Current Status and Challenges Associated with Tick-Borne Pathogens and Diseases: Where Do We Stand?

Lyme Borreliosis (LB), caused by Borrelia burgdorferi sensu lato (s [...].

Anti-Treponema pallidum IgA response as a potential diagnostic marker of syphilis.

OBJECTIVES: Serological tests for syphilis detect mainly total Ig, IgM or IgG antibodies. We aimed to evaluate the specific IgA response in syphilis patients according to disease stage. METHODS: A serum IgA-enzyme immunoassay was developed using commercially available microplates coated with recombinant treponemal antigens and an anti-IgA-conjugate. To define a cut-off, we used 91 syphilis positive and 136 negative sera previously defined by the rapid plasma reagin and the Treponema pallidum particle agglutination results. Then we determined the intra- and inter-assay precisions, diagnostic sensitivity according to the clinical stage (in 66, 55 and 42 sera from primary, secondary and latent syphilis patients, respectively) and specificity (in 211 sera from people with conditions different to syphilis). IgA values were further measured in 71 sera from patients with previously treated syphilis. RESULTS: The newly developed IgA-enzyme immunoassay showed a good discrimination between negative and positive samples with intra- and inter-assay variation coefficients <20%. The sensitivity was 80.3% (95% CI, 70.0-90.6), 100.0% (95% CI, 99.1-100.0) and 95.2% (95% CI, 87.6-100.0) in primary, secondary and latent syphilis, respectively, and the specificity was 98.1% (95% CI, 96.0-100.0). Further, IgA values were negative in 61.3% (38/62) of patients with previously treated syphilis. DISCUSSION: Our findings suggest serum IgA as a sensitive and specific marker of syphilis and its detection could be used as a screening assay for active infection. Further evaluation is needed in prospective longitudinal field studies.

Whole genome sequences of Treponema pallidum subsp. endemicum isolated from Cuban patients: The non-clonal character of isolates suggests a persistent human infection rather than a single outbreak.

Bejel (endemic syphilis) is a neglected non-venereal disease caused by Treponema pallidum subsp. endemicum (TEN). Although it is mostly present in hot, dry climates, a few cases have been found outside of these areas. The aim of this work was the sequencing and analysis of TEN isolates obtained from "syphilis patients" in Cuba, which is not considered an endemic area for bejel. Genomes were obtained by pool segment genome sequencing or direct sequencing methods, and the bioinformatics analysis was performed according to an established pipeline. We obtained four genomes with 100%, 81.7%, 52.6%, and 21.1% breadth of coverage, respectively. The sequenced genomes revealed a non-clonal character, with nucleotide variability ranging between 0.2-10.3 nucleotide substitutions per 100 kbp among the TEN isolates. Nucleotide changes affected 27 genes, and the analysis of the completely sequenced genome also showed a recombination event between tprC and tprI, in TP0488 as well as in the intergenic region between TP0127-TP0129. Despite limitations in the quality of samples affecting breadth of sequencing coverage, the determined non-clonal character of the isolates suggests a persistent infection in the Cuban population rather than a single outbreak caused by imported case.

Sexually transmitted diseases during the COVID-19 pandemic: A focus on syphilis and gonorrhoea in Cuba.

OBJECTIVES: The implementation of social confinement measures to reduce the spread of coronavirus disease 2019 (COVID-19) has limited the clinical and laboratory diagnosis of sexually transmitted diseases (STDs) and their transmission. This study aims to explore the epidemiological behaviour of two notifiable STDs during the first 6 months of the COVID-19 pandemic in Cuba. STUDY DESIGN: This is an exploratory study of the incidence rates of syphilis and gonorrhoea during the COVID-19 pandemic in Cuba. METHODS: This study compares national weekly surveillance data on syphilis and gonorrhoea incidence rates in Cuba with the timeline of implemented social confinement measures. RESULTS: Incidence rates for both STDs were declining before the first case of COVID-19 was identified in Cuba. Following the confirmation of COVID-19 in Cuba and the implementation of social confinement measures, the decreasing incidence rates of syphilis and gonorrhoea continued. When social measures were subsequently relaxed, there was an increase in the incidence of syphilis, but incidence levels of gonorrhoea remained at a plateau. CONCLUSIONS: The results of this study suggest that the social measures introduced to curb the spread of COVID-19 influenced the sexual risk behaviours of individuals and subsequently modified the incidence of syphilis and gonorrhoea. It is necessary to continue epidemiological surveillance for longer periods of time and to explore it at the local, regional and national levels to better understand STDs in times of COVID-19.

