Species typing of Cuban Trichomonas vaginalis virus by RT-PCR, and association of TVV-2 with high parasite adhesion levels and high pathogenicity in patients.

The viral infection of the parasite with T. vaginalis virus (TVV) may have important implications for trichomonal virulence. In this study we identified the TVV species isolated from Cuban T. vaginalis, using specie specific Reverse Transcriptase-PCR. Of the 37 clinical isolates studied, 21 were infected with TVV, 6 contained TVV-1, 12, TVV- 2 and 3 were co-infected with TVV-1 and -2. The strains infected with TVV showing highest adhesion level in comparison to not infected strains, with high statistical significance. The strains infected only with TVV-2 showing highest adhesion level in comparison to strains infected with TVV-1, with high statistical significance. The parasites classified as mild symptomatic are infected only with TVV-1, however the severe only with TVV-2. According to our results, it seems that only two TVV species are infecting the Cuban isolates. Further studies using higher number of strains should be conducted in order to corroborate these results.

Mycoplasma hominis in Cuban Trichomonas vaginalis isolates: association with parasite genetic polymorphism.

Trichomonas vaginalis can be naturally infected with intracellular Mycoplasma hominis. This bacterial infection may have implications for trichomonal virulence and disease pathogenesis. The objective of the study was to report the presence of M. hominis in Cuban T. vaginalis isolates and to describe the association between the phenotype M. hominis infected with RAPD genetic polymorphism of T. vaginalis. The Random Amplified Polymorphic DNA (RAPD) technique was used to determine genetic differences among 40 isolates of T. vaginalis using a panel of 30 random primers and these genetic data were correlated with the infection of isolates with M. hominis. The trees drawn based on RAPD data showed no relations with metronidazole susceptibility and significantly association with the presence of M. hominis (P=0.043), which demonstrates the existence of concordance between the genetic relatedness and the presence of M. hominis in T. vaginalis isolates. This result could point to a predisposition of T. vaginalis for the bacterial enters and/or survival.

Double-stranded RNA viral infection of Trichomonas vaginalis and correlation with genetic polymorphism of isolates.

Trichomonas vaginalis can be infected with double-stranded RNA (dsRNA) viruses known as T. vaginalis virus (TVV). This viral infection may have important implications for trichomonal virulence and disease pathogenesis. The objective of this study was to determine the possible correlation between the T. vaginalis genetic polymorphism and the isolate infection with TVV. The Random Amplified Polymorphic DNA (RAPD) technique was used to determine genetic differences among 37 isolates of T. vaginalis using a panel of 30 random primers and these genetic data were correlated with the infection of isolates with TVV. The trees drawn based on RAPD data showed significantly association with the presence of TVV (P = 0.028) demonstrating the existence of concordance between the genetic relatedness and the presence of TVV in T. vaginalis isolates. This result could point to a predisposition of T. vaginalis for the viral enters and/or survival.

Genetic Diversity of Trichomonas Vaginalis Clinical Isolates According to Restriction Fragment Length Polymorphism Analysis of the 60-kDa Proteinase Gene.

INTRODUCCTION: Trichomonas vaginalis is a highly prevalent parasitic that causes the sexually transmitted disease trichomoniasis with some serious health complications. More understanding about genetic features of the parasite can be helpful in the study of the pathogenesis, epidemiology of the infection and drug susceptibility. For this end, we conducted analysis of a fragment (23 kDa) of the p60 of T. vaginalis gene. MATERIAL AND METHODS: The restriction fragment length polymorphism (RFLP) methods was used. RESULT AND DISCUSSION: RFLP analysis showed the difference between T. vaginalis isolates from symptomatic and asymptomatic patients, suggesting a relation between the genetic identity of the isolates and their clinical manifestations.

Antimalarial activity from three ascidians: an exploration of different marine invertebrate phyla.

