Recent advances in diagnostic technology: applications in autoimmune and infectious diseases.

Biomarkers are used ubiquitously as indicators of biological health. The development of genomic and proteomic multiplex technologies have enormously amplified biomarker discovery and application to diagnostic and therapeutic decisions in clinical practice. New technologies are now available that simultaneously identify a wide spectrum of biomarkers and save time and costs. Multiplexed assays can be coupled to other disease specific indicators (i.e., cytokines, single nucleotide polymorphisms) in order to get more powerful information. However, there is an urgent need for validation/standardization of the new assays before they are adopted into clinical diagnostics. It is worthy to note a new assay, T cell interferon gamma release (TIGRAs), which has recently been introduced in the diagnosis of latent tuberculosis infection. It seems to perform better than tuberculin skin test in patients with inflammatory rheumatic diseases. In this review, we focus on advantages and limits of novel approaches to the detection of autoantibody profiles in autoimmune diseases or pathogen signatures in microbiology.

Peripheral blood neutrophils from hepatitis C virus-infected patients are replication sites of the virus.

BACKGROUND AND OBJECTIVE: Hepatitis C virus (HCV) is able to cause not only acute and chronic liver disease, but also immunologic and hematologic disorders. In order to clarify the extra-hepatic tropism of HCV, and to understand the pathogenetic mechanisms of HCV infection, we evaluated viral replication in peripheral blood mononuclear cells. DESIGN AND METHODS: The presence of genomic and antigenomic (replicative) forms of HCV in B- and T-lymphocytes, monocytes, and polymorphonuclear leukocytes (PML) was determined by reverse transcriptase-polymerase chain reaction in 54 HCV-RNA positive patients and, as control groups, in 10 patients who had recovered from HCV infection without evidence of serum HCV-RNA, and in 10 HCV-negative subjects. RESULTS: In HCV-RNA positive patients, the genomic RNA was found in 94% of B-cells, in 14% of T-cells, in 40% of monocytes and in 77% of PML, while only 1 of the HCV-RNA negative subjects showed positivity in B-cells. The anti-genomic form of HCV-RNA was found in 52% of B-cells, in 3% of monocytes, and in 31% of PML. By contrast, it was never detected in T-cells and in HCV-RNA negative subjects. Neither genomic nor anti-genomic forms were found in HCV-negative cases. INTERPRETATION AND CONCLUSIONS: These data suggest that PML are replication sites of HCV. Whether the infection occurs at the level of the stem cells or subsequently during myeloid cell differentiation is, as yet, unknown. The absence of correlation between the presence of replicative forms and any clinical and/or laboratory data opens the question of the role of HCV replication in extra-hepatic sites.

HCV genotypes and cryoglobulinemia.

OBJECTIVE: To evaluate the HCV genotype distribution in subjects affected by cryoglobulinemia in order to verify its possible role in the pathogenesis of the disease and to provide the clinician with a useful datum for therapy. METHODS: Nested PCR with universal and type-specific primers was used for the genotyping. RESULTS: Genotype I (1a) was never present in cryoglobulinemia, while it was present in 7 (4.3%) patients with chronic hepatopathy and in 4 (10.8%) asymptomatic patients. Type II (1b) was present in 28 (58.3%) and in 8 (47.1%) cryoglobulinemic patients with and without hepatopathy, respectively, in 106 (64.6%) patients with chronic hepatitis; in one patient with acute hepatitis; and in 14 (37.9%) asymptomatic patients. Type III (2a) was present in 2 (4.2%) and 2 (11.8%) cryoglobulinemic patients with and without hepatopathy, respectively; in 1 (0.6%) patient with chronic hepatopathy; and in 2 (5.4%) asymptomatic subjects. Type IV (2b) was present in 1 (2.1%) and in 2 (11.8%) cryoglobulinemic patients with and without hepatopathy, respectively; in 5 (3%) patients with chronic hepatopathy; and in 1 (2.7%) asymptomatic subject. Coinfections were present in 42 cases: 6 (12.5%) cryoglobulinemia with hepatopathy, 4 (23.5%) cryoglobulinemia without hepatopathy, 25 (15.3%) chronic hepatopathy, and in 7 (18.9%) asymptomatic subjects. For 41 (15.4%) strains typing was not possible. Eight of the "untypable" strains and 3 strains from patients with coinfection proved to belong to a new genotype. CONCLUSIONS: Genotype II (1b) was the most frequent in patients with and without cryoglobulinemia; genotype I (1a) was absent in all 65 patients with cryoglobulinemia, in whom, however, as in the subjects without cryoglobulinemia, all the other genotypes could be found. An interferon-resistant genotype characterized by an elevated homology with Simmonds' type 2c (rare genotype) was present.

Low-grade malignant lymphoma, hepatitis C virus infection, and mixed cryoglobulinemia.

Because a close relationship has been established between mixed cryoglobulinemia and hepatitis C virus (HCV) infection, the clinical, histologic, and virologic findings of 31 patients affected by mixed cryoglobulinemia have been determined. HCV infection was investigated by the presence of anti-HCV antibodies and by polymerase chain reaction (PCR) amplification of the 5' untranslated region (5'UTR), and the genotype of HCV was also determined according to Okamoto et al (J Gen Virol 73:673, 1992). A bone marrow (BM) biopsy was performed in all patients, and liver and kidney biopsies were performed when indicated. The prevalence of anti-HCV antibodies was high (83.9%); polymerase chain reaction amplification of the 5' untranslated region was positive in 26 subjects (83.9%), and Core region amplification in 26 of 27 subjects (96.2%). A high prevalence of genotype II was found (76.6%). Chronic liver disease was present in 15 (48%) patients. BM biopsy specimens showed the presence of low-grade non-Hodgkin's lymphomas in 12 cases (38.7%), whereas, in 11 patients (35.5%), the BM infiltration was not monoclonal (reactive). Mixed cryoglobulinemia is closely associated with HCV infection. Apparently, only 1 patient was not infected by the virus. Several HCV genotypes are involved in the pathogenesis of mixed cryoglobulinemia. The disease is associated with a high prevalence of low-grade non-Hodgkin's lymphomas.

