Inhibition of bacterial FMN transferase: A potential avenue for countering antimicrobial resistance.

Antibiotic resistance is a challenge for the control of bacterial infections. In an effort to explore unconventional avenues for antibacterial drug development, we focused on the FMN-transferase activity of the enzyme Ftp from the syphilis spirochete, Treponema pallidum (Ftp_Tp). This enzyme, which is only found in prokaryotes and trypanosomatids, post-translationally modifies proteins in the periplasm, covalently linking FMN (from FAD) to proteins that typically are important for establishing an essential electrochemical gradient across the cytoplasmic membrane. As such, Ftp inhibitors potentially represent a new class of antimicrobials. Previously, we showed that AMP is both a product of the Ftp_tp-catalyzed reaction and an inhibitor of the enzyme. As a preliminary step in exploiting this property to develop a novel Ftp_Tp inhibitor, we have used structural and solution studies to examine the inhibitory and enzyme-binding properties of several adenine-based nucleosides, with particular focus on the 2-position of the purine ring. Implications for future drug design are discussed.

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.

Neurosyphilis: a series of 178 cases at the 3rd-level hospital of Marrakesh (Morocco).

Neurosyphilis (NS) is an infection of the central nervous system (CNS), caused by Treponema pallidum. Up to 4-10% of patients with untreated syphilis may develop NS which still constitutes a health challenge. The aim of this study is to analyze epidemiological, clinical, paraclinical, therapeutic, and progression profiles of NS in the south of Morocco. Authors analyzed retrospectively 178 files of patients with neurosyphilis, collected in the Neurology Department of Marrakesh over 25 years from January 1994 to March 2019. In our study, the mean age was 46 years (17-75 years). The number of males was dominant (87.6%). The most common presentation was meningoencephalitis. Four cases were atypical (late congenital syphilis, amyotrophic lateral sclerosis, neurosyphilis associated with neuro-Behcet's disease, and acute polyradiculoneuropathy). All patients had positive TPHA and VDRL serologies in blood; CSF-TPHA was positive in all patients, and CSF-VDRL was positive in 64.0% of patients. CT scan and brain MRI showed cortical atrophy in the majority of cases. One hundred seventy-seven patients were treated with intravenous injection of aqueous penicillin G. Neurosyphilis is still a significant medical problem in developing countries, and its occurrence in HIV infection is the reason for a growing number of new cases in developed countries. Given the frequent atypical manifestations of the disease, screening for neurosyphilis should be considered in all patients with neurological or psychiatric symptoms.

Efficacy of linezolid on Treponema pallidum, the syphilis agent: A preclinical study.

BACKGROUND: Penicillin G, the current standard treatment for syphilis, has important drawbacks, but virtually no preclinical or clinical studies have been performed to identify viable alternatives. We tested, both in vitro and in vivo, three marketed antibiotics with adequate pharmacological properties to treat syphilis. METHODS: We used an in vitro culturing system of T. pallidum to perform drug susceptibility testing and applied quantitative PCR targeting the tp0574 gene to measure bacterial growth. To confirm in vivo efficacy, fifteen rabbits were infected intradermally with T. pallidum at eight sites each and randomly allocated to an experimental treatment (linezolid, moxifloxacin, clofazimine) or a control arm (benzathine penicillin G [BPG], untreated). The primary outcome was treatment efficacy defined as the time to lesion healing measured from the date of treatment start. Secondary outcomes were absence of treponemes or treponemal mRNA in injection sites, absence of seroconversion, and cerebrospinal fluid (CSF) abnormalities and negative rabbit infectivity tests (RIT). FINDINGS: Linezolid showed in vitro bactericidal activity at concentrations of 0.5 µg/mL or higher. When administered orally to experimentally infected rabbits, it induced healing of early lesions at a time similar to BPG (hazard ratio 3.84; 95% CI 2.05-7.17; p < 0.0001 compared to untreated controls). In linezolid-treated animals, dark-field microscopy and qPCR assessment showed no presence of treponemes after day 3 post-treatment start, serologic test did not convert to positive, CSF had no abnormalities, and RIT was negative. Moxifloxacin and clofazimine failed to inhibit bacterial growth in vitro and could not cure the infection in the rabbit model. INTERPRETATION: Linezolid, a low-cost oxazolidinone, has in vitro and in vivo activity against T. pallidum, with efficacy similar to BPG in treating treponemal lesions in the animal model. Our findings warrant further research to assess the efficacy of linezolid as an alternative to penicillin G to treat syphilis in human clinical trials. FUNDING: European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (Grant agreement No. 850450).

Is There a Resistance Threshold for Macrolide Consumption? Positive Evidence from an Ecological Analysis of Resistance Data from Streptococcus pneumoniae, Treponema pallidum, and Mycoplasma genitalium.

