Serological diagnosis of acute delta hepatitis

Sixty-three intravenous drug addicts with HBsAg positive hepatitis were studied to evaluate the diagnostic usefulness of hepatitis delta virus (HDV) markers for diagnosis of acute HDV infection. Patients were tested for HBsAg, anti-HBc-IgM, and anti-HD-IgM by radioimmunoassay (RIA), and for hepatitis delta antigen (HD-Ag) by a commercial enzyme-linked immunoassay (ELISA). At least two serum samples at a mean interval of 4 wk were examined from each patient. HDV markers were found in 41 cases. In the first serum sample (obtained within 1-5 wk after onset of illness) HD-Ag was found in 32 cases and was the only HDV marker in 22; in the remaining 10 cases, HD-Ag was found along with total anti-HD, and in 6 of them anti-HD-IgM was also detected. Five additional patients were only positive for total anti-HD, and anti-HD of the IgM class was the only marker in one patient. HD-Ag was found more often in the patients studied during the first 2 wk of illness. In the second serum sample, HD-Ag was never the only marker detected, seven patients were still positive, and in all of them anti-HD was also present. Thirty patients were only positive for anti-HD. Seroconversion from HD-Ag to anti-HD occurred in 20 of 22 (91%) patients. The results suggest that HD-Ag determination by ELISA in the initial serum sample, during the first 2 wk of illness, may be the most sensitive test for the diagnosis of acute delta infection, and that seroconversion to anti-HD usually occurs after the sixth week of illness.     

Serological markers of posttransfusion hepatitis C viral infection.

Serological markers for hepatitis C virus (HCV) infection were measured in serial samples from 14 posttransfusion chronic non-A, non-B hepatitis patients by a semiquantitative dot blot immunoassay. The assay detected antibodies to HCV by use of recombinant proteins that represent putative HCV capsid (core), nonstructural protein 3 (NS3) (33c), and NS4 (c100) epitopes. Seroconversion to anti-HCV antibodies (anti-HCV) was detected in all patients. The average time to active antibody production detected by any of the recombinant proteins was 13.8 (range, 3.6 to 22.0) weeks posttransfusion or 4.6 (range, -4.5 to 13.4) weeks after the first biochemical marker of illness. Anti-HCV were detected earliest by the core antigen in most cases; however, the patterns of anti-HCV responses varied significantly among individuals. Overall, the addition of the core and NS3 antigens to the assay enabled the detection of the antibody response 4 to 5 weeks earlier than did the addition of the c100 antigen, the sole antigen used in current screening tests in the United States. Passively transferred antibodies were detected by at least one antigen in early posttransfusion samples from 12 patients and decayed below detectable levels for all antigens in only 2 patients. Antibodies to all three gene products were evident in the last sample from all five patients monitored for greater than 3 years from transfusion indicating the persistence of antibodies in patients with chronic illness. Our data show that the period following the onset of hepatitis during which anti-HCV are not detected by current screening assays can be greatly shortened by the detection of anti-HCV responses by a combination of core, NS3, and c100 antigens.     

Serological techniques for diagnosis of fungal infection

This review summarizes recent developments in the serodiagnosis of candidiasis, aspergillosis, cryptococcosis, histoplasmosis, blastomycosis, coccidioidomycosis, mucormycosis and sporotrichosis. A number of studies have substantiated the presence of circulating antigens in invasive candidiasis, invasive aspergillosis, disseminated histoplasmosis and coccidioidomycosis, and immunoassays for antigen detection provide moderate sensitivity but high specificity for disease. Improved detection may result mainly from repeated serum or concentrated urine samplings rather than from the development of more sensitive immunoassays. Immunoblot analysis of the serological response is a useful tool for the identification of immunogenic fungal components that elicit a specific antibody response in invasive disease. This method, and others, have been successfully applied to the study of the immune response to several fungi, includingCandida, Aspergillus andRhizopus.     

Co-expression of markers for hepatitis delta and hepatitis B viruses in human liver

Delta hepatitis (HDV) infection can only occur in the presence of hepatitis B (HBV) infection, as HDV requires a coat of HBV surface antigen (HBsAg) for assembly of complete virus. A number of studies have examined the variation of HBV markers in serum and liver during establishment of HDV infection, but none has systematically examined the relationship between the two viruses in individual hepatocytes. Liver biopsies from five patients with HDV/HBV infection were stained for HBsAg, HBV core antigen (HBcAg) and hepatitis D (delta) antigen (HDAg). Double immunostaining was performed with a combination of indirect immunoperoxidase and alkaline phosphatase/antialkaline phosphatase techniques. HDV and HBV antigens were expressed in all five liver biopsies. Co-localization of HBsAg was seen in up to 39% of HDAg positive cells, and HBcAg in up to 8% of HDAg positive cells. HBcAg was detectable in approximately 9% of HBsAg positive cells, and HBsAg in approximately 12% of HBcAg positive cells. HDV can replicate without HBV but ultimately requires HBV to produce complete virus and subsequently infect other cells. In this study the majority of HDV positive cells did not appear to contain HBV markers. This might suggest delta virus replication without assembly, or possibly sequential production/assembly of the virus.     

