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PubMed2001

Quantitative assay for group M (subtype A-H) and group O HIV-1 RNA detection in plasma.

A quantitative HIV-1 test is described based on a competitive RT-PCR assay combined with a sandwich hybridization as a detection system. The internal RNA standard (IS) was designed specifically to be competitive during the amplification and during the hybridization step. Sample viral load determination was carried out with one RT-PCR in the presence of 10(3) IS copies. The HIV-1 copy number was calculated by reference to an external standard curve performed on known and increasing amounts of the reference HIV-1 (Ref HIV-1) RNA co-amplified with a constant amount of the IS RNA. The assay had a linear range from 10(1) to 10(6) HIV-1 copies. HIV-1 strains belonging to the different subtypes from group M, but also group O, were all detected. Absolute quantification of purified HIV-1 RNA copies gave identical results as the AMPLICOR HIV-1 Monitor assay. The quantification of patient's samples was evaluated according to different criteria such as dynamic range, sensitivity, efficacy of material recovery, reproducibility and convenience of sample handling. The microplate format of the assay combined with the colorimetric detection provides a convenient tool and fulfills the requirement for routine molecular diagnostic laboratories.

HIV Infections↗
PubMed1986

Measles virus RNA detected in Paget's disease bone tissue by in situ hybridization.

Morphological and immunocytological studies have demonstrated the presence of paramyxovirus antigens in Paget's bone disease tissue and in particular antigens related to measles virus and respiratory syncytial virus. To examine the relationship between measles virus and Paget's bone disease we used in situ hybridization and a cloned measles virus DNA probe specific for the nucleocapsid protein to detect and locate measles virus RNA sequences in Paget's bone tissue. In five patients with the disease, measles virus RNA sequences were detected not only in 80 to 90% of the multinucleated osteoclasts where there is morphological and immunocytological evidence of measles virus activity but also in 30 to 40% of mononucleated bone cells, mainly osteoblasts, osteocytes, fibroblasts and lympho-monocytes. In contrast, no hybridization was observed in bone tissue from three control patients without signs of Paget's bone disease. These results indicate that the host cell range for measles virus in Paget's disease is more widespread than has been supposed. They also demonstrate the usefulness of the in situ hybridization method to detect viral genetic information in cells where viral antigenic activity is not detectable. These observations further support the hypothesis that measles virus is involved in the pathogenesis of Paget's bone disease.

Bone and Bones↗
PubMed1996

Peripheral blood tyrosinase messenger RNA detection and survival in malignant melanoma.

BACKGROUND: The most widely accepted criteria for the evaluation of prognosis of malignant melanoma are histopathologic and clinical presentation. No currently available laboratory tests provide additional prognostic information. It has recently been suggested that reverse transcription and polymerase chain reaction (RT-PCR)-based detection of tyrosinase messenger RNA (mRNA) in peripheral blood might be useful in the early detection of circulating tumor cells, since tyrosinase is thought to be a melanocyte-specific marker. PURPOSE: To further evaluate the clinical relevance of this potential marker, we examined peripheral blood samples from patients with malignant melanoma in different stages of disease for the presence of tyrosinase mRNA. METHODS: Total cellular RNA was extracted from heparinized peripheral blood cells from 64 patients with malignant melanoma, from five healthy control subjects, and from four patients with other cancers using the RNAzol A method. For analysis of tyrosinase mRNA, RT-PCR was performed as previously described by Smith et al.; the sensitivity of this assay was tested using RNA extracted from human melanoma cells (SK-mel 1 and SK-mel 3 cell lines) serially diluted with peripheral blood obtained from healthy control subjects. Two additional human melanoma cell lines (SK-mel 30 and RPMI-7951) served as positive controls for RT-PCR detection of tyrosinase mRNA. Overall patient survival curves were constructed using Kaplan-Meier estimates. RESULTS: Tyrosinase mRNA was detected by RT-PCR assay of all four of the established melanoma cell lines tested. Nine of the 64 patients with malignant melanoma were found to have detectable tyrosinase mRNA in their peripheral blood cells (tyrosinase-positive patients). The 16 patients with localized primary melanoma did not have detectable tyrosinase mRNA in their peripheral blood cells. Among the 48 patients with metastatic disease, all 27 patients who exhibited no evidence of disease progression were tyrosinase negative. Notably, all nine tyrosinase-positive patients had visceral metastases and were found to exhibit disease progression at the time of the sampling. Four of the nine tyrosinase-positive patients were also found to test negative at times without evidence of progressive disease; one patient became negative after achieving stable disease and three became positive for tyrosinase transcripts on disease progression. The probability of survival from time of sampling was significantly lower in the nine tyrosinase-positive patients when tested versus the 23 patients with comparable disease but without detectable tyrosinase mRNA (two-sided; P < or = .05). CONCLUSIONS: The results of this study demonstrate that the detection of tyrosinase mRNA in cells in the peripheral blood by RT-PCR may be a useful prognostic marker for predicting tumor progression and poor clinical outcome in patients with malignant melanoma.

