Evaluation of a rapid diagnostic test for measles IgM detection; accuracy and the reliability of visual reading using sera from the measles surveillance programme in Brazil, 2015.

Laboratory-based case confirmation is an integral part of measles surveillance programmes; however, logistical constraints can delay response. Use of RDTs during initial patient contact could enhance surveillance by real-time case confirmation and accelerating public health response. Here, we evaluate performance of a novel measles IgM RDT and assess accuracy of visual interpretation using a representative collection of 125 sera from the Brazilian measles surveillance programme. RDT results were interpreted visually by a panel of six independent observers, the consensus of three observers and by relative reflectance measurements using an ESEQuant Reader. Compared to the Siemens anti-measles IgM EIA, sensitivity and specificity of the RDT were 94.9% (74/78, 87.4-98.6%) and 95.7% (45/47, 85.5-99.5%) for consensus visual results, and 93.6% (73/78, 85.7-97.9%) and 95.7% (45/47, 85.5-99.5%), for ESEQuant measurement, respectively. Observer agreement, determined by comparison between individuals and visual consensus results, and between individuals and ESEQuant measurements, achieved average kappa scores of 0.97 and 0.93 respectively. The RDT has the sensitivity and specificity required of a field-based test for measles diagnosis, and high kappa scores indicate this can be accomplished accurately by visual interpretation alone. Detailed studies are needed to establish its role within the global measles control programme.

A Monovalent Chimpanzee Adenovirus Ebola Vaccine Boosted with MVA.

BACKGROUND: The West African outbreak of Ebola virus disease that peaked in 2014 has caused more than 11,000 deaths. The development of an effective Ebola vaccine is a priority for control of a future outbreak. METHODS: In this phase 1 study, we administered a single dose of the chimpanzee adenovirus 3 (ChAd3) vaccine encoding the surface glycoprotein of Zaire ebolavirus (ZEBOV) to 60 healthy adult volunteers in Oxford, United Kingdom. The vaccine was administered in three dose levels--1×10(10) viral particles, 2.5×10(10) viral particles, and 5×10(10) viral particles--with 20 participants in each group. We then assessed the effect of adding a booster dose of a modified vaccinia Ankara (MVA) strain, encoding the same Ebola virus glycoprotein, in 30 of the 60 participants and evaluated a reduced prime-boost interval in another 16 participants. We also compared antibody responses to inactivated whole Ebola virus virions and neutralizing antibody activity with those observed in phase 1 studies of a recombinant vesicular stomatitis virus-based vaccine expressing a ZEBOV glycoprotein (rVSV-ZEBOV) to determine relative potency and assess durability. RESULTS: No safety concerns were identified at any of the dose levels studied. Four weeks after immunization with the ChAd3 vaccine, ZEBOV-specific antibody responses were similar to those induced by rVSV-ZEBOV vaccination, with a geometric mean titer of 752 and 921, respectively. ZEBOV neutralization activity was also similar with the two vaccines (geometric mean titer, 14.9 and 22.2, respectively). Boosting with the MVA vector increased virus-specific antibodies by a factor of 12 (geometric mean titer, 9007) and increased glycoprotein-specific CD8+ T cells by a factor of 5. Significant increases in neutralizing antibodies were seen after boosting in all 30 participants (geometric mean titer, 139; P<0.001). Virus-specific antibody responses in participants primed with ChAd3 remained positive 6 months after vaccination (geometric mean titer, 758) but were significantly higher in those who had received the MVA booster (geometric mean titer, 1750; P<0.001). CONCLUSIONS: The ChAd3 vaccine boosted with MVA elicited B-cell and T-cell immune responses to ZEBOV that were superior to those induced by the ChAd3 vaccine alone. (Funded by the Wellcome Trust and others; ClinicalTrials.gov number, NCT02240875.).

Serum anti-tetanus and measles antibody titres in Ugandan children aged 4 months to 6 years: implications for vaccine programme.

