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Recent studies demonstrated that human papillomavirus (HPV) specific immunoglobulins (IgG) are present and detectable in non-invasively collected first-void urine (FVU) samples. As IgG levels in urine are low, we evaluated the potential of a highly sensitive HPV16-specific assay based on time-resolved fluorescence, DELFIA, and compared it with three immunoassays, GST-L1-MIA, M4ELISA, and M9ELISA. A total of 225 paired serum and FVU samples from two cohorts of healthy female volunteers were analyzed. Strong Spearman rank correlations between HPV16-specific IgG results measured with DELFIA, M4ELISA, GST-L1-MIA, and M9ELISA were found for both sample types (r s > 0.80). Additionally, total human IgG results, determined in all samples using HTRF human IgG kit and BioPlex Pro™ Human Isotyping Assay, were compared. Moderate correlations between total human IgG concentrations in FVU samples were found for the two total IgG assays (r s ≥ 0.42, p < 0.0001), while correlations for serum were non-significant. In conclusion, the HPV16-DELFIA assay is usable for detecting HPV16-specific antibodies in FVU and serum samples. As total human IgG remains an interesting parameter for the normalization of HPV-specific IgG in FVU, the accuracy of both assays needs to be validated further., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests. A.V. is a co-founder and former board member of Novosanis (Subsidiary of OraSure Technologies Inc, Wijnegem, Belgium), a spin-off company of the University of Antwerp, and was a minority shareholder until January 2019. The University of Antwerp received grants from Merck, GSK, Hologic, Abbott, Roche, and Cepheid to support the HPV Prevention and Control Board. The University of Antwerp received a project grant and honoraria fee for lectures, presentations, and speaker bureaus from Merck. Other authors declare that they have no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

First-void urine (FVU) samples, containing human papillomavirus (HPV)-specific IgG from female genital tract secretions, provide a non-invasive option for disease monitoring and vaccine impact assessment. This study explores the utility of FVU for IgG quantification, exploring stability and compatibility with DNA preservation methods, alongside various IgG enrichment methods. Healthy female volunteers provided FVU and serum samples. FVU was collected with or without urine conservation medium (UCM) and stored under different conditions before freezing at -80 °C. Four IgG enrichment methods were tested on FVU samples. All samples were analyzed using three total human IgG quantification assays and an in-house HPV16-specific IgG assay. Samples stored with UCM buffer had higher total and HPV16-specific IgG concentrations (p ≤ 0.01) and IgG remained stable for at least 14 days at room temperature. Among IgG enrichment methods, Amicon filtration (AM) and AM combined with Melon Gel purification (AM-MG) provided similar HPV16-IgG concentrations, correlating strongly with serum levels. Protein G magnetic beads methods were incompatible with time-resolved fluorescence-based assays. This study highlights FVU as a reliable and convenient sample for IgG quantification, demonstrating stability for at least 14 days at room temperature and compatibility with UCM DNA preservation. It emphasizes the need to select appropriate IgG enrichment methods and confirms the suitability of both AM and AM-MG methods, with a slightly better performance for AM-MG., (© 2024. The Author(s).)

Vaccine-induced human papillomavirus (HPV) antibodies originating from cervicovaginal secretions were recently shown to be detectable in first-void (FV) urine. This presents a novel opportunity for noninvasive sampling to monitor HPV antibody status in women participating in large epidemiological studies and HPV vaccine trials. With a view towards method optimization, this study compared the measurement of HPV antibodies in FV urine using a multiplex L1/L2 virus-like particles (VLP)-based ELISA (M4ELISA) with previously reported results using a glutathione S-transferase (GST)-L1-based immunoassay (GST-L1-MIA). We tested 53 paired FV urine and serum samples from 19- to 26-year-old healthy women, unvaccinated (n = 17) or vaccinated with either the bivalent or quadrivalent HPV-vaccine during adolescence (n = 36). HPV6/11/16/18 antibodies were measured using M4ELISA and compared with GST-L1-MIA results. Inter-assay and inter-specimen correlations were examined using the Spearman's rank test (rs). As expected, lower HPV antibody concentrations were found in FV urine than in serum. Vaccinated women had significantly higher HPV6/11/16/18 antibody levels in both FV urine and serum compared with those unvaccinated (M4ELISA; FV urine P = .0003; serum P ≤ .0001). HPV antibody levels in FV urine and serum showed a significant positive correlation (M4ELISA anti-HPV6/11/16/18, r s  = 0.85/0.86/0.91/0.79, P ≤ .001). Despite assay differences, there was moderate to good correlation between M4ELISA and GST-L1-MIA (FV urine anti-HPV6/11/16/18, r s  = 0.86/0.83/0.89/0.53, P ≤  .0001; serum anti-HPV6/11/16/18, r s  = 0.93/0.89/0.94/0.75, P ≤ .0001). FV urine HPV antibody detection is comparable with both assays, further supporting this noninvasive sampling method as a possible option for HPV vaccine assessment. Approaches to improve the sensitivity and larger studies are warranted to determine the feasibility of FV urine for vaccine-induced HPV antibody detection., (© 2020 The Authors. Journal of Medical Virology published by Wiley Periodicals, Inc.)

The potential of first-void (FV) urine as a non-invasive method to monitor human papillomavirus (HPV) vaccination has been reported, mainly focusing on urine as a sample to assess HPV DNA. Besides HPV DNA, vaccine-induced HPV antibodies originating from cervicovaginal secretions were recently shown to be detectable in FV urine as well. This presents a novel opportunity for non-invasive sampling to monitor HPV antibody status in women participating in large epidemiological studies and HPV vaccine trials. The simultaneous assessment of both HPV infection and immunogenicity on a non-invasive, readily obtained sample is particularly attractive., (Copyright © 2020 Pattyn, Van Keer, Téblick, Van Damme and Vorsters.)

