Sensitivity Of Nucleic Acid Sequence-based Amplification Research Articles

Application and evaluation of nucleic acid sequence-based amplification, PCR and cryptococcal antigen test for diagnosis of cryptococcosis

BackgroundCryptococcosis is a major opportunistic invasive mycosis in immunocompromised patients, but it is also increasingly seen in immunocompetent patients. In the early stages of cryptococcosis, limitations of the detection method may hinder the diagnosis. A molecular diagnostic technique based on nucleic acid sequence-based amplification (NASBA) method was developed to fulfil the need for efficient diagnosis of cryptococcosis.MethodsWe compared the diagnostic performance of NASBA, PCR and cryptococcal antigen (CrAg) test (colloidal gold method) in clinical samples from 25 cryptococcosis patients (including 8 cryptococcal meningoencephalitis and 17 pulmonary cryptococcosis) who were categorized as proven cases (n = 10) and probable cases (n = 15) according to the revised EORTC/MSG definitions. 10 patients with non-Cryptococcus infection and 30 healthy individuals were categorized as control group.ResultsThe lowest detection limit of NASBA was 10 CFU/mL, and RNA of non-target bacteria or fungi was not amplified. The sensitivity of NASBA, PCR and colloidal gold method was 92.00% (95% CI 72.50–98.60%), 64.00% (95% CI 42.62–81.29%), 100.00% (95% CI 83.42–100.00%), and the specificity was 95.00% (95% CI 81.79–99.13%), 80.00% (95% CI 63.86–90.39%) and 82.50% (95% CI 66.64–92.11%) respectively. The highest specificity (97.50%), accuracy (95.38%) and k value (0.90) were achieved when both NASBA and colloidal gold results were positive.ConclusionsNASBA is a new alternative detection method for cryptococcosis which is both accurate and rapid without expensive equipment and specialised personnel. It may be used as a tool for confirming current infection as well as monitoring the effectiveness of antifungal treatment. The use of NASBA to detect Cryptococcus RNA in blood samples is of great significance for the diagnosis of pulmonary cryptococcosis. The combination of NASBA and colloidal gold can improve the diagnostic accuracy of cryptococcosis.

Retrospective Comparison of Nucleic Acid Sequence–Based Amplification, Real-Time PCR, and Galactomannan Test for Diagnosis of Invasive Aspergillosis

Invasive aspergillosis is a life-threatening infection in immunocompromised patients, and treating these infections at an early stage is often crucial for a favorable outcome. Early diagnosis, however, remains challenging. We performed a retrospective comparison of three methods: real-time quantitative PCR (qPCR), nucleic acid sequence-based amplification (NASBA), and galactomannan enzyme-linked immunosorbent assay (GM-ELISA); these detect circulating Aspergillus DNA, RNA, and galactomannan, respectively. Blood samples from 80 patients at high risk for invasive aspergillosis were tested by each assay. The sensitivity of NASBA, qPCR, and GM-ELISA was 76.47% (95% CI, 58.4-88.6%), 67.65% (95% CI, 49.4-82.0%), and 52.94% (95% CI, 35.4-69.8%), respectively, and the specificity was 80.43% (95% CI, 65.6-90.1%), 89.13% (95% CI, 75.6-95.9%), and 80.43% (95% CI, 65.6-90.1%), respectively. We also evaluated the efficiency of the three tests in various combinations. Perfect specificity (100%; 95% CI, 90.4-100%) and perfect positive predictive value (100%; 95% CI, 77.1-100%) were achieved by combining NASBA and qPCR testing in series. Testing with both NASBA and qPCR in parallel was the most sensitive and had the highest Youden index. Our data support the great potential of NASBA and qPCR, singly or in combination, for diagnosis of invasive aspergillosis in high-risk populations.

