Development and evaluation of a loop-mediated isothermal amplification (LAMP) assay for rapid detection of Actinobacillus pleuropneumoniae based the dsbE-like gene

This paper reports on the development and validation of a loop-mediated isothermal amplification assay (LAMP) for the rapid and specific detection of Actinobacillus pleuropneumoniae (A. pleuropneumoniae). A set of six primers were designed derived from the dsbE-like gene of A.pleuropneumoniae and validate the assay using 9 A. pleuropneumoniae reference/field strains, 132 clinical isolates and 9 other pathogens. The results indicated that positive reactions were confirmed for all A. pleuropneumoniae strains and specimens by LAMP at 63ºC for 60 min and no cross-reactivity were observed from other non-A.pleuropneumoniae including Haemophilus parasuis, Escherichia coli, Pasteurella multocida, Bordetella bronchiseptica, Streptococcus suis, Salmonella enterica, Staphylococcus, porcine reproductive and respiratory syndrome virus (PRRSV), and Pseudorabies virus. The detection limit of the conventional PCR was 10² CFU per PCR test tube, while that of the LAMP was 5 copies per tube. Therefore, the sensitivity of LAMP was higher than that of PCR. Moreover, the LAMP assay provided a rapid yet simple test of A. pleuropneumoniae suitable for laboratory diagnosis and pen-side detection due to ease of operation and the requirement of only a regular water bath or heat block for the reaction.

A facile, branched DNA assay to quantitatively measure glucocorticoid receptor auto-regulation in T-cell acute lymphoblastic leukemia / 癌症

Glucocorticoid (GC) steroid hormones are used to treat acute lymphoblastic leukemia (ALL) because of their pro-apoptotic effects in hematopoietic cells. However, not all leukemia cells are sensitive to GC, and no assay to stratify patients is available. In the GC-sensitive T-cell ALL cell line CEM-C7, auto-up-regulation of RNA transcripts for the glucocorticoid receptor (GR) correlates with increased apoptotic response. This study aimed to determine if a facile assay of GR transcript levels might be promising for stratifying ALL patients into hormone-sensitive and hormone-resistant populations. The GR transcript profiles of various lymphoid cell lines and 4 bone marrow samples from patients with T-cell ALL were analyzed using both an optimized branched DNA (bDNA) assay and a real-time quantitative reverse transcription-polymerase chain reaction assay. There were significant correlations between both assay platforms when measuring total GR (exon 5/6) transcripts in various cell lines and patient samples, but not for a probe set that detects a specific, low abundance GR transcript (exon 1A3). Our results suggest that the bDNA platform is reproducible and precise when measuring total GR transcripts and, with further development, may ultimately offer a simple clinical assay to aid in the prediction of GC-sensitivity in ALL patients.

HIV testing technologies after two decades of evolution.

Over the past two decades, HIV diagnostics have been essential in detecting and monitoring infection, and continue to play a major role in saving lives throughout the world. As technology evolved, screening, confirmatory, and HIV monitoring assays have been improved and offer better alternatives to address blood screening, surveillance, diagnosis, and patient management. Molecular methods are critical in detecting early infection and for managing patients on anti retroviral therapy whose viral infection may become resistant to therapy. In addition, modifications to conventional methods have introduced new assays, such as sensitive/less sensitive (detuned) assays that can estimate when someone was infected, thereby providing a useful tool for epidemiologic incidence estimates and enrollment into specific intervention programmes for recently infected persons. Many of the newly evolving technologies are essential for use in resource-limited countries because they can address cost issues, limited infrastructure, and a lack of formally trained personnel. Newer rapid HIV kits can be stored in a wide range of temperatures (2-30 degrees C) to address cold-chain issues, can use easily-collected fingerstick blood and oral fluids, and have one-step procedures that are relatively foolproof. Manual CD4 lymphocyte count assays that require only a light microscope and haemacytometer and more simple assays to estimate viral load are appropriate for developing countries where sophisticated instrumentation cannot be supported. Technologic advances with HIV diagnostics continue to address outstanding and new issues associated with diagnosis and the monitoring of infection by providing more simplified, cost-effective, and accurate testing throughout the world.

