Interferon-gamma administration after abdominal surgery rescues antigen-specific helper T cell immune reactivity.

Antigen-induced activation of T cells is determined by many factors. Among these factors are (i) the number of T-cell receptors (TCRs) triggered by TCR ligands on antigen-presenting cells (APCs), and (ii) the intrinsic cellular threshold for activation. T-cell receptor triggering is optimized by adhesion molecules that form the interaction site between T cells and APCs, i.e. the immunological synapse. In addition, signals through co-stimulatory molecules lower the intrinsic T-cell activation threshold. Immunosuppressive agents and traumatic events such as major operative procedures change physiological T-cell responses. Depressed immune functions after surgery are presumed to render patients more susceptible to pathogens. Interferon-gamma (IFNgamma) is a type II homodimeric cytokine with multiple immunostimulatory properties. Several studies have been performed to assess the effects of IFNgamma treatment in patients in need of increased immune reactivity. However, until now, the effect of IFNgamma on human antigen specific CD4(pos) T-cell reactivity after surgically-induced immunosuppression has not been reported. Therefore, a comparative trial of recombinant human (rh) IFNgamma versus placebo in patients after abdominal surgery was initiated. Antigen-specific helper T cell immune reactivity was assessed by antigen-induced cytokine production, intracellular cytokine staining and flow cytometry. A single dose of rhIFNgamma rescued down-modulation of antigen-specific CD4(pos) T-cell reactivity, concomitant with an up-regulation of TCR-ligands on antigen-presenting cells. Selected patients may benefit from the immunostimulatory properties of rhIFNgamma administration in vivo.

Molecular diagnosis of visceral herpes zoster.

Patients with disseminated herpes zoster may present with severe abdominal pain that results from visceral involvement of varicella-zoster-virus infection. In the absence of cutaneous eruptions of herpes zoster, visceral herpes zoster is extremely difficult to diagnose. This diagnostic difficulty has the potential to cause devastating delays in treatment. We report a case series of four patients with visceral herpes zoster in whom large concentrations of DNA from varicella zoster virus were detectable in blood by PCR before signs of infection appeared on the skin, thus enabling early diagnosis and treatment.

Elevation of systemic markers related to cardiovascular diseases in the peripheral blood of periodontitis patients.

BACKGROUND: Periodontitis is a common, often undiagnosed, chronic infection of the supporting tissues of the teeth, epidemiologically associated with cardiovascular diseases. Since C-reactive protein (CRP) and other systemic markers of inflammation have been identified as risk factors for cardiovascular diseases, we investigated whether these factors were elevated in periodontitis. METHODS: Consecutive adult patients with periodontitis (localized n = 53; generalized n = 54), and healthy controls (n = 43), all without any other medical disorder, were recruited and peripheral blood samples were taken. RESULTS: Patients with generalized periodontitis and localized periodontitis had higher median CRP levels than controls (1.45 and 1.30 versus 0.90 mg/L, respectively, P = 0.030); 52% of generalized periodontitis patients and 36% of the localized periodontitis patients were sero-positive for interleukin-6 (IL-6), compared to 26% of controls (P= 0.008). Plasma IL-6 levels were higher in periodontitis patients than in controls (P = 0.015). Leukocytes were also elevated in generalized periodontitis (7.0 x 10(9)/L) compared to localized periodontitis and controls (6.0 and 5.8 x 10(9)/L, respectively, P= 0.002); this finding was primarily explained by higher numbers of neutrophils in periodontitis (P= 0.001). IL-6 and CRP correlated with each other, and both CRP and IL-6 levels correlated with neutrophils. The current findings for periodontitis were controlled for other known factors associated with cardiovascular diseases, including age, education, body mass index, smoking, hypertension, cholesterol, and sero-positivity for CMV, Chlamydia pneumoniae, and Helicobacter pylori. CONCLUSIONS: Periodontitis results in higher systemic levels of CRP, IL-6, and neutrophils. These elevated inflammatory factors may increase inflammatory activity in atherosclerotic lesions, potentially increasing the risk for cardiac or cerebrovascular events.

Detection and quantitation of hepatitis C virus RNA in feces of chronically infected individuals.

Hepatitis C virus (HCV) RNA was detected and quantified in human fecal specimens with the Roche COBAS AMPLICOR system adapted by us for fecal specimens. HCV RNA could be detected in the feces of four of six (67%) patients chronically infected with HCV, with loads up to about 2.8 x 10(5) copies/ml of feces. The same HCV genotypes were observed in feces and plasma as determined by direct sequencing of the 5' untranslated region.