IntroductionCrimean-Congo haemorrhagic fever (CCHF) is a tick-borne disease in Africa, Asia, the Balkan peninsula, the south-east of Europe and the Middle East, with mortality rates of 3-30%. Transmission can also occur through contact with infected animals or humans.AimThis observational, prospective case series aimed to investigate detectable viral genomic RNA in whole-body fluids and antibody dynamics in consecutive daily samples of patients diagnosed with CCHF until discharge from hospital.MethodsWe tested 18 patients and 824 swabs and sera with RT-PCR and 125 serum samples serologically.ResultsThe longest duration until clearance of viral RNA was 18 days from serum collection and 18, 15, 13, 19 and 17 days, respectively, from nasal, oral, genital (urethral or vaginal) and faecal swab, and urine. In seven patients, viral load decreased in serum at the same time as it increased in urine or persisted at the same logarithmic values. Despite clearance in serum, viral RNA was detected in faeces and genital swabs in two and three patients, respectively. Viral clearance from body fluids occurred earlier than from serum in eight patients on ribavirin treatment. The shortest seroconversion time was 3 days after symptom onset for IgM and IgG. Seroconversion of IgG occurred until Day 14 of symptoms.ConclusionWe report persistence of viral RNA in urine, faeces and genital swabs despite serum clearance. This may indicate a need for extending isolation precautions, re-evaluating discharge criteria and transmission risk after discharge, and considering oral swabs as a less invasive diagnostic alternative.

Background: Dengue is an important arboviral disease with about 100 million dengue cases per year, of which, ~5% result in severe disease. Clinical differentiation of dengue from other acute febrile illnesses (AFI) is difficult, and diagnostic blood tests are costly. We evaluated the utility of anti-DENV IgM in urine to identify dengue cases among AFI patients enrolled in a clinical study., Methods: Between May 2012-March 2013, 1538 study participants with fever for ≤7 days were enrolled, a medical history was obtained, and serum and urine specimens were collected. Serum was tested for DENV RNA and anti-DENV IgM. Urine was tested for anti-DENV IgM, and its sensitivity and specificity to detect sera laboratory-positive dengue cases were calculated. We evaluated if urine anti-DENV IgM positivity early (≤5 days post-illness onset [DPO]) and late (6-14 DPO) in the clinical course was associated with dengue severity., Results: Urine anti-DENV IgM sensitivity and specificity were 47.4% and 98.5%, respectively, when compared with serum anti-DENV IgM ELISA results, and 29.7% and 91.1% when compared with serum rRT-PCR results. There was no correlation between urine anti-DENV IgM positivity and patient sex or pre-existing chronic disease. Early in the clinical course, a significantly higher proportion of those who developed dengue with warning signs had anti-DENV IgM in their urine when compared to those without warning signs (20.4% vs. 4.3%). There was no difference in the proportion with urine anti-DENV IgM positivity between severity groups late in the clinical course., Conclusion: While detection of urine anti-DENV IgM lacked adequate diagnostic sensitivity, it is a highly specific marker for laboratory-positive dengue, and its presence early in the clinical course may distinguish those with more severe disease. Further assessment of urine anti-DENV IgM by DPO is warranted to determine its utility as an early diagnostic (and possibly prognostic) marker for dengue., Competing Interests: The authors have declared that no competing interests exist.

Rapid point-of-care testing is a megatrend in infectious disease diagnosis. We have introduced a homogeneous immunoassay concept, which is based on the simultaneous binding of antigen and protein L to a given immunoglobulin molecule. The complex formation is detected utilizing time-resolved Förster resonance energy transfer between antigen-attached donor and acceptor-labeled protein L, hence the name LFRET. Here, we demonstrate that urine can be used as a sample matrix in LFRET-based serodiagnostics. We studied urine samples collected during the hospitalization and recovery of patients with acute Puumala orthohantavirus (PUUV) infection. We compared PUUV antibody-specific LFRET signals in urine to those in plasma, and found excellent correlation in the test outcomes The LFRET test from urine was positive in 40/40 patients with acute PUUV infection. PUUV causes a mild form of hemorrhagic fever with renal syndrome, characterized by acute kidney injury and proteinuria. Immunofluorescence and western blotting demonstrated PUUV-IgG and -IgA in urine, however, the presence of intact immunoglobulins did not fully explain the LFRET signals. We purified free light chains (FLCs) from both urine and serum of healthy volunteers and patients with acute PUUV infection, and verified the presence of antigen-specific FLCs. Antigen-specific FLCs provide a new means for non-invasive antibody detection and disease diagnosis., Competing Interests: The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. Some of the authors are listed as inventors in a patent (EP3111218B1, https://patents.google.com/patent/EP3111218B1/en) owned by University of Helsinki.

Background: Monitoring HPV antibodies non-invasively would be a major advantage for large epidemiological studies and follow-up of vaccinees., Objectives: This study investigated the presence of HPV-specific antibody transudates from systemic circulation in first-void urine of (un)vaccinated subjects and the agreement with paired sera., Study Design: In this case-control study, 55 paired first-void urine and serum samples were included from 19- to 26-year-old women, unvaccinated (n = 19) or vaccinated (n = 36) with the bi- or quadrivalent HPV vaccine during adolescence (NCT02714114). Human IgA, total human IgG, and HPV6/11/16/18-Ig(M/G/A) were measured in paired samples., Results: Significant positive Spearman rank correlations (r s ) were found in HPV-specific antibody levels between paired samples (HPV6: r s  = 0.777; HPV11: r s  = 0.757; HPV16: r s  = 0.876; HPV18: r s  = 0.636 (p < 0.001)). In both first-void urine and serum, significantly higher HPV6/11/16/18 antibody levels were observed in vaccinated compared with unvaccinated women (p ≤ 0.017)., Conclusions: The present study provides the first proof that vaccine-induced HPV antibodies are detectable in the first-void urine of young women. Moreover, significant positive correlations were observed between HPV6/11/16/18-antibodies in first-void urine and paired sera. Further optimization and validation are required to demonstrate its potential use in epidemiological studies and follow-up of HPV vaccination., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: A range of different neurological manifestations has been reported in fetuses and adults after Zika virus (ZIKV) infection., Objective: We describe a detection of the ZIKV in the brain tissue from a multiple sclerosis (MS) patient with acute disseminated encephalomyelitis (ADEM)-like event in Rio de Janeiro, Brazil., Methods: Biological samples collected during the hospitalization were tested by serology and molecular diagnostic for various infectious agents. Histopathological analysis was performed using the anti-flavivirus group 4G2 monoclonal antibody, anti-ZIKV non-structural 1 (NS1) monoclonal antibody, and anti-CD4, CD8, and CD11b antibodies., Results: Anti-ZIKV IgM and IgG antibodies were positive in the serum and urine. A brain biopsy showed ZIKV protein in brain cells and T CD8 infiltration in brain tissue., Conclusion: Our data describe the coexistence of a recent central nervous system (CNS) ZIKV infection accompanied by a severe ADEM-like syndrome outcome in a patient with clinical history of MS. A de novo immune response concomitant with ZIKV infection might be involved in the mechanism of the ADEM-like syndrome and response to immunotherapy. The present report reinforces the importance of providing the differential diagnosis of acute episodes of MS exacerbation in an environment prone to ZIKV expression.

