Sensitivity Of Enzyme Immunoassay Research Articles

Development and Application of a Sensitive, Second Antibody Format Enzymeimmunoassay (EIA) for Estimation of Plasma FSH in Mithun (Bos frontalis)

Mithun (Bos frontalis) is a semi-wild rare ruminant species. A simple sensitive enzymeimmunoassay suitable for assaying FSH in the blood plasma of mithun is not available which thereby limits our ability to understand this species reproductive processes. Therefore, the aim of this article was to develop a simple and sensitive enzymeimmunoassay (EIA) for estimation of FSH in mithun plasma and apply the assay to understand the estrous cycle and superovulatory process in this species. To accomplish this goal, biotinylated FSH was bridged between streptavidin-peroxidase and immobilized antiserum in a competitive assay. Forty microlitre mithun plasma was used directly in the EIA. The FSH standards were prepared in hormone free plasma and ranged from 5–1280 pg/well/40 μL. The sensitivity of EIA was 5 pg/well FSH, which corresponds to 0.125 ng/mL plasma and the 50% relative binding sensitivity was 90 pg/well/40 μL. Although the shape of the standard curve was not influenced by different plasma volumes viz. 40 and 80 μL, a slight drop in the OD450 was observed with the increasing volume of plasma. Parallelism tests conducted between the endogenous mithun FSH and bovine FSH standards showed good homology between them. Plasma FSH estimated using the developed EIA and commercially available FSH EIA kit in the same samples were correlated (r = 0.98) and showed linearity. Both the Intra- and inter-assay CV were below 6%. Recovery of known concentrations of added FSH showed linearity (r = 0.99). The developed EIA was further validated biologically by estimating FSH in cyclic cows for the entire estrous cycle, in mithun heifers administered with GnRH analogues and in mithun cows during superovulatory treatment with FSH. In conclusion, the EIA developed for FSH determination in mithun blood plasma is simple and highly sensitive for estimation of mithun FSH in all physiological conditions.

Immune status of health care workers to measles virus: evaluation of protective titers in four measles IgG EIAs

BackgroundFollowing the recognition of a measles case in a hospital in The Netherlands, health care workers (HCW) from the premises were screened by a commercial enzyme immunoassay (EIA) for measles IgG to identify persons at risk for measles. At least 10% of the HCW were tested measles IgG-negative. As this was considered an unusually high proportion, we hypothesized suboptimal sensitivity of EIAs, especially in medical personnel that had vaccine-induced immunity rather than antibodies resulting from natural infection. ObjectivesTo determine (vaccine-induced) measles immunity in HCW, using different EIAs compared to the plaque reduction neutralization (PRN) test, the best surrogate marker for vaccine efficacy and immune protection. Study designSera from HCW were tested for measles IgG antibodies in three commercial EIAs, in a bead-based multiplex immunoassay (MIA) and in the PRN test, and evaluated against age and vaccination history of the HCW. ResultsOf the 154 HCW, born between 1960 and 1995, 153 (99.4%) had protective levels of measles antibodies (PRN>120mIU/ml). The three EIAs failed to detect any measles IgG antibodies in approximately 10% of the HCW, while this percentage was approximately 3% for the MIA. Negative IgG results rose to 19% for individuals born between 1975 and 1985, pointing to an age group largely representing vaccinated persons from the first measles vaccination period in The Netherlands. ConclusionThe results show limitations in the usefulness of current EIA assays for determining protective measles antibodies in persons with a vaccination history.

