial detection was demonstrated with 3 gram-negative and 2 gram-positive bacteria. Species identification was performed via analysis of the hypervariable amplicons. In summary, we have developed a universal diagnostic phenotyping technique that assays for the susceptibility of drug-resistant septicemia with the speed of PCR. The real-time PCR antibiogram achieves detection, susceptibility testing, minimum inhibitory concentration determination, and identification in less than 24 hours.     

Application of quantitative real-time PCR for rapid identification of Bacteroides fragilis group and related organisms in human wound samples

Our goal was to establish a quantitative real-time PCR (QRT-PCR) method to detect Bacteroides fragilis group and related organisms from clinical specimens. Compared to conventional anaerobic culture, QRT-PCR can provide accurate and more rapid detection and identification of B.?fragilis group and similar species. B.?fragilis group and related organisms are the most frequently isolated anaerobic pathogens from clinical samples. However, culture and phenotypic identification is quite time-consuming. We designed specific primers and probes based on the 16S rRNA gene sequences of Bacteroides caccae, Bacteroides eggerthii, B.?fragilis, Bacteroides ovatus, Bacteroides stercoris, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroides vulgatus, Odoribacter splanchnicus (Bacteroides splanchnicus), Parabacteroides distasonis (Bacteroides distasonis) and Parabacteroides merdae (Bacteroides merdae), and detected these species by means of QRT-PCR in 400 human surgical wound infection samples or closed abscesses. The target bacteria were detected from 31 samples (8%) by culture, but from 132 samples (33%) by QRT-PCR (p-value?相似文献   

A rapid and quantitative method for the detection of Leptospira species in human leptospirosis

Prompt laboratory diagnosis of leptospirosis infection facilitates patient management and initiation of therapy. A cost effective real-time PCR assay using SYBR Green I was developed for detection of pathogenic leptospires in serum specimens. Specific PCR products were obtained only with DNA of pathogenic Leptospira genomospecies. LightCycler PCR ability to distinguish between species was possible using melting curves, providing an approach for identification with a specific Tm assigned to a single species or set of species. Assay sensitivity was approximately 50 leptospires/ml, corresponding to one to two genome copies in a PCR mixture. Fifty-one patients who had clinical symptoms consistent with leptospirosis were tested both with a previously described rrs amplification and our real-time assay. Our LFB1 real-time assay confirmed the diagnosis for 25 patients (49%, 25/51) and revealed an estimated density of 8.0x10(1)-3.9x10(4) leptospires/ml of blood. The total assay time for 12 clinical samples from sample to data analysis was less than 3 h. These data illustrate the potential of our LFB1 real-time assay for the rapid detection of leptospires in serum samples and their subsequent quantification in a single run.     

Relative Sensitivity of Conventional and Real-Time PCR Assays for Detection of SFG Rickettsia in Blood and Tissue Samples from Laboratory Animals

Studies on the natural transmission cycles of zoonotic pathogens and the reservoir competence of vertebrate hosts require methods for reliable diagnosis of infection in wild and laboratory animals. Several PCR-based applications have been developed for detection of infections caused by Spotted Fever group Rickettsia spp. in a variety of animal tissues. These assays are being widely used by researchers, but they differ in their sensitivity and reliability. We compared the sensitivity of five previously published conventional PCR assays and one SYBR green-based real-time PCR assay for the detection of rickettsial DNA in blood and tissue samples from Rickettsia- infected laboratory animals (n = 87). The real-time PCR, which detected rickettsial DNA in 37.9% of samples, was the most sensitive. The next best were the semi-nested ompA assay and rpoB conventional PCR, which detected as positive 18.4% and 14.9% samples respectively. Conventional assays targeting ompB, gltA and hrtA genes have been the least sensitive. Therefore, we recommend the SYBR green-based real-time PCR as a tool for the detection of rickettsial DNA in animal samples due to its higher sensitivity when compared to more traditional assays.     

PCR-based diagnostics for anaerobic infections

Conventional methods to identify anaerobic bacteria have often relied on unique clinical findings, isolation of organisms, and laboratory identification by morphology and biochemical tests (phenotypic tests). Although these methods are still fundamental, there is an increasing move toward molecular diagnostics of anaerobes. In this review, some of the molecular approaches to anaerobic diagnostics based on the polymerase chain reaction (PCR) are discussed. This includes several technological advances in PCR-based methods for the detection, identification, and quantitation of anaerobes including real-time PCR which has been successfully used to provide rapid, quantitative data on anaerobic species on clinical samples. Since its introduction in the mid-1980s, PCR has provided many molecular diagnostic tools, some of which are discussed within this review. With the advances in micro-array technology and real-time PCR methods, the future is bright for the development of accurate, quantitative diagnostic tools that can provide information not only on individual anaerobic species but also on whole communities.     

