How Useful is COVID-19 Antibody Testing - A Current Assessment for Oncologists.

The coronavirus disease 2019 (COVID-19) pandemic due to infection by a new human coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has seriously disrupted the provision of oncology services and their uptake. Antibody testing, both at an individual level and of populations, has been widely viewed to be a key activity for guiding the options for treatment of high-risk individuals, as well as the implementation of safe control of infection measures. Ideally, the detection of a specific antibody should signify that all individuals tested have been infected by SARS-CoV-2 and that in the case of specific IgG that they are immune to further infection. This would enable SARS-CoV-2-infected individuals to be appropriately managed and healthcare workers shown to be immune to return to work where they would no longer pose a risk to their patients or be at risk themselves. Unfortunately, this is not the case for COVID-19, where it has been shown that immunity may not be protective, and seroconversion delayed or absent. The variability in antibody test performance, particularly that of lateral flow assays, has caused confusion for the public and healthcare professions alike. Many antibody test devices have been made available without independent evaluations and these may lack both adequate sensitivity and specificity. This review seeks to educate healthcare workers, particularly those working in oncology, of the current benefits and limitations of SARS-CoV-2 antibody testing.

Differentiation between wild-type and vaccines strains of varicella zoster virus (VZV) based on four single nucleotide polymorphisms.

Varicella-zoster virus (VZV) infection (chickenpox) results in latency and subsequent reactivation manifests as shingles. Effective attenuated vaccines (vOka) are available for prevention of both illnesses. In this study, an amplicon-based sequencing method capable of differentiating between VZV wild-type (wt) strains and vOka vaccine is described. A total of 44 vesicular fluid specimens collected from 43 patients (16 from China and 27 from the UK) with either chickenpox or shingles were investigated, of which 10 had received previous vaccination. Four sets of polymerase chain reactions were set up simultaneously with primers amplifying regions encompassing four single nucleotide polymorphisms (SNPs), '69349-106262-107252-108111'. Nucleotide sequences were generated by Sanger sequencing. All samples except one had a wt SNP profile of 'A-T-T-T'. The sample collected from a patient who received vaccine 7-10 days ago, along with VZV vaccine preparations, Zostavax and Baike-varicella gave a SNP profile 'G-C-C-C'. The results show that this method can distinguish vaccine-derived virus from wt viruses from main four clades, (clades 1-4) and should be of utility worldwide.

The differences in short- and long-term varicella-zoster virus (VZV) immunoglobulin G levels following varicella vaccination of healthcare workers measured by VZV fluorescent-antibody-to-membrane-antigen assay (FAMA), VZV time-resolved fluorescence immunoassay and a VZV purified glycoprotein enzyme immunoassay.

Healthcare workers (HCWs) reporting no history of varicella frequently receive varicella vaccination (vOka) if they test varicella-zoster virus (VZV) immunoglobulin G (IgG) negative. In this study, the utilities of VZV-IgG time-resolved fluorescence immunoassay (VZV-TRFIA) and a commercial VZV-IgG purified glycoprotein enzyme immunoassay (gpEIA) currently used in England for confirming VZV immunity have been compared to the fluorescent-antibody-to-membrane-antigen assay (FAMA). A total of 110 HCWs received two doses of vOka vaccine spaced 6 weeks apart and sera collected pre-vaccination (n = 100), at 6 weeks post-completion of vaccination (n = 86) and at 12-18 months follow-up (n = 73) were analysed. Pre-vaccination, by FAMA, 61·0% sera were VZV IgG negative, and compared to FAMA the sensitivities of VZV-TRFIA and gpEIA were 74·4% [95% confidence interval (CI) 57·9-87·0] and 46·2% (95% CI 30·1-62·8), respectively. Post-completion of vaccination the seroconversion rate by FAMA was 93·7% compared to rates of 95·8% and 70·8% determined by VZV-TRFIA and gpEIA, respectively. At 12-18 months follow-up seropositivity rates by FAMA, VZV-TRFIA and gpEIA were 78·1%, 74·0% and 47·9%, respectively. Compared to FAMA the sensitivities of VZV-TRFIA and gpEIA for measuring VZV IgG following vaccination were 96·4% (95% CI 91·7-98·8) and 74·6% (95% CI 66·5-81·6), respectively. Using both FAMA and VZV-TRFIA to identify healthy adult VZV susceptibles and measure seroconversion showed that vOka vaccination of HCWs is highly immunogenic.