Comparison of diagnostic test performance in a population of high risk young adults versus a general population presenting with influenza

BackgroundUpper respiratory tract infection (URI) is a well-documented cause of morbidity, extra expense and lost training time among basic military trainees (BMTs). ObjectivesThe goal of this study is to better understand how influenza diagnostic tests perform in the BMT population, and how this performance differs from the general population. Study designLaboratory test data was collected in a prospective study that enrolled Department of Defense beneficiaries presenting to medical facilities in San Antonio, TX with URI symptoms between January 2005 and March 2011. Three laboratory tests for influenza were performed during the study period: polymerase chain reaction (PCR), enzyme immunoassay (EIA), and viral culture. Patients were grouped into BMT and non-BMT populations and the tests from each of these populations were compared for statistical differences. Similar comparisons were made with various sub-groups to include: influenza A versus influenza B, and influenza A subtypes: (H1N1) versus (H3N2) versus (H1N1)pdm09. ResultsAmong 4448 participants enrolled, 466 (10.5%) tested positive for influenza. Sensitivity of viral culture differed between BMTs and non-BMTs: 63% versus 41% (p<0.01). There was no difference in the sensitivity of PCR or EIA between the two populations. The sensitivities of viral culture, EIA and PCR were higher in those infected with influenza A than in those infected with influenza B. The sensitivity of viral culture was significantly higher in (H1N1)pdm09 subtype cases. ConclusionsViral culture performed better in BMTs than in non-BMTs. These differences are likely attributable to the younger age of the BMTs.

Comparison of Illumigene, Verigene, and Amplivue for Rapid Molecular Detection of Clostridium difficile in Pediatric Stool Specimens

Background: Pediatric Clostridium difficile infection (CDI) has been steadily rising over the past decade and is associated with increased morbidity and mortality. Rapid and accurate diagnostic testing is important in medical management. Due to the low sensitivity of enzyme immunoassays, and the complexity of culture based methods, many labs are utilizing newer molecular techniques for direct detection of nucleic acid in stool. Methods: 59 prospective stool specimens from 57 pediatric patients (aged 4 months to 19 years) with suspected CDI were tested over a 4 month period. Three FDA-cleared molecular platforms, Meridian Illumigene, Nanosphere Verigene, and Quidel AmpliVue, were compared. All samples had a consistency rating (CR) from 1 - 5 (watery - solid) assigned and were refrigerated prior to testing. Those with a CR=5 were excluded. Six positive specimens were frozen for 3 months then retested, and two of those were serially frozen, thawed, and retested 5 more times over 5 months. Results: All 3 platforms agreed for 55 (93%) specimens. There were 17 positive and 38 negative results. Five positive specimens were called BI/NAP1/027 by Verigene. Of the 4 discrepant results, 3 (2 CR=3, 1 CR=2) were invalid by Illumigene and negative by the other platforms. One sample (CR=4) was Verigene indeterminate, Illumigene positive, and AmpliVue negative. All frozen specimens gave correct results on all platforms. Conclusions: These data suggest that each platform offers a viable solution for rapid diagnosis of CDI in children. Additionally, long term frozen samples can be tested reliably.

Disparate detection outcomes for anti-HCV IgG and HCV RNA in dried blood spots

Dried blood spots (DBS) expedite the collection, storage and shipping of blood samples, thereby facilitating large-scale serologic studies. We evaluated the sensitivity of anti-HCV IgG testing and HCV-RNA quantitation using freshly prepared and stored DBS derived from HCV-infected patients.Protocols for elution were optimized using DBS prepared from plasma of 52 HCV-infected persons and 51 uninfected persons (control DBS), then applied to DBS from 33 chronic hepatitis C patients that had been stored at −20°C for 5 years (stored DBS). Control and stored DBS, and their corresponding plasma, were processed for anti-HCV IgG testing using the VITROS chemiluminescence assay (CIA) and the HCV 3.0 enzyme immunoassay (EIA) (Ortho-Clinical Diagnostics), and for HCV RNA quantitation by quantitative (q) RT-PCR. HCV genotyping was conducted by nucleotide sequencing.The sensitivity of CIA and EIA in control DBS was 92% and 90%, respectively, compared to 100% and 97%, respectively, in stored DBS. The sensitivity of HCV RNA detection was 88% in control DBS, compared to 36% in stored DBS. Specificity was 100% for all the assays in both control and stored DBS. Genotypes 1, 2 and 3 were detected in 16 (62%), 6 (23.1%), and 4 (15.3%) samples, respectively. Sequences generated from DBS and their corresponding plasma samples were identical.Whereas the sensitivity of anti-HCV IgG detection in stored DBS was equivalent to that in recently prepared DBS, the sensitivity of HCV RNA detection was markedly lower in stored DBS compared to recently prepared DBS. Stored DBS may be reliably used for anti-HCV detection but for HCV-RNA-based testing freshly prepared DBS is preferable to stored DBS.

