Predicting progression of HIV disease: usefulness of acid-dissociated p24 antigen.

To ascertain whether immune complex dissociation (ICD) improves the value of p24 antigen as a prognostic marker for progression of HIV infection, 53 patients were followed over a 3-year period, including at least one visit per year. All had CD4+ counts at entry > 400/mm3; progressors (n = 18) were defined as having CD4+ counts < 200/mm3 and nonprogressors (n = 35) as having CD4+ counts still > 400/mm3 at the end of follow-up. Serum specimens were collected at each annual visit and assayed for p24 antigen with and without ICD treatment. At entry, the percentage of progressors positive for ICD p24 antigen was significantly higher than the percentage of positive nonprogressors (39% versus 3%, p < 0.01). The sensitivity of p24 antigen over all visits in terms of predicting the progression increased from 61% before ICD to 83% after. The specificity of p24 antigen in terms of predicting progression decreased from 97% before ICD to 89% after. The relative risk of progression in individuals positive for p24 antigen was 6.7 before ICD and increased after ICD to 12.7. When evaluating the respective prognostic value of the p24 antigen and of the ICD p24 antigen, only ICD p24 was significant (RR 10.2, 95% CI 2.2-46.9). ICD p24 antigen appears to be a marker of progression that may be detected earlier than p24 antigen without ICD.

Evaluation of newly developed microparticle enzyme immunoassays for the detection of HCV antibodies.

The newly developed anti-HCV assays AxSYM HCV version 3.0 and IMx HCV version 3.0 were evaluated with regard to their precision, sensitivity and specificity in comparison to the HCV EIA 3.0 (Abbott GmbH, Wiesbaden, Germany). Precision testing was undertaken using five positive controls with different anti-HCV levels for each assay. Specificity was estimated by testing 4383 blood donor specimens. The supplemental assay Matrix HCV 2.0 (Abbott GmbH, Wiesbaden, Germany) was used to confirm repeatedly reactive results. Samples which had been found to be positive or indeterminate by Matrix HCV 2.0 were tested by qualitative polymerase chain reaction after reverse transcription (RT-PCR, Amplicor HCV test, Roche Diagnostic Systems, Basel, Switzerland). To determine sensitivity, 20 commercially available seroconversion panels were tested. Based on supplemental testing, the apparent specificities of AxSYM HCV version 3.0, IMx HCV version 3.0 and HCV EIA 3.0 were estimated to be 99.84, 99.98 and 99.80%, respectively. In seroconversion panel testing, AxSYM and IMx HCV version 3.0 detected seroconversion in up to 12/20 panels earlier and in up to 1/20 cases later than the comparison EIA. The highest sensitivity was shown in AxSYM HCV version 3.0, followed by IMx HCV version 3.0 and HCV EIA 3.0. Based on the improved seroconversion sensitivity and specificity, the AxSYM and IMx HCV version 3.0 assays appear to be suitable for detecting HCV antibodies in blood donor testing and other routine laboratory assessments.

Restriction of T cell receptor V beta repertoire in melanoma metastasis responding to immunotherapy.

Restriction of the T cell receptor repertoire suggesting ongoing specific immune mechanisms has recently been described in melanoma tissue by several groups of investigators. The functional relevance for immunotherapy of melanoma, however, has not been established. We studied the T cell receptor repertoire in two melanoma metastases of a patient with a mixed response to immunotherapy. Expression of T cell receptor V beta regions was determined by subgroup-specific semiquantitative RNA polymerase chain reaction (PCR). In the regressing skin lesion a restricted expression of the T cell receptor repertoire and overexpression of three V beta subgroup genes was found; no restriction was present in the simultaneously progressing skin lesion of the same patient, compared with peripheral blood lymphocytes. Comparison of T cell receptor V beta gene expression in two metastatic lesions of a patient with simultaneously growing skin metastases, who did not receive immunotherapy, revealed only minor differences. These observations show for the first time an association between restricted T cell receptor repertoire and responsiveness of melanoma to immunotherapy and suggest a role of T cells using the overexpressed V beta genes for the cytokine-induced tumour regression.

Presenting XML-based medical discharge letters according to CDA.

UNLABELLED: The HL7 Clinical Document Architecture (CDA) is an important XML-based standard for the representation of clinical documents. OBJECTIVES: The use of Markup Languages could satisfy the demands of involved healthcare staff as well as the needs of patients, to receive an overview of the patient's treatment during the hospital stay. The standardization efforts of different groups dealing with this problem have demonstrated progress, but have not, as yet, achieved a routinely usable result. In particular, differentiating information according to a hierarchical order has not been published to date. METHODS: A retrospective analysis of 60 discharge letters from a cardiology ward (ward A) as well as 60 discharge letters from a gastroenterology ward (ward B) were extracted from the central hospital information system, by taking every fifth discharge letter issued over a one year period. RESULTS: An XML-based prototype for medical discharge letters has been put in place representing the required information units and information elements. By means of an XSL-stylesheet, a detailed representation of the conventional discharge letter has been produced that is platform independent and permits the recurrent use of information units. CONCLUSIONS: Through the introduction of definitions like information elements and information units, progress in the development of CDA level two and three might be realized. We present a method by which discharge letters can be used by an Internal Medicine Department. This concept is implemented in a XML-based prototype allowing a special view on XML data to generate this document type.