To study the antibody response to tetanus toxoid and measles by age following vaccination in children aged 4 months to 6 years in Entebbe, Uganda. Serum samples were obtained from 113 children aged 4-15 months, at the Mother-Child Health Clinic (MCHC), Entebbe Hospital and from 203 of the 206 children aged between 12 and 75 months recruited through the Outpatients Department (OPD). Antibodies to measles were quantified by plaque reduction neutralisation test (PRNT) and with Siemens IgG EIA. VaccZyme IgG EIA was used to quantify anti-tetanus antibodies. Sera from 96 of 113 (85.0%) children attending the MCHC contained Measles PRNT titres below the protective level (120 mIU/ml). Sera from 24 of 203 (11.8%) children attending the OPD contained PRNT titres 0.15 IU/ml by EIA, a level considered protective. The overall concentration of anti-tetanus antibody was sixfold higher in children under 12 months compared with the older children, with geometric mean concentrations of 3.15 IU/ml and 0.49 IU/ml, respectively. For each doubling in age between 4 and 64 months, the anti-tetanus antibody concentration declined by 50%. As time since the administration of the third DTP vaccination doubled, anti-tetanus antibody concentration declined by 39%. The low measles antibody prevalence in the children presenting at the MCHC is consistent with the current measles epidemiology in Uganda, where a significant number of measles cases occur in children under 1 year of age and earlier vaccination may be indicated. The consistent fall in anti-tetanus antibody titre over time following vaccination supports the need for further vaccine boosters at age 4-5 years as recommended by the WHO.

Monoclonal antibodies to the haemagglutinin HA1 subunit of the pandemic influenza A/H1N1 2009 virus and potential application to serodiagnosis.

In order to provide specific serological reagents for pandemic influenza A/H1N1 2009 virus, monoclonal antibodies (Mabs) to recombinant haemagglutinin component HA1 (rHA1) were generated after fusing spleen cells from a mouse immunized with rHA1 protein derived from influenza strain A/California/06/09 H1N1 with a mouse myeloma cell line. Five hybridoma clones secreting Mabs specific for the rHA1 protein derived from pandemic influenza A/H1N1 2009 and not for rHA1 from seasonal H1N1 influenza strains A/Brisbane/59/07 and A/Solomon Islands/03/06 were identified by EIA. Mabs 7H4, 9A4, and 9E12 were reactive in Western blots with full length rHA and/or rHA1 subunit derived from A/California/06/09 strain. Only Mab 1F5 inhibited haemagglutination of turkey red blood cells with recombinant NIBRG-121 virus derived from A/California/07/09, but did not react in Western blots. Immunostaining of MDCK cells infected with NIBRG-121 was localized to the membrane/cytoplasm for four of the reactive Mabs. The differing reactivity of the Mabs in Western blots, immunostaining, EIA, and haemagglutination inhibition assay suggest that at least four of the five Mabs recognize different epitopes on HA1 of the pandemic influenza A/H1N1 2009 virus. Ferret antisera to pandemic influenza A/H1N1 2009 (A/England/195/09 and A/California/07/09 strains) and sera from human subjects vaccinated with Influenza A (H1N1) 2009 Monovalent Vaccine (CELTURA®, Novartis Vaccines, Germany), inhibit binding of 1F5-HRP to biotinylated rHA1 derived from A/California/06/09 in a competitive EIA, suggesting that the epitope recognized by this Mab also evokes an antibody response in infected ferrets and vaccinated humans.

A point-of-care test for measles diagnosis: detection of measles-specific IgM antibodies and viral nucleic acid.

OBJECTIVE: To evaluate the performance of a newly developed point-of-care test (POCT) for the detection of measles-specific IgM antibodies in serum and oral fluid specimens and to assess if measles virus nucleic acid could be recovered from used POCT strips. METHODS: The POCT was used to test 170 serum specimens collected through measles surveillance or vaccination programmes in Ethiopia, Malaysia and the Russian Federation: 69 were positive for measles immunoglobulin M (IgM) antibodies, 74 were positive for rubella IgM antibodies and 7 were positive for both. Also tested were 282 oral fluid specimens from the measles, mumps and rubella (MMR) surveillance programme of the United Kingdom of Great Britain and Northern Ireland. The Microimmune measles IgM capture enzyme immunoassay was the gold standard for comparison. A panel of 24 oral fluids was used to investigate if measles virus haemagglutinin (H) and nucleocapsid (N) genes could be amplified by polymerase chain reaction directly from used POCT strips. FINDINGS: With serum POCT showed a sensitivity and specificity of 90.8% (69/76) and 93.6% (88/94), respectively; with oral fluids, sensitivity and specificity were 90.0% (63/70) and 96.2% (200/208), respectively. Both H and N genes were reliably detected in POCT strips and the N genes could be sequenced for genotyping. Measles virus genes could be recovered from POCT strips after storage for 5 weeks at 20-25 °C. CONCLUSION: The POCT has the sensitivity and specificity required of a field-based test for measles diagnosis. However, its role in global measles control programmes requires further evaluation.

