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Concept: Serology

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BACKGROUND: The anti-JC virus (JCV) antibody status has been introduced to stratify patients with multiple sclerosis (MS) for higher or lower risk of progressive multifocal leukoencephalopathy (PML). OBJECTIVE: To assess the potential utility of anti-JCV antibody levels for earlier diagnosis or prediction of PML. METHODS: An analytically validated antibody assay was used to determine serological status, normalised optical density values, and dilution titres for anti-JCV antibodies. The method was applied to stored sera of 1157 patients with MS including five cases of PML, all enrolled in the Swedish pharmacovigilance study for natalizumab (NAT). Anticytomegalovirus (CMV) and antivaricella-zoster (VZV) antibody levels served as controls. RESULTS: Prior to treatment with NAT, anti-JCV antibody levels were stable in the anti-JCV positive patients. During therapy, a slight decrease in anti-JCV and anti-VZV antibody levels, but not anti-CMV antibody levels, was observed. All five patients who developed PML showed a mild to moderate increase in anti-JCV antibody levels at time of PML diagnosis; pre-PML samples suggested that this increase might start already prior to diagnosis of PML. CONCLUSIONS: Treatment initiation with NAT may lead to a slight decrease in anti-JCV and anti-VZV antibody levels, suggestive of a mild suppressive effect of NAT on antibody levels. Our findings in five cases of PML demonstrate that the onset of PML can be accompanied by increasing anti-JCV antibodies in serum. Monitoring of anti-JCV antibody levels could potentially be used as a tool for prediction or earlier diagnosis of PML during NAT treatment for MS. Further studies are warranted.

Concepts: Antibody, Density, Multiple sclerosis, ELISA, Progressive multifocal leukoencephalopathy, JC virus, Serology, Natalizumab

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The aim of this study was to evaluate if the use of antitransglutaminase (tTG) and antiendomysium (EM) antibodies has modified the profile of coeliac disease (CD) in children.

Concepts: Cohort study, Epidemiology, Serology

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BACKGROUND: With declining vectorial transmission, Chagas disease predominantly affects adults nowadays. The efficacy of nifurtimox in the chronic phase in adult patients is poorly known, particularly in regions where there is no risk of reinfection. Recommendations for treatment outcome assessment rely on serological follow-up. We evaluated the serological and parasitological response to nifurtimox in a cohort of adult patients three years post-treatment in Switzerland. METHODS: Patients treated with nifurtimox in 2008 during a cross-sectional study in Geneva, Switzerland, were contacted for follow-up in 2011. Two ELISAs and a rapid immunochromatographic test were used to test 2008 and 2011 serum samples simultaneously. In addition, conventional and real-time PCR were performed on 2011 samples. RESULTS: Thirty-seven (84.1%) of 44 eligible patients, predominantly female, middle-aged, Bolivians at the indeterminate stage, were enrolled. All 2011 ELISA and immunochromatographic tests were positive. Twenty-eight (75.7%) patients presented a lower optical density (OD) in 2011 compared to 2008. This OD difference was significant in both commercial (P < 0.001) and in-house (P = 0.002) ELISAs. Agreement between the two ELISAs was low (Kappa = 0.469). All patients had negative conventional PCR results but one (2.7%) was positive with real-time PCR. CONCLUSION: Our results highlight the inadequacy of serology for assessing response in adults, three years after treatment. In our cohort, 97.3% had results that could either indicate treatment failure or persistant humoral response despite treatment. The lack of accurate early post-treatment tests of cure prevents appropriate patients information and councelling. New follow-up tests are needed to assess treatments efficacy given the large adult population in need of antiparasitic therapy.

