Sensitivity Of Reverse Transcriptase Polymerase Chain Reaction Research Articles

Diagnostic accuracy of screening tests for patients suspected of COVID-19, a retrospective cohort study

Background The diagnostic gold standard for Coronavirus-2019 disease (CoViD-19) is reverse transcriptase-polymerase chain reaction (RT-PCR). However, its sensitivity might be suboptimal. The current study aims to investigate predictive factors for false-negative nasopharyngeal RT-PCR in CoViD-19 patients. Additionally, the specificity and sensitivity of RT-PCR on the nasopharyngeal swab, serology and chest computerized-tomography (CCT) as a screening tool for the diagnosis of CoViD-19 were investigated. Methods Medical records of patients admitted at the university hospital UZ Brussel during the CoViD-19 epidemic were reviewed. A group of CoViD-19 patients with false-negative RT-PCR was identified through scrupulous examination of medical records. Serological testing was performed through chemiluminescent microparticle assay. Results Eighteen CoViD-19 patients with ‘false negative’ RT-PCR were identified and compared to 51 ‘true positives’. Logistic regression for prediction of ‘false negative’ RT-PCR found significantly higher serology results at hospitalization and more intensive care unit admission in the group with false-negative testing. In a cohort of 228 patients, the sensitivity of RT-PCR for the diagnosis of CoViD-19 was 85%. The sensitivity of serology was 86% and its specificity 92%. Chest computerized-tomography (CCT) showed a sensitivity of 93%, its specificity was 62%. By combining RT-PCR and serology results any ‘false negative’ could be excluded. Conclusions In this cohort, the sensitivity and specificity of RT-PCR and serology for the diagnosis of CoViD-19 were high and comparable. CCT had the highest sensitivity and confirmed its efficacy as a screening tool. CoViD-19 patients, who have a more severe presentation, might have negative RT-PCR and positive serology results.

Differential clinic in children infected by SARS-CoV-2, traceability of contacts and cost-effectiveness of diagnostic tests: Cross-sectional observational study

IntroductionGiven the possible coexistence of infection by the SARS-CoV-2 with other seasonal infections, the aim is to identify differential symptoms. The role of children in intrafamily contagion and the sensitivity of reverse transcriptase polymerase chain reaction (RT-PCR) in an area with low community transmission has been studied. Material and methodsCross-sectional observational study. Patients between 0–15 years studied by RT-PCR technique due to clinical suspicion of infection by SARS-CoV-2 virus in the months of March-May 2020. Survey on symptoms and contacts. Determination of Anti-SARS-CoV-2 antibodies at least 21 days after the RT-PCR test. Results126 patients were included, 33 with confirmed infection and age mean 8.4 years (95% CI 6.8–10.5) higher than not infected. Fever was the most common and with greater sensitivity. The differences found were a greater frequency of anosmia (P=.029) and headache (P=.009) among children infected with a specificity of 96.7% and 81.5% respectively. There were no differences in the duration of the symptoms. 81.8% of those infected were probably infected in the nucleus 85.2% by a parent who worked outside the home. The sensitivity of RT-PCR was 70.9% and its negative predictive value 91.1%. ConclusionsThe clinical picture is nonspecific and the symptoms more specific difficult to detect in younger children. Children had a reduced role in the intrafamily transmission. The sensitivity of RT-PCR could be related to a less contagiousness in children after one week of infection.

Diagnostic Performance of CT and Reverse Transcriptase Polymerase Chain Reaction for Coronavirus Disease 2019: A Meta-Analysis.

