Real-time polymerase chain-reaction detection of pathogens is feasible to supplement the diagnostic sequence for urinary tract infections

To evaluate, in a prospective pilot study, the feasibility of identifying pathogens in urine using real-time polymerase chain reaction (PCR), and to compare the results with the conventional urine culture-based procedures.

Cost and mortality prediction using polymerase chain reaction pathogen detection in sepsis: evidence from three observational trials

Delays in adequate antimicrobial treatment contribute to high cost and mortality in sepsis. Polymerase chain reaction (PCR) assays are used alongside conventional cultures to accelerate the identification of microorganisms. We analyze the impact on medical outcomes and healthcare costs if improved adequacy of antimicrobial therapy is achieved by providing immediate coverage after positive PCR reports.

Primer extension based quantitative polymerase chain reaction reveals consistent differences in the methylation status of the MGMT promoter in diffusely infiltrating gliomas (WHO grade II-IV) of adults

Diffusely infiltrating gliomas (WHO grade II-IV) are the most common primary brain tumours in adults. These tumours are not amenable to cure by surgery alone, so suitable biomarkers for adjuvant modalities are required to guide therapeutic decision-making. Epigenetic silencing of the O(6)-methylguanine-DNA methyltransferase (MGMT) gene by promoter methylation has been associated with longer survival of patients with high-grade gliomas who receive alkylating chemotherapy; and molecular testing for the methylation status of the MGMT promoter sequence is regarded as among the most relevant of such markers. We have developed a primer extension-based assay adapted to formalin-fixed paraffin-embedded tissues that enables quantitative assessment of the methylation status of the MGMT promoter. The assay is very sensitive, highly reproducible, and provides valid test results in nearly 100% of cases. Our results indicate that oligodendrogliomas, empirically known to have a relatively favourable prognosis, are also the most homogeneous entities in terms of MGMT promoter methylation. Conversely, astrocytomas, which are more prone to spontaneous progression to higher grade malignancy, are significantly more heterogeneous. In addition, we show that the degree of promoter methylation correlates with the prevalence of loss of heterozygosity on chromosome arm 1p in the oligodendroglioma group, but not the astrocytoma group. Our results may have potentially important implications for clinical molecular diagnosis.

Comparison of real-time polymerase chain reaction and DNA-strip technology in microbiological evaluation of periodontitis treatment

The impact of a semiquantitative commercially available test based on DNA-strip technology (microIDent®, Hain Lifescience, Nehren, Germany) on diagnosis and treatment of severe chronic periodontitis of 25 periodontitis patients was evaluated in comparison with a quantitative in-house real-time PCR. Subgingival plaque samples were collected at baseline as well as at 3, 6, and 12 months later. After extracting DNA, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, and several other periodontopathogens were determined by both methods. The results obtained by DNA-strip technology were analyzed semiquantitatively and additionally quantitatively by densitometry. The results for the 4 major periodontopathogenic bacterial species correlated significantly between the 2 methods. Samples detecting a high bacterial load by one method and negative by the other were always found in less than 2% of the total samples. Both technologies showed the impact of treatment on microflora. Especially the semiquantitative DNA-strip technology clearly analyzed the different loads of periodontopathogens after therapy and is useful in microbial diagnostics for patients in dental practices.

Detecting and resolving position-dependent temperature effects in real-time quantitative polymerase chain reaction

Real-time quantitative polymerase chain reaction (qPCR) depends on precise temperature control of the sample during cycling. In the current study, we investigated how temperature variation in plate-based qPCR instruments influences qPCR results. Temperature variation was measured by amplicon melting analysis as a convenient means to assess well-to-well differences. Multiple technical replicates of several SYBR Green I-based qPCR assays allowed correlation of relative well temperature to quantification cycle. We found that inadequate template denaturation results in an inverse correlation and requires increasing the denaturation temperature, adding a DNA destabilizing agent, or pretreating with a restriction enzyme. In contrast, inadequate primer annealing results in a direct correlation and requires lowering the annealing temperature. Significant correlations were found in 18 of 25 assays. The critical nature of temperature-dependent effects was shown in a blinded study of 29 patients for the diagnosis of Prader-Willy and Angelman syndromes, where eight diagnoses were incorrect unless temperature-dependent effects were controlled. A method to detect temperature-dependent effects by pairwise comparisons of replicates in routine experiments is presented and applied. Systematic temperature errors in qPCR instruments can be recognized and their effects eliminated when high precision is required in quantitative genetic diagnostics and critical complementary DNA analyses.

