Evaluation of different nucleic acid amplification techniques for the detection of M. pneumoniae, C. pneumoniae and Legionella spp. in respiratory specimens from patients with community-acquired pneumonia.

The number of pathogens involved in community-acquired pneumonia, with varying susceptibilities to antimicrobials, is numerous constituting an enormous challenge for diagnostic microbiology. Differentiation of infections due to Streptococcus pneumoniae and those due to Mycoplasma pneumoniae, Chlamydophila pneumoniae, or L. pneumophila as well as those due to viruses is essential to allow correct decisions concerning the antibiotics to be administered. The sensitivity and specificity of real-time simplex and multiplex nucleic acid sequence-based amplification (NASBA), and simplex PCR were compared for the detection of M. pneumoniae, C. pneumoniae and Legionella spp. in respiratory specimens from hospitalized and outpatients with community-acquired pneumonia (CAP). Two hundred fifty one respiratory specimens were collected from 147 patients with CAP. NASBA was done using the NucliSens Basic Kit (bioMérieux). PCR for M. pneumoniae and C. pneumoniae was done as described earlier [Ieven, M., Ursi, D., Van Bever, H., Quint, W., Niesters, H. G. M., and Goossens, H. 1996. Detection of Mycoplasma pneumoniae by two polymerase chain reactions and role of M. pneumoniae in acute respiratory tract infections in pediatric patients. J. Infect. Dis. 173, 1445-14452.; Ursi, D., Ieven, M., Van Bever, H. P., and Goossens, H. 1998. Construction of an internal control for the detection of Chlamydia pneumoniae by PCR. Mol. Cellul. Probes. 12, 235-238.]. A real-time PCR was developed to detect L. pneumophila whereas a real-time NASBA was designed to detect Legionella spp. All samples with discordant results were re-analysed. Compared to an expanded gold standard the sensitivities of the different techniques, were 77.8%, 100%, and 100% for detection of M. pneumoniae; and 50%, 100%, and 50% for detection of L. pneumophila by PCR, real-time simplex NASBA, and real-time multiplex NASBA, respectively. C. pneumoniae was detected in two samples only. Simplex real-time NASBA proved to be more sensitive than simplex PCR and was also more sensitive than real-time multiplex NASBA, as previously found with spiked clinical specimens. It's practical attractiveness pleads for further optimalisation of the multiplex approach.

Development of real-time multiplex nucleic acid sequence-based amplification for detection of Mycoplasma pneumoniae, Chlamydophila pneumoniae, and Legionella spp. in respiratory specimens.

Real-time multiplex isothermal nucleic acid sequence-based amplification (NASBA) was developed to detect Mycoplasma pneumoniae, Chlamydophila pneumoniae, and Legionella spp. in respiratory specimens using the NucliSens Basic Kit (bioMérieux, Boxtel, The Netherlands). Oligonucleotide primers were derived from the M. pneumoniae, C. pneumoniae, and Legionella pneumophila 16S rRNA. For real-time detection, molecular beacons were used. Specificity was established on a panel of bacterial strains. The analytical sensitivity of the assay was determined by testing dilutions of wild-type in vitro-generated RNA in water and dilutions of reference strains in lysis buffer or added to pools of respiratory specimens. Subsequently, a limited number of M. pneumoniae-, C. pneumoniae-, and L. pneumophila-positive and -negative clinical specimens were analyzed. Specific detection of the 16S rRNA of the three organisms was achieved. The analytical sensitivity of the multiplex NASBA on spiked respiratory specimens was slightly diminished compared to the results obtained with the single-target (mono) real-time assays. We conclude that the proposed real-time multiplex NASBA assay, although less sensitive than the real-time mono NASBA assay, is a promising tool for the detection of M. pneumoniae, C. pneumoniae, and Legionella spp. in respiratory specimens, regarding handling, speed, and number of samples that can be analyzed in a single run.

