TRUGENE sequencing versus INNO-LiPA for sub-genotyping of HCV genotype-4

Hepatitis C virus genotypes and subtypes determination is an important factor for understanding the epidemiology of the virus, in the pre-treatment evaluation of the patients and in defining better treatment strategies. In the present study, we compared two commercially available assays for HCV genotyping: the reverse hybridization based Innogenetics INNO-LiPA HCV II and the direct sequencing by TRUGENE assay. The study included 31 HCV-RNA positive Egyptian patients; 18 patients with chronic active hepatitis, 8 with HCC, and 5 with cirrhosis. Using the TRUGENE genotyping test, all the samples had genotype 4 (100%) and subtyped as 4a in 18/31(58%), 4c in 10/31 (32%), 4e in 1/31 (3%), 4a/c in 1/31 (3%), and 4g in 1/31 (3%). Using the INNO-LiPA assay, 30 samples had genotype 4 (97%), and 1 sample had genotype 1e (3%). One sample showed mixed infection with type 4f and type 1. Only six samples were subtypable by INNO-LiPA, three were genotype 4c/d, and the other three were 4f, 4e, and 1e. Seven samples gave reactivity in the INNO-LiPA of lines 5, 6, 16, 17, 18, which are considered untypable by the interpretation chart but considered to be a rare HCV genotype 4 by the manufacturer. At the genotype level, there was a 97% concordance between TRUGENE sequencing and INNO-LiPA, but at the subtype level the concordance rate was 3% only. We conclude that the TRUGENE genotyping assay is a reliable test for HCV genotyping for the detection of major types and subtypes detection, while INNO-LiPA is a good test at the genotype level but unreliable for subtyping especially in the Egyptian population. This is mainly due to the high diversity of genotype 4, which is the most prevalent genotype in Egypt.     

Direct comparison of hepatitis C virus genotypes tested by INNO-LiPA HCV II and TRUGENE HCV genotyping methods.

BACKGROUND: Hepatitis C virus (HCV) is a major cause of chronic liver disease worldwide. It is associated with the development of end-stage liver disease and hepatocellular carcinoma. Studies have shown that patients infected with different genotypes of HCV may respond to interferon-ribavirin therapy differently and thus HCV genotype information is very important in helping physicians to better managing their patients. OBJECTIVES: Compare the end results of HCV typing of the two commercially available tests. STUDY DESIGN: TRUGENE Genotyping test (Visible Genetics) was used to analyze clinical specimens obtained from North America. The 5' NC was amplified with the Roche COBAS Amplicor HCV Monitor Test. Amplification products were blinded and genotyped by the TRUGENE HCV 5'NC method. Genotype results were compared with those obtained by the reverse hybridization based INNO-LiPA HCV II (Innogenetics) assay. Additional sequencing of the NS5B region was done to resolve discrepancies. RESULTS AND CONCLUSIONS: Among the total of 110 consecutively collected serum specimens submitted for HCV genotyping, 108/110 could be typed by the sequencing method and 107/110 were typable by LiPA HCV II method. Our experiences with the tests suggest that at type level, HCV genotype results are 100% concordant between the two tests studied for those 106 specimens successfully typed by both methods. More sensitive amplification, such as qualitative PCR, is needed to test specimens with viral load lower than 20000 IU/ml. Both tests can be easily adapted by a clinical diagnostic laboratory.     

Improvement of hepatitis C virus (HCV) genotype determination with the new version of the INNO-LiPA HCV assay

Hepatitis C virus (HCV) isolates have been classified into six main genotypes. Genotyping methods, and especially the widely used line probe assay (LiPA), are frequently based on the 5'-untranslated region (5'UTR). However, this region is not appropriate for discriminating HCV strains at the subtype level and for distinguishing many genotype 6 samples from genotype 1. We investigated the capacity of a novel LiPA (Versant HCV Genotype 2.0 assay) based on the simultaneous detection of 5'UTR and Core regions for genotypes 1 and 6 to provide correct HCV genotypes (characterized with a phylogenetic analysis) in a set of HCV strains mainly encountered in Western countries. The improvement was assessed by comparing the results to those obtained with the previous version of the assay. Of the 135 tested samples, 64.7% were concordant for genotype group and subtype with sequencing reference results using the Versant HCV Genotype 2.0 assay versus 37.5% with the previous version. The yield was mainly related to a better characterization of genotype 1, since the accuracy, tested in 62 genotype 1 samples, increased from 45.2% with the first version to 96.8% with the new one. However, this new version necessitates a specific PCR and could no longer be used after 5'UTR PCR used for current HCV infection diagnosis. Moreover, the information provided by 5'UTR hybridization is not reliable for correctly identifying the diversity within genotypes 2 and 4. Thus, the Versant HCV Genotype 2.0 assay remains a useful tool for clinical practice when only the discrimination between major HCV genotypes is necessary.     

