J. F. L. Weel

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

ObjectiveTo 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. DesignRetrospective study with 394 HIV-1-infected patients initiating HAART at a single university clinic. MethodsLiver 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. ResultsOf 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. ConclusionsHIV-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.

Effect of Helicobacter pylori eradication on gastritis in relation to cagA: A prospective 1-year follow-up study

BACKGROUND & AIMSnWhether Helicobacter pylori eradication resolves intestinal metaplasia and atrophy and whether infection with cagA+ H. pylori is related to a specific clinical outcome are not known. The aim of this study was to investigate the role of H. pylori eradication on the course of intestinal metaplasia (IM) and atrophy in relation to cagA.nnnMETHODSnIn a large prospective study, the cagA status of H. pylori isolated from consecutive dyspeptic patients was related to clinical outcome before and 1 year after successful eradication of H. pylori. At pretreatment and 4-6 weeks and on average 1 year after eradication therapy, the degree of gastritis and the status of H. pylori were assessed by culture and histopathology.nnnRESULTSnSpecimens of cagA+ H. pylori were recovered from 122 of 155 (79%) patients infected with H. pylori. Pretreatment degrees of gastritis activity, superficial epithelial damage, IM, and atrophy were significantly greater in patients infected with cagA+ H. pylori (P < 0.001). After successful eradication of H. pylori, a significant improvement of activity of gastritis and superficial epithelial damage occurred (P < 0.001), but the degree of IM and atrophy did not change, irrespective of the cagA status.nnnCONCLUSIONSnThe usefulness of H. pylori eradication to revert precancerous lesions such as IM and atrophy after 1-year follow-up is questionable.

Quasispecies Development of Helicobacter pylori Observed in Paired Isolates Obtained Years Apart from the Same Host

Helicobacter pylori isolates show greater genetic diversity than other bacterial species studied, but the basis for this phenomenon is unknown. Whether detectable genomic mutation appears within an H. pylori population during persistent colonization was investigated. Paired H. pylori populations obtained across 7- to 10-year intervals from 13 patients were characterized by use of methods including polymerase chain reaction (PCR) genotyping for cagA, vacA, iceA, recA, and IS605; random arbitrarily primed DNA (RAPD)-PCR and amplified fragment length polymorphism (AFLP) analysis; and ELISA, to determine Lewis phenotypes. Genotyping, including recA sequence analysis, revealed that initial and follow-up populations represented the same population in 11 patients (85%). Nevertheless, distinct dissimilarities were shown within each of these 11 pairs by both RAPD-PCR and AFLP analyses. During follow-up, Lewis-y levels, but not Lewis-x levels, decreased significantly. The changes detected by RAPD-PCR and AFLP indicate that genetic drift occurs within H. pylori populations over the course of years of colonization of a single host.

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.

Fate of the major outer membrane protein P.IA in early and late events of gonococcal infection of epithelial cells

We investigated the fate of the major outer membrane protein of Neisseria gonorrhoeae, P.IA, during gonococcal infection of Chang conjunctiva epithelial cells by using immunoelectron microscopy. Probing of P.IA epitopes with mono- and polyclonal antibodies revealed variable, fixation-dependent P.IA epitope exposure in the gonococci used as an inoculum in the infection experiments. Detection of invariable exposed P.IA epitopes in cryosections of infected epithelial cells with a polyclonal antiserum revealed unaltered P.IA exposure on the bacterial membranes during early attachment of the bacteria to the eukaryotic cells. Upon entry of the bacteria into the host cells, however, labelling was extended to membraneous structures that intercalated between the bacteria and the host cell surface, and, occasionally, to the host cell plasma membrane. The latter observation is consistent with the suggested active role of P.I. in the uptake process (as shown in 1985 by E.C. Gotschlich). Once inside the epithelial cells, both morphologically intact and disintegrating bacteria could be distinguished. The disintegration of the bacteria was accompanied by a loss of P.IA immunoreactivity.

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.