The trade-off between accuracy and accessibility of syphilis screening assays.

The availability of rapid and sensitive methods to diagnose syphilis facilitates screening of pregnant women, which is one of the most cost-effective health interventions available. We have evaluated two screening methods in Tanzania: an enzyme immunoassay (EIA), and a point-of-care test (POCT). We evaluated the performance of each test against the Treponema pallidum particle agglutination assay (TPPA) as the reference method, and the accessibility of testing in a rural district of Tanzania. The POCT was performed in the clinic on whole blood, while the other assays were performed on plasma in the laboratory. Samples were also tested by the rapid plasma Reagin (RPR) test. With TPPA as reference assay, the sensitivity and specificity of EIA were 95.3% and 97.8%, and of the POCT were 59.6% and 99.4% respectively. The sensitivity of the POCT and EIA for active syphilis cases (TPPA positive and RPR titer ≥1/8) were 82% and 100% respectively. Only 15% of antenatal clinic attenders in this district visited a health facility with a laboratory capable of performing the EIA. Although it is less sensitive than EIA, its greater accessibility, and the fact that treatment can be given on the same day, means that the use of POCT would result in a higher proportion of women with syphilis receiving treatment than with the EIA in this district of Tanzania.

English

The diagnostic efficacy of Ortho VITROS chemiluminescence assay (CIA) in detecting antibodies to the hepatitis C virus (HCV) and the clinical significance of specimens with low sample-to-cut off (S/Co) ratio was analysed, comparing the positive rate for CIA in 5,550 consecutive outpatients with enzyme-immunoassay (EIA). In parallel testing, 43 samples (0.8%) were low positive by CIA (S/Co ratio from 1.0 to 8.0) but negative by EIA. No samples CIA negative/EIA positive were found. Among EIA negative results we found 22 RIBA positive or indeterminate, yielding CIA sensitivities of 100% and EIA sensitivity of 97.8%. None of the 33 samples with CIA S/Co ratios of &le; 2.0 and only 3 (10.7%) with S/Co ratios of between 2.1 and 8.0 were found to be RIBA positive. Instead, the majority of samples with S/Co ratios &le; 8.0 (55.7%) were recombinant immunoblotting assay (RIBA) negative.&nbsp; HCV RNA and/ or clinical evidence of HCV infection was not found in any of the 12 indeterminate cases examined with S/Co ratios &le; 2. We suggest to report them as &ldquo;Borderline&rdquo;, with the recommendation to follow up in the future. &nbsp; Key words:&nbsp;HCV infection, RIBA, HCV-RNA, CIA, EIA.

Performance of Clostridium difficile Toxin Enzyme Immunoassay and Nucleic Acid Amplification Tests Stratified by Patient Disease Severity

Many clinical laboratories in the United States are transitioning from toxin enzyme immunoassays (EIA) to nucleic acid amplification tests (NAATs) as the primary diagnostic test for Clostridium difficile infection (CDI). While it is known that the analytical sensitivity of the toxin EIA is poor, there are limited clinical data on the performance of these assays for patients with mild or severe CDI. Two hundred ninety-six hospital inpatients with diarrhea and clinical suspicion for CDI were tested prospectively by toxin EIA, by C. difficile NAAT, and with a reference standard toxigenic culture. Following completion of laboratory testing, retrospective chart reviews were performed to stratify patients into mild and severe disease groups based on clinical criteria using a standard point-based system. One hundred forty-three patients with CDI confirmed by toxigenic culture were evaluated in this study. Among the patients with mild CDI, 49% tested positive by toxin EIA and 98% tested positive by NAAT. Among patients with severe CDI, 58% tested positive by toxin EIA and 98% tested positive by NAAT. Increased CDI disease severity was not associated with an increased sensitivity of EIA (P = 0.31). These data demonstrate that toxin EIA performs poorly both for patients with severe CDI and for those with mild CDI and support the routine use of NAAT for the diagnosis of CDI. The presence of stool toxin measured by EIA does not correlate with disease severity.

