Specific detection of Treponema pallidum in clinical samples: validation of a qPCR assay combining two genomic targets.

OBJECTIVES: We evaluated a real-time quantitative PCR (qPCR) for detection of the Treponema pallidum (TP) genome in clinical samples through simultaneous detection of two genomic targets. METHODS: We performed qPCR with TaqMan technology using two TP genes, polA and tpp47, as targets, with an internal positive control. The qPCR assay was compared with syphilis diagnosis based on a combination of clinical examination, serological results and inhouse nested PCR (nPCR). Samples were analysed at the National Reference Center for STIs at Cochin Hospital in Paris. RESULTS: In total, from October 2010 to December 2016, 320 documented clinical samples (mucosal and cutaneous swabs) were collected from patients with or without syphilis attending STI centres in France. The qPCR had an overall sensitivity of 89% (95% CI 85.1% to 92.1%), a specificity of 100%, a positive predictive value of 100% and a negative predictive value of 88% (95% CI 84.3% to 91.5%). The agreement between qPCR and nPCR results was 94% (κ=0.88, 95% CI 0.83 to 0.93). Calibration of the qPCR assay, by cloning both the polA and tpp47 genes, defined the detection threshold as 1 copy/µL of DNA elution. CONCLUSIONS: We validated a new qPCR for detecting the TP genome in clinical samples with excellent sensitivity and specificity. The cloning of polA and tpp47 genes for calibration would be interesting in the evaluation of bacterial loads in samples.

Comparison of molecular and serological assays on cerebrospinal fluid for the diagnosis of neurosyphilis.

BACKGROUND: Many assays are available on cerebrospinal fluid (CSF) for the diagnosis of neurosyphilis (NS) but there is no 'gold standard'. OBJECTIVES: The aim of this study was to evaluate different molecular and serological assays used in NS. METHODS: We evaluated two PCR assays and three serological techniques in parallel on CSF samples collected between 2019 and 2020 from patients suspected of NS. RESULTS: The study included 143 patients comprising 30 early NS, 7 late NS and 106 patients without a diagnosis of NS. All patients with NS were symptomatic and had either neurological (67.6%) or ophthalmological signs (54.1%). The qPCR and nPCR assays had overall sensitivities (Se) of 41% and 27%, respectively; with each an overall specificity (Sp) of 100%. VDRL had a Se of 51% and a Sp of 92%. Immunoblot had a Se of 62% and a Sp of 85%. Finally, treponemal tests (TT) had a Se of 96% and a Sp of 69%. CONCLUSIONS: Our study confirms the excellent specificity of molecular techniques allowing to avoid overdiagnosis of NS, and thus, unjustified intensive antibiotic therapy protocols. CSF TT, although not very specific, has an excellent Se confirming that there is almost never NS with negative CSF TT. VDRL and immunoblot tests have better overall diagnostic performance. However, none of these techniques has sufficient diagnostic performance to represent a 'gold standard'. Thus, the diagnosis of NS relies on a combination of clinical and biological parameters with the association of PCR with serology, associating VDRL and immunoblot, in CSF.

Surveillance of Antibiotic Resistance Genes in Treponema Pallidum Subspecies Pallidum from Patients with Early Syphilis in France.

Benzathine penicillin G (BPG) is the reference treatment for early syphilis, but shortages have recently been reported, highlighting a need for the validation of alternative treatments. The aim of this study was to evaluate the genomic resistance of Treponema pallidum subspecies pallidum (TPA) to macrolides and doxycycline in France. Swabs from genital, anal, oral and cutaneous lesions were obtained from 146 patients with early syphilis in France. They were screened for mutations conferring resistance to macrolides and doxycycline by nested PCR and sequencing. Resistance to macrolides was detected in 85% of the isolates, but no point mutations conferring doxycycline resistance were detected. These findings confirm that, in France, resistance to macrolides is widespread. Moreover, we confirmed the absence of genomic resistance to doxycycline in the TPA strains. Therefore, doxycycline could be safely recommended as an alternative to BPG for the treatment of early syphilis.

Prognostic Factors in Syphilitic Uveitis.