Value of a novel Neisseria meningitidis--specific polymerase chain reaction assay in skin biopsy specimens as a diagnostic tool in chronic meningococcemia

BACKGROUND: Chronic meningococcemia (CM) is a diagnostic challenge. Skin lesions are frequent but in most cases nonspecific. Polymerase chain reaction (PCR)-based diagnosis has been validated in blood and cerebrospinal fluid for acute Neisseria meningitidis infection, in patients in whom routine microbiologic tests have failed to isolate the bacteria. In 2 patients with CM, we established the diagnosis by a newly developed PCR-based approach performed on skin biopsy specimens. OBSERVATIONS: Two patients presented with fever together with systemic and cutaneous manifestations suggestive of CM. Although findings from blood cultures remained negative, we were able to identify N meningitidis in the skin lesions by a newly developed PCR assay. In 1 patient, an N meningitidis strain of the same serogroup was also isolated from a throat swab specimen. Both patients rapidly improved after appropriate antibiotherapy. CONCLUSIONS: To our knowledge, we report the first cases of CM diagnosed by PCR testing on skin biopsy specimens. It is noteworthy that, although N meningitidis-specific PCR is highly sensitive in blood and cerebrospinal fluid in acute infections, our observations underscore the usefulness of PCR performed on skin lesions for the diagnosis of chronic N meningitidis infections. Whenever possible, this approach should be systematically employed in patients for whom N meningitidis infection cannot be confirmed by routine microbiologic investigations.

Potential clinical utility of polymerase chain reaction in microbiological testing for sepsis

OBJECTIVES: To evaluate the potential improvement of antimicrobial treatment by utilizing a new multiplex polymerase chain reaction (PCR) assay that identifies sepsis-relevant microorganisms in blood. DESIGN: Prospective, observational international multicentered trial. SETTING: University hospitals in Germany (n = 2), Spain (n = 1), and the United States (n = 1), and one Italian tertiary general hospital. PATIENTS: 436 sepsis patients with 467 episodes of antimicrobial treatment. METHODS: Whole blood for PCR and blood culture (BC) analysis was sampled independently for each episode. The potential impact of reporting microorganisms by PCR on adequacy and timeliness of antimicrobial therapy was analyzed. The number of gainable days on early adequate antimicrobial treatment attributable to PCR findings was assessed. MEASUREMENTS AND MAIN RESULTS: Sepsis criteria, days on antimicrobial therapy, antimicrobial substances administered, and microorganisms identified by PCR and BC susceptibility tests. RESULTS: BC diagnosed 117 clinically relevant microorganisms; PCR identified 154. Ninety-nine episodes were BC positive (BC+); 131 episodes were PCR positive (PCR+). Overall, 127.8 days of clinically inadequate empirical antibiotic treatment in the 99 BC+ episodes were observed. Utilization of PCR-aided diagnostics calculates to a potential reduction of 106.5 clinically inadequate treatment days. The ratio of gainable early adequate treatment days to number of PCR tests done is 22.8 days/100 tests overall (confidence interval 15-31) and 36.4 days/100 tests in the intensive care and surgical ward populations (confidence interval 22-51). CONCLUSIONS: Rapid PCR identification of microorganisms may contribute to a reduction of early inadequate antibiotic treatment in sepsis.

Evaluation of the diagnostic value of serologic microagglutination testing and a polymerase chain reaction assay for diagnosis of acute leptospirosis in dogs in a referral center.