BackgroundRecent studies have suggested that chest computed tomography (CT) scans could be used as a primary screening or diagnostic tool for coronavirus disease 2019 (COVID-19) in epidemic areas.PurposeTo perform a meta-analysis to evaluate diagnostic performance measures, including predictive values, of chest CT and initial reverse transcriptase-polymerase chain reaction (RT-PCR).Materials and MethodsMEDLINE and Embase were searched from January 1, 2020 to April 3, 2020 for studies on COVID-19 that reported the sensitivity and/or specificity of CT scans and/or RT-PCR assays. The pooled sensitivity and specificity were estimated by using random-effects models. The actual prevalence (i.e., the proportion of confirmed patients among those tested) in eight countries was obtained from web sources, and the predictive values were calculated. Meta-regression was performed to reveal the effect of potential explanatory factors on the diagnostic performance measures.ResultsThe pooled sensitivity was 94% (95% CI: 91%, 96%; I2=95%) for chest CT and 89% (95% CI: 81%, 94%; I2=90%) for RT-PCR. The pooled specificity was 37% (95% CI: 26%, 50%; I2=83%) for chest CT. The prevalence of COVID-19 outside China ranged from 1.0% to 22.9%. For chest CT scans, the positive predictive value (PPV) ranged from 1.5% to 30.7%, and the negative predictive value (NPV) ranged from 95.4% to 99.8%. For RT-PCR,the PPV ranged from 47.3% to 96.4%, while the NPV ranged from 96.8% to 99.9%. The sensitivity of CT was affected by the distribution of disease severity, the proportion of patients with comorbidities, and the proportion of asymptomatic patients (all p < 0.05). The sensitivity of RT-PCR was negatively associated with the proportion of elderly patients (p = 0.01).ConclusionOutside of China where there is a low-prevalence of COVID-19 (1-22.9%), chest CT screening of patients with suspected disease had low positive predictive value (1.5-30.7%).

ODF4, MAGEA3, and MAGEB4: Potential Biomarkers in Patients with Transitional Cell Carcinoma

Background:This study aimed to evaluate the diagnostic value of outer dense fiber 4 (ODF4), melanoma-associated antigen A3 (MAGEA3), and MAGEAB4 mRNAs in transitional cell carcinoma (TCC), using a small amount of cell reverse transcriptase-polymerase chain reaction (RT-PCR) on urinary exfoliated cells.Methods:We recruited a total of 105 suspected TCC patients and 54 sex- and age-matched non-TCC controls. The candidates’ genetic expression patterns were investigated with RT-PCR, while reverse transcription quantitative PCR was applied to quantify and compare each mRNA level between cases and control groups.Results:The sensitivity of ODF4, MAGEA3, and MAGEAB4 RT-PCR was 54.8%, 63%, and 53.4%, whereas the specificity was 73.7%, 86%, and 94.7%, respectively. Combining ODF4, MAGEA3, and MAGEAB4 RT-PCR offered a relatively higher sensitivity (83.6%).Conclusion:RT-PCR with ODF4, MAGEA3, and MAGEAB4 on urinary exfoliated cells could provide clinicians with a promising method to improve TCC diagnosis, especially in the case of gross hematuria and catheterization. The method used here is non-invasive, simple and convenient, and unlike cytology, it does not rely directly on expert professional opinions. These features can be of particular importance to the management of TCC patients in whom regular and lifelong surveillance is required.

Comparative analysis of rabies virus detection tests

Rabies is one of the oldest recognised fatal encephalitis of warm-blooded animals and has worldwide prevalence. The causative agent is a neurotropic RNA virus belonging to the family Rhabdoviridae and genus Lyssavirus. Correct and prompt diagnosis of rabies is important for the post-exposure prophylaxis/measures in human and animals. Total 20 samples (12 brains and 8 saliva) were collected from dead and live animalsnamely dog, buffalo, cow, goat, donkey and hyena for detecting rabies virus. Brain samples were collected after post-mortem and were examined by Seller's staining technique, immunochromatographic assay, Direct fluorescent antibody test (d-FAT) and Reverse transcriptase polymerase chain reaction (RT-PCR) To diagnose rabies in live animals, saliva samples were examined by immunochromatographic test and its efficacy was compared with RT-PCR. Relative sensitivity of Seller's staining technique, immunochromatographic test and RT-PCR in comparison with d-FAT was found 16.66%, 100% and 90.9%, respectively, while specificity was found 100% for all the tests. Sensitivity and specificity of immunochromatographic test to detect rabies virus from saliva were found 80% and 100%, respectively, when compared to RT-PCR.