Development of conventional and real-time NASBA for the detection of Legionella species in respiratory specimens.

Isothermal nucleic acid sequence-based amplification (NASBA) was applied to detect Legionella 16S rRNA. The assay was originally developed as a Legionella pneumophila conventional NASBA assay with electrochemiluminescence (ECL) detection and was subsequently adapted to a L. pneumophila real-time NASBA format and a Legionella spp. real-time NASBA using molecular beacons. L. pneumophila RNA prepared from a plasmid construct was used to assess the analytical sensitivity of the assay. The sensitivity of the NASBA assay was 10 molecules of in vitro wild type L. pneumophila RNA and 0.1-1 colony-forming units (CFU) of L. pneumophila. In spiked respiratory specimens, the sensitivity of the NASBA assays was 1-10000 CFU of L. pneumophila serotype 1 depending on the background. After dilution of the nucleic acid extract prior to amplification, 1-10 CFU of L. pneumophila serotype 1 could be detected with both detection methods. Finally, 27 respiratory specimens, well characterized by culture and PCR, collected during a L. pneumophila outbreak, were tested by conventional and real-time NASBAs. All 11 PCR positive samples were positive by conventional NASBA, 9/11 and 10/11 were positive by L. pneumophila real-time NASBA and Legionella spp. real-time NASBA, respectively.

Development of conventional and real-time nucleic acid sequence-based amplification assays for detection of Chlamydophila pneumoniae in respiratory specimens.

Isothermal nucleic acid sequence-based amplification (NASBA) was applied to the detection of Chlamydophila pneumoniae 16S rRNA by using the NucliSens basic kit (bioMérieux, Boxtel, The Netherlands). The assay was originally developed as a conventional NASBA assay with electrochemiluminescence detection and was subsequently adapted to a real-time NASBA format by using a molecular beacon. C. pneumoniae RNA prepared from a plasmid construct was used to assess the analytical sensitivity of the assay. The sensitivity of the NASBA assay was 10 molecules of in vitro wild-type C. pneumoniae RNA and 0.1 inclusion-forming unit (IFU) of C. pneumoniae. In spiked respiratory specimens, the sensitivity of the C. pneumoniae NASBA assay varied between 0.1 and 1 IFU/100 mul sample, depending on the type of specimen. Finally, conventional and real-time NASBA were applied to respiratory specimens previously tested by PCR. A 100% concordance between the test results was obtained.

Detection of rhinoviruses by tissue culture and two independent amplification techniques, nucleic acid sequence-based amplification and reverse transcription-PCR, in children with acute respiratory infections during a winter season.

Five hundred seventeen consecutive nasopharyngeal aspirates were collected between October 1998 and May 1999 for episodes of acute respiratory tract infections in children presenting at the University Hospital of Antwerp. Culture and nucleic acid amplification techniques--nucleic acid sequence-based amplification (NASBA) and reverse transcription-PCR (RT-PCR)--were applied to detect rhinoviruses (RVs). Other respiratory viruses were detected by immunofluorescence (IF) analysis of the specimens and IF analysis of shell vial cultures. Among the 517 specimens, 219 viral agents were identified. They were, in decreasing order, rhinoviruses (93 [18.0%]), respiratory syncytial virus (76 [14.7%]), adenoviruses (16 [3.1%]), influenza viruses (15 [2.9%]), enteroviruses (15 [2.9%]), and herpes simplex virus (4 [0.8%]). For the evaluation of rhinovirus detection, culture positivity and/or a positive reaction in the two independent amplification methods was used as an expanded "gold standard." Based on this standard, the sensitivity, specificity, positive predictive value, and negative predictive value of culture were 44.7, 100, 100, and 99.8%, and those of NASBA and RT-PCR were 85.1, 98.3, 83.3, and 98.5% and 82.9, 93.4, 55.7, and 98.2%, respectively. NASBA and RT-PCR produced comparable results and were significantly more sensitive than virus culture. RVs showed the highest incidence in acute respiratory tract infections in children.

