Syphilis, caused by Treponema pallidum subsp. pallidum (TPA), remains an important public health problem with an increasing worldwide prevalence. Despite recent advances in in vitro cultivation, genetic variability of this pathogen during infection is poorly understood. Here, we present contemporary and geographically diverse complete treponemal genome sequences isolated directly from patients using a methyl-directed enrichment prior to sequencing. This approach reveals that approximately 50% of the genetic diversity found in TPA is driven by inter- and/or intra-strain recombination events, particularly in strains belonging to one of the defined genetic groups of syphilis treponemes: Nichols-like strains. Recombinant loci were found to encode putative outer-membrane proteins and the recombination variability was almost exclusively found in regions predicted to be at the host-pathogen interface. Genetic recombination has been considered to be a rare event in treponemes, yet our study unexpectedly showed that it occurs at a significant level and may have important impacts in the biology of this pathogen, especially as these events occur primarily in the outer membrane proteins. This study reveals the existence of strains with different repertoires of surface-exposed antigens circulating in the current human population, which should be taken into account during syphilis vaccine development.

Molecular identification of Treponema pallidum subsp. endemicum, the agent of bejel, in Cuban patients diagnosed with syphilis indicates the clear limitations of a diagnosis based exclusively on serology, geographical occurrence, clinical symptoms and anamnestic data. This finding has important implications for Global Public Health Systems, including paradigm changes regarding the location of endemic outbreaks, clinical aspects and transmission of this neglected disease.

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Objective To describe and analyze the clinical and epidemiological status in 28 confirmed cases of human leptospirosis at the main public hospital of Cordoba. Methods Between 2012 and 2013, we conducted an active surveillance at the main hospital of Cordoba to establish the etiologic diagnosis of the undifferentiated tropical febrile illness (UTFI) cases. UTFI is defined as a fever without an infection focus in the initial physical examination or in basic laboratory tests. Patients in acute phase were accompanied by prodromal symptoms, including myalgia, arthralgia, headache, asthenia, chills, icterus, dyspnea, abdominal pain, rash, and nausea. Samples were collected on admission and at discharge. Clinical and epidemiological data were collected for each patient. Microscopic agglutination test (MAT) was performed. Results The 28 leptospirosis cases presented the following gender distribution: male (n=24) and female (n=4). The duration of hospitalization was 10.39 days. The main symptoms and clinical manifestations were fever, headache and nausea, vomiting, and abdominal pain, all of which occurred in up to 60% of patients. Of the 28 cases studied, 4 were fatal. The most frequent infecting serogroups were Ballum and Canicola. Conclusion Leptospirosis is a common cause of undifferentiated tropical febrile illness in Colombia; it is important to establish ongoing and accurate surveillance for acute febrile illness to facilitate the detection of cases of leptospirosis.

Leptospirosis is a neglected disease causing severe infections in humans and animals. Due in part to misdiagnosis, this infectious disease results in nearly 60,000 deaths per year around the globe. This study represents the first effort to describe the diversity of pathogenic Leptospira in Cuba based on whole-genome sequencing. We have collected nineteen whole-blood samples from patients that were diagnosed as having leptospirosis between 2008 and 2012 in Cuba. In addition, we have enhanced our sample set by three historical strains that were used for the development of a human vaccine in 1990s. The Leptospira strains were grown and serotyped by the microscopic agglutination test, and the draft genomes were generated by NGS (Illumina). Subsequently, the core genomes were analyzed and compared to the genetic data available from other Caribbean islands and countries in Central America. Core genome Multi-locus Sequence Typing (cgMLST) revealed four different core genome clonal groups (cgCGs), with the highest number of samples belonging to L. interrogans, followed by L. borgpetersenii and L. kirschneri. All cgCGs that were found in Cuba have been also identified from multiple origins across the globe, except in neighbor countries and Central America. Serotyping divided the samples into the serogroups Canicola, Ballum and Pomona. The most frequent cgCGs, cgCG28, associated with serogroup Canicola, and cgCG15, associated with serogroup Ballum, have also been identified from samples isolated from dogs, rodents, and pigs; suggesting that these hosts represent the major source of human infection in Cuba. The vaccine strains did not significantly differ from the recent patient isolates. However, the increasing prevalence of samples belonging to the serogroup Ballum combined with the fact that the available vaccine in Cuba represents inactivated Leptospira belonging to serogroups other than Ballum, should be a valuable information for the National and Regional Leptospirosis Control Programs.

Rodríguez I, Burri C, Noda AA, Douet V, Gern L. Multiplex PCR for molecular screening of Borrelia burgdorferi sensu lato, Anaplasma spp. and Babesia spp. Ann Agric Environ Med. 2015; 22(4): 642-646. doi: 10.5604/12321966.1185767 Abstract Introduction. Ticks transmit a great variety of pathogenic microorganisms to humans and animals. The detection of tickborne pathogens (TBP) is mainly by molecular techniques based on polymerase chain reactions (PCR). Objective. To design and evaluate a multiplex PCR for the molecular screening of zoonotic TBP for exploratory studies. Material and methods. Control DNA from reference strains, DNA from experimentally-infected biological specimens, and from Rhipicephalus sanguineus ticks collected from domestic and homeless dogs were used. A multiplex PCR assay to detect the presence of Borrelia burgdorferi sensu lato, Anaplasma spp. and Babesia spp. was designed and optimized using primers previously reported for B. burgdorferi sensu lato and Anaplasma spp., while for Babesia spp. they were designed in silico. The multiplex PCR was evaluated on the DNA from biological samples. Results. A new set of specific primers for Babesia spp. was designed. Adjustment of the master mix reactive concentrations and amplification conditions for the multiplex PCR allowed the successful amplification of the specific amplicons for each microbial group from the control DNA and experimentally-infected biological specimens. The efficiency of the multiplex PCR amplifying three DNA targets was confirmed. Individual and co-infection of Anaplasma spp. and Babesia spp. were detected in the R. sanguineus ticks from dogs. Conclusions. A multiplex PCR assay for the screening of three TBP is available. By using it, B. burgdorferi sensu lato, Anaplasma spp. and Babesia spp. can be detected accurately in one PCR reaction.

Syphilis, caused by the spirochete Treponema pallidum subspecies pallidum, is a rising global public health concern and laboratory diagnostics remain challenging. Especially during early disease, rapid and accurate diagnosis is crucial to ensure patients and their contacts receive timely treatment to eradicate infection and prevent further transmission. In this prospective observational study, we evaluated the performance of polymerase chain reaction (PCR) and serological testing for the diagnosis of primary syphilis by evaluating anogenital swabs and sera from 178 Cuban patients presenting with ulcers. Three different PCR assays were evaluated targeting polA, tpp47 and 16S rDNA loci. Sera were evaluated with venereal disease research laboratory (VDRL) and T. pallidum hemagglutination (TPHA) assays. Assuming both methods were confirmatory, our data showed that PCR and serology did not correlate well (agreement = 52.3%, kappa 0.0512, 95% CI −0.0928–0.1951, p = 0.496). The sensitivities, specificities, positive and negative predictive values of the PCR assays were 76.1%, 100%, 100% and 57.9%, respectively, while the values for serology were 62.5%, 100%, 100% and 45.2%, respectively. The combination of PCR and serology can offer valuable information for the diagnosis of syphilis in patients presenting with anogenital ulceration avoiding further clinical complications and disease transmission.

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