BACKGROUND: Detection of the viral load of Human Immunodeficiency Virus type 1 (HIV-1) RNA is important for clinical decision making and for determining the prognosis of HIV-infected patients. The aim of the study is to compare the performance of real-time RT-PCR (COBAS AmpliPrep/COBAS TaqMan HIV-1, CAP/CTM, Roche Diagnostics) and the Nucleic Acid Sequence Based Amplification (NucliSens EasyQ HIV-1, NucliSens, BioMerieux) methods for testing Korean HIV-infected patients. METHODS: Among the specimens that were requested to undergo HIV-1 RNA viral load detection from 2005 to 2006, 153 specimens were selected based on the status of the specimens. The CAP/CTM and NucliSens tests were performed according to the manufacturer's instruction. RESULTS: HIV-1 RNA was detected by both tests in 93 specimens. Among the remainder, CAP/CTM detected HIV-1 RNA in 10 specimens, while the same specimens showed results lower than the detection limit with using the NucliSens. Though the results were appropriately correlated (r=0.85, P<0.0001), the mean differences between the two test results were -0.1321 log(10) IU/mL on the Bland-Altman test. CONCLUSION: The methodologic difference or the presence of a HIV subtype may affect the agreement between the two tests. The standardization of methods and the establishment of a linear range for the individual laboratory results may be helpful to obtain accurate test results.
Decision Making ; HIV ; HIV-1 ; Humans ; Limit of Detection ; Prognosis ; RNA ; Self-Sustained Sequence Replication ; Viral Load

BACKGROUND: Enteroviruses are the most frequent etiologic agents of aseptic meningitis and are estimated to be the cause of 70% to 90% of viral meningitis cases. Enterovirus diagnosis can be difficult because clinical features vary according to patient immunity and age. The purpose of this study was to evaluate the performance of the real-time nucleic acid sequence-based amplification (NASBA) assay compared to that of the real-time nested RT-PCR assay for enterovirus detection. METHODS: This study was performed on 96 patients suspected of aseptic meningitis based on clinical features. RNA was extracted using NucliSENS EasyMAG and real-time NASBA assay was performed using NucliSENS EasyQ Enterovirus and NucliSENS EasyQ Basic 2. We also executed in-house real-time nested RT-PCR assay for RNA extracted via QIAamp Viral RNA Mini. RESULTS: The positive rate of real-time NASBA assay was 45.8% for enterovirus detection. The positive rate of first real-time reverse transcription PCR was 22.9% and the second real-time PCR was 57.3%. The concordant rate of the real-time NASBA assay and first real-time reverse transcription PCR was 75.0%. The concordant rate of the real-time NASBA assay and second real-time PCR was 86.5%. CONCLUSION: The detection of enteroviruses using the real-time NASBA assay is less prone to cross-contamination and is simple, without the need for reverse transcription. We conclude that the NASBA assay is an effective method for the rapid diagnosis of aseptic meningitis.
Enterovirus ; Humans ; Meningitis, Aseptic ; Meningitis, Viral ; Polymerase Chain Reaction ; Real-Time Polymerase Chain Reaction ; Reverse Transcription ; RNA ; RNA, Viral ; Self-Sustained Sequence Replication

OBJECTIVE: In order to improve the diagnosis of invasive aspergillosis (IA) and to establish the monitoring guideline of treatment in neutropenic febrile patients, we evaluated and compared nucleic acid sequence-based amplification (NASBA) with galactomannan-enzyme immunosorbent assay (GM-EIA), and beta-glucan assay. We also determined the tentative cutoff value of NASBA for the presumptive diagnosis of IA. METHODS: Blood samples were collected twice a week from 55 patients with hematologic malignancy during neutropenic fever after chemotherapy or hematopoietic stem cell transplantation. NASBA was carried out by NucliSens kit (BioMerieux) and ECL detector (Sewang Medical, Seoul). GM-EIA was performed by using a sandwich immunocapture ELISA (Platelia Aspergillus, BioRad). beta- glucan was detected using the G test. The tentative cutoff value of NASBA was determined through receiver- operator characteristic (ROC) curve. RESULTS: Total 164 blood samples were tested by three non-culture methods. Based on of EORTC/MSG criteria, 18 out of 55 cases were found to belong to the category of possible to proven cases of IA (1 proven, 2 probable, 15 possible cases). The mean value of NASBA in the IA group was significantly larger than that in the non-IA group (41,665.98 vs 29688.40, respectively, P<0.05). beta- glucan assay showed little concordance to the result of GM-EIA or NASBA. The cutoff value of NASBA determined from ROC curve was 30,000. CONCLUSION: Further study is necessary to determine sensitivity and specificity of NASBA and GM-EIA, and to assess their usefulness for early detection of IA. NASBA cutoff value of 30,000 can be a useful marker for the presumptive diagnosis of IA, and it should be stringently evaluated in the future study.
Aspergillosis* ; Aspergillus ; Diagnosis* ; Drug Therapy ; Enzyme-Linked Immunosorbent Assay ; Fever ; Hematologic Neoplasms ; Hematopoietic Stem Cell Transplantation ; Humans ; ROC Curve ; Self-Sustained Sequence Replication* ; Sensitivity and Specificity