A 27-year-old pregnant woman who was positive for anti-cytomegalovirus (CMV) antibodies gave birth to a congenitally CMV-infected neonate at 40 weeks of gestation. According to strain-specific serological analysis, which is able to determine the two types of CMV glycoprotein H (gH), the mother possessed anti-gH(To) antibodies only, but the neonate possessed anti-gH(AD) and anti-gH(To) antibodies at 4 weeks after birth. As the anti-gH(To) IgG was decreased in the neonate at 8 months post-delivery, these antibodies are thought to have been transferred from the mother as maternal antibodies. The anti-gH(AD) IgG level was maintained in the child even after 8 months post-delivery. Congenital infection with a CMV gH(AD) type strain was confirmed by strain-specific real-time PCR using a urine specimen from the child. On the other hand, anti-gH(AD) IgG was not detected even after 8 months post-delivery in a maternal specimen. The mother only produced antibodies against the CMV strain identified as the primary infection, which is characteristic of original antigenic sin., (Copyright © 2018 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Objective: We evaluated cytomegalovirus (CMV) immunoglobulin M (IgM) titer in pregnant women with primary infection as a predictive factor for congenital infection., Study Design: Maternal CMV antibody screening during the first trimester was conducted prospectively at 16 centers in Japan between September 2013 and 2015. Women with confirmed maternal primary infection underwent testing for fetal congenital infection, and we investigated the positive predictive value of CMV IgM titer levels for congenital infection in women with a low IgG avidity., Results: We identified 6 (8.6%) cases of congenital infection among 70 pregnant women with positive/borderline IgG, positive IgM and IgG avidity index ⩽35.0% and 11 (39.3%) among 28 women with IgG and/or IgM seroconversion. IgM titer level ⩾6.00 index showed the highest positive predictive value (17.1%)., Conclusion: High titer of CMV IgM during the first trimester in pregnant women with primary infection is a risk factor for congenital infection.

Natalizumab greatly reduces inflammatory relapses in multiple sclerosis (MS) by blocking the integrin-mediated leukocyte traffic to the brain, but less is known about its effects on the systemic immunity. We measured 48 cytokines/chemokines in sera from 19 natalizumab-treated MS patients. Serum concentrations of both anti-(IL-10, IL1ra) and pro-inflammatory (IL7, IL16) molecules decreased after 21-month treatment, without associations to unbalanced Th2/Th1cytokine ratios, clinical responses, and blood/urine replication of polyomavirus JC (JCPyV). No patient developed the JCPyV-related progressive multifocal leukoencephalopathy (PML), the major risk factor of natalizumab therapy. Our data suggest that natalizumab has marginal impact on the systemic immunity., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Sixty-three natalizumab-treated patients with relapsing multiple sclerosis were screened for JC polyomavirus (JCV) viruria. Urinary-positive patients were longitudinally sampled for up to 24 weeks. Using methods that distinguish encapsidated virus from naked viral DNA, 17.5 % of patients were found to excrete virus, consistent with the prevalence of urinary excretion in the general population. Unexpectedly, urinary excretion was predominantly seen (>73 %) in patients with high JC antibody index (≥2.0). Active JCV infection, therefore, tends to occur in natalizumab patients that carry a high risk factor for the development of disease, directly linking JC infection to the risk factors for PML development., Competing Interests: M.H.W. is the president and chief executive officer of Inhibikase Therapeutics, Inc. and a significant shareholder in Inhibikase Therapeutics, Inc.; D.H. has no conflict to report.

Hendra virus (HeV) is an important emergent virus in Australia known to infect horses and humans in certain regions of the east coast. Whilst pteropid bats ("flying foxes") are considered the natural reservoir of HeV, which of the four mainland species is the principal reservoir has been a source of ongoing debate, particularly as shared roosting is common. To help resolve this, we sampled a colony consisting of just one of these species, the grey-headed flying fox, (Pteropus poliocephalus), at the southernmost extent of its range. Using the pooled urine sampling technique at approximately weekly intervals over a two year period, we determined the prevalence of HeV and related paramyxoviruses using a novel multiplex (Luminex) platform. Whilst all the pooled urine samples were negative for HeV nucleic acid, we successfully identified four other paramyxoviruses, including Cedar virus; a henipavirus closely related to HeV. Collection of serum from individually caught bats from the colony showed that antibodies to HeV, as estimated by a serological Luminex assay, were present in between 14.6% and 44.5% of animals. The wide range of the estimate reflects uncertainties in interpreting intermediate results. Interpreting the study in the context of HeV studies from states to the north, we add support for an arising consensus that it is the black flying fox and not the grey-headed flying fox that is the principal source of HeV in spillover events to horses.

The kinetics of dengue virus (DENV)-specific IgA antibody in urine and the potential correlation with disease severity remain elusive. In this study, 262 serial urine samples from 78 laboratory-confirmed patients were assayed by a commercial immunoglobulin A (IgA) kit against DENV. All cases were classified into dengue fever (DF) and severe dengue (SD) according to the 2009 WHO/TDR guideline. The total positive rate of IgA in urine was 59%. DENV-specific IgA was detected in urine from day 2 to day 13 after the onset of illness in DF patients; While for SD patients, anti-DENV IgA could be detected till day 14. The positive rate of IgA in patients with secondary infection was higher than that in patients with primary infection. Importantly, during 4-7 days after the onset of illness, the IgA positive rate of SD patients was significantly higher than that of DF patients. Especially, the intensity of IgA signal in SD patients was obviously stronger than that in DF patient at the recovery stage. Overall, our results suggested that the existence of DENV-specific IgA antibodies in urine might be a warning sign for the severity of disease and its measurement might provide valuable guidance for proper patient management.

