simple and rapid method for the detection of HIV-1/HCV in co-infected patients

Due to some limitations of serological methods in diagnosis of patients infected with HIV-1 [human immunodeficiency virus] and HCV [hepatitis C virus], it is profoundly important to use molecular methods for the detecting of these infectious agents. However, the most significant problems are the exorbitant cost of these methods and the need of a thermocycler which is an expensive instrument. The current research recruits a multiplex nucleic acid sequence base amplification [NASBA] in order to simultaneously detect HIV-1 and HCV genomes in patients' plasma samples. Sensitivity and specificity of this method have been evaluated using clinical samples. A multiplex NASBA assay for simultaneous detection of HCV and HIV-1 by the use of specific primers were designed and validated. A well-conserved region in the HIV-1 pol gene and 5'-NCR of HCV genome were used. A total of 40 samples of HIV-1 [20 samples] and HCV [20 samples] were used in the NASBA assay. The specificity and sensitivity of the assay were evaluated. Our results have demonstrated that the primers used in the assay had no interrelation with each other and other possible interfering agents in the assay. The analytical sensitivity of the assay for both HIV-1 and HCV was determined to be 1000 copies/mL and the clinical sensitivity and specificity were 93.3% and 100%, respectively. By exploiting this multiplex NASBA assay, it is possible to detect HIV-1 and HCV infection/co-infection in patients' plasma with a suitable sensitivity and specificity. Furthermore, due to its simplicity and multiplexing feature, it could be used in limited access laboratories in a cost-effective manner.

Cervical cytology, HPV DNA and mRNA - a comparative study in 162 patients / Citologia cervicovaginal, DNA HPV e RNAm - estudo comparativo em 162 pacientes

In contrast to the general improvement of the socioeconomic status of the Brazilian population, pathologies that are characteristic of poor health assistance persist. Among those, cervical cancer (CC) is emblematic; it still presents a persistently high incidence. Objective: to compare the performance of cervical cytology to HPV DNA and mRNA detection methods in 162 patients undergoing routine gynecological clinical practice. Methods:a total of 162 patients attended during routine gynecological examination in a private clinic in Florianópolis, Santa Catarina, Brazil, had cervical samplescollected and processed for cytopathological and molecular tests, conventional PCR and NASBA. Positive samples positive for HPV DNA were submittedto Type-Specific PCR (TS-HPV PCR). Patients with altered smears were submitted to colposcopy and biopsy. Results: among the 162 samples, 19.8%(32/162) had altered smears, being 4/32 classified as ASC-H, 9/32 as ASC-US, 9/32 as LSIL and 10/32 as HSIL. Biopsies revealed nine cases of CIN I,nine CIN II and one CIN III, while seven were negative for cervical neoplasia. Overall, HPV DNA was detected in 38.3% (62/162) of the samples and HPV E6/E7 mRNA expression was found in 13.6% (22/162). Using TS-HPV PCR, HPV 16 was the most frequent type, found in 8% of the samples (5/62).Considering CIN2+ the gold-standard, cytology had 38.5% of specificity. Sensitivity and specificity of HPV-DNA PCR and NASBA were, respectively,100% and 60%; 18.7% and 68.7%. Conclusion: mRNA E6/E7 expression was not a highly specific or sensitive marker for prevalent cervical disease while HPV DNA may be used for cervical cancer screening only in conjunction to more specific adjuvant tests.

Em contraste com a melhora geral da situação socioeconômica da população brasileira, patologias que são características de uma deficiente assistência à saúde persistem. Entre elas, o câncer cervical (CC) é emblemático, ainda apresentando uma persistente alta incidência. Objetivo: avaliaro desempenho da citologia e de métodos de detecção de DNA e RNAm de HPV em 162 pacientes submetidas a prática clínica ginecológica de rotina.Métodos: cento e sessenta e duas pacientes atendidas em uma clínica particular de Florianópolis, Santa Catarina, Brasil, tiveram amostras cervicais coletadas e processadas para estudo citopatológico e molecular; PCR convencional e NASBA. Amostras positivas para o DNA do HPV foram submetidas àPCR tipo-específica (PCR HPV-TE). Resultados: entre as 162 amostras, 19,8% (32/162) apresentaram esfregaços alterados, sendo 4/32 classificadas comoASC-H, 9/32 como ASC-US, 9/32 como LSIL e 10/32 como HSIL. Biópsias revelaram nove casos de NIC I, nove casos de NIC II e um caso de NIC III. ODNA do HPV foi detectado em 38,3% (62/162) das amostras. Expressão de E6/E7 (RNAm) foi encontrada em 13,6% (22/162) das amostras. Utilizando a PCR tipo-específica (HPV-TE), o HPV 16 foi o tipo mais frequente, encontrado em 8% (5/62) das amostras HPV+. Considerando NIC 2+ o padrão-ouro,a especificidade da citologia foi de apenas 38,5%, enquanto a sensibilidade e a especificidade da PCR DNA e RNAm foram, respectivamente, 100% e 60%;18,7% e 68,7%. Conclusão: a expressão de E6/E7 RNAm não se mostrou um marcador altamente específico ou sensível para doença cervical prevalente,enquanto o DNA HPV pode ser utilizado para rastreamento apenas em conjunto com testes adjuvantes mais específicos.

Detection of Enterovirus using Real-Time Nucleic Acid Sequence-based Amplification / 대한임상미생물학회지

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.

Comparison of Two Commercial Real-time Nucleic Acid Amplification Methods for Detecting the Viral Load of Human Immunodeficiency Virus Type 1 / 대한수혈학회지

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.

