E. Quirós

Detection of mycobacterial DNA in papulonecrotic tuberculid lesions by polymerase chain reaction.

Tuberculids are a heterogenous group of cutaneous lesions. Recent discoveries of M. Tuberculosis DNA in these lesions by PCR suggest that M. tuberculosis could play a role in their pathogenesis. The aim of this study was to demonstrate the presence of M. tuberculosis DNA by polymerase chain reaction in papulonecrotic tuberculid lesions. Skin biopsy specimens from ten patients with papulonecrotic tuberculid lesions (histopathologic features) were studied. All of them tested solidly positive in a tuberculin intradermal test. A gene‐amplification PCR, using primers capable of amplifying DNA in the M. tuberculosis complex, was performed to detect M. tuberculosis DNA in the lesions. A 285‐bp sequence specific of M. tuberculosis complex was amplified and confirmed by Southern‐blot hybridation with a 32 p 5′‐labelled internal probe. No inhibitors were detected in the negative PCR samples. The PCR technique makes the detection of mycobacterial DNA in tuberculids a possibility, and therefore provides a rational basis for antituberculous therapy and for the clinical management of these disorders. J. Clin. Lab. Anal. 14:133–135, 2000.

Human immunodeficiency virus type-1 can be detected in monocytes by polymerase chain reaction.

Lymphocytes and monocytes from 25 patients infected with human immunodeficiency virus type-1 (HIV-1)--13 asymptomatic, seven with the AIDS-related complex (ARC) and five with the acquired immunodeficiency syndrome (AIDS)--were lysed and subjected to PCR with three primer pairs: SK38/SK39 (gag), SK68/SK69 (env) and SK29/SK30 (LTR). Amplified DNA was solution-hybridised with 32P-labelled probes (SK19, SK70 and SK31, respectively) and detected by PAGE-autoradiography. HIV-1 DNA was detected as follows. Asymptomatic patients: monocytes--gag 61.5%, env 100%, LTR 0%; lymphocytes--gag 100%, env 92.3%, LTR 53.84%. ARC patients: monocytes--gag 71.4%, env 57.1%, LTR 0%; lymphocytes--gag 100%, env 71.4%, LTR 71.4%. AIDS patients: monocytes--gag 80.0%, env 100%, LTR 0%; lymphocytes--gag 100%, env 60%, LTR 60%. The presence of HIV-1 DNA was confirmed in the monocyte fraction. In this cell subset, the env gene-directed primers were the most effective for amplification, whereas the LTR gene-directed primers failed to amplify HIV-1 DNA. The different pattern of amplification found in monocytes may suggest that these cells could be infected by a genetic variant of the virus.

Immune complex p24 antigen: A new prognostic marker in human immunodeficiency virus infection