The risk of developing progressive multifocal leukoencephalopathy (PML), as a consequence of infection/reactivation with JC virus (JCV), is consistent in natalizumab-treated multiple sclerosis (MS) patients, with 430 cases of PML reported so far. The risk of PML is higher in JCV seropositive patients, and it is recommended that only MS patients without JCV antibodies should be enrolled in the treatment postulating that they do not have JCV infection.We have studied forty-two natalizumab-treated MS patients, and urine and blood were collected monthly for up to 60 months. JCV and BK virus (BKV) DNA presence was verified using quantitative real-time PCR assays, and serum anti-JCV antibodies were measured with the Stratify and/or Stratify DxSelect tests.JCV and BKV DNA were not found in the blood samples, whereas they were found at least once in the urine of 21 of 42 (50 %) and of 25/42 (59.5 %) patients, respectively. JCV DNA urinary shedding increased up to month 24 of natalizumab treatment (45.2 %), and the effect of time was significant for JCV (p = 0.04), but not for BKV (p = 0.39). JCV viruria and seropositivity did not completely correlate, since three patients shedding JCV DNA in the urine were seronegative according to the serological tests.The results indicated that natalizumab therapy may increase the rate of JCV urinary shedding. Additionally, we confirmed that the identification of JCV carriers cannot solely rely on serological tests, but sensitive methods for viral DNA detection should be adopted to more precisely identify the truly JCV uninfected cases.

Background: Dengue laboratory diagnosis is essentially based on detection of the virus, its components or antibodies directed against the virus in blood samples. Blood, however, may be difficult to draw in some patients, especially in children, and sampling during outbreak investigations or epidemiological studies may face logistical challenges or limited compliance to invasive procedures from subjects. The aim of this study was to assess the possibility of using saliva and urine samples instead of blood for dengue diagnosis., Methodology/principal Findings: Serial plasma, urine and saliva samples were collected at several time-points between the day of admission to hospital until three months after the onset of fever in children with confirmed dengue disease. Quantitative RT-PCR, NS1 antigen capture and ELISA serology for anti-DENV antibody (IgG, IgM and IgA) detection were performed in parallel on the three body fluids. RT-PCR and NS1 tests demonstrated an overall sensitivity of 85.4%/63.4%, 41.6%/14.5% and 39%/28.3%, in plasma, urine and saliva specimens, respectively. When urine and saliva samples were collected at the same time-points and tested concurrently, the diagnostic sensitivity of RNA and NS1 detection assays was 69.1% and 34.4%, respectively. IgG/IgA detection assays had an overall sensitivity of 54.4%/37.4%, 38.5%/26.8% and 52.9%/28.6% in plasma, urine and saliva specimens, respectively. IgM were detected in 38.1% and 36% of the plasma and saliva samples but never in urine., Conclusions: Although the performances of the different diagnostic methods were not as good in saliva and urine as in plasma specimens, the results obtained by qRT-PCR and by anti-DENV antibody ELISA could well justify the use of these two body fluids to detect dengue infection in situations when the collection of blood specimens is not possible.

Background: Urine is an important source for the detection of infections caused by CMV in stem cell transplant patients. Currently, there is no agreement about the type of urine specimen. In order to investigate which is the better specimen type for quantitative detection of CMV, we compared the results from urine supernatant and sediment from the same patients., Methods: Seventy urine specimens were collected from patients with hematological disorders or solid tumors. After performing shell vial culture, residual urine specimens were centrifuged. Then, 10 mL of each urine supernatant and sediment were taken and immediately frozen at -70 degrees C. Afterwards, archived urine specimens were thawed at room temperature and CMV-quantitative PCR was performed on both the supernatant and sediment fraction of urine. The results from each patient were reviewed for CMV antigenemia, blood shell vial culture, CMV-IgM or IgG, and clinical symptoms., Results: CMV-qPCR results for the urine sediment fraction revealed a significant difference (p = 0.012) between the active CMV infection group and the latent CMV infection group. In addition, receiver operating characteristic curves for active CMV infection revealed that CMV-qPCR using urine sediment produced more accurate results than urine supernatant., Conclusions: These findings suggest that the sediment fraction of urine is a more suitable specimen in CMV-qPCR testing.

Objective: To explore the cost-effectiveness of the diagnosis of congenital cytomegalovirus (CMV) infection by fluorescent quantitative polymerase chain reaction (FQ-PCR) in neonates., Methods: Serum CMV immunoglobulin M (CMV-IgM) and CMV-IgG were detected using ELISA in 610 neonates aged less than 14 days. CMV DNA content was detected by FQ-PCR. The cost-effectiveness analysis was then performed., Results: The positive rate of FQ-PCR in neonates with positive CMV-IgM was 42.9% (15/35), while, 2.9% (16/547) in neonates with positive CMV-IgG. The mean logarithm values of CMV DNA in neonates with positive CMV-IgM were higher than those in neonates with positive CMV-IgG (5.79±1.24 vs 4.11±0.87; P<0.01). The costs of the diagnosis of CMV infection by FQ-PCR were 256 RMB/case in neonates with positive CMV-IgM, and 3 760 RMB/case in neonates with positive CMV-IgG., Conclusions: The CMV DNA content in neonates with positive CMV-IgM is higher than that in neonates with positive CMV-IgG. Diagnosis of congenital CMV infection by FQ-PCR in neonates with positive CMV-IgG is not suitable for large scale epidemiological survey because of high cost-effectiveness ratio.

Objective: Analyses were conducted to determine the clinical utility of measuring JC virus (JCV) DNA in blood or urine of natalizumab-treated multiple sclerosis (MS) patients to predict the risk of progressive multifocal leukoencephalopathy (PML)., Methods: A total of 12,850 blood and urine samples from nearly 1,400 patients participating in natalizumab clinical trials were tested for JCV DNA using a commercially available quantitative polymerase chain reaction (qPCR) assay. A subset of these samples was also tested using a more sensitive qPCR assay developed at the National Institutes of Health (NIH)., Results: At the time natalizumab dosing was suspended, JCV DNA was detected in plasma by the commercial assay in 4 of 1,397 (0.3%) patients; the NIH assay confirmed these positive samples and detected JCV DNA in an additional 2 of 205 (1%) patients who tested negative with the commercial assay. None of these 6 JCV DNA positive patients developed PML. In a 48-week study testing the safety of natalizumab redosing, JCV DNA was detected in plasma of 6 of 1,094 (0.3%) patients, none of whom developed PML. Urine at baseline and week 48 was assessed in 224 patients; 58 (26%) were positive at baseline, and 55 (25%) were positive after 48 weeks of natalizumab, treatment. JCV DNA was not detected in peripheral blood mononuclear cells from any of these 1,094 patients before or after natalizumab treatment. In 5 patients who developed PML, JCV DNA was not detected in blood at any time point before symptoms first occurred., Interpretation: Measuring JCV DNA in blood or urine with currently available methods is unlikely to be useful for predicting PML risk in natalizumab-treated MS patients.

