Real-time PCR Assay For Diagnosis Research Articles

Clinical evaluation of a real-time PCR assay for diagnosis of Helicobacter pylori infection and antibiotic resistance.

Helicobacter pylori (H. pylori) is a globally prevalent bacterium that increases the risk of developing various gastrointestinal diseases, including gastric adenocarcinoma. This study aimed to evaluate the performances of real-time PCR assay in detecting H. pylori infection, as well as clarithromycin and levofloxacin resistance, in both stool and gastric biopsy specimens. Stool and gastric biopsy specimens were collected from patients within one to three days post-hospitalization. All patients were analyzed for H. pylori infection and resistance to clarithromycin and levofloxacin using a real-time PCR based molecular assay. 169 patients (83 males) with a mean age of 43.6±13.1 years were included in the study. The prevalence of H. pylori was 89.9% (152/169) in stool and 90.5% (153/169) in gastric biopsy samples. The molecular diagnostics employed in this study exhibited a sensitivity of 99.3% and a specificity of 100%, resulting in a diagnostic accuracy rate of 99.6%. Resistance to clarithromycin was 36.1% (61/169) in stool and 44.4% (75/169) in gastric biopsy samples. The molecular tests for clarithromycin resistance demonstrated a sensitivity of 96.8% and a specificity of 86.8%, with an overall diagnostic accuracy of 90.5%. Furthermore, resistance to levofloxacin was 22.5% (38/169) and 26.6% (45/169) in stool and gastric biopsy samples, respectively. The molecular test demonstrated a sensitivity of 80.9% and a specificity of 94.3%, resulting in a diagnostic accuracy of 90.5%. The implementation of real-time PCR-based screening for H. pylori infection and resistance to clarithromycin and levofloxacin in the stool may enhance the success rate of eradication therapy.

Deciphering Intermediate Nugent scores: Utility of Real-time PCR for Bacterial Vaginosis diagnosis

PurposeTo optimize real-time PCR assays for diagnosis of Bacterial Vaginosis (BV) and determine cut-off loads by ROC analysis for Gardnerella vaginalis, Atopobium vaginae and Lactobacillus spp. as compared to Nugent scoring (Gold standard) in clinical samples. ResultsOut of 125 women, 34 were positive, 26 intermediate and 65 negative for BV by Nugent scoring. All three real-time PCR assays were found to be highly sensitive & specific and AUC suggested excellent diagnostic accuracy. An optimal cut-off was >9.45 × 103 copies/ ml, >3.34 × 103 copies/ ml & ≤ 18.63 × 103 copies/ ml for G. vaginalis, A. vaginae and Lactobacillus spp. respectively, in BV positives. Gram staining and qPCR were discordant only in patients with intermediate scores (n = 26) where qPCR identified 15 (57.69%) as positive and 11 (42.3%) as negative. ConclusionPCR-based molecular BV diagnosis is more accurate and can be used for deciphering intermediate Nugent scores.

Clinical performance of a quantitative pan-genus Leishmania Real-time PCR assay for diagnosis of cutaneous and visceral leishmaniasis

Leishmaniasis is a complex vector-borne disease caused by various Leishmania species, affecting humans and animals. Current diagnostic methods have limitations, leading to potential misdiagnosis. Therefore, there is an urgent need for specific and sensitive diagnostic tools. We evaluated the sensitivity of a quantitative real-time PCR (qPCR) assay targeting the 18S gene in diverse clinical sample matrices. The assay showed a wide dynamic range and a limit of detection (LoD) of 1 parasite equivalent per milliliter (eq-p/mL) for all tested species. It exhibited high specificity for Leishmania DNA, with no amplification against other microorganisms. When applied to samples from patients with visceral and cutaneous leishmaniasis, the qPCR assay provided results that matched the reference methods and allowed estimation of parasite burdens. This assay holds promise for diagnosing and monitoring leishmaniasis by offering high sensitivity, specificity, and the ability to estimate parasitemia. Further studies are needed to enhance Leishmania molecular diagnostics and expand their coverage for improved clinical impact.

Tracing the Negative Results of Multiplex Real-Time PCR Assay for Diagnosis of Bacterial Pediatrics Meningitis.