Concepts: Antibody, Epidemiology, Infectious disease, Polymerase chain reaction, Chagas disease, Serology, Antiparasitic, Nifurtimox

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BACKGROUND: Australia uses a protocol combining human rabies immunoglobulin (HRIG) and rabies vaccine for post-exposure prophylaxis (PEP) of rabies and Australian bat lyssavirus (ABLV), with the aim of achieving an antibody titre of ≥0.5 IU/ml, as per World Health Organization (WHO) guidelines, as soon as possible. METHODOLOGYPRINCIPAL FINDINGS: We present the course of PEP administration and serological testing for four men with complex requirements. Following dog bites in Thailand, two men (62 years old, 25 years old) received no HRIG and had delayed vaccine courses: 23 days between dose two and three, and 18 days between dose one and two, respectively. Both seroconverted following dose four. Another 62-year-old male, who was HIV-positive (normal CD4 count), also suffered a dog bite and had delayed care receiving IM rabies vaccine on days six and nine in Thailand. Back in Australia, he received three single and one double dose IM vaccines followed by another double dose of vaccine, delivered intradermally and subcutaneously, before seroconverting. A 23-year-old male with a history of allergies received simultaneous HRIG and vaccine following potential ABLV exposure, and developed rash, facial oedema and throat tingling, which was treated with a parenteral antihistamine and tapering dose of steroids. Serology showed he seroconverted following dose four. CONCLUSIONSSIGNIFICANCE: These cases show that PEP can be complicated by exposures in tourist settings where reliable prophylaxis may not be available, where treatment is delayed or deviates from World Health Organization recommendations. Due to the potentially short incubation time of rabies/ABLV, timely prophylaxis after a potential exposure is needed to ensure a prompt and adequate immune response, particularly in patients who are immune-suppressed or who have not received HRIG. Serology should be used to confirm an adequate response to PEP when treatment is delayed or where a concurrent immunosuppressing medical condition or therapy exists.

Concepts: HIV, Immune system, Antibody, Rabies, Serology, Mononegavirales, Lyssavirus, Seroconversion

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Infection with Leishmania donovani is typically asymptomatic, but a significant number of individuals may progress to visceral leishmaniasis (VL), a deadly disease that threatens 200 million people in endemic areas. While diagnosis of acute VL has been simplified by the use of cost-effective confirmatory serological tests, similar standardized tools are not widely available for detecting asymptomatic infection which can be 4-20 times more prevalent than active disease. A simple and accurate serological test capable of detecting asymptomatic L. donovani infection will be useful for surveillance programs targeting VL control and elimination. To address this unmet need, we evaluated recombinant antigens for their ability to detect serum antibodies in 104 asymptomatic L. donovani infected individuals (qualified as positive for L. donovani-specific antibodies by direct agglutination test; DAT) from the VL hyperendemic Mymensingh district of Bangladesh. The novel proteins rKR95 and rTR18 possessed the greatest potential and detected 69% of DAT positive individuals, with rKR95 being more robust in reactivity. Agreement in results with individuals with high DAT responses, who are more likely to progress to VL disease, was 74%. When considered along with rK39, a gold standard antigen used to confirm clinical diagnosis of VL but which is now becoming widely used for surveillance, rKR95 and rTR18 conferred a sensitivity of 84% based on a theoretical combined estimate. Our data indicate that incorporating rKR95 and rTR18 with rK39 in serological tests amenable to rapid or high-throughput screening could enable simple and accurate detection of asymptomatic infection. Such tests will be important tools to measure L. donovani infection rates, a primary goal in surveillance and a critical measurement with which to assess elimination programs.

Concepts: Immune system, Antibody, Epidemiology, Leishmaniasis, Visceral leishmaniasis, Leishmania, Serology, Direct agglutination test

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A field trial was conducted to investigate the impact of oral vaccination of free-living badgers against natural-transmitted Mycobacterium bovis infection. For a period of three years badgers were captured over seven sweeps in three zones and assigned for oral vaccination with a lipid-encapsulated BCG vaccine (Liporale-BCG) or with placebo. Badgers enrolled in Zone A were administered placebo while all badgers enrolled in Zone C were vaccinated with BCG. Badgers enrolled in the middle area, Zone B, were randomly assigned 50:50 for treatment with vaccine or placebo. Treatment in each zone remained blinded until the end of the study period. The outcome of interest was incident cases of tuberculosis measured as time to seroconversion events using the BrockTB Stat-Pak lateral flow serology test, supplemented with post-mortem examination. Among the vaccinated badgers that seroconverted, the median time to seroconversion (413 days) was significantly longer (p = 0.04) when compared with non-vaccinated animals (230 days). Survival analysis (modelling time to seroconversion) revealed that there was a significant difference in the rate of seroconversion between vaccinated and non-vaccinated badgers in Zones A and C throughout the trial period (p = 0.015). For badgers enrolled during sweeps 1-2 the Vaccine Efficacy (VE) determined from hazard rate ratios was 36% (95% CI: -62%- 75%). For badgers enrolled in these zones during sweeps 3-6, the VE was 84% (95% CI: 29%- 97%). This indicated that VE increased with the level of vaccine coverage. Post-mortem examination of badgers at the end of the trial also revealed a significant difference in the proportion of animals presenting with M. bovis culture confirmed lesions in vaccinated Zone C (9%) compared with non-vaccinated Zone A (26%). These results demonstrate that oral BCG vaccination confers protection to badgers and could be used to reduce incident rates in tuberculosis-infected populations of badgers.