p24 antigen (p24 Ag) is usually not found in patients in the asymptomatic phase of HIV infection, mainly because it is bound to anti-p24 antibodies (p24 Ab), with which it forms immune complexes. Nine asymptomatic patients positive for HIV-1 antibodies (stage II CDC criteria) treated with rα-interferon were followed (7.5+/-0.5 months), and a basal and final sample were tested for p24 Ag immune complexes (p24 Ag-IC), free p24 Ag, p24 Ab, CD4 count, β2-microglobulin, properdin B factor, and C3 and C4 complement fractions. p24 Ag-IC was detected in 55.5% of the samples, whereas free p24 Ag was detected in 5.5%. The finding of no change or an increase in p24 Ag-IC serum levels may be considered a prognostic marker. We believe that p24 Ab cannot be used as a prognostic marker unless p24 Ag-IC detection is simultaneously evaluated. In der asymptomatischen Phase der HIV-Infektion läßt sich das p24 Antigen (p24 Ag) meist nicht nachweisen; dies ist vor allem darauf zurückzuführen, daß es an anti-p24 Antikörper (p24 Ab) gebunden ist und mit ihnen Immunkomplexe bildet. Bei neun anti-HIV-1 positiven, asymptomatischen Patienten (Stadium II nach CDC Kriterien), die mit r-alpha-Interferon behandelt wurden, wurden Verlaufsbeobachtungen (7,5 +/- 0,5 Monate) durchgeführt. Basis- und Abschlußuntersuchungen auf p24 Ag Immunkomplexe (p24 Ag-IC), freies p24 Ag, p24 Ab, CD4 Zellzahlen, β2-Mikroglobulin, Properdin B Faktor und Komplementfraktionen C3 und C4 wurden dabei vorgenommen. In 55,5% der Proben fand sich p24 Ag-IC, in 5,5% freies p24 Ag. Die Beobachtung gleichbleibender oder ansteigender Werte von p24 Ag-IC im Serum hat möglicherweise prognostische Bedeutung. Unserer Ansicht nach kann der p24 Ab nicht als prognostischer Marker eingesetzt werden, wenn nicht zugleich die Bestimmung von p24 Ag-IC erfolgt.Summaryp24 antigen (p24 Ag) is usually not found in patients in the asymptomatic phase of HIV infection, mainly because it is bound to anti-p24 antibodies (p24 Ab), with which it forms immune complexes. Nine asymptomatic patients positive for HIV-1 antibodies (stage II CDC criteria) treated with rα-interferon were followed (7.5+/-0.5 months), and a basal and final sample were tested for p24 Ag immune complexes (p24 Ag-IC), free p24 Ag, p24 Ab, CD4 count, β2-microglobulin, properdin B factor, and C3 and C4 complement fractions. p24 Ag-IC was detected in 55.5% of the samples, whereas free p24 Ag was detected in 5.5%. The finding of no change or an increase in p24 Ag-IC serum levels may be considered a prognostic marker. We believe that p24 Ab cannot be used as a prognostic marker unless p24 Ag-IC detection is simultaneously evaluated.ZusammenfassungIn der asymptomatischen Phase der HIV-Infektion läßt sich das p24 Antigen (p24 Ag) meist nicht nachweisen; dies ist vor allem darauf zurückzuführen, daß es an anti-p24 Antikörper (p24 Ab) gebunden ist und mit ihnen Immunkomplexe bildet. Bei neun anti-HIV-1 positiven, asymptomatischen Patienten (Stadium II nach CDC Kriterien), die mit r-alpha-Interferon behandelt wurden, wurden Verlaufsbeobachtungen (7,5 +/- 0,5 Monate) durchgeführt. Basis- und Abschlußuntersuchungen auf p24 Ag Immunkomplexe (p24 Ag-IC), freies p24 Ag, p24 Ab, CD4 Zellzahlen, β2-Mikroglobulin, Properdin B Faktor und Komplementfraktionen C3 und C4 wurden dabei vorgenommen. In 55,5% der Proben fand sich p24 Ag-IC, in 5,5% freies p24 Ag. Die Beobachtung gleichbleibender oder ansteigender Werte von p24 Ag-IC im Serum hat möglicherweise prognostische Bedeutung. Unserer Ansicht nach kann der p24 Ab nicht als prognostischer Marker eingesetzt werden, wenn nicht zugleich die Bestimmung von p24 Ag-IC erfolgt.

Gbv-C RNA presence in several high-risk groups of Spain

GB virus C subtype (GBV-C) seems to share the same routes of transmission as other parenteral transmitted viruses. We have evaluated the prevalence of GBV-C in 247 patients with potential risk for GBV-C infection and in 91 healthy blood donors. The presence of GBV-C RNA was examined by polymerase chain reaction in serum samples. The 23.6% of parenteral drug users were GBV-C positive, 36.3% of them were also HIV infected. Moreover, 22.5 and 19% of sera from patients with HBV and HCV chronic hepatitis, respectively, but without apparent risk factors seemed GBV-C infected. Finally, the 6% of patients on hemodialysis were also positive. Therefore, these results suggest that GBV-C is transmitted by parenteral routes but other non-parenteral routes shared with HBV or HCV must be considered.

Helicobacter pylori seroepidemiology in risk groups

Helicobacter pylori is associated with peptic ulcer and chronic active gastritis. The response to infection can be determined by measuring serum titers of anti-H.pylori antibodies. We compared antibody liters in 612 serum samples from 570 individuals considered at risk forH. pylori infection, 170 of them are control sera from 110 adults and 60 children with no gastric alterations. The study groups were 93 institutionalized mentally handicapped children, 40 heterosexual couples, 101 HIV-sero-positive patients, 86 patients with chronic renal failure and 40 individuals (20 adults and 20 children) with symptoms associated with gastritis or gastro duodenal ulcer disease. In the adult and child control groups, 33.5% and 11.6% of the individuals had circulating anti-H.pylori antibodies. Significantly more adults (80%) and children (75%) with gastric symptoms had detectable circulating antibody titers. Elevated titers were also found in institutionalized children and in adults with renal failure.