Detection of Mycoplasma pneumoniae by real-time nucleic acid sequence-based amplification.

Real-time isothermal nucleic acid sequence-based amplification (RT-NASBA) was applied to the detection of Mycoplasma pneumoniae. In vitro-generated M. pneumoniae RNA was used to assess the sensitivity of the assay. The 95% hit rate was 148 molecules of M. pneumoniae RNA in the amplification and 10(4) molecules of in vitro-generated RNA after nucleic acid extraction. The sensitivity of the RT-NASBA and the conventional NASBA assays corresponded to 5 color-changing units (CCU) of M. pneumoniae. In spiked throat swabs, nasopharyngeal aspirates, bronchoalveolar lavages, and sputum, the sensitivity of both NASBA assays corresponded to 5 to 50 CCU of M. pneumoniae. A total of 17 clinical specimens positive for M. pneumoniae by PCR were also positive by conventional NASBA, but one specimen was negative by RT-NASBA. These results indicate that the sensitivity of detection of M. pneumoniae by RT-NASBA in respiratory samples might be slightly reduced compared to that by conventional NASBA. However, the real-time assay is superior in speed and ease of handling.

Improved detection of rhinoviruses by nucleic acid sequence-based amplification after nucleotide sequence determination of the 5' noncoding regions of additional rhinovirus strains.

The isothermal nucleic acid sequence-based amplification (NASBA) system was applied for the detection of rhinoviruses using primers targeted at the 5' noncoding region (5' NCR) of the viral genome. The nucleotide sequence of the 5' NCRs of 34 rhinovirus isolates was determined to map the most conserved regions and design more appropriate primers and probes. The assay amplified RNA extracted from 30 rhinovirus reference strains and 88 rhinovirus isolates, it did not amplify RNA from 49 enterovirus isolates and other respiratory viruses. The assay allows one to discriminate between group A and B rhinoviruses. Sensitivities for the detection of group B and group A rhinoviruses was 20 and 200 50% tissue culture infective doses, respectively.

Application of NucliSens Basic Kit for the detection of Mycoplasma pneumoniae in respiratory specimens.

A commercially available nucleic acid sequence-based amplification (NASBA) NucliSens Basic Kit (NBK) assay for the detection of Mycoplasma pneumoniae 16S rRNA in respiratory specimens was developed and compared to standard NASBA and PCR assays previously developed in our laboratory. The specificity and sensitivity of the NBK assay was comparable to the specificity and sensitivity of the corresponding standard NASBA assay. The NBK offers standardized reagents for the development of a NASBA assay for the detection of M. pneumoniae in respiratory specimens and is easily adaptable to other amplification targets.

Detection of Mycoplasma pneumoniae in spiked clinical samples by nucleic acid sequence-based amplification.

Isothermal nucleic acid sequence-based amplification (NASBA) was applied to the detection of Mycoplasma pneumoniae. M. pneumoniae RNA prepared from a plasmid construct was used to assess the sensitivity of the assay, and an internal control for the detection of inhibitors was constructed. The sensitivity of the NASBA assay was 10 molecules of wild-type M. pneumoniae RNA generated in vitro and 5 color-changing units (CCU) of M. pneumoniae. An appropriate specimen preparation procedure was developed: after protease treatment of the respiratory specimens, guanidine thiocyanate lysis solution (4.7 M guanidine thiocyanate [Sigma-Aldrich NV], 46 mM Tris-HCl [Merck, Darmstadt, Germany], 20 mM EDTA [Sigma-Aldrich NV], 1.2% [wt/vol] Triton X-100 [Sigma-Aldrich NV], pH 6.2.) was added. With spiked throats, nasopharyngeal aspirates, bronchoalveolar lavage specimens, and sputum specimens, the sensitivity of the NASBA assay in the presence of the internal control was 2 x 10(4) molecules of in vitro-generated RNA or 5 CCU of M. pneumoniae. The sensitivity of the NASBA assay was comparable to that of a PCR targeted to the P1 adhesin gene. Fifteen clinical specimens positive for M. pneumoniae by PCR were also positive by NASBA. These results indicate that the sensitivity of detection of M. pneumoniae in spiked respiratory samples by NASBA is high. Together with the use of the internal control, the assay merits evaluation as a diagnostic tool.