头颈部放疗患者口腔假丝酵母菌感染的快速检测

目的通过对传统培养法和PCR法在假丝酵母菌感染检出率的比较,拟探索一种能够早期、快速、高效检测头颈部放疗患者假丝酵母菌感染的方法。方法收集120名头颈部放疗患者唾液,分别应用假丝酵母菌显色培养基（CHROMagar）进行分离、培养和鉴定;同时提取基因组DNA,通过假丝酵母菌通用引物、特异性引物、改良引物进行PCR扩增,结果与假丝酵母菌表型进行对比。结果与传统培养法相比,PCR法检出率更高（χ2=47.672,P=0.000）;改良特异性引物D扩增的检出率为77%,高于通用引物B（χ2=7.702,P=0.006）和特异性引物C（χ2=12.522,P=0.001）。结论本研究证实PCR技术耗时短,阳性检出率高,可用于头颈部肿瘤放疗患者假丝酵母菌感染的快速检测。     

Detection of Candida species by polymerase chain reaction (PCR) in blood samples of experimentally infected mice and patients with suspected candidemia

In this study, we have established and evaluated a genus-specific polymerase chain reaction (PCR) and species-specific nested PCRs for the detection of Candida species in blood samples of neutropenic mice and patients suspected of candidemia. DNA segments of the gene encoding cytochrome P450 L1A1 were targeted for amplification by using genus and species-specific primers. As compared to the genus-specific PCR, the species-specific nested PCRs improved the sensitivity by 10 times with the detection limit < 10 yeast cells. Of the 18 blood samples tested daily over a period of 8 days following Candida albicans infection in neutropenic mice, four samples were positive by genus-specific PCR and 11 were positive by species-specific nested PCR. The PCR results were correlated with culture findings obtained on blood samples. Two of the three blood culture-positive samples were positive by genus-specific PCR and all the three with species-specific nested PCR. Among 15 mice, which were negative by blood culture but had C. albicans isolated from visceral organs, 2 and 8 mice yielded positive results by genus-specific PCR and species-specific nested PCR, respectively. Consistent with the results of the animal study, species-specific nested PCR yielded much higher positivity as compared to culture (52.2% versus 21.2%) in patients suspected for candidemia. Moreover, 8 specimens which were negative for Candida by genus-specific PCR became positive by species-specific nested PCR. No correlation was apparent between PCR positivity and Candida antigen titers. The results suggest that nested PCR is a sensitive technique for the detection of Candida species from blood samples, and thus it may have application in the diagnosis of suspected cases of candidemia and candidiasis.     

Periodontal pathogens: a quantitative comparison of anaerobic culture and real-time PCR

Periodontitis is a multi-factorial chronic inflammatory and destructive disease of the tooth-supporting tissues. Quantitative anaerobic culture techniques have been used for microbial diagnosis of the different forms of the disease. The aim of this study was to compare real-time PCR with quantitative anaerobic culture for detection and quantification of 5 prominent periodontal pathogens. Real-time PCR assays with the 16s rRNA genes of Actinobacillus actinomycetemcomitans, Prevotella intermedia, Tannerella forsythensis, Peptostreptococcus micros and Fusobacterium spp. were developed. The PCR was validated on pure cultures of various bacterial strains. Subsequently, subgingival plaque samples from 259 adult patients with periodontitis were analyzed with quantitative anaerobic culture and real-time PCR. A standard curve for DNA quantification was created for each primer-probe set based on colony-forming units equivalents. All bacterial species were correctly identified. The lower limits of detection by PCR varied between 1-50 colony-forming units equivalents depending on the species. No cross-reactivities with heterologous DNA of other bacterial species were observed. Real-time PCR results showed a high degree of agreement with anaerobic culture results. Real-time PCR is a reliable alternative for diagnostic quantitative anaerobic culture of subgingival plaque samples.     

Quantitative detection of Legionella pneumophila in water samples by immunomagnetic purification and real-time PCR amplification of the dotA gene

A new real-time PCR assay was developed and validated in combination with an immunomagnetic separation system for the quantitative determination of Legionella pneumophila in water samples. Primers that amplify simultaneously an 80-bp fragment of the dotA gene from L. pneumophila and a recombinant fragment including a specific sequence of the gyrB gene from Aeromonas hydrophila, added as an internal positive control, were used. The specificity, limit of detection, limit of quantification, repetitivity, reproducibility, and accuracy of the method were calculated, and the values obtained confirmed the applicability of the method for the quantitative detection of L. pneumophila. Moreover, the efficiency of immunomagnetic separation in the recovery of L. pneumophila from different kinds of water was evaluated. The recovery rates decreased as the water contamination increased (ranging from 59.9% for distilled water to 36% for cooling tower water), and the reproducibility also decreased in parallel to water complexity. The feasibility of the method was evaluated by cell culture and real-time PCR analysis of 60 samples in parallel. All the samples found to be positive by cell culture were also positive by real-time PCR, while only eight samples were found to be positive only by PCR. Finally, the correlation of both methods showed that the number of cells calculated by PCR was 20-fold higher than the culture values. In conclusion, the real-time PCR method combined with immunomagnetic separation provides a sensitive, specific, and accurate method for the rapid quantification of L. pneumophila in water samples. However, the recovery efficiency of immunomagnetic separation should be considered in complex samples.     

Detection of Brucella canis from inguinal lymph nodes of naturally infected dogs by PCR

The aim of this study was to standardize and evaluate a PCR assay for the detection of Brucella canis (B. canis) in lymph node samples of naturally infected dogs. The performance of the PCR was compared with the results of bacteriological culture as reference method. Forty-eight inguinal lymph node samples were collected from 48 dogs (18 males and 30 females) that died in the city's pound in the years 2007-2008 and were examined by microbiological culture and the PCR assay. B. canis was isolated from 4 (8.3%) of 48 lymph node samples. Forty-four (91.7%) of the samples were bacteriological culture negative. B. canis DNA was directly detected from all culture positive lymph node samples (n = 4) by PCR. All of the culture negative samples were confirmed as negative by PCR. When the culture method was used as a gold standard, sensitivity and specificity of the PCR assay were found to be 100%. The limit of PCR detection of B. canis DNA was 1.4 × 10¹ CFU/g at least. In conclusion, the PCR assay has been shown to have a diagnostic performance equal to bacteriological culture for detection of B. canis. By a non-hazardous protocol for laboratory workers, the assay can be performed in one day.