The death because of meningitis remains high in some parts of the world. It is important to know the specific cause of meningitis because the treatment differs depending on the cause. This study aimed to trace the false-negative results of multiplex RT-PCR to detect Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria meningitidis serogroup by two different molecular methods. In this study, the CSF of the suspicious pediatric for acute bacterial meningitis among children aged 1 month to 14 years who are admitted to the hospitals in four cities of a certain region of Iran was collected. S. pneumoniae, H. influenzae, and N. meningitidis in CSF samples were detected by single-tube multiplex RT-PCR and specific RT-PCR with a probe on the same specimens. In this cross-sectional study, 506 CSF samples were collected during one year. The multiplex RT-PCR can detect 3.3% and 2.2% of S. pneumoniae and H. influenzae, respectively. N. meningitidis was not detected. The CSF analysis was abnormal in 53% of 506 patients. On the other hand, 11.5%, 4.8%, and 4.1% of S. pneumoniae, H. influenzae, and N. meningitidis were identified, respectively, by specific RT-PCR assay, exactly on the same specimens. Various types of PCR can be used for pathogen identification. As we change the type of PCR in our study, we could approximately increase 15% our positive results and also consequently decrease our false-negative responses.

Performance evaluation of automated real-time PCR assays for diagnosis of TB in HIV infected individuals

Performance evaluation of automated real-time PCR assays for diagnosis of TB in HIV infected individuals

Evaluation of a real-time PCR assay for diagnosis of schistosomiasis japonica in the domestic goat

BackgroundSchistosomiasis japonica is an infectious disease caused by Schistosoma japonicum that seriously endangers human health. Domestic animals have important roles in disease transmission and goats are considered a primary reservoir host and source of infection. The prevalence and intensity of schistosomiasis infections have significantly decreased in China, and a more sensitive, specific detection method is urgently needed. The aim of this study was to develop a real-time PCR assay for accurate detection of S. japonicum infection in goats.MethodsA real-time PCR method for detecting schistosomiasis japonica in goats was developed by amplification of a specific S. japonicum DNA fragment, and validated using a total of 94 negative and 159 positive plasma and serum samples collected in our previous study of S. japonicum infection. Both plasma and serum samples were evaluated by real-time PCR and enzyme-linked immunosorbent assay (ELISA). In addition, 120 goat plasma samples from an S. japonicum-endemic area (Wangjiang) and 33 from a non-endemic region (Weihai) were collected and evaluated using our method.ResultsThe sensitivity and specificity of the real-time PCR for detecting infected samples were 98.74% (157/159, 95% CI: 95.53–99.85%) and 100% (94/94, 95% CI: 96.15–100%), respectively. For the ELISA, sensitivity and specificity were 98.11% (156/159, 95% CI: 94.59–99.61%) and 90.43% (85/94, 95% CI: 82.60–95.53%), respectively. Further, we found positivity rates for S. japonicum infection in Wangjiang and Weihai of 8.33% (10/120, 95% CI: 4.07–14.79%) and 0% (0/33, 95% CI: 0–10.58%), respectively.ConclusionsThe results of this study indicate that our real-time PCR method exhibits higher sensitivity and specificity than ELISA and is a useful method for detection of S. japonicum infection in goats.

Applicability of commercial multiplex kit for the diagnosis of: a multicenter investigation Clostridium difficile infections

Objective To evaluate the performance of Xpert C. difficile multiplex real-time PCR assay for diagnosis of Clostridium difficile infections in Chinese hospital settings. Methods This study was performed in Huashan Hospital, Ruijin Hospital, Beijing Hospital, Nanfang Hospital and Sir Run Run Shaw Hospital using a standard study protocol. Unique unformed stools from patients with acute hospital-acquired diarrhea were simultaneously analyzed by toxigenic anaerobic cultures and the Xpert C. difficile assay. All specimens displaying discordant results between the Xpert assay and toxigenic culture were sent for Sanger tcdB gene sequencing. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), total concordance rate, and 95% confidence interval (CI) were calculated before and after resolution of discordant results using SAS 9.3. Results A total of 745 stool specimens were collected and 46 were excluded due to failure of C. difficile recovery. The remaining 699 specimens were included. Compared to the results of toxigenic culture, the sensitivity, specificity, PPV, and NPV of Xpert C. difficile assay were 94.1% (144/153)(95%CI:89.1%-97.3%), 93.2% (509/546)(95%CI:96.7%-99.2%), 79.6% (144/181)(95%CI: 72.9%-85.2%)and 98.3% (509/518)(95%CI: 96.7%-99.2%), respectively. Both methods had a Kappa of 0.819. Xpert C. difficile assay showed sensitivity of 98.4%(62/63) (95% CI: 90.3%-99.9%) and specificity of 93.2%(509/546) (95% CI: 90.8%-95.2%) for toxin A-negative toxin B-positive strains. After the discordant results resolved by tcdB gene sequencing, PCR assay provided better performance with high sensitivity, specificity, positive predictive value, and negative predictive value [98.8% (171/173), 98.1% (516/526), 94.5% (171/181) and 99.6% (516/518), respectively]. Conclusions Compared to the results of toxigenic culture, the sensitivity, specificity and NPV of Xpert C. difficile assay were 94.1% (144/153) and 93.2% (509/546), respectively. With the results available within 1 h, Xpert C. difficile assay provides prompt and precise laboratory diagnosis in Chinese clinical settings. Key words: Clostridium difficile; Clostridium infections; Real-time polymerase chain reaction