If we were to keep macrolide consumption below a certain threshold, would this reduce the probability of macrolide resistance emerging? No study that we are aware of has addressed this question. We, therefore, assessed at a country level if there was a macrolide consumption threshold for the selection of a prevalence of macrolide resistance of over 5% in Streptococcus pneumoniae, Treponema pallidum, and Mycoplasma genitalium. In this ecological-level analysis, we found evidence for a macrolide consumption threshold of 1.3 defined daily doses per 1,000 inhabitants per day (DID) for M. genitalium, 1.8 DID for T. pallidum, and 2.3 DID for S. pneumoniae. Our results provide further motivation for macrolide stewardship campaigns that strive to reduce macrolide consumption to levels below at least 2 DID.

A case of primary syphilis characterised by lymphadenitis with abscess formation treated with only antibiotic without surgical excision of lymph node.

Syphilis is an increasingly common infectious disease caused by the bacterium Treponema pallidum. Atypical clinical presentations occur that may delay its diagnosis and treatment. Regional enlargement of lymph nodes is seen in both primary and secondary stages. Such lymph nodes very rarely become abscesses. Antibiotics should be administered in this situation; however, if this fails, the lymph nodes should be surgically excised.

Treponema pallidum in female sex workers from the Brazilian Marajó Archipelago: prevalence, risk factors, drug-resistant mutations and coinfections.

BACKGROUND: Female sex workers (FSWs) are an especially vulnerable group for syphilis and other sexually transmitted infection (STIs). This study determined the prevalence of syphilis in FSWs and factors associated with this disease in the Marajó Archipelago (northern Brazil), as well as the frequency of point mutations (A2058G and A2059G) in the 23S rRNA gene of Treponema pallidum and coinfections with hepatitis B virus (HBV), hepatitis C virus (HCV) and hepatitis D virus (HDV). METHODS: FSWs were diagnosed using a rapid qualitative test and the isolates were evaluated for the presence of point mutations by real-time PCR. Blood samples with T. pallidum were tested for the presence of HBV, HCV and HDV by ELISA and confirmed by real-time PCR. The factors associated with syphilis were identified using Poisson regression models. RESULTS: Overall, 41.1% FSWs tested positive for syphilis and 23.5% were infected with strains having A2058G/A2059G point mutations. HBV (23.0%) and HCV (8.1%) were detected among FSWs with syphilis. Six factors were associated with syphilis: low levels of education, reduced income, drug use, unprotected sex, a lengthy career in prostitution and a lack of regular medical check-ups. CONCLUSIONS: These findings indicate an urgent need for implementation of effective strategies to diagnose, prevent and treat syphilis, as well as other STIs, in this Brazilian region.

A novel peptide nucleic acid- and loop-mediated isothermal amplification assay for the detection of mutations in the 23S rRNA gene of Treponema pallidum.

Introduction. Macrolides could be a potential alternative treatment for Treponema pallidum infections in patients; however, macrolide-resistant T. pallidum is spreading rapidly worldwide.Hypothesis/Gap Statement. There are presently no alternatives to serological tests for syphilis that can be used to evaluate therapeutic effects due to the fact that T. pallidum cannot be cultured in vitro.Aim. In this study, we constructed a method for rapidly identifying T. pallidum and confirming macrolide resistance by using loop-mediated isothermal amplification (LAMP) with peptide nucleic acids (PNAs).Methodology. A set of LAMP primers was designed to span nucleotide positions 2058 and 2059 in 23S rRNA. A PNA clamping probe was also designed to be complementary to the wild-type sequence (A2058/A2059) and positioned to interfere with both the annealing of the 3' end of the backward inner primer and the concomitant extension. Prior to the LAMP assay, swab samples from suspected syphilitic lesions were boiled for DNA extraction.Results. The assay had an equivalent detection limit of 1.0×101 copies/reaction and showed specificity against 38 pathogens. In the presence of a 4 µM PNA probe, LAMP amplified up to 1.0×101 copies/reaction using plasmids harbouring the complementary mutant sequences (A2058G or A2059G), whereas amplification was completely blocked for the wild-type sequence up to a concentration of 1.0×103 copies/reaction. For the 66 PCR-positive clinical specimens, the overall detection rate via LAMP was 93.9 % (62/66). Amplification was successful for all 53 mutant samples and was incomplete for all nine WT samples by the PNA-mediated LAMP assays.Conclusion. We developed a PNA-mediated LAMP method that enabled us to rapidly identify T. pallidum and determine its macrolide susceptibility via a culture-independent protocol.

Immunization with nontreponemal antigen alters the course of experimental syphilis in the rabbit model.