Is it appropriate to use fixed assay cut-offs for estimating seroprevalence?

Population seroprevalence can be estimated from serosurveys by classifying quantitative measurements into positives (past infection/vaccinated) or negatives (susceptible) according to a fixed assay cut-off. The choice of assay cut-offs has a direct impact on seroprevalence estimates. A time-resolved fluorescence immunoassay (TRFIA) was used to test exposure to human parvovirus 4 (HP4). Seroprevalence estimates were obtained after applying the diagnostic assay cut-off under different scenarios using simulations. Alternative methods for estimating assay cut-offs were proposed based on mixture modelling with component distributions for the past infection/vaccinated and susceptible populations. Seroprevalence estimates were compared to those obtained directly from the data using mixture models. Simulation results showed that when there was good distinction between the underlying populations all methods gave seroprevalence estimates close to the true one. For high overlap between the underlying components, the diagnostic assay cut-off generally gave the most biased estimates. However, the mixture model methods also gave biased estimates which were a result of poor model fit. In conclusion, fixed cut-offs often produce biased estimates but they also have advantages compared to other methods such as mixture models. The bias can be reduced by using assay cut-offs estimated specifically for seroprevalence studies.

Comparison of a commercial Varicella Zoster glycoprotein IgG enzyme immunoassay with a reference time resolved fluorescence immunoassay (VZV TRFIA) for measuring VZV IgG in sera from pregnant women, sera sent for confirmatory testing and pre and post vOka vaccination sera from healthcare workers.

BACKGROUND: Recently, a commercial, standardised VZV IgG glycoprotein EIA, Binding Site VaccZyme™VZV glycoprotein IgG low level EIA (VaccZyme™EIA) has become available. The VaccZyme™EIA is more robust and user friendly than the reference VZV time-resolved fluorescence immunoassay (VZV TRFIA). OBJECTIVES: To assess the usefulness of the VaccZyme™EIA in the diagnostic laboratory by comparing VZV IgG levels generated by both assays on serum panels representing, non-vaccinated, and vOka vaccinated populations. STUDY DESIGN: Sera from non-vaccinated individuals were tested; 248 from pregnant women, 117 from various patient groups referred to the Virus Reference Department for confirmatory VZV IgG testing and 102 from healthcare workers enrolled in a study (ROVE) of antibody/IgG response to vOka. From the ROVE study, 282 post vaccination sera were tested; 108 and 101 collected at six weeks post first and second doses of vOka, respectively, and 73 collected at 18 month follow-up. RESULTS: Sensitivities and specificities (equivocals treated as negatives) of the VaccZyme™EIA for sera from pregnant women were 97.8% (95% CI: [94.6%, 99.4%]) and 96.8% (95% CI: [89.0%, 99.6%]), respectively, and for sera referred for confirmatory testing were 81.2% (95% CI: [71.2%, 88.8%]) and 96.9% (95% CI: [83.8%, 99.9%]), respectively, and for ROVE baseline sera were 54.2% (95% CI: [32.8%, 74.4%]) and 100% (95% CI: [95.4%, 100.0%]), respectively. For the post vOka serum panels sensitivities of the VaccZyme™EIA ranged from 65.3% (95% CI: [50.4%, 78.3%]) to 80.4% (95% CI: [71.1%, 87.8%]). Specificities were all 100%. Correlation with VZV TRFIA was high and agreement varied between the serum panels tested. CONCLUSIONS: VaccZyme™EIA is recommended for detecting VZV IgG in sera from non-vaccinated populations; however, caution is advised when measuring post vOka VZV IgG levels.