Serological diagnostics of hepatitis E virus infection

Development of accurate diagnostic assays for the detection of serological markers of hepatitis E virus (HEV) infection remains challenging. In the course of nearly 20 years after the discovery of HEV, significant progress has been made in characterizing the antigenic structure of HEV proteins, engineering highly immunoreactive diagnostic antigens, and devising efficient serological assays. However, many outstanding issues related to sensitivity and specificity of these assays in clinical and epidemiological settings remain to be resolved. Complexity of antigenic composition, viral genetic heterogeneity and varying epidemiological patterns of hepatitis E in different parts of the world present challenges to the refinement of HEV serological diagnostic assays. Development of antigens specially designed for the identification of serological markers specific to acute infection and of IgG anti-HEV specific to the convalescent phase of infection would greatly facilitate accurate identification of active, recent and past HEV infections.     

Serological evidence for hepatitis E virus infection in Israel

Israel is suspected to be endemic for hepatitis E virus (HEV) because of its geographic location and the large-scale immigration from endemic countries. Although no cases of local HEV infection have been diagnosed, a serological survey would provide indirect evidence for such infection. We examined sera from 1,416 healthy subjects, including 1,139 Jews from various regions of Israel and 277 Arabs, most of whom reside in the West Bank of the Jordan River. In addition, we tested 13 non-A, non-B, and non-C viral hepatitis patients. Sera were screened for antibody to hepatitis E virus (anti-HEV) by a newly developed enzyme immunoassay (EIA) and by immuno-blots for both IgG and IgM anti-HEV activity. Positive samples were confirmed by neutralization. The seroprevalence found by EIA was 2.81% and 1.81% in the Jewish and Arab populations, respectively. More than a 2-fold higher prevalence in males compared to females and an increase with age were found in both populations. However, these differences were nonsignificant. The geographical distribution was even throughout the country, except for two clusters of 3 and 4 seropositive individuals possibly reflecting past foci of infection. Eight of 37 ElA-positive sera were positive for IgG, and 3 were positive for IgM by the immunoblot assay. Among hepatitis patients (9 acute and 4 chronic), one patient with chronic hepatitis was positive for both IgG and IgM. Our study provides indirect evidence that Israel is endemic for HEV. The lack of outbreaks may be attributed to generally good hygienic conditions and a controlled potable water supply, while unrecognized sporadic cases may be due to the unavailability of diagnostic tests. © 1995 Wiley-Liss, Inc.     

Serological analysis of duck hepatitis B virus infection

A radioimmunoassay was developed to detect duck hepatitis B virus surface antigen and antibody; viraemia (DHBV DNA or DHBsAg) was detected in all ducks inoculated within 3 weeks post-hatch, and persistent infection developed in 93% of birds in this group. In contrast, only 80% and 60% of ducks inoculated 4- and 6-weeks post-hatch respectively developed viraemia, and approximately 70% of the viraemic ducks became carriers. Markers of viraemia were undetected in ducks inoculated 8 weeks post-hatch and in uninfected controls. A typical anti-DHBs seroconversion developed subsequently in 2 of 4 birds that showed transient viraemia, and antibody also developed in 3 of 7 ducks inoculated 4-8 weeks post-hatch that showed no viraemia. However, gene amplification by the polymerase chain reaction demonstrated DHBV DNA in ducks from the latter group suggesting that the antibody did not result from passive vaccination. Thus, increased resistance to infection develops with increasing age that may be related to several factors including host immunity. This model may help elucidate similar age-related features of human hepatitis B virus infections.     

Elimination of hepatitis delta virus infection after loss of hepatitis B surface antigen in patients with chronic delta hepatitis

The aim of this study was to evaluate whether patients with chronic hepatitis delta virus (HDV) infection treated with alpha interferon and subsequent loss of hepatitis B surface antigen (HBsAg) eliminate HDV. HDV RNA was detected in 26 of 28 patients with chronic delta hepatitis using the polymerase chain reaction. Seventeen patients in whom HDV RNA was detected were treated with alpha interferon; in 65%, HDV RNA remained detectable during treatment or reappeared after stopping therapy whereas in three patients HDV RNA remained absent (17.5%). HDV RNA became and remained undetectable in serum and liver of two of these three patients who lost HBsAg from serum and in one patient who was intermittently HBsAg negative during therapy. After loss of HBsAg, hepatitis B virus (HBV) DNA was still detectable in the liver, but not HBV RNA, indicating absent or very low HBV replication. Three patients were lost to follow up (17.5%). Two nontreated patients with chronic HDV infection also lost HBsAg during follow up; HDV RNA also became undetectable in their serum. Thus, HDV replication does not persist after the loss of HBsAg. Clearance of HBsAg may be a useful guide to when therapy can be stopped. © 1994 Wiley-Liss, Inc.     

