Comparison of sequence analysis and INNO-LiPA HBV DR line probe assay in patients with chronic hepatitis B.

The aim of this study was to compare direct sequence analysis of partial HBV pol gene and Inno-LiPA HBV DR in serum samples of 120 chronic hepatitis B patients sent to the Clinical Microbiology Laboratory of Ege University Hospital because of lamivudine resistance. Sequence analysis was performed on ABI Prism 310 Genetic Analyzer. Comparison of Inno-LiPA and sequence results obtained by double-blind evaluation showed full agreement (both at rt180 and rt204) in 58.8% of samples. Visually rechecking of the electropherograms increased this rate to 68.3% Codon based rates are 81.7% and 75.8% at rt180 and rt204 respectively. LiPA detected variants in additional 12 (10%) samples, but missed one variant sample (both rt180 and rt204) and one sample was indeterminate due to poor probe binding. LiPA allows determination of mixed variants and seems to be more sensitive and simple for routine testing even though sequence analysis is still the gold standard for detecting new variants.

[Evaluation of the agreement of results obtained from ABI Prism 7000 and 7700 sequencers in the quantification of hepatitis B virus DNA]. / Hepatit B virus DNA kantitasyonunda ABI Prism 7000 ile 7700 sekans saptama cihazlarinin sonuçlarinin birbiri ile uygunlugunun arastirilmasi.

There are different methods and systems for quantification of HBV-DNA in clinical virology laboratories. The aim of this study was to evaluate the agreement of the polymerase chain reaction (PCR) protocol with ABI Prism 7000 instrument (PE Biosystems) which was designed and optimised for ABI Prism 7700 (PE Biosystems). Serum samples obtained from 168 chronic hepatitis B patients were treated with "High Pure Viral Nucleic Acid Kit" (Roche Applied Science, USA), and MagnaPure LC isolation station (Roche Applied Science, Germany) was used for HBV-DNA isolation. Real time PCR procedure which amplifies pre-S gene of HBV genome was performed. Amplification and detection steps of all samples were performed with ABI Prism 7700 and 7000 Sequence Detection Systems. Among 168 samples, results of 124 serum samples were found to be in dynamic ranges of the tests. The results of these 124 samples obtained from ABI 7000 and ABI 7700 were concordant. Among the rest of 44 samples; one yielded higher than 10(10) copies/mL with two of the systems; six samples gave results higher than 10(10) copies/mL only with 7700; thirty samples were found negative with both of the systems; seven samples were positive (320-1220 copies/mL) with 7000 but negative with 7700. As a result this PCR protocol can be used in ABI 7000 system according to viral quality control (VQC) results. However, since the results of samples with HBV-DNA less than 1 x 10(6) copy/ml were discordant with the results obtained by ABI 7700 system, it can be concluded that different systems must not be used for the management and monitoring of the same patient.

TT virus infection and genotype distribution in blood donors and a group of patients from Turkey.

BACKGROUND: TT virus (TTV) DNA has been found in a large proportion of patients with different forms of non-A-G hepatitis, however the clinical importance is unclear. We aimed to determine the genotypes of TTV isolates found in blood donors and different patient groups from the western part of Turkey. MATERIALS AND METHODS: TT DNA was investigated in serum samples of 91 volunteer blood donors (BD), 105 thalassemia (TH) patients, ten patients with fulminant hepatitis (FH) and 16 hemodialysis (HD) patients by heminested PCR using primers NG059, NG061 and NG063 from the ORF1 region. 39 isolates were genotyped by analyzing the partial sequence of ORF1. RESULTS: TTV DNA was found in 75% of HD, 80% of FH, 61% of TH patients and in 51.6% of BD. Among the sequenced isolates, 14 (35.9%) belonged to genotype 1 (G1) and 25 (64.1%) belonged to genotype 2 (G2). Among the G2 sequences, 22 were grouped as G2c. CONCLUSION: TTV infection was common in the population studied, even with moderately sensitive primers. G2 was the major genotype of the studied population without any significant differences in distribution between various patient groups and BD.

Cord formation in MB/BacT medium is a reliable criterion for presumptive identification of Mycobacterium tuberculosis complex in laboratories with high prevalence of M. tuberculosis.

We evaluated cord formation in MB/BacT broth as a rapid method for presumptive identification of the Mycobacterium tuberculosis complex. Kinyoun acid-fast-stained smears from 370 positive MB/BacT bottles were examined for the presence of serpentine cording. The smears were examined independently by two observers. Observer 1 (the supervisor of the mycobacteriology laboratory) examined all of the smears while observer 2 (a clinical microbiologist not familiar with acid-fast bacillus [AFB] microscopy) examined 148 randomly chosen smears that were read by observer 1 without knowledge of which smear was which. The sensitivity, specificity, and positive and negative predictive values of cording for the presumptive identification of M. tuberculosis read by observer 1 were 88.2, 97.4, 99.2, and 69.7%, respectively. These values were reported at 90.6, 52.3, 82.8, and 69. 7%, respectively, by observer 2. Our laboratory prevalence of M. tuberculosis among positive cultures was 78% during the time this study was conducted. At the time of positive signal of the MB/BacT bottles, the broth of the bottles had sufficient cell mass to allow for observation of the presence or absence of serpentine cording. The presence of cords in MB/BacT broth is a reliable criterion for rapid, predictive identification of the M. tuberculosis complex for laboratories with a high proportion of the M. tuberculosis complex when the smears are examined by a microbiologist who has experience with AFB staining.

Use of nucleic acid probes for identification of Mycobacterium tuberculosis directly from MB/BacT bottles.

The feasibility of using nucleic acid probes directly from positive MB/BacT broth to identify mycobacteria was determined in this study. A total number of 2,727 specimens were cultured into the MB/BacT (Organon Teknika) automated system and on conventional Loweinstein-Jensen (LJ) slants. The Gen-Probe AccuProbe culture identification tests (DNA probes) were used on samples from bottles which were identified as positive for mycobacteria by MB/BacT. Samples of positive MB/BacT broth (0.1 ml) were used directly in the broth culture method for the DNA probes as published by Gen-Probe. Centrifugation of the contents of the bottle was not done prior to probe testing. The number of mycobacteria detected by MB/BacT and LJ was 253 (221 isolates of M. tuberculosis and 32 isolates of mycobacteria other than M. tuberculosis [MOTT]). A total of 96.4% (213 of 221) of the bottles growing M. tuberculosis produced a positive direct DNA probe result for M. tuberculosis complex. One hundred percent (16 of 16) of the bottles growing M. gordonae produced a positive direct DNA probe result for M. gordonae. A total of 3.6% (8 of 221) of the bottles growing M. tuberculosis did not yield a positive direct DNA probe result for M. tuberculosis complex. The testing of subcultures made onto solid media from the positive bottles by AccuProbe identified six of these eight M. tuberculosis isolates. Two (0.9%) M. tuberculosis isolates gave a negative result for the M. tuberculosis probe test applied on the MB/BacT broth and its subculture. The rest of the positive MB/BacT bottles growing MOTT (16 of 32) were negative for M. gordonae, M. avium, M. intracellulare, and M. kansasii probes. The sensitivity and specificity of AccuProbe for the identification of M. tuberculosis and M. gordonae directly from MB/BacT broth were 96.4 and 100% for M. tuberculosis and 100 and 100% for M. gordonae, respectively. The direct testing of positive MB/BacT broth by AccuProbe, without prior centrifugation, allows for the accurate and rapid identification of M. tuberculosis and M. gordonae.