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.

Macrolide-resistant Mycoplasma genitalium infections in Cuban patients: an underestimated health problem.

BACKGROUND: The increasing prevalence of macrolide resistant Mycoplasma genitalium is a major concern worldwide. In Cuba, several cases of clinical treatment failure with 1 g single dose and extended azithromycin regimen have been detected and the aim of the present investigation was to retrospectively determine the prevalence of macrolide-resistance mediating mutations (MRMM) in M. genitalium-positive samples conserved at the Cuban National Reference Laboratory of Mycoplasma Research between 2009 and 2016. METHODS: A total of 280 positive DNA extracts were analysed by a 5' nuclease assay for detection of M. genitalium MRMM. Ten urogenital specimens from patients with azithromycin treatment failure and MRMM were inoculated in Vero cell to obtain the isolates for subsequent determination of antimicrobial susceptibility. RESULTS: The overall prevalence of MRMM was 32%. No MRMM was detected in samples collected between 2009 and 2013 but since 2014 a dramatic increase to 90% (95% CI, 76-96%) in 2016 was seen. Three new M. genitalium isolates were isolated in Vero cell cultures and confirmed phenotypic resistance to macrolides in a cell-culture assisted susceptibility test. Preliminary observations suggest that combination therapy with levofloxacin and doxycycline may represent an affordable option for treatment of macrolide resistant M. genitalium infections. CONCLUSIONS: This investigation showed the rapid emergence and high prevalence of MRMM in M. genitalium-infected patients in Cuba and confirmed the phenotypic resistance in isolates carrying MRMM. We suggest that Cuban guidelines for sexually transmitted infections are modified to include testing for M. genitalium and detection of MRMM in patients with failure of syndromic treatment, to ensure that in these cases, the treatment will be guided by etiologic diagnosis.

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.

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.

Imported cases of cutaneous leishmaniasis in Cuba, 2017: role of human movement.

BACKGROUND: Leishmaniasis is a vector-borne disease caused by several species from genus Leishmania. An increase in the number of cases related to human movement has been informed in the last years. Due to the increase of suspicious leishmaniasis cases arriving in Cuba during 2017, a general analysis is presented herein. METHODS: Clinical samples were collected from 5 patients suspicious of leishmaniasis, received from January to December 2017 at the Institute of Tropical Medicine Pedro Kourí, Cuba. Skin lesion samples were analyzed using different diagnostic assays: direct smear, histological examination, and molecular analysis for species identification. Epidemiological and demographic data were requested from each case and analyzed. Treatment and follow up of patient was also performed. RESULTS: Five cases were confirmed as Leishmania infection according to microscopic observation and molecular methods results. PCR-18S, PCR-N/RFLP and PCR-F/RFLP identified the following species: L. panamensis (2 cases), L. braziliensis (1 case), L.panamensis/L.guyanensis (1 case), L. mexicana complex (1 case). In treated patients, drugs were well tolerated, cure were documented and no relapse have been currently reported (3 years later). CONCLUSIONS: Clinical characteristics, demographic data, and epidemiological features of infection for each case evidence the potential risk related with travel to endemic areas of leishmaniasis. KEYWORKS: Cutaneous leishmaniasis, Epidemiology, Imported cases.

Kaposi's sarcoma-associated herpesvirus load in asymptomatic contacts of Cuban epidemic KS patients.

To evaluate the pathogenic mechanisms and transmission routes involved in KSHV infection in 22 Cuban individuals who maintained close contact with epidemic KS patients, real-time PCR was used to quantify KSHV-DNA in clinical samples of plasma, saliva and peripheral blood mononuclear cells (PBMC). KSHV-DNA was detected in 72.7% (16/22) of the contacts. The highest levels of KSHV load were detected in saliva, followed by PBMC (average log copies/100 ng DNA = 1.28 and 1.12), while significantly lower levels were detected in plasma (average log copies/ml = 0.37). Two of three intra-domiciliary and two serodiscordant sexual contacts of AIDS-KS patients were infected with KSHV. The rate of KSHV-DNA detection in saliva and PBMC samples in men who have sex with men (MSM) was significantly higher than in heterosexuals (HT) (p = 0.014). MSM were more likely to harbor KSHV-DNA in saliva when compared with HT individuals (OR 4.33; 95% CI 1.117-16.8). These results emphasize that, in Cuba, KSHV horizontal transmission through saliva may occur, although homosexual behavior may predispose an individual to KSHV acquisition. Even in the absence of disease, KSHV could cause an asymptomatic systemic infection in individuals who maintain close contact with AIDS-KS patients.

