Redefining the treponemal history through pre-Columbian genomes from Brazil.

The origins of treponemal diseases have long remained unknown, especially considering the sudden onset of the first syphilis epidemic in the late 15th century in Europe and its hypothesized arrival from the Americas with Columbus' expeditions1,2. Recently, ancient DNA evidence has revealed various treponemal infections circulating in early modern Europe and colonial-era Mexico3-6. However, there has been to our knowledge no genomic evidence of treponematosis recovered from either the Americas or the Old World that can be reliably dated to the time before the first trans-Atlantic contacts. Here, we present treponemal genomes from nearly 2,000-year-old human remains from Brazil. We reconstruct four ancient genomes of a prehistoric treponemal pathogen, most closely related to the bejel-causing agent Treponema pallidum endemicum. Contradicting the modern day geographical niche of bejel in the arid regions of the world, the results call into question the previous palaeopathological characterization of treponeme subspecies and showcase their adaptive potential. A high-coverage genome is used to improve molecular clock date estimations, placing the divergence of modern T. pallidum subspecies firmly in pre-Columbian times. Overall, our study demonstrates the opportunities within archaeogenetics to uncover key events in pathogen evolution and emergence, paving the way to new hypotheses on the origin and spread of treponematoses.

Immunohistochemical detection of Treponema pallidum in skin samples with clinical and histopathological correlations and Warthin-Starry staining critical analysis.

BACKGROUND: Syphilis in its different phases may be a difficult diagnosis in clinical and histopathological grounds. OBJECTIVES: The present study objectives were to evaluate the detection and tissue distribution of Treponema pallidum in skin lesions of syphilis. METHODS: A blinded diagnostic accuracy study was performed with immunohistochemistry and Warthin-Starry silver staining in skin samples from patients with syphilis and other diseases. Patients attended two tertiary hospitals between 2000 and 2019. Prevalence ratios (PR) and 95% confidence intervals (95% CI) were calculated for the association between immunohistochemistry positivity and clinical-histopathological variables. RESULTS: Thirty-eight patients with syphilis and their 40 biopsy specimens were included in the study. Thirty-six skin samples were used as non-syphilis controls. The Warthin-Starry technique was unable to accurately demonstrate bacteria in all samples. Immunohistochemistry showed spirochetes only in skin samples from patients with syphilis (24/40) with 60% sensitivity (95% CI 44.8‒75.2). Specificity was 100% and accuracy, 78.9% (95% CI 69.8‒88.1). Most cases had spirochetes in both dermis and epidermis and there was a high bacterial load. STUDY LIMITATIONS: Correlation between immunohistochemistry and clinical or histopathological characteristics was observed but was limited statistically due to the small sample size. CONCLUSIONS: Spirochetes were promptly seen in an immunohistochemistry protocol, which can contribute to the diagnosis of syphilis in skin biopsy samples. On the other hand, the Warthin-Starry technique showed to be of no practical value.

In Vitro Isolation of Treponema pallidum subsp. pallidum From Fresh and Frozen Needle Aspirates of Primary Experimental Syphilis Lesions.

ABSTRACT: Isolation of Treponema pallidum subsp. pallidum strains still relies on rabbit intratesticular inoculation of clinical samples. In this article, we report an alternative isolation approach based on the inoculation of fresh and frozen needle aspirates of primary experimental lesions into culture plates suitable for in vitro propagation of the syphilis agent.

A sad portrait of congenital syphilis in the State of Rio de Janeiro. Case report with Polymerase Chain Reaction (PCR) of scrapings from skin lesions and placenta fragment positive for Treponema pallidum