Detection ofClostridium difficileas a Routine Diagnosis: Comparison of Real-Time PCR and Enzyme ImmunoassayThe Authors’ Reply

To the Editor We read with great interest the article about the comparison for 3 kinds of toxin enzyme immunoassay (EIA) as a screening test by Reller and colleagues.1 As described in the article, EIA for toxin A and toxin B has been widely used for the diagnosis of Clostridium difficile infection owing to its simplicity and short turnaround time.2 However, although the sensitivity of EIA has been reported to be improved in dry and wet tests, problems associated with its relatively low sensitivity still have been reported, and many large laboratories are now replacing or keeping pace with polymerase chain reaction (PCR)-based methods. Therefore, in a study we planned to compare the most popular 2 methods for detecting C difficile , EIA and real-time PCR. This study was conducted at Kyung Hee University Hospital, a tertiary teaching hospital in Seoul, Korea, from October 2010 to February 2011, using 291 fresh diarrheal stools. All specimens were immediately tested by culture and VIDAS C difficile toxins A & B assay using an automated VIDAS immunoanalyzer (bioMerieux, Marcy l’Etoile, France), …

Cryptosporidiosis: comparison of three diagnostic methods and effects of storage temperature on detectability of cryptosporidia in cattle faeces

Three diagnostic methods (a modified Ziehl-Neelsen staining technique (MZN), a negative staining with carbol fuchsine (CF) and a commercial enzyme immunoassay (EIA) kit, ProSpecT® Cryptosporidium Microplate Assay (Remel, Lenexa, KS, USA)) for detection of Cryptosporidium oocysts in cattle faeces were compared regarding sensitivity and suitability under routine laboratory conditions, with particular emphasis on sample storage. In the 103 faecal samples examined, cryptosporidia infections were detected significantly more often by EIA (p<0.05; n=76) than by MZN (n=65) if ten random fields were evaluated microscopically, but not if the whole coverslip was scanned. In contrast, sensitivities of EIA and CF (n=69) did not differ significantly. Results were obtained very rapidly by CF. However, the hands-on time of CF is comparable to EIA, while MZN is more time consuming. EIA is more expensive than CF and MZN but easy to perform and to evaluate and does not need considerably experienced staff in contrast to CF and MZN. Moreover, 45 faecal samples stored for up to 27 days at different temperatures (+6°C, +16°C, +30°C, +40°C) were examined. The sensitivity of microscopic detection of oocysts in stained smears (CF, MZN) decreased in a temperature and time-dependent manner, while EIA results were not influenced by sample storage at any temperature.

Rotavirus infection in a tertiary hospital: laboratory diagnosis and impact of immunization on pediatric hospitalization

Background and objectivesRotavirus (RV) is the main etiological agent of diarrhea in childhood; its laboratory diagnosis is crucial to guide the clinical management and prevention of its spread. RV immunization was introduced in Brazilian 6-month-old children in 2006. The present study was aimed to evaluate three methodologies used for human RV detection in stool samples obtained from patients hospitalized due to gastroenteritis in a teaching hospital and report the impact of RV immunization in hospitalization by diarrhea. Methods293 stool samples collected in the 2001-2008 period were analyzed by enzyme immunoassay (EIA), latex agglutination (LA) and polyacrylamide gel electrophoresis (PAGE). ResultsRotavirus was detected in 34.8% of samples by LA assay, 28.3% of samples by EIA assay and in 25.6% of samples by PAGE assay. Considering the PAGE method as gold standard, the sensitivity, specificity and accuracy of EIA were 94.6%, 94.4% and 94.5%, and to LA were 82.6%, 81.6% and 81.9%, respectively. ConclusionsThese results indicate that antigen detection by EIA is a rapid, sensitive and specific method, and could be used in large-scale applications for screening stool samples suspected of RV infection. This study showed decreased incidence of RV infection in hospitalized children prior to the implementation of the national immunization program against RV.