Genotyping of Treponema pallidum in Cuba (2018-2019): Increased Circulation of Recombinant Genotype and No New Treponema pallidum Subspecies endemicum Infection Among Syphilis Patients.

OBJECTIVES: This study aimed to determine the allelic profiles of Treponema pallidum in patients confirmed with syphilis in Cuba (2018-2019) and to explore mutations leading to macrolide and tetracycline resistance. METHODS: Multilocus sequence typing and polymerase chain reaction of rrn loci (23S and 16S rDNA), followed by Sanger sequencing, were used to define the allelic profile of TPA and resistance mutations, respectively. RESULTS: Allelic profile 1.3.1 and the recombinant profile were identified, with 15.7.3 having an increased frequency. We did not detect the presence of the T. pallidum subspecies endemicum among syphilis patients, as in previous reports. A high frequency of macrolide-resistant strains and the absence of mutations potentially causing tetracycline resistance were found. CONCLUSIONS: Understanding the current status of treponemal infection in Cuban patients provides insights into the syphilis epidemiology.

A first insight into the genomic diversity of Leptospira strains isolated from patients in Cuba.

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.

Multilocus Sequence Typing of Treponema pallidum subsp. pallidum in Cuba From 2012 to 2017.

BACKGROUND: The increased prevalence of syphilis in Cuba prompted us to map the circulating Treponema pallidum subsp. pallidum allelic profiles in this geographic region. METHODS: Samples were collected from 2012 to 2017, from 83 male patients with ulcers or skin lesions, and were examined using multilocus sequence typing. Additionally, we analyzed the 23S rDNA and 16S rDNA regions for the presence of possible mutations leading to macrolide and tetracycline resistance. RESULTS: Among 94% of fully typed strains, we found 7 different allelic profiles, of which 4 had not been previously described. More than 87% of patients were infected with the T. pallidum SS14-like group and only 8.2% with T. pallidum Nichols-like group. As in other countries, the 1.3.1 allelic profile (ie, SS14-like) was the most common. In addition, 1 of the newly described allelic profiles represents T. pallidum strains that arose by recombination events between members of different T. pallidum subgroups. More than 90% of patients were infected with treponemes harboring the A2058G mutation. However, we found no potential tetracycline-resistant T. pallidum mutations. CONCLUSIONS: Our results suggest that, in Cuba, tetracycline antibiotics could be used to treat syphilis in penicillin-allergic patients instead of macrolides.

Etiology of Genital Ulcer Disease in Male Patients Attending a Sexually Transmitted Diseases Clinic: First Assessment in Cuba.

BACKGROUND: Sexually transmitted diseases (STDs) and in particular genital ulcer disease (GUD) have a major impact on morbidity and mortality in developing countries. The World Health Organization recommends the use of syndromic guidelines for the treatment of sexually transmitted infections (STIs) in resource-constrained countries. Surveillance of autochthonous etiologies provides epidemiological information contributing to the prevention and treatment of STIs. We investigated the etiology and factors associated with GUD among male patients attending a STD clinic in Havana, Cuba. METHODS: Swabs from genital ulcers of 113 male patients, collected from May 2012 to June 2015, were analyzed using PCR for herpes simplex virus types 1 and 2, Treponema pallidum, Haemophilus ducreyi, and Chlamydia trachomatis. We also investigated the clinical and epidemiological characteristics associated with the presence of these pathogens in GUD. RESULTS: At least one of the pathogens was detected in 70% of patients. The occurrence of the pathogens was herpes simplex virus type 2 (HSV-2) (51.3%), T. pallidum (29.2%), and C. trachomatis (1.8%). Co-infections occurred as follows: T. pallidum-HSV-2 (10.6%), C. trachomatis-HSV-2 (0.9%) and C. trachomatis-T. pallidum (0.9%). Herpes simplex virus type 1 and H. ducreyi were not detected. Ages 15 to 40 years, HIV-positive serostatus, and no condom use were significant risk factors for the presence of HSV-2 in genital ulcers. CONCLUSIONS: Our preliminary results highlight the predominance of HSV-2 and T. pallidum as the leading GUD etiologies in the study population and identified risk factors associated with HSV-2. This information should help to inform guidelines for better management of GUD in Havana, Cuba.