A survey of liver pathology in needle biopsies from HBsAg and anti-HBe positive individuals.

AIMS: To use laboratory data and liver biopsies, prospectively obtained from hepatitis B surface antigen (HBsAg) and anti hepatitis B e antigen (anti-HBe) positive patients, for the assessment of: (1) the relation between biopsy length/number of portal tracts and sampling error; (2) the relation between the severity of piecemeal necrosis and the new grading terminology (minimal, mild, moderate, and severe chronic hepatitis); and (3) liver pathology, which has not been studied in patients with this specific serological profile. METHODS: The study group (n = 174) included 104 patients with normal aminotransferase concentrations and no cases with clinically apparent cirrhosis. The specimen length and number of portal tracts were measured at light microscopy examination. Sampling error analysis was related to the discrepancies between aminotransferase concentrations versus histological grade. Detailed histological scorings were undertaken by the reference pathologist and compared with laboratory and hepatitis B virus (HBV) DNA precore sequence data. RESULTS: Sampling error seemed to be a constant feature, even for biopsies > or = 20 mm, but increased dramatically in biopsies < 5 mm long and/or containing less than four portal tracts. Between 25% and 30% of biopsies, graded as "mild" or "moderate" activity showed features of moderate and severe piecemeal necrosis, respectively. Ten per cent of the patients with normal aminotransferase values had stage III-IV hepatic fibrosis, and 20% had piecemeal necrosis. Only cytoplasmic, not nuclear, core antigen expression was a strong predictor of high hepatitis B viraemia. There was no association between precore stop codon mutations, grade/stage of liver disease, and hepatitis B core antigen (HBcAg) expression. CONCLUSIONS: The specimen available for light microscopical examination should be > 5 mm long and should contain more than four portal tracts. In addition, the new grading terminology might give the clinician an inappropriately mild impression of the severity of piecemeal necrosis. Furthermore, even in the presence of normal aminotransferase concentrations, considerable liver pathology can be found in 10-20% of HBsAg and anti-HBe positive individuals; such pathology is not associated with the occurrence of precore stop codon mutations.

Quantitation of varicella-zoster virus DNA in whole blood, plasma, and serum by PCR and electrochemiluminescence.

We describe a highly sensitive assay for quantitation of varicella-zoster virus (VZV) DNA in blood, involving PCR amplification, solution hybridization with Tris-(2, 2'-bipyridine)-ruthenium(II) chelate-labeled probes, and measurement by electrochemiluminescence (ECL). Extraction and amplification efficiencies were monitored by the inclusion of internal control (IC) DNA, mimicking the VZV target, in the DNA extraction. Viral DNA load was calculated from the ratio of VZV and IC ECL signals. The lower limit of sensitivity was 20 VZV DNA copies/ml of plasma or serum and 80 copies/ml of whole blood. In reconstruction experiments, expected and calculated VZV DNA loads were in excellent accordance. Blood specimens from 42 VZV-infected patients were tested for the presence of VZV DNA and showed detection rates of 86% in patients with varicella and 81% in patients with herpes zoster. In specimens obtained during the first week after onset of the rash, detection rates were 100 and 89%, respectively. Viral DNA was detected in all immunocompromised patients with herpes zoster, emphasizing the risk of disseminated disease in this patient group. VZV DNA load was similar in patients with varicella and multidermatomal herpes zoster and lower in patients with unidermatomal zoster. Despite the cell-associated nature of the virus, VZV DNA was detected in serum and plasma at high copy numbers, and at similar frequencies compared to whole-blood specimens. Quantitation of VZV DNA in blood is of potential importance for diagnosis and clinical management of VZV-infected patients. Plasma and serum provide convenient matrices for this purpose.

Development of virus-specific CD4(+) T cells during primary cytomegalovirus infection.