Evaluation of diagnostic kits and reagents-a viewpoint from the industry.

BACKGROUND AND OBJECTIVES: Assuring the performance characteristics of in vitro diagnostics (IVDs) is a major objective of product evaluations. This includes using sets of well planned and controlled trials in order to: (i) analyze product performance characteristics; (ii) validate design specifications; and (iii) assess product safety. Performance parameters like sensitivity, specificity, precision, robustness etc. are assessed in order to assure that the product design consistently meets performance specifications upon manufacturing. Documentation on product performance can also be used for pre-market approval submissions which are required in some European countries. THE CONTEXT FOR IVD PRODUCT EVALUATIONS: Internal quality policies have the following impact on the design of evaluation trials: (i) use of at least three production lots (with final specifications); (ii) performed under field conditions and GLP; (iii) performed at different sites; (iv) analyzing clinical specimens having statistically significant numbers; (v) use of validated statistical methods/software (SAS); (6) quality-assuring documentation. External expectations influencing product evaluations include the following aspects: (i) state-of-the-art performance; (ii) technical standards like ISO, EN, DIN; (iii) Customers' needs; and (iv) regulatory requirements for the approval of IVDs. IVD PRODUCT EVALUATIONS AT ABBOTT DIAGNOSTICS: Based on a quality system (ISO 9001), product evaluations are a major constituent in a phased development and manufacturing process of IVDs prior to market entry. Product evaluations address particular performance goals which relate to aspects outlined above including regulatory requirements. Depending on the assay type, they are displayed in absolute and relative features, e.g. (i) intra- and interassay variation below 10% for precision; (ii) no false-negatives among certified positive specimens for sensitivity; (iii) a 'true' specificity > 99.75% (lower limit of 95% CI); and (4) earlier detection of seroconversion by the 'new' assay vs. the 'old' assay etc. The basic design and the assessment of performance characteristics is described by giving particular examples.

Analysis of the HSV-1 strain 17 DNA polymerase gene reveals the expression of four different classes of pol transcripts.

We have investigated the structure and the expression of transcripts of the HSV-1 strain 17 DNA polymerase gene (pol) by various mapping methods including cDNA cloning. The majority of mature pol transcripts is strictly colinear with the pol gene. But additionally, pol cDNAs show a defined heterogeneity in respect to their 5'-terminal regions and can be divided into four classes with characteristic differences; (i) class 1 represents the major transcript (pol-R1) with initiation at HSV-1 positions 62,605-62,610, (ii) class 2 initiates about 70 bp downstream, (iii) class 3 is generated by splicing the short open reading frame (SORF) to a 5'-truncated part of the long open reading frame (LORF) which results in a partially different coding potential, and (iv) class 4 starts 120 bp upstream of the major initiation site in the central part of the origin of replication (oriL). S1 and Exo VII nuclease and RNase protection assays as well as primer extension analyses confirm the classification regarding the genuine structure of pol mRNAs and the differential usage of transcriptional start sites. Furthermore, the transcript classes can be distinguished from each other by their kinetics of appearance/disappearance in the cytoplasm: The first transcription of the pol gene is indicated by the predominant presence of class 2 and class 4 mRNAs at 2 hr postinfection (h.p.i.), followed by an increase of class 1 transcripts up to 4 h.p.i. and a parallel decrease of class 2 mRNAs. These data suggest that expression of the pol gene is finely regulated already at the transcriptional and/or posttranscriptional level prior to the translation of pol mRNAs.

Cell cycle-dependent expression of nuclear matrix proteins of Ehrlich ascites cells studied by in vitro translation.

A combination of methods was used to study the cell cycle-dependent expression of nuclear matrix proteins of Ehrlich ascites cells: Separation of asynchronous cells growing in vivo into fractions of G1-, S- and G2- phase cells by centrifugal elutriation with less than 10% cross-contamination. Isolation of poly(A+) RNA populations from total cytoplasmic RNA by affinity chromatography on messenger affinity paper (mAP). In vitro translation of poly(A+) RNA from asynchronous and phase synchronous cells. Immunoprecipitation of in vitro synthesized nuclear matrix proteins by a monoclonal antibody with anti-lamin specificity (PKB8) and by a polyspecific anti-nuclear matrix serum (AMS5) followed by analysis of immunoprecipitated materials on SDS-polyacrylamide gels. The results indicate that mRNAs for nuclear matrix-associated proteins including the lamins B and C are either exclusively or at least predominantly present in the cytoplasm of cells in S phase suggesting a high rate of in vivo synthesis of these proteins during S phase. This is consistent with an anticipated biological function of the nuclear matrix which is considered to organize parental and newly synthesized DNA in higher order structures.

Mitral valve endocarditis: an uncommon cause of myocardial infarction.