OBJECTIVE To determine the diagnostic value of a serologic microagglutination test (MAT) and a PCR assay on urine and blood for the diagnosis of leptospirosis in dogs with acute kidney injury (AKI). DESIGN Cross-sectional study. Animals-76 dogs with AKI in a referral hospital (2008 to 2009). PROCEDURES Dogs' leptospirosis status was defined with a paired serologic MAT against a panel of 11 Leptospira serovars as leptospirosis-associated (n = 30) or nonleptospirosis-associated AKI (12). In 34 dogs, convalescent serologic testing was not possible, and leptospirosis status was classified as undetermined. The diagnostic value of the MAT single acute or convalescent blood sample was determined in dogs in which leptospirosis status could be classified. The diagnostic value of a commercially available genus-specific PCR assay was evaluated by use of 36 blood samples and 20 urine samples. RESULTS Serologic acute testing of an acute blood sample had a specificity of 100% (95% CI, 76% to 100%), a sensitivity of 50% (33% to 67%), and an accuracy of 64% (49% to 77%). Serologic testing of a convalescent blood sample had a specificity of 92% (65% to 99%), a sensitivity of 100% (87% to 100%), and an accuracy of 98% (88% to 100%). Results of the Leptospira PCR assay were negative for all samples from dogs for which leptospirosis status could be classified. CONCLUSIONS AND CLINICAL RELEVANCE Serologic MAT results were highly accurate for diagnosis of leptospirosis in dogs, despite a low sensitivity for early diagnosis. In this referral setting of dogs pretreated with antimicrobials, testing of blood and urine samples with a commercially available genus-specific PCR assay did not improve early diagnosis.

Establishing optimal quantitative-polymerase chain reaction assays for routine diagnosis and tracking of minimal residual disease in JAK2-V617F-associated myeloproliferative neoplasms: a joint European LeukemiaNet/MPN&MPNr-EuroNet (COST action BM0902) study.

Reliable detection of JAK2-V617F is critical for accurate diagnosis of myeloproliferative neoplasms (MPNs); in addition, sensitive mutation-specific assays can be applied to monitor disease response. However, there has been no consistent approach to JAK2-V617F detection, with assays varying markedly in performance, affecting clinical utility. Therefore, we established a network of 12 laboratories from seven countries to systematically evaluate nine different DNA-based quantitative PCR (qPCR) assays, including those in widespread clinical use. Seven quality control rounds involving over 21,500 qPCR reactions were undertaken using centrally distributed cell line dilutions and plasmid controls. The two best-performing assays were tested on normal blood samples (n=100) to evaluate assay specificity, followed by analysis of serial samples from 28 patients transplanted for JAK2-V617F-positive disease. The most sensitive assay, which performed consistently across a range of qPCR platforms, predicted outcome following transplant, with the mutant allele detected a median of 22 weeks (range 6-85 weeks) before relapse. Four of seven patients achieved molecular remission following donor lymphocyte infusion, indicative of a graft vs MPN effect. This study has established a robust, reliable assay for sensitive JAK2-V617F detection, suitable for assessing response in clinical trials, predicting outcome and guiding management of patients undergoing allogeneic transplant.

Comparison of fecal culture and F57 real-time polymerase chain reaction for the detection of Mycobacterium avium subspecies paratuberculosis in Swiss cattle herds with a history of paratuberculosis

BACKGROUND: Bovine paratuberculosis is an incurable chronic granulomatous enteritis caused by Mycobacterium avium subspecies paratuberculosis (MAP). The prevalence of MAP in the Swiss cattle population is hard to estimate, since only a few cases of clinical paratuberculosis are reported to the Swiss Federal Food Safety and Veterinary Office each year.Fecal samples from 1,339 cattle (855 animals from 12 dairy herds, 484 animals from 11 suckling cow herds, all herds with a history of sporadic paratuberculosis) were investigated by culture and real-time polymerase chain reaction (PCR) for shedding of MAP. RESULTS: By culture, MAP was detected in 62 of 445 fecal pools (13.9%), whereas PCR detected MAP in 9 of 445 pools (2.0%). All 186 samples of the 62 culture-positive pools were reanalyzed individually. By culture, MAP was grown from 59 individual samples (31.7%), whereas PCR detected MAP in 12 individual samples (6.5%), all of which came from animals showing symptoms of paratuberculosis during the study. Overall, MAP was detected in 10 out of 12 dairy herds (83.3%) and in 8 out of 11 suckling cow herds (72.7%). CONCLUSIONS: There is a serious clinically inapparent MAP reservoir in the Swiss cattle population. PCR cannot replace culture to identify individual MAP shedders but is suitable to identify MAP-infected herds, given that the amount of MAP shed in feces is increasing in diseased animals or in animals in the phase of transition to clinical disease