Clinical characteristics and outcomes of patients with primary lung adenocarcinoma harboring ALK rearrangements detected by FISH, IHC, and RT-PCR.

EML4-ALK is a new driver gene of non-small cell lung cancer and a target of crizotinib. The objectives of this study were to determine the frequency of ALK rearrangements in a large cohort of patients with primary lung adenocarcinoma and to analyze the association of ALK rearrangements with clinicopathological characteristics and clinical outcomes. The roles of fluorescence in situ hybridization (FISH), Ventana immunohistochemistry (IHC), and reverse transcriptase polymerase chain reaction (RT-PCR) in the detection of ALK rearrangements were evaluated. The ALK rearrangement was detected in 430 specimens from individual patients with primary lung adenocarcinoma using FISH and Ventana IHC based on tissue microarrays. The EGFR status was detected in all of the specimens through DNA sequencing. An RT-PCR was performed on 200 of the specimens and confirmed by sequencing. Of the 430 patients, 46 (10.7%) harbored ALK rearrangements. The ALK rearrangements were associated with a younger age and the EGFR wild type in comparison with ALK-negative patients. The sensitivity and specificity of the Ventana IHC were 100% and 98.2%, respectively, and the concordance rate between the FISH and the Ventana IHC was 98.4%. The sensitivity and specificity of RT-PCR were 95.5% and 87.0%, respectively, and the concordance rate between the FISH and the RT-PCR was 89.0%. The Cox analysis indicated that an early stage and EGFR-activating mutations were independently associated with a longer OS. This study demonstrated that ALK rearrangements are associated with a younger age and the EGFR wild type rather than with other clinicopathological factors. Although the FISH and Ventana IHC have better concordance, and RT-PCR is a more sensitive method and can identify different variants or partners, the IHC and RT-PCR need to be further evaluated in clinical trials to identify their roles in guiding patients’ targeted therapy using crizotinib.

A comprehensive analysis of breakpoint cluster region-abelson fusion oncogene splice variants in chronic myeloid leukemia and their correlation with disease biology.

BACKGROUND:BCR-ABL fusion oncogene is a hallmark of Chronic Myeloid Leukemia (CML). It results due to translocation between chromosome 22 and chromosome 9 [t (9; 22)(q34; q11)]. It gives rise to translation of a 210 KDa chimeric protein (p210), leading to enhanced tyrosine kinase activity and activation of leukemogenic pathways, ultimately causing onset of CML. In case of CML, the classic fusions are b2a2 or b3a2, fusing exon 13 (b2) or exon 14 (b3) of BCR, respectively, to exon 2 (a2) of ABL. The type of BCR-ABL transcripts are thought to be have different prognosis and hence useful in clinical decision-making. The frequencies of different fusion oncogenes associated with leukemia can vary in different ethnic groups and geographical regions due to interplay of genetic variation in different ethnic populations, diverse environmental factors and living style. Moreover, earlier relevant studies from our region were carried out in small subset of patients. Therefore, objective of this study was to find out frequencies of different BCR-ABL splice variants in larger subset of CML patients.METHODS:A nested reverse transcriptase polymerase chain reaction (RT-PCR) was established to detect BCR-ABL splice variants in 130 CML patients. Sensitivity of RT-PCR and ability to detect BCR-ABL fusion gene in least possible time was studied.RESULTS:BCR-ABL detection using our optimized RT-PCR protocol could be completed in 8 hours, starting from RNA extraction to Gel electrophoresis. Sensitivity of RT-PCR assay was of the order of 10−6. Out of 130 Pakistani patients, 83 (63.84%) expressed b3a2 while 47 (36.15%) expressed b2a2 transcript.CONCLUSION:Our RT-PCR was proved to be very quick to detect BCR-ABL fusion oncogene in CML patients within one working day. Because of its sensitivity, it can be used to monitor complete molecular response in CML. BCR-ABL RT-PCR and BCR-ABL splice variants frequency in our study differs from other ethnic groups. It shows that ethnic and geographical differences exist in BCR-ABL splice variant frequency, which may have a profound effect on disease biology as well as implications in prognosis and clinical management of BCR-ABL positive leukemias.