Overexpression of human virus surface glycoprotein precursors induces cytosolic unfolded protein response in Saccharomyces cerevisiae.

BACKGROUND: The expression of human virus surface proteins, as well as other mammalian glycoproteins, is much more efficient in cells of higher eukaryotes rather than yeasts. The limitations to high-level expression of active viral surface glycoproteins in yeast are not well understood. To identify possible bottlenecks we performed a detailed study on overexpression of recombinant mumps hemagglutinin-neuraminidase (MuHN) and measles hemagglutinin (MeH) in yeast Saccharomyces cerevisiae, combining the analysis of recombinant proteins with a proteomic approach. RESULTS: Overexpressed recombinant MuHN and MeH proteins were present in large aggregates, were inactive and totally insoluble under native conditions. Moreover, the majority of recombinant protein was found in immature form of non-glycosylated precursors. Fractionation of yeast lysates revealed that the core of viral surface protein aggregates consists of MuHN or MeH disulfide-linked multimers involving eukaryotic translation elongation factor 1A (eEF1A) and is closely associated with small heat shock proteins (sHsps) that can be removed only under denaturing conditions. Complexes of large Hsps seem to be bound to aggregate core peripherally as they can be easily removed at high salt concentrations. Proteomic analysis revealed that the accumulation of unglycosylated viral protein precursors results in specific cytosolic unfolded protein response (UPR-Cyto) in yeast cells, characterized by different action and regulation of small Hsps versus large chaperones of Hsp70, Hsp90 and Hsp110 families. In contrast to most environmental stresses, in the response to synthesis of recombinant MuHN and MeH, only the large Hsps were upregulated whereas sHsps were not. Interestingly, the amount of eEF1A was also increased during this stress response. CONCLUSIONS: Inefficient translocation of MuHN and MeH precursors through ER membrane is a bottleneck for high-level expression in yeast. Overexpression of these recombinant proteins induces the UPR's cytosolic counterpart, the UPR-Cyto, which represent a subset of proteins involved in the heat-shock response. The involvement of eEF1A may explain the mechanism by which only large chaperones, but not small Hsps are upregulated during this stress response. Our study highlights important differences between viral surface protein expression in yeast and mammalian cells at the first stage of secretory pathway.

Development and evaluation of a rapid immunochromatographic test for mumps-specific IgM in oral fluid specimens and use as a matrix for preserving viral nucleic acid for RT-PCR.

The use of oral fluid specimens for diagnosis of mumps by detection of virus-specific IgM or nucleic acid is now well established. The utility of oral fluids would be improved further if testing could be performed closer to the patient. A near patient test (NPT) for the detection of mumps-specific IgM in oral fluid specimens was developed and evaluated using 196 oral fluid specimens from suspected cases of mumps and measles. Compared to enzyme immunoassay (EIA), the sensitivity, specificity, positive and negative predictive value of the mumps IgM NPT were 79.5%, 100%, 100%, and 72.6%, respectively. On oral fluid specimens with a test to negative control optical density ratio (T/N) > or =10 in the EIA, sensitivity of the NPT increased to 95.2%. To determine whether viral nucleic acid from oral fluid was preserved on the NPT strips used for IgM detection, reverse transcription-polymerase chain reaction (RT-PCR) was performed on 58 oral fluids before and after testing on NPT strips. In RT-PCR-positive oral fluids, mumps virus was detected in 19/21 (90.5%) specimens extracted from NPT strips. Mumps virus RNA was not detected in 37/37 (100%) NPT strips from mumps RT-PCR-negative oral fluid specimens. Mumps IgM NPT is rapid and simple to perform, with an acceptable level of performance for confirmation of a clinical diagnosis outside of the laboratory. The NPT strip is also a suitable matrix for preserving nucleic acid, enabling virus-specific RT-PCR to be performed. J. Med. Virol. 82:485-493, 2010. (c) 2010 Wiley-Liss, Inc.

Evaluation of a commercial assay for the detection of mumps specific IgM antibodies in oral fluid and serum specimens.