Latin American Clinical Practice Guidelines on the Systemic Treatment of Psoriasis SOLAPSO - Sociedad Latinoamericana de Psoriasis (Latin American Psoriasis Society).

This Clinical Practice Guideline on the systemic treatment of Psoriasis includes the recommendations elaborated by a panel of experts from the Latin American Psoriasis Society SOLAPSO, who assessed the quality of the available evidence using the GRADE system and the PICO process to guide the literature search. To answer each question, the experts discussed the results of randomized controlled trials, observational studies and metanalysis evaluating the interventions identified (non-biologics, biologics and phototherapy) in different populations of patients with moderate to severe plaque-psoriasis, which was summarized in Tables ad-hoc. The main end-points considered to assess efficacy were PASI 50, 75, 90 and 100, PGA 0-1 and significant improvement of health-related quality of life. Specific adverse events, either severe or leading to treatment interruption, were also evaluated. The 31 recommendations included in this CPG follow the structure proposed by GRADE: direction (for or against) and strength (strong or weak). The goal of this CPG is to improve the management of patients with psoriasis by recommending interventions of proved benefit and providing a reference standard for the treating physician. Adhering to the contents of this CPG does not guarantee therapeutic success. The final decision on the specific treatment is the responsibility of the physician based on the individual circumstances and considering the values, the preferences and the opinions of the patient or caregivers.

Mycoplasma genitalium infections in Cuba: surveillance of urogenital syndromes, 2014-2015.

Mycoplasma genitalium is an emerging sexually transmitted pathogen implicated in urethritis in men and several inflammatory reproductive tract syndromes in women. The prevalence of M. genitalium infections in Cuban patients with urogenital syndromes is unknown. The aim of this study was to analyse the prevalence of M. genitalium infection in sexually-active Cuban men and women with urogenital syndromes as a part of aetiological surveillance of urogenital syndromes in Cuba. Samples from men and women with urogenital syndromes submitted to the Mycoplasma Reference Laboratory for mycoplasma diagnosis from 1 January 2014 to 1 June 2015 were analysed by polymerase chain reaction (PCR) for detection of M. genitalium. A total of 971 samples were received and processed. Of the patients tested, 5.7% (47/824) of women and 27.9% (41/147) of men were positive for M. genitalium. This paper presents the largest study of M. genitalium infections among Cuban patients with urogenital syndromes and is Cuba's first M. genitalium survey. We suggest that M. genitalium should be considered in the Cuban sexually transmitted infection management protocols as an important pathogen, particularly in men.

Imported leishmaniasis cases in Cuba (2006-2016): what have we learned.

BACKGROUND: Leishmaniasis is a neglected parasitic disease caused by Leishmania spp., which is not endemic in Cuba. However, several factors (such as human activities, climate changes, and tourism) have led to an increase in the number of leishmaniasis cases in all regions, raising diagnosis and surveillance issues. We aim to present the retrospective analysis of 16 human cases suspicious of leishmaniasis, which were received during 2006-2016 for diagnosis at the Department of Parasitology from the Institute of Tropical Medicine Pedro Kourí, Cuba. METHODS: Clinical samples were collected and analyzed via different diagnostic assays, including direct smear, cultivation, histological analysis, and molecular analysis. Epidemiology and background of infection, clinical features, sex and age from each patient was recorded. RESULTS: From the 16 suspicious cases, 5 cases were confirmed for Leishmania infection, based on at least two positive results using different methods: PCR-based diagnosis [18S rRNA (5/5), hsp20 gene (4/5), hsp70 gene (3/5)], histopathology evaluation (2/3), parasite cultivation (2/3), or direct smears (2/3). L. braziliensis and L. mexicana were identified as the involving species in two cases, according to hsp70 PCR-RFLP protocols. Demographic and clinical features, as well as treatment and follow up, are described for every case. CONCLUSIONS: The combination of parasitological and molecular methods allowed proper diagnosis of imported leishmaniasis cases in Cuba. The utility and advantages of molecular diagnosis assays in non-endemic countries like Cuba are discussed.

Human papillomavirus infection in anal intraepithelial lesions from HIV infected Cuban men.