Adult↗
PubMed2002

Highly sensitive hepatitis C virus RNA detection methods: molecular backgrounds and clinical significance.

After treatment of chronic hepatitis C virus (HCV), infection with interferon-alpha+/-ribavirin in a considerable proportion of patients with a virologic end-of-treatment response (ETR), a relapse during follow-up is observed. The biological background of relapse is unknown. Either antiviral therapy leads to a complete replication arrest and latent virions survive until the end-of-treatment or replication is not completely suppressed. For the latter hypothesis, residual HCV RNA should be detectable dependent on the sensitivity of the HCV RNA assay. In patients from different studies, serum or plasma samples from the end-of-treatment were investigated for the presence of HCV RNA with a standard polymerase chain reaction (PCR)-based assay and retested for comparison with a new highly sensitive transcription-mediated amplification (TMA)-based assay. Generally, in patients with virologic non-response or sustained virologic response (SR), no differences were observed between PCR- and TMA-based assays. In patients with relapse after standard interferon-alpha therapy with or without ribavirin, HCV RNA was detected in 33-36% of end-of-treatment samples previously negative by PCR-based assays. The lower rate of HCV RNA detected by TMA (7%) in end-of-treatment samples from patients after treatment with pegylated interferon-alpha 2a is most likely explained by the longer half-life of pegylated interferon-alpha in comparison with standard interferon-alpha. HCV RNA is detectable at the end-of-treatment by TMA in 7-36% of patients who were HCV RNA negative by reverse transcriptase (RT)-PCR-based methods. These PCR-negative/TMA-positive patients have to be considered as non-responders (NR) with a very low HCV RNA viral load. Future studies will show whether these patients benefit from prolonged antiviral therapy.

Hepacivirus↗
PubMed2002

High-throughput HBV DNA and HCV RNA detection system using a nucleic acid purification robot and real-time detection PCR: its application to analysis of posttransfusion hepatitis.

BACKGROUND: A high-throughput detection system was developed for HBV DNA and HCV RNA. METHODS: A combination of real-time detection PCR using an automated system (PRISM 7700, PE Biosystems, Foster City, CA) and automatic viral nucleic acid extraction (BioRobot 9604, Qiagen, Hilden, Germany) was used as the high-throughput detection system. An internal control for HBV DNA detection was also developed. RESULTS: Testing of 96 samples for HBV and HCV was completed within 5 hours. The sensitivity of this system almost equals that of the manual method using nested PCR. The addition of an internal control for HBV detection did not affect the sensitivity of the method and confirmed the accuracy of results. It was possible to quantify HBV in HBV+ samples that contain more than 500 genome equivalents per mL. We started using this system from June 1999 for testing stored donor and patient samples to analyze cases of posttransfusion hepatitis and identified three HBV+ donations that were implicated in posttransfusion hepatitis B. CONCLUSION: The high-throughput detection system is a useful tool for HBV DNA and HCV RNA detection because it enables rapid and reliable testing of a large number of samples.

Automation↗
PubMed2005

A heuristic approach for detecting RNA H-type pseudoknots.