Although virus-specific CD4(+) T cells have been characterized extensively in latently infected individuals, it is unclear how these protective T-cell responses develop during primary virus infection in humans. Here, we analyzed the kinetics and characteristics of cytomegalovirus-specific (CMV-specific) CD4(+) T cells in the course of primary CMV infection in kidney transplant recipients. Our data reveal that, as the first sign of specific immunity, circulating CMV-specific CD4(+) T cells become detectable with a median of 7 days after first appearance of CMV-DNA in peripheral blood. These cells produce the T helper 1 type (Th1) cytokines IFNgamma and TNFalpha, but not the T helper 2 type (Th2) cytokine IL4. In primary CMV infection, the vast majority of these circulating virus-specific T cells have features of recently activated naive T cells in that they coexpress CD45RA and CD45R0 and appear to be in the cell cycle. In contrast, in people who have recovered from CMV infection earlier in life, virus-specific T cells do not cycle and express surface markers characteristic of memory T cells. After the initial rise, circulating virus-specific CD4(+) T cells decline rapidly. During this phase, a strong rise in IgM and IgG anti-CMV antibody titers occurs, concomitant with the reduction of CMV-DNA in the circulation.

Measles in a Dutch hospital introduced by an immuno-compromised infant from Indonesia infected with a new virus genotype.

A fatal measles case in an immunocompromised Indonesian child was associated with nosocomial transmission to health care workers. The virus isolated proved to represent a new genotype within clade G.

Detection and quantitation of human cytomegalovirus DNA in faeces.

The development and performance of a robust and sensitive PCR assay are described for the detection and quantitation of human cytomegalovirus DNA in human faecal specimens. In this assay, CMV DNA was purified by an optimised DNA extraction protocol together with internal control DNA that monitored both DNA extraction efficiency and PCR efficiency. The lower detection limit of the assay was reached at about 100 CMV particles per ml of (25-50%) faecal suspension. CMV DNA could be quantitated in the range of about 300-100000 molecules per ml of faecal suspension. CMV DNA loads obtained in clinical faeces specimens suggest that the assay can be used to monitor the efficacy of antiviral treatment. Reconstruction experiments that monitored the efficiency of DNA extraction of a preliminary DNA extraction protocol, showed low DNA yields for 9% of the specimens (n = 78). In all cases, low DNA extraction efficiency seemed to be due to a component present in faeces that prevented DNA binding to silica particles, presumably by competitive binding. Choosing the right ratio of silica particles to faeces specimen solved this problem. Similarly, reconstruction experiments showed that the strong PCR inhibition that was observed in 8% of the specimens could effectively be relieved by the inclusion of alpha-casein in the PCR mixtures.

Hepatitis B and C virus co-infection and the risk for hepatotoxicity of highly active antiretroviral therapy in HIV-1 infection.

OBJECTIVE: To investigate the risk of hepatotoxicity after initiation of protease inhibitor-containing highly active antiretroviral therapy (HAART) for HIV-1 infected patients with chronic hepatitis B virus (HBV) or hepatitis C virus (HCV) co-infection. DESIGN: Retrospective study with 394 HIV-1-infected patients initiating HAART at a single university clinic. METHODS: Liver enzyme elevation (LEE) was defined as alanine aminotransferase or aspartate aminotransferase at least five times the upper limit of normal and an absolute increase of > 100 U/l. Relative risks for time to LEE were estimated using Cox proportional hazards models. RESULTS: Of 394 patients 7% were hepatitis B surface antigen (HBsAg)-positive and 14% were anti-HCV-positive. Patients with chronic hepatitis had a higher risk for LEE compared with patients without co-infection: 37% versus 12% respectively. After adjustment for higher baseline transaminases, the presence of HBsAg or anti-HCV remained associated with an increased risk of LEE - relative risk 2.78 (95% confidence interval, 1.50-5.16) and 2.46 (95% confidence interval, 1.43-4.24) respectively. In patients with LEE, transaminases declined whether HAART was continued or modified. Of patients with chronic HBV infection 38% lost HBeAg or developed anti-HBe after initiation of HAART, and one seroconverted from HBsAg-positive to anti-HBs-positive. However, there was no clear relationship with LEE. CONCLUSIONS: HIV-1-infected patients co-infected with HBV or HCV were at considerably higher risk of developing LEE when HAART was initiated compared with patients without co-infection, but it is usually not necessary to modify antiretroviral therapy.

Adenoviruses from human immunodeficiency virus-infected individuals, including two strains that represent new candidate serotypes Ad50 and Ad51 of species B1 and D, respectively.