Follow-up of pregnant women exposed to chicken pox: an audit of relationship between level of antibody and development of chicken pox.

The purpose of this study was to validate through natural exposure a cut-off level of varicella zoster IgG as protective against infection with varicella zoster virus (VZV). Laboratory testing to determine VZV immune status of pregnant women exposed to varicella is recommended. Quantitative assays are now available which are sensitive and specific. More than 200 consecutive requests for screening in pregnant patients with recent varicella contacts were followed-up by questionnaire. DiaSorin LIAISON and VZV time resolved fluorescence immuno assay (VZV TRFIA) were used to measure VZV antibody level. One hundred fifty out of 209 (72%) questionnaires were returned; 14 patients developed varicella, 129 did not and seven were not known. Patients who had been given VZIG and developed varicella on follow-up had a mean antibody level before VZIG of 28 mIU/ml and 62 mIU/ml, by LIAISON and TRFIA, respectively. The mean IgG level of those that did not develop varicella was 885 and 866 mIU/ml by LIAISON and TRFIA, respectively. Those with levels <100 mIU/ml were more likely to develop chicken pox than those with levels >100 mIU/ml (relative risk of 10.4 for LIAISON and 8.8 for TRFIA). On the basis of the relatively small numbers in this study, quantitative assays, using a 100mIU/ml cut-off, can differentiate between those who are susceptible and those who are protected against exposure, however follow-up studies should include sampling for VZV DNA and IgM.

Evaluation of the time resolved fluorescence immunoassay (TRFIA) for the detection of varicella zoster virus (VZV) antibodies following vaccination of healthcare workers.

Determination of varicella zoster virus (VZV) immunity in healthcare workers without a history of chickenpox is important for identifying those in need of vOka vaccination. Post immunisation, healthcare workers in the UK who work with high risk patients are tested for seroconversion. To assess the performance of the time-resolved fluorescence immunoassay (TRFIA) for the detection of antibody in vaccinated as well as unvaccinated individuals, a cut-off was first calculated. VZV-IgG specific avidity and titres six weeks after the first dose of vaccine were used to identify subjects with pre-existing immunity among a cohort of 110 healthcare workers. Those with high avidity (≥ 60%) were considered to have previous immunity to VZV and those with low or equivocal avidity (<60%) were considered naive. The former had antibody levels ≥ 400 mIU/mL and latter had levels < 400 mIU/mL. Comparison of the baseline values of the naive and immune groups allowed the estimation of a TRFIA cut-off value of > 130 mIU/mL which best discriminated between the two groups and this was confirmed by ROC analysis. Using this value, the sensitivity and specificity of TRFIA cut-off were 90% (95% CI 79-96), and 78% (95% CI 61-90) respectively in this population. A subset of samples tested by the gold standard Fluorescence Antibody to Membrane Antigen (FAMA) test showed 84% (54/64) agreement with TRFIA.

Characterization of Treponema pallidum particle agglutination assay-negative sera following screening by treponemal total antibody enzyme immunoassays.

Following a laboratory audit, a significant number of Treponema pallidum particle agglutination assay (TPPA)-negative sera were identified when TPPA was used as a confirmatory assay of syphilis enzyme immunoassay (EIA) screening-reactive sera (SSRS). Sera giving such discrepant results were further characterized to assess their significance. A panel of 226 sera was tested by the Abbott Murex ICE Syphilis EIA and then by the Newmarket Syphilis EIA II. TPPA testing was performed on 223 sera. Further testing by the Venereal Disease Research Laboratory (VDRL) test, the Mercia Syphilis IgM EIA, the fluorescent treponemal antibody (FTA-ABS) assay, and INNO-LIA immunoblotting was undertaken in discrepant cases. One hundred eighty-seven of 223 (83.8%) SSRS were TPPA reactive, while 26 (11.6%) sera which were reactive in both the ICE and Newmarket EIAs were nonreactive by TPPA. The majority (68%) of the TPPA-discrepant sera were from HIV-positive patients and did not represent early acute cases, based on previous or follow-up samples, which were available for 22/26 samples. FTA-ABS testing was performed on 24 of these sera; 14 (58.3%) were FTA-ABS positive, and 10 (41.7%) were FTA-ABS negative. Twenty-one of these 26 sera were tested by INNO-LIA, and an additional 4 FTA-ABS-negative samples were positive. In this study, significant numbers (18/26) of SSRS- and TPPA-negative sera were shown by further FTA-ABS and LIA (line immunoblot assay) testing to be positive. The reason why certain sera are negative by TPPA but reactive by treponemal EIA and other syphilis confirmatory assays is not clear, and these initial findings should be further explored.