Marcadores virologicos no convencionales en pacientes infectados con el virus de la inmunodeficiencia humana: ADN HIV-T, ADN HIV-2LTR y ARN de HIV / Non conventional virological markers in HIV-infected patients: T-HIV DNA, 2LTR-HIV DNA and HIV RNA

La terapia antirretrovial de alta eficacia (TAAE) induce una reducción marcada y persisatente de la viremia plasmática, contribuvendo a disminuir la mortalidad de los pacientes HIV-positivos. Así, la carga viral (CV) es el método de referencia para evaluar la eficacia terapéutica. Sin embargo, aun en presencia de una TAAE eficiente no se ha logrado la erradicación viral. En este estudio analizamos la presencia del ADN total de HIV (ADN HIV-T), del ADN no integrado con 2LTR (ADN HIV-2LTR) y del ARN de HIV, en un grupo de 55 pacientes HIV-positivos en distintos estadios clínicos, con y sin TAAE, mediante ensayos de PCR con revelado colorimétrico en microplaca, optimizados en nuestro laboratorio. La sensibilidad clínica de ARN del HIV fue evaluada con el bDNA, resultando del 74% y del 64%, respectivamente, con una concordancia del 85%. Este ensayo podría utilizado en el seguimiento de pacientes bajo TAAE. EI ADN HIV-2LTR resultó positivo en el 54% aunque estuvo ausente en pacientes con elevada CV. Este marcador se considereba un producto lábil y su presencia se asociaba a infección reciente. Sin embargo, actuales evidencias ponen en discusión su estabilidad por lo que su significado clínico debe ser reconsiderado. La ausencia del ADN HIV-2LTR en pacientes con CV detectable puede relacionarse con la heterogeneidade de la secuencia utilizada para su detección. EI ADN HIV-T estuvo presente en el 100% de las muestras y resultaría relevante como marcador de remisión cuando se dispongan de terapias que efectivamente erradiquen la infección.

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.

The Clinical Significance of Quantitation of HBV DNA in Serum: Comparison of the Branched-DNA Assay with the Second-Generation Digene Hybrid Capture Assay and Long-term Observation / 대한간학회지

BACKGROUNDS/AIMS: Serum levels of hepatitis B virus (HBV) DNA are direct measures of viral replication in hepatocytes. We compared branched-DNA assay (QuantiplexTM, bDNA) and Hybridization Capture II (HCII), and evaluated the clinical significance of HBV DNA quantitation in the natural course of HBV infection. METHODS: We analyzed results of bDNA in 324 serum samples from 83 untreated male patients with chronic hepatitis B. Mean follow up period was 11.8 years. HCII was also performed in 157 arbitrarily selected samples. RESULTS: HBV DNA levels measured with two assays were very similar (r2=0.893, p<0.0001). HBV DNA detection rate of HCII was 6.4% higher than that of bDNA in HBeAg positive samples. HBV DNA detection rate was higher in cases with higher ALT. Among 73 patients with initial diagnosis of chronic hepatitis, 38 patients (52.1%) experienced progression to cirrhosis, and hepatocellular carcinoma (HCC) was developed in 9 (12.3%). HCC was developed in 5 (50.0%) of 10 patients with initial diagnosis of cirrhosis. While HBeAg positive rates during chronic hepatitis, cirrhosis and HCC were 57.8%, 55.0% and 14.3%, respectively (p=0.006), HBV DNA detection rates were 70.6%, 64.0% and 42.9%, respectively (p=0.08). Especially in HCC, the discrepancy between HBeAg positive rate and HBV DNA detection rate may suggest the appearance of variants which cannot produce HBeAg. CONCLUSION: Both HCII and bDNA were similar HBV DNA quantitation assays for clinical use. HBV DNA level correlated with the severity of liver disease. Screening tests for HCC should be recommended in patients whose HBeAg is negative and have detectable HBV DNA.