Quantitative competitive NASBA for measuring mRNA expression levels of the immediate early 1, late pp67, and immune evasion genes US3, US6 and US11 in cells infected with human cytomegalovirus.

Different cell types were infected with human cytomegalovirus (HCMV) and RNA expression dynamics were analyzed by quantitative NASBA assays for IE1 (UL123), pp67 (UL65) and the immune evasion genes (US3, US6 and US11). The quantitative NASBA assays gave reproducible quantification in the range of 10(3)-10(6) RNA copies for IE1 and pp67 RNA, from 3x10(3) to 1x10(6) RNA copies for US6 and US11 RNA, and from 1x10(4) to 1x10(6) RNA copies for US3 RNA. SMC, HAEC and HUVEC cells infected with an, in endothelial cells, propagated HCMV strain (VHL/E) showed similar RNA expression dynamics for the analyzed genes. Expression of all genes studied was observed within the first 4 h post-infection. The first gene for which expression could be detected was IE1, followed by US3, US11, pp67 and US6. Fibroblasts infected with HCMV strain AD169 showed a different RNA expression pattern for US3. Translation of the mRNA studied was demonstrated by detection of the proteins 48 h post-infection by immunofluorescence.

Sustained virological response in chronic hepatitis C patients after a 6- and a 36-month interferon-alpha2b treatment schedule: a multicenter, randomized, controlled study.

BACKGROUND: In patients with chronic hepatitis C (HCV) Interferon-alpha (IFN) treatment for 12-18 months is more effective than 6 months in inducing a sustained virological response. METHODS: In a multicenter, randomized, controlled trial, 88 patients with chronic HCV were enrolled (47 treated with IFN-alpha2b and 41 constituted an untreated control group). Treatment consisted of 5 million units (MU) IFN thrice a week (tiw) for 8 weeks and subsequently 2.5 MU IFN tiw for 16 weeks ('standard treatment'). After week 24 ('long-term treatment'), in virological non-responders treatment was continued using 5 MU IFN tiw for up to week 156, whereas in virological responders IFN was discontinued. In case of a virological relapse, treatment with 5 MU IFN tiw was restarted and continued up to week 156. RESULTS: Sustained virological response rate was 6/47 (13%) after standard treatment and increased to 19/47 (40%) after long-term treatment (McNemar paired test; P = 0.002). Of the 18 patients with a breakthrough or relapse during or after standard treatment, 14 (78%) became sustained virological responders upon long-term treatment. Of the 4 patients who did not have a sustained virological response after long-term treatment, 3 did not receive complete treatment due to side effects and/or non-compliance. In patients who failed to respond to standard treatment, no virological response was observed during long-term treatment. In the control group, no spontaneous clearance of HCV was observed. CONCLUSIONS: Long-term IFN (re)treatment enhanced the virological sustained response rate significantly and was particularly effective in patients with a breakthrough or relapse following standard treatment.

Analytical performance and clinical utility of a nucleic acid sequence-based amplification assay for detection of cytomegalovirus infection.