Comparison of the performance of the LIAISON VZV-IgG and VIDAS automated enzyme linked fluorescent immunoassays with reference to a VZV-IgG time-resolved fluorescence immunoassay and implications of choice of cut-off for LIAISON assay.

BACKGROUND: Determination of Varicella Zoster virus (VZV) immune status in pregnant women without history of chickenpox is important in identifying those who genuinely need VZV immune globulin prophylaxis following significant exposure to chickenpox or shingles. Immune status testing requires highly sensitive and specific immunoassays for timely and accurate results. OBJECTIVES: To compare the performance of DiaSorin LIAISON and Biomerieux VIDAS VZV-IgG assays with reference to a VZV-IgG time-resolved fluorescence immunoassay (TRFIA). STUDY DESIGN: A panel of sera collected from 65 pregnant contacts of VZV and 62 individuals tested for VZV immunity was tested in all three assays. Dose-response curves were generated using International Standards W1044 and 90/690. RESULTS: Sensitivity and specificity of VIDAS compared to VZV-TRFIA was 54.5% and 97.9% respectively and for LIAISON compared to VZV-TRFIA was 67% and 100% respectively. Both assays correlated well with TRFIA with R2 correlation coefficients of 0.79 and 0.76 respectively. Dose-response curves showed both Standards behaved in a similar manner in each assay. For VIDAS, the test cut-off value of 0.9 correlated with 275-280mIU/ml and for LIAISON a cut-off value of 150mIU/ml correlated with 208-219mIU/ml. CONCLUSIONS: By dose-response data and in comparison with TRFIA, LIAISON is more sensitive and specific than VIDAS.

Application of a noninvasive oral fluid test for detection of treponemal IgG in a predominantly HIV-infected population.

The performance of a time-resolved fluorescence immunoassay (TRFIA) for detection of treponemal IgG from oral fluid specimens has been assessed in a predominantly HIV-infected population. Serological investigation is the method of choice for confirming clinical suspicion of syphilis; however, in the primary stage of disease, direct detection of treponemes in lesion fluid or Treponema pallidum DNA is recommended because of the reduced sensitivity of serological tests. There may be occasions when blood for serological investigation is difficult to obtain due to individual patient preference or logistical necessity to improve participation in screening initiatives, particularly in outreach situations. Collection of oral fluid for detection of treponemal antibody may prove an attractive alternative and, with this in mind, an oral fluid assay for detection of treponemal IgG was developed. Time-resolved fluorescence was used to detect treponemal IgG extracted from commercially available oral fluid collection devices. Paired serum and saliva samples were obtained from 210 individuals, 101 of whom were diagnosed with syphilis on the grounds of medical examination confirmed by serological testing. Oral fluid specimens from 14 subjects were rejected because they contained insufficient control antibody or were inhibitory. The population tested was predominantly men who have sex with men, many of whom were HIV infected. The overall sensitivity and specificity of the oral fluid assay was 95.8 and 86.1%, respectively, based on the 5th percentile of the positive results, and 93.7 and 91.1%, respectively, based on a cutoff derived by mixture model analysis. For individuals with primary syphilis, the optimum sensitivity of the oral fluid assay was 87.5%, whereas in those with disease classified as secondary syphilis and early latent syphilis, the sensitivity of the oral fluid assay was 100 and 94.7%, respectively. The oral fluid assay is a useful alternative to serological testing in certain situations, and further development of this technology to enable detection of treponemal IgM should increase its sensitivity for detecting cases of primary syphilis.

