Virginia Capó

Human papillomavirus infection of the esophagus. A clinicopathologic study with demonstration of papillomavirus antigen by the immunoperoxidase technique

Papillomaviruses are known to be oncogenic in animals. In humans they are associated with benign squamous tumors (verruca, condylomata acuminata, and papillomas) in a variety of body sites. Human papillomavirus (HPV) infection of the esophagus, however, has not previously been documented. Recent reports of condylomatous changes in esophageal epithelium adjacent to esophageal carcinoma and the sporadic descriptions of esophageal papillomas in the literature for many years, lend credence to the assumption that HPV may affect the squamous mucous membrane of the esophagus. In the current study 75 cases, including 2 papillomas and 73 focal epithelial hyperplasia of the esophagus, were examined for histologic evidence of HPV infection as characterized by the presence of koilocytosis, giant and multinucleated cells, dyskeratosis, hyperkeratosis, acanthosis, papillomatosis, and anisonucleosis. Thirteen of the cases—the 2 papillomas and 11 of the focal epithelial hyperplasias—contained distinctive histologic evidence of HPV infection. The presence of HPV antigens was demonstrated by immunoperoxidase (IMPO) in the 4 of the 13 cases (31%). In the remaining cases the IMPO was equivocal in two and negative in seven.

Trichinella spiralis: vascular endothelial growth factor is up-regulated within the nurse cell during the early phase of its formation.

The L1 larval stage of Trichinella spiralis induces modification in a portion of striated skeletal muscle cell resulting in the formation of the nurse cell. This specialized host cell is completely encased in a capsule composed mainly of collagen type IV and type VI, which, in turn, is surrounded by a unique rete of vessels whose formation begins on around day 12 after intracellular infection. We investigated the possibility that vascular endothelial growth factor (VEGF) may be up-regulated during nurse cell formation by employing immunohistochemistry and in situ hybridization on synchronously infected mouse muscle tissue. Both VEGF mRNA and VEGF peptide were detected in the developing nurse cell cytoplasm from day 7 up to 16 mo after infection. In addition, VEGF was also detected in cells in the area immediately surrounding the nurse cell on days 15 and 17. On the basis of these results, we propose that hypoxia is induced by T. spiralis within the developing nurse cell some time prior to the up-regulation of VEGF, perhaps as early as day 7. We further propose, on the basis of the continued presence of VEGF in nurse cell cytoplasm, that a constant state of hypoxia cell is maintained.

Molecular diagnosis of Toxoplasma gondii infection in cerebrospinal fluid from AIDS patients

BackgroundToxoplasmic encephalitis (TE) is one of the most common opportunistic infections in immunocompromised patients. In Cuba, despite the highly active antiretroviral therapy, TE is still the most important cause of cerebral mass lesions in patients infected with the human immunodeficiency virus (HIV). The detection of Toxoplasma gondii by PCR may facilitate the diagnosis and follow-up of TE in acquired immunodeficiency syndrome (AIDS) patients by direct identification of parasite DNA in clinical samples. The aim of the present study was to evaluate a rapid PCR method using the B1 gene to detect T. gondii in cerebrospinal fluid (CSF) samples from patients with suspected TE.MethodsCSF samples from AIDS and HIV-negative patients were analyzed. Patients were divided into two groups according to the Centre for Disease Control and Prevention (CDC) criteria for AIDS-related TE: AIDS patients with suspected neurotoxoplasmosis and AIDS and HIV-negative patients with other confirmed neurological diseases but no suspicions of TE. Predictive values, diagnostic accuracy, sensitivity and specificity of the PCR B1 method were calculated.ResultsThe results obtained from 190 patients showed that this assay has a good sensitivity and specificity (83.3% and 95.7%, respectively) for the diagnosis of TE in AIDS patients.ConclusionPCR using the B1 gene and B22/B23 set of primers is a single, rapid and reliable method that may be valuable for discrimination between toxoplasmosis and other central nervous system (CNS) diseases.