Adenovirus (Ad) isolates from a large number of human immunodeficiency virus (HIV)-infected individuals were compared serologically and genetically with Ad isolates from immunocompetent patients. Between 1982 and 1994, stool and urine samples from 137 subjects with AIDS hospitalized in The Netherlands yielded 143 Ad strains. Forty additional Ad strains were obtained from 35 HIV-positive patients in Manchester, United Kingdom, in 1992 and 1993. Of these 183 HIV-associated Ad strains, 84% belonged to species D and 3% belonged to species C. These strains were compared with 2,301 Ad strains collected during general diagnostic examinations in The Netherlands from 1973 to 1992. Of the latter strains, 5% belonged to species D and 49% belonged to species C. Two of the Ads isolated from fecal specimens of AIDS patients represent new serotypes: candidate Ad serotype 50 (prototype strain, Wan) of subspecies B1 and candidate Ad serotype 51 (prototype strain, Bom) of species D. The DNA restriction enzyme patterns of strains Wan and Bom differed from the patterns of all established prototypes.

Kawasaki disease: a maturational defect in immune responsiveness.

Kawasaki disease (KD), an acute febrile disease in children of unknown etiology, is characterized by a vasculitis that may result in coronary artery aneurysms (CAAs). In new patients with KD, a selective and prolonged T cell unresponsiveness to activation via the T cell antigen receptor CD3 was observed, whereas proliferation to other stimuli was intact. This "split T cell anergy" delineated KD from other pediatric infections and autoimmune diseases and correlated with CAA formation (P<.001). A transient immune dysfunction was also suggested by an incomplete responsiveness to measles-mumps-rubella (MMR) vaccination in patients with KD versus controls (P<.0001; odds ratio, 15.6; 95% confidence interval, 4.8-51.1), which was overcome by revaccination(s). The reduced responsiveness to MMR in patients with KD suggests a subtle and predetermining immune dysfunction. An inherent immaturity to clear certain antigens may be an important cause that precipitates KD and the immune dysregulation during acute disease.

Influence of highly active antiretroviral therapy on the development of CMV disease in HIV positive patients at high risk for CMV disease.

BACKGROUND/AIMS: In the pre-HAART era, HIV positive patients with CD4+ cell counts below 50 cells x10(6)/l, and those with detectable cytomegalovirus (CMV) DNA in their peripheral blood, were considered to be at high risk for the development of CMV disease. With the start of highly active antiretroviral therapy (HAART), a restoration of immune function occurred in these patients, and as a consequence patients became less vulnerable to CMV disease. Since it is not exactly known how HAART influences CMV viral load in peripheral blood and the incidence of CMV disease in high risk HIV positive patients a group of patients was followed before and after initiation of HAART. METHODS: 29 HIV positive patients, seen in the first 3 months of 1996 at the AIDS clinic of the Academic Medical Centre, at high risk for development of CMV disease (positive CMV DNA assay in blood and/or CD4+ cell count below 50 cells x10(6)/l), not receiving anti-CMV maintenance therapy, were included in a prospective cohort study. HAART was started in the second trimester of 1996. Patients were evaluated for the occurrence of CMV retinitis, or CMV disease elsewhere, comparing the incidence of CMV events before and after the start of HAART. Following the introduction of HAART, CD4+ cell counts and quantitative polymerase chain reaction (PCR) for CMV DNA in blood were monitored in all patients who remained alive and were not receiving anti-CMV maintenance therapy (n=22). Follow up was performed until August 1998; the mean follow up after the start of HAART was 14.9 months (range 8-22 months). RESULTS: In the pre-HAART period four patients developed CMV disease, and four died (without clinically manifest CMV disease). After the start of HAART no patient developed CMV disease or died. With HAART, the mean CD4+ cell counts increased from 34 cells x10(6)/l to 194 cells x10(6)/l at the end of follow up. CMV DNA could be detected in the blood of 11 patients. Quantification showed a decline in the amount of detectable DNA during follow up. At the last examination only one patient showed a positive PCR assay. This was the only patient with a CD4+ cell count remaining below 100 cells x10(6)/l. CONCLUSION: In HIV positive patients at high risk of CMV retinitis, either with a positive CMV PCR assay in blood and/or with CD4+ cell counts below 50 cell x10(6)/l, HAART causes a dramatic decrease in the occurrence of CMV disease. This decrease is paralleled by an increase in CD4+ cell count, and a decrease in the amount of CMV DNA in the blood, which was below detection levels in all patients with CD4+ cell counts above 100 cells x10(6)/l.

A highly sensitive assay for detection and quantitation of human cytomegalovirus DNA in serum and plasma by PCR and electrochemiluminescence.