Effectiveness of dual and triple antiretroviral therapy in the treatment of HIV-infected children

OBJETIVOS: Como iniciar a terapia anti-retroviral é uma questão amplamente discutida no manejo de crianças infectadas pelo HIV. O objetivo deste estudo foi comparar a efetividade da terapia dupla e tríplice em uma coorte de crianças infectadas pelo HIV. MÉTODO: Este estudo foi realizado em um serviço de referência para assistência à criança infectada da Faculdade de Medicina da UFMG. Foram incluídas crianças que iniciaram o primeiro regime anti-retroviral entre janeiro de 1998 e dezembro de 2000, com seguimento até dezembro de 2001. O evento final para análise foi a primeira falha terapêutica ou óbito. RESULTADOS: Foram analisados 101 pacientes, sendo 58 (57,4 por cento) e 43 (42,6 por cento) com terapia dupla e tríplice, respectivamente. Não houve diferença entre os grupos quanto ao sexo, idade, contagem de linfócitos CD4+ e carga viral basal. A média de duração da terapia dupla foi de 26,3 meses (IC95 por cento 21,3-31,3) e da terapia tríplice, de 34,3 meses (IC95 por cento 29,2-39,5 por cento). Falha terapêutica ocorreu em 33 (56,9 por cento) pacientes em terapia dupla e 11 (25,6 por cento) em terapia tríplice (log rank 5,03; p = 0,025). O risco relativo de falha para terapia dupla foi 2,2 vezes maior (IC = 1,3-3,9). O percentual de linfócitos T CD4+ inicial foi preditor de risco para falha terapêutica (p = 0,001). Pacientes em terapia tríplice apresentaram maior redução da carga viral (p = 0,001). CONCLUSÃO: A terapia tríplice permaneceu eficaz por mais tempo e apresentou melhor resposta virológica do que a terapia dupla nesta coorte de crianças infectadas pelo HIV, justificando a sua escolha como regime preferencial de tratamento.

OBJECTIVE: The use of antiretroviral therapy in HIV-infected children has been a widely discussed issue. The aim of this study was to compare the effectiveness of dual nucleoside analogue reverse transcriptase inhibitor (NRTI) regimens and three-drug regimens [2NRTI+ non-nucleoside reverse transcriptase inhibitor (NNRTI) or protease inhibitor (PI)] in a cohort of HIV-infected children. METHODS: The study was carried out in a referral center for the management of infected children, which is affiliated with the School of Medicine of Universidade Federal de Minas Gerais (UFMG). Those children whose antiretroviral therapy was implemented between January 1998 and December 2000 and who were followed up until December 2001 were included in the study. Therapeutic failure or death was regarded as the endpoint in our analysis. RESULTS: A total of 101 patients were assessed, 58 (57.4 percent) on dual therapy and 43 (42.6 percent) on triple therapy. No statistically significant difference was observed between the groups in terms of gender, age, CD4+ count and baseline viral load. The average duration of dual therapy was 26.3 months (95 percentCI 21.3-31.3) and that of triple therapy was 34.3 months (95 percentCI 29.2-39.5 percent). There was therapeutic failure in 33 (56.9 percent) patients on dual therapy and in 11 (25.6 percent) patients on triple therapy (log rank = 5.03; p = 0.025). The relative risk of therapeutic failure of the dual therapy was 2.2 times higher (95 percentCI = 1.3-3.9). The percentage of initial CD4+ T cells was a predictor of risk for therapeutic failure (p = 0.001). Patients on triple therapy showed a more remarkable reduction in their viral load (p = 0.001). CONCLUSION: Triple therapy was efficient for a longer time period and showed better virologic response than dual therapy in this cohort of HIV-infected children. Therefore, triple therapy should be the treatment of choice.

Application of nucleic acid sequence-based amplification in detection of cytomegalovirus mRNA of recipients after allogeneic peripheral blood stem cell transplantation / 中国实验血液学杂志

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.

Evaluation of Nucleic Acid Sequence-Based Amplification (NASBA) for Diagnosis and Monitoring of Invasive Aspergillosis : A Preliminary Report

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.

Evaluation of Nucleic Acid Sequence-Based Amplification (NASBA) for Diagnosis and Monitoring of Invasive Aspergillosis : A Preliminary Report

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.

Significant differences between plasma HIV-1 RNA assays in HIV-1 subtype E infected patients treated with antiretroviral therapy.

A total of 72 HIV-1 infected Thai patients treated with didanosine (ddI) or stavudine (d4T) plus ddI at the time of interim analysis were analyzed. Sixty patients (83%) carried subtype E documented by HIV-1 V3 serotyping. HIV-1 RNA levels were measured using three commercial viral load assays. At baseline (n = 57), Quantiplex 2.0 and NucliSens 2.0 showed mean log10 HIV-1 RNA of 0.7 log10 or 5 fold lower than Amplicor 1.5 (mean 4.29 versus 5.0 log10, respectively, p < 0.001). At week 20 of treatment (n = 29), HIV-1 RNA levels were detected in 55.2%, 31%, and 33.5% of subjects tested by Amplicor 1.5, Quantiplex 2.0, and NucliSens 2.0, respectively. In conclusion: plasma HIV-1 RNA analyses showed comparable values with Quantiplex 2.0 and NucliSens 2.0 assays. In contrast, Amplicor 1.5 resulted in approximately 5 folds higher HIV-1 RNA levels and a 25% higher rate of detection of plasma HIV-1 RNA as compared to the other two assays. As the current goal of therapy is to suppress plasma viral load below the detection limit of the assays, the significant differences between the assays may influence antiretroviral efficacy evaluation and management.