Risk Factors for Ebola Virus Persistence in Semen of Survivors in Liberia.

BACKGROUND: Long-term persistence of Ebola virus (EBOV) in immunologically privileged sites has been implicated in recent outbreaks of Ebola virus disease (EVD) in Guinea and the Democratic Republic of Congo. This study was designed to understand how the acute course of EVD, convalescence, and host immune and genetic factors may play a role in prolonged viral persistence in semen. METHODS: A cohort of 131 male EVD survivors in Liberia were enrolled in a case-case study. "Early clearers" were defined as those with 2 consecutive negative EBOV semen test results by real-time reverse-transcription polymerase chain reaction (rRT-PCR) ≥2 weeks apart within 1 year after discharge from the Ebola treatment unit or acute EVD. "Late clearers" had detectable EBOV RNA by rRT-PCR >1 year after discharge from the Ebola treatment unit or acute EVD. Retrospective histories of their EVD clinical course were collected by questionnaire, followed by complete physical examinations and blood work. RESULTS: Compared with early clearers, late clearers were older (median, 42.5 years; P < .001) and experienced fewer severe clinical symptoms (median 2, P = .006). Late clearers had more lens opacifications (odds ratio, 3.9 [95% confidence interval, 1.1-13.3]; P = .03), after accounting for age, higher total serum immunoglobulin G3 (IgG3) titers (P = .005), and increased expression of the HLA-C*03:04 allele (0.14 [.02-.70]; P = .007). CONCLUSIONS: Older age, decreased illness severity, elevated total serum IgG3 and HLA-C*03:04 allele expression may be risk factors for the persistence of EBOV in the semen of EVD survivors. EBOV persistence in semen may also be associated with its persistence in other immunologically protected sites, such as the eye.

Detection of Lymphogranuloma Venereum-Associated Chlamydia trachomatis L2 Serovars in Remnant Rectal Specimens Collected from 7 US Public Health Laboratories.

ABSTRACT: The frequency of lymphogranuloma venereum or invasive Chlamydia trachomatis infection with serovar L1, L2, or L3 is unknown in the United States. While no diagnostic test is commercially available, we used a laboratory-developed test and detected lymphogranuloma venereum-associated serovar L2 in 14% of 132 remnant C. trachomatis-positive rectal swabs.

Use of real-time PCR as an alternative to conventional genotyping methods for the laboratory detection of lymphogranuloma venereum (LGV).

Lymphogranuloma venereum (LGV) can be differentiated from non-LGV chlamydial infection using Sanger sequencing or molecular assays, including those that are commercially-available internationally. Here, we describe the performance of a rapid real-time PCR (RT-PCR)-based strategy in differentiating Chlamydia trachomatis infections associated with LGV or non-LGV serovars. One hundred three rectal swabs, previously genotyped using Sanger sequencing of the ompA gene as a reference method, were tested in the RT-PCR assays. All non-LGV specimens were correctly identified, but the RT-PCR failed to detect 1 LGV specimen, resulting in a sensitivity of 87.5% for the non-LGV/LGV RT-PCR assay. Additional performance characteristics (e.g., specificity, accuracy, and reproducibility) were all between 93% and 100% with a limit of detection ≤100 copies/reaction. Thus, this rapid RT-PCR method for LGV detection in clinical specimens is comparable to the reference method.

Development of a Multiplex Bead Assay To Detect Immunoglobulin G Antibodies to Babesia duncani in Human Serum.

Babesia duncani is the causative agent of babesiosis in the western United States. The indirect fluorescent antibody (IFA) assay is the diagnostic test of choice for detection of B. duncani-specific antibodies. However, this test requires parasitized red blood cells harvested from infected hamsters, and test results are often difficult to interpret. To simplify serological testing for B. duncani, a proteomics approach was employed to identify candidate immunodiagnostic antigens. Several proteins were identified by electrospray ionization mass spectrometric analysis, and four recombinant protein constructs were expressed and used in a multiplex bead assay (MBA) to detect B. duncani-specific antibodies. Two antigens, AAY83295.1 and AAY83296.1, performed well with high sensitivities and specificities. AAY83295.1 had a higher sensitivity (100%) but lower specificity (89%) than AAY83296.1, which had a sensitivity of 90% and a specificity of 96%. Combining these two antigens did not improve the performance of the assay. This MBA could be useful for diagnosis, serosurveillance, and blood donor screening for B. duncani infection.