Simplified PCR protocols for INNO-LiPA HBV Genotyping and INNO-LiPA HBV PreCore assays.

BACKGROUND: INNO-LiPA HBV Genotyping (LiPA HBV GT) and INNO-LiPA HBV PreCore (LiPA HBV PC) are commercially available assays for hepatitis B virus (HBV) characterization. These assays are labor-intensive and may be prone to exogenous DNA contamination due to their use of nested PCR amplification procedures and lack of contamination control measures. OBJECTIVE: Standardized, single-round INNO-LiPA PCR amplification protocols incorporating uracil N-glycosylase and automated sample processing by the MagNA Pure LC instrument were evaluated. STUDY DESIGN: HBV standards containing 10,000, 1000, 100, 10, and 0 IU/mL were analyzed to determine the analytical sensitivity and reproducibility of these modified procedures. One hundred clinical serum specimens with viral titers ranging from 390 to 16,900,000 IU/mL were tested by modified LiPA HBV GT, while 34 specimens with viral titers ranging from 378 to 11,600,000 IU/mL were tested by modified LiPA HBV PC. RESULTS: Modified LiPA HBV GT and LiPA HBV PC each yielded analytical sensitivities of 100% at an HBV DNA level of 1000 IU/mL and 90% at a level of 100 IU/mL. Among clinical specimens, success rates for both INNO-LiPA procedures were > or =94%. CONCLUSIONS: Both modified INNO-LiPA procedures were sensitive and reproducible, with improved efficiency and suitability for routine laboratory use.     

Medullary thyroid carcinoma with poor differentiation and atypical radiographic pattern of metastasis

A diagnosis of sporadic medullary thyroid carcinoma (MTC) is complicated. On first diagnosis it may present with distant metastasis. There has been inconsistency regarding metastatic MTC tissue expression of calcitonin, its tumor marker. Adding to the difficulty is the fact that the radiographic pattern of pulmonary metastasis from MTC may vary substantially among patients. Herein is reported the case of a 73-year-old man who presented with two ill-defined pulmonary opacities, clinically resembling primary lung carcinoma. MTC was diagnosed on histopathology of tissue obtained from a total thyroidectomy. The pulmonary biopsy specimens were confirmed to be MTC metastasis on positive immunohistochemical staining of chromogranin-A and synaptophysin, even though only a few cells were stained for calcitonin. To the authors' knowledge this is the first reported case of MTC presenting initially as complex pulmonary metastasis with weakened expression of calcitonin in the metastatic lesion.     

Effects of HCV treatment on cytokine expression during HCV/HIV coinfection.

There is growing evidence that cytokine expression is linked to hepatitis C virus (HCV) pathogenesis and treatment response rates among HCV-monoinfected persons. However, because of the profound effects of human immunodeficiency virus (HIV) coinfection on HCV, it is not clear if these observations are also true for HCV/HIV-coinfected individuals. Serum expression of the proinflammatory cytokines interleukin-8 (IL-8) and tumor necrosis factor-alpha (TNF-alpha) and the fibrogenic cytokine transforming growth factor-beta1 (TGF-beta1) were measured in HCV/HIV-coinfected persons at baseline and at week 24 of HCV therapy. Higher levels of IL-8 and TGF-beta were demonstrated among nonwhite subjects at baseline. Increases in TNF-alpha and IL-8 expression were found at week 24 of HCV therapy, suggesting that enhanced proinflammatory cytokine production may occur during HCV treatment. However, cytokine levels were not predictive of HCV virologic, biochemical, or histologic response. Although previous studies conducted among HCV-monoinfected individuals have suggested that cytokine levels could predict the virologic response to therapy, no such associations were observed among HCV/HIV-coinfected persons, suggesting that they may respond differently to treatment than do their HCV-monoinfected counterparts.     