Rapid Stool-Based Diagnosis of Clostridium difficile Infection by Real-Time PCR in a Children's Hospital

Clostridium difficile is a major cause of nosocomial antibiotic-associated infectious diarrhea and pseudomembranous colitis. Detection of C. difficile by anaerobic bacterial culture and/or cytotoxicity assays has been largely replaced by rapid enzyme immunoassays (EIA). However, due to the lack of sensitivity of stool EIA, we developed a multiplex real-time PCR assay targeting the C. difficile toxin genes tcdA and tcdB. Stool samples from hospitalized pediatric patients suspected of having C. difficile-associated disease were prospectively cultured on cycloserine-cefoxitin-fructose agar following alcohol shock. Six testing modalities were evaluated, including stool EIA, culture EIA, and real-time PCR (tcdA and tcdB) of cultured isolates and stool samples. Real-time PCR detection was performed with tcdA and tcdB gene-specific primers and hydrolysis probes using the LightCycler platforms (Roche Diagnostics, Indianapolis, IN). A total of 157 samples from 96 pediatric patients were analyzed. The sensitivities of stool real-time PCR and stool EIA were 95% and 35%, respectively, with a specificity of 100% for both methods. The lower limit of detection of the stool real-time PCR was 30 CFU/ml of stool sample per reaction for tcdA and tcdB. This study highlights the poor performance of stool toxin EIAs in pediatric settings. Direct detection of C. difficile toxin genes in stool samples by real-time PCR showed sensitivity superior to that of stool and culture EIAs and performance comparable to that of real-time PCR assay of cultured isolates. Real-time PCR of DNA from stool samples is a rapid and cost-effective diagnostic modality for children that should facilitate appropriate patient management and halt the practice of serial testing by EIA.

Detection of Cryptococcus by conventional, serological and molecular methods

The rising incidence of cryptococcosis in India is posing a serious threat. Due to lack of sensitive methods for diagnosis, high morbidity and mortality are associated with the disease. Early diagnosis is essential to prevent serious complications. Therefore, we attempted to find highly sensitive and specific detection methods. A comparative evaluation of the detection of cryptococcosis was done by conventional (direct microscopy and culture) and rapid diagnostic [latex agglutination test (LAT), enzyme immunoassay (EIA) and PCR] methods. The study was done on 359 samples from 52 positive patients and 30 negative controls in an Indian set-up. Evaluation was done for cerebrospinal fluid (CSF), serum and urine separately. The diagnostic value of the tests was assessed in pre-treatment samples, and follow-up tests were also done on samples obtained after initiation of treatment. PCR had the highest sensitivity, followed by EIA and LAT, both before and after treatment. The positive detection by LAT, EIA and PCR was the longest in CSF (>90 days), followed by serum ( approximately 65 days) then urine ( approximately 45 days) after initiation of treatment. Our results indicated that the sensitivity and specificity of PCR and EIA were comparable in urine, CSF and serum for diagnosis of cryptococcosis.

Preparation and characterization of monoclonal antibodies to the cholera toxin

Monoclonal antibodies to cholera toxin were obtained. They do not cross-react with the termolabile toxin (LT) of Escherichia coli, ricin, diphtherial toxin, staphylococcus enterotoxins of SEA, SEB, SEI, SEG, or the lethal factor and protective antigen of the anthrax toxin. Pairs of antibodies for the quantitative measurement of the cholera toxin in sandwich enzyme immunoassay (EIA) were selected. The detection limit of the toxin is 0.2 ng/ml for plate EIA and 0.44 ng/ml for microchip EIA. The presence of milk, broth, or surface water in the toxin samples does not reduce the sensitivity of EIA.