Sensitivity of detection of rhinoviruses in spiked clinical samples by nucleic acid sequence-based amplification in the presence of an internal control

The objectives of the study were to determine the sensitivity of Nucleic Acid Sequence-Based Amplification (NASBA) for the detection of rhinovirus (RV) serotype 15 in water and in spiked respiratory specimens in the presence of a newly developed internal control (IC). The sensitivity of NASBA on RNA was 10 molecules per reaction. The sensitivity of RV NASBA on RV-15 in water and in respiratory specimens was equal to that in tissue culture. Addition of 10 4 molecules of rhinovirus internal control (RV IC) did not affect the sensitivity. Viral RNA should be extracted from clinical specimens as rapidly as possible after collection, since loss of detectable RNA occurs after 2 h. NASBA allows a sensitive detection of RV RNA in spiked respiratory specimens in the presence of an internal control.

A prospective study comparing cytomegalovirus antigenemia, DNAemia and RNAemia tests in guiding pre-emptive therapy in thoracic organ transplant recipients

We evaluated the usefulness of DNAemia and mRNAemia tests in guiding the pre-emptive therapy against cytomegalovirus (CMV) infections in thoracic organ transplant recipients using antigenemia test as the reference. Seven lung (LTR) and 14 heart (HTR) transplant recipients were prospectively monitored for CMV by antigenemia, DNAemia (Cobas Amplicor PCR Monitor) and pp67-mRNAemia (NASBA) tests. However, only the antigenemia test guided pre-emptive therapy with cut-off levels of >or=2 and >or=5-10 pp65-positive leukocytes/50 000 leukocytes in the LTRs and HTRs, respectively. CMV DNAemia was detected in 26/28 (93%) and RNAemia in 17/28 (61%) of the CMV antigenemias requiring antiviral therapy (P = 0.01). Optimal DNAemia levels (sensitivity/specificity) estimated from receiver-operating characteristic curve to achieve maximal sum of sensitivity and specificity were 400 (75.9/92.7%), 850 (91.3/91.3%) and 1250 (100/91.5%) copies/ml for the antigenemia of 2, 5 and 10 pp65-positive leukocytes, respectively. The sensitivities of nucleic acid sequence-based amplification (NASBA) were 25.9%, 43.5% and 56.3% in detecting the same cut-off levels of antigenemia. In thoracic organ transplant recipients, the Cobas PCR assay is comparable with the antigenemia test in guiding pre-emptive therapy against CMV infections when threshold levels of over 5 pp65-antigen-positive leukocytes are used as the reference. In contrast, the low sensitivity of NASBA limits its usefulness in the guidance of pre-emptive therapy.

Development and evaluation of a nucleic acid sequence based amplification (NASBA) protocol for the detection of enterovirus RNA in cerebrospinal fluid samples

A nucleic acid sequence based (NASBA) assay for the generic detection of enterovirus RNA in cerebrospinal fluid (CSF) samples was developed and compared with an established reverse transcription/nested polymerase chain reaction (PCR) protocol. The sensitivity of NASBA followed by detection of amplicons with a biotinylated oligonucleotide probe was ≤ ten copies of enterovirus RNA, indicating that enterovirus NASBA achieves a similar sensitivity as nested PCR. Moreover, NASBA detected a panel of 22 different serotypes of the species poliovirus, human enterovirus A, human enterovirus B and human enterovirus C completely. For evaluating NASBA as a diagnostic tool, 61 CSF samples of patients suffering from aseptic meningitis were tested in parallel with NASBA and nested PCR. NASBA detected enterovirus RNA in four CSF samples, two of these were also positive by nested PCR and two other CSF samples were positive only by nested PCR (in total six positive samples). All other 55 of 61 CSF samples were concordantly enterovirus negative by both methods. In conclusion, the more simple to handle ‘one step’ NASBA is as sensitive as nested PCR and may be used as an alternative method for the detection of enterovirus RNA in CSF samples. Enterovirus NASBA is a ‘one step’ RNA amplification procedure that is less prone to cross-contamination compared to a three step nested PCR.