Research progress on the nucleic acids diagnosis of rabies virus / 医学研究生学报

Rabies is a deadly zoonos caused by rabies virus, the case fatality almost is 100%following suffering from this dis-ease.Recently, the incidence of rabies tends to increase in China.A rapid, accurate, economical, simple and convenient, easy to be widely used method for rabies virus detection in laboratory is urgently needed.This paper reviews the nucleic acids diagnosis techniques of rabies virus from the nucleic acids detection methods based on PCR, NASBA, LAMP, Genechips and analyzes the advantages and disadvantagesof the above-mentioned methods, which provides a reference for rapid laboratory diagnosis of rabies virus to efficently prevent and control rabies.

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.

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.

A prospective study of enterovirus infection in Thai infants presenting as sepsis.

Objective: To study prospectively the prevalence, clinical presentations and laboratory findings of enterovirus (EV) infection in infants under 3 months of age who present as a sepsis-like syndrome. Method: All infants less than 3 month of age admitted as a sepsis-like syndrome to King Chulalongkorn Memorial Hospital between April 2003 and February 2004 were included. Patients who were immunocompromised or who had been admitted for longer than 14 days before developing symptoms were excluded. A detailed history, physical and laboratory findings were recorded and analyzed. Specimens of blood and cerebrospinal fluid were tested for enteroviruses using Nucleic Acid Sequence-Based Amplification (NASBA). Patients were followed to determine the clinical outcome and duration of hospitalization. Results: Of 56 infants, thirty-six were admitted to the pediatric wards and 20 had been hospitalized since birth in the neonatal intensive care unit (NICU) or nursery wards. Enterovirus infection was diagnosed in 13 (36.1 %) of the patients admitted to the pediatric wards and none in the group of NICU/nursery patients. The most common clinical presentations were high grade fever (92 %), rashes (77 %) and lethargy (54 %) as compared to fever (78.3 %), poor feeding (60.9 %) and lethargy (56.5 %) in the EV negative group. Ten (76.9 %) of the enterovirus positive infants had evidence of central nervous system (CNS) involvement as evidenced by the presence of EV RNA in cerebrospinal fluid (CSF) or CSF pleocytosis plus EV RNA in blood and/or CSF. Nevertheless, CSF pleocytosis was found in only 7 infants (53.8 %). Average duration of illness was 3.2 days as compared to 3.5 days in the nonenteroviral group with similar clinical features. All enterovirus positive patients had an uncomplicated recovery. Ten (76.9 %) received parenteral antibiotics for a mean of 5 days (versus 4.8 days in enterovirus negative group). The average length of stay was 8.1 days as compared to 15 days in enterovirus negative group. Conclusion: Enterovirus infections are important causes of a sepsis-like syndromes in infants under 3 months of age. Most enterovirus infected patients presented with fever without localizing signs and rashes. Detection of enterovirus RNA by NASBA in serum and/or CSF represents a rapid method for the diagnosis of enterovirus infection in infants presenting with a sepsis-like syndrome.

Comparison of the Real-time Nucleic Acid Sequence-based Amplification (RTi-NASBA) with Conventional NASBA, and Galactomannan Assay for the Diagnosis of Invasive Aspergillosis

We compared a real time-nucleic acid sequence-based amplification (RTi-NASBA) with conventional NASBA, galactomannan enzyme immunosorbent assay (GMEIA), and Mycology Study Group of the European Organization for Research and Treatment of Cancer (EORTC/MSG) criteria for the diagnosis of invasive aspergillosis (IA). From May 2004 to May 2005, blood samples (314 in total) were collected twice a week from 78 patients with hematologic diseases during neutropenic fever after chemotherapy or hematopoietic stem cell transplantation. Results were compared with each other on the basis of EORTC/ MSG criteria. The cutoff of conventional NASBA was set to be 3.5; GM 0.5; RTi-NASBA, 20% above the negative control. There were 22 patients with IA (7 probables and 15 possibles) and 56 patients with nonfungal infection. The Kappa statistic for RTi-NASBA versus conventional NASBA was 0.80 (0.66-0.82; p<0.001) indicating that there was fairly good accordance between two tests. RTi-NASBA showed sensitivity 0.96, specificity 0.43, positive- and negative-predictive value 0.40 and 0.96, respectively. GM showed good specificity (0.98), while the sensitivity (0.45) was poor. When we use the combination of GM with either of two NASBAs, the sensitivity was improved up to 100%. In conclusion, RTi-NASBA could be a good alternative to the conventional one for the screening of IA.

Nucleic Acid Sequence-Based Amplification and Its Applications in Viral Diagnosis / 中国生物工程杂志

Nucleic acid sequence-based amplification(NASBA) is a sensitive,isothermal,transcription-based amplification system specifically designed for the detection of RNA targets,which could amplify templete RNA in 2h under isothermal condition at about 42?C and without any special equipment.NASBA is now widely applicated in diagnosis of many pathogenic microorganism.It is mainly about principles and applications of NASBA in viral diagnosis.

Detection of HCV RNA in Plasma/Serum from Donors by NASBA / 中国输血杂志

Objective To analyse the necessity and feasibility of HCV RNA detection for donor screening.Methods Plasma from 50 donors was mixed into a minipool, the nucleic acid was extracted by NucliSens Extractor and amplified by NASBA, the amplification products were detected by ECL. A performance control and a positive control were coupled with the samples through storage, nucleic acid extraction, amplification and detection.Results Two specimens were found HCV RNA positive in the 10000 anti HCV negative samples.Conclusion HCV RNA screening of plasma minipools by NASBA may prevent hepatitis C virus infection.