Varying abilities of recombinant polypeptides from different regions of hepatitis E virus ORF2 and ORF3 to detect anti‐HEV immunoglobulin M

Following infection with hepatitis E virus (HEV), anti-HEV immunoglobulin (Ig) M is thought to develop before anti-HEV IgG and to be a better marker for differentiating between the acute and convalescent phases of infection. In order to select polypeptides for improved detection of anti-HEV IgM, six and three overlapping polypeptides from open reading frames (ORFs) 2 and 3, respectively, of HEV genotypes 1 and 4 were expressed as fusion proteins in Escherichia coli. The reactivities of the polypeptides with anti-HEV IgM were evaluated using immunoblotting and enzyme immunoassays (EIAs). The data indicated that polypeptides from the N-terminus of ORF3 and middle region of ORF2 were weakly or not reactive with anti-HEV IgM, while those from the remaining regions of ORF2 and ORF3 contained reactive epitopes. Anti-HEV IgM against the N- or C-terminus of ORF2 appeared earlier and disappeared faster than that against polypeptides from the C-terminus of ORF3, based on serum samples from rhesus monkeys infected experimentally, and from patients infected naturally, with HEV. The N- and C-terminal polypeptides from ORF2 complemented one another in detecting anti-HEV IgM and EIA sensitivity was improved significantly with a combination of these polypeptides. The reactivities of ORF2 polypeptides from genotypes 1 and 4 were similar but that of ORF3 differed with sera from monkeys infected by the two genotypes. Thus, a combination of N- and C-terminal polypeptides of ORF2 from one genotype may be effective in EIAs to detect anti-HEV IgM.

Of gastro and the gold standard: evaluation and policy implications of norovirus test performance for outbreak detection

BackgroundThe norovirus group (NVG) of caliciviruses are the etiological agents of most institutional outbreaks of gastroenteritis in North America and Europe. Identification of NVG is complicated by the non-culturable nature of this virus, and the absence of a diagnostic gold standard makes traditional evaluation of test characteristics problematic.MethodsWe evaluated 189 specimens derived from 440 acute gastroenteritis outbreaks investigated in Ontario in 2006–07. Parallel testing for NVG was performed with real-time reverse-transcriptase polymerase chain reaction (RT2-PCR), enzyme immunoassay (EIA) and electron microscopy (EM). Test characteristics (sensitivity and specificity) were estimated using latent class models and composite reference standard methods. The practical implications of test characteristics were evaluated using binomial probability models.ResultsLatent class modelling estimated sensitivities of RT2-PCR, EIA, and EM as 100%, 86%, and 17% respectively; specificities were 84%, 92%, and 100%; estimates obtained using a composite reference standard were similar. If all specimens contained norovirus, RT2-PCR or EIA would be associated with > 99.9% likelihood of at least one test being positive after three specimens tested. Testing of more than 5 true negative specimens with RT2-PCR would be associated with a greater than 50% likelihood of a false positive test.ConclusionOur findings support the characterization of EM as lacking sensitivity for NVG outbreaks. The high sensitivity of RT2-PCR and EIA permit identification of NVG outbreaks with testing of limited numbers of clinical specimens. Given risks of false positive test results, it is reasonable to limit the number of specimens tested when RT2-PCR or EIA are available.

P.057 Development of a duplex real-time PCR (AB rt-PCR) for the detection of two DNA respiratory viruses: AdV and HBoV

Background: Adenovirus-associated hemorrhagic cystitis (HC) has become a recognized sequel of immunosuppression. The diagnosis of viral infection is usually determined by viral cultures. Objectives: Analysis of different diagnostic methods for adenovirus (AdV) detection in bone marrow transplant patients with hemorrhagic cystitis. Study design: We describe a prospective study for AdV detection in the urine of patients with hematuria in the first 100 days after bone marrow transplant (BMT), comparing different laboratory techniques, PCR, enzyme immunoassay (EIA) and conventional culture. Results: A total of 143 urine samples were analyzed, 75 collected in the pre-transplant period with and without hematuria and 68 post-transplant, only with microscopic or macroscopic hematuria. After BMT, hematuria occurred in 38.9% of patients, being more frequent in unrelated donor transplants. AdV was isolated in one pre-transplant patient without symptoms and in three post-transplant patients with HC grades 3 and 4 (severe), who were in month 2 or 3 post-transplant. Compared to culture as the gold standard, the accuracy, specificity and sensitivity of EIA were 95, 30 and 100% and for PCR were 63, 100 and 60%, respectively. Conclusions: We concluded that despite technical difficulties and the long time that elapsed before results were obtained, cell culture still remains the best method for adenovirus detection in the urine of patients with hemorrhagic cystitis.