MOTIVATION: RNA H-type pseudoknots are ubiquitous pseudoknots that are found in almost all classes of RNA and thought to play very important roles in a variety of biological processes. Detection of these RNA H-type pseudoknots can improve our understanding of RNA structures and their associated functions. However, the currently existing programs for detecting such RNA H-type pseudoknots are still time consuming and sometimes even ineffective. Therefore, efficient and effective tools for detecting the RNA H-type pseudoknots are needed. RESULTS: In this paper, we have adopted a heuristic approach to develop a novel tool, called HPknotter, for efficiently and accurately detecting H-type pseudoknots in an RNA sequence. In addition, we have demonstrated the applicability and effectiveness of HPknotter by testing on some sequences with known H-type pseudoknots. Our approach can be easily extended and applied to other classes of more general pseudoknots. AVAILABILITY: The web server of our HPknotter is available for online analysis at http://bioalgorithm.life.nctu.edu.tw/HPKNOTTER/ CONTACT: cllu@mail.nctu.edu.tw, chiu@cc.nctu.edu.tw

Algorithms↗
PubMed2001

[The role of hepatitis C virus RNA detection in various substrates of patients with chronic hepatitis C].

Puncture biopsy of the liver and blood count were made in 72 patients with chronic hepatitis C (CHC). Morphological alterations in the liver were assessed by Knodell index. The blood serum, lymphocytes and hepatic tissue were examined for a genome form of hepatitis C virus (HCV) RNA, blood lymphocytes and hepatic tissue--for a relevant replication form. HCV RNA was detected using "nested" RT-PCR. Only 26% patients had symptoms of asthenovegetative and dyspeptic syndromes. Normal alaninaminotransferase (ALT) level was observed in 24% patients, the rest had it high. HCV RNA was encounted more frequently in hepatic tissue than lymphocytes or serum (83, 68 and 46%, respectively). A replication form of HCV RNA was present in hepatic tissue of 31% patients and was absent in the lymphocytes. The incidence of the RNA detection was not related either to the disease symptoms or morphological alterations in hepatic tissue. The occurrence of the genome and replication forms in hepatic tissue does not correlate to ALT level. HCV RNA occurs more often in the serum, blood lymphocytes and in three substrates simultaneously in patients with hyperalatemia.

Adolescent↗
PubMed1998

Supplementary anti-hepatitis C virus (HCV) testing with 2nd and 3rd generation recombinant immunoblot assay and matrix applied to enzyme immunoassay positive sera and comparison with HCV-RNA detection.

A series of 118 serum samples tested positive in the anti-hepatitis C virus (HCV) 2nd-generation enzyme immunoassay (EIA-2), and indeterminate (93 samples) or negative (25 samples) in the supplementary 2nd-generation recombinant immunoblot assay (RIBA-2). These sera were further evaluated by three additional tests: 3rd-generation RIBA (RIBA-3), MATRIX, and AMPLICOR HCV-PCR. For the 93 RIBA-2 indeterminate serum samples, the results of the immunoassays had a concordance of 69%. Twenty-one and 34 samples remained anti-HCV indeterminate in the RIBA-3 test and the MATRIX, respectively. Among the 25 RIBA-2-negative samples, only seven samples remained anti-HCV negative, while five samples tested anti-HCV positive in both RIBA-3 and MATRIX. The reactivity of the RIBA-3 antigen NS5 was not crucial for the result of any sample. Positive to negative contradictions between the results of MATRIX and RIBA-3 were never observed. Altogether, the MATRIX tested a significantly lower number of samples anti-HCV negative than did the RIBA-3. HCV RNA was detectable in 54/93 RIBA-2 indeterminate and 7/25 RIBA-2 negative samples. High percentages of PCR positive results among RIBA-3-indeterminate and among MATRIX-indeterminate samples indicate an increased possibility of detecting HCV RNA if at least one antigen is reactive. The type of antigen, the pattern of antigen reactivity, or the level of reactivity had no prognostic value in predicting the presence of HCV RNA. Our findings show the necessity of being cautious in the interpretation of RIBA-2-negative results.

Antibodies, Viral↗
PubMed2004

Influence of hemodialysis procedure on HCV RNA detection in serum and peripheral blood mononuclear cells.

AIM: To assess whether hemodialysis procedure induces qualitative or quantitative changes in hepatitis C virus (HCV) RNA. METHODS: We obtained blood samples in the 10 HCV RNA-positive patients of our hemodialysis unit before (sample I) and 5 min after a dialysis session (sample II), and before the next dialysis session (sample III). HCV RNA was tested by PCR in serum and peripheral blood mononuclear cells (PBMC). Serum viral load was measured by branched-DNA assay. RESULTS: Serum HCV RNA was positive in samples I, II and III of the 10 patients. PBMC HCV RNA was detected in samples I, II and III of seven patients. Mean viral load was 1.43+/-0.99 Meq genome/mL in sample I, 0.86+/-0.40 Meq genome/mL in sample II and 1.27+/-0.56 Meq genome/mL in sample III. CONCLUSIONS: HCV load was low in most HCV RNA-positive patients. It had a downward trend during dialysis procedure but HCV RNA remained detectable in all serum samples and in most PBMC samples. Therefore, qualitative HCV RNA seems to be better than viral load to assess HCV infection in hemodialysis patients.