Quantitative RT-PCR Analysis of the MOZ-CBP Fusion Transcript in Therapy-related Acute Myeloid Leukemia With t(8;16)(p11;p13)

We developed a real time reverse transcriptase polymerase chain reaction (RT-PCR) assay system for detecting the MOZ-CBP fusion transcript and used it to monitor minimal residual disease (MRD) status in a patient with therapy related acute myeloid leukemia (t-AML) harboring t(8;16)(p11;p13). Expression of the MOZ-CBP fusion transcript was determined by RT-PCR analysis of the patient's bone marrow at the time of diagnosis. Thereafter, real time RT-PCR was used to evaluate MRD levels throughout the entire course of treatment. The sensitivity of quantitative RT-PCR for the MOZ-CBP fusion transcript was 10(-5). Below this level, MRD was classified as negative. Real time RT-PCR of the bone marrow after induction therapy showed the reduction of MOZ-CBP transcript to approximately 10(-3) level when compared to the diagnostic sample. MRD was classified as negative (< 10(-5) compared with that in the bone marrow at diagnosis) after 5 courses of chemotherapy, a level that was maintained post-allo-hematopoietic stem cell transplantation. Real time RT-PCR of the MOZ-CBP transcript is a useful tool for assessing MRD status for a patient with therapy related acute myeloid leukemia who was initially predicted to have a poor prognosis.

Role of RT-PCR and FISH in diagnosis and monitoring of acute promyelocytic leukemia

Patients with a presence of Promyelocytic Leukemia-Retinoic Acid Receptor Alpha (PML-RARA) genes rearrangement predict a favorable response to all-trans retinoic acid (ATRA), and a significant improvement in survival. Therefore, establishing the presence of PML-RARA rearrangement is important for optimal patient management. The objective of this study is to compare and assess the role of fluorescent in situ hybridization (FISH) and reverse transcriptase polymerase chain reaction (RT-PCR) in the diagnosis and long-term monitoring of Acute Promyelocytic Leukemia (APL). We compared 145 samples received at different interval of times to analyze the sensitivity of RT-PCR and FISH. The failure rate for RT-PCR was 4% at baseline, 13% at induction, and 0% at the end of consolidation. And for FISH it was 8% at baseline, 38% at induction, and 66% at the end of consolidation. The predictive values of relapse in the patients who were positive and negative by RT-PCR, at the end of induction, were 60% and 3%, respectively, and at end of consolidation it was 67% and 4%, respectively. On the other hand the predictive values of relapse in patients who were positive and negative by FISH at end of induction were 57% and 6%, respectively; while at end of consolidation it was 14% who were negative by FISH. Both RT-PCR and FISH are important for the diagnosis of APL cases, as both techniques complement each other in the absence or failure of any one of them. However, RT-PCR is more sensitive than FISH for the detection of minimal residual disease in the long-term monitoring of these patients. The present study shows that the predictive value of relapse is more associated with minimal residual disease (MRD) results by RT-PCR than that by FISH.

Comparison of a novel molecular assay with touch imprint and permanent histology of sentinel lymph nodes in early-stage breast cancer.