Long-term effects of alpha-interferon therapy for type II mixed cryoglobulinemia.

BACKGROUND: Several reports showed that mixed cryoglobulinemia (MC) is closely associated with hepatitis C virus (HCV) infection. Since several authors reported the efficacy of alpha-interferon in the treatment of MC, we investigated the long-term effects of this drug on clinical, hematological and virological parameters in a group of 18 patients (13 women and 5 men, mean age 56 +/- 11 years) affected by MC. METHODS: A bone marrow biopsy was performed in all patients, and a liver biopsy was obtained in those with biochemical signs of chronic liver disease. The presence of HCV-RNA in serum was assessed by detection of anti-HCV antibodies and by PCR amplification of the 5' untranslated region of HCV. All patients followed the same treatment schedule: three million units of recombinant interferon alpha-2b s.c., three times a week for 1 year. RESULTS: In 5 cases bone marrow histology showed the presence of a monoclonal lymphocytic infiltrate. Liver biopsies were performed in 13 (72%) of the patients and a chronic liver disease was found in all 13. Anti-HCV antibodies were present in 17 (95%) subjects. HCV-RNA was detected in all cases (100%) before therapy. Five (28%) patients achieved a complete response and 9 (50%) a partial response, while the others (4 cases, 22%) showed minor responses. Four patients cleared the virus and obtained a complete remission of the MC. CONCLUSIONS: HCV may be a cause of MC. The disease is associated with a high incidence of monoclonal lymphocytic infiltrate of the bone marrow. Alpha-interferon seems to be an effective agent for the treatment of MC.

Laboratory diagnosis of rotavirus infection in diarrhoeal patients by immunoenzymatic and latex-agglutination assays.

One hundred stool samples from children with acute diarrhoea were examined by six commercial latex and immunoenzymatic assays for the diagnosis of rotavirus infection in four different laboratories. Samples were also analyzed by solid-phase immune electron microscopy using a rabbit anti-group A rotavirus antiserum. With electron microscopy as a basis for comparison, sensitivity and specificity for the latex and ELISA assays varied from 91.1 to 92.9% and from 94.2 to 99.4%, respectively. Statistically significant differences were revealed in the confirmation rate of electron microscopy-negative samples between different commercial assays. Significant variability was also found between results obtained by the laboratories taking part in the study.

Phenotypic profile and functional characteristics of human gamma and delta T cells during acute toxoplasmosis.

Gamma and delta (gamma delta) T-cell receptor lymphocytes are increased during acute toxoplasmosis. These cells are BB3+ CD45RO+ CD8-. Purified gamma delta T cells failed to proliferate in response to Toxoplasma gondii antigen (stimulation index, 1.4 +/- 0.6) but were responsive to phytohemagglutinin stimulation (stimulation index, 20.8 +/- 1.9). Natural-killer-like cytotoxicity was strongly acquired only after in vitro culture of purified gamma delta T cells with recombinant interleukin 2 (40% +/- 7% specific lysis). Our data show that gamma delta T-cell receptor T cells with a peculiar phenotype are increased during human acute T. gondii infection.

Borreliosis risk after tick bite in north-eastern Italy.

Lyme Borreliosis is an infectious disease caused by the Borrelia burgdorferi spirochete transmitted to man by a tick bite. The aim of this study was to assess the predictive value of tick bites for the diagnosis of Borreliosis in a sample of 266 subjects residing in an endemic area of north-eastern Italy. In the serological diagnosis of Borreliosis, positive and negative predictive values of tick bites were found to be 24% and 88% respectively.

Immunologic abnormalities related to antigenaemia during HIV-1 infection.

The expression of phenotypic markers on T and B lymphocytes in long-term human immunodeficiency virus type 1 (HIV-1) seropositive, antigen negative patients, in seropositive, antigen positive individuals without AIDS and in seronegative intravenous drug abusers was examined by two colour flow cytometry. Seropositive, antigen positive patients showed decreased CD4+ lymphocyte numbers, causing lower CD4/CD8 ratios when compared to seropositive, antigen negative subjects. While CD4 CDw29+ (4B4) lymphocytes are selectively reduced in seropositive, antigen negative individuals, both CD4 CDw29+ and CD4 CD45R+ (2H4) lymphocytes are decreased when antigenaemia is present. An increased percentage of CD3 HLA DR+ activated T lymphocytes and of CD20+ (B1) Leu 8 negative activated B cells was seen in HIV-1 seropositive antigen positive patients. These results demonstrate that, in long-term seropositive individuals, antigenaemia is associated with peculiar phenotypic changes of lymphocyte subsets.

[Detection of bacteremia by biphasic and radiometric methods]. / Rilevamento di batteriemia con metodo bifasico e radiometrico.

During one year period our laboratory carried out 859 hemocultures. These have been evaluated with two methods: a conventional biphasic method (Castaneda bottles), and the automated radiometric method (Bactec System). 185 cultures were obtained with one or both methods. Of these 3.5% were considered contaminated, therefore the clinically significant isolation rate was 16.2%. Of these 86.4% was recovered by biphasic system and 79.8% by Bactec System. The recovery time to positivity and spectrum of isolates were similar for the two methods. Although there were substantially more contaminants isolated in the Vacutainer-Bactec System.