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Introduction. Antibody testing for evidence of a recent Bordetella pertussis infection by estimating anti-pertussis toxin immunoglobulin G (anti-PT-IgG) titres by enzyme-linked immunosorbent assays is often recommended for those with a cough lasting more than 14 days. Interpreting results varies, with studies recommending different anti-PT-IgG titre thresholds for assigning positivity. In England, early work looking at antibody titre distributions for samples submitted from April 2010 to July 2012 found an optimal threshold of greater than 70 IU ml -1 for good sensitivity, specificity and positive predictive value. Aim. The aim of this study is to use the same mixture modelling technique to determine if the 70 IU ml -1 threshold remains appropriate when assessing data before, during and after the outbreak of pertussis in 2011-2012. Methods. We reviewed titres for all serology-tested samples in England between 1 July 2008 to 30 June 2022. IgG titres were used to calculate the positivity based on the current threshold of 70 IU ml -1 , the median duration of cough for individuals who tested positive and, through mixture modelling, the sensitivity, specificity, positive and negative predictive values (PPV and NPV) of assay thresholds. Results. Positivity rates increased from 21.7 % prior to the outbreak to 30.3 % during the outbreak and dropped to 25.1 % post-outbreak; similar to estimates from the mixture model of 20.5, 33.3 and 28.7 %, respectively. Although the estimated sensitivity dropped during and after the outbreak when applying the 70 IU ml -1 threshold, the PPV remained high and therefore no change to this threshold is warranted. Conclusion. Mixture modelling is a useful tool to establish thresholds, but reassessment should also be done when there have been changes to prevalence and/or testing regimes to determine whether there have been any changes in sensitivity, specificity, PPV, and NPV and whether the threshold should be revised.

Introduction. Diphtheria is a potentially life-threatening infection and remains endemic in many low- and middle-income countries (LMICs). A reliable, low-cost method for serosurveys in LMICs is warranted to estimate the accurate population immunity to control diphtheria. Hypothesis/Gap Statement. The correlation between the ELISA results against diphtheria toxoid and the gold standard diphtheria toxin neutralization test (TNT) values is poor when ELISA values are <0.1 IU ml -1 , which results in inaccurate estimates of susceptibility in populations when ELISA is used for measuring antibody levels. Aim. To explore methods to accurately predict population immunity and TNT-derived anti-toxin titres from ELISA anti-toxoid results. Methodology. A total of 96 paired serum and dried blood spot (DBS) samples collected in Vietnam were used for comparison of TNT and ELISA. The diagnostic accuracy of ELISA measurement with reference to TNT was assessed by area under the receiver operating characteristic (ROC) curve (AUC) and other parameters. Optimal ELISA cut-off values corresponding to TNT cut-off values of 0.01 and 0.1 IU ml -1 were identified by ROC analysis. A method based on the multiple imputation approach was also applied to estimate TNT measurements in a dataset that only included ELISA results. These two approaches were then applied to ELISA results previously generated from 510 subjects in a serosurvey in Vietnam. Results. The ELISA results on DBS samples showed a good diagnostic performance compared to TNT. The cut-off values for ELISA measurement corresponding to the TNT cut-off values of 0.01 IU ml -1 were 0.060 IU ml -1 in serum samples, and 0.044 IU ml -1 in DBS samples. When a cut-off value of 0.06 IU ml -1 was applied to the 510 subject serosurvey data, 54 % of the population were considered susceptible (<0.01 IU ml -1 ). The multiple imputation-based approach estimated that 35 % of the population were susceptible. These proportions were much larger than the susceptible proportion estimated by the original ELISA measurements. Conclusion. Testing a subset of sera by TNT combined with ROC analysis or a multiple imputation approach helps to adjust ELISA thresholds or values to assess population susceptibility more accurately. DBS is an effective low-cost alternative to serum for future serological studies for diphtheria.

Introduction. Current serological diagnosis of pertussis is usually performed by ELISA, which is typically performed in larger diagnostic or reference laboratories, requires trained staff, and due to sample batching may have longer turnaround times. Hypothesis and Aim. A rapid point-of-care (POC) assay for pertussis serology would aid in both the diagnosis and surveillance of the disease. Methodology. A quantitative lateral flow (LF)-based immunoassay with fluorescent Eu-nanoparticle reporters was developed for the detection of anti-pertussis toxin (PT) and adenylate cyclase toxin (ACT) antibodies from oral fluid samples ( N =100), from suspected pertussis cases with respiratory symptoms. Results. LF assay results were compared to those obtained with anti-PT IgG oral fluid ELISA. For an ELISA cut-off value of 50 arbitrary units, the overall agreement between the assays was 91/100 (91 %), the sensitivity was 63/70 (90 %) and the specificity was 28/30 (93 %). No ACT-specific antibodies were detected from oral fluid samples; however, the signal readout positively correlated to those patients with high anti-PT IgG antibodies. Conclusion. The developed LF assay was a specific, sensitive and rapid test for serological diagnosis of pertussis with anti-PT antibodies and is a suitable POC test using oral fluid samples.