642 Background: Current intraoperative assay of the sentinel lymph nodes (SLN) either by touch imprint or frozen section has high false-negative rates. We aimed to study validity of a novel reverse transcriptase-polymerase chain reaction (RT-PCR) assay compared to intraoperative touch preps as well as final histology. Methods: Pts with BrCa underwent SLN Mapping (M). Alternate slices of the SLNs were examined for mammoglobin and cytokeratin 19 (CK19) by RT-PCR assay, with the remainder of the SLNs processed and evaluated pathologically with four H&E and one CK immunostain. Results: Seventy-eight pts were studies. Nine (11.5%) DCIS, 57 (73.1%) T1, 11 (14.1%) T2 and 1 (1.3%) had T3 tumor. Overall nodal metastasis was 25% with 4 pts having positive micrometastasis (0.2-2 mm). In the included 69 pts (excluding DCIS), sensitivity of RT-PCR was 81.3% compared to 50% for touch prep. RT-PCR carried a negative predictive value of 94.5% (52/55 pts) compared to 86.8% (53/61) with touch preps. RT-PCR identified one p...

Laboratory diagnosis of mumps in a partially immunized population: The Nova Scotia experience.

In 2007, Atlantic Canada experienced a large outbreak of mumps predominately in university students who had received a single dose of measles, mumps and rubella vaccine. The present study describes the performance characteristics of reverse transcriptase polymerase chain reaction (RT-PCR) on buccal and urine specimens and immunoglobulin M (IgM) serology in this partially immune population. Patients presenting with symptoms suspicious for mumps had a serum, urine and a buccal swab collected for diagnostic testing. Persons were classified as a 'confirmed' case according to the Public Health Agency of Canada's definition. Sera were tested using an enzyme-linked immunoassay. Detection of mumps virus in buccal swabs and urine samples was performed by RT-PCR. A subset of 155 cases and 376 non-cases that had all three specimens submitted was used for calculating the performance characteristics. The sensitivity of RT-PCR on buccal swabs, urine specimens and IgM serology were 79%, 43% and 25%, respectively. The specificity of RT-PCR on buccal swabs, urine specimens and IgM serology was 99.5%, 100% and 99.7%, respectively. Only 12 of 134 (9%) patients had positive urine specimens in the presence of negative oral swabs. RT-PCR on buccal swabs is the ideal specimen for diagnosis. Testing an additional urine sample in an outbreak setting did not increase the diagnostic yield significantly, but doubled testing volume and cost. In addition, the data suggest that, in this partially immune group, IgM serology has little value in the diagnosis of acute infection.

Pilot Study to Evaluate MYCN Expression as a Neuroblastoma Cell Marker to Detect Minimal Residual Disease by RT-PCR

This pilot study was performed to determine whether MYCN expression warrants further investigation as a tumor marker to detect low levels of residual neuroblastoma (NB). Seven NB cell lines and 30 bone marrow (BM) samples from patients with high-risk NB were analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) for MYCN expression, and for the established NB marker tyrosine hydroxylase. MYCN was expressed in all 7 NB cell lines, but not in normal peripheral blood, CD34 cells, or BM. In dilution studies using cell lines with or without DNA amplification of MYCN, 1 NB cell in 10 to 10 nucleated blood cells was detectable by RT-PCR. MYCN was identified in all 21 BM samples in which tumor cells were identified by histologic examination, including 4 samples in which tyrosine hydroxylase was not detected. Additionally, expression of both markers was detected in 5 samples that were negative by histology but presumably contained low levels of tumor cells, consistent with the greater sensitivity of RT-PCR compared with morphologic methods. Detection of MYCN RNA was independent of MYCN DNA amplification status. The selective expression of MYCN in tumor cells, and the sensitivity of detection of MYCN by RT-PCR noted in this and other studies, supports further evaluation of MYCN as a NB marker for molecular detection of minimal residual disease.