First Report of the 23S rRNA Gene A2058G Point Mutation Associated With Macrolide Resistance in Treponema pallidum From Syphilis Patients in Cuba.

This study aimed to assess the presence of macrolide-resistant Treponema pallidum subtypes in Havana, Cuba. Samples from 41 syphilis patients were tested for T. pallidum 23S rRNA gene mutations. Twenty-five patients (61%) harbored T. pallidum with the A2058G mutation, which was present in all 8 subtypes that were identified. The A2059G mutation was not detected.

First molecular evidence of Coxiella burnetii infecting ticks in Cuba.

Coxiella burnetii is the causative agent of Q fever. In order to explore the occurrence of C. burnetii in ticks, samples were collected from horses, dogs and humans living in a Cuban occidental community. The species most commonly recovered were Amblyomma mixtum (67%), Rhipicephalus sanguineus s.l. (27%) and Dermacentor nitens (6%). Specific IS1111 PCR and amplicon sequencing allowed the identification of C. burnetii DNA in A. mixtum collected from a domestic horse. These findings, for first time in Cuba, indicate the need for an in-depth assessment of the C. burnetii occurrence in hosts and humans at risk of infection.

Multiplex PCR for molecular screening of Borrelia burgdorferi sensu lato, Anaplasma spp. and Babesia spp.

INTRODUCTION: Ticks transmit a great variety of pathogenic microorganisms to humans and animals. The detection of tick-borne 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.

High sensitive PCR method for detection of pathogenic Leptospira spp. in paraffin-embedded tissues.

This study describes the development and application of a new PCR assay for the specific detection of pathogenic leptospires and its comparison with a previously reported PCR protocol. New primers were designed for PCR optimization and evaluation in artificially-infected paraffin-embedded tissues. PCR was then applied to post-mortem, paraffin-embedded samples, followed by amplicon sequencing. The PCR was more efficient than the reported protocol, allowing the amplification of expected DNA fragment from the artificially infected samples and from 44% of the post-mortem samples. The sequences of PCR amplicons from different patients showed >99% homology with pathogenic leptospires DNA sequences. The applicability of a highly sensitive and specific tool to screen histological specimens for the detection of pathogenic Leptospira spp. would facilitate a better assessment of the prevalence and epidemiology of leptospirosis, which constitutes a health problem in many countries.

HIGH SENSITIVE PCR METHOD FOR DETECTION OF PATHOGENIC Leptospira spp. IN PARAFFIN-EMBEDDED TISSUES / PCR altamente sensible para la detección de Leptospira spp. patógenas en tejidos embebidos en parafina

This study describes the development and application of a new PCR assay for the specific detection of pathogenic leptospires and its comparison with a previously reported PCR protocol. New primers were designed for PCR optimization and evaluation in artificially-infected paraffin-embedded tissues. PCR was then applied to post-mortem, paraffin-embedded samples, followed by amplicon sequencing. The PCR was more efficient than the reported protocol, allowing the amplification of expected DNA fragment from the artificially infected samples and from 44% of the post-mortem samples. The sequences of PCR amplicons from different patients showed >99% homology with pathogenic leptospires DNA sequences. The applicability of a highly sensitive and specific tool to screen histological specimens for the detection of pathogenic Leptospira spp. would facilitate a better assessment of the prevalence and epidemiology of leptospirosis, which constitutes a health problem in many countries.

El presente estudio describe el desarrollo y aplicación de un nuevo ensayo de PCR para la detección específica de leptospiras patógenas y su comparación con un protocolo reportado previamente. Se diseñaron nuevos cebadores para la optimización y evaluación de la PCR en tejidos embebidos en parafina infectados artificialmente. La PCR se aplicó además a muestras de tejidos embebidos en parafina y se realizó la secuenciación del amplicón resultante. La PCR diseñada fue más eficiente que el protocolo reportado, permitiendo la amplificación del fragmento de ADN esperado en las muestras infectadas artificialmente y del 44% de las muestras post mortem. Se secuenciaron 10 amplicones provenientes de pacientes diferentes. La aplicabilidad de una herramienta altamente sensible y específica en la búsqueda de leptospiras patógenas en especímenes histopatológicos podría facilitar una mejor valoración de la prevalencia y la epidemiología de la leptospirosis, la que constituye un problema de salud en disímiles países.