A nucleic acid sequence-based amplification (NASBA) assay for qualitative detection of human cytomegalovirus (CMV) pp67 mRNA was evaluated in a multicenter study. Negative results were obtained for all specimens from 50 CMV-seronegative and 50 CMV-seropositive low-risk whole-blood donors. No interference with CMV mRNA amplification was observed in the testing of 288 specimens containing various potential interfering substances, nonspecifically reacting substances (including mRNA from other herpesviruses), and three anticoagulants. A total of 95% (50 of 51) of CMV-positive (cell culture- and antigenemia immunofluorescence [AG-IFA]-positive) clinical specimens were positive by the NASBA assay. Results from different operators over multiple testing days were consistent for each of four panel members containing different concentrations of CMV mRNA, indicating the reproducibility of the assay. The estimated 95% reliable upper detection limit of the assay was 600 mRNA copies; the lower limit of detection was less than 25 mRNA copies. The clinical utility of the assay was evaluated with longitudinally collected specimens from solid-organ transplant patients (n = 21). A total of 98% (81 of 83) of the specimens from CMV-negative patients were negative by the NASBA assay, while 90% (10 of 11) of patient specimens that were positive by cell culture or AG-IFA were positive by the NASBA assay. Positive NASBA assay results were obtained earlier than AG-IFA or cell culture results for 55% of the patients and at the same time for the remainder of the patients (45%). The overall agreement between the NASBA assay and current reference tests was 86% when active CMV infection was present. These studies indicate that the CMV pp67 mRNA NASBA assay has reproducible and sensitive performance characteristics that should enable more rapid diagnosis of CMV infection.

Characterization of the quantitative HCV NASBA assay.

A quantitative nucleic acid sequence-based amplification assay (NASBA-QT) for detection of hepatitis C virus RNA (HCV-RNA) was evaluated and compared with the HCV branched-DNA (bDNA) assay (Chiron Corporation) and the HCV MONITOR assay (Roche Diagnostic Systems). For this evaluation five panels were designed: (1) serial dilutions of genotype 1b in-vitro HCV-RNA; (2) standards of in-vitro HCV-RNA genotypes 1a, 1b, 2, 3, 4, and 5; (3) a proficiency panel consisting of 12 HCV-RNA positive plasma samples of different genotypes and HCV-RNA concentrations and a genotype 1a and 1b 3-fold dilution series; (4) a panel of 67 HCV-RNA positive plasma samples obtained from patients with HCV infection and (5) an HCV-RNA positive control sample, diluted 50-fold in 25 different HCV-RNA negative plasma samples. The quantitative detection limit was found to be 10(3) copies per 100 microl and the qualitative detection limit 10(2.3) per 100 microl. The amplification efficiency was independent of the plasma matrix, but dependent on the HCV genotype. The HCV NASBA-QT assay was more than 10 times as sensitive as the bDNA assay while the quantitative results of both assays were highly concordant. The HCV NASBA-QT assay was comparable in sensitivity with the HCV MONITOR assay, but the HCV MONITOR assay yielded consistently lower values. It is concluded that the HCV NASBA-QT assay is a reliable assay for quantitative HCV-RNA detection in various settings.

Early detection of human cytomegalovirus infection after kidney transplantation by nucleic acid sequence-based amplification.

BACKGROUND: The early detection of human cytomegalovirus infection after organ transplantation is a prerequisite for effective antiviral therapy. We evaluated the diagnostic value of monitoring the viral immediate-early (IE) 1 mRNA expression in blood leukocytes by nucleic acid sequence-based amplification (NASBA). METHODS: Nucleic acids were isolated from 489 blood samples collected from 42 kidney transplant recipients and subjected to amplification by IE NASBA. The IE NASBA results were compared to those from pp67 NASBA, pp65 antigenemia, cell culture (DEAFF and CPE), and serology. RESULTS: IE NASBA proved to be the most sensitive assay which detected the onset of both primary and secondary cytomegalovirus infection significantly earlier than the other assays. CONCLUSIONS: The early detection of cytomegalovirus infection with IE NASBA would enable the start of effective antiviral therapy at an early state of infection to prevent cytomegalovirus disease in patients at risk.

Diagnostic value of nucleic-acid-sequence- based amplification for the detection of cytomegalovirus infection in renal and liver transplant recipients.