Diagnostic value of reverse-transcriptase polymerase chain reaction for MAGE families expression in malignant thoracic pleural effusions

9533 Background: The identification of malignant cells in effusions by conventional cytology is hampered by its limited sensitivity. Previous studies show that the MAGE genes families are only expressed in the malignant cells and not expressed in normal tissues except testis and placenta. The aim of this study was to improve tumor cell detection in effusions by reverse-transcriptase polymerase chain reaction (RT-PCR) for MAGE families expression. Methods: A total of 22 thoracic effusions from patients with lung cancer and 18 thoracic effusions from patients without an evidence of malignancy were included in this study. All effusion specimens were evaluated in parallel by cytology, RT-PCR for MAGE families expression and pleural biopsy under video assisted thoracoscopy. To improve the sensitivity of RT-PCR, we combined the results of RT-PCR for MAGE-1, 2, 3 and 4 expressions. Anyone of them expressed in the effusion was recorded as MAGE positive. Results: Tumor cell detection by cytology, RT-PCR for MAGE e...

Detection of peritoneal micrometastasis by reverse transcriptase‐polymerase chain reaction for heparanase mRNA and cytology in peritoneal wash samples

Peritoneal dissemination is the most common pattern of metastasis in advanced gastric carcinoma with serosal invasion. In the present study, we have reported the clinical relevance of a new diagnostic method with reverse transcriptase-polymerase chain reaction (RT-PCR) assay using heparanase as a target gene for detection of free cancer cells in peritoneal washes. Intraoperative peritoneal washes were obtained from 48 patients who underwent surgery for gastric cancer. RT-PCR analysis with primers specific for heparanase and conventional cytological examination were performed subsequently. Heparanase RT-PCR was capable of detecting at least 10 tumor cells in 10 ml peritoneal wash fluid. There was no detectable heparanase expression in normal gastric epithelial cells and peritoneal wash samples from patients with benign disease. Twenty-five (52%) patients with gastric cancer had a detectable heparanase expression. Its positive rate was 100% and 59% for the cases with clinically evident peritoneal metastasis and serosal invasion, respectively, both of which are higher than that of cytology. Heparanase mRNA detected in peritoneal lavaged fluid might indicate the presence of free cancer cells in peritoneal cavity. The high sensitivity of RT-PCR based heparanase assay made it a candidate molecular marker for detecting peritoneal micrometastasis.

Estimation of specificity and sensitivity of three diagnostic tests for infectious salmon anaemia virus in the absence of a gold standard

Reverse transcriptase-polymerase chain reaction (RT-PCR), virus isolation (VI) and indirect fluorescent antibody test (IFAT) are three tests currently used by the salmon industry to identify fish infected with the infectious salmon anaemia virus (ISAV). However, very limited information is available on the sensitivity and specificity of these methods. In order to evaluate these tests in fish representing a range of farmed Atlantic salmon populations, five laboratories participated in a blind study of 400 kidney samples from four groups of fish with different prevalences of ISAV. Each laboratory used its own testing protocols. Estimates of the specificity of each test were determined directly from a population assumed to be free of infection. Indirect estimates of the sensitivity and specificity of each test were obtained using maximum likelihood estimation of a latent class model (i.e. no gold standard test result available). There was a substantial difference in sensitivity and specificity of RT-PCR among the three laboratories using this test. If only the best results for the RT-PCR tests are taken into account, the maximum likelihood estimates obtained from this study suggest RT-PCR and VI are of similar high sensitivity (range 92-100%), IFAT is the least sensitive method (range 65-76%) while the three tests have similar high specificities (range 96-100%). The results of the study suggest: (1) RT-PCR tests should be standardized before they are used as a diagnostic test for prevention and control of ISAV, (2) the sensitivity of VI was higher than expected and (3) IFAT has a low sensitivity but might be a good screening test because of its low cost.

