Quality assurance in RT-PCR-based BCR/ABL diagnostics--results of an interlaboratory test and a standardization approach.

Here we describe the results of an interlaboratory test for RT-PCR-based BCR/ABL analysis. The test was organized in two parts. The number of participating laboratories in the first and second part was 27 and 20, respectively. In the first part samples containing various concentrations of plasmids with the ela2, b2a2 or b3a2 BCR/ABL transcripts were analyzed by PCR. In the second part of the test, cell samples containing various concentrations of BCR/ABL-positive cells were analyzed by RT-PCR. Overall PCR sensitivity was sufficient in approximately 90% of the tests, but a significant number of false positive results were obtained. There were significant differences in sensitivity in the cell-based analysis between the various participants. The results are discussed, and proposals are made regarding the choice of primers, controls, conditions for RNA extraction and reverse transcription.

Nucleotide sequence of a new HLA-DQB1 allele, DQB1*03033.

HLA-genotyping by sequencing of the corresponding polymerase chain reaction (PCR) product allow the identification of a new HLA-DQB1 allele, DQB1*03033. To confirm the finding the entire exon 2 was sequenced.

Clinical relevance of hepatitis G virus (HGV) infection in heart transplant patients.

To investigate whether the recently discovered hepatitis G virus (HGV) influences the clinical outcome of heart transplant recipients under immunosuppression, we determined the prevalence of HGV infections correlated with liver function and survival in 51 patients. Presence of HGV RNA and anti-E2, a marker for resolved HGV infection, were serially tested in sera from patients before and after heart transplantation (HTX) by nested RT-PCR and ELISA. Four of 51 (7.8%) patients before transplantation, and 22 of 50 patients (44%) after transplantation showed signs of persistent or resolved HGV infection. HGV infection was not associated with impairment of liver function or with patient survival. In summary, presence of HGV infection does not influence the clinical outcome in heart transplant patients.

Parameters predicting response to alpha-interferon treatment in chronic hepatitis C.

BACKGROUND/AIMS: Sustained response to alpha-interferon treatment for chronic hepatitis C is seen in only 25% of cases. Therefore, it is desirable to define pretreatment factors predicting responders. MATERIALS AND METHODS: Forty-nine patients with chronic hepatitis C were treated with a standard alpha-interferon regimen (3 x 3 MU s.c./week). Demographic, biochemical and immunological parameters, and HCV genotypes were obtained prior to initiation of treatment and evaluated for their value in predicting response to alpha-interferon therapy. RESULTS: Response, as defined by normalization of ALT, was 71% during interferon therapy and sustained response after discontinuation of interferon 24.5%. Patients infected with HCV-genotype 1b had significantly more often "community-acquired" disease. Their outcome was worse with a response rate of 44% during therapy and a sustained response of 12.5%, as compared to 87% and 27% respectively in patients infected with genotypes other than 1b. On multivariate analysis, absence of cirrhosis, HCV-genotype other than 1b, higher ALT levels and higher numbers of CD8 positive liver infiltrates were found to be predictors of response during alpha-interferon therapy. CONCLUSION: Response to alpha-interferon therapy seems to be influenced both by viral virulence factors and by the intensity of the host immune response to HCV.

[Value of polymerase chain reaction (PCR) in diagnosis of tuberculosis and mycobacterium infections caused by ubiquitous mycobacteria]. / Stellenwert der Polymerase-Kettenreaktion (PCR) in der Diagnostik der Tuberkulose und der Mykobakteriosen durch ubiquitäre Mykobakterien.

The rise in both incidence and the number of multi-drug-resistant strains has made tuberculosis an alarming health problem even in some developed countries. Moreover, due to the HIV pandemic an increase in mycobacterial diseases caused by ubiquitous mycobacteria has been observed. The microbiological standard procedures are critical diagnostic tools, and a more rapid detection of mycobacteria by new laboratory techniques would be helpful. We investigated the role of the polymerase chain reaction (PCR) in the detection of M. tuberculosis complex and of ubiquitous mycobacteria. 580 clinical specimens obtained from 525 patients were examined. The majority of sampled material was bronchoalveolar lavage fluid. Based on cultural identification of mycobacteria, the incidence of tuberculosis in our patients was 3.4%. For detection of M. tuberculosis complex the insertion sequence IS 6110 was used. Relative to cultural identification, diagnostic specificity of PCR was 99.5%, and sensitivity was 66.7%, respectively. In 3.0% of the total patient group and in 8.8% of HIV-infected patients an infection by ubiquitous mycobacteria was found. Ubiquitous mycobacteria were detected using the hypervariable region of the 16S-rRNA-gene, and species-identification was done by sequence analysis. For ubiquitous mycobacteria, sensitivity of PCR was 60.0% relative to cultural identification and 62.5% relative to a proven disease. PCR is a useful method in the rapid diagnosis of pulmonary tuberculosis with excellent specificity but lack of sensitivity.

Virus inactivation and prevalence of GBV-C in haemophiliacs.