A 39 year old woman presented with acute anterior myocardial infarction. At coronary angiography the distal left anterior descending coronary artery (LAD)was occluded despite otherwise normal coronary arteries. The LAD was successfully recanalized using PTCA. Subsequently, a transesophageal echocardiogram revealed vegetations and a significant incompetence of the mitral valve. Blood cultures identified out enterococcus faecalis. Despite intra-venous antibiotic treatment guided by sensitivity testing, the patient ultimately required elective mitral valve replacement. During a prior outpatient diagnostic work-up of fever/malaise, the diagnosis of infective endocarditis was not made.This case conveys two main messages: 1) because the history and physical sings of bacterial endocarditis can be subtle or non-specific, the first step to diagnose infective endocarditis is to include it in the differential diagnosis. 2) Percutaneous coronary intervention is an effective treatment of septic embolic occlusion of a major coronary artery.

Serum anti-p24 antibody concentration has a predictive value on the decrease of CD4 lymphocyte count higher than acid-dissociated p24 antigen.

We studied the prognostic value on the decrease of CD4 lymphocyte count of anti-p24 antibody (ab) titer and compared this value with that of polyclonal and monoclonal p24 (ag) titer before and after immune complex dissociation (ICD); 53 human immunodeficiency virus (HIV)-infected patients having CD4+ counts above 400/mm3 when first examined were followed up over a 3-year period including at least four visits; HIV disease progressors (n = 18) were defined as having CD4+ counts below 200/mm3 and non-progressors (n = 35) as having CD4+ counts still above 400/mm3 at the end of follow-up. Sera were collected at each visit and assayed for p24 ag with and without ICD and for anti-p24 ab titer. The mean anti-p24 ab titer of progressors and of non-progressors at entry was significantly different. A threshold of anti-p24 ab titer indicating a HIV progression was determined at 1/300. The proportion of individuals with an anti-p24 ab titer lower than 1/300 at least once during the study period was 34% in non-progressors and 94% in progressors. The difference between progressors and non-progressors at entry was significant with monoclonal p24 ag without ICD and more significant with monoclonal p24 ag after ICD. The marker having the highest predictive value was the anti-p24 ab titer, then monoclonal p24 ag with ICD, then polyclonal p24 ag with ICD. Anti-p24 ab is an earlier and stronger marker of the decrease of CD4 lymphocyte count than p24 ag even after ICD.

Sense and antisense transcripts of human papillomavirus type 16 in cervical cancers.

Human papillomavirus type 16 (HPV-16) transcription was analysed in one squamous cervical carcinoma by cDNA cloning and DNA sequencing, and in eight additional squamous cervical carcinomas and 11 precancerous lesions by RNA-RNA in situ hybridization. The nucleotide sequences of the cDNA clones revealed structures of early HPV-16 mRNAs (E6*-E7-E1 E4-E5) in agreement with data reported for other premalignant and malignant tumours. cDNA clones possibly representing viral RNA of antisense orientation were also detected. These RNAs included sequences of the upstream regulatory region, part of the early and the late region of the genome. In three of eight squamous cervical carcinomas examined by in situ hybridization, signals specific for viral antisense RNA were also found. The antisense RNAs had a predominantly nuclear localization. Viral antisense RNA could not be detected in any of 11 HPV-16-positive premalignant lesions. The expression of HPV antisense RNA is likely to be linked to viral integration into the host genome. The possible effects of viral antisense transcription with regard to tumour progression remain to be determined.

Even individuals considered as long-term nonprogressors show biological signs of progression after 10 years of human immunodeficiency virus infection.

Despite a decade of human immunodeficiency virus (HIV) seropositivity, a few individuals termed as long-term nonprogressors (LTNPs) maintain a stable CD4+ T-cell count for a period of time. The aim of this study was to establish, through the sequential determination of all known predictors of HIV disease, the proportion of such patients having stringent criteria of true long-term nonprogression. Among 249 individuals who were HIV-infected and prospectively followed up over a 10-year period (1985 to 1995), 12 having a CD4+ T-cell count greater than 500/microL (LTNP I group) and 9 having a CD4+ T-cell count less than 500 but stable over time (LTNP II group) after at least 10 years of infection without intervention of antiviral therapy, were studied over the entire follow-up period. The plasma HIV RNA copy number and the serum concentrations of p24 antigen, each anti-HIV antibody, neopterin, beta-2-microglobulin, Immunoglobulin (Ig) G and IgA were determined every 18 months over the study period. Cellular and plasma viremias were cross-sectionaly assayed in all 21 patients. Only two patients had strictly no marker of progression over the follow-up period. They were the only ones who had, over the 10-year period, a viral copy number too low to be detected. The other patients had a viral copy number higher than 400/mL at at least one visit and increasing over the follow-up period, and they evidenced one or more markers of virological or immunological deterioration. Cellular viremia was positive in all patients but two, while plasma viremia was negative in all but one. The population of individuals termed as LTNPs is not virologically and immunologically homogeneous. The majority present biological signs of HIV disease progression. A new pattern of true LTNP can be drawn through stringent criteria based on the whole known predictors. This pattern appears to be rare in HIV-positive population.