Introduction: Congenital syphilis is a serious public health problem that causes high rates of intrauterine morbidity and mortality, revealing flaws and weaknesses in the health system. Objective: to report a case of congenital syphilis in a university hospital in the Center-South Region of the State of Rio de Janeiro, Brazil. Case report: A pregnant woman, aged between 19 and 23 years old, carrying a Pregnant Woman's Handbook with a record of seven prenatal consultations and a note of the serological reaction for positive syphilis, but without any treatment, hospitalized at the University Hospital of Vassouras (RJ), in labor, gave birth to a newborn (NB) with a clinical picture and serological test of congenital syphilis. The NB required care in an intensive care unit and was discharged 28 days after birth. Scraping of skin lesions of the NB and placenta was performed for analysis by molecular biology (PCR in house) and genetic material of Treponema pallidum was detected. Conclusion: Congenital syphilis is a serious outcome of syphilis during pregnancy, consuming high financial resources and significant emotional distress for the mother, father, the whole family, as well as for the health teams. Our case report was the first that we are aware of in Brazil with a diagnosis by PCR for positive Treponema pallidum of skin scraping and placental fragment. It also showed poor quality prenatal care, a common factor in most cases of CS in our reality

Intrathecal Synthesis Index of Specific Anti-Treponema IgG: a New Tool for the Diagnosis of Neurosyphilis.

Neurosyphilis (NS) diagnosis is challenging because clinical signs are diverse and unspecific, and a sensitive and specific laboratory test is lacking. We tested the performance of an antibody index (AI) for intrathecal synthesis of specific anti-Treponema IgG by enzyme-linked immunosorbent assay (ELISA) for NS diagnosis. We conducted a retroprospective monocentric study including adults with neurological symptoms who had serum and cerebral spinal fluid (CSF) samples collected between 2006 and 2021. Two NS definitions were used. NS1 included patients with neurological symptoms, positive Treponema pallidum particle agglutination (TPPA) serology, and CSF-TPPA of ≥320, as well as CSF-leukocytes of >5 cells/mm3 and/or CSF-protein of >0.45 g/L and/or a reactive CSF-VDRL/RPR test. NS2 included patients with acute ocular and/or otologic symptoms, positive TPPA serology, and a response to NS treatment. Controls were patients with central nervous system disorders other than neurosyphilis. Anti-Treponema pallidum IgG were measured simultaneously in serum and CSF, and AI was calculated according to Reiber diagram. We assessed the AI test area under the curve (AUC), sensitivity/specificity, and estimated positive and negative predictive values. In total, 16 NS1 patients, 11 NS2 patients, and 71 controls were included. With an AI of ≥1.7 as a positive test for NS diagnostic, specificity was 98.6% (95% confidence interval [CI 95%] of 92.4 to 100.0) and sensitivity was 81.3% (CI 95% of 54.4 to 96.0) for NS1 and 98.6% (CI 95% 92.4 to 100.0) and 27.3% (CI 95% 6.0 to 61.0), respectively, for NS2. Positive and negative predictive values were >95% for NS1 and >85% for NS2, for prevalence above and below 20%. Measuring an AI for intrathecal synthesis of specific anti-Treponema pallidum IgG is a new promising tool highly specific for NS diagnosis. IMPORTANCE In the context of a lack of a gold standard for the diagnosis of neurosyphilis due to either nonspecific or nonsensitive tests, we present in this article a new promising tool highly specific for NS diagnosis. This new test involves measuring an intrathecal synthesis index of specific anti-Treponema IgG by ELISA.

Analysis of DNA extraction methods for detection of Treponema pallidum: A comparison of three methods.

Syphilis is a sexually transmitted disease caused by Treponema pallidum. DNA amplification methods have started to be used to facilitate diagnosis at different stages of the disease. The success of such methodologies depends on obtaining DNA from clinical samples in adequate quantity and quality for molecular reactions. There are many DNA extraction kits, but often the molecular analysis process is unfeasible due to its cost and access to imported products. Thus, this study aimed to analyze three methods of extracting DNA from Treponema pallidum from ulcers of patients investigated for syphilis. The three methods, an in house one (sonication) and two commercial ones (LGC, Brazil) and the PureLink Genomic DNA Mini Kit (Thermo Fisher Scientific, USA) were compared to the sequencing of these samples, which were used as a reference. Each method was evaluated based on the detection of T. pallidum by PCR using the tpp47 gene as a target for amplification, DNA quantification and method execution time. When compared to the sequencing, the sensitivity and agreement of the PureLink, sonication and LGC methods to extracted DNA were 100% (K = 1.0), 96.5% (K = 0.96) and 72.4% (K = 0.694), respectively. Specificity was 100% with the three methods. The sonication method was the closest in concentration of DNA to the PureLink method with a similar degree of purity, besides having the lowest cost-benefit ratio. It can be an interesting option for laboratories that work with reduced costs, since it is much more financially viable.