A new putative hepatitis virus has recently been discovered and termed GB virus C (GBV-C). We investigated the prevalence of this virus among 50 haemophiliacs treated with non-virus-inactivated clotting factor concentrates prior to 1985 and 21 haemophiliacs treated exclusively with virus-inactivated clotting factor concentrates. In the first group the prevalence of GBV-C based on PCR and ELISA was 46%. In the second group the prevalence of GBV-C was similar to that of healthy blood donors (5%). We therefore conclude that GBV-C is reliably inactivated by modern virucidal methods such as vapour heating, pasteurization and treatment with solvent/detergent mixtures.

Hepatitis C virus infections in dialysis units: prevalence of HCV-RNA and antibodies to HCV.

HCV infection causes serious complications in dialysis patients that lead to problems in management of patients in dialysis units. Determination of HCV-RNA is at present essential for monitoring the course of HCV infection. Reports concerning HCV-RNA in dialysis patients are mostly from Asian dialysis units; therefore, an analysis of dialysis patients in Europe was undertaken. From 1515 patients 2630 blood samples were screened for HCV-RNA and anti-HCV. Two-thirds of patients positive in an anti-HCV test containing a mixture of three antigens (EIA-II, Ortho) were further analysed for antibodies against these individual antigens. From 523 patients multiple samples were tested. Related on dialysis units from which all the attending patients were tested, mean prevalence of HCV-RNA was 8.4%, of anti-HCV 13.2%. Concerning all plasma samples from dialysis patients sent to our laboratory for investigation of HCV-RNA and anti-HCV the prevalence of HCV-RNA was 21.9%, of anti-HCV 23.1%, HCV-RNA was present in 76% of anti-HCV positive patients and in 4.1% of anti-HCV negative patients (1.3% of single and 6.8% of multiple tested patients). Acute and chronic infections with self-limited, persistent or intermittent viraemia were observed with changes and fluctuations of both HCV markers. With the exception of differences in onset of antibody production in some patients following acute infections, there were no major differences of dialysis patients compared to patients without dialysis treatment as far as antibody spectrum and detectability of HCV-RNA were concerned.

The chronic myelocytic cell line K 562 contains minor (m) as well as major (M) ber/abl fusion mRNAs.

By searching for additional chimeric bcr/abl transcripts in K 562 cells characterized by major (M) bcr/abl fusions, a new mRNA, a minor (m) bcr/abl transcript, was detected. A practical implication of this finding is that the K 562 cell line can be used as positive control for the detection by the polymerase chain reaction of both types of transcripts for the diagnosis of Philadelphia chromosome associated leukemias.

Anti-LKM-1 antibodies determined by use of recombinant P450 2D6 in ELISA and western blot and their association with anti-HCV and HCV-RNA.

Several subtypes of anti-liver-kidney microsome antibodies (LKM) are known. LKM-1 antibodies associated with autoimmune chronic active hepatitis recognize P450 2D6, a cytochrome P450 mono-oxygenase. The frequent association of anti-LKM-1 antibodies and hepatitis C virus (HCV) infections and the probable existence of an infectious and autoimmune form of anti-LKM-1-associated hepatitis, requiring different therapeutical strategies, necessitates the exact determination of anti-LKM-1 specificities. Therefore, we compared various antibody tests (immunofluorescence, ELISA with recombinant P450 2D6, and Western blot with recombinant and natural antigens and agargel double diffusion) with sera of 27 anti-LKM-1-positive chronic active hepatitis (CAH) patients, with 61 sera harbouring anti-mitochondrial antibodies, 100 sera each from HCV-RNA-positive and HCV-RNA-negative patients, and 50 sera of healthy persons. Western blot techniques using recombinant MS2-polymerase P450 2D6 fusion protein were found to be the most sensitive and specific method for anti-LKM-1 antibody determination in routine laboratory. The recently recognized association of anti-LKM-1 antibody and HCV infection was confirmed by the results of this study. In anti-HCV and HCV-RNA-positive patients with anti-LKM-1 antibodies there was a preponderance of males with higher mean age and lower antibody titres. The results support the hypothesis of the existence of an autoimmune as well as an infectious (HCV triggered) subgroup of anti-LKM-1-positive hepatitis.

[Rapid diagnosis of tuberculosis by the polymerase chain reaction (PCR)]. / Tuberkulose-Schnelldiagnostik mit der Polymerase-Ketten-Reaktion (PCR).