Genotype↗
PubMed2006

Prolonged viral RNA detection in blood and lymphoid tissues from coxsackievirus B4 E2 orally-inoculated Swiss mice.

The spreading of viral RNA within Swiss Albino mice orally inoculated with coxsackievirus B4 E2 strain (CVB4 E2) was studied by using RT-PCR and semi-nested-RT-PCR methods. Viral RNA was detected in various organs: pancreas, heart, small intestine, spleen, thymus, and blood at various postinfectious (p.i.) times ranging from 8 hr to 150 days. Our results show that (i) outbred mice can be infected with CVB4 E2 following an oral inoculation, which results in systemic spreading of viral RNA, (ii) CVB4 E2 infection can be associated with a prolonged detection of viral RNA in spleen, thymus and blood, up to 70 days p.i. and further in other organ tissues.

Administration, Oral↗
PubMed1998

In situ reverse transcriptase-polymerase chain reaction: an innovative tool for hepatitis C virus RNA detection and localisation, and for quantification of infected cells.

In situ reverse transcriptase-polymerase chain reaction is a novel and exciting technique which couples the extremely high sensitivity of DNA amplification with the advantages of in situ hybridisation, allowing the preservation of cell morphology and the localisation of the positive signal within intact cells. In this brief report, we analyse and discuss the application of such a technique to the study of hepatitis C virus (HCV) infection, the most common cause of chronic liver disease worldwide. Moreover, given the lymphotropism of this virus, we describe here our own approach to detect and localise HCV RNA within intact peripheral blood mononuclear cells and to quantify the relative number of infected cells.

Hepacivirus↗
PubMed1998

Hepatitis E virus RNA detection in serum and feces specimens with the use of microspin columns.

This report describes the use of microspin columns for extraction of hepatitis E virus (HEV) RNA from stool and serum specimens for reverse transcription-polymerase chain reaction (RT-PCR) and compares this method with the glass powder method. The microspin column method was found to be 1- to 2-log more sensitive in detecting HEV RNA than the glass powder method and had better reproducibility. The microspin column method also detected HEV RNA in a larger number of specimens than the glass powder method from among a panel of serum and stool specimens. Use of this method may allow better assessment of viremia and fecal excretion in patients and experimental animals infected with HEV.

Animals↗
PubMedDate not supplied

HCV RNA detection in parotid gland biopsy in a patient with chronic hepatitis C virus liver disease.

We report a case of a 75-year-old woman admitted to our Unit for chronic sialadenitis, characterized by histologic evidence of B and T lymphocyte infiltrate; laboratory and histologic tests revealed chronic hepatitis C, without autoimmune markers. We found hepatitis C virus (HCV) RNA in liver, in parotid gland, in saliva and in peripheral blood mononuclear cells, but not in serum. HCV RNA was detected by PCR technique. To our knowledge this is the first case of detection of HCV RNA in parotid gland. This finding raises the question about the relationship between sialadenitis and viral HCV infection.

Aged↗
PubMedDate not supplied

HCV-RNA detection using different PCR methods in sera, cryoglobulins and peripheral blood mononuclear cells of patients with mixed cryoglobulinemia.

OBJECTIVE: The hepatitis C virus (HCV) is frequently associated with mixed cryoglobulinemia (MC), and a number of authors have reported the presence of anti-HCV antibodies and HCV-RNA in the blood of MC patients. The presence of the HCV genome in the blood cells of individuals infected by HCV may correlate with the etiopathology of MC. We investigated the presence of HCV-related sequences in the sera, cryoglobulins and peripheral blood mononuclear cells (PBMC) of patients with MC and of individuals with type C chronic active hepatitis (CAH). METHODS: 39 patients with MC, 11 non-cryoglobulinemic HCV-positive individuals with CAH, and 2 anti-HCV negative controls were included in the study. The presence of HCV-RNA was detected by nested RT-PCR and by a commercial kit. The PCR was performed by amplifying the 5'-non coding region (5'-NCR) of HCV. RESULTS: HCV-RNA was detected in the sera and cryoglobulins of about 90% of the patients; the commercial kit showed a higher sensitivity than nested PCR. One MC patient showed HCV-RNA only in the cryoglobulins. HCV-RNA was present in the PBMC of 14 of the 20 (70%) MC patients analyzed. No differences in serum and PBMC HCV-RNA positivity were found between MC patients and controls. CONCLUSION: Our results confirm the spread of HCV infection among patients with MC. HCV-RNA is present in the serum, cryoglobulins and PBMC of a large proportion of MC patients. The prevalence of HCV-RNA in the PBMC of MC patients and controls did not differ significantly; this may suggest a tropism of HCV for PBMC regardless of the presence of cryoglobulinemia.