Detection of Toxoplasma gondii in cerebrospinal fluid from AIDS patients by nested PCR and rapid identification of type I allele at B1 gene by RFLP analysis

Highly active antiretroviral therapy (HAART) has decreased the incidence of opportunistic infections in the central nervous system (CNS) in AIDS patients. However, toxoplasmic encephalitis (TE) still represents the most common cerebral mass lesion in patients infected with human immunodeficiency virus (HIV). The aim of this study was to evaluate nested PCR-B1 using cerebrospinal fluid (CSF) to detect Toxoplasma gondii DNA for the diagnosis of TE. A total of 114 samples were evaluated, and 33/44 samples from patients with TE were positive by PCR (sensitivity 75%), demonstrating the diagnostic usefulness of PCR technique. PCR-B1 products were analyzed by restriction fragment length polymorphism (RFLP) in 30 samples. Only type I allele at B1 was identified in these samples according banding patterns. This is the first report of evaluation of S1-AS1/S2-AS2 set of primers in more than 100 clinical samples as well as the first genotyping study of T. gondii in Cuba.

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 Kaposis 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.

Conventional polymerase chain reaction for the diagnosis of neurotoxoplasmosis: comparison of three sets of primers for the B1 gene using CSF samples

Polymerase chain reaction (PCR) has made a significant improvement in the diagnosis of toxoplasmic encephalitis (TE). Nevertheless, a wide variety of targets and primers has been used in different assays, and few comparative studies had been carried out. The aim of the present study was to compare the efficiency of 3 conventional PCR methods by using 3 sets of primers targeting the repetitive B1 gene in the diagnosis of TE. Diagnostic sensitivity and specificity of PCR and nested-PCR protocols were assessed for 207 (nested-PCR/T1-T4), 200 (nested-PCR/S1-AS1), and 206 (PCR/B22-B23) cerebrospinal fluid (CSF) samples, including AIDS and HIV-negative patients. The diagnostic sensitivity of PCR and nested-PCR assays was 50.85%, 68.97%, and 72.41% for T1-T4, S1-AS1, and B22-B23, respectively. The diagnostic specificity was high for all the assays showing values between 95% and 97%. In general, the best results were obtained for the B22-B23 set of primers, suggesting their usefulness compared with 2 nested-PCR protocols and showing that this simple and rapid strategy may be the preferred one for the diagnosis of TE in AIDS patients.

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.

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

BackgroundLeishmaniasis 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.MethodsClinical 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.ResultsFrom 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.ConclusionsThe 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.

Multiplex PCR to Detect T. gondii Infection based on B1 Gene and 529 bp Repetitive Element

Polymerase Chain Reaction (PCR) has made a significant improvement in the diagnosis of toxoplasmosis. Nevertheless, a wide variety of targets and primers has been evaluated in different independent assays, and only few comparative studies have been performed. Even when the B1 gene and the 529 repetitive element have been the most studied markers, there no concluding remarks respect the best. Objetive: The aim of the present study was to design a multiplex PCR assay to simultaneously detect these two markers in a single multiplex PCR to diagnose T. gondii infection. Methods: Specific PCR primers targeting the B1 gene and 529 repetitive element were evaluated. After a careful optimization of multiplex PCR process, analytical sensitivity and specificity were evaluated. The usefulness of multiplex assay was evaluated using DNA from 8 T. gondii reference strains, corresponding to the three principal genotypes, and 70 clinical samples (blood) from AIDS patients with and without Toxoplasmic encephalitis (TE). Results: Our preliminar results show that multiplex PCR assay is able to amplify both targets in using DNA from one parasite in the mix PCR reaction. Unspecific reactions for other microorganisms were not observed. The diagnostic sensitivity of multiplex assay in blood, according to the Centers for Disease Control and Prevention (CDC) criteria, was 86.6% while the diagnostic specificity was 100%. Only one sample was positive to one marker (B1 gene) in the multiplex reaction. Conclusion: This multiplex PCR method is the first multiplex PCR evaluating the detection of T. gondii DNA in TE cases and it proved to be rapid, enough sensitive, highly specific and inexpensive respect to perform independent assays for each marker, real time PCR or nested PCR assays.