OBJECTIVE: In order to improve the diagnosis of invasive aspergillosis (IA) and to establish the monitoring guideline of treatment in neutropenic febrile patients, we evaluated and compared nucleic acid sequence-based amplification (NASBA) with galactomannan-enzyme immunosorbent assay (GM-EIA), and beta-glucan assay. We also determined the tentative cutoff value of NASBA for the presumptive diagnosis of IA. METHODS: Blood samples were collected twice a week from 55 patients with hematologic malignancy during neutropenic fever after chemotherapy or hematopoietic stem cell transplantation. NASBA was carried out by NucliSens kit (BioMerieux) and ECL detector (Sewang Medical, Seoul). GM-EIA was performed by using a sandwich immunocapture ELISA (Platelia Aspergillus, BioRad). beta- glucan was detected using the G test. The tentative cutoff value of NASBA was determined through receiver- operator characteristic (ROC) curve. RESULTS: Total 164 blood samples were tested by three non-culture methods. Based on of EORTC/MSG criteria, 18 out of 55 cases were found to belong to the category of possible to proven cases of IA (1 proven, 2 probable, 15 possible cases). The mean value of NASBA in the IA group was significantly larger than that in the non-IA group (41,665.98 vs 29688.40, respectively, P<0.05). beta- glucan assay showed little concordance to the result of GM-EIA or NASBA. The cutoff value of NASBA determined from ROC curve was 30,000. CONCLUSION: Further study is necessary to determine sensitivity and specificity of NASBA and GM-EIA, and to assess their usefulness for early detection of IA. NASBA cutoff value of 30,000 can be a useful marker for the presumptive diagnosis of IA, and it should be stringently evaluated in the future study.
Aspergillosis* ; Aspergillus ; Diagnosis* ; Drug Therapy ; Enzyme-Linked Immunosorbent Assay ; Fever ; Hematologic Neoplasms ; Hematopoietic Stem Cell Transplantation ; Humans ; ROC Curve ; Self-Sustained Sequence Replication* ; Sensitivity and Specificity

The study was purposed to investigate diagnostic value of late-mRNA detection by nucleic acid sequence-based amplification (NASBA) technique for human cytomegalovirus (HCMV) infection of the recipients after allogeneic peripheral blood stem cell transplantation (allo-PBSCT) and to evaluate the clinical significance for guiding antiviral therapy. 352 samples were collected from 128 transplant patients after allo-PBSCT. A molecular biological diagnostic technique--NASBA was used to detect human cytomegalovirus (HCMV) late mRNA encoding the viral structural protein PP67 (UL65) expression in peripheral blood of recipients after allo-PBSCT, and the detected results were compared with HCMV DNA detection by PCR. The sensitivity, specificity and early diagnostic value of HCMV mRNA detection for HCMV disease were evaluated. The results showed that out of 352 detected blood specimens from 84 patients 183 specimens (51.99%) were positive of HCMV DNA by PCR, 105 specimens (29.83%) were positive of HCMV mRNA by NASBA. 45 patients were infected by HCMV. The sensitivity and specificity of HCMV DNA and HCMV mRNA for detecting HCMV disease were 95.56% (43/45), 93.33% (42/45) and 60.24% (50/83), 97.59% (81/83). The results of specificity showed significant difference between two groups of HCMV mRNA and HCMV DNA (P < 0.05). It is concluded that the detection of late-mRNA of HCMV by NASBA technique is rapid, sensitive and specific detection for HCMV active infection. The detected result correlates with clinical symptoms. It can monitor HCMV infection of allo-PBSCT transplanted recipients and provide indication to antiviral therapy.
Adolescent ; Adult ; Child ; Cytomegalovirus ; isolation & purification ; Cytomegalovirus Infections ; diagnosis ; virology ; Female ; Hematologic Neoplasms ; therapy ; Humans ; Male ; Peripheral Blood Stem Cell Transplantation ; adverse effects ; RNA, Viral ; analysis ; Self-Sustained Sequence Replication