PURPOSE: To identify predictors of treatment success in syphilitic uveitis (SU). DESIGN: Retrospective multicentric analysis of patients treated for SU. PARTICIPANTS: A total of 95 eyes (66 patients, mean [standard deviation] aged 49 [12.5] years, 31 [47%] of whom were human immunodeficiency virus [HIV]+) were analyzed. METHODS: Activity of SU was assessed at 1 week and 1 month after treatment onset, and at last follow-up. Improvement was defined by a ≥2-step decrease of both anterior chamber and vitreous haze inflammation levels, and by the size reduction in chorioretinal lesions. MAIN OUTCOME MEASURES: Recovery was defined as the resolution of inflammation in all anatomic structures at 1 month. RESULTS: Panuveitis and posterior uveitis were the most frequent findings. Inflammatory parameters were higher in HIV+ patients. Recovery was reported in 65% and 85% of eyes at 1 month and at last follow-up, respectively. In multivariate analysis, after adjusting for initial best-corrected visual acuity and the antimicrobial treatment regimen, clinical improvement at 1 week (corrected risk ratios [cRR], 3.5 [2.3-3.8]; P = 0.001) was predictive of recovery at 1 month, whereas the use of periocular dexamethasone injections (cRR, 0.05 [0.02-0.6]; P = 0.01) and methylprednisolone pulses negatively affected the outcomes of eyes. CONCLUSIONS: Early improvement is the strongest predictor of ophthalmological recovery in SU.

Clinical and Biological Characteristics of 40 Patients With Neurosyphilis and Evaluation of Treponema pallidum Nested Polymerase Chain Reaction in Cerebrospinal Fluid Samples.

BACKGROUND: Syphilis remains a significant public health problem. We conducted a prospective study to define more precisely the clinical and biological characteristics of patients with neurosyphilis (NS), and we assessed the diagnostic value of nested polymerase chain reaction (PCR) testing for Treponema pallidum in cerebrospinal fluid (CSF) samples. METHODS: From 2001 to 2013, we included 40 patients (90% men; 45% infected with human immunodeficiency virus) with NS, defined as syphilis with neurological and/or ophthalmological symptoms and CSF abnormalities. RESULTS: Thirty patients (75%) had early, 5 (12.5%) had late, and 5 had meningovascular NS. Twenty-four patients (80%) with early NS had ophthalmological symptoms, 14 (47%) had neurological symptoms, and 8 (26%) had both. All patients with meningovascular NS had only neurological symptoms. All patients with late NS had neurological symptoms, and 2 (40%) also had ocular symptoms. Ophthalmological symptoms were present in 65% of all patients with NS, and neurological symptoms in 60%. Seventeen patients (42.5%) had CSF white blood cell counts >20/µL (mean, 57/µL), and 27 (67.5%) had high CSF protein levels (>0.5 g/L; mean value, 1 g/L). CSF PCR results were positive in 42%, and CSF VDRL results in 30%. The nested PCR assay had an overall sensitivity of 42.5%, a specificity of 97%, a positive predictive value of 77%, and a negative predictive value of 86%. CONCLUSIONS: Early NS is the most frequent presentation, with an overrepresentation of polymorphous ophthalmological symptoms. PCR is highly specific and of potential value when used with other biological parameters.

Molecular subtyping of Treponema pallidum in Paris, France.

Two major Treponema pallidum subtypes, 14 d/g and 14 d/f, were identified in a population of 119 patients with syphilis in Paris, France, characterized by a high proportion of men who have sex with men. A new subtype named 11 q/j was characterized, and a reinfection case was determined in 1 patient having consecuitve syphilis infection at 19-month interval.

The epidemiological and clinical presentation of syphilis in a venereal disease centre in Paris, France. A cohort study of 284 consecutive cases over the period 2000-2007.