Performance of a time-resolved fluorescence immunoassay for measuring varicella-zoster virus immunoglobulin G levels in adults and comparison with commercial enzyme immunoassays and Merck glycoprotein enzyme immunoassay.

Highly sensitive and specific, quantitative assays are needed to detect varicella-zoster virus (VZV) immunoglobulin G in human sera, particularly for determining immune status and response following vaccination. A time-resolved fluorescence immunoassay (TRFIA) has been developed, and its performance was compared to that of two commercial enzyme immunoassays (EIAs) and Merck glycoprotein EIA (gpEIA). The TRFIA had equivalent sensitivity (97.8%) and high specificity (93.5%) in relation to gpEIA. A commercial (Behring) EIA compared favorably with TRFIA in terms of sensitivity (98.4%) but had lower specificity (80.7%). Another commercial EIA (Diamedix) had high specificity (97.1%) but low sensitivity (76.4%) compared to TRFIA if equivocal test results were treated as negative for VZV antibody. A novel feature of the TRFIA was that the cutoff was generated using population mixture modeling and was expressed in mIU/ml, as the assay was calibrated using the British standard VZV antibody.

An oral fluid test for syphilis.

We have developed a time-resolved fluorescence immunoassay to detect antibodies to Treponema pallidum recombinant antigens in oral fluid specimens. Using an 'Oracol' swab, oral fluid was collected from 34 subjects with a serological diagnosis of syphilis and 97 seronegative controls. Using a cut-off of three standard deviations over control mean, the sensitivity and specificity of the assay in all subjects with positive syphilis serology was 76.5% and 97.9%, respectively. In those with early syphilis, the sensitivity and specificity of the assay was 100% and 97.9%. In a non-outbreak situation, screening clinic attendees for syphilis using oral fluid specimens is potentially useful when collection of blood is not practicable. In addition, it may have much to offer in outreach projects and epidemiological investigations.

The seroepidemiology of Bordetella pertussis infection in Western Europe.

High titres of pertussis toxin (PT) antibody have been shown to be predictive of recent infection with Bordetella pertussis. The seroprevalence of standardized anti-PT antibody was determined in six Western European countries between 1994 and 1998 and related to historical surveillance and vaccine programme data. Standardized anti-PT titres were calculated for a series of whole-cell and acellular pertussis vaccine trials. For the serological surveys, high-titre sera (> 125 units/ml) were distributed throughout all age groups in both high- (> 90%) and low-coverage (< 90%) countries. High-titre sera were more likely in infants in countries using high-titre-producing vaccines in their primary programme (Italy, 11.5%; Western Germany, 13.3%; France, 4.3%; Eastern Germany, 4.0%) compared to other countries (The Netherlands, 0.5%; Finland, 0%). Recent infection was significantly more likely in adolescents (10-19 years old) and adults in high-coverage countries (Finland, The Netherlands, France, East Germany), whereas infection was more likely in children (3-9 years old) than adolescents in low-coverage (< 90%; Italy, West Germany, United Kingdom) countries. The impact and role of programmatic changes introduced after these surveys aimed at protecting infants from severe disease by accelerating the primary schedule or vaccinating older children and adolescents with booster doses can be evaluated with this approach.

Sero-epidemiology of Bordetella pertussis in England and Wales.

The incidence of pertussis infection can be estimated in the population by defining a single high titre of anti pertussis toxin (PT) immunoglobulin G (IgG) antibody predictive of recent infection. Sera samples collected in 1986, 1996 or annually between 1987 and 1998 were tested for anti-PT IgG antibody. In 1996, the age-adjusted prevalence of pertussis infection was 1.2% and was higher in children than in adults. Amongst samples collected annually, older age and female sex, but none of the temporal variables, were associated with a serologically defined pertussis infection. There is an important incidence of infection in the population, which is greater amongst children than adults, but there is only limited evidence of a correlation with epidemic cycles.