Evaluation of a new commercial real-time PCR assay for diagnosis of Pneumocystis jirovecii pneumonia and identification of dihydropteroate synthase (DHPS) mutations

The PneumoGenius® real-time PCR assay is a new commercial multiplex real-time PCR method, which detects the Pneumocystis mitochondrial ribosomal large subunit (mtLSU) and two dihydropteroate synthase (DHPS) point mutations. To evaluate the clinical performance of this new real-time PCR assay we tested 120 extracted DNA samples from bronchoalveolar lavage specimens. These set of extracted DNA samples had already tested positive for Pneumocystis and patients had been classified in probable and unlikely PCP in a previous study. To evaluate de accuracy of the DHPS mutant's identification, an “in house” PCR and sequencing was performed. The sensitivity and specificity of PneumoGenius® PCR in discriminating between probable and unlikely Pneumocystis pneumonia (PCP) were 70% and 82% respectively. PneumoGenius® PCR was able to genotype more samples than “in house” DHPS PCR and sequencing. The same DHPS mutations were observed by both methods in four patients: two patients with a single mutation in position 171 (Pro57Ser) and two patients with a double mutation in position 165 (Thr55Ala) and in position 171 (Pro57Ser). A low rate of P. jirovecii (4.5%) harboring DHPS mutations was found, comparable to rates observed in other European countries. The PneumoGenius® real-time PCR is a suitable real-time PCR for PCP diagnosis and detection of DHPS mutants. The added value of DHPS mutation identification can assist in understanding the role of these mutations in prophylaxis failure or treatment outcome.

Correction for Hilbert et al., Development and Validation of a Highly Accurate Quantitative Real-Time PCR Assay for Diagnosis of Bacterial Vaginosis.

Volume 54, no. 4, p. [1017–1024][1], 2016. Page 1021, first paragraph of Discussion, lines 13 to 17: The sentence beginning “Although” should read “It is important to note that in one study specimens from subjects with intermediate flora were not analyzed (18) and in the other study the

Utilization of a real-time PCR assay for diagnosis of Babesia microti infection in clinical practice

Babesiosis is an emerging tick-borne disease mainly caused Babesia microti, a protozoan that infects erythrocytes. Microscopic examination of blood smears is the current gold standard for detection of Babesia infection, but this diagnostic test has several limitations. We developed and assessed the clinical utilization of a multiplex real-time PCR assay targeting the 18S rRNA gene of B. microti and the human gapdh gene. The limit of detection of this PCR assay was approximately 1–3parasites/μl of blood. The assay showed a diagnostic sensitivity and probable specificity of 100% based on testing 145 retrospective and 185 prospective blood specimens from controls and patients with confirmed babesiosis. Notably, the PCR assay was more sensitive than blood smear examination in patients during and following anti-babesia drug therapy. Our study suggests that PCR testing is as good or better than a blood smear for detection of B. microti in routine clinical practice. PCR testing may confirm the presence of babesiosis in patients whose level of infection is too low for reliable microscopic detection.

An approach for interlaboratory comparison of conventional and real-time PCR assays for diagnosis of human leishmaniasis

Protozoa of the Leishmania genus are transmitted to humans by the bite of infected sandflies, and are the causative agents of leishmaniasis which ranges from cutaneous to visceral clinical forms. The definitive diagnosis of leishmaniasis has relied traditionally on parasite demonstration, either by microscopy or culture; in the last years, diagnosis based on PCR methods has overcome some drawbacks of traditional methods, increasing sensitivity and allowing using less invasive sampling for diagnosis. However, there are not defined protocols and almost each laboratory applies its own in-house method. Although there are several studies comparing the performance of different methods within the same laboratory, those addressing interlaboratory comparison are scarce, in spite of the growing number of collaborative projects between partners from different leishmaniasis endemic and non-endemic countries. In this work we propose a protocol for interlaboratory comparison of conventional and real-time PCR methods involving four participant laboratories from four different endemic regions in four continents; the protocol includes a quality control step and reduces the variability among the samples tested by each participant. A panel of 77 samples from human origin and 9 from different parasite strains was blindly tested by the participants, aiming to assess the sensitivity of the different methods as well as their usefulness for species identification. Real-time PCR methods targeting the kDNA minicircles returned the highest sensitivity, while both PCR targeting ITS-1 and further HaeIII digestion and a combined algorithm including hsp70 PCR and restriction fragment length polymorphism analysis were the most appropriate approaches for species identification.