Objectives: The kinetics of three serological markers (IgM, IgA, and IgG) in serum, saliva, and urine samples from adult patients with primary or secondary dengue infection were studied., Design: Serum, saliva, and urine samples were collected from 22 patients with clinical and confirmed dengue 3 virus infection during the outbreak in Havana City in 2001. They were tested by capture IgM (MAC-ELISA), IgA (AAC-ELISA), and IgE (EAC-ELISA) and IgG ELISA inhibition method (EIM) to detect specific dengue antibodies., Results: Similar kinetics were observed in IgM, IgA, and IgG antibodies in saliva and IgA and IgG in urine samples from secondary cases compared with kinetics in serum samples, although the values were lower. No IgG antibody was detected in saliva and urine samples in primary cases and IgM antibody was not detected in urine samples from either primary or secondary infection. All secondary cases were positive for IgG in saliva and urine samples at day 7. The kinetics of specific IgE antibodies in primary and secondary cases were different., Conclusions: The kinetics of three serological markers (IgM, IgA, and IgG) in serum, saliva, and urine samples from adult patients with primary or secondary dengue 3 virus infection were studied for the first time, showing its behavior and usefulness in dengue virus diagnosis. The specific IgE could play a role as a serological marker in secondary infections.

Rapid and reliable laboratory diagnosis of cytomegalovirus (CMV) infection in transplanted patients becomes more significant considering possible prevention or moderation of CMV disease. The aim of this study was comparison of CMV isolation in cell culture and CMV detection by PCR in blood and urine samples of transplanted patients. The study comprised 21 patients undergoing immunosuppressive therapy after renal or bone marrow transplantation. MRC-5 cell culture was used for viral isolation. Inoculated cultures were maintained for 4 weeks or until appearance of specific cytopathic effect (CPE). Primers used in PCR were specific for the sequence within egzon 4 of immediate-early 1 (IE-1) gene. Presence of anti-CMV IgG and IgM antibodies was determined by ELISA. PCR was more sensitive for blood (4/21) and urine samples (3/19) than cell culture method--blood (0/21) urine (2/19). Statistically significant correlation was not shown between presence of CMV(determined by PCR) in blood and urine of the same patient.

Simian foamy virus (SFV) infection and the subsequent immune response are not well characterized. Blood plasma, saliva, and urine were obtained from four humans and nine chimpanzees persistently infected with chimpanzee-type SFV for an unknown length of time. SFV-specific immunoglobulin G (IgG) antibodies, but not IgA antibodies, against the Gag and Bet proteins were detected, by Western blotting, in all sample types from infected humans and chimpanzees. Overall, chimpanzee samples had higher anti-SFV IgG titers than humans. These results provide a first comparative evaluation of SFV-specific host mucosal humoral immunity in infected humans and chimpanzees that is characterized by a predominant IgG response and a virtually absent IgA response.

Little information is available on the prevalence of retrovirus infections in populations of non-human primates living in their natural habitats. To gain such information, methods were developed to detect antibodies to simian T-lymphotropic virus type 1 (STLV-1) in urine from wild chimpanzees. Samples from more than 74 chimpanzees living in three communities in the Taï National Park, Côte d'Ivoire, were analysed. The prevalence of STLV-1 antibodies in adults and adolescents was significantly higher (35/49, 71.4 %) than that in infant and juvenile chimpanzees (3/31, 9.7 %).

Objectives: To evaluate the utility of urinary assessment for epidemiological studies of rubella, we measured anti-rubella virus immunoglobulin G (anti-RV IgG) using samples from pediatric patients with initial rubella infection, healthy volunteers who received a prophylactic inoculation of live rubella vaccine, and 3 years-old children undergoing a health examination at a community health center., Methods: Blood and urine samples were collected from 12 of spontaneous rubella cases treated at 7 local pediatric clinics during acute and convalescent stages. In addition, blood and urine samples were collected from 17 healthy volunteers receiving prophylactic rubella vaccination immediately before, and 3 and 6-7 weeks after vaccination. Urine samples for anti-RV IgG measurement were also collected from 740 children 3 years of age at Odawara Community Health Center after obtaining informed consent from their parents. In addition, a questionnaire survey of the past history of prophylactic vaccinations was conducted. Serum titers of anti-RV antibody were measured using VIDAS Rubella-IgG and IgM (bioMerieux Japan Ltd.) and urinary titers of anti-RV IgG by ELISA (Otsuka Pharmaceutical Co., Ltd.)., Results: 1) The sensitivity and specificity for anti-RV IgG measurement in urine were 99.4% and 100%, respectively. 2) Six of 12 cases suspected of rubella infection were confirmed as initial rubella infection, and showed significantly increased anti-RV IgG titers in convalescent sera. Anti-RV IgG titers were also increased in the urine specimens. 3) In 17 subjects who received prophylactic inoculation with live rubella vaccine, serum titers of anti-RV IgG were increased 6-7 weeks after vaccination and anti-RV IgG was also detected in urine samples from all cases. 4) Urine samples from 80.9% of the children were positive for anti-RV IgG. In addition, 81.7% of the 698 cases, whose parents completed the questionnaire had received prophylactic inoculation with live rubella vaccine, confirmed by the vaccination records in maternal and child health handbooks. Furthermore, urine samples from 12.5% of children who had not received prophylactic live rubella vaccination were positive for anti-RV IgG., Conclusions: The results of this study suggest that increased antibody titers after spontaneous rubella infection and prophylactic vaccination can be confirmed by measuring antibody titers in the urine. The results also suggest that urine sampling is useful for epidemiological studies of rubella because collection is simple, even from children.