Estimation of the repeatability and reproducibility of three diagnostic tests for infectious salmon anaemia virus

Reverse transcriptase polymerase chain reaction (RT-PCR), virus isolation (VI) and indirect fluorescent antibody tests (IFAT) are three assays currently used by the salmon industry to identify fish infected with infectious salmon anaemia virus (ISAV). However, no data are available on the repeatability (within-laboratory consistency) and reproducibility (between-laboratory consistency) of these assays and very limited information is available on the effect of freezing samples on test results. In order to evaluate these assays, five laboratories participated in a blinded study of 400 kidney samples representing four populations of farmed Atlantic salmon with different prevalence of ISAV. Each laboratory used its own testing protocols. Repeatability and reproducibility were evaluated using kappa as the measure of agreement. The effect of freezing was evaluated using the McNemar test. Freezing did not affect VI but improved the sensitivity of RT-PCR. The repeatability and reproducibility of VI was almost perfect. There was a substantial difference in repeatability of RT-PCR among the three laboratories with kappa ranging from 0.5 to 0.96. The repeatability for RT-PCR was generally low. The repeatability of IFAT was moderate when the results were analysed using 1+ and above as a positive result. The results of the study show the need to standardize the protocol and interpretation of RT-PCR.

Comparison of three rapid detection systems for type A influenza virus on tracheal swabs of experimentally and naturally infected birds

The present paper reports of the comparison between three rapid virus detection systems and virus isolation (VI) from pooled tracheal swabs collected from naturally and experimentally infected birds with a low pathogenicity avian influenza virus of the H7N3 subtype. The relative sensitivity, specificity and agreement (K value) were calculated for a commercial antigen capture enzyme immunoassay (AC-EIA) and for two nucleic acid detection tests, a one-step reverse transcriptase-polymerase chain reaction (RT-PCR) and a real-time RT-PCR (RRT-PCR), both targeting the M gene. The results indicate that in experimentally infected turkeys VI was positive from the pooled tracheal swabs collected from day 3 to day 10. One-step RT-PCR was able to detect influenza RNA from samples collected from day 3 to day 12, while RRT-PCR amplified influenza RNA in swabs collected from day 3 to day 15. The AC-EIA test yielded positive results between day 5 and day 10 post-infection. On field samples, the K value between the AC-EIA and VI tests was 0.82. Compared with VI, the relative sensitivity of this test was 88.9% (CI95=85.2–92.6) and the relative specificity was 95.7% (CI95=93.7–97.7). The K value between the RT-PCR and VI tests was 0.88. Compared with virus isolation, the relative sensitivity of the one-step RT-PCR was 95.6% (CI95=93.1–98.0) and the relative specificity was 96.3% (CI95=94.4–98.1). The K value between the RRT-PCR and VI tests was 0.92. Compared with virus isolation, the relative sensitivity and specificity of RRT-PCR was 93.3% (CI95=90.4–96.3) and 98.4% (CI95=97.2–99.6), respectively. Generally speaking, comparison between virus isolation, the AC-EIA test and the two nucleic acid detection methods indicated excellent agreement. Data obtained from both experimental and field study suggest a higher sensitivity of the PCR-based methods compared with the AC-EIA. The economical and practical implications of using one of the rapid tests as an alternative to VI during an avian influenza epidemic are discussed.

Detection of nonmelanoma skin cancer micrometastases in lymph nodes by using reverse transcriptase-polymerase chain reaction for keratin 19 mRNA.

A new sensitive method for the detection of skin cancer micrometastases in lymph nodes is based on amplification of keratin 19 (K19) mRNA by reverse transcriptase-polymerase chain reaction (RT-PCR). To compare results of RT-PCR with those of histological examination in terms of the detection rate of skin cancer micrometastases. Twenty-six lymph nodes obtained from 13 patients with squamous cell carcinoma (SCC), eccrine porocarcinoma and Paget's disease were investigated by histological examination (haematoxylin and eosin sections) and RT-PCR. RT-PCR was performed on extracted RNA by using K19 primer pairs. RT-PCR products were visualized by ethidium bromide staining and confirmed by non-radioactive hybridization with K19-specific probes. All of 10 histologically positive lymph nodes yielded the expected 460-bp band. Of the 16 histologically negative lymph nodes, one (6%) was found by RT-PCR to express K19 mRNA, indicating the presence of micrometastases which could not be detected by histological examination. A serial dilution study using RNA extracted from SCC cells mixed with RNA extracted from normal lymph node cells showed a detection sensitivity of K19 RT-PCR of 10-5 micro g cancer cell RNA in 1 micro g lymph node RNA. Nested RT-PCR showed a detection sensitivity of one tumour cell in 106 lymphocytes. These results demonstrate the usefulness of K19 RT-PCR for the detection of skin cancer micrometastases in lymph nodes.