Treponema pallidum genome sequencing from six continents reveals variability in vaccine candidate genes and dominance of Nichols clade strains in Madagascar.

In spite of its immutable susceptibility to penicillin, Treponema pallidum (T. pallidum) subsp. pallidum continues to cause millions of cases of syphilis each year worldwide, resulting in significant morbidity and mortality and underscoring the urgency of developing an effective vaccine to curtail the spread of the infection. Several technical challenges, including absence of an in vitro culture system until very recently, have hampered efforts to catalog the diversity of strains collected worldwide. Here, we provide near-complete genomes from 196 T. pallidum strains-including 191 T. pallidum subsp. pallidum-sequenced directly from patient samples collected from 8 countries and 6 continents. Maximum likelihood phylogeny revealed that samples from most sites were predominantly SS14 clade. However, 99% (84/85) of the samples from Madagascar formed two of the five distinct Nichols subclades. Although recombination was uncommon in the evolution of modern circulating strains, we found multiple putative recombination events between T. pallidum subsp. pallidum and subsp. endemicum, shaping the genomes of several subclades. Temporal analysis dated the most recent common ancestor of Nichols and SS14 clades to 1717 (95% HPD: 1543-1869), in agreement with other recent studies. Rates of SNP accumulation varied significantly among subclades, particularly among different Nichols subclades, and was associated in the Nichols A subclade with a C394F substitution in TP0380, a ERCC3-like DNA repair helicase. Our data highlight the role played by variation in genes encoding putative surface-exposed outer membrane proteins in defining separate lineages, and provide a critical resource for the design of broadly protective syphilis vaccines targeting surface antigens.

Global phylogeny of Treponema pallidum lineages reveals recent expansion and spread of contemporary syphilis.

Syphilis, which is caused by the sexually transmitted bacterium Treponema pallidum subsp. pallidum, has an estimated 6.3 million cases worldwide per annum. In the past ten years, the incidence of syphilis has increased by more than 150% in some high-income countries, but the evolution and epidemiology of the epidemic are poorly understood. To characterize the global population structure of T. pallidum, we assembled a geographically and temporally diverse collection of 726 genomes from 626 clinical and 100 laboratory samples collected in 23 countries. We applied phylogenetic analyses and clustering, and found that the global syphilis population comprises just two deeply branching lineages, Nichols and SS14. Both lineages are currently circulating in 12 of the 23 countries sampled. We subdivided T. p. pallidum into 17 distinct sublineages to provide further phylodynamic resolution. Importantly, two Nichols sublineages have expanded clonally across 9 countries contemporaneously with SS14. Moreover, pairwise genome analyses revealed examples of isolates collected within the last 20 years from 14 different countries that had genetically identical core genomes, which might indicate frequent exchange through international transmission. It is striking that most samples collected before 1983 are phylogenetically distinct from more recently isolated sublineages. Using Bayesian temporal analysis, we detected a population bottleneck occurring during the late 1990s, followed by rapid population expansion in the 2000s that was driven by the dominant T. pallidum sublineages circulating today. This expansion may be linked to changing epidemiology, immune evasion or fitness under antimicrobial selection pressure, since many of the contemporary syphilis lineages we have characterized are resistant to macrolides.

Detection of Treponema pallidum by immunocytochemistry of cervical smear: A case report.

Syphilis is an infectious disease caused by Treponema pallidum (T. pallidum). A cervical smear is useful when screening for sexually transmitted diseases; however, T. pallidum is not detected in the usual Papanicolaou smear. We report the detection of T. pallidum by immunocytological examination of a cervical smear. A 22-year-old woman presented with nephrotic syndrome. On admission, we performed screening tests for infections, and her serology was positive for syphilis. A Papanicolaou cervical smear (Thin-Prep) showed slight nuclear enlargement, nuclear irregularity, and mild hyperchromasia in the superficial cells, but no organism was detected. T. pallidum was detected in the remaining specimen using immunocytochemistry. We also detected the T. pallidum DNA in a cervical biopsy specimen by polymerase chain reaction (PCR). Our findings suggest that immunocytological examination and PCR assay examination are useful tests for syphilis diagnosis.