The present study pertains to the evaluation of urine as a specimen for detection of anti-hepatitis A virus (anti-HAV) antibodies. Immunoglobulin M (IgM), IgG, and IgA capture enzyme-linked immunosorbent assays for hepatitis A were performed on paired serum and urine specimens collected from hepatitis A patients (n = 92), healthy individuals (n = 100), non-A hepatitis patients (n = 70), and patients with nonhepatic diseases (n = 64, including 37 renal disease patients). Hepatitis A patients seropositive for anti-HAV IgM showed 95.65% uropositivity. No false-positive reactions were observed in control groups. The uropositivity of anti-HAV IgM persisted during the convalescent phase of the disease. Anti-HAV IgG uropositivity correlated well with corresponding seropositivity in all groups (P > 0.05 for each). No significant difference between the proportions of serum and urine positivity for anti-HAV IgA was noted (P > 0.05 for each). Using seroreactivity as a "gold standard," the sensitivity and specificity for anti-HAV IgM, anti-HAV IgG, and anti-HAV IgA tests with urine as a specimen were found to be 95.65 and 100%, 97.76 and 76.47%, and 92.23 and 88.18%, respectively. Urine appears to be comparable to serum for diagnosis of recent and past infection with hepatitis A.

MMR (measles-mumps-rubella) immunization in Japan was suspended in 1993 due to the high incidence of mumps meningitis as a complication. As a result, immunization coverage for rubella still remains at the 50-60% level in Japan. One way to increase the coverage rate is to increase the frequency of immunization. We calculated the predicted positivity rate of the antibody and cost and the benefits is three models of double vaccination, i.e., vaccination twice. The first model consists of simply two identical vaccinations. The second model consists of two vaccinations with mass vaccination at school for the second immunization. The third model consists of two vaccinations with screening of the urinary antibody for rubella in the second immunization. To calculate the predicted values we used coefficients from Ibara City. The predicted positivity rates and cost increases ranged from 60% to 90% and from 7.3 billion to 12.8 billion yen from the first to third models, respectively. Screening for the urinary antibody should be much cheaper than the presumed price because more than a million subjects will be screened. Since it would cost less than half the price, the third model should be best for the positivity rate of the antibody and cost and benefits. Therefore, we think that third model is the best correction until MMR immunization can be reintroduced.

Background: In Japan rubella vaccination is generally done once during a lifetime, and the vaccination rate decreased after a revised vaccination law in 1995. History of rubella or vaccination may still be unreliable. Testing for rubella antibody is significant to prevent the occurrence of congenital rubella syndrome. However, the collection of blood samples to detect antibodies from young children is invasive and difficult., Methods: For this study we obtained 853 matched serum and urine samples from 904 healthy students 10 or 14 years of age in the Ibara and Yoshii districts of Okayama, Japan, for a comparison of antibodies for rubella in the matched samples. The serum and urine antibodies were measured with hemagglutination-inhibition and enzyme-linked immunosorbent assays, respectively, and with our urine-based antibody test., Results: The sensitivity, specificity and concordance rates of this urine-based antibody test were 96, 99 and 97% based on the serum antibody results of both assays. The coefficiency was 0.627 between the titers of the urinary and serum antibodies by the enzyme-linked immunosorbent assay. The urinary antibodies were stable for at least 5 months at 4 degrees C and 25 degrees C., Conclusions: Urine-based assay methods are helpful not only because they avoid the invasive approach of venipuncture but also because unprocessed urine specimens can be used and urinary antibody is stable for a long period. Therefore this test is suitable for screening. In addition protective amounts of rubella antibody in blood can be reliably assessed by means of urine samples.

Human endogenous retrovirus (HERV)-like sequences are normal inherited elements that constitute several hundredths of the human genome. The expression of genes located within these elements can occur as a consequence of several different events, including persistent inflammation or genotoxic events. Antibodies to endogenous retroviral gene products have been found in a number of infectious, chronic, and malignant diseases, suggesting a role in disease initiation and progression. We studied human immunodeficiency virus type 1 (HIV-1)-infected patients for evidence of urine antibody to a HERV peptide and investigated correlates with clinical and laboratory parameters. Forty-three HIV-1-infected patients in documented asymptomatic, symptomatic, or AIDS stages of disease and 21 age- and gender-matched, uninfected controls were tested for antibody to HERV-related peptide 4.1. Urine specimens were examined in a blinded fashion with the Calypte Biomedical Corp. experimental enzyme immunoassay for antibody to peptide 4.1. Results were compared with demographic data, medical history, clinical state of disease, and results of other laboratory tests. Thirty-six percent of the asymptomatic (Centers for Disease Control and Prevention [CDC] category A) and 81.3% of both the symptomatic (CDC category B) and AIDS (CDC category C) patients were positive for antibody to HERV-related peptide 4.1. None of the controls were positive. In this study, antibodies to HERV-related peptide 4.1 were found more frequently in patients with advanced stages (categories B and C) of HIV-1 disease than in those patients with an earlier stage (category A) of HIV disease. In HIV patients, severe immunosuppression, defined as having had at least one opportunistic infection, correlated with the expression of antibody to a HERV-related peptide.

Background: Noninvasive methodologies provide alternatives to diagnostic blood tests and have high patient acceptance, increased safety, and reduced costs. Such tests may supplement or replace blood diagnostic assays currently in use., Methods: Using a licensed urine-based test for antibody to HIV-1, we performed 25 991 HIV-1 urine antibody enzyme immunoassay (EIA) screening tests [confirmable by HIV-1 Western blot (WB)] on paired urine and blood specimens obtained from high- and low-risk HIV-1 subjects collected at six sites representative of the US population., Results: Using HIV-1 urine EIA tests confirmed by urine Western blot, a compartmentalized immune response (urine positive/serum negative) occurred in 0.24% of a cohort of 11 896 subjects. In the same cohort, specimens that were urine negative/serum positive occurred in 0.17% of subjects. In a second study of 25 991 subjects that included 859 high-risk individuals, the false-positive urine EIA frequency (urine WB negative or indeterminate) was 1.3%. This false-positive frequency in the high-risk cohort was attributed, in part, to an IgA antibody response. We tabulated urine and serum indeterminate reactivities and examined their possible causes. Data are presented showing that antibodies from a seroindeterminate HIV-1vau group O subject were reactive in urine EIA and urine WB tests. An analysis of the HIV-1vau strain group O env nucleotide sequence disclosed a high frequency of homology with human chromosome 7q31, a fragile site implicated in many human malignancies., Conclusions: These results demonstrate the utility of urine for alternative HIV-1 antibody testing and provide new insights into the pathogenesis of HIV-1 infection and into potential application of this approach in investigation of other microbial pathogens and toxic compounds.

