Validation of a clinical assay for botulinum neurotoxins through mass spectrometric detection.

Botulism is a paralytic disease due to the inhibition of acetylcholine exocytosis at the neuromuscular junction, which can be lethal if left untreated. Botulinum neurotoxins (BoNTs) are produced by some spore-forming Clostridium bacteria. The current confirmatory assay to test for BoNTs in clinical specimens is the gold-standard mouse bioassay. However, an Endopep-MS assay method has been developed to detect BoNTs in clinical samples using benchtop mass spectrometric detection. This work demonstrates the validation of the Endopep-MS method for clinical specimens with the intent of method distribution in public health laboratories. The Endopep-MS assay was validated by assessing the sensitivity, robustness, selectivity, specificity, and reproducibility. The limit of detection was found to be equivalent to or more sensitive than the mouse bioassay. Specificity studies determined no cross-reactivity between the different serotypes and no false positives from an exclusivity panel of culture supernatants of enteric disease organisms and non-toxigenic strains of Clostridium. Inter-serotype specificity testing with 19 BoNT subtypes was 100% concordant with the expected results, accurately determining the presence of the correct serotype and the absence of incorrect serotypes. Additionally, a panel of potential interfering substances was used to test selectivity. Finally, clinical studies included clinical specimen stability and reproducibility, which was found to be 99.9% from a multicenter evaluation study. The multicenter validation study also included a clinical validation study, which yielded a 99.4% correct determination rate. Use of the Endopep-MS method will improve the capacity and response time for laboratory confirmation of botulism in public health laboratories.

Evaluation of the WHO/CDC Syphilis Serology Proficiency Programme to support the global elimination of mother-to-child transmission of syphilis: an observational cross-sectional study, 2008-2015.

OBJECTIVES: Syphilis morbidity is high among pregnant women in lower income countries with limited laboratory capacity. We evaluated a long-standing global Syphilis Serology Proficiency Programme (SSPP) that supports testing quality in national reference laboratories to determine if participation affects congenital syphilis elimination strategies. DESIGN: In this observational cross-sectional study, we calculated coverage on type, frequency and quality of syphilis testing reported by laboratories enrolled in the SSPP from 2008 to 2015. We used country-reported data to WHO on four congenital syphilis (CS) indicators and World Bank country economic data to compare coverage and completeness of reporting of indicators in lower income countries with and without an SSPP-enrolled laboratory. PARTICIPANTS: From 2008-2015, 78 laboratories from 51 countries participated in >1 SSPP evaluation; 56% were national reference laboratories, of which most (93%) participated for >3 years and 11 (22%) in all 24 cycles. RESULTS: Median proficiency performance score was >95% regardless of test conducted. Of the 51 countries with an SSPP-enrolled laboratory, 22 (43%) were lower-income countries, of which 21 reported CS data during 2008-2015. Comparing CS data from 87 (90% of total) lower income countries with and without an SSPP-enrolled laboratory, countries with an SSPP-laboratory had stronger reporting on antenatal syphilis testing (p=0.04). For 2015, an estimated 74% of prenatal syphilis tests and 63% of positive tests reported to WHO from countries with an SSPP-enrolled laboratory. CONCLUSION: The SSPP has focused well on national reference laboratories, but has been only partially successful in recruiting laboratories from lower income countries. The finding that over half of syphilis infections in pregnant women living in countries with SSPP-enrolled laboratories suggests wide reach of the current quality assurance programme. However, reach could expand with focussed recruitment of laboratories from lower income countries.

Development of a syphilis serum bank to support research, development, and evaluation of syphilis diagnostic tests in the United States.

The Centers for Disease Control and Prevention's (CDC) Division of STD Prevention, in collaboration with the Association of Public Health Laboratories (APHL), is developing a nationally available syphilis serum repository for research of Food and Drug Administration (FDA)-cleared or investigational syphilis diagnostic assays in the United States. State and local public health laboratories (PHL) submitted de-identified residual sera with information on collection date, volume, storage conditions, freeze-thaw cycles, PHL serology results, reported syphilis stage and demographic details if available. Previous test results were blinded and sera (N = 152 reported syphilis stage, N = 131 unknown status) were tested at CDC using five FDA-cleared and one investigational syphilis tests. Treponemal and nontreponemal test sensitivity ranged from 76.3-100% and 63.2-100%, respectively, among staged specimens. The conventional treponemal assays showed high concordance of 95.4%. By providing syphilis stage and comprehensive serological test data, developed repository may serve as a valuable resource for diagnostic test validation studies.