The role of nontreponemal antibodies in the Treponema pallidum infection course is unclear. We investigated the effect of immunization with nontreponemal antigen on T. pallidum-challenged rabbits. Nontreponemal antigen was injected intravenously into rabbits in the nontreponemal group (n = 12) to elicit antibodies (≥1:64), and normal saline-injected rabbits were used as controls (n = 12). Then, rabbits were challenged with 106T. pallidum per site along their back. Lesion development was observed, and the injection sites were biopsied for mRNA analysis every week. Six rabbits from both groups were euthanized at 14 d and 28 d. The popliteal lymph nodes were extracted to assess infectivity using a rabbit infectivity test. The maximum lesion diameters were not different between the two groups (12.4 ± 0.9 mm in the nontreponemal group vs. 12.5 ± 1.0 mm in the control group, P = 0.386), but the time to maximum diameter appearance was delayed by approximately 4 d in the nontreponemal group (14.4 ± 1.6 d vs. 10.8 ± 1.9 d, P = 0.000). There were no significant differences in the proportions of lesions (58/60 (96.7%) vs. 59/60 (98.3%), P = 0.500) or ulcers (55/60 (91.7%) vs. 57/60 (95.0%), P = 0.359) between the two groups. An ulcer development delay of 5 d was observed in the nontreponemal group (19.3 ± 2.0 d vs. 14.0 ± 1.8 d, P = 0.000). IL-2 and IFN-γ mRNA expression in the nontreponemal group was significantly higher than that in the control group at 7 d and 14 d post-challenge. flaA mRNA expression and the rabbit infectivity test positive rate were not different between the two groups. Immunization with nontreponemal antigen altered the syphilis course in rabbits, resulting in delayed maximal lesion diameter and ulcer development, but it could not inhibit the spread of T. pallidum from primary lesion sites to viscera.

Phase II trial evaluating the clinical efficacy of cefixime for treatment of active syphilis in non-pregnant women in Brazil (CeBra).

BACKGROUND: Syphilis is a sexually and vertically transmitted infection caused by the bacteria Treponema pallidum for which there are few proven alternatives to penicillin for treatment. For pregnant women infected with syphilis, penicillin is the only WHO-recommended treatment that will treat the mother and cross the placenta to treat the unborn infant and prevent congenital syphilis. Recent shortages, national level stockouts as well as other barriers to penicillin use call for the urgent identification of alternative therapies to treat pregnant women infected with syphilis. METHODS: This prospective, randomized, non-comparative trial will enroll non-pregnant women aged 18 years and older with active syphilis, defined as a positive rapid treponemal and a positive non-treponemal RPR test with titer ≥1:16. Women will be a, domized in a 2:1 ratio to receive the oral third generation cephalosporin cefixime at a dose of 400 mg two times per day for 10 days (n = 140) or benzathine penicillin G 2.4 million units intramuscularly based on the stage of syphilis infection (n = 70). RPR titers will be collected at enrolment, and at three, six, and nine months following treatment. Participants experiencing a 4-fold (2 titer) decline by 6 months will be considered as having an adequate or curative treatment response. DISCUSSION: Demonstration of efficacy of cefixime in the treatment of active syphilis in this Phase 2 trial among non-pregnant women will inform a proposed randomized controlled trial to evaluate cefixime as an alternative treatment for pregnant women with active syphilis to evaluate prevention of congenital syphilis. TRIAL REGISTRATION: Trial identifier: www.Clinicaltrials.gov, NCT03752112. Registration Date: November 22, 2018.

Molecular diagnostic of syphilis. / Diagnóstico molecular de la sífilis.

Syphilis is a sexually transmitted infection caused by Treponema pallidum subsp. pallidum with an increasing incidence in Spain and in the rest of the world. Diagnosis is based mainly on serology, since direct diagnosis by dark field microscopy presents difficulties that limit its widespread use. Molecular biology techniques can be a useful tool for diagnosis in primary and secondary syphilis, although not all types of samples show the same behaviour. These techniques are also useful for the diagnosis of congenital syphilis. They are not recommended, however, for neurosyphilis, due to the low sensitivity of polymerase chain reaction in cerebrospinal fluid. These techniques have been used to study the controversial origin of syphilis, and, through the enhanced Centers for Disease Control method, to perform typing, which helps to elucidate the epidemiology of this infection. Finally, molecular techniques can detect mutations related to macrolide resistance, which are present in a very high percentage of infections.

Are nontreponemal tests suitable for monitoring syphilis treatment efficacy? Evidence from rabbit infection models.