Assessment and utility of 2 Chlamydia trachomatis Pgp3 serological assays for seroprevalence studies among women in the United States.

Two plasmid gene protein (Pgp3)-based serological assays, the Pgp3-ELISA and multiplex bead assay (Pgp3-MBA), were compared and used to estimate seropositivity of Chlamydia trachomatis (CT) among females 14 to 39 years old participating in the National Health and Nutrition Examination Survey between 2013-2016. Of the 2,201 specimens tested, 502 (29.5%, 95% CI 27.6-31.5) were positive using Pgp3-ELISA and 624 (28.4%, 95% CI 26.5-30.3) were positive using Pgp3-MBA. The overall agreement between the assays was 87.7%. Corresponding nucleic acid amplification test (NAAT) results were available for 1,725 specimens (from women 18-39 years old); of these, 42 (2.4%, 95% CI 1.8-3.3) were CT NAAT-positive. Most of the CT NAAT-positive specimens had corresponding positive serological assay results; 33 (78.6%, 95% CI 62.8-89.2) were Pgp3-ELISA-positive and 36 (85.7%, 95% CI 70.8-94.1) were Pgp3-MBA-positive. Although Pgp3-ELISA and Pgp3-MBA demonstrated equivalent performance in this study, an advantage of the Pgp3-MBA over Pgp3-ELISA is that it is well suited for high sample throughput applications.

Whole-Genome Enrichment and Sequencing of Chlamydia trachomatis Directly from Patient Clinical Vaginal and Rectal Swabs.

Chlamydia trachomatis, an obligately intracellular bacterium, is the most prevalent cause of bacterial sexually transmitted infections (STIs) worldwide. Numbers of U.S. infections of the urogenital tract and rectum have increased annually. Because C. trachomatis is not easily cultured, comparative genomic studies are limited, restricting our understanding of strain diversity and emergence among populations globally. While Agilent SureSelectXT target enrichment RNA bait libraries have been developed for whole-genome enrichment and sequencing of C. trachomatis directly from clinical urine, vaginal, conjunctival, and rectal samples, public access to these libraries is not available. We therefore designed an RNA bait library (34,795 120-mer probes based on 85 genomes, versus 33,619 probes using 74 genomes in a previous one) to augment organism sequencing from clinical samples that can be shared with the scientific community, enabling comparison studies. We describe the library and limit of detection for genome copy input, and we present results of 100% efficiency and high-resolution determination of recombination and identical genomes within vaginal-rectal specimen pairs in women. This workflow provides a robust approach for discerning genomic diversity and advancing our understanding of the molecular epidemiology of contemporary C. trachomatis STIs across sample types, geographic populations, sexual networks, and outbreaks associated with proctitis/proctocolitis among women and men who have sex with men.IMPORTANCEChlamydia trachomatis is an obligate intracellular bacterium that is not easily cultured, which limits our understanding of urogenital and rectal C. trachomatis transmission and impact on morbidity. To provide a publicly available workflow for whole-genome target enrichment and sequencing of C. trachomatis directly from clinical urine, vaginal, conjunctival, and rectal specimens, we developed and report on an RNA bait library to enrich the organism from clinical samples for sequencing. We demonstrate an increased efficiency in the percentage of reads mapping to C. trachomatis and identified recombinant and identical C. trachomatis genomes in paired vaginal-rectal samples from women. Our workflow provides a robust genomic epidemiologic approach to advance our understanding of C. trachomatis strains causing ocular, urogenital, and rectal infections and to explore geo-sexual networks, outbreaks of colorectal infections among women and men who have sex with men, and the role of these strains in morbidity.