Four hundred blood sera from a cattle production unit were tested for BLV-(Bovine Leukaemia Virus) antibody with IP (Institut Porquier) and SB (Svanova Biotech) ELISA kits. Seventy-seven cattle with BLV-antibody (19.25%) and 77 without the antibody were used. No significant difference was found between O.D. of sera of PL+ (Persistent Lymphocytosis Positive) and PL- (Negative) cattle. The mean O.D. of urine samples of 77 seropositive cattle was significantly higher than that of 77 seronegative cattle (P < 0.01). There were also differences between urine O.D.s of seropositive (PL+) and seropositive (PL-) groups of cattle with IP (P < 0.05) and SB (P < 0.01) kits. All the results revealed the presence of BLV-antibody in the urine of the cattle without any urinary dysfunction.

A clinical evaluation of a new, purified, heat-treated equine rabies immunoglobulin (PHT-Erig), F(ab')2 preparation, was carried out in Thailand and in the Philippines-two countries where rabies is endemic. An initial prospective, randomised, controlled trial (Study 1), compared the safety and pharmacokinetics (serum concentrations of rabies antibodies) after administration either of PHT-Erig or of a commercially-available, equine rabies immune globulin (Erig PMC). A second trial (Study 2) simulated post-exposure rabies prophylaxis by using a reference cell culture vaccine, the purified Vero-cell rabies vaccine (PVRV), administered in association with either Erig PMC or PHT-Erig. In Study 1, 27 healthy, Thai adults received a 40 IU kg(-1) dose of either Erig PMC (n = 12) or PHT-Erig (n = 15) via the intramuscular (i.m.) route; half of the dose was injected into the deltoid area and the other half into the buttocks. Serum for rabies antibody determination and F(ab')2 concentration was collected at hours (H) 0, 6 and 12, and on day (D) 2, 3, 4, 6, 8, 10, 12 and 15. Both products were safe, with no serious adverse events, and in particular, no anaphylactic reactions or serum sickness was reported. A statistical comparison of the pharmacokinetic parameters did not demonstrate bioequivalence of the two products. Nonetheless, the relative bioavailability of 93% and the similar absorption rates suggest the pharmacokinetic profiles of Erig and PHT-Erig are similar. The antibody level in either group were low throughout the 15-day study period. The geometric mean titer (GMT) values ranged from group 0.027-0.117 IU ml(-1) in the Erig group and from 0.029 to 0.072 IU ml(-1) in the PHT-Erig. There was no significant difference between the evolution of GMT values for the two groups. In Study 2, 71 healthy volunteers received 40 IU kg(-1) via the intramuscular route of either Erig PMC (n = 36) or PHT-Erig (n = 35) on D0, in association with five doses of PVRV on D0, D3, D7, D14 and D28. The safety evaluation was performed during the 28-day follow-up and serum samples for anti-rabies antibody titration were collected on D0 (before injection) D3, D7, D14 and D28. No serious reactions were reported in either group. In particular, no immediate (anaphylactic type) or delayed (serum sickness) allergic reactions were observed. Over the 28-day follow-up period, GMT profiles of the two groups were statistically equivalent. On D14, 100% of the subjects had protective antibody titers (anti-rabies antibodies > or = 0.5 IU ml(-1), which is the WHO-recommended level of seroconversion), and Erig PMC and PHT-Erig were indistinguishable according to the clinical definition chosen. On D28, the GMT values were 33.2 IU ml(-1) (95% CI, 23.8-46.1 IU ml(-1)) in the Erig PMC/PVRV group and 31.4 IU ml(-1) (95% confidence interval, CI, 23.4-42.2 IU ml(-1)) in the PHT-Erig/PVRV group, showing evidence of adequate vaccine-induced antibody responses in both groups. The increased purity, the heat-treatment step introduced in the manufacturing process of PHT-Erig, and the good clinical results substantiate the use of this new generation, purified equine F(ab')2 preparation in the post-exposure prophylaxis of rabies.

Urine and serum samples from 89 healthy volunteers and three healthy individuals who underwent rubella vaccination were tested for immunoglobulin G (IgG), IgA, and IgM to rubella virus (RV) by enzyme-linked immunosorbent assay methods. Subjects with positive (n = 68) or negative (n = 21) results for serum IgG were exactly the same as those with the corresponding results for urinary IgG. Both urinary and serum IgG levels remained elevated from the 3rd or 4th week after vaccination until the end of the study. Both urinary IgA and serum IgM levels tended to increase rapidly between the 3rd and 5th week and then gradually decrease until the end of the study, but the levels of both remained positive except for one sample each at the end (26th week). On the other hand, the ratio of anti-RV IgA titer to anti-RV IgG titer in urine (urinary anti-RV IgA/IgG ratio) increased rapidly between the 3rd and 4th week after vaccination and then rapidly returned to the ratio levels of the subjects positive for serum IgG from among the healthy volunteers. In summary, detection of urinary anti-RV IgG should be useful for screening for previous RV infection, and measurement of urinary anti-RV IgA/IgG ratio might be useful for diagnosing recent infection.

Sensitive and specific methods are needed to diagnose respiratory virus infections using body fluids such as urine that, unlike blood samples, are readily obtained by non-invasive means. Immunoglobulin G antibody capture enzyme-linked immunosorbent assays were developed for detection of antibody rises to respiratory syncytial virus and influenza A/Taiwan (H1N1) after initial quantification and adjustment of urinary IgG concentration. Of 24 elderly subjects whose sera were assayed by the complement fixation test for antibody to RSV, seven had convalescent titres > or = 32, and five had > or = 4-fold rises in titre. Acute and convalescent urines for six of these seven subjects were tested for virus-specific urinary IgG by GACELISA. Four of four persons with > or = 4-fold rises in CFT had urine ELISA convalescent to acute ratios of > or = 1.8 whereas two subjects with convalescent CF titres > 16, but no increase in serum antibody titre, had urine convalescent/acute ratios of 1.0. Ten subjects with > or = 4-fold rises in CFT or HI antibodies to influenza A/Taiwan had urine ELISA ratios of > or = 1.4 when samples taken on the day of influenza vaccination and 16 days later were compared. These preliminary observations demonstrate clinically significant rises in respiratory pathogen antibody levels between acute and convalescent urine samples, provided that total urinary IgG concentrations are quantified and then standardised.