Base Sequence↗
PubMed1998

Further study of hepatitis C virus RNA detection in formalin-fixed, paraffin-embedded liver tissues by ligation-dependent polymerase chain reaction.

Ligation-dependent polymerase chain reaction (LD-PCR) is a novel, simple and sensitive PCR assay that could be useful for the molecular diagnosis of various viral infections. This assay uses two capture probes for RNA isolation and two hemiprobes for the subsequent PCR. To investigate its usefulness in the field of pathological diagnosis, further study of this new PCR technology to detect hepatitis C virus (HCV) RNA was carried out in formalin-fixed, paraffin-embedded (FFPE) liver tissues and the results compared with those obtained by the reverse transcription (RT)-PCR method. We analyzed routinely processed FFPE liver specimens of surgical origin from 13 anti-HCV-seropositive Japanese patients with hepatocellular carcinoma (HCC). As a result, HCV-RNA was detected in FFPE liver specimens in 12 of the tested patients (92.3%) by the LD-PCR method without the use of reverse transcription. In contrast, HCV-RNA was detectable in eight out of 13 cases (61.5%) by the RT-PCR method. These results indicated that LD-PCR was useful for HCV-RNA detection in routinely processed FFPE liver specimens.

Adult↗
PubMed2005

The importance of tick-borne encephalitis virus RNA detection for early differential diagnosis of tick-borne encephalitis.

BACKGROUND: Tick-borne encephalitis virus (TBEV) is one of the most important causes of human viral infections of the central nervous system in Europe. Currently, the diagnosis of TBE is based on the demonstration of specific antibodies in patient's serum, which appear only several weeks after the infection. OBJECTIVE: To determine how successfully can viral RNA be detected by RT-PCR in the samples of body fluids of patients with TBE prior to and after the appearance of antibodies. STUDY DESIGN: Serum, whole blood and CSF samples from 34 patients with a serologically confirmed TBE were collected. Samples were tested for the presence of TBEV RNA by using RT-PCR method. RESULTS: Viral RNA was detected in all blood and serum samples collected before the development of antibodies. After the appearance of IgM antibodies, the number of positive samples dropped by at least one third. After the development of IgG antibodies, only 3% of serum and 16% of blood samples tested positive for viral RNA. Samples of cerebrospinal fluid were shown to be inappropriate for the molecular diagnosis of TBE using this assay, since only one sample (10%) that was collected in the sero-negative phase of disease was found positive by the PCR assay. CONCLUSIONS: RT-PCR is an efficient method for an early detection of TBEV in blood and serum samples collected prior to the appearance of antibodies. This method can be of valuable use for a differential diagnosis of TBEV infection in patients with febrile illness after a tick bite, particularly in regions where more than one tick-transmitted diseases are endemic.

Adolescent↗
PubMed1996

Quantification of Plasmodium falciparum sporozoites by ribosomal RNA detection.

We used sequences specific to the small subunit ribosomal RNA (SSU rRNA) of the sporogonic stages of Plasmodium falciparum to design a reserve transcriptase-polymerase chain reaction (RT-PCR) assay that can detect 0.1 sporozoites in total RNA purified from potentially infected mosquitoes. We made a synthetic RNA that is amplified in the RT-PCR by the same primers as the parasite SSU rRNA and that serves as an internal control and competitive quantitation standard. We calibrated the assay for quantitation of sporozoites by making a standard curve with RNA from purified and counted sporozoites. The assay accurately measured sporozoite number with a linear range of at least three orders of magnitude in a single reaction. Some application and limitations of the assay are discussed.

Animals↗