Derivative chromosome 9 deletions in chronic myeloid leukaemia: interpretation of atypical D-FISH pattern

BACKGROUND/AIMS: New molecular cytogenetic techniques are increasingly applied as a routine investigative tool in haematological malignancies, both at diagnosis and subsequent monitoring. This report describes the interpretation of atypical signal patterns encountered using BCR-ABL dual colour dual fusion fluorescence in situ hybridisation (D-FISH) translocation probes in chronic myeloid leukaemia (CML). METHODS: Interphase FISH experiments were carried out using BCR-ABL D-FISH probes in 46 patients with CML at diagnosis and during subsequent disease monitoring. Atypical hybridisation signal patterns were characterised by molecular cytogenetic techniques and correlated with conventional karyotyping. RESULTS: Two patients showed atypical interphase D-FISH patterns with one orange, one green, and one fusion (1O1G1F) signal. The presence of BCR-ABL gene fusion was documented by a dual colour single fusion (S-FISH) probe. The submicroscopic deletion of the ABL-BCR fusion gene on the derivative chromosome 9 in these cases was subsequently characterised by metaphase FISH on relocated G banded metaphases. CONCLUSIONS: Atypical interphase D-FISH patterns should not be interpreted in isolation and should be considered in conjunction with other cytogenetic or molecular genetic investigations.     

Distinct serum immunoglobulins pattern in Egyptian patients with chronic HCV infection analyzed by nephelometry

Hepatitis C has emerged as a major worldwide public health problem. The host immune response to HCV infection is composed of both a non-specific immune response, including interferon (IFN) production and natural killer (NK) cell activity, and a virus-specific immune response, including humoral and cellular components. Susceptibility to infection has been related to immunological disturbances. Several studies have provided experimental evidence of disorders of both cellular and humoral immunity. The present study was carried out to evaluate the serum immunoglobulins level (IgG, IgM, IgA) and IgG-subclasses (IgG1-4) in chronic hepatitis C patients in comparison with healthy control patients. This study included 50 patients with biochemical, serologic, virologic, and histologic evidence of chronic hepatitis C. Total IgG, IgA, and IgM were assayed by nephelometry. IgG subclasses were assayed using human IgG subclasses enzyme immunoassay. The results showed a significant increase of total serum IgG and IgM levels found in patients with chronic HCV compared with the healthy control patients (P < 0.001 for each). There was a statistically significant difference in the IgG subclasses (IgG1 to IgG4) between the patients and controls (P < 0.001 for each). On the other hand, no significant difference was found between patients and healthy controls in IgA level (P = 0.4). The normal total serum immunoglobulins pattern is apparently shifted in chronic hepatitis C infection in the Egyptian patients. This pattern may include an ethnic or biologic background and could be used in the differentiation of the patients with minimal liver disease.     

Mucin-producing atypical bronchial carcinoid.

In a case of atypical mucin-producing bronchial carcinoid in a 40-year-old man covert metastases were present at the time of lobectomy and were revealed at necropsy--a feature highlighting the malignant potential of the atypical carcinoid.     

Effects of corticosteroids on HCV infection.

The risk factors for clinical recurrent hepatitis C in liver transplant recipients are not clearly defined. It has been suggested that the corticosteroids included in the treatments of patients undergoing allograft rejection might induce acute hepatitis by increasing HCV replication. In this study we investigated the effects of corticosteroid boluses on HCV viremia in liver allograft recipients treated for acute rejection. Since we had previously developed a model of HCV replication in peripheral blood mononuclear cells (PBMC) in vitro, we also studied the effects of corticosteroids on HCV replication in vitro. A transient peak of HCV viremia was observed in patients treated with corticosteroid boluses for an acute allograft rejection. In the cell cultures, corticosteroids induced an increase of the total amount of viral RNA detectable. Our results demonstrate that corticosteroids induce an increase of hepatitis C virus replication in vivo and in vitro.     

Quantitation of HCV RNA with the microwell plate and COBAS Amplicor HCV Monitor assays.

BACKGROUND: We performed the Roche Amplicor Monitor hepatitis C virus (HCV) v2.0 microwell plate (MWP) assay for 2 years prior to bringing a COBAS instrument into the lab. Before reporting any results from the automated COBAS Amplicor Monitor HCV v2.0, we compared quantitative data with results on the same specimens from the manual Amplicor Monitor HCV. OBJECTIVE: To determine if the COBAS Amplicor Monitor assay yielded quantitative results that were comparable with those given by the manual Amplicor Monitor HCV. STUDY DESIGN: We tested 145 specimens with both assays. Specimens were chosen on the basis of having fallen within the linear range of the first assay. RESULTS: The log(10) mean (+/-standard deviation) for all 145 specimens was 5.976 (+/-0.597) for the COBAS and 6.142 (+/-0.597) for the MWP assay. When plotted as 145 pairs of numbers (log MWP result vs. log COBAS result), the linear regression line was displaced slightly downward from the line of equivalence by 0.1 log at the lower end and by 0.2 log at the upper end indicating the COBAS result was somewhat lower than the MWP result across the full range of the assay. The mean of the difference of the manual method and the COBAS for all 145 specimens was 0.166 log(10). A subset of 45 specimens for which we had HCV genotype data was analyzed separately. This set of specimens (of which 33 were genotype 1) also showed excellent concordance between the automated and manual methods. The two trendlines, one for genotype 1 and the other for genotypes 2, 3 and 4, were superimposable and thus the quantitative results were apparently not influenced by the genotypes, although the numbers were small (six HCV genotype 2, five HCV genotype 3 and one HCV genotype 4). CONCLUSIONS: We conclude that the automated Roche COBAS Amplicor Monitor v2.0 yields results that are comparable with the manual Amplicor Monitor assay for HCV genotype 1 and possibly also for genotypes 2, 3 and 4.     