Development of a new real-time polymerase chain reaction assay to detect Duck adenovirus A DNA and application to samples from Swiss poultry flocks.

Between 2008 and 2012, commercial Swiss layer and layer breeder flocks experiencing problems in laying performance were sampled and tested for infection with Duck adenovirus A (DAdV-A; previously known as Egg drop syndrome 1976 virus). Organ samples from birds sent for necropsy as well as blood samples from living animals originating from the same flocks were analyzed. To detect virus-specific DNA, a newly developed quantitative real-time polymerase chain reaction method was applied, and the presence of antibodies against DAdV-A was tested using a commercially available enzyme-linked immunosorbent assay. In 5 out of 7 investigated flocks, viral DNA was detected in tissues. In addition, antibodies against DAdV-A were detected in all of the flocks.

Allele-specific polymerase chain reaction diagnostic test for the functional MDR1 polymorphism in dogs

The major multidrug transporter P-glycoprotein (Pgp) contributes to the barrier function of several tissues and organs, including the brain. In a subpopulation of Collies and seven further dog breeds, a 4 base pair deletion has been described in the Pgp-encoding MDR1 gene. This deletion results in the absence of a functional form of Pgp and loss of its protective function. Severe intoxication with the Pgp substrate ivermectin has been attributed to the genetically determined lack of Pgp. An allele-specific polymerase chain reaction (PCR)-based screening method has been developed to detect the mutant allele and to determine if a dog is homozygous or heterozygous for the mutation. Based on this validation, the allele-specific PCR proved to be a robust, reproducible and specific tool, allowing rapid determination of the MDR1 genotype of dogs of at risk breeds using blood samples or buccal swabs.

Cloning of a protease gene family of Fasciola hepatica by the polymerase chain reaction

Degenerate oligonucleotide primers derived from conserved cysteine protease sequences were used in the reverse transcription polymerase chain reaction to amplify seven different cysteine protease cDNA clones, Fcp1-7, from RNA isolated from adult Fasciola hepatica. Five of the amplified F. hepatica sequences showed homology to the cathepsin L type and two were more related to the cathepsin B type. Southern blot analysis suggests that some members of this protease gene family are present in multiple copies. Northern blot analysis revealed differences in the levels of steady state mRNA expression for some of these proteases. The 5' and the 3' regions of Fcp1 were amplified using the rapid amplification of cDNA ends PCR protocol (RACE-PCR) and an additional clone was obtained by screening a lambda gt10 cDNA library using Fcp1 as a probe. The Fcp1 cDNA fragment was also subcloned in the expression vector pGEX and expressed as a glutathione-S-transferase (GST) fusion protein in Escherichia coli. Antibodies, raised in rabbits against the GST:Fcp1 fusion protein, were used in western blot analysis to examine expression in different life-cycle stages of F. hepatica. In extracts from adult and immature parasites, the immune serum recognised predominantly two proteins of 30 kDa and 38 kDa. In other parasite stages, proteins of different molecular weight were recognised by the anti-GST:Fcp1 antiserum, indicating stage-specific gene expression or processing of Fcp1. In gelatine substrate gel analysis, strong proteolytic activity could be detected at 30 kDa, but not at 38 kDa, suggesting that the 30 kDa protein represents the mature enzyme and the 38 kDa protein the proenzyme.