Triagem neonatal para infecção chagásica congênita: aplicação de análise de classe latente para avaliação dos testes diagnósticos

The present study had the aim of evaluating conventional serum tests that are used in neonatal screening for Chagas disease, with a discussion on the statistical methods available. A random sample among 23,308 newborns who were screened for congenital Chagas disease was studied using the following three tests: enzyme immunoassay, indirect immunofluorescence and indirect hemagglutination. The data were analyzed by different statistical methodologies: latent class analysis, Kappa test and relative sensitivity analysis. Using latent class analysis, enzyme immunoassay had the highest sensitivity (48.6%), followed by indirect immunofluorescence (39.8%) and indirect hemagglutination (23.2%). The kappa value was 0.496. The ratio between the sensitivities of enzyme immunoassays and indirect immunofluorescence tests was 92% [0.74;1.13]. Latent class analysis was not found to be adequate for sensitivity and specificity determination, but it provided important data about the equivalence of the tests, corroborated by relative sensitivity analysis. The results showed that enzyme immunoassaying of dry blood samples can be used as safely as the indirect immunofluorescence test.

A new method of concentrating hepatitis B virus (HBV) DNA and HBV surface antigen: an application of the method to the detection of occult HBV infection

The risk of post-transfusion hepatitis B virus (HBV) infection has been reduced after the implementation of HBV nucleic acid amplification technology (NAT). However, the problem of HBV DNA-positive and HBV surface antigen (HBsAg)-negative occult HBV infections remains to be solved. This is in part due to the HBV DNA load being too low to detect these occult HBV infections using mini-pool NAT. In Japan, the assay for the antibody against the HBV core antigen (anti-HBc) has not completely excluded occult HBV infection. To solve this problem, we have developed a new method of concentrating HBV DNA and HBsAg simultaneously to increase the sensitivity of detection tests. Virus concentration is achieved by the enhancement of the agglutination of viruses using poly-L-lysine in the presence of a bivalent metal. Poly-L-lysine-coated magnetic beads are used to shorten the time of each step of the concentration procedure. Seventy-seven anti-HBc-positive and HBsAg-negative donations were examined. HBsAg and anti-HBc were tested by enzyme immunoassay (EIA) (AxSYM; Abbott) and haemagglutination inhibition test (Japanese Red Cross), respectively. HBV surface antigen and HBV DNA levels were concentrated up to four- to sevenfold. Using this method, 35 of the 77 anti-HBc-positive and HBsAg-negative donors were HBV DNA-positive by individual NAT and a further five donors became HBV DNA-positive by HBV concentration. Twenty-seven of 40 occult HBV infections became HBsAg-positive by HBsAg concentration. Our new method of concentrating HBV and HBsAg increased the sensitivities of EIA and HBV NAT, and enabled us to detect 27 of 40 occult HBV infections by HBsAg EIA.

