Is there added value from using three serial samples when surveying the occurrence of intestinal parasites in children?

BACKGROUND: Surveys for intestinal parasitic infections (IPIs) often involve samples from three sampling dates analysed by various microscopy techniques. However, analysis of three samples per individual is a huge burden on time and resources. We compared the value from analysing three or fewer samples. METHODS: In this cross-sectional study, three faecal samples were collected every other day from 332 children from two locations in Guantanamo province, Cuba. Samples were analysed by wet mount with Lugol stain, Willis flotation method and Kato-Katz thick smear. RESULTS: Most parasites were detected by wet mount, although helminth eggs not found by wet smear were detected by the Willis flotation method (in particular) and Kato-Katz smear. Blastocystis spp. was the most commonly detected parasite (about 65%), then Giardia duodenalis and then Entamoeba spp. Although analysis of two stool samples significantly increased occurrence data for Blastocystis, this was not so for the other parasites. For none of the protozoan parasites were results from analysing three samples significantly higher than results from analysing just two samples. CONCLUSIONS: Analysing two faecal samples by wet mount and the Willis flotation method provides useful data for estimating the prevalence of IPIs in relatively high prevalence settings. Analysing further samples provides limited additional information and adds an extra burden in terms of time and resources.

Identification of two potential aetiological agents of chronic diarrhoea in an immunocompromised patient in Cuba using conventional and molecular diagnostic techniques.

The aetiology of diarrhoea in a patient in Cuba with HIV was investigated. Although molecular diagnostics are still not used in many under-resourced settings, here traditional methods were supported by use of PCR. This approach enabled detection of a dual infection (Cystoisospora belli and Enterocytozoon bieneusi), the latter of which was not identified by microscopy with Didier's trichromic staining.

A retrospective study of Taenia spp. in Cuban patients: what does molecular analysis tell us?

Taeniosis is a neglected disease, particularly in developing countries, and is caused by infection with the adult tapeworm of either Taenia solium, Taenia saginata, and Taenia asiatica. Of these, T. solium is of primary concern due to the potential for cysticercosis should T. solium eggs be ingested. In Cuba, all cases of taeniosis are assumed to be caused by T. saginata, although some cases of cysticercosis have been documented. It is therefore important to gain further insights regarding the species causing taeniosis in Cuba, especially as diagnostic records indicate an increasing incidence, with the highest number of cases reported in 2020. In this study, we analysed 37 Taenia-positive faecal samples (or proglottids isolated from faecal samples) from the period 2001 until 2020 from all regions of the country. Genomic DNA was extracted from the samples, which had been stored in 10% formalin, using the QIAamp Tissue Kit. Species identification was carried out by duplex real-time PCR targeting the mitochondrial DNA. All cases were found to be T. saginata, and sequence analysis of three isolates confirmed the identification of this species. Our data do not provide any evidence that T. solium currently occurs in Cuba. However, given the relatively low number of samples analysed here, that the parasite may be imported with visitors or travellers who have been in endemic countries, and that taeniosis has relatively mild symptoms and thus infected patients may not seek medical attention, we recommend species determination for all taeniosis cases reported in Cuba.

Frecuencia de infección por cestodos en el Laboratorio Nacional de Referencia de Parasitismo Intestinal-IPK, Cuba, 2010-2018 / Frequency of cestode infection at the Intestinal Parasitic Disease National Reference Laboratory of Pedro Kourí Institute in Cuba