European Sero-Epidemiology Network: standardisation of the assay results for pertussis.

A standardisation process was developed in order to compare and harmonize serological results of pertussis toxin (PT) antibody measurements performed by laboratories using different technical procedures for detection. This involved the development of a common panel, of sera by a designed reference centre, the distribution of the panel to each participating laboratory for testing with their routine methods, the comparison of the obtained results to those of the reference centre, and the calculation of standardisation equations by regressing the quantitative results against those of the reference centre. As a cut-off indicative of protection against pertussis has not yet been defined, a particular emphasis was laid upon achieving standardisation of high titre results that would allow epidemiological evaluations based on the estimation of the incidence of recent infections rather than on the traditional approach of determining the population immunity profile. A generally good agreement was achieved between the participating laboratories, all using ELISA procedures very similar in many crucial aspects, and standardisation equations were produced useful to enable inter-country comparison during the next stages of the European Sero-Epidemiology Network (ESEN) project concerning the serological surveillance of immunity to pertussis in Europe.

Measurement of IgG antibodies to Chlamydia trachomatis by commercial enzyme immunoassays and immunofluorescence in sera from pregnant women and patients with infertility, pelvic inflammatory disease, ectopic pregnancy, and laboratory diagnosed Chlamydia psittaci/Chlamydia pneumoniae infection.

BACKGROUND: Screening for Chlamydia trachomatis specific antibodies is valuable in diagnosing asymptomatic pelvic inflammatory disease (PID) and tubal damage following repeated episodes of PID. The assays in current use are unsuitable for screening large numbers of samples so there is a need to develop more suitable assays. AIMS: To compare the performance of several commercial C trachomatis enzyme immunoassays (EIAs) (SeroCT, C tracho(pep), Medac p-EIA, Vircell and Labsystems C trachomatis IgG EIAs) using major outer membrane protein (MOMP), an inactivated organism EIA (Genzyme Virotech EIA), and a genus specific EIA (Platelia Chlamydia IgG) with the whole cell inclusion immunofluorescence (WIF) assay. In addition, to adapt, using time resolved fluorescence technology, the assay showing the highest correlation with WIF. METHODS: Ninety sera from patients presenting with ectopic pregnancies, 187 sera from those with a variety of types of infertility, 33 sera from cases of PID where a fourfold rise in WIF titre occurred, and 90 sera from antenatal clinic attenders were tested. A panel of 36 sera from laboratory diagnosed cases of Chlamydia psittaci/Chlamydia pneumoniae infection was also tested. RESULTS: The Genzyme Virotech EIA showed the highest rank correlation coefficient (0.82) with WIF, particularly at high WIF titres. The MOMP specific assays varied in their correlation with WIF, with rank correlation coefficients ranging from 0.70 (Medac p-EIA) to 0.80 (Vircell EIA). The Genzyme Virotech assay showed poor specificity (5.6%; 95% confidence interval (CI), 0.68% to 18.7%)--it was reactive with 34 of the panel of 36 C psittaci/C pneumoniae positive sera. The MOMP based EIAs showed high specificity, particularly the Medac p-ELISA (97.2%; 95% CI, 85.5% to 99.9%)--only one serum was reactive. In view of the good correlation between WIF and the Genzyme Virotech EIA, a time resolved fluorescence immunoassay (TRFIA) was developed using the Genzyme Virotech antigen. Using an appropriate cut off the TRFIA assay showed excellent correlation with WIF. CONCLUSIONS: The TRFIA assay may be useful as a screening assay, possibly in conjunction with one of the highly specific EIAs studied (for example, Medac p-EIA) to confirm the antibody specificity of sera selected by the screening assay.