Method for Detection of Extraneous Active Bovine Viral Diarrhoea Virus and Classical Swine Fever Virus in Animal Viral Vaccines by RT-PCR, Which Amplify Negative-strand Viral RNA in Infected Cells

An oligonucleotide sense primer, Pst324α, was designed and used for synthesizing cDNA from negative-strand viral RNA in infected cells and used for rapid detection of active extraneous bovine viral diarrhoea virus (BVDV) and classical swine fever virus (CSFV) in animal viral vaccines by culturing a sample in cells followed by reverse transcriptase–polymerase chain reaction (RT-PCR). Active and inactivated viruses of BVDV No. 12–43 strain and CSFV GPE−strain were inoculated to bovine testicle and swine testicle cells for incubation. After the complete extraction of RNA from these cells, cDNA was synthesized using Pst324α, and PCR was carried out using primers 324 and 326 (novel RT-PCR). Amplification of novel RT-PCR products was observed in cells infected with active viruses but not in cells inoculated with inactivated viruses, inoculums and cultured media after incubation. This novel RT-PCR was able to amplify viral sequences from cells infected with only a small number of infectious particles (less than 10 TCID50) at three days postinoculation and was as sensitive as the general RT-PCR using a random primer and the interference and immunofluorescent antibody (FA) methods. The results of experiments on detection of BVDV RNA from vaccines contaminated with active and inactivated BVDV showed that the sensitivity of the novel RT-PCR was almost the same as the sensitivities of the interference and FA methods. These results suggest that the novel RT-PCR is easier and more rapid than the interference method for detection of active BVDV and that the novel RT-PCR is a reliable means for detection of active extraneous BVDV for quality control of animal vaccines.

REAL TIME REVERSE TRANSCRIPTASE POLYMERASE CHAIN REACTION OF URINARY CYTOKERATIN 20 DETECTS TRANSITIONAL CELL CARCINOMA CELLS

We evaluate the diagnostic use of cytokeratin 20 messenger (m) RNA quantitation in urine as a marker of urothelial transitional cell carcinoma using the real time reverse transcriptase polymerase chain reaction (RT-PCR). Spontaneously voided urine was obtained from 47 patients with urothelial transitional cell carcinoma (carcinoma group), 19 other urological diseases (noncarcinoma group) and 27 healthy volunteers (control group). Quantification of cytokeratin 20 was performed with mRNA extracted from urine samples with primers and hybridization probes specific for cytokeratin 20 on a LightCycler instrument (Roche Diagnostics Corp., Indianapolis, Indiana). This method allowed reproducible quantitation of 10 to 106 cytokeratin 20 expressing colon carcinoma cells per 107 peripheral blood leukocytes, comparable to the sensitivity of conventional RT-PCR with a wide linear measuring range. Cytokeratin 20 mRNA values in the carcinoma group (mean 35,850) were significantly higher than noncarcinoma (171) and control groups (4.55, p <0.0001 and <0.0001, respectively). Urinary cytokeratin 20 mRNA values significantly correlated with tumor grade, urinary cytological class, immunostaining pattern and depth of tumor invasion. Sensitivity and specificity of real time RT-PCR with a cutoff value of 15 were 81% and 83%, whereas those of conventional cytology were 28% and 100%, respectively. These results indicate that real time cytokeratin 20 RT-PCR is a sensitive, quantitative, rapid and specific method to detect free cancer cells in the urine, with good potential for monitoring recurrence of urothelial transitional cell carcinoma.