Shotgun metagenomic screening to improve the retrospective diagnosis of undocumented intrauterine infections on embedded placenta samples: Lessons from a brucellosis case.

BACKGROUND: Microbiological diagnosis of intrauterine infections (IIU) still relies on bacteriological cultures or targeted DNA amplification lacking in sensitivity. Shotgun metagenomics (SMg) is an emerging unbiased molecular approach that makes it possible to sequence all the nucleic acids from any sample. It had never previously been used for IIU. METHODS: We here report the case of a patient with an unexplained IIU and fetal loss that could be documented by a combined SMg/microbiological approach, leading to the diagnosis of maternal brucellosis. RESULTS: A 31-year-old woman presented with an undocumented IIU with fetal loss at 24 weeks of gestation. Culture-based work-up failed to identify the pathogen involved. Paraffin-embedded placenta sample was retrospectively analyzed by SMg. Brucella spp nucleic acids were detected, and subacute maternal brucellosis was confirmed by targeted PCR and serological testing. CONCLUSION: This case provides grounds for further utilization of SMg for the microbiological diagnosis of unexplained obstetrical infections.

Use of a Multiplex PCR Assay To Assess the Presence of Treponema pallidum in Mucocutaneous Ulcerations in Patients with Suspected Syphilis.

We evaluated the utility of the commercial Allplex genital ulcer real-time PCR multiplex assay for detecting Treponema pallidum, herpes simplex virus 1 (HSV-1) and 2 (HSV-2), and Chlamydia trachomatis serovar L (lymphogranuloma venereum [LGV]) DNA in mucosal and genital ulcers in the context of suspected syphilis. In total, 374 documented genital and mucosal ulcers from patients with and without syphilis presenting at several sexually transmitted infection (STI) centers in France from October 2010 to December 2016 were analyzed at the National Reference Center (CNR) for Bacterial STIs at Cochin Hospital in Paris. T. pallidum subsp. pallidum detection results were compared with the final diagnosis based on a combination of clinical examination, serological results, and in-house nested PCR (nPCR). Detections of HSV and LGV were validated against reference methods. We found that 44.6% of the 374 samples tested were positive for T. pallidum subsp. pallidum, 21% for HSV, and 0.8% for LGV. No positive results were obtained for 30.7% of samples, and 4.8% presented coinfections. For T. pallidum subsp. pallidum detection, the overall sensitivity was 80% (95% confidence interval [CI], 76.1 to 84.1%), specificity was 98.8% (95% CI, 97.7 to 99.9%), positive predictive value was 98.8% (95% CI, 97.7 to 99.9%) and negative predictive value was 80.2% (95% CI, 76.2 to 84.2%), with a rate of concordance with the reference method of 92.5% (k = 0.85). This PCR multiplex assay is suitable for T. pallidum subsp. pallidum detection in routine use and facilitates the simultaneous rapid detection of a broad panel of pathogens relevant in a context of suspected syphilis lesions.

Congenital syphilis: A prospective study of 22 cases diagnosed by PCR.

Congenital syphilis (CS) is caused by Treponema pallidum infection in utero. There is a need to develop new tools to diagnose CS: the diagnostic value of PCR is difficult to assess. The aim of this study was to describe the clinical and laboratory characteristics of mothers and infants with CS as diagnosed by PCR tests on various maternal and neonatal samples. PATIENTS AND METHODS: We included all infants epidemiologically linked to a mother diagnosed with syphilis whose samples were referred to the Syphilis Reference Center, and for whom at least one positive PCR result was obtained. RESULTS: Twenty-two mother-infant pairs (8.3%) with assay performed on samples from one to four different anatomic sites were included between February 2011 and April 2018. Seven mothers (31.8%) were born abroad, fifteen (68.2%) presented psychological and/or social problems, eight (36.4%) had not been screened for syphilis prior to delivery, and eleven (50%) were referred from French overseas departments or territories, or from the Paris region. Six infants (27.3%) were stillborn and six were born preterm, while fifteen infants (68.2%) presented clinical features of CS. All infants born preterm were symptomatic. Infant VDRL/RPR titer was no greater than four times that in the mother's serum, except in two cases. DISCUSSION: Lack of antenatal care, social disadvantage and psychological issues were common. There is a need for enhanced surveillance both in the French overseas departments/territories and in the Paris region. A larger study is required to assess the sensitivity and specificity of PCR. The best site for sampling has yet to be established. We recommend the collection of as many samples as possible to avoid underdiagnosis of CS.

Evaluation of a PCR test for detection of treponema pallidum in swabs and blood.

Syphilis diagnosis is based on clinical observation, serological analysis, and dark-field microscopy (DFM) detection of Treponema pallidum subsp. pallidum, the etiological agent of syphilis, in skin ulcers. We performed a nested PCR (nPCR) assay specifically amplifying the tpp47 gene of T. pallidum from swab and blood specimens. We studied a cohort of 294 patients with suspected syphilis and 35 healthy volunteers. Eighty-seven of the 294 patients had primary syphilis, 103 had secondary syphilis, 40 had latent syphilis, and 64 were found not to have syphilis. The T. pallidum nPCR results for swab specimens were highly concordant with syphilis diagnosis, with a sensitivity of 82% and a specificity of 95%. Reasonable agreement was observed between the results obtained with the nPCR and DFM methods (kappa = 0.53). No agreement was found between the nPCR detection of T. pallidum in blood and the diagnosis of syphilis, with sensitivities of 29, 18, 14.7, and 24% and specificities of 96, 92, 93, and 97% for peripheral blood mononuclear cell (PBMC), plasma, serum, and whole-blood fractions, respectively. HIV status did not affect the frequency of T. pallidum detection in any of the specimens tested. Swab specimens from mucosal or skin lesions seemed to be more useful than blood for the efficient detection of the T. pallidum genome and, thus, for the diagnosis of syphilis.