Unusual erythematous plaque with white scales, a case of acquired syphilis in a child and literature review.

BACKGROUND: Syphilis in children is uncommon with the mode of infection for this rare condition likely being congenital or acquired. While most acquired cases of syphilis in children result from sexual abuse, children can also be infected with syphilis through kissing, breastfeeding, sharing of daily necessities or pre-chewed food. Here, we report a case of acquired secondary syphilis in a child due to consumption of pre-chewed-food and provide a review of the literature on the characteristics of acquired syphilis in preschool children. CASE PRESENTATION: A 3-year-old girl presented with erythematous plaques and scales on her head, neck, and thighs as well as flat red papules with a moist, well circumscribed surface covered with a grayish-white film. The grandmother who cared for the girl was in the habit of pre-chewing food before giving it to the girl. The child and grandmother tested positive for RPR. The girl, who was not sexually abused, was diagnosed with acquired secondary syphilis, resulting from the transmission of pre-chewed food from her grandmother. CONCLUSIONS: Our case report and literature review reveal that close contact among family members can result in the transmission of syphilis. We recommend that pre-chewing food should be discouraged by caregivers when caring for their children to avoid disease transmission.

Unusual clinical manifestation and challenging serological interpretation of syphilis: insights from a case report.

BACKGROUND: The clinical manifestations of recent syphilis can be variable, with typical and atypical patterns. Several conditions may cause atypical clinical aspects, including human immunodeficiency virus (HIV) co-infection. Besides the clinical features, co-infections may completely alter syphilis serological tests, causing interpretative difficulties and diagnostic delays. Aim of the work is to describe the difficulties encountered during the diagnostic evaluation of atypical skin manifestations and of the serology for syphilis of an HIV-infected patient who had contracted it several times. CASE PRESENTATION: In 2020, a 52-year old HIV-positive bisexual male patient was admitted to our department with a 4-month history of moderately itchy cutaneous lesions localized at his neck, trunk and arms. In 2013, the patient presented with a classic syphilitic roseola of the trunk and a secondary syphilis was diagnosed, with increased levels of rapid plasma reagin (RPR), Treponema pallidum hemagglutination assay (TPHA), anti-Treponema pallidum IgM and IgG Index. A second episode occurred in 2018, as a primary syphilis with multiple ulcerative lesions of the penis, and increased levels of RPR, IgG and IgM. In 2019, a further episode of secondary syphilis was treated with Doxycycline. In 2020, erythematous and papular lesions with vesicular components and urticarial erythema multiforme (EM)-like lesions were present at the neck, trunk and arms. Serological tests and Nucleic Acid Amplification Test (NAAT) for Treponema Pallidum were performed, as well as a cutaneous biopsy with histological and immunohistochemical evaluation of one lesion. NAAT was negative for T. pallidum. Serological test results were discordant with a new syphilis infection, showing only increased levels of RPR and anti-Treponema IgG. The cutaneous biopsy revealed a non specific histological pattern, while the immunohistochemical evaluation with anti-spirochetal antibodies was mandatory for the diagnosis of recent syphilis, showing clusters of rod-shaped elements, some of which with spiral form, focally present at the epidermis and adnexal structures. CONCLUSIONS: Nowadays, syphilis may present with atypical clinical and serological features. Physicians should be aware of these possible alterations and consider syphilis even in case of uncommon clinical aspect and unclear serological tests. Cutaneous biopsy and immunohistochemical exam may be mandatory for the diagnosis.

Diagnostic tools for neurosyphilis: a systematic review.

PURPOSE: Increasing incidences of syphilis highlight the preoccupation with the occurrence of neurosyphilis. This study aimed to understand the current diagnostic tools and their performance to detect neurosyphilis, including new technologies and the variety of existing methods. METHODS: We searched databases to select articles that reported neurosyphilis diagnostic methods and assessed their accuracy, presenting sensitivity and specificity values. Information was synthesized in tables. The risk of bias was examined using the Cochrane Handbook for Systematic Reviews of Diagnostic Test Accuracy recommendations. RESULTS: Fourteen studies were included. The main finding was a remarkable diversity of tests, which had varied purposes, techniques, and evaluation methodologies. There was no uniform criterion or gold standard to define neurosyphilis. The current basis for its diagnosis is clinical suspicion and cerebrospinal fluid analysis. There are new promising tests such as PCR tests and chemokine measurement assays. CONCLUSIONS: The diagnosis of neurosyphilis is still a challenge, despite the variety of existing and developing tests. We believe that the multiplicity of reference standards adopted as criteria for diagnosis reveals the imprecision of the current definitions of neurosyphilis. An important next step for the scientific community is to create a universally accepted diagnostic definition for this disease.