Conventional and real-time PCR for the identification of Fusarium fujikuroi and Fusarium proliferatum from diseased rice tissues and seeds

Fusarium fujikuroi is a species of the Gibberella fujikuroi species complex (GFSC) and the causal agent of bakanae disease on rice. Even if F. fujikuroi is the most abundant Fusarium species found on rice, other species can also be isolated from rice, such as F. proliferatum. Multiple alignment of translation elongation factor (TEF) gene sequences of different Fusarium spp., showed a deletion of six nucleotides in F. fujikuroi sequence and a two nucleotide polymorphism in the same region of F. proliferatum sequence. These elements of variability were used to develop a conventional and Real-Time PCR assay for diagnosis. The species specific primer pairs (Fuji1F/TEF1R and Proli1F/TEF1R) gave a product of 179 and 188 bp for F. fujikuroi and F. proliferatum respectively. Primer specificity was confirmed by analyzing the DNA of the most representative species of the GFSC and 298 strains of Fusarium spp. isolated from rice plants and seeds in Italy. The specific primers were also successfully used to detect fungal presence directly from infected rice tissues and seeds, providing a rapid tool for the early detection of pathogen contamination.

Comparison of loop-mediated isothermal amplification (LAMP) and real-time PCR method targeting a 529-bp repeat element for diagnosis of toxoplasmosis

Loop-mediated isothermal amplification (LAMP) is a simple method that can amplify DNA with high specificity, sensitivity, and rapidity. In this study, we compared the performance of LAMP and real-time PCR assays for diagnosis of toxoplasmosis. We designed a real-time PCR assay targeting a 529bp element repeated 200–300 times in the Toxoplasma gondii genome. The detection limits of the LAMP and real-time PCR assays were 10fg/μL and 1fg/μL of T. gondii DNA, respectively. Conventional PCR, LAMP, and real-time PCR methods were applied to detect T. gondii DNA in blood samples from 284 pigs and 292 sheep. Positive results were obtained with 0.4%, 3.2%, and 4.2% of the pig samples and 3.8%, 17.1%, and 17.8% of the sheep samples with conventional PCR, LAMP, and real-time PCR analyses, respectively. The real-time PCR assay provided the most sensitive diagnosis of toxoplasmosis, but the LAMP assay has potential as an alternative tool for detection of T. gondii in the field.

Comparative performance of the Roche COBAS Amplicor assay and an in-house real-time PCR assay for diagnosis of Chlamydia trachomatis infection

This study investigated the comparative performance of the Amplicor assay and an in-house semi-automated, multiplex real-time PCR for the diagnosis of genital chlamydial infection. Four different assays, the COBAS Amplicor CT test (Amplicor PCR), in-house real-time PCR (IHRT-PCR), in-house nested cryptic plasmid PCR and in-house nested major outer membrane protein PCR, were performed on genital swabs from 1000 consecutive patients attending a genitourinary medicine clinic. The samples were designated true positive if Chlamydia trachomatis DNA was detected by at least two of the four above-mentioned assays while a sample was defined as true negative if C. trachomatis DNA was detected in only one or none of the assays. By this criterion, there were 129 true positive and 871 true negative samples for C. trachomatis DNA in this cohort. Amplicor PCR designated 144 samples positive: 128 (89%) of 144 samples were true positive and 16 (11%) were false positive. IHRT-PCR detected 126 of 129 true positive samples and did not generate any false positive results. The sensitivity of IHRT-PCR was comparable with, and specificity was higher than, Amplicor PCR for the diagnosis of genital chlamydial infection.

Advantages of real-time PCR assay for diagnosis and monitoring of canine leishmaniosis

The aim of the present study is to highlight the advantages of real-time quantitative PCR intended to aid in the diagnosis and monitoring of canine leishmaniosis. Diagnosis of canine leishmaniosis is extremely challenging, especially in endemic areas, due to the diverse and non-specific clinical manifestations, and due to the high seroprevalence rate in sub-clinical dogs. Veterinarian clinicians are usually confronted with cases that are compatible with the disease, and with several diagnostic tests, sometimes with contradictory results. We have developed a new TaqMan assay, targeting the kinetoplast, applied to 44 samples of bone marrow aspirate or peripheral blood. The dynamic range of detection of Leishmania DNA was established in 7 logs and the limit of detection is 0.001 parasites in the PCR reaction. At the time of diagnosis parasitemia ranges from less than 1 to 10 7 parasites/ml. The ability to quantify the parasite burden allowed: (i) to elucidate the status of positive dogs by conventional PCR, although larger studies are necessary to clarify the dividing line between infection and disease, (ii) to estimate the kinetics of the parasite load and the different response to the treatment in a follow-up and (iii) to validate blood as less invasive sample for qPCR. The continuous data provided by real-time qPCR could solve the dilemma for the clinician managing cases of canine leishmaniosis by differentiating between Leishmania-infected dogs or dogs with active disease of leishmaniosis.