Rapid Nucleic Acid Amplification Tests Research Articles

The clinical impact of rapid nucleic acid amplification tests for detection of M. tuberculosis

The clinical impact of rapid nucleic acid amplification tests for detection of M. tuberculosis

PL05.2 New Technologies in STI Diagnosis and Control: Promising Future

BackgroundOver 350 million (M) prevalent cases of curable STIs are estimated worldwide: 100 M chlamydia (CT), 36M gonorrhoea (NG), 187 M trichomonas, and 36 M cases of syphilis. 34 M...

Development and Comparison of a Rapid Isothermal Nucleic Acid Amplification Test for Typing of Herpes Simplex Virus Types 1 and 2 on a Portable Fluorescence Detector

We have developed a rapid and simple molecular test, the IsoGlow HSV Typing assay, for the detection and typing of herpes simplex virus (type 1 and 2) from genital or oral lesions. Clinical samples suspended in viral transport mediums are simply diluted and then added to a helicase-dependent amplification master mix. The amplification and detection were performed on a portable fluorescence detector called the FireFly instrument. Detection of amplification products is based on end-point analysis using cycling probe technology. An internal control nucleic acid was included in the amplification master mix to monitor the presence of amplification inhibitors in the samples. Because the device has only two fluorescence detection channels, two strategies were developed and compared to detect the internal control template: internal control detected by melting curve analysis using a dual-labeled probe, versus internal control detection using end-point fluorescence release by a CPT probe at a lower temperature. Both have a total turnaround time of about 1 hour. Clinical performance relative to herpes viral culture was evaluated using 176 clinical specimens. Both formats of the IsoGlow HSV typing assay had sensitivities comparable to that of the Food and Drug Administration-cleared IsoAmp HSV (BioHelix Corp., Beverly MA) test and specificity for the two types of HSV comparable to that of ELVIS HSV (Diagnostic Hybrids, Athens, OH).

Low Prevalence of Chlamydia trachomatis Infection in Non-Urban Pregnant Women in Vellore, S. India

ObjectiveTo determine the prevalence and risk factors for Chlamydia trachomatis (CT) infection in pregnant women and the rate of transmission of CT to infants.MethodsPregnant women (≥28 weeks gestation) in Vellore, South India were approached for enrollment from April 2009 to January 2010. After informed consent was obtained, women completed a socio-demographic, prenatal, and sexual history questionnaire. Endocervical samples collected at delivery were examined for CT by a rapid enzyme test and nucleic acid amplification test (NAAT). Neonatal nasopharyngeal and conjunctival swabs were collected for NAAT testing.ResultsOverall, 1198 women were enrolled and 799 (67%) endocervical samples were collected at birth. Analyses were completed on 784 participants with available rapid and NAAT results. The mean age of women was 25.8 years (range 18–39 yrs) and 22% (95% CI: 19.7–24.4%) were primigravida. All women enrolled were married; one reported >one sexual partner; and six reported prior STI. We found 71 positive rapid CT tests and 1/784 (0.1%; 95% CI: 0–0.38%) true positive CT infection using NAAT.ConclusionsTo our knowledge, this is the largest study on CT prevalence amongst healthy pregnant mothers in southern India, and it documents a very low prevalence with NAAT. Many false positive results were noted using the rapid test. These data suggest that universal CT screening is not indicated in this population.

Isothermal Amplification Using a Chemical Heating Device for Point-of-Care Detection of HIV-1

BackgroundTo date, the use of traditional nucleic acid amplification tests (NAAT) for detection of HIV-1 DNA or RNA has been restricted to laboratory settings due to time, equipment, and technical expertise requirements. The availability of a rapid NAAT with applicability for resource-limited or point-of-care (POC) settings would fill a great need in HIV diagnostics, allowing for timely diagnosis or confirmation of infection status, as well as facilitating the diagnosis of acute infection, screening and evaluation of infants born to HIV-infected mothers. Isothermal amplification methods, such as reverse-transcription, loop-mediated isothermal amplification (RT-LAMP), exhibit characteristics that are ideal for POC settings, since they are typically quicker, easier to perform, and allow for integration into low-tech, portable heating devices.Methodology/Significant FindingsIn this study, we evaluated the HIV-1 RT-LAMP assay using portable, non-instrumented nucleic acid amplification (NINA) heating devices that generate heat from the exothermic reaction of calcium oxide and water. The NINA heating devices exhibited stable temperatures throughout the amplification reaction and consistent amplification results between three separate devices and a thermalcycler. The performance of the NINA heaters was validated using whole blood specimens from HIV-1 infected patients.ConclusionThe RT-LAMP isothermal amplification method used in conjunction with a chemical heating device provides a portable, rapid and robust NAAT platform that has the potential to facilitate HIV-1 testing in resource-limited settings and POC.

A rapid and simple isothermal nucleic acid amplification test for detection of herpes simplex virus types 1 and 2

Background A simple and rapid IsoAmp ® HSV assay has been developed for qualitative detection of herpes simplex virus (HSV) types 1 and 2 from genital lesions. Sample preparation involved a simple dilution step and the diluted specimens were directly added to the device and amplified by isothermal helicase-dependent amplification (HDA). Amplification products were then detected by a DNA strip embedded in a disposable cassette without any instrument. The total test turn-around time is less than 1.5 h from specimen processing to result reporting. Objectives To evaluate the analytical and clinical performance of the IsoAmp ® HSV assay as well as the robustness and reproducibility of the assay. Study design The analytical sensitivity of the IsoAmp ® HSV assay was determined using both HSV-1 and HSV-2. Clinical performance was evaluated using 135 frozen specimens collected from patients with suspected HSV infection in genital area. Results The analytical sensitivity of the assays was 5.5 and 34.1 copies/reaction for HSV-1 and HSV-2 respectively with a 95% confidence interval. When the herpes viral culture was used as the reference standard, the clinical sensitivity and specificity of the IsoAmp ® HSV assay were 100.0% and 96.3% respectively. The inter-laboratory reproducibility achieved an overall 97.5% agreement by testing a total of 80 blinded HSV-1 samples among five laboratories. Conclusion Adequate analytical and clinical performance of the IsoAmp ® HSV assay was demonstrated. This assay is simple to perform and has acceptable inter-laboratory reproducibility.