OBJECTIVES: We aimed to characterize kinetics of non-treponamal antibody titres during the natural course of syphilis and explore their roles in monitoring syphilis treatment efficacy. METHODS: Sixty New Zealand white male rabbits were challenged with Nichols or Amoy Treponema pallidum strains, and the rapid plasma reagin (RPR) test was performed to quantify non-treponemal antibody titres during the infection course. Viable T. pallidum in the challenged rabbits was assessed with rabbit infectivity tests. RESULTS: The RPR titres of the Nichols or Amoy strain between no benzathine penicillin G (BPG) and BPG treatment subgroups displayed a similar trend: first ascending and then descending. Compared with baseline, the proportions of fourfold decline in RPR titres in the Nichols or Amoy group presented a similar result on days 30, 60 and 180 between the no BPG and BPG treatment subgroups (0%, 0/5; 80%, 4/5; 100%, 5/5; vs. 0%, 0/5; 80%, 4/5; 100%, 5/5; p 0.999; 0%, 0/5; 80%, 4/5; 80%, 4/5; vs. 40%, 2/5; 100%, 5/5; 100%, 5/5; p 0.098, respectively). Compared with the maximum baseline titre, the proportion of fourfold decline in PRR titre also showed a similar result in the two groups on days 30, 60 and 180 between the no BPG and the BPG treatment subgroups (0%, 0/5; 100%, 5/5; 100%, 5/5, vs. 40%, 2/5; 100%, 5/5; 100%, 5/5; p 0.129; 0%, 0/5; 100%, 5/5; 100%, 5/5, vs. 80%, 4/5; 100%, 5/5; 100%, 5/5; p 0.091, respectively. Moreover, regardless of whether the RPR titres presented a fourfold decline, viable T. pallidum could be detected in untreated rabbits' lymph nodes at 30, 60 and 180 days post infection, while viable T. pallidum was not detected in any of the treated rabbits' lymph nodes. CONCLUSIONS: The RPR titre increased and then decreased (even became negative) during the natural course of syphilis, similar to that seen after BPG treatment. The RPR tetre is thus a questionable indicator of syphilis treatment efficacy.

The "Scourge of the Renaissance". A Short Review About Treponema pallidum infection.

BACKGROUND: There is not a time in the history when epidemics did not loom large: infectious diseases have always had civilisation and evolution-altering consequences. Throughout history, there have been a number of pandemics: cholera, bubonic plague, influenza, smallpox are some of the most brutal killers in human history. Historical accounts of pandemics clearly demonstrate that war, unhygienic conditions, social and health inequality create conditions for the transmission of infectious diseases, and existing health disparities can contribute to unequal morbidity and mortality. The Renaissance was a period of European cultural, artistic, political and economic "rebirth" following the Middle Ages, but it was also the time when new infectious disease appeared, such as Syphilis. The epidemic spread of Syphilis began between the late 15th century and early 16th century due to the increased migration of peoples across Europe. The rapid spread of venereal syphilis throughout Europe suggests the introduction of a disease into a population that had not previously been exposed. Syphilis is a type of treponematosis, which includes syphilis, bejel, yaws, and pinta, but, while syphilis is venereal disease, the others are nonvenereal. Syphilis was, at the beginning, a disease of great severity due to its novelty, as the population had no time to gain any immunity against this venereal disease. METHODS: The purpose of this study is to investigate the origin of syphilis and the evolution of the treatments from the empiric means to the discovery of penicillin, but also to understand how this venereal disease has largely influenced human lifestyle and evolution. CONCLUSION: The first of the three hypotheses about its origins is the Columbian hypothesis, which states that Columbus's crew acquired syphilis from Native Americans and carried it back to Europe in 1493 A. D. On the contrary, the second hypothesis (pre-Columbian) asserts that syphilis was present in Europe long before Columbus's voyage and was transferred to the New World by Columbus's men. The Unitarian theory argues that syphilis, bejel, yaws, and pinta are not separate diseases but they represent syndromes caused by slightly different strains of one organism. Nowadays, Syphilis' origin is still uncertain and remains controversial. However, the large impact on the social behavior and international public health is an important reason to investigate about its origins and how to prevent the transmission.

Enhanced molecular typing and macrolide and tetracycline-resistance mutations of Treponema pallidum in Barcelona.

Aim: To describe the molecular types of Treponema pallidum and the proportion of macrolide and tetracycline resistance mutations in Barcelona. Materials & methods: Molecular type was determined using the Enhanced-CDC Typing system and antibiotic resistance was determined by sequencing the 23S and 16S rRNA genes. Results: A total of 183 patients were enrolled and 213 specimens (99 ulcers, 114 bloods) were collected. Sixty-two (70.5%) of 88 ulcers and 0 (0%) of bloods T. pallidum-DNA containing samples were fully typed. Up to 21 different strain types were identified (14d/g in 27.4%; 14f/g in 14.5%). Macrolide resistance mutations were present in 95% and tetracycline in 0%. Conclusion: Several different strains co-exist in Barcelona with a high proportion of macrolide resistance and absence of tetracycline resistance.