Since 2000, the incidence of syphilis has risen in developed countries. An updated knowledge of syphilis features is the key for early diagnosis and treatment. Our objective was to appraise the clinical and epidemiological presentation of syphilis in Paris, France. A retrospective monocentric descriptive study of 284 consecutive syphilis cases was conducted in a venereal disease centre (Paris, France), over the period 2000-2007. Epidemiological, clinical and microbiological data were systematically collected, using standardized medical forms. Overall, 95% of the cases occurred in men (271/284), 83% in men having sex with men (MSM) (231/278), 58% in patients having more than 10 partners/year (138/240) and 19% in patients who never use a condom (49/253). At least one STD has been previously diagnosed in 79% (220/279) of the cases. In 50.5% of the cases (142/281), HIV serology was positive. Most patients had primary (82/279, 29%) or secondary (125/279, 45%) syphilis. The most frequent physical signs in primary and secondary syphilis were, respectively, a genital chancre (63/82, 77%) and a diffuse exanthema (108/125, 86%). Syphilis occurs chiefly in MSM, often in HIV-positive patients. Many patients never use condoms. These data will help provide the basis for the development of national information and screening campaigns.

Multi-locus sequence typing of Treponema pallidum subsp. pallidum present in clinical samples from France: Infecting treponemes are genetically diverse and belong to 18 allelic profiles.

Treponema pallidum subsp. pallidum, the causative agent of sexually transmitted syphilis, detected in clinical samples from France, was subjected to molecular typing using the recently developed Multilocus Sequence Typing system. The samples (n = 133) used in this study were collected from 2010-2016 from patients with diagnosed primary or secondary syphilis attending outpatient centers or hospitals in several locations in France. Altogether, 18 different allelic profiles were found among the fully typed samples (n = 112). There were five allelic variants identified for TP0136, 12 for TP0548, and eight for TP0705. Out of the identified alleles, one, seven, and three novel alleles were identified in TP0136, TP0548, and TP0705, respectively. Partial allelic profiles were obtained from 6 samples. The majority of samples (n = 110) belonged to the SS14-like cluster of TPA isolates while 7 clustered with Nichols-like isolates. Patients infected with Nichols-like samples were more often older (p = 0.041) and more often diagnosed with secondary syphilis (p = 0.033) compared to patients infected with SS14-like samples. In addition, macrolide resistance caused by the A2058G mutation was found to be associated with allelic profile 1.3.1 or with strains belonging to the 1.3.1 lineage (p<0.001). The genetic diversity among TPA strains infecting the European population was surprisingly high, which suggests that additional studies are needed to reveal the full genetic diversity of TPA pathogens infecting humans.

Human Treponema pallidum 11q/j isolate belongs to subsp. endemicum but contains two loci with a sequence in TP0548 and TP0488 similar to subsp. pertenue and subsp. pallidum, respectively.

BACKGROUND: Treponema pallidum subsp. endemicum (TEN) is the causative agent of endemic syphilis (bejel). An unusual human TEN 11q/j isolate was obtained from a syphilis-like primary genital lesion from a patient that returned to France from Pakistan. METHODOLOGY/PRINCIPAL FINDINGS: The TEN 11q/j isolate was characterized using nested PCR followed by Sanger sequencing and/or direct Illumina sequencing. Altogether, 44 chromosomal regions were analyzed. Overall, the 11q/j isolate clustered with TEN strains Bosnia A and Iraq B as expected from previous TEN classification of the 11q/j isolate. However, the 11q/j sequence in a 505 bp-long region at the TP0488 locus was similar to Treponema pallidum subsp. pallidum (TPA) strains, but not to TEN Bosnia A and Iraq B sequences, suggesting a recombination event at this locus. Similarly, the 11q/j sequence in a 613 bp-long region at the TP0548 locus was similar to Treponema pallidum subsp. pertenue (TPE) strains, but not to TEN sequences. CONCLUSIONS/SIGNIFICANCE: A detailed analysis of two recombinant loci found in the 11q/j clinical isolate revealed that the recombination event occurred just once, in the TP0488, with the donor sequence originating from a TPA strain. Since TEN Bosnia A and Iraq B were found to contain TPA-like sequences at the TP0548 locus, the recombination at TP0548 took place in a treponeme that was an ancestor to both TEN Bosnia A and Iraq B. The sequence of 11q/j isolate in TP0548 represents an ancestral TEN sequence that is similar to yaws-causing treponemes. In addition to the importance of the 11q/j isolate for reconstruction of the TEN phylogeny, this case emphasizes the possible role of TEN strains in development of syphilis-like lesions.