BACKGROUND: An IgM antibody capture radioimmunoassay (MACRIA) has been used in the UK Reference Laboratory for the detection of mumps specific IgM antibodies in oral fluid and serum samples. The method has proved both sensitive and specific and has been used for the surveillance of mumps since 1994. The use of radioactive labels has restricted the use of MACRIA to specialised laboratories and alternative, non-isotopic, sensitive assays capable of detecting the low levels of specific antibodies in oral fluid have not been available. Recently, a novel mumps specific IgM capture enzyme immunoassay (MACEIA) utilising recombinant mumps nucleoprotein (rMuVN) and monoclonal antibodies to the nucleoprotein, produced by Microimmune Limited, has been developed for use with both serum and oral fluid samples. OBJECTIVES: In this study, we have evaluated the performance of the Microimmune MACEIA for both serum and oral fluid against specimens tested by MACRIA. STUDY DESIGN: The panel consisted of matched serum and oral fluid specimens from 137 cases of suspected mumps received in the Virus Reference Department for routine investigation from March 2003 to October 2004. RESULTS: The sensitivity, specificity, positive and negative predictive value of the Microimmune MACEIA on serum samples compared to MACRIA were 98.8%, 100.0%, 100.0% and 98.5%, respectively. The sensitivity, specificity, positive and negative predictive value of the Microimmune MACEIA on oral fluid samples compared to Microimmune MACEIA and MACRIA consensus results on the paired serum samples were 90.3%, 97.6%, 98.3% and 87.1%, respectively. CONCLUSIONS: The Microimmune MACEIA was found to be a rapid, sensitive and specific alternative to MACRIA for the detection of mumps specific IgM in sera and oral fluids.

Detection of anti-pertussis toxin IgG in oral fluids for use in diagnosis and surveillance of Bordetella pertussis infection in children and young adults.

Bordetella pertussis infection is being increasingly recognized as a cause of prolonged, distressing cough (without whooping symptoms) in children and young adults. Diagnosis of infection in this population is important for treatment and surveillance purposes, and may also prove useful in reducing transmission to unvaccinated babies, for whom disease can be fatal. Serum IgG titres against pertussis toxin (PT) are routinely used as a marker of recent or persisting B. pertussis infection. However, collection of serum from young children is difficult, and compliance amongst these subjects to give samples is low. To circumvent these problems, an IgG-capture ELISA capable of detecting anti-PT IgG in oral fluid was devised. The assay was evaluated by comparison to a serum ELISA, using 187 matched serum and oral fluid samples from children (aged 5-16 years) with a history of prolonged coughing, whose serum anti-PT titre had already been determined (69 seropositive, 118 seronegative). The results showed that, using a cutoff of 70 arbitrary units (AU), the oral fluid assay detected seropositive subjects with a sensitivity of 79.7% [95% confidence interval (CI) 68.3-88.4] and a specificity of 96.6% (95% CI 91.5-99.1). Thus, oral fluid titres of >or=70 AU would possess a positive predictive value of 76.2-93.2% for pertussis amongst children with chronic coughs when used as a surrogate for the serum ELISA (assuming disease prevalence of 12-37%). This oral fluid ELISA will greatly assist in the convenience of B. pertussis disease diagnosis and surveillance.

Chlamydia trachomatis Pgp3 Antibody Persists and Correlates with Self-Reported Infection and Behavioural Risks in a Blinded Cohort Study.

Chlamydia trachomatis (Ct) serological studies in populations could help monitor changes in lifetime cumulative risk of infection. We developed a double-antigen sandwich ELISA based on the Ct-specific Pgp3 antigen, then tested blind stored sera from over 800 participants in a New Zealand birth cohort from Dunedin at ages 26, 32 and 38. The double-antigen sandwich ELISA was more sensitive than our previously characterised indirect Pgp3 ELISA. Pgp3 antibody was detected more often in women compared to men and correlated with increasing numbers of sexual partners, self-reported Ct, and younger age at sexual debut in both women and men. At age 26, 24.1% (99/411) of women were Pgp3 seropositive, as were 79.5% (35/44) of those reporting Ct infection; Pgp3 antibody persisted to age 38 in 96.5% (83/86). In men at age 26, the figures were 10.7% (47/442) and 25.0% (6/24), respectively, with high (83.9%) antibody persistence to age 38. At age 38, among those Pgp3 seropositive, 63.3% of women and 83.1% of men had not reported Ct infection. Thus, Ct-specific Pgp3 antibody was detected in most women reporting Ct infection and correlated with risk of infection in those who did not, with most infections remaining undetected. As this antibody persisted for at least twelve years in 96% of these women, serology could be used to evaluate Ct prevention programmes among women.