Blood sera and concentrated urine were tested for antibodies to HFRS virus. 18 patients were in acute period and 16 had suffered from HFRS 16 years before. Indirect IFA test revealed antibodies in urine of 16 (88.9%) patients with HFRS. Urine antibodies disappeared for 9-22 days. There were no antibodies in the urine of patients with the history of HFRS, though it was registered in high titre in the blood serum. Thus, if the patient has antibodies to HFRS both in the blood and urine, it says about acute stage of the disease and can be used for earlier diagnosis of HFRS.

Fetal infection with human cytomegalovirus (HCMV) is the leading viral cause of brain damage among newborns at birth or later in life. Efforts to screen newborns routinely for shedding of the virus by immunoassay have been hampered by inhibitors in urine, reportedly the host protein beta2-microglobulin (beta 2m). An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of HCMV antigen in which the reactivity was not affected by the presence of beta 2m, but nevertheless inhibition was observed when urine samples with high levels of virus were tested. The presence of antibodies to HCMV was demonstrated in these urine samples by antibody ELISA and immunoblot using the major antigenic protein of HCMV (pp150) expressed in Escherichia coli; this offers an alternative explanation for the inhibition in ELISA. The presence of HCMV antibodies correlated significantly with congenital HCMV infection (as detected by tissue culture isolation of virus from urine samples of newborns), especially with asymptomatic cases (sensitivity 70%; specificity 94%). The data indicate a local (renal) immune response to HCMV in congenitally infected children, which may have future diagnostic applications.

Human immunodeficiency virus (HIV) can be transmitted through infected seminal fluid or vaginal or rectal secretions during heterosexual or homosexual intercourse. To prevent mucosal transmission and spread to the regional lymph nodes, an effective vaccine may need to stimulate immune responses at the genitourinary mucosa. In this study, we have developed a mucosal model of genital immunization in male rhesus macaques, by topical urethral immunization with recombinant simian immunodeficiency virus p27gag, expressed as a hybrid Ty virus-like particle (Ty-VLP) and covalently linked to cholera toxin B subunit. This treatment was augmented by oral immunization with the same vaccine but with added killed cholera vibrios. Polymeric secretory immunoglobulin A (sIgA) and IgG antibodies to p27 were induced in urethral secretions, urine, and seminal fluid. This raises the possibility that the antibodies may function as a primary mucosal defense barrier against SIV (HIV) infection. The regional lymph nodes which constitute the genital-associated lymphoid tissue contained p27-specific CD4+ proliferative and helper T cells for antibody synthesis by B cells, which may function as a secondary immune barrier to infection. Blood and splenic lymphocytes also showed p27-sensitized CD4+ T cells and B cells in addition to serum IgG and IgA p27-specific antibodies; this constitutes a third level of immunity against dissemination of the virus. A comparison of genito-oral with recto-oral and intramuscular routes of immunization suggests that only genito-oral immunization elicits specific sIgA and IgG antibodies in the urine, urethra, and seminal fluid. Both genito-oral and recto-oral immunizations induced T-cell and B-cell immune responses in regional lymph nodes, with preferential IgA antibody synthesis. The mucosal route of immunization may prevent not only virus transmission through the genital mucosa but also dissemination and latency of the virus in the draining lymph nodes.

Serum, milk and urine specimens were taken from 15 bovine leukaemia virus (BLV)-positive and 20 BLV-negative cattle which had been determined previously to be infected or not by the use of a monoclonal enzyme-linked immunosorbent assay (ELISA). An ELISA was performed on the samples for the detection of IgG1 antibodies to the BLV surface glycoprotein, gp 51. The three types of samples had parallel optical density (OD) values apart from three urine samples which, although accepted as negative for anti-BLV antibodies, had numerically higher ODS than those of control BLV-negative animals. Therefore, detection of IgG1 antibodies against BLV in the urine of naturally infected animals could be an indication for the use of urine for diagnosis of BLV infection.

Using a commercial diagnostic preparation for indirect IFA test, 135 urine samples from 50 patients with HFRS were examined at different periods of the disease. Antibodies were demonstrable in all urine specimens from HFRS patients for 13 days. In 14-20 days they could be detected in half of the patients, and no antibodies could be demonstrated since the 21st day on. The results of urine examination from healthy subjects and some patients with other clinical diagnoses were negative same as controls with normal antigen. The dynamics of antibody titres in the patients' urine differed from that in the blood and was considered as "decreasing" similarly as the clinical disease. The antibody excretion in the urine coincided with the period of renal structure damage and stopped when the normal renal function was restored. The data are discussed from the point of view of the pathogenesis and diagnosis. Special attention was paid to the possibility and advantages of early HFRS diagnosis by antibody determinations in the patients' urine.

Since 1976, sera obtained serially from 10,218 pregnant women during the first, second, and third trimesters of gestation and cord sera were tested for CMV complement-fixing (CF) and immunofluorescent (IF) antibodies. CMV IgG-IF antibody was positive in 9,735/10,218 (95%) in the first trimester, and a significant rise of CF antibodies during pregnancy was found in 70/9,206 (0.76%) of the seropositive group and in 5/438 (1.14%) of the seronegative group. IgM antibody was found in 6/9,206 (0.06%) of seropositive women during the first trimester and in 7/70 (10.0%) of seropositive mothers with CF antibody rise and in 4/5 of seroconverted mothers of the seronegative group, suggesting that the incidence of primary infection with CMV during pregnancy was approximately 1% of susceptible women. All the mothers with immune response had infants with neither viruria nor IgM antibody in the cord blood, whereas seropositive mothers without an immune response had infants with viruria (7/1,826; 0.4%) or with IgM antibody in the cord blood (6/9,136; 0.06%). None of these 13 babies, shedding CMV or with IgM IF antibody, had physical or mental retardation. CMV IgG-IF antibody was present in almost 80% of infants between 7 and 12 months of age in 1988, suggesting that perinatal or postnatal CMV infection may occur in infants born to seropositive mothers in 70-80% of pregnancies.