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.

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.

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.

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.

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.

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.

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.

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.

Diagnóstico serológico de sífilis en pacientes cubanos con VIH/SIDA / Serological diagnosis of syphilis in HIV/AIDS Cuban

Se utilizó la prueba no treponémica de detección rápida de reaginas plasmáticas y la prueba treponémica de hemaglutinación de Treponema pallidum, en la detección de la infección por T. pallidum en 60 hombres que presentaban la infección VIH/SIDA con diagnóstico clínico-epidemiológico de sífilis. Se confirmó que 30 por ciento presentaba sífilis reciente adquirida sintomática o latente y que 10 por ciento poseía marcadores de infección pasada tratada o de sífilis tardía adquirida latente, mientras que en 60 por ciento restante no se detectó reactividad serológica. Se realizó, además, un estudio de seroprevalencia de anticuerpos reagínicos por detección rápida de reaginas plasmáticas en 59 mujeres con VIH/SIDA, utilizando como control 67 mujeres negativas a este virus, todas sin síntomas compatibles con la infección sifilítica. Se obtuvo que 20,3 y 11,9 por ciento, respectivamente, mostraban reactividad, lo que estableció un diagnóstico probable de sífilis o una serorresitencia a una sífilis anterior. Estos resultados muestran una fuerte asociación entre sífilis y VIH/SIDA y que ambas enfermedades pueden coexistir en un mismo paciente