Exploration of an Efficient Simultaneous Molecular Detection Method of HIV, HCV, and Syphilis from a Single Dried Blood Spot.

OBJECTIVE: The aim of the present study was to evaluate the performance of the simultaneous detection of HIV-1 RNA, HIV-1 DNA, and HCV RNA using one dried blood spot (DBS) as an alternative sample to plasma. METHOD: A total of 571 paired DBS/plasma samples were collected from men who have sex with men (MSM) and injection drug users (IDUs), and serological and molecular assays were performed. Using plasma results as the reference standard, the performance of DBS tests for HIV-1 RNA, HIV-1 DNA, and HCV RNA was evaluated. Pearson's correlation coefficients and Bland-Altman analysis were performed to assess the correlation and concordance between DBS and plasma. RESULTS: Among paired plasma/DBS samples with detectable HIV-1 RNA and HCV RNA, five samples (5/32) were not detectable in DBS, while measurable HIV-1 RNA levels were present in plasma (1.44 to 3.99 log 10 copies/mL). There were two samples (2/94) with undetectable HCV RNA in DBS, while measurable HCV RNA levels were present in plasma (-5 to 5.99 log 10 copies/mL). The correlation between HIV-1 RNA light chain variable region (VL) values obtained from plasma and DBS showed that r = 0.683 ( P < 0.01), n = 27 and r = 0.612 ( P < 0.01), n = 89 in HCV RNA. Bland-Altman analysis revealed that in HIV-1 RNA, the mean (± SD) difference between HIV-1 RNA in plasma and DBS was 1.00 ± 1.01 log 10 copies/mL, and all samples were within ± 1.96 SD (-0.97 to 2.97 log 10 copies/mL) for DBS. The mean difference (± SD) in HCV RNA was 0.15 ± 1.08 log 10 copies/mL, and 94.38% (84/89) were within ± 1.96 SD (-1.96 to 2.67 log 10 copies/mL). Overall, HIV-1 RNA and HCV RNA levels obtained from a DBS were lower than those obtained from plasma. HIV-1 DNA in a DBS showed concordant results with HIV-1 RNA in plasma. HIV-1 DNA RT-PCR using a DBS showed acceptable performance. CONCLUSION: The performance of the simultaneous detection of HIV-1 RNA, HIV-1 DNA, and HCV RNA using one DBS was acceptable. DBS, as an alternative sample to plasma, may be a viable option for the simultaneous detection of HIV-1 RNA, HIV-1 DNA, and HCV RNA in resource-limited settings or for individuals living in areas that are difficult to access.

Treponema pallidum detection in lesion and non-lesion sites in men who have sex with men with early syphilis: a prospective, cross-sectional study.