Recent research suggests that marine organisms may produce compounds with activity against malaria parasites. Of a total of 27 aqueous extracts from different marine species, collected on the northwest Cuban coast, 20 were considered as showing no significant activity against Plasmodium falciparum F32, with minimum inhibitory concentrations (MIC) >500 microg/ml, while seven extracts (MIC < or =500 microg/ml) were selected for further investigation by determining their selectivity indices and in vivo antimalarial activity. Three species of tunicates were chosen, as more than 50% reduction of P. berghei parasitaemia was produced after administration of 250 or 500 mg/kg of their crude extracts into infected mice. The aqueous extracts of Microcosmus goanus, Ascidia sydneiensis and Phallusia nigra were partitioned between water and n-butanol; the organic phases inhibited P. falciparum growth by 50% at concentrations of 17.5 microg/ml, 20.9 microg/ml and 29.4 microg/ml respectively. In general, these results are similar to those of most ethnobotanical surveys. Further chemical studies are being undertaken in order to isolate new antimalarial compounds from these Caribbean tunicates.

Lack of knowledge regarding the microscopic diagnosis of malaria by technicians of the laboratory network in Luanda, Angola.

INTRODUCTION: Malaria is still one of the most important public health problems worldwide. The diagnosis of this disease is still mainly based on thick blood films.  OBJECTIVE: To evaluate the knowledge about malaria diagnosis of the technicians of the public health network in Luanda, Angola, by means of a survey.  MATERIALS AND METHODS: This survey was carried out in three phases. In the first one, open interviews were done to technicians related with the different procedures for malaria diagnosis. In the second one, a preliminary questionnaire was prepared and evaluated. In the third phase, a definitive questionnaire was applied to 120 technicians from Luanda between April and July, 2013. The proportions of correct and incorrect answers were compared for every question of the survey.  RESULTS: Significantly higher proportions of incorrect answers (p<0.05) were found in the questions related to clinical manifestations, 68/52 (p<0.05), species of Plasmodium according to geographical areas, 76/44 (p<0.05), the type of granulations according to species, 96/24 (p<0.01), the class of microscope magnifying glasses used to observe the thick smear, 105/15 (p<0.01), the thick smear report, 76/44 (p<0.01), the time and preparation of different stain solutions, 81/39 (p<0.01), and the number of parasites counted per 200 leukocytes, 96/24 (p<0.01).  CONCLUSIONS: Various failures for the microscopic diagnosis of malaria were observed amongst the evaluated technicians. These results will be useful as a baseline study before applying an educational intervention aimed to improve the quality of malaria diagnosis in Luanda's laboratory network.

Double-stranded RNA viral infection in Cuban Trichomonas vaginalis isolates.

Trichomonas vaginalis can be infected with double-stranded RNA (dsRNA) viruses designated T. vaginalis virus (TVV), which may have important implications for trichomonal virulence and disease pathogenesis. We tested for TVV in 40 fresh T. vaginalis isolates from Cuban patients by total extraction of nucleic acids (DNA and RNA). TVV was detected in 22 (55%) of the 40 T. vaginalis isolates. This gives an estimate of the infection rate of Cuban T. vaginalis isolates by the dsRNA virus. Future research should focus on the association between trichomonosis symptoms and the presence of TVV.

Characterization of Specific RAPD Markers of Virulence in Trichomonas vaginalis Isolates.

BACKGROUND: As for human trichomoniasis the host-parasite relationship is very complex, and the broad ranges of clinical symptoms are unlikely be attributable to a single pathogenic mechanism. Specific Random Amplified Polymorphic DNA (RAPD) markers of 490 bp, 720 bp and 460 bp using the primers Tv-5, OPA-6 and OPA-11, respectively, were reported. This was the first description of possible genetic virulence markers of the infection by T. vaginalis. The aim of this study was to characterize the specific RAPD markers in order to elucidate their importance on virulence of this illness. METHODS: The selected specific RAPD fragments were cloned and sequenced. The obtained sequences were compared by the BLAST algorithm. RESULTS: The nucleotide sequence of the Tv-5490 RAPD marker exhibited significant similarity to T. vaginalis hypothetical G3 leucine rich repeat (LRR) family protein (e-value: 6e-14) and Giardia lamblia leucine rich repeat protein 1 virus receptor protein (e-value: 6e-14 and 2e-12) ; however, the OPA-6720 and OPA-11460 showed no significant similarity with any coding published sequence. All the evaluated strains showed the presence of the LRR gene. CONCLUSION: These results demonstrate a possible role of this gene in the virulence of T. vaginalis and in the parasite infection with Trichomonas virus as a possible virus receptor. Further analysis of this gene and encoded protein will allow determining the role that they play in the isolates virus susceptible or resistant phenotypes.