BACKGROUND: An association between HPV infection and progression to anal squamous intraepithelial lesions (ASIL) has been established, specifically in high-risk populations such as HIV-infected men. In this population, anal cancer is one of the most common non-AIDS-defining malignancies. METHODS: A cross-sectional study to detect anal lesions and HPV infection was performed. Anal mucosa samples were collected from 56 HIV-infected men from Cuba. The cytological diagnosis was done according to Bethesda 2001 System. HPV DNA detection was determined by qPCR for six high-risk HPV types and end point PCR for low-risk HPV types (6 and 11). The end point PCR with nucleotide sequencing technique was achieved to detect other genotypes of HPV not included in the qPCR in those samples negative for HPV- 6 and 11 or negative for the six genotypes identified in the qPCR. RESULTS: Cytological diagnosis identified 53 of 56 (95%) men with abnormal anal cytology. Among those, 26% (14/53) had atypical squamous cells of undetermined significance (ASC-US), 4% (2/53) had atypical squamous cells of undetermined significance cannot exclude high-grade lesions (ASC-H), 64% (34/53) had low-grade squamous intraepithelial lesions (LSIL), and 6% (3/53) had high-grade squamous intraepithelial lesions (HSIL). HPV DNA was detected in 89% (50/56) of men and 79% had at least one of the high-risk HPV types. HPV- 16 was the most common genotype (52%), while HPV-18 was the most frequently detected genotype in men with HSIL. We found statistically significant differences in the HPV viral loads with respect to the cytology results (p = 0.0006) and that the practice of receptive anal sex was a risk factor for anal HPV infection (p = 0.032). CONCLUSION: This study shows a high prevalence of ASIL and high-risk HPV infections in the study group and is the first study showing the distribution of HPV genotypes in HIV infected Cuban men with abnormal anal cytology. This information may be of importance for local decision makers to improve prevention strategies, including the introduction of HPV vaccine in Cuba.

Antimicrobial Susceptibility Patterns of Recent Cuban Mycoplasma genitalium Isolates Determined by a Modified Cell-Culture-Based Method.

Isolation of Mycoplasma genitalium from clinical specimens remains difficult and few strains are available for antimicrobial susceptibility testing. We describe the antimicrobial susceptibility of M. genitalium strains grown in Vero cell culture with first- and second- line antibiotics, using a modified cell-culture-based method. Macrolide- and -fluoroquinolone resistance determinants were detected by sequencing of the 23S and parC genes, respectively. Seven strains were examined, including three new, genetically distinct M. genitalium strains isolated from endocervical and urethral swab specimens from Cuban patients together with four reference strains isolated from specimens collected from men in Denmark, Sweden and Australia. Azithromycin was the most active drug against two of the Cuban M. genitalium strains with MICs values of 0.008 mg/liter, however, one strain was macrolide resistant with an MIC of >8 mg/liter, and the A2059G resistant genotype. Ciprofloxacin was the least active antimicrobial drug and moxifloxacin was the most active fluoroquinolone against the new clinical strains, although an MIC of 1 mg/l was found for two strains. However, no relevant parC mutations were detected. MICs for tetracyclines were 0.5-4 mg/liter. Although the number of Cuban strains was low, the results suggest that a single-dose azithromycin treatment could be ineffective, and that a second-line treatment with moxifloxacin, should become an option in Cuba. To our knowledge, this is the first report of isolation and antibiotic susceptibility testing of M. genitalium strains from the Latin-American region, and the first detection of macrolide resistance in such strains.

Molecular epidemiology and KSHV K1 subtypes in a Cuban AIDS-Kaposi's sarcoma population.

Kaposi's sarcoma-associated herpesvirus (KSHV) is detected consistently in Kaposi's sarcoma (KS). Because of its dramatic sequence variation, the K1 gene has been used to classify KSHV. We found a diverse array of KSHV subtypes A1, A2, A3, A5, B1, B2, and C3 in 23 Cuban KS samples containing several novel sporadic insertions/deletions in subtypes A and C. The molecular epidemiology of the KSHV subtypes seems to reflect the unique mixed ethnic background of the Cuban population.

Molecular and histological tools to diagnose an imported case of American cutaneous leishmaniasis in Cuba.