The polymerase chain reaction (PCR) was used for the detection of Mycobacterium tuberculosis DNA. More than 2000 different clinical specimens were analyzed by this assay. The efficiency of two different methods for processing the DNA from biological material was analyzed. DNA amplification was done according to standard protocols by amplifying a segment of 402 bp of the 65 kD mycobacterial gene, electrophoretic separation of the amplification product followed by Southern transfer and hybridization with a Mycobacterium tuberculosis-specific probe or by a semi-nested amplification procedure in which the initial amplification product was reamplified by a second round with a Mycobacterium tuberculosis-specific primer. The specificity of primers and probe for mycobacterial DNA was proven by testing 70 of class-I microorganisms, as well as 20 different strains and own isolates of Mycobacterium tuberculosis and 67 strains of 25 different MOTTs. Some of the amplification products were sequenced. The clinical relevance of the results and the sensitivity of the PCR method were confirmed by simultaneous quantitative bacterial culture from the same clinical specimens. The results of conventional culture method received after 8 to 10 weeks culture time correlated with the results from PCR obtained within 12 hours in 95.4% in the semi-nested amplification procedure. The discrepancy of 4.6% was caused by positive results of PCR and negative cultures which might be due to the higher sensitivity of PCR compared to culture technique. The results show that PCR may be used for detection of Mycobacterium tuberculosis in clinical specimens. The specificity can be regarded as largely proven, advantages are velocity and sensitivity.(ABSTRACT TRUNCATED AT 250 WORDS)

[Hepatitis C virus: sequence homology of a European isolate and divergence from the prototype]. / Hepatitis-C-Virus: Sequenzhomologie europäischer Isolate und Divergenz zum Prototyp.

The polymerase chain reaction (PCR) detected specific hepatitis C viral (HCV) RNA sequences in liver biopsies from two patients with chronic hepatitis, in the tissue of a liver implantate, in plasma from four chronic non-A, non-B hepatitis (NANBH) patients and, for the first time, in an infectious anti-D-immunoglobulin preparation. A comparison of the viral sequences coding for a region for the nonstructural NS3 protein from the liver tissues revealed only a very small degree of sequence divergence on the cDNA as well as on the amino acid level (between 0 and 5%). The sequence similarities of the RNA isolated from plasma of the four chronic NANBH patients and the anti-D-immunoglobulin preparation were partly somewhat lower but altogether also high (between 90 and 100%). In contrast, all eight cDNA and amino acid sequences exhibited a significantly higher degree of divergence in comparison with the HCV prototype sequence (between 29 and 32%) than among themselves (between 0 and 10%). This unexpected high sequence similarity of the eight European isolates and their low homology to the Northamerican prototype sequence is indicative for the existence of different types of HCV. This will be important not only for epidemiological studies but also for the development of effective diagnostic procedures and vaccines. Concerning the pathogenesis of NANBH, a double infection or a helper mechanism has to be considered: in addition to the C virus, sequences of an other virus particle were found in the infectious IgG preparation as well as in the liver biopsies.

[The demonstration of hepatitis B virus DNA with the polymerase chain reaction]. / Nachweis von Hepatitis-B-Virus-DNA mit der Polymerase-Kettenreaktion.

Results with the polymerase chain reaction and conventional DNA hybridizing technique (dot-blot) for the detection of hepatitis B virus (HBV) DNA were compared for 439 patients. In 261 patients who were positive for HBs antigen (Ag), HBV DNA was demonstrated with the polymerase reaction in all of the 69 HBe-Ag positive sera (dot-blot 56%), as well as in 81 (62%) of 131 anti-HBe positive sera (dot-blot 5%), in 29 (48%) of 61 HBe marker negatives (dot-blot 3%) and in six (29%) of 21 delta positive sera. Among HBs-Ag negative patients, HBV DNA was detected in six (22%) of 27 anti-HBc positive sera, but not in sera (n = 50) which were both anti-HBc and anti-HBs positive, as well as in HBV marker negative patients with liver disease (n = 30) and healthy controls (n = 50). If infectiousness is to be checked or in case of double infection, the more sensitive polymerase chain reaction is to be recommended for the detection of HBV DNA. HBe-Ag positive patients must be considered as infectious: tests for HBV DNA can in general be omitted.

Improved sexual function during recombinant human erythropoietin therapy.

In seven male haemodialysis patients, living with a constant sexual partner, sexual life (self-administered questionnaire) and sex hormones were evaluated before and 3 or 10 months after commencement of recombinant human erythropoietin (rHuEpo) therapy respectively. Haematocrit increased from 22.2 +/- 2.1% to 30.6 +/- 1% with a maintenance dose of 140 +/- 55 U/kg per week. Two patients were hypertensive before and after rHuEpo therapy; antihypertensive medication was kept constant during the study. Self-reported sexual function improved in four of seven patients, including libido and erection. This was paralleled by improved indices of wellbeing (physical fitness, mental alertness). In contrast, serum values of sexual hormones (testosterone or oestradiol) and of basal and stimulated (LHRH/TRH), prolactin and LH, and FSH were not significantly changed during rHuEpo therapy.

Neutropenic colitis: evaluation with computed tomography.

The computed tomography findings of 10 patients with neutropenic colitis are described and illustrated. Seven of these patients had leukemia, one had lymphocytic lymphoma, and two had systemic lupus erythematosus. All patients had colon wall thickening which was either isodense with the normal bowel tissue or showed areas of intramural low density. Air in the thickened bowel wall was seen in six patients. These computed tomography findings in neutropenic patients with fever, abdominal pain, and diarrhea should suggest the diagnosis in most instances, resulting in prompt treatment of this usually life-threatening entity.