Concepts: Immune system, Vaccine, Vaccination, Tuberculosis, Mycobacterium, Mycobacterium bovis, Bacillus Calmette-Guérin, Serology

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Bovine besnoitiosis is considered an emerging chronic and debilitating disease in Europe. Many infections remain subclinical, and the only sign of disease is the presence of parasitic cysts in the sclera and conjunctiva. Serological tests are useful for detecting asymptomatic cattle/sub-clinical infections for control purposes, as there are no effective drugs or vaccines. For this purpose, diagnostic tools need to be further standardized. Thus, the aim of this study was to compare the serological tests available in Europe in a multi-centred study. A coded panel of 241 well-characterized sera from infected and non-infected bovines was provided by all participants (SALUVET-Madrid, FLI-Wusterhausen, ENV-Toulouse, IPB-Berne). The tests evaluated were as follows: an in-house ELISA, three commercial ELISAs (INGEZIM BES 12.BES.K1 INGENASA, PrioCHECK Besnoitia Ab V2.0, ID Screen Besnoitia indirect IDVET), two IFATs and seven Western blot tests (tachyzoite and bradyzoite extracts under reducing and non-reducing conditions). Two different definitions of a gold standard were used: (i) the result of the majority of tests (‘Majority of tests’) and (ii) the majority of test results plus pre-test information based on clinical signs (‘Majority of tests plus pre-test info’). Relative to the gold standard ‘Majority of tests’, almost 100% sensitivity (Se) and specificity (Sp) were obtained with SALUVET-Madrid and FLI-Wusterhausen tachyzoite- and bradyzoite-based Western blot tests under non-reducing conditions. On the ELISAs, PrioCHECK Besnoitia Ab V2.0 showed 100% Se and 98.8% Sp, whereas ID Screen Besnoitia indirect IDVET showed 97.2% Se and 100% Sp. The in-house ELISA and INGEZIM BES 12.BES.K1 INGENASA showed 97.3% and 97.2% Se; and 94.6% and 93.0% Sp, respectively. IFAT FLI-Wusterhausen performed better than IFAT SALUVET-Madrid, with 100% Se and 95.4% Sp. Relative to the gold standard ‘Majority of test plus pre-test info’, Sp significantly decreased; this result was expected because of the existence of seronegative animals with clinical signs. All ELISAs performed very well and could be used in epidemiological studies; however, Western blot tests performed better and could be employed as a posteriori tests for control purposes in the case of uncertain results from valuable samples.

Concepts: Antibody, Epidemiology, Infection, Type I and type II errors, Apicomplexa, Western blot, Transmission and infection of H5N1, Serology

