Objectives: The burden of SARS-CoV-2 infection in people living with HIV (PLHIV) in South Sudan is unknown. Methods: We conducted a cross-sectional seroprevalence survey of SARS-CoV-2 immunoglobulin (Ig) G antibodies and other diseases of public health importance (strongyloidiasis, toxoplasmosis) in PLHIV in South Sudan during April 1, 2020-April 30, 2022. We used a multiplex SARS-CoV-2 immunoassay to detect IgG antibodies targeting the SARS-CoV-2 spike, receptor binding domain, and nucelocapsid (N) proteins, and antigens for other pathogens (Strongyloides stercoralis and Toxoplasma gondii). Results: Among 3518 samples tested, seroprevalence of IgG antibodies to SARS-CoV-2 spike protein and receptor binding domain 591 and nucleocapsid ranged from 1.4% (95% confidence interval [CI]: 0.9-2.1%) in April-June 2020 to 53.3% (95% CI: 49.5-57.1%) in January-March 2022. The prevalence of S. stercoralis IgG ranged between 27.3% (95% CI: 23.4-31.5%) in October-December 2021 and 47.2% (95% CI: 37.8-56.8%) in July-September 2021, and, for T. gondii IgG, prevalence ranged from 15.5% (95% CI: 13.3-17.9%) in April-June 2020 to 36.2% (95% CI: 27.4-46.2%) July-September 2021. Conclusions: By early 2022, PLHIV in South Sudan had high rates of SARS-CoV-2 seropositivity. Surveillance of diseases of global health concern in PLHIV is crucial to estimate population-level exposure and inform public health responses. © 2024 The Authors

In the United States, the general laboratory method for diagnosing pertussis, caused by Bordetella pertussis, is real-time PCR (rt-PCR) targeting insertion sequence 481 (IS481). Other Bordetella species (parapertussis, holmesii, and bronchiseptica) can also cause a pertussis-like syndrome, and some commercial laboratory assays include the insertion sequence 1001 (pIS1001) that can detect B. parapertussis/B. bronchiseptica (BppBb). Because IS481 exists in B. pertussis and B. holmesii, current commercial assays cannot differentiate these two species. We used a multiplex rt-PCR assay containing species-specific targets to Bordetella to evaluate clinical specimens detected as B. pertussis/B. holmesii (BpBh) or BppBb by commercial laboratories. A sample of 3,984 clinical specimens positive for IS481 or pIS1001 from two commercial laboratories during 2012-2019 were re-tested at CDC. Agreement of Bordetella species between the CDC and commercial laboratory assays, and the proportion of commercial laboratory specimens that were non-B. pertussis by CDC's assay was assessed. Overall agreement in Bordetella species detection and identification between the CDC and commercial lab assays was 85%. Agreement for identifying B. pertussis was 87% for 3,663 BpBh specimens and 98% for identifying B. parapertussis in 310 BppBb specimens. CDC's assay detected B. holmesii in 55/3,984 (1.4%) specimens. Most discrepant results (410/490, 82%) were BpBh specimens interpreted as indeterminate B. pertussis at CDC. We found a small portion of B. holmesii in a sample of IS481-positive clinical specimens originally identified by commercial laboratory rt-PCR assays, suggesting that commercial PCR assays are a reliable diagnostic tool for correctly identifying Bordetella species in most patients with suspected pertussis. IMPORTANCE: When testing specimens collected from patients with suspected pertussis, large-scale commercial laboratories in the United States employ an IS481-based assay that cannot differentiate between Bordetella pertussis and Bordetella holmseii. The level of B. holmesii causing pertussis-like illness in the United States is not well-understood given that only B. pertussis is nationally notifiable. After re-testing with a multiplex, species-specific rt-PCR assay, our data show low levels of B. holmesii identified in a sample of IS481-positive clinical specimens originally identified by commercial laboratory rt-PCR assays. These results reinforce the validity of large-scale commercial rt-PCR testing as a reliable diagnostic tool for pertussis in the United States.

Respiratory diphtheria, characterized by a firmly adherent pseudomembrane, is caused by toxin-producing strains of Corynebacterium diphtheriae, with similar illness produced occasionally by toxigenic Corynebacterium ulcerans or, rarely, Corynebacterium pseudotuberculosis While diphtheria laboratory confirmation requires culture methods to determine toxigenicity, real-time PCR (RT-PCR) provides a faster method to detect the toxin gene (tox). Nontoxigenic tox-bearing (NTTB) Corynebacterium isolates have been described, but impact of these isolates on the accuracy of molecular diagnostics is not well characterized. Here, we describe a new triplex RT-PCR assay to detect tox and distinguish C. diphtheriae from the closely related species C. ulcerans and C. pseudotuberculosis Analytical sensitivity and specificity of the assay were assessed in comparison to culture using 690 previously characterized microbial isolates. The new triplex assay characterized Corynebacterium isolates accurately, with 100% analytical sensitivity for all targets. Analytical specificity with isolates was 94.1%, 100%, and 99.5% for tox, Diph_rpoB, and CUP_rpoB targets, respectively. Twenty-nine NTTB Corynebacterium isolates, representing 5.9% of 494 nontoxigenic isolates tested, were detected by RT-PCR. Whole-genome sequencing of NTTB isolates revealed varied mutations putatively underlying their lack of toxin production, as well as eight isolates with no mutation in tox or the promoter region. This new Corynebacterium RT-PCR method provides a rapid tool to screen isolates and identify probable diphtheria cases directly from specimens. However, the sporadic occurrence of NTTB isolates reinforces the viewpoint that diphtheria culture diagnostics continue to provide the most accurate case confirmation.