BACKGROUND: Leishmaniasis represents a polymorphous group of diseases caused by around 20 different species of Leishmania parasite. Increases in the number of cases of leishmaniasis reported as a consequence of the growth in travel and migration are of concern to epidemiologists and are diagnostically challenging in non-endemic areas. METHODS: Molecular and histological analyses of a paraffin-embedded skin biopsy were used in parallel to detect Leishmania parasites in a Cuban woman with suspicious lesions arriving in Cuba from Venezuela. Primers based on the 18S fragment of ribosomal ribonucleic acid (rRNA) and heat shock protein 70 genes (hsp70) were used for molecular detection. RESULTS: Histological studies detected the presence of the parasite. A small fragment of Leishmania DNA was amplified by polymerase chain reaction (PCR) targeting the 18S fragment using, for the first time, nucleic acid obtained from paraffin-embedded tissue as a template. Amplification of a larger fragment from the hsp70 gene did not occur. CONCLUSIONS: The detection of Leishmania DNA from paraffin-embedded tissue by means of 18S-targeted PCR is a feasible approach to diagnosis. In combination with classical methods such as histology, the molecular detection of the parasite was demonstrated to be useful in confirming Leishmania infection in a traveler.

[Detection of Leishmania spp. based on the gene encoding HSP20]. / Detección de Leishmania spp. en base al gen que codifica la proteína HSP20.

OBJECTIVES: Explore a new target for molecular diagnosis of Leishmania. MATERIALS AND METHODS: We evaluated the utility of the gene that encodes the heat shock protein 20-kDa (Hsp20) for detecting Leishmania by polymerase chain reaction (PCR). PCR was normalized and analytical parameters were determined, as well as the validity and diagnostic accuracy, and concordance with the PCR - 18S. PCR-Hsp20 with DNA was obtained from a group of clinical samples from different sources. RESULTS: The analytical parameters were adequate. The sensitivity obtained was 86% and the specificity was 100%. The concordance with the reference method was good (κ = 0.731), which supports its potential use for diagnosis. The possibility of subsequent identification of the species by sequencing the amplified product gives an additional advantage. CONCLUSIONS: The usefulness of this gene as a new target for the detection of Leishmania was demonstrated. Because of its potential, it is recommended to improve the sensitivity of the method and to evaluate it in different endemic regions.

Kaposi's Sarcoma and Human Herpesvirus 8 in Cuba: evidence of subtype B expansion.

OBJECTIVE: To evaluate the temporal distribution (1991-2009) and associated variation of KSHV subtypes in Cuba. METHOD: Phylogenetic characterization based on the KSHV K1 gene was performed using 90 KSHV positive samples. RESULTS: Molecular characterization confirmed the prevalence of a wide range of KSHV subtypes (A: n=48 [A5=12]; C: n=15; B: n=22; and E: n=5). In the current study, we observed a significant increase in HHV-8 subtype B after 2004 (p=0.0063). This Subtype B in Cuba was associated with: heterosexual behaviour (OR: 3.63, CI: 1,2-10,98; p=0.03), with the antecedent of acquiring HIV/KSHV in Africa (p=0.0003), with nodular stage of KS lesions (OR 4.2, CI: 1.1 to 15.7; p=0.04). CONCLUSION: Our study is the first to report KSHV Subtype B expansion in Cuba, that might be reflective of a change in human behavioural pattern.

Simultaneous quantification of human herpesvirus 8 DNA by real time PCR in different tissues of HIV infected cuban patients with Kaposi's sarcoma.

In Cuba, previous reports have shown an increase of epidemic KS, reaching a total of 120 cases by the end of 2007, despite the use of HAART. To evaluate and compare the role of human herpes virus 8 (HHV-8) viral loads in different compartments of AIDS-related Kaposi's sarcoma (AIDS-KS) patients real-time polymerase chain reaction (RT-PCR) was used to determine the genome copy number of HHV-8 in plasma, saliva, tissue and peripheral blood mononuclear cells (PBMC) of 49 AIDS-KS patients. Overall, 98% of AIDS-KS patients harbored detectable HHV-8. HHV-8 could be detected in 91.6% of KS tissue lesions showing the highest viral load (median log = 3.14 copies/100 ng DNA) followed by saliva and PBMC which were positive in 78%, and 69.2%; respectively. In contrast, HHV-8 was detected in only 37% of plasma samples, which also showed lower viral loads. Men who had sex with men (MSM) were more likely to have three-times higher HHV-8 genome copies in KS lesions when compared with tissues from heterosexuals individuals (OR 3; 95% CI 1.1 to 12.5). These results emphasize the systemic nature of HHV-8-infection and demonstrate the possible role of saliva in HHV-8 transmission among MSM.