Soft tissue Ewing sarcoma--peripheral primitive neuroectodermal tumor with atypical clear cell pattern shows a new type of EWS-FEV fusion transcript.

This study describes a new case of Ewing sarcoma (ES)-peripheral primitive neuroectodermal tumor (pPNET) with unusual phenotype and fusion gene structure. The tumor located in the inguinal area of a 15-year-old boy showed a highly aggressive behavior with hematogenous metastases after intensive chemotherapy and bone marrow transplant, causing death 28 months after diagnosis. The tumor displayed a clear cell pattern, and several neuroectodermal markers proved positive both in the original tumor and in xenografts. This neuroectodermal character was confirmed by electron microscopy. Moreover, cytogenetically the tumor has an unusual chromosomal rearrangement, t(2;22)(q13;q22,t(3;18)(p21;q23); representing a new EWS-FEV fusion type in which exon 7 of EWS gene is fused with exon 2 of FEV gene. This is the third published study of an ES-pPNET showing EWS-FEV fusion described, but it is the first study of a tumor with the aforementioned fusion points. These findings support the genetic and morphologic heterogeneity existing within the group of ES-pPNET tumors.     

An atypical form of mucolipidosis III.

We report two sibs showing a very mild form of mucolipidosis III with no clinical signs but isolated involvement of the hip and very mild abnormalities of the spine. This indicates that a storage disease, in particular mucolipidosis III, should be considered in any case of isolated bilateral hip dysplasia. The differences from other reported atypical variants of mucolipidosis III are discussed.     

Evaluation of the INNO-LiPA HBV genotyping assay for determination of hepatitis B virus genotype

Specific genotypes of hepatitis B virus (HBV) are increasingly recognized for their clinical significance and association with particular viral mutations. Although many HBV genotyping methods exist, there has been no standardized or commercially available method for direct molecular typing of the HBV genome. A newly available line probe assay (INNO-LiPA HBV Genotyping assay; Innogenetics N.V., Ghent, Belgium) that allows the identification of HBV genotypes A to G was assessed by comparison with pre-S1/pre-S2 sequence analysis of the isolates in 188 serum specimens. All seven genotypes were detected by the line probe assay (LiPA), and complete concordance between LiPA and sequence analysis was observed for 152 specimens (81%). LiPA was able to detect 19 mixed genotype infections not detected by amplicon sequencing, which for the most part were confirmed by cloning and sequencing of the pre-S1/pre-S2 amplicon. Four specimens had discrepant results between the two methods, and five specimens had indeterminate results by LiPA. The HBV DNA in four specimens was unable to be amplified by the nested INNO-LiPA HBV DR amplification primers; however, the HBV DNA in six specimens unable to be genotyped by sequencing was clearly genotyped by LiPA. The INNO-LiPA HBV Genotyping assay appears to be useful for the rapid genotyping of HBV, particularly for the sensitive detection of mixed genotype infections.     

Evaluation of an atypical HIV type 1 antibody. Serologic pattern leading to detection of HIV type 2 infection in North America

The variability and discordance of human immunodeficiency virus type 1 (HIV-1) antibody enzyme immunoassay determinations on serial specimens derived, to our knowledge, from the first documented case of HIV-2 infection in North America are described. The initial specimen was weakly reactive, but two subsequent serum specimens were both nonreactive by enzyme immunoassay. All specimens were indeterminate for HIV-1 antibody by HIV-1 Western blot analysis. Serum HIV-2 antibody was demonstrated by enzyme immunoassay using whole virus lysate, HIV-2-specific synthetic peptide assays, and HIV-2 Western blot analysis. Human immunodeficiency virus type 2 genomic sequences were demonstrated in peripheral blood mononuclear cells using gene amplification technology. Human immunodeficiency virus type 2, isolated from peripheral blood lymphocytes, had typical morphologic features of lenti-virus by electron microscopy. Western blot analysis and other specific assays should be considered in individuals with clinical evidence suggesting HIV infection who are nonreactive for HIV-1 antibody by enzyme immunoassay or who have atypical reactivity patterns.