In-house Nucleic Acid Amplification Tests Research Articles

The Application of 3base™ Technology to Diagnose Eight of the Most Clinically Important Gastrointestinal Protozoan Infections.

Globally, over 3.5 billion people are infected with intestinal parasites each year, resulting in over 200,000 deaths. Three of the most common protozoan pathogens that affect the gastrointestinal tract of humans are Cryptosporidium spp., Giardia intestinalis, and Entamoeba histolytica. Other protozoan agents that have been implicated in gastroenteritis in humans include Cyclospora cayetanensis, Dientamoeba fragilis, Blastocystis hominis, and the microsporidia Enterocytozoon bieneusi and Encephalitozoon intestinalis. Genetic Signatures previously developed a 3base™ multiplexed Real-Time PCR (mRT-PCR) enteric protozoan kit (EP001) for the detection of Giardia intestinalis/lamblia/duodenalis, Cryptosporidium spp., E. histolytica, D. fragilis, and B. hominis. We now describe improvements to this kit to produce a more comprehensive assay, including C. cayetanensis, E. bieneusi, and E. intestinalis, termed EP005. The clinical performance of EP005 was assessed using a set of 380 clinical samples against a commercially available PCR test and other in-house nucleic acid amplification tests where commercial tests were not available. All methods provided at least 90% agreement. EP005 had no cross-reactivity against 82 organisms commonly found in the gut. The EP005 method streamlines the detection of gastrointestinal parasites and addresses the many challenges of traditional microscopic detection, resulting in cost savings and significant improvements in patient care.

Meta-analysis of the diagnostic performance characteristics of three commercial and one in-house nucleic acid amplification tests for malaria screening

Abstract Background A meta-analysis of previously performed evaluation studies of nucleic acid amplification testing (NAT) approaches for the screening for and differentiation of Plasmodium spp. using identical samples was performed to comparatively assess their suitability for the routine diagnostic setting. Methods Three commercial NATs for malaria (one loop-mediated isothermal amplification [LAMP] assay, two real-time polymerase chain reactions [PCRs]) and one in-house real-time PCR were comparatively assessed with a collection of 1020 well-characterized ethylenediaminetetraacetic acid (EDTA) blood samples from patients with suspected or confirmed malaria. Results Altogether 765 (75%) concordantly negative and 223 (21.9%) concordantly positive results of the four molecular tests were obtained, while discordant results were seen in 32 (3.1%) instances. For genus-specific assays, the observed sensitivity and specificity ranges were 96.4%–98.4% and 99.6%–99.9%, and for species-specific assays, 94.0%–97.6% and 99.6%–100%, respectively. Falsely negative molecular test results comprised microscopically negative samples, samples at the microscopic detection threshold and quantitatively less abundant species in mixed infections. Conclusions Excellent test characteristics of all assessed assays with only minor differences encourage molecular malaria screening with genus- and species-specific NAT with discrepancies only within the borderline range of their detection thresholds.

Nonspecific Amplification of Human DNA by Streptococcus pneumoniae LytA Primer

Background: Determination of various analytical parameters is essential for the validation of primers used for in-house nucleic acid amplification tests. While standardising a high-resolution melt analysis (HRMA) for detection of Streptococcus pneumoniae in acute pyogenic meningitis, we encountered non-specific amplification of certain base pair sequences of human DNA by Centers for Disease Control & Prevention, USA recommended S. pneumoniae LytA primer. Materials and Methods: HRMA was standardised using DNA extracted from an ATCC strain of S. pneumoniae using SP LytA F373 primer and Type-it HRMTM polymerase chain reaction kit in Rotor-Gene Q Thermal Cycler according to the manufacturer’s instructions. Specificity of the primers was determined in dry and wet laboratory experiments against diverse related and unrelated microbial pathogens by HRMA and on DNA extracted from unspiked clinical samples negative for SP DNA. Sensitivity was determined by calculating lower limit of detection threshold in experiments with spiked samples. The amplicon from spiked experiments was sequenced and analysed through Gene Bank. Results: Our dry/wet laboratory experiments showed two separate curves and different Tm values indicating certain non-specific amplification by the primer. Basic Local Alignment Search Tool (BLAST) analysis of the amplicon obtained in the spiked experiment showed sequences of human chromosome 20 associated with Homo sapiens protein tyrosine phosphatase, receptor type T gene. The problem was resolved by stopping the reaction at 30th Ct cycle and observing the Tm values. Conclusion: Since HRMA is done without a specific probe, one should be aware of non-specific amplifications while using primers for HRMA of human clinical samples.