Laboratory testing for hepatitis C

Serological diagnosis of patients infected with the hepatitisC virus (HCV) can be performed using two categories of tests:indirect tests, which detect antibodies against HCV; and directtests, which detect, quantify, or characterize components ofthe viral particle, such as HCV RNA testing and testing fordetection of the HCV core antigen.Anti-HCV antibodies are usually detected using third- andfourth-generation immunoenzymatic assays – enzymeimmunoassay (EIA)/enzyme-linked immunosorbent assay(ELISA) 3 and EIA/ELISA 4, respectively – which containHCV core antigens and HCV nonstructural genes. Thespecificity of the EIA tests available on the market that detectanti-HCV was determined to be higher than 99%, whereastheir sensitivity, which was more difficult to determine due tothe lack of gold standard tests with high sensitivity, was 95-99% [1]. However, false-positive results for anti-HCV canoccasionally occur, especially in populations with prevalencerates below 10% [2-4].There are many reasons why laboratories do not routinelyuse a supplementary test based on immunoblot analysis, suchas the recombinant immunoblot assay, to complement thediagnosis of HCV infection. In addition to the high cost ofsuch a test, the lack of laboratory standards that can evaluateits performance and interpretation, in conjunction with itsactual accuracy, is among the principal reasons. Furthermore,this type of test does not distinguish past from presentinfection, and its use is only indicated for confirmation of EIAresults.In contrast, the use of nucleic acid testing (NAT) makes itpossible to differentiate between viremic and nonviremicindividuals by detection of HCV RNA, allowing the clinician adifferentiated approach to anti-HCV-positive individuals.However, there can be situations in which HCV RNA is notdetected (negative HCV RNA) and the individual has activeinfection with HCV. This can occur in individuals in whomanti-HCV antibody titers are high and RNA titers are low [5].Therefore, HCV RNA might not be detectable in certainindividuals in the acute phase of the disease. However, thesefindings are transient, and chronic infection can develop [6].In addition, HCV RNA intermittent positivity has beenobserved in individuals chronically infected with HCV [6-8].Negativity of HCV RNA results can indicate resolved infection.In 15 to 25% of those anti-HCV positive individuals whoacquired the infection after 45 years of age, the infectionresolves spontaneously. This percentage increases to 40-45%in those who acquired the HCV infection in childhood oryoung adulthood [9].Different tests based on polymerase chain reaction (PCR)have been developed to directly detect the viral particle. Onecharacteristic of real-time PCR is amplification coupled withdetection, which allows the evaluation of the number of viralgenomes at the onset of and throughout the reaction.Qualitative detection of HCV RNA by reverse transcriptase(RT)-PCR is generally accepted as the most sensitive andstandardized test to date [10,11]. Nevertheless, there is variabilityamong the results from different laboratories, as evidenced bythe use of international panels of proficiency. The accuracyand reliability of the results are directly related to the laboratoryprocedures adopted in the performance of the tests [12]. Thelack of preliminary care in sample collection, in conjunctionwith the time involved in preparing and separating the samples,can result in incorrect results. It is extremely important that alllaboratory procedures comply with Good Laboratory Practiceand strictly follow the protocols standardized by themanufacturers of the diagnostic kits and reagents.The gold standard consists of the careful use of NAT,standardized for detection of HCV RNA, together with EIAs(specificity in conjunction with sensitivity).An alternative to aid diagnosis is the use of the ratiobetween optical density and cut-off value (OD/COV) or thesample/cut-off ratio as an indicator of the true positivity ofthe test. Studies carried out in Brazil show that, in EIAs,reagents with OD/COV greater than 3 are repeatedly associatedwith 100% true-positive results (positive predictive value) andpresent approximately 92% positivity for HCV RNA by RT-PCR [13]. In terms of the population studied, the positivepredictive value is increased when accompanied by riskfactors, high levels of alanine aminotransferase (ALT), or liverdisease.In immunocompetent patients, EIAs present excellentreproducibility; however, in hemodialyzed orimmunocompromised patients, EIA sensitivity is significantlyreduced [14].In low-risk populations, such as blood donors, or inrandom population screening, i.e., in populations that do notpresent risk factors for the acquisition of HCV infection,negative EIA results are sufficient to rule out the presence ofHCV. However, false-positive results can occur in thesepopulations. In such cases, a qualitative study of HCV RNAshould be performed to confirm the diagnosis.In high-risk populations, when there is clinical suspicion ofHCV infection, positive EIA results confirm the exposure toHCV. A qualitative study of HCV RNA should be performed todistinguish individuals with chronic infection from those whohave eliminated the HCV spontaneously.In patients with chronic hepatitis of unknown cause andnegative anti-HCV EIA results, especially inimmunocompromised patients [14], a qualitative study of HCVRNA should be performed. The presence of HCV RNA confirmsthe diagnosis, although a negative result does not rule outHCV infection. In such cases, it is recommended that a newHCV RNA study be performed six months after the first study.Detection of the HCV core antigen by EIA can be an alternativefor early diagnosis of HCV infection.