Serum hepatitis B virus-DNA in chronic hepatitis B and delta infection

Hepatitis B-associated delta agent, a defective RNA virus requiring helper functions of hepatitis B virus (HBV), has been shown to interfere with HBV replication. Low titers of serum hepatitis B surface antigen, absence of hepatitis B e antigen, and low levels of stainable hepatitis B core antigen in liver cells usually seen in chronic delta infection are indirect evidences of such an interference. Measurement of serum HBV-DNA by hybridization with phosphorus 32-labeled HBV-clone DNA is the most sensitive method currently available to detect HBV replication. Using this method, we found that only two of 13 patients with chronic delta infection showed serum HBV-DNA positivity in comparison with seven of 14 patients who had chronic hepatitis B without delta infection. These two groups were matched for hepatitis B e antigen status and liver histopathology. Thus, we report direct evidence of delta agent interfering with the replication of the helper (HBV) virus.     

Differential diagnosis of viral hepatitis based on hepatitis viral markers

192 patients of acute viral hepatitis (AVH) from three different hospitals of Madras metropolitan area during November 1985 to January 1986 were investigated for serologic markers of hepatitis A virus (anti HAVIgM) and hepatitis B virus (HBsAg, HBeAg, anti HBcIgM and anti HBs) by Enzyme linked immunosorbent assay (ELISA). While the overall pattern of AVH in Madras as revealed from the study showed Hepatitis A to be 36.4%, Hepatitis B 34.4% and Non-A Non-B 29.1%, the pattern differed significantly when areawise categorisation was done. The major AVH type in Government General Hospital was Hepatitis B (48.9%). While it was hepatitis A (46.9%) in Government Stanley Hospital and Non-A Non-B (40.0%) in Military Hospital. Using anti HBcIgM marker of Hepatitis B Virus and anti HAVIgM it was possible to make out that 13.5% of the cases, currently suffering from hepatitis A were either HBV carriers (8.3%) or cases convalescing from a previous Hepatitis B attack (5.3%). Various combinations of HBV markers positivity were observed and their diagnostic significance inferred.     

Serological and molecular genetic markers of hepatitis C virus in infected donors

The frequency of hepatitis C virus (HCV) markers was determined in donors; the spectrum and activity of specific antibodies (anti-HCV), the distribution of virus genotypes, and HCV RNA concentrations were studied in virus carrier donors. The activity of antibodies in HCV RNA-negative donors was significantly lower than that in HCV RNA-positive donors (p < or = 0.001). There was a statistically significant difference in antibody activities in donors infected with genotype 1b as compared with those infected with genotype 3a (p < 0.001). However, no correlation was found between the concentration of a virus genome and the activity of specific antibodies. The risk for obtaining infected blood donations was determined during plasma screening by enzyme immunoassay (EIA). Our investigations have indicated that the frequency of serological window period donations is one case per 74750 test plasma units and that of HCV RNA-positive donations with low antibody positivity coefficients, which are frequently detectable as seronegative during screening for laboratory errors, is one case per 37375 test units. A combination of EIA and polymerase chain reaction has shown to minimize the risk of contamination of donor plasma with HCV markers.     

Evaluation of enzyme immunoassay for anti-HBc IgM in the diagnosis of acute hepatitis B virus infection

Corzyme-MTM (Abbott Laboratories, North Chicago, IL), a newly introduced kit for the measurement of serum IgM antihepatitis B core antigen by enzyme immunoassay, was evaluated for the diagnosis of acute B-viral hepatitis (AVH-B). The study included 175 acute viral hepatitis patients with transient hepatitis B surface antigen (HBsAg). Sera from 160 were tested on multiple occasions until their HBsAg cleared. IgM anti-HBc was found in 171 of 175 patients (98.4%) during the acute phase. The serum samples from 42 patients with liver biopsy-proven chronic active hepatitis, type B (CAH-B), and 18 patients with persistent hepatitis, type B (PH-B), were analyzed for the presence of IgM anti-HBc, using the same technic. None of the sera from 42 patients with CAH-B and only 2 of the 18 patients with PHB had IgM anti-HBc. Thus, the measuring IgM anti-HBc using Corzyme-M kit is helpful in the diagnosis of AVH-B and in the discrimination of acute from chronic HBV infections.     

Evolution of hepatitis delta virus RNA during chronic infection.