Quantitation of Hepatitis C Virus RNA by Competitive RT-PCR and DNA-ELISA Method / 대한간학회지

BACKGROUND/AIMS: Quantitation of Hepatitis C Virus (HCV) RNA in serum is important for monitoring the response to interferon-alpha therapy in patients with chronic hepatitis C. Several commercial assays are recently available, but they are expensive and cannot be used as a gold standard. METHODS: An in-house competitive reverse transcription-polymerase chain reaction (cRT-PCR) was developed and validated. The procedure involves the construction of a mutant and wild type HCV RNA internal standard (IS), cRT-PCR, and colorimetric detection with DNA-ELISA. A standard curve was obtained and used for final HCV RNA quantitation. RESULTS: The standard curve was linear over the range of 1x104 to 5x107 copies/mL of the HCV RNA standard (r=0.976). This in-house cRT-PCR was comparable with the branched DNA (bDNA) assay (Quantiplex HCV 2.0, Chiron, USA) with positive correlation between the two tests (r=0.735). CONCLUSION: The quantitation of HCV RNA by in-house cRT-PCR and DNA ELISA was more sensitive and had wider range of detection compared to bDNA assay. This assay is useful for follow-up of HCV RNA concentration after interferon-alpha therapy.

Comparison of Hybrid Capture System, Hybrid Capture II and Quantiplex HBV DNA Assay for Quantitation of Hepatitis B Virus DNA / 대한임상병리학회지

BACKGROUND: Quantitative measurement of hepatitis B virus (HBV) DNA in serum allows monitoring of HBV replication and assessing responses to antiviral treatment, which is not possible with serological markers for HBV infection. However, most assays developed for the quantitative measurement of HBV DNA have not been standardized. Therefore, we tried to compare the performance of three commercial quantitative methods for the measurement of HBV DNA. METHODS: One hundred consecutive sera with request for HBV DNA quantitation were tested with Hybrid Capture System (HCS), Hybrid Capture II (HC-II) and Quantiplex HBV DNA Assay (bDNA Assay) to evaluate the detection rate, the concordance rate and the correlation of the quantitative results measured by each method. In addition, nested polymerase chain reaction (PCR) was performed for qualitative detection of HBV DNA. RESULTs: Concordance rate was 87% for all three methods, 91% for HCS and HC-II, 94% for HCS and bDNA Assay, and 89% for HC-II and bDNA Assay. HBV DNA quantities measured by three methods showed significant correlation between HCS and HC-II (R=0.88, P<0.0001), HCS and bDNA Assay (R=0.82, P<0.0001), and HC-II and bDNA Assay (R=0.95, P<0.0001). Thirteen sera of discrepant results and 29 of 39 sera of negative results by all three methods showed PCR positivity. CONCLUSIONS: Three quantitative methods for the measurement of HBV DNA showed relatively high concordance rate and good correlation. However, the results by bDNA Assay increased more rapidly than HCS and HC-II as the amounts of HBV DNA in the sample increased that the concentrations of HBV DNA measured by bDNA Assay were two or three times higher than those measured by HCS or HC-II at high HBV DNA concentration range. Thus, further studies are necessary to develop more standardized methods.