Genetic variability between Trichomonas vaginalis isolates and correlation with clinical presentation.

The random amplified polymorphic DNA (RAPD) technique was used to determine genetic differences among isolates of Trichomonas vaginalis and these genetic data were correlated with patient records. A panel of 10 random primers was used to determine the type and extent of intraspecific polymorphism in 40 isolates of T. vaginalis. All primers detected DNA polymorphism among isolates. Numerical analysis of 124 RAPD amplified bands generated by these 10 primers were carried out with the unweighted pair group methods analysis (UPGMA) using Jaccard's Similarity Coefficient and data were used to construct a dendrogram. Four main groups can be distinguished by RAPD data, these groups coincide with four different patient categories (asymptomatic and symptomatic: light, moderate, and severe infection). These patients did not have any concomitant vaginal infection. Each of the four groups can be characterized by specific genetic markers, but a specific 490bp marker was found to be specific for all symptomatic isolates, not the asymptomatic isolates. This is the first description of a possible virulence marker for T. vaginalis. Further studies will be necessary to ascertain the importance and function of this genetic marker in clinical infection.

Optimization of random amplified polymorphic DNA techniques for its use in genetic studies of Trichomonas vaginalis isolates.

The random amplified polymorphic DNA (RAPD) technique is a simple method to detect DNA polymorphism. Several factors can affect the amplification profiles causing the presence of false bands and assay non-reproducibility. In this study, we analyzed the effect of changing concentrations of the primer, template DNA and Taq DNA polymerase with the goal of determining their optimum concentration for the standardization of the RAPD technique for genetic studies of Trichomonas vaginalis, a parasite that is of major epidemiological relevance in Cuba.

Setting a colorimetric assay with MTT for assessment of trichomonicidal activity.

Trichomonas vaginalis infection is associated with important problems of public health, including the spreading of other sexual transmitted infections. The existence of clinical resistant isolates metronidazole and tinidazole, the drugs approved for the treatment of trichomoniasis, points to the necessity of continue searching for trichomonicidal substances. Here we optimize a colorimetric assay with MTT to assess trichomonas viability. The absence of ascorbic acid and cysteine in the culture medium was indispensable to perform the assay, as these compounds spontaneously reduce the MTT. Linearity of absorbance was verified versus trichomonas counts. Medium inhibitory concentration of metronidazole was determined using the sigmoidal Emax model, by comparing the absorbance of test cultures with controls. The obtained value was in the range of published data. The test would be used for the evaluation of trichomonicidal activity of chemical compounds and natural products.

Genetic characterization of three Cuban Trichomonas vaginalis virus. Phylogeny of Totiviridae family.

Trichomonas vaginalis can be infected with double stranded RNA (dsRNA) viruses known as T. vaginalis virus (TVV). This viral infection may have important implications for trichomonal virulence and disease pathogenesis. In this study we identified and genetic characterized three strains of TVVs isolated from T. vaginalis in Cuba. The three new predicted sequences of capsid protein and RNA-dependent RNA polymerase amounted to the previously determined 20 TVV sequences and other 21 viruses of Totiviridae family were used for a phylogenetic analysis. Four distinct monophyletic clades are shown in a phylogenetic tree. One corresponds with TVVs, other with Victorivirus, Leishmaniavirus and Eimeria brunetti virus and, other with viruses of the genus Totivirus and the last with Giardiavirus. The E. brunetti virus is identified in the phylogenetic tree as independent taxon between Leishmaniavirus and Victorivirus isolates, most closely related to Victorivirus. TVV constitute a monophyletic cluster distinguishable from all other viruses in Totiviridae family. This result suggested that TVV may be grouped in a separated genus and not inside of Giardiavirus. TVVs appear to be more closely related to protozoan viruses in the genus Leishmaniavirus and to fungal viruses in the genus Victorivirus than to other protozoan and fungal viruses in Giardiavirus and Totivirus. Among TVVs, four main groups can be recognized within Trichomonasvirus cluster, which correspond with the previous species classification proposed. Further studies, with more TVV strains, especially TVV3 and 4 strains, are needed in order to determine the phylogenetic relationship among Trichomonasvirus genus and specifically if TVV2 and 3 each also constitute a well-delimited group.

Toxocariasis in Cuba: a literature review.