We describe a diagnostic PCR assay (D-PCR) and a quantitative PCR assay (Q-PCR) for the detection of human cytomegalovirus (CMV) in plasma and serum. In the D-PCR, DNA was purified from plasma or serum together with internal control (IC) DNA, which monitored both DNA extraction efficiency and PCR efficiency. DNA was subjected to PCR with a single primer pair, and the amount of PCR products was determined by electrochemiluminescence (ECL) in the QPCR System 5000 (Perkin-Elmer) after hybridization with Tris (2,2'-bipyridine) ruthenium (II) chelate-labeled probes. The lower limit of sensitivity of the D-PCR was reached at about 25 CMV particles/ml. Even with extremely low DNA inputs (four molecules of IC DNA/200 microl of plasma), very high yields (near 100%) were reached. DNA extracted from specimens that were CMV positive by the D-PCR was subsequently used in the Q-PCR, which was similar to the D-PCR. The viral load was calculated directly from the ratio of CMV and IC signals obtained by ECL. The Q-PCR assay is quantitative in the range of 100 to 150,000 copies of CMV/ml, independent of the anticoagulant. Interassay variation, intra-assay variation, and interspecimen variation were about 25%, suggesting that the Q-PCR will reliably detect fourfold differences in viral load. Comparison of paired serum and plasma specimens from CMV-infected individuals showed that serum CMV loads were frequently more than 10-fold lower than plasma CMV loads.

Improved silica-guanidiniumthiocyanate DNA isolation procedure based on selective binding of bovine alpha-casein to silica particles.

DNA purified from clinical cerebrospinal fluid and urine specimens by a silica-guanidiniumthiocyanate procedure frequently contained an inhibitor(s) of DNA-processing enzymes which may have been introduced by the purification procedure itself. Inhibition could be relieved by the use of a novel lysis buffer containing alpha-casein. When the novel lysis buffer was used, alpha-casein was bound by the silica particles in the first step of the procedure and eluted together with DNA in the last step, after which it exerted its beneficial effects for DNA-processing enzymes. In the present study we have compared the novel lysis buffer with the previously described lysis buffer with respect to double-stranded DNA yield (which was nearly 100%) and the performance of DNA-processing enzymes.

Comparison of PCR, culture, and serological tests for diagnosis of Mycoplasma pneumoniae respiratory tract infection in children.

For diagnosis of Mycoplasma pneumoniae infection we compared two rapid tests, PCR and the immunoglobulin M immunofluorescence assay (IgM IFA), with culture and the complement fixation test (CFT), in a prospective study among 92 children with respiratory tract infection and 74 controls. Based on positivity of culture and/or CFT as the diagnostic criterion, nine patients (10%) were diagnosed with M. pneumoniae infection. All patients positive by culture were also positive by PCR. In all controls cultures, PCRs, and serological assays were negative, except in one with a positive IgM IFA. The IgM IFA had a low positive predictive value of 50%. Only a combination of PCR (seven patients) and CFT (seven patients) allowed diagnosis of all cases.

Relation between laboratory test results and histological hepatitis activity in individuals positive for hepatitis B surface antigen and antibodies to hepatitis B e antigen.

BACKGROUND: Hepatitis B surface antigen (HBsAg) and antibodies to hepatitis B e antigen (anti-HBe) commonly coexist, and laboratory tests are often requested to assess histological hepatitis activity. An optimum panel of tests has not been found and the usefulness of hepatitis B virus (HBV) DNA assays in this context has not been established. We assessed various blood tests to find which best predicted hepatitis activity. METHODS: Routine plasma biochemical liver tests and serum HBV DNA (hybridisation and PCR assays) were assessed prospectively in 123 patients positive for HBsAg and anti-HBe. We scored histological hepatitis activity (hepatitis activity index) and determined whether chronic active hepatitis (chronic hepatitis with portal and periportal lesions) was present. We analysed the relation between laboratory data and the hepatitis activity index or risk of chronic active hepatitis by multiple regression and multiple logistic regression, respectively. FINDINGS: The analyses provided models for predicting either the hepatitis activity index or the risk of chronic active hepatitis. Aspartate aminotransferase was the most important test in the two models. The contribution of HBV DNA and other assays, especially alanine-aminotransferase activity, were of no practical importance. INTERPRETATION: Because screening by aspartate-aminotransferase activity could not be improved by the addition of other assays or HBV DNA, patients positive for HBsAg and anti-HBe could be screened for chronic active hepatitis with a single assay and counselling of patients can be improved if proper reference values are used.