A Prospective Study of Therapeutic Effect of 6 Months Trial with Lamivudine in Patients with Chronic Viral Hepatitis B / 대한간학회지

BACKGROUND/AIMS: The purpose of this study was to evaluate the effectiveness of lamivudine treatment in patients with chronic liver disease caused by chronic infection of hepatitis B virus (HBV). METHODS: Thirty-ive patients with chronic infection of HBV were included in this study who were diagnosed at Hanyang University Hospital from January 1998 to January 1999. They received 150mg of lamivudine per oral once daily for 6 months with follow-p of liver function test, serum HBV DNA and serologic markers for hepatitis B virus every two months. Lamivudine was well tolerated. Eight patients underwent liver biopsies before entering the study and follow-p biopsies were done at 5 patients. RESULTS: Out of all 35 patients, chronic hepatitis patients histologically confirmed were 8, chronic hepatitis patients clinically diagnosed were 25 and liver cirrhosis patients clinically diagnosed were 2. The mean age was 35.7 years. Male-female ratio was 2.2:1. There was no hepatitis B surface antigen (HBsAg) negative seroconversion. The HBeAg loss rate was 26.9%(7/26) and HBeAg seroconversion rate was 10.7%(3/28) at the end of follow-p. Ten patients were anti-Be positive prior to treatment, 3 of them became anti-Be negative at the end of follow-p. Five patients underwent follow-p liver biopsies, in which histologic improvements were shown in 4 cases. Serum replicative HBV DNA by bDNA assay was decreased in all patients and HBV DNA was undetectable in 52.9%(9/17) at the end of treatment. Out of the 15 patients with abnormal alanine aminotransferase (ALT) levels at baseline, ALT level in 7 patients(46.7%) was normalized at treatment completion. Pretherapy ALT level was the only predictive factor for loss of HBeAg by stepwise logistic regression analysis(odds ratio : 1.0208) (95% Confidence Interval : 1.0023 ~ 1.0396) (p value=0.0271). CONCLUSIONS: Lamivudine induced sustained suppression of HBV replication during treatment in all patients. In treating patients with lamivudine, who had chronic liver disease due to chronic infection of HBV, the improvement of liver function test and suppression of viral replication appeared early and was sustained during the 6months treatment. This, in turn, may induce histological improvement as well. Pretherapy ALT level was the only predictive determinant for HBeAg loss during lamivudine therapy, and that should be kept in mind in selecting patients for treatment.

Quantification of Serum Hepatitis C Virus in Patients with Chronic C Viral Liver Disease / 대한임상병리학회지

BACKGROUND: The quantification of hepatitis C virus (HCV) is useful in diagnosis and monitoring of HCV infection. We evaluated clinical usefulness of HCV quantification and two quantification methods using different assay principles. METHODS: HCV RNA quantities and liver function were measured in patients with different disease severity using bDNA assay (QuantiplexTM, Chiron, USA). HCV RNA loads were quantified at the time of pre/post-interferon treatment in some of them using RT-PCR hybridization assay (AMPLICORTM, Roche, USA). These two quantification methods were also compared. RESULTS: HCV RNA loads showed no significant difference according to disease severity (group I, 3.8 5.3 MEq/mL; group II, 3.8 7.4 MEq/mL; group III, 5.9 13.0 MEq/mL; P=0.181) or interferon response (complete responders, 1.5 105/mL; partial or non responders, 2.2 105/mL; P=0.670). But HCV viral loads decreased at 6th month after interferon treatment (P=0.063) and correlated poorly with liver function tests. The bDNA assay correlated well with the RT-PCR hybridization method (r2=0.854). CONCLUSIONS: The quantificaion of HCV RNA is useful in following up treatment effect but not in predicting therapeutic failure or assessment of disease severity. HCV RNA quantities are independent of liver function. The bDNA assay showed good correlation with the RT-PCR hybridization method.

Comparison of Molecular Biologic Methods for Detecting HBV-DNA in the Sera which Showed Both Hepatitis B Surface Antigen and Antibody Positivity / 대한임상병리학회지