Human toxocariasis (HT) is a zoonotic disease caused by infection with the larval stage of Toxocara canis, the intestinal roundworm of dogs. Infection can be associated with a wide clinical spectrum varying from asymptomatic to severe organ injury. While the incidence of symptomatic human toxocariasis appears to be low, infection of the human population is widespread. In Cuba, a clear overview on the status of the disease is lacking. Here, we review the available information on toxocariasis in Cuba as a first step to estimate the importance of the disease in the country. Findings are discussed and put in a broader perspective. Data gaps are identified and suggestions on how to address these are presented. The available country data suggest that Toxocara infection of the definitive dog host and environmental contamination with Toxocara spp. eggs is substantial, but information on HT is less conclusive. The availability of adequate diagnostic tools in the country should be guaranteed. Dedicated studies are needed for a reliable assessment of the impact of toxocariasis in Cuba and the design of prevention or control strategies.

La insuficiencia de conocimientos para el diagnóstico microscópico de la malaria en técnicos de la red de laboratorios de Luanda, Angola / Lack of knowledge regarding the microscopic diagnosis of malaria by technicians of the laboratory network in Luanda, Angola

Introducción. La malaria, o paludismo, continúa siendo uno de los más importantes problemas de salud pública a nivel mundial. La gota gruesa sigue siendo la técnica de referencia para su diagnóstico. Objetivo. Evaluar los conocimientos para el diagnóstico de la malaria de técnicos del sector de la salud en Luanda, Angola, mediante una encuesta. Materiales y métodos. La preparación de la encuesta transcurrió en tres etapas: primero, se hicieron entrevistas abiertas a los técnicos en torno a los diferentes procedimientos para el diagnóstico microscópico de la malaria; en una segunda etapa, se preparó el cuestionario preliminar, y en la tercera, se elaboró el cuestionario definitivo. La encuesta se hizo en un grupo de 120 técnicos de la red nacional de salud entre los meses de abril y julio de 2013. Se comparó la proporción de respuestas correctas e incorrectas considerando como significativo un valor de p<0,05. Resultados. Las respuestas incorrectas predominaron sobre las correctas, de forma estadísticamente significativa (p<0,05), en las preguntas sobre: las manifestaciones clínicas (68 Vs . 52); las especies de Plasmodium según zonas geográficas (76 Vs . 44); el tipo de granulaciones según la especie parasitaria (96 Vs . 24); el tipo de lente para visualizar la gota gruesa (105 Vs . 15); la forma de hacer un informe del resultado de la microscopía (76 Vs . 44); el tiempo y la concentración de las diferentes soluciones que se utilizan (81 Vs . 39), y el número de parásitos por 200 leucocitos (96 Vs . 24). Conclusiones. Se demostró la insuficiencia de conocimientos para el diagnóstico microscópico de la malaria. Estos resultados servirán de base para el desarrollo de las herramientas de una intervención educativa orientada a mejorar la calidad del diagnóstico de la malaria en la red de laboratorios de Luanda.

Introduction: Malaria is still one of the most important public health problems worldwide. The diagnosis of this disease is still mainly based on thick blood films. Objective: To evaluate the knowledge about malaria diagnosis of the technicians of the public health network in Luanda, Angola, by means of a survey. Methods: This survey was carried out in three phases. In the first one, open interviews were done to technicians related with the different procedures for malaria diagnosis. In the second one, a preliminary questionnaire was prepared and evaluated. In the third phase, a definitive questionnaire was applied to 120 technicians from Luanda between April and July, 2013. The proportions of correct and incorrect answers were compared for every question of the survey. Results: Significantly higher proportions of incorrect answers (p<0.05) were found in the questions related to clinical manifestations, 68/52 (p<0.05), species of Plasmodium according to geographical areas, 76/44 (p<0.05), the type of granulations according to species, 96/24 (p<0.01), the class of microscope magnifying glasses used to observe the thick smear, 105/15 (p<0.01), the thick smear report, 76/44 (p<0.01), the time and preparation of different stain solutions, 81/39 (p<0.01), and the number of parasites counted per 200 leukocytes, 96/24 (p<0.01). Conclusions: Various failures for the microscopic diagnosis of malaria were observed amongst the evaluated technicians. These results will be useful as a baseline study before applying an educational intervention aimed to improve the quality of malaria diagnosis in Luanda´s laboratory network.