To evaluate the diagnostic value of nucleic-acid-sequence-based amplification (NASBA) for the detection of cytomegalovirus (CMV) infection in transplant recipients, we compared immediate early 1 (IE1) and late pp67 mRNA detection by NASBA with the antigenemia assay, PCR and viral culture in 72 renal transplant (RTx) recipients and with antigenemia and serology in 25 liver transplant (LTx) recipients. Antigenemia, viral culture and pp67 NASBA were almost equivalent for the detection of CMV in RTx recipients. In LTx recipients, antigenemia detected more positive samples and more positive recipients compared to pp67 NASBA. In RTx recipients, PCR detected more positive samples and positive recipients compared to pp67 NASBA, antigenemia and viral culture. Also the first day of detection was slightly earlier for PCR. However, IE1 NASBA was the most sensitive test and detected 96% of all positive samples and positive transplant recipients. In addition, IE1 NASBA preceded PCR and all other positive results. This makes IE1 NASBA a very attractive screening test for the early detection of CMV infection.

Monitoring of Chlamydia trachomatis infections after antibiotic treatment using RNA detection by nucleic acid sequence based amplification.

AIM: To investigate the value of RNA detection by nucleic acid sequence based amplification (NASBA) for the monitoring of Chlamydia trachomatis infections after antibiotic treatment. METHODS: Cervical smears (n = 97) and urine specimens (n = 61) from 25 C trachomatis positive female patients were analysed for the presence of C trachomatis 16S ribosomal RNA (rRNA) by NASBA and C trachomatis plasmid DNA by the polymerase chain reaction (PCR) before and up to five weeks after antibiotic treatment. RESULTS: Chlamydia trachomatis RNA was found in all cervical smears taken before antibiotic treatment (n = 24) and in two smears taken one week after antibiotic treatment; no C trachomatis RNA was detected after two weeks or more. In contrast, C trachomatis DNA was found in all such specimens before treatment, and 21 of 25, six of 21, and five of 20 smears were found to be positive at one, two, and three weeks after treatment, respectively. After four weeks, only one of six smears was positive, and this smear had been negative in the two preceding weeks. Of the 61 urine samples investigated, C trachomatis DNA and C trachomatis RNA were found in all before treatment (n = 15), whereas one week after treatment four of 15 were C trachomatis DNA positive and C trachomatis RNA was detected in one sample only. CONCLUSIONS: These data show that RNA detection by NASBA can be used successfully to monitor C trachomatis infections after antibiotic treatment. Furthermore, it might be possible to use urine specimens as a test of cure because neither C. trachomatis DNA or RNA could be detected two weeks or more after treatment.

Stability of hepatitis C virus RNA during specimen handling and storage prior to NASBA amplification.

The influence of different anticoagulants and pre-amplification storage conditions on the stability of hepatitis C virus (HCV)-RNA, as detected by the quantitative HCV NASBA assay (NASBA-QT), was studied. The HCV-RNA load remained stable for at least 15 months when serum or plasma samples (EDTA and heparin) were directly frozen at -70 degrees C in lysis buffer. At 4 degrees C, the HCV-RNA load in serum or plasma stored with lysis buffer did not decline for at least 14 days. At 30 degrees C, however, the load declined significantly after 7 days. When clotted, whole blood was stored at 4 degrees C, the HCV-RNA load was stable for 72 h. However, when EDTA-anticoagulated whole blood was stored at 4 degrees C, the HCV-RNA load declined significantly after 48 h. In paired plasma and serum samples at baseline the HCV-RNA levels were similar. Heparin did not influence the efficiency of the HCV NASBA-QT assay. Clotted blood as well as EDTA or heparin anticoagulated blood can be used for quantifying HCV-RNA using the NASBA-QT assay. Blood samples should be stored at 4 degrees C after collection and serum or plasma separated within 24 h. Preferably, after separation, samples should be frozen in lysis buffer at -70 degrees C until NASBA-QT analysis.