Evaluation of a Rapid Syphilis Test in an Emergency Department Setting in Detroit, Michigan.

BACKGROUND: Syphilis transmission can be prevented by prompt diagnosis and treatment of primary and secondary infection. We evaluated the performance of a point-of-care rapid syphilis treponemal (RST) test in an emergency department (ED) setting. METHODS: Between June 2015 and April 2016, men aged 18 to 34 years seeking services in a Detroit ED, and with no history of syphilis, were screened for syphilis with the RST test, rapid plasma reagin (RPR) test, and Treponema pallidum particle agglutination assay (TP-PA). A positive reference standard was both a reactive RPR and a reactive TP-PA. We compared test results in self-reported men who have sex with men (MSM) to non-MSM. RESULTS: Among 965 participants, 10.9% of RST tests were reactive in MSM and only 1.5% in non-MSM (P < 0.001). Sensitivity of the RST test was 76.9% and specificity was 99.0% (positive predictive value, 50.0%) compared with the positive reference standard. Three discordant specimens found negative with the RST test but positive with the reference standard had an RPR titer of 1:1, compared with 10 specimens with concordant positive results that had a median RPR titer of 1:16. The RST sensitivity was 50.0% (positive predictive value, 68.4%) compared to the TP-PA test alone. Among men seeking care in an ED, the RST detected 76.9% of participants with a reactive RPR and TP-PA. CONCLUSIONS: The RST test detected all of the participants with an RPR titer ≥1:2 but less than 20% of participants with a positive TP-PA and negative RPR. The RST test was useful to detect a high proportion of participants with an active syphilis in an urban ED.

Laboratory Evaluation of a Commercially Available Rapid Syphilis Test.

Serological diagnosis of syphilis depends on assays that detect treponemal and nontreponemal antibodies. Laboratory certification and trained personnel are needed to perform most of these tests, while high costs and long turnaround time can hinder treatment initiation or linkage to care. A rapid treponemal syphilis test (RST) that is simple to perform, accessible, and inexpensive would be ideal. The Syphilis Health Check (SHC) assay is the only Food and Drug Administration (FDA)-cleared and Clinical Laboratory Improvement Amendments (CLIA)-waived RST in the United States. In this study, 1,406 archived human serum samples were tested using SHC and traditional treponemal and nontreponemal assays. Rapid test results were compared with treponemal data alone and with a laboratory test panel consensus defined as being reactive by both treponemal and nontreponemal assays for a given specimen, or nonreactive by both types of assays. The sensitivity and specificity of the SHC assay compared with treponemal tests alone were 88.7% (95% confidence interval [CI], 86.2 to 90.0%) and 93.1% (95% CI, 90.0 to 94.9%), respectively, while comparison with the laboratory test panel consensus showed 95.7% (95% CI, 93.6 to 97.2%) sensitivity and 93.2% (95% CI, 91.0 to 95.1%) specificity. The data were further stratified based on age, sex, pregnancy, and HIV status. The sensitivity and specificity of the SHC assay ranged from 66.7% (95% CI, 46.0 to 83.5%) to 91.7% (95% CI, 87.7 to 94.7%) and 88% (95% CI, 68.8 to 97.5%) to 100% (95% CI, 47.8 to 100%), respectively, across groups compared to traditional treponemal assays, generally increasing for all groups except the HIV-positive (HIV+) population when factoring in the laboratory test panel consensus. These data contribute to current knowledge of the SHC assay performance for distinct populations and may guide use in various settings.

A Nonhuman Primate Model for Rectally Transmitted Syphilis.

Among men who have sex with men (MSM), those with a diagnosis of syphilis or other rectal sexually transmitted infections (STIs) are at a higher risk for human immunodeficiency virus acquisition, which is concerning given the large increase in recently reported syphilis cases in the United States. We have developed the first nonhuman primate model for rectally transmitted syphilis by exposing simian/human immunodeficiency virus-infected and naive rhesus macaques to Treponema pallidum in the rectum. All animals showed mucosal lesions, systemic dissemination, and seroconversion (treponemal antibodies). This model would be valuable for studying the manifestations of and interventions for T. pallidum infection, with and without human immunodeficiency virus coinfection.