Antimalarial activity of new acridinone derivatives.

Malaria is one of the major threats concerning world public health. Resistance to the current antimalarial drugs has led to searches for new antimalarial compounds. Acridinone derivatives have recently demonstrated to be active against malaria parasite. We focused our attention on synthesized new acridinone derivatives, some of them resulting with high antiviral and trypanocidal activity. In this study new derivatives of 10-alyl-, 10-(3-methyl-2-butenyl)- and 10-(1,2-propadienyl)-9(10H)-acridinone were evaluated for their antimalarial activity against Plasmodium falciparum. To assess the selectivity, cytotoxicity was assessed in parallel against human MRC-5 cells. Inhibition of ß-hematin formation was determined using a spectrophotometric assay. Mitochondrial bc(1) complexes were isolated from yeast and bovine heart cells to test acridinone inhibitory activity. This study resulted in the identification of three compounds with submicromolar efficacy against P. falciparum and without cytotoxic effects on human cellular line. One compound, IIa (1-fluoro-10-(3-methyl-2-butenyl)-9(10H)-acridinone), can be classified as hit for antimalarial drug development exhibiting IC(50) less than 0.2 µg/mL with SI greater than 100. In molecular tests, no relevant inhibitory activity was obtained for our compounds. The mechanism of acridinones antimalarial action remains unclear.

Fascioliasis: can Cuba conquer this emerging parasitosis?

Fascioliasis, an emerging parasitic infection, impacts significantly on both veterinary and human health worldwide. Endemic foci are not limited only to areas of extensive livestock farming, but owing to the parasite's abilities to colonise new intermediate hosts and adapt to new environments, also occur in other places, including Cuba. In Cuba, despite a high prevalence of fascioliasis in livestock, and the widespread occurrence of two potential intermediate hosts, human infection has decreased steadily over the past 10 years. In other parts of the world, human fascioliasis is apparently becoming more frequent. Problems in counteracting the spread of fascioliasis, and approaches used in Cuba to limit zoonotic transmission are discussed, with emphasis on diagnostic and treatment problems, malacological initiatives, and the importance of an integrated control programme. Such programmes may be of benefit in other countries where the prevalence of human fascioliasis is increasing, and lessons may perhaps be learned from the Cuban approach.

[Identification of breeding sites of Anopheles sp. during part of the dry season in Jigawa, Nigeria]. / Identificación de los sitios de cría de Anopheles sp. durante parte de la estación seca en el estado de Jigawa, Nigeria.

A study was conducted in the state of Jigawa, Republic of Nigeria, from November to December in the dry season, where malaria is one of the main morbidity and mortality causes particularly in under 5 years-old children and pregnant women. This state had two climate seasons: dry from October to May and rainy from June to September. A total of 112 water bodies were sampled and just 18 in nine local governments were positive to mosquitoes. Breeding sites for Anopheles were rice fields, small holes in land, animal footsteps, small ponds, flooded pasture fields and water treatment dam, among others, to amount to 10 sites. Contrary to what has always been reported about the presence of Anopheles in clean waters, they were also breeding in highly polluted waters containing human faeces and garbage and located in open sewers.

Segunda encuesta nacional de infecciones parasitarias intestinales en Cuba, 2009 / Second national survey of intestinal parasitic infections in Cuba, 2009