MEASUREMENTS OF INVASION BY ANTIBODY LABELING AND ELECTRON MICROSCOPY

Publisher Summary This chapter outlines and discusses practical strategies and technical details for successful immunomicroscopic measurement of bacterial invasion in monolayers of cells. The advantages of microscopic analysis of infected eukaryotic cells are that it provides direct information about the number of adherent and ingested bacteria at the single cell level, and that it enables localization of the bacteria within specific cells or cellular compartments independent of their viability. This approach is different from and complements intracellular survival assays, which involve the selection and counting of the viable intracellular bacteria isolated from a batch of host cells. The basis of immunomicroscopic assessment of bacterial invasion is the differential staining of bacteria that are internalized from those that remain on the cell surface using immunoreagents. The methods to achieve this include double-fluorochrome immunolabeling and immunogold–silver staining procedures for use in combination with bright-field light microscopy, fluorescence microscopy, and confocal scanning microscopy. The principle of the measurement of bacterial invasion by antibody labeling using light microscopy is that antibodies cannot pass through the plasma membrane of eukaryotic cells and, therefore, bind only to extracellular adherent bacteria and not to intracellular bacteria. This enables exclusive immunolabeling of the extracellular microorganisms. Counterstaining of the intracellular bacteria then provides the desired differential staining of adherent and ingested bacteria.

Treatment of Helicobacter pylori infection with low or high dose omeprazole combined with amoxycillin and the effect of early retreatment

Background: Cure rates of H. pylori infection, using dual therapy with omeprazole and amoxycillin, vary considerably and the efficacy of retreatment with this regimen in the case of initial failure is controversial. Therefore, we conducted a large prospective double‐blind randomized trial, studying the efficacy of low vs. high dose omeprazole in dual therapy and of early retreatment with the same regimens.

Effect of specimen collection techniques, transport media, and incubation of cultures on the detection rate of Helicobacter pylori

Culture and histologic examination are considered “gold standard” methods for the detection ofHelicobacter pylori, but discrepancies may occur with either method. Failure to detectHelicobacter pylori may be due to sampling error, inappropriate transport or culture media, or insufficient duration of the incubation period. Rates of detection ofHelicobacter pylori by culture and histopathologic examination of gastric mucosal biopsy specimens were determined in 102 consecutive dyspeptic patients. In a separate group of 60 patients, rates of detection ofHelicobacter pylori by culture of antral brushings and the length of incubation required in selective and nonselective culture media were studied. In the first group of 102 patients, the combination of culture and histologic examination detected 54Helicobacter pylori-positive patients, whereas the separate techniques each detected 51Helicobacter pylori-positive patients. In the second group of 60 patients evaluated by culture of antral brushings, the rate of detection ofHelicobacter pylori was 25 of 60 and was similar for culture (25/60) and histologic examination (25/60). In the second group the length of incubation required to detectHelicobacter pylori was different for selective and nonselective media. In nonselective media, incubation of up to ten days was required to detect allHelicobacter pylori infections, whereas in selective media seven days was sufficient. Rates of detection ofHelicobacter pylori by culture, histopathologic examination and culture from brushings were similar, whereas the combination of culture and histopathologic examination achieved a superior rate of detection. The incubation period required for the detection ofHelicobacter pylori by culture was a minimum of seven days and was dependent on the culture medium used.

The use of immunogold-silver staining to study antigen variation and bacterial entry into eukaryotic cells by conventional light microscopy

Immunogold-silver staining is a sensitive staining technique that enables the visualisation of the presence of individual antigens by conventional light microscopy. The application of this method to detect the antigenic heterogeneity of bacterial surface components and also the localisation of intracellular or extracellular bacteria is described. The latter application involved selective immuno-silver staining of the extracellular bacteria and counterstaining of the intracellular bacteria and the eukaryotic cells by crystal violet. The efficacy of the assay was confirmed by transmission electronmicroscopy of the silver-stained specimens. Immunogold-silver staining was shown to be useful for studying bacterial antigen variation and the uptake of bacteria by eukaryotic cells.