Prevalence of Urogenital Mycoplasma genitalium Infection, United States, 2017 to 2018.

ABSTRACT: During the 2017-2018 National Health and Nutrition Examination Survey, urine samples from participants aged 14 to 59 years were tested for Mycoplasma genitalium infection. Overall prevalence was 1.7% (95% confidence interval [CI], 1.1%-2.7%). Prevalence was similar between males (1.8% [95% CI, 0.9%-3.1%]) and females (1.7% [95% CI, 0.8%-3.0%]).

Genomic Surveillance and Improved Molecular Typing of Bordetella pertussis Using wgMLST.

Multilocus sequence typing (MLST) provides allele-based characterization of bacterial pathogens in a standardized framework. However, classical MLST schemes for Bordetella pertussis, the causative agent of whooping cough, seldom reveal diversity among the small number of gene targets and thereby fail to delineate population structure. To improve the discriminatory power of allele-based molecular typing of B. pertussis, we have developed a whole-genome MLST (wgMLST) scheme from 225 reference-quality genome assemblies. Iterative refinement and allele curation resulted in a scheme of 3,506 coding sequences and covering 81.4% of the B. pertussis genome. This wgMLST scheme was further evaluated with data from a convenience sample of 2,389 B. pertussis isolates sequenced on Illumina instruments, including isolates from known outbreaks and epidemics previously characterized by existing molecular assays, as well as replicates collected from individual patients. wgMLST demonstrated concordance with whole-genome single nucleotide polymorphism (SNP) profiles, accurately resolved outbreak and sporadic cases in a retrospective comparison, and clustered replicate isolates collected from individual patients during diagnostic confirmation. Additionally, a reanalysis of isolates from two statewide epidemics using wgMLST reconstructed the population structures of circulating strains with increased resolution, revealing new clusters of related cases. Comparison with an existing core genome (cgMLST) scheme highlights the stable gene content of this bacterium and forms the initial foundation for necessary standardization. These results demonstrate the utility of wgMLST for improving B. pertussis characterization and genomic surveillance during the current pertussis disease resurgence.

Estimating Recommended Gonorrhea and Chlamydia Treatment Rate Using Linked Medical Claims, Prescription, and Laboratory Data in US Private Settings.

BACKGROUND: The Centers for Disease Control and Prevention (CDC) recommends specific regimens for chlamydia and dual therapy for gonorrhea to mitigate antimicrobial-resistant gonorrhea in the CDC 2015 sexually transmitted disease treatment guidelines. Only limited studies examining adherence to these recommendations have been conducted at private practices in the United States. METHODS: We used the OptumLabs Data Warehouse, a comprehensive, longitudinal data asset with deidentified persons with linked commercial insurance claims and clinical information, to identify persons aged 15 to 60 years who had valid nucleic acid amplification testing results demonstrating urogenital or extragenital gonorrhea or chlamydia in 2016 to 2018. We defined valid laboratory results as positive or negative. We then assessed the time of their first positive test result and the type of treatment within 30 days to determine if there was evidence in the claims record that the CDC-recommended treatment was provided. We defined presumed treatment if the date of treatment was before the date of the positive test result within 30 days. RESULTS: Among 6476 patients with positive gonorrhea test results and 26,847 patients with positive chlamydia test results only, 34.8% and 64.2% had evidence of receiving the CDC-recommended therapy, respectively. Approximately 11.6% of patients with positive gonorrhea test results with recommended dual treatment and 7.1% of patients with positive chlamydia test results only with recommended chlamydia treatment were presumptively treated. CONCLUSION: Analysis of treatment claims and medical records from private settings indicated low rates of recommended gonorrhea and chlamydia treatment. Validation of treatment claims is needed to support further quality of care interventions based on these data.

Detection and Characterization of Diphtheria Toxin Gene-Bearing Corynebacterium Species through a New Real-Time PCR Assay.

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.