Introducción: en Cuba las parasitosis intestinales continúan siendo endémicas, con una mayor frecuencia en zonas rurales y montañosas. Después de haber transcurrido casi 25 años desde la última encuesta nacional, se hizo necesario hacer una nueva encuesta para conocer los índices de prevalencia de las parasitosis intestinales y comparar los resultados obtenidos entre ambos estudios. Tal conocimiento resultaría de gran valor para elaborar estrategias de salud y el diseño de programas de control de las infecciones parasitarias intestinales. Objetivo: determinar los índices actuales de prevalencia de las parasitosis intestinales, y comparar los resultados con los de la encuesta anterior luego de haber transcurrido 25 años. Métodos: se realizó un estudio descriptivo de corte trasversal durante los meses de mayo a agosto de 2009 en una muestra de la población cubana. A cada uno se le recogió una muestra de heces que fue analizada por el método de examen directo, la técnica de concentración de Willis y el examen de Kato-Katz; también se le llenó un cuestionario. Resultados: al comparar los resultados de las encuestas nacionales de infecciones parasitarias intestinales realizadas en 1984 y 2009, se encontró que en general disminuyeron las infecciones por parásitos, tanto los helmintos como los protozoos patógenos, aunque los infectados con comensales aumentaron su frecuencia en la de 2009. También se produjo una disminución de las frecuencias de infecciones por todas las especies de geohelmintos, Trichuris trichiura, Ascaris lumbricoides, ancylostomídeos, y Strongyloides stercoralis, así como por los protozoos patógenos Giardia lamblia y Entamoeba histolytica/E. dispar, y los comensales: Endolimax nana y Entamoeba coli en 2009. La única especie de parásito intestinal que aumentó su frecuencia con respecto a la encuesta de 1984 fue Enterobius vermicularis. En ambos estudios la frecuencia de infección con parásitos o comensales fue mayor en el grupo de 5 a 14 años (edad escolar). Conclusiones: al comparar los resultados de las encuestas nacionales de infecciones parasitarias intestinales realizadas en 1984 y 2009, se encontró que en general disminuyeron las frecuencias de infecciones con parásitos intestinales. El hallazgo en ambos estudios de una frecuencia mayor de infección con parásitos o comensales en el grupo de 5 a 14 años (edad escolar), respalda la recomendación de poner énfasis en los programas de control para las parasitosis intestinales en este grupo de edad.

Introduction: the intestinal parasitic infections are still endemic in Cuba, with a higher frequency in rural and mountain regions. Twenty five years after the last national survey, it deemed necessary to carry out a new national survey in order to know the prevalence of intestinal parasitic infections and to compare the obtained results between both studies. That knowledge would be valuable to work out strategies of health and to design a control program for intestinal parasitic infections in Cuba. Objective: to determine the current prevalence of intestinal parasitic infections in Cuba and to compare these results with those obtained from the previous survey after a 25 year-period. Methods: a cross sectional study was conducted from May to August of 2009 in a sample of Cuban population. A stool sample was collected from each individual, which was analyzed by direct examination, Willis' brine flotation method and the Kato-Katz thick smear technique. Additionally, a questionnaire was administered. Results: the comparison between 1984 and 2009 surveys showed a general decrease of frequencies of intestinal parasitic infections caused by both helminths and pathogenic protozoa; however, the percentage of infections with commensal protozoa increased in 2009. In this last survey, there was observed decline of frequencies of infections with soil transmitted species, Trichuris trichiura, Ascaris lumbricoides, hookworm, and Strongyloides stercoralis and the pathogenic protozoa: Giardia lamblia, Entamoeba histolytica/E. dispar, and the commensals: Endolimax nana and Entamoeba coli. The intestinal parasite Enterobius vermicularis was the only parasitic species that increased the frequency of infections with respect to the 1984 survey. The frequencies of parasitic and commensal infections in both studies were higher in the 5-14 y age group (school age). Conclusions: the comparison between the intestinal parasitic infections surveys of 1984 and 2009 showed a reduction in the frequencies of intestinal parasitic infections in the last survey. The finding in both studies of a higher frequency of pathogenic parasitic infections and commensal infections in the 5-14 y age group (school age) supports the recommendation of making emphasis on the control programs for intestinal parasitic infections in this age group.

Use of in vitro cytoadherence assays in the comparative study of the virulence of isolates of Trichomonas vaginalis.

This study describes the use of bromo-deoxiuridine, a non-radioactive analogue of thymidine, to determine the adhesion of 40 T. vaginalis isolates, as compared with the clinical manifestations found in patients. Parasite-labeling was optimized and label detection by immunoassay was carried out. Bromo-deoxiuridine at 10 microM for 16 h produced the highest sensitivity. Once optimized, the assay was able to detect between 3.12 x 10(3) and 4 x 10(5) parasites, with a detection limit of 9.14 x 10(2), which is lower than with the use of [3H]-thymidine. The variation coefficients were 2.65% for repeatability and 3.8% for reproducibility. A correlation coefficient of 0.97 was found between the pattern curves obtained by both labeling procedures. The level of adhesion to HeLa cells was directly proportional to the severity of the clinical manifestations (P < 0.05).