The complete RNA sequences of hepatitis delta virus (HDV) isolated at three different time points from a chronic delta hepatitis patient were determined. These time points represented three different periods of clinical flare-ups. The sequence analysis showed that these three different HDV isolates evolved at a rate ranging from 3.0 x 10(-2) to 3.0 x 10(-3) substitutions/nucleotide/year, depending on the period of infection. The evolution rates appeared to correlate with the changes of clinical pictures of hepatitis, i.e., the more drastic the change in the symptom of hepatitis was, the more nucleotide changes were detected. Except during the transition from the acute phase to chronic phase of delta hepatitis, when there was a much larger number of changes in HDV RNA sequence, the overall evolution rate of HDV RNA in the chronic phase appeared to be similar to those of other RNA viruses. Sequence relationship of these HDV RNAs suggested that acute exacerbations in chronic delta hepatitis were associated with the evolution of the persistently infected HDV, rather than resulting from new viral infections. However, some of the mutations were not cumulative, suggesting that HDV isolated at a later time was not directly evolved from the immediately previous one. Thus, HDV at any time point was a mixture of viruses with slight sequence variations, and a specific HDV RNA species was selected from this virus population under different environments. These findings indicate that HDV RNA is heterogeneous and evolves at a fast rate. The evolution rates in different parts of HDV RNA also varied. The evolution rate of HDV RNA determined here was higher than the ones determined previously from partial RNA sequences of two Japanese HDV isolates.     

Serological diagnosis of Mycoplasma pneumoniae infection by enzyme immunoassay.

Antibodies against Mycoplasma pneumoniae antigen obtained by Tween-ether treatment from purified M. pneumoniae were measured by means of enzyme-linked immunosorbent assay (ELISA). Paired sera from 19 patients with pneumonia and from 13 patients with acute pancreatitis with a significant rise in complement fixing antibodies against M. pneumoniae were studied. Single sera from healthy 1-year-old children were used as controls. High levels of IgG and IgM class antibodies were seen in sera from patients with pneumonia while most patients with acute pancreatitis and all the children showed low levels of antibodies. The results indicate that ELISA using Tween-ether treated M. pneumoniae antigen could be used successfully in the specific laboratory diagnosis of M. pneumoniae infection.     

Serological diagnosis of Epstein-Barr virus infection: Problems and solutions

Serological tests for antibodies specific for Epstein-Barr virus (EBV) antigens are frequently used to define infection status and for the differential diagnosis of other pathogens responsible for mononucleosis syndrome. Using only three parameters [viral capsid antigen (VCA) IgG, VCA IgM and EBV nuclear antigen (EBNA)-1 IgG],it is normally possible to distinguish acute from past infection: the presence of VCA IgM and VCA IgG without EBNA-1 IgG indicates acute infection, whereas the presence of VCA IgG and EBNA-1 IgG without VCA IgM is typical of past infection. However, serological findings may sometimes be difficult to interpret as VCA IgG can be present without VCA IgM or EBNA-1 IgG in cases of acute or past infection, or all the three parameters may be detected simultaneously in the case of recent infection or during the course of reactivation. A profile of isolated EBNA-1 IgG may also create some doubts. In order to interpret these patterns correctly, it is necessary to determine IgG avidity, identify anti-EBV IgG and IgM antibodies by immunoblotting, and look for heterophile antibodies, anti-EA (D) antibodies or viral genome using molecular biology methods. These tests make it possible to define the status of the infection and solve any problems that may arise in routine laboratory practice.     

血液炎性标志物用于关节假体感染诊断的研究与应用

背景：人工关节置换作为一种成熟的治疗方法现已在国内外广泛应用,关节假体感染是人工关节置换后严重的并发症之一,感染后将给患者带来严重后果。目前,对假体感染的诊断还没有一项临床或实验室检查在灵敏度、特异度和精确度上达到令人满意的程度。 目的：讨论并总结人工关节置换后假体感染的相关血液炎性标志物诊断方法。 方法：由第一作者用计算机检索中国期刊全文数据库(CNKI：2002至2012年)和PubMed(2002至2012年)数据库,检索词分别为“关节置换、假体感染、诊断、实验室检查”和“joint replacement prosthesis infection, diagnosis, laboratory tests”。从寻找关节置换后假体感染的临床表现及相关诊断方法上进行相关介绍及总结。共检索到153篇文章,按纳入和排除标准对文献进行筛选,共纳入30篇文章。 结果与结论：白细胞介素6血清水平对于诊断关节假体感染准确性最高,之后依次是C-反应蛋白,红细胞沉降率,白细胞计数。然而,常规检查白细胞介素6条件还是有限,进一步评估白细胞介素6和其他细胞因子在不同的患者身上诊断关节假体感染的准确性还需要更深入的研究。