Detection of Clostridium difficile in Fecal Specimens: a Comparative Evaluation of Nucleic Acid Amplification Test and Toxigenic Culture.

The available data regarding Clostridium difficile infections (CDIs) in developing countries are scarce. This may be related in part to the complexity of anaerobic bacterial culture and/or cytotoxicity assays of C. difficile. Here, we evaluated the diagnostic efficacy of PCR in comparison with toxigenic culture for direct detection of conserved genes as well as toxin genes of C. difficile in fecal specimens of patients with clinical symptoms of CDI. Loose or soft feces from 171 patients suspected of having C. difficile associated diarrhea (CDAD) were subjected to DNA extraction, PCR of cdu-2, cdd-3, gdh, tpi, tcdA/B, and toxigenic culture (TC). Limit of detection (LoD) was defined as the lowest concentration of DNA at which the target gene was amplified via PCR. The Kappa agreement between two diagnostic tests was calculated. The in-house extraction method extracted DNA successfully as confirmed by amplification of conserved genes of C. difficile. LoD of PCR for total DNA was 0.064 ng/μL. Only 10 specimens were positive for C. difficile via both PCR and TC. Among 10 identified C. difficile strains, 8 were tcdA+B+, but 2 were tcdA-B+. A very good agreement was observed between TC as reference method and PCR (κ = 1). Despite the high concordance between PCR and TC, this in-house nucleic acid amplification test can be used to identify symptomatic patients who harbor high amounts of bacteria. This procedure allows primary and same day diagnosis of C. difficile, and clinical laboratories in low-income countries may adopt the method for sample extraction and PCR assay at least for symptomatic patients.

A preliminary evaluation of a new GeneXpert (Gx) molecular point-of-care test for the detection of Trichomonas vaginalis

ObjectivesGlobal concerns regarding the prevalence, asymptomatic nature and burden of disease associated with Trichomonas vaginalis (TV) continue. The lack of a portable molecular point-of-care assay to detect this infectious disease...

The Presence of Chlamydia Is Associated With Increased Leukocyte Counts and Pain Severity in Men With Chronic Pelvic Pain Syndrome

To examine the role of urinary chlamydial infection in patients with chronic prostatitis and/or chronic pelvic pain syndrome (CP-CPPS). We recruited men aged 18-55 years over 4 years. We excluded patients with acute urethritis and any acute genitourinary infections. The participants evaluated their CP-CPPS-like symptoms with the Korean version National Institutes of Health-Chronic Prostatitis Symptom Index questionnaires. We measured white blood cell (WBC) counts in expressed prostatic secretion (EPS). In-house nucleic acid amplification test for Chlamydia trachomatis and Neisseria gonorrhoeae detection and WBC counts were performed from the patient's first-voided urine. A total of 765 eligible men were classified into 3 groups: 196 in non-CP-CPPS, 410 in noninflammatory CP-CPPS, and 159 in inflammatory CP-CPPS groups. The chlamydia-infected men showed higher pain, poor quality of life (QOL), and total scores in National Institutes of Health-Chronic Prostatitis Symptom Index questionnaires than the negative men (P = .041 for pain; P = .043 for QOL, and P = .027 for total). Multivariate analysis found that urinary chlamydial infection increased the risk of WBC count ≥16 in EPS (adjusted odds ratio [OR], 2.189; 95% confidence interval [CI], 1.021-4.690; P = .044) and WBC count between 2 and 4 in urine (OR, 5.227; 95% CI, 2.503-10.918; P = .001). In addition, chlamydial infection also increased the risk of inflammatory CP-CPPS than the non-CP-CPPS group (OR, 2.448; 95% CI, 1.010-5.932; P = .044), whereas the patients with noninflammatory CP-CPPS were not affected (OR, 1.6557; 95% CI, 0.738-3.717; P = .221). Urinary chlamydial infection increased the pain scores and WBC counts in EPS and worsens the QOL in the patients with CP-CPPS.