Introduction. In 2009, the World Health Organization (WHO) established the Global Invasive Bacterial Vaccine Preventable Disease (IB-VPD) Surveillance Network (GISN) to monitor the global burden and aetiology of bacterial meningitis, pneumonia and sepsis caused by Haemophilus influenzae (Hi), Neisseria meningitidis (Nm) and Streptococcus pneumoniae (Sp). Hypothesis/Gap Statement . The GISN established an external quality assessment (EQA) programme for the characterization of Hi, Nm and Sp by culture and diagnostic PCR. Aim. To assess the performance of sentinel site laboratories (SSLs), national laboratories (NLs) and regional reference laboratories (RRLs) between 2014 and 2019 in the EQA programme. Methodology. Test samples consisted of bacterial smears for Gram-staining, viable isolates for identification and serotyping or serogrouping (ST/SG), plus simulated cerebrospinal fluid (CSF) samples for species detection and ST/SG by PCR. SSLs and NLs were only required to analyse the slides for Gram staining and identify the species of the live isolates. RRLs, and any SLs and NLs that had the additional laboratory capacity, were also required to ST/SG the viable isolates and analyse the simulated CSF samples. Results. Across the period, 69-112 SS/NL labs and eight or nine RRLs participated in the EQA exercise. Most participants correctly identified Nm and Sp in Gram-stained smears but were less successful with Hi and other species. SSLs/NLs identified the Hi, Nm and Sp cultures well and also submitted up to 56 % of Hi, 62 % of Nm and 33 % of Sp optional ST/SG results each year. There was an increasing trend in the proportion of correct results submitted over the 6 years for Nm and Sp. Some SSLs/NLs also performed the optional detection and ST/SG of the three organisms by PCR in simulated CSF from 2015 onwards; 89-100 % of the CSF samples were correctly identified and 76-93 % of Hi-, 90-100 % of Nm- and 75-100 % of Sp-positive samples were also correctly ST/SG across the distributions. The RRLs performed all parts of the EQA to a very high standard, with very few errors across all aspects of the EQA. Conclusion. The EQA has been an important tool in maintaining high standards of laboratory testing and building of laboratory capacity in the GISN.

In Western Europe, the incidence of both respiratory and cutaneous diphtheria, caused by toxin-producing Corynebacterium diphtheriae , Corynebacterium ulcerans or Corynebacterium pseudotuberculosis , has been low over the past few decades thanks to the use of an effective vaccine and a high level of vaccination coverage. However, the disease has still not been eradicated and continues to occur in all of Europe. In order to prevent sequelae or a fatal outcome, diphtheria antitoxin (DAT) should be administered to suspected diphtheria patients as soon as possible, but economic factors and issues concerning regulations have led to poor availability of DAT in many countries. The European Centre for Disease Prevention and Control and World Health Organization have called for European Union-wide solutions to this DAT-shortage. In order to illustrate the importance of these efforts and underline the need for continued diphtheria surveillance, we present data on all registered cases of toxigenic and non-toxigenic C. diphtheriae , C. ulcerans and C. pseudotuberculosis in Belgium during the past decade, up to and including 2017.

Introduction. Diphtheria is caused by toxigenic strains of Corynebacterium diphtheriae , Corynebacterium ulcerans and Corynebacterium pseudotuberculosis . For diagnostic purposes, species identification and detection of toxigenic strains (diphtheria toxin ( tox ) - positive strains) is typically performed using end-point PCR. A faster quadruplex real-time PCR (qPCR) was recently developed (De Zoysa et al . J . Med . Microbiol. 2016;65(12):1521-1527). Aims. We aimed to improve the quadruplex method by adding a 16S rRNA gene target as an internal processing control, providing confirmation of the presence of bacterial DNA in the assays, thus avoiding the possibility of false-negative reporting. Methodology. Universal 16S rRNA gene primers and a probe were defined. The novel method was tested using 36 bacterial isolates and 17 clinical samples. Experimental robustness to temperature and reagent concentration variations was assessed. Results. The method allows detection of the tox gene and distinguishing C. diphtheriae (including the newly described species Corynebacterium belfantii ) from C. ulcerans and C. pseudotuberculosis . Complete diagnostic specificity, sensitivity and experimental robustness were demonstrated. The lower limit of detection for C. diphtheriae , C. ulcerans and tox targets was 1.86 genome copies per 5 µl reaction volume. The method was successfully used on two distinct qPCR technologies (LightCycler 480, Roche Diagnostics and Rotor-Gene Q, Qiagen) and in two laboratories (Institut Pasteur, Paris, France and Public Health England - National Infection Service, London, UK). Conclusion. This work describes validation of the improved qPCR quadruplex method and supports its implementation for the biological diagnosis of diphtheria.

To evaluate the role of Bordetella pertussis infection, 96 otherwise healthy 7- to 17-year-old subjects who were suffering from a cough lasting from 2 to 8 weeks were prospectively recruited. At enrolment, a nasopharyngeal swab and an oral fluid sample were obtained to search for pertussis infection by the detection of B. pertussis DNA and/or an elevated titre of anti-pertussis toxin IgG. Evidence of pertussis infection was found in 18 (18.7 %; 95 % confidence interval, 11.5-28.0) cases. In 15 cases, the disease occurred despite booster administration. In two cases, pertussis was diagnosed less than 2 years after the booster injection, whereas in the other cases it was diagnosed between 2 and 9 years after the booster dose. This study used non-invasive testing to show that pertussis is one of the most important causes of long-lasting cough in school-age subjects. Moreover, the protection offered by acellular pertussis vaccines currently wanes more rapidly than previously thought.