BACKGROUND: Syphilis transmission is increasing, and precisely how Treponema pallidum is transmitted sexually from person to person is unclear. We aimed to determine the frequency of T pallidum shedding from potentially asymptomatic sites and the stage of infection at which shedding is most frequent in men who have sex with men (MSM), who have been disproportionately affected by syphilis. METHODS: We did a prospective, cross-sectional study in MSM recruited from Melbourne Sexual Health Centre (Melbourne, VIC, Australia). Men were eligible if they were aged 18 years or older, reported sex with men during the past 12 months, and had laboratory confirmed primary, secondary, or early latent syphilis, consistent with Australian definitions. Primary and secondary syphilis lesions were swabbed and non-lesion samples were collected via oral rinse, oral cavity swab, anal canal swab, urine, and semen. Samples were tested for T pallidum using PCR assays targeting polA (lesion and non-lesion samples) and 47 kDa (non-lesion samples only) gene targets. The primary outcome was the proportion of men with T pallidum detected from potentially asymptomatic sites-namely, the mouth, anus, urethra, and semen. FINDINGS: Between Nov 30, 2015, and May 23, 2019, 246 MSM were screened for inclusion, of whom 200 had serologically confirmed early syphilis and were included in the study: 54 (27%) of 200 had primary syphilis, 93 (47%) had secondary syphilis, and 53 (27%) had early latent syphilis. T pallidum DNA was detected in 48 (24%; 95% CI 18·3-30·5) of 200 men by oral rinse or oral lesion swab, or both, of whom 24 had no oral lesions. Oral T pallidum detection was most frequent in those with secondary syphilis compared with those at other stages of disease (41 [44%] of 93 vs seven [7%] of 107; p<0·0001), and in men with rapid plasma reagin titres of 1/64 or higher compared with those with lower titres (37 [32%] of 117 vs 11 [13%] of 83; p=0·0026). T pallidum was detected by anal canal swab or anal lesion swab, or both, in 45 (23·0%; 95% CI 17·3-29·5) of 196 men with available samples, of whom ten had no anal lesion. Furthermore, T pallidum was detected in urine samples of 12 (6·1%, 3·2-10·3) of 198 men and in semen samples from six (12·0%, 4·5-24·3) of 50 men who provided samples. Among the 93 men with secondary syphilis, 69 (74%) had T pallidum detected at any site, and 24 (26%) had detection at two or more separate sites. Among the 54 men with primary syphilis, 49 (91%) had T pallidum detected at any site, and 11 (20%) had detection at two or more separate sites. Among the 53 men with early latent syphilis, four (8%) had T pallidum detected at any site and none had T pallidum detected at two or more separate sites. INTERPRETATION: Unrecognised oral and anal shedding of T pallidum occurs in MSM with early syphilis, most frequently in those with secondary syphilis, suggesting secondary syphilis is the most infectious stage and that earlier detection and treatment of syphilis to prevent progression to the secondary stage might improve syphilis control. Future research is needed to ascertain the contribution of shedding of T pallidum from non-lesion sites to transmission of syphilis. FUNDING: Australian National Health and Medical Research Council.

Cauliflower-shaped lesions on a young woman's vulva.

A 20-year-old young woman with cauliflower-shaped lesions on her vulva was misdiagnosed as condyloma acuminatum. Further evaluation revealed positive serological tests for syphilis. On histological examination, and numerous plasma cells were seen to infiltrate the dermis heavily. The cauliflower-shaped lesions on vulva completely disappeared after benzathine penicillin treatment. Cauliflower-shaped lesions are a rare manifestation of secondary syphilis.

Epidemiological study in Brazilian women highlights that syphilis remains a public health problem.

Syphilis, an infectious disease considered a global public health concern, can cause stillbirths and neonatal deaths. This highlights the importance of continuous surveillance studies among women of reproductive age. A cross-sectional study was carried out to analyze the prevalence and risk factors associated with Treponema pallidum infection in women assisted by primary health care units in Dourados, a city located in Mato Grosso do Sul State, Brazil, which borders Paraguay. A questionnaire was applied to a population-based sample, blood samples were collected for syphilis testing and multivariable analyses were performed to screen associations with T. pallidum infection. The prevalence of T. pallidum infection was 6.04%. Bivariate analysis showed that women referring multiple sexual partners (c2: 6.97 [p=0.014]), income less 2 minimal wages (c2: 15.93 [p=0.003]), who did not have high school (c2: 12.64 [p=0.005]), and reporting history of STIs (c2: 7.30 [p=0.018]) are more likely to have syphilis. In the multivariate analysis, a highest prevalence ratio was observed in women with income less than 2 minimal wages (PR: 0.96 [95% CI: 0.85 - 0.97]), and who did not have high school (PR: 0.94 [95% CI: 0.90 - 0.98]). In addition, 80% of the women reported irregular use of condoms and 63.89% declared having sexual intercourses with multiple partners, which creates more opportunities for the transmission of the infection. These results highlight the need for healthcare systems to implement initiatives to monitor syphilis screening and the commitment of patients and their sexual partners to the treatment in order to achieve a decrease of new cases.