P71 Validation of an in house nucleic acid amplification (NAATs) PCR test for Trichomonas vaginalis

BackgroundTrichomonas vaginalis, a flagellated protozoan, is transmitted through sexual contact that commonly manifests itself as symptomatic in more women than men. However, the true prevalence of infection and the proportion...

Reply to “Routine Confirmation of Positive Nucleic Acid Amplification Test Results for Neisseria gonorrhoeae Is Not Necessary”

In various settings worldwide, many different commercially available FDA-approved nucleic acid amplification tests (NAATs), as well as other commercially available NAATs plus in-house NAATs, are utilized in the diagnosis of Neisseria gonorrhoeae. The performance characteristics of these NAATs

Enhanced Viral Etiological Diagnosis of Respiratory System Infection Outbreaks by Use of a Multitarget Nucleic Acid Amplification Assay

A study was undertaken to assess the utility of the xTAG respiratory viral panel (RVP) for enhanced laboratory investigation of respiratory outbreaks. Specimens (n = 1,108) from 244 suspected respiratory virus outbreaks in 2006 and 2007 in Alberta, Canada, were included in the study. Testing by direct fluorescent antigen detection (DFA) and various in-house nucleic acid amplification tests (NATs) for common respiratory viruses provided an etiological diagnosis in 177 outbreaks (72.5%), with 524 samples testing positive (47.3%) for a respiratory virus. Two hundred samples from 51 unresolved outbreaks were further tested by RVP retrospectively. Fifty-eight samples from 30 unresolved outbreaks had a respiratory virus detected by RVP (47 picornavirus-positive, 9 coronavirus-positive, and 2 influenza virus A-positive samples). Overall, detection of a viral etiological agent was achieved in 90.8% of outbreaks using a combination of DFA, NATs, and RVP. Use of RVP enhances the laboratory investigation of respiratory virus outbreaks and facilitates appropriate patient and outbreak management.

Comparison of the Luminex xTAG Respiratory Viral Panel with In-House Nucleic Acid Amplification Tests for Diagnosis of Respiratory Virus Infections

Detection of respiratory viruses using sensitive real-time nucleic acid amplification tests (NATs) is invaluable for patient and outbreak management. However, the wide range of potential respiratory virus pathogens makes testing using individual real-time NATs expensive and laborious. The objective of this study was to compare the detection of respiratory virus targets using the Luminex xTAG respiratory viral panel (RVP) assay with individual real-time NATs used at the Provincial Laboratory of Public Health, Calgary, Alberta, Canada. The study included 1,530 specimens submitted for diagnosis of respiratory infections from December 2006 to May 2007. Direct-fluorescent-antigen-positive nasopharyngeal samples were excluded from this study. A total of 690 and 643 positives were detected by RVP and in-house NATs, respectively. Kappa correlation between in-house NATs and RVP for all targets ranged from 0.721 to 1.000. The majority of specimens missed by in-house NATs (96.7%) were positive for picornaviruses. Samples missed by RVP were mainly positive for adenovirus (51.7%) or respiratory syncytial virus (27.5%) by in-house NATs and in general had low viral loads. RVP allows for multiplex detection of 20 (and differentiation between 19) respiratory virus targets with considerable time and cost savings compared with alternative NATs. Although this first version of the RVP assay has lower sensitivity than in-house NATs for detection of adenovirus, it has good sensitivity for other targets. The identification of picornaviruses and coronaviruses and concurrent typing of influenza A virus by RVP, which are not currently included in our diagnostic testing algorithm, will improve our diagnosis of respiratory tract infections.