Prevalence of Mycoplasma pneumoniae Infection in Malagasy Children.

BACKGROUND: Childhood community-acquired pneumonia is a leading cause of childhood morbidity in low-income countries. The etiologic agents are usually Staphylococcus aureus, Streptococcus pneumoniae and Mycoplasma pneumoniae. M. pneumoniae was recognized as a cofactor in asthmatic disease. High asthma prevalence was reported in Madagascar. Our aim was to clarify the prevalence of M. pneumoniae infection in this country and its relationship with asthma. METHODS: A prospective study was conducted in 351 children (from 2 to 16 years of age) from January 2012 to December 2014. According to the clinical symptoms, children were enrolled in 3 groups: "control group" (CG, n = 106), "asthma group" (n = 129) and "pneumonia group" (n = 116). The IgG and IgM M. pneumoniae status was evaluated by an enzyme-linked immunosorbent assay. Clinical signs of infection, socioeconomic data and antimicrobial treatment were recorded. RESULTS: The overall prevalence of M. pneumoniae infection was 18.2%. The multivariate analysis demonstrated that M. pneumoniae infection was significantly more frequent in the CG [pneumonia group vs. CG: odds ratio = 0.45 (0.21-0.91), P = 0.037 and asthma group vs. CG: odds ratio = 0.39 (0.18-0.87), P = 0.021]. The C-reactive protein value was significantly higher in children with M. pneumonia-positive serology (85 vs. 61 mg/L, P = 0.03). Of note, 99 (41%) children received antibiotics before attending. CONCLUSIONS: We report a prevalence of 18.2% for M. pneumoniae infection in children in Madagascar. The prevalence of M. pneumoniae infection was higher in the control patients than in asthmatic ones.

[Syphilis and pregnancy]. / Syphilis et grossesse.

Syphilis is a sexually transmitted disease responsible for a congenital severe infection. Congenital syphilis is complicated by fetal loss/neonatal death (50%), prematurity (25%) and major long term sequelae in other surviving children (20%). Every woman delivering a stillborn after 20WG should be tested for syphilis. Early screening is the cornerstone of prevention, and should be repeated in women at higher risk of contamination. Maternal management relies on early benzathine penicillin administration. Neonatal management relies on early diagnosis and prompt adequate penicillin therapy.

Clinical and serologic baseline and follow-up features of syphilis according to HIV status in the post-HAART era.

There is a lack of large studies appraising the effect of the human immunodeficiency virus (HIV) on the course of syphilis since the advent of highly active antiretroviral therapy (HAART). We aimed to appraise the effect of HIV on clinical and serologic features of syphilis at baseline and during follow-up in the post-HAART era.We designed a retrospective cohort study of consecutive syphilis cases, diagnosed between 2000 and 2007, in an academic venereal disease center. Data were collected using standardized medical forms. Patients were treated according to the European guidelines. Serologic failure was defined as either a 4-fold rise in Venereal Disease Research Laboratory (VDRL) titers 30-400 days posttreatment or a lack of 4-fold drop in VDRL titers at 270-400 days posttreatment.Among 279 syphilis cases with informative baseline clinical and serologic data, HIV infection was significantly associated with men having sex with men, French origin, multiple partners, lesser usage of condom, history of sexually transmitted disease, early syphilis, anal primary chancre, and cutaneous eruption. Median baseline titer from the Treponema pallidum hemagglutination assay (TPHA) was higher in HIV-infected patients (p = 0.02).Among 144 informative syphilis cases, there was a nonsignificant trend for a lower rate of serologic response among HIV-positive patients (91.8% vs. 98.3%, p = 0.14). Serologic failure was significantly associated with a history of previous syphilis (p < 0.05). The median delay to serologic response was similar in HIV-positive (117 d) and in HIV-negative (123 d) patients (p = 0.44).We conclude that for patients under HAART treatment, the effect of HIV on serologic response to syphilis treatment is likely minimal or absent.