Detección de Leishmania spp. en base al gen que codifica la proteína HSP20 / Detection of Leishmania spp. based on the gene encoding HSP20

Objetivos. Explorar una nueva diana para el diagnóstico molecular de Leishmania. Materiales y métodos. Se evaluó la utilidad del gen que codifica la proteína de choque térmico de 20kDa (hsp20) para la detección de Leishmania por medio de la reacción en cadena de la polimerasa (PCR).Se normalizó la PCR y se determinaron los parámetros analíticos, así como la validez y seguridad diagnóstica y la concordancia con la PCR-18S. Se evaluó la PCR-hsp20 con ADN obtenido de un grupo de muestras clínicas de distinta procedencia. Resultados. Los parámetros analíticos resultaron adecuados. La sensibilidad obtenida fue de 86% y la especificidad del 100%, la concordancia con el método de referencia resultó buena (ƙ=0,731), lo que apoya su posible uso para el diagnóstico. La posibilidad de identificación posterior de la especie mediante secuenciación del producto amplificado le confiere una ventaja adicional. Conclusiones. Se demuestra la utilidad de este gen como una nueva diana para la detección del género Leishmania. Debido a su potencial, se recomienda mejorar la sensibilidad del procedimiento y realizar su evaluación en diversas regiones endémicas.

Objectives. Explore a new target for molecular diagnosis of Leishmania. Materials and methods. We evaluated the utility of the gene that encodes the heat shock protein 20-kDa (Hsp20) for detecting Leishmania by polymerase chain reaction (PCR). PCR was normalized and analytical parameters were determined, as well as the validity and diagnostic accuracy, and concordance with the PCR - 18S. PCR-Hsp20 with DNA was obtained from a group of clinical samples from different sources. Results. The analytical parameters were adequate. The sensitivity obtained was 86% and the specificity was 100%. The concordance with the reference method was good (Ƙ = 0.731), which supports its potential use for diagnosis. The possibility of subsequent identification of the species by sequencing the amplified product gives an additional advantage. Conclusions. The usefulness of this gene as a new target for the detection of Leishmania was demonstrated. Because of its potential, it is recommended to improve the sensitivity of the method and to evaluate it in different endemic regions.

Molecular epidemiology of Kaposi's sarcoma herpesvirus (KSHV) in Cuban and German patients with Kaposi's sarcoma (KS) and asymptomatic sexual contacts.

Kaposi's sarcoma (KS) shows a distinct geographical and ethnic distribution. The variable K1 gene serves to differentiate the KSHV subtypes A-E, M, N, and Q. Phylogenetic characterization of 19 classical and epidemic German KS specimens revealed the Eurasian KSHV subtypes C (n = 13, including 6 classical KS) and A (n = 6), while 27 Cuban specimens showed a variety of different subtypes (A: n = 16, 4 being A5; C: n = 8; B: n = 2; and the new subtype E: n = 1). Three pairs of isolates from KS patients and peripheral blood mononuclear cells (PBMC) of their sexual partners without KS were studied for the first time and found identical, strongly arguing for sexual transmission of KSHV in this unique cohort. The unique ethnic background of the Cuban population may explain the variety of different KSHV strains.

[Serological diagnosis of syphilis in Cuban HIV/AIDS patients]. / Diagnóstico serológico de sífilis en pacientes Cubanos con VIH/SIDA.

The non-treponemic fast detection test of plasmatic reagines (RPR) and the treponemic hemagglutination test of Treponema pallidum were used in the detection of infection due to T. pallidum in 60 males presenting HIV/AIDS infection with clinicoepidemiological diagnosis of syphilis. It was confirmed that 30% presented recently acquired symptomatic or latent syphilis and that 10% had markers of past treated infection or of late acquired latent syphilis, whereas in the other 60% no serological reactivity was detected. A seroprevalence study of reaginic antibodies was conducted by RPR in 59 HIV/AIDS females, using 67 women negative to this virus as controls. All of them had no symptoms compatible with the syphilitic infection. It was concluded that 20.3% and 11.9%, respectively, presented reactivity, which established a probable diagnosis of syphilis or a seroresistance to a previouos syphilis. These results showed a close association between syphilis and HIV/AIDS and that both diseases may coexist in a same patient.