In-house nucleic acid amplification tests for the detection of Mycobacterium tuberculosis in sputum specimens: meta-analysis and meta-regression.

BackgroundMore than 200 studies related to nucleic acid amplification (NAA) tests to detect Mycobacterium tuberculosis directly from clinical specimens have appeared in the world literature since this technology was first introduced. NAA tests come as either commercial kits or as tests designed by the reporting investigators themselves (in-house tests). In-house tests vary widely in their accuracy, and factors that contribute to heterogeneity in test accuracy are not well characterized. Here, we used meta-analytical methods, including meta-regression, to identify factors related to study design and assay protocols that affect test accuracy in order to identify those factors associated with high estimates of accuracy.ResultsBy searching multiple databases and sources, we identified 2520 potentially relevant citations, and analyzed 84 separate studies from 65 publications that dealt with in-house NAA tests to detect M. tuberculosis in sputum samples. Sources of heterogeneity in test accuracy estimates were determined by subgroup and meta-regression analyses. Among 84 studies analyzed, the sensitivity and specificity estimates varied widely; sensitivity varied from 9.4% to 100%, and specificity estimates ranged from 5.6% to 100%. In the meta-regression analysis, the use of IS6110 as a target, and the use of nested PCR methods appeared to be significantly associated with higher diagnostic accuracy.ConclusionEstimates of accuracy of in-house NAA tests for tuberculosis are highly heterogeneous. The use of IS6110 as an amplification target, and the use of nested PCR methods appeared to be associated with higher diagnostic accuracy. However, the substantial heterogeneity in both sensitivity and specificity of the in-house NAA tests rendered clinically useful estimates of test accuracy difficult. Future development of NAA-based tests to detect M. tuberculosis from sputum specimens should take into consideration these findings in improving accuracy of in-house NAA tests.

Nucleic acid amplification tests in the diagnosis of tuberculous pleuritis: a systematic review and meta-analysis.

BackgroundConventional tests for tuberculous pleuritis have several limitations. A variety of new, rapid tests such as nucleic acid amplification tests – including polymerase chain reaction – have been evaluated in recent times. We conducted a systematic review to determine the accuracy of nucleic acid amplification (NAA) tests in the diagnosis of tuberculous pleuritis.MethodsA systematic review and meta-analysis of 38 English and Spanish articles (with 40 studies), identified via searches of six electronic databases, hand searching of selected journals, and contact with authors, experts, and test manufacturers. Sensitivity, specificity, and other measures of accuracy were pooled using random effects models. Summary receiver operating characteristic curves were used to summarize overall test performance. Heterogeneity in study results was formally explored using subgroup analyses.ResultsOf the 40 studies included, 26 used in-house ("home-brew") tests, and 14 used commercial tests. Commercial tests had a low overall sensitivity (0.62; 95% confidence interval [CI] 0.43, 0.77), and high specificity (0.98; 95% CI 0.96, 0.98). The positive and negative likelihood ratios for commercial tests were 25.4 (95% CI 16.2, 40.0) and 0.40 (95% CI 0.24, 0.67), respectively. All commercial tests had consistently high specificity estimates; the sensitivity estimates, however, were heterogeneous across studies. With the in-house tests, both sensitivity and specificity estimates were significantly heterogeneous. Clinically meaningful summary estimates could not be determined for in-house tests.ConclusionsOur results suggest that commercial NAA tests may have a potential role in confirming (ruling in) tuberculous pleuritis. However, these tests have low and variable sensitivity and, therefore, may not be useful in excluding (ruling out) the disease. NAA test results, therefore, cannot replace conventional tests; they need to be interpreted in parallel with clinical findings and results of conventional tests. The accuracy of in-house nucleic acid amplification tests is poorly defined because of heterogeneity in study results. The clinical applicability of in-house NAA tests remains unclear.