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Fourth-generation human immunodeficiency virus-1 (HIV-1) screening assays have improved sensitivity, but vary in performance characteristics. The purpose of this study was to evaluate four different fourth-generation HIV-1 assays. These assays included the AxSYM HIV Ag/Ab Combo (Abbott diagnostics, Delkenheim, Germany), ARCHITECT HIV Ag/Ab Combo (Abbott), Elecsys 2010 HIV Combi (Roche Diagnostics GmbH, Mannheim, Germany), and Elecsys HIV Combi PT (Roche). A total of 1,306 samples that included 1,225 clinical samples and 81 samples consisting of seroconversion panels, an HIV-1 p24 antigen sensitivity panel, and dilution series of HIV-1 lysates and HIV-1 antibodies were tested. All of the assays had sensitivities of 100% on clinical samples. The specificities of the AxSYM, ARCHITECT, Elecsys 2010 HIV Combi, and Elecsys HIV Combi PT were 99.6, 99.6, 99.0, and 99.5%, respectively. Of the 81 samples with different levels of HIV antigen or antibody and/or subtypes, Elecsys HIV Combi PT and ARCHITECT HIV Ag/Ab Combo showed better analytical sensitivities than the other two assays. In summary, the performance characteristics of AxSYM, ARCHITECT, and Elecsys HIV Combi PT were comparable and satisfactory for clinical samples. ARCHITECT HIV Ag/Ab Combo and Elecsys HIV Combi PT have the higher analytical sensitivities, and would be preferable for reducing the window period. J. Med. Virol. 84:1884-1888, 2012. © 2012 Wiley Periodicals, Inc.

Concepts: HIV, Immune system, Antibody, HIV/AIDS, Subtypes of HIV, ELISA, Serology, Roche Diagnostics

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BACKGROUND: Primary CMV infections in pregnancy are usually asymptomatic and only detected by serology. Estimating the onset of infection is a major diagnostic goal, since primary infections around conception and in early gestation hold a higher risk for congenital disease than those in later pregnancy. OBJECTIVES: To assess the ability of serological supplementary CMV assays to date the onset of primary infection. STUDY DESIGN: From our routine diagnosis we identified 61 pregnant women (n=188 serum samples) with precisely determined onset of CMV primary infection either by IgG seroconversion (n=24) or by significant IgG antibody rise (n=37). One hundred and forty-seven sera were investigated using the VIDAS(®) CMV IgG avidity EIA (BioMèrieux) and 83 sera using the recomBlot CMV IgG with avidity (Mikrogen). RESULTS: Both assays proofed to be reliable in terms of timing the onset of CMV primary infection. An avidity index (AI) in the VIDAS avidity EIA of <40% indicated primary infection within the last 20 weeks (positive predictive value 93.4%; 99/106), whereas an intermediate AI excluded primary infection within the last 12 weeks (negative predictive value 88.2%; 15/17). The recomBlot showed high reliability (PPV 96.9%; 31/33) for timing the onset of infection within the last 14 weeks. Avidity testing by blot however could not be interpreted in 11 of 47 sera (23.4%). CONCLUSION: For timing the onset of infection (before or in early pregnancy) CMV avidity testing is most helpful if carried out within the first trimester up to the beginning of second trimester.

Concepts: Immune system, Antibody, Pregnancy, Cytomegalovirus, Serology

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Diagnosis of active Helicobacter pylori infection in intellectually disabled (ID) children is problematic because they are unable to cooperate with performance of invasive tests. In this study, the non-invasive methods of measuring serum IgG antibody concentrations and performing stool antigen tests were used to screen for H. pylori infection in ID children. Eighty-seven children with intellectual disabilities were studied. The amount of serum IgG antibody against H. pylori was measured by the ELISA method. Stool samples were examined using an amplified IDEIA HpStAR kit. To assess categorical variables, X(2) , Fisher’s exact and Kappa tests were used. The stool antigen tests showed that 93.1% of the children had H. pylori antigen and the serology test that 85.1% of children were positive for H. pylori IgG antibodies. Agreement between results of H. pylori stool antigen (HpSA) testing and IgG antibody serology was 82.8%; however, according to the kappa measure of agreement this agreement is not statistically significant (value, 0.128; P = 0.19). Discordant results were observed for 15 children (17.2%): 11 (12.6%) who were positive on HpSA test but negative by serology and 4 (4.6%) who were IgG seropositive but had negative HpSA tests. This study showed a notably higher rate of H. pylori infection in ID children than has been reported by others for non-ID children from the same geographical area. The HpSA test is a valid method for primary screening for H. pylori infection in ID children; it detects the specific antigens shed during active infections and has less cross-reactivity than serological tests that detect antibodies. HpSA is a sensitive non-invasive method for detecting infection in ID children and may serve as an accurate alternative to serology.

Concepts: Immune system, Antibody, Bacteria, Fc receptor, Gastroenterology, ELISA, Helicobacter pylori, Serology