BACKGROUND: Serologic markers are used to screen and diagnose the hepatitis B virus infection. In endemic area of hepatitis B, the coexistence of HBsAg and anti-HBs was frequently observed. This finding is unusual and difficult to interpret. In this study, we performed three molecular biologic assays-polymerase chain reaction (PCR), chemiluminescent molecular hybridization assay (CMHA), branched DNA (bDNA) nucleic acid hybridization assay- to detect HBV-DNA in the sera which showed both HBsAg and anti-HBs positivity. To define the patients` exact clinical conditions, we analysed the characteristics of the patients according to their diagnoses, other serologic markers and clinical findings. METHODS: HBsAg and anti-HBs were detected by EIA (Enzygnost, Behringwerke, Germany) from clinical specimens of Yonsei University College of Medicine Severance Hospital collected In the period between January 1996 and December 1996. Eighty three specimens from Severance Hospital and twenty two specimens from Health Care Center were randomly selected and were subjected to HBV PCR, HBV CMHA and HBV bDNA assay for the presence of HBV-DNA. RESULTS: The patients were arbitrarily divided into 4 groups on the basis of the optical density values of enzyme immunoassay results. Group I (high HBsAg and high antral-HBs) consisted of 6 cases; group II (high HBsAg and low anti-HBs) consisted of 70 cases, group III (low HBsAg and high anti-HBs) consisted of 1 case; group IV (low HBsAg and low antral-HBs) consisted of 6 cases. Among 83 cases, the positive rate was 51.8% (43 cases) using PCR method, 53.0% (44 cases) using CMHA, 60.2% (50 cases) using bDNA assay. HBeAg and anti-HBc IgM were helpful to predict the presence of HBV-DNA in the sera. CONCLUSIONS: More than half of the patients who showed both HBsAg and anti-HBs positivity were positive for HBV-DNA by molecular biologic methods. In contrast, no one whose serologic markers with only anti-HBc positivity with out HBsAg and anti-HBs positivity showed HBV-DNA positive in the sera from Health Care Center. Taken together, the management and follow-up of the patients of both HBsAg and anti-HBs positivity could be greatly aided by combined adoption of any one molecular biologic assay of HBY-DNA with other serologic markers such as HBeAg and anti-HBc IgM.

Quantitation of Hepatitis B Virus DNA in Sera of HBsAg-Positive Patients Using a Branched DNA Signal Amplification Assay / 대한임상병리학회지

BACKGROUND: Several studies have demonstrated that quantitation of hepatitis B virus (HBV) DNA in sera of HBsAg-positive patients is more useful test for the assessment of infectivity and for the evaluation of disease status than previously utilized numerous serological markers and qualitative polymerase chain reaction for the detection of HBV DNA. We tried to measure serum HBV DNA using a branched DNA (bDNA) signal amplification assay, which is recently introduced and known to be a simple and nonradioisotopic method. METHODS: Total forty patients with HBsAg were randomly selected and serum HBV DNA was measured with duplication using bDNA signal amplification assay (QUANTIPLEXTM HBV DNA ASSAY, Chiron, USA). Quantitation was determined from a standard curve and expressed as HBV DNA equivalents/mL (Eq/mL; 1 Eq = 1 molecule of the primary HBV DNA standard). Serum HBeAg, aspartate aminotransferase (AST), alanine aminotransferase (ALT) , and soluble interleukin-2 receptor (sIL-2R) were compared with HBV DNA. RESULTS: Serum HBV DNA was quantitated in 13 patients (32.5%) (range 6.4x106-7.4x109 Eq/mL, mean 1.8x109 Eq/mL, CV 8.1%). All eleven patients (100%) with both HBsAg and HBeAg an4 2 of 29 patients (6.9%) with HBsAg but not with HBeAg showed measurable HBV DNA (p < 0.001). In addition, serum levels of AST, ALT, and sIL-2R were significantly higher in HBV DNA measured patients compared with those of unmeasured patients. CONCLUSIONS: Above results show that more than half the HBsAg-positive patients do not have enough HBV DNA which is measurable with boNA signal amplification assay but all of HBeAg-positive patients and some of HBeAg-negative patients do. In addition, HBV DNA quantitation might be correlated with the disease activity in HBsAg-positive patients because serum levels of AST, ALT, and sIL-2R are higher in patients measured with HBV DNA than unmeasured.