Bronchoalveolar lavage for pandemic influenza A (H1N1)v pneumonia in critically ill patients

Dear Editor, Microbiological diagnosis of pandemic influenza A (H1N1)v pneumonia is based on rapid and sensitive nucleic acid amplification test of respiratory samples [1], mainly nasopharyngeal aspirate or, alternatively, nasal and pharyngeal swabs. False negatives can be obtained due to errors in collection, inadequate handling or transport, but also because of low viral load in the specimen. Over 4 months, 18 patients with confirmed influenza virus A (H1N1)v infection were admitted to a medical surgical ICU of a community hospital. Of these, 15 patients presented with severe pneumonia. At ICU admission, respiratory specimens were obtained from the upper respiratory tract. The results of reverse transcriptase polymerase chain reaction (RT-PCR) were positive in 12 cases and negative in 3. Due to high clinical suspicion of the disease, the diagnostic study was extended with other samples. Based on the hypothesis that more extensive viral replication exists in the lower respiratory tract, bronchoalveolar lavage (BAL) was considered. Fiberoptic bronchoscopy is a safe procedure in critically ill patients without absolute contraindications, but in cases of severe hypoxemia, it is not recommended. One of the three patients presented hypoxemia despite high PEEP, and in this case the diagnosis was achieved by serology. Two cases are presented here in which BAL was performed due to a high clinical suspicion of influenza A (H1N1)v pneumonia despite negative RT-PCR results from upper respiratory samples. Case 1: Patient with morbid obesity and epilepsy was admitted to the emergency department with fever, dry cough, and myalgia of 5 days of duration, and required ICU admission due to respiratory failure and the presence of bilateral infiltrates on chest radiograph. At admission the patient was intubated for controlled mechanical ventilation. RT-PCR of nasopharyngeal exudate and aspirate performed at ICU admission tested negative for influenza A (H1N1)v. BAL performed 4 days after ICU admission was positive. Case 2: Young smoker patient was admitted to the pneumology ward with fever, diarrhea, nonproductive cough, and progressive dyspnea. During admission the patient developed respiratory failure and was admitted to the ICU. Chest radiograph showed bilateral alveolar infiltrates, and RT-PCR tests of upper respiratory samples for influenza A (H1N1)v performed at admission and 4 days later were negative. On the 5th day after ICU admission, a BAL was performed, obtaining a positive result. The incidence of false negatives in critically ill patients was documented in a subgroup of 21 such patients in Australia and New Zealand [2]. BAL samples were positive in all patients, but upper respiratory tract samples tested positive in only 17 of 21 patients. Rello and colleagues documented a 10% false negative rate for nasopharyngeal swabs [3]. Based on these findings and the presented cases, negative results of RT-PCR of upper respiratory samples can not rule out the diagnosis of pandemic influenza A (H1N1)v pneumonia [4]. We did not report viral load, as in cases of influenza A (H1N1)v with severe pneumonia, the natural history of viral shedding from the respiratory tract is uncertain. Well-conducted studies are needed to confirm the hypothesis that viral load is higher in the lower respiratory tract than in the

Pitfalls in Diagnosis of Pandemic (Novel) A/H1N1 2009 Influenza

Laboratory diagnosis of influenza is important for treatment, surveillance, infection control, chemoprophylaxis, and monitoring of resistance. Rapid and sensitive nucleic acid amplification tests have widely replaced virus isolation as the reference standard ([9][1]). However, despite improvements

Rapid Antigen Testing Compares Favorably with Transcription-Mediated Amplification Assay for the Detection of Trichomonas vaginalis in Young Women

Diagnosis of Trichomonas vaginalis (TV) infection is limited by imperfect testing methods. Newer tests, such as rapid antigen and nucleic acid amplification tests, are often compared with culture, which is not widely used but is more sensitive than wet mount. We assessed the sensitivity and specificity of 4 tests for the identification of TV using 3 statistical approaches. Sexually active adolescent women aged 14-21 years (n=330) were recruited from a teen health center and emergency department. Vaginal swabs were tested for TV using wet mount, culture (InPouch TV; Biomed Diagnostics), rapid antigen testing (OSOM TV; Genzyme Diagnostics), and transcription-mediated amplification testing (TMA; APTIMA TV analyte specific reagents; Gen-Probe). TV was detected in 61 participants (18.5%). Compared with a composite reference standard (i.e., any TV test with positive results), the sensitivities of wet mount, culture, rapid antigen testing, and TMA were 50.8%, 75.4%, 82%, and 98.4%, respectively. Using latent class analysis, the sensitivity of wet mount (56%) was significantly lower than that of other tests, and the sensitivities of culture and rapid antigen testing were similar (83% and 90%, respectively); specificity was 100% for each of these 3 methods. TMA had a sensitivity of 98.2% and a specificity of 98%. Tests performed equally well regardless of whether the participant had bleeding or other infections. The sensitivities of the rapid antigen test and TMA were comparable (92.5% and 97.5%, respectively) in women who had vaginal symptoms. Wet mount alone is insufficient for the reliable diagnosis of TV infection in women. TMA and rapid antigen tests are highly sensitive and specific, and both are superior to wet mount. Rapid antigen testing is equivalent to culture, and it compares favorably with the sensitivity of TMA for the detection of TV.