Diagnosis of HIV-1 infection by PCR with two primer pairs

We have used two different primer pairs to assess HIV-1 infection of peripheral blood mononuclear cells (PBMC) by polymerase chain reaction (PCR). The study was carried out on 150 individuals: 50 seronegative individuals without risk behaviours for HIV-1 infection, 50 individuals with risk-behaviours but seronegative for HIV-1 and 50 patients with risk-behaviours who were HIV-1 seropositive. Discordances were found between the two primer pairs (SK38/39; SK68/69) in 3 cases. In the non-risk seronegative group, one specimen was scored positive with only one primer pair (SK38/SK39); all the samples belonging to seropositive individuals were found to be positive for HIV-1 DNA using both primer pairs; in the seronegative at risk group 2 samples were positive with only one primer pair (SK38/SK39), and 4 samples were found positive by both primer pairs (SK38/39 & SK68/69). Our study demonstrates that discrepant results take place with relatively high frequency; we propose that all specimens should be tested twice using at least two different primer pairs.

GB virus C in patients with liver disease of unknown etiology.

To assess the prevalence of GBV‐C in patients suffering unknown liver disease we have investigated the GBV‐C‐RNA in serum of 54 patients: 10 with acute and 32 with chronic non‐A‐E hepatitis (16 active and 16 persistent), 10 with hepatocellular carcinoma, 2 diagnosed with hepatic fulminant failure, and 91 healthy blood donors (control). PCR with primers from NS3 helicase region was performed and the product was identified by a double strand DNA enzyme immunoassay. GBV appears to infect 40 and 31% of acute or chronic non‐A‐E hepatitis respectively. Also the GBV genome was found in 1 in 10 samples of hepatocarcinoma, in 2 cases of fulminant hepatitis, and in 1 in 91 of the control group. In spite of these results the role of GBV in the etiology of liver diseases has to be analyzed in more comprehensive studies. J. Clin. Lab. Anal. 14:70–72, 2000.

Serum hepatitis B virus DNA detection with S- and C-region-directed probes

Developments in molecular biology have offered a wide range of nucleic acid probes to detect the genome of hepatitis B virus (HBV). We have tested the ability of two enzyme-linked (alkaline phosphatase) probes to detect HBV-DNA. These hybridise with the S and C regions of the genome of HBV and are used to determine the clinical significance of detecting the two regions. A total of 66 serum samples from patients at different stages of HBV infection was examined. HBV-DNA was detected with at least one of the probes in 17 (85%) patients with HBeAg-positive chronic hepatitis, five (26.3%) with anti-HBe-positive chronic hepatitis and six (66.6%) with acute hepatitis. Although both probes were able to detect as little as 10 pg/ml (2.86 x 10(6) g.E./ml) of a full length HBV-DNA standard, the C-region-directed probe did not react in one patient with acute hepatitis, two with HBeAg-positive and three with anti-HBe-positive chronic hepatitis. When C-region-directed probes are used for diagnostic purposes, results should always be accompanied by hybridisation with probes directed against other regions showing less variability (e.g. S region).

HGV RNA sequences in liver and PBMC.

HGV has been identified in patients with chronic hepatitis of unknown aetiology and the possibility that HGV may be the cause has been raised. We have analysed liver biopsies and PBMC (peripheral blood mononuclear cells) from 80 patients with chronic hepatitis and HGV-RNA in serum by PCR. In ten patients HGV-RNA was detected in liver, in five patients it was detected in PBMC and seven were positive in both specimens by PCR. Whether this agent resides and replicates in hepatocytes remains controversial and needs more studies.HGV has been identified in patients with chronic hepatitis of unknown aetiology and the possibility that HGV may be the cause has been raised. We have analysed liver biopsies and PBMC (peripheral blood mononuclear cells) from 80 patients with chronic hepatitis and HGV-RNA in serum by PCR. In ten patients HGV-RNA was detected in liver, in five patients it was detected in PBMC and seven were positive in both specimens by PCR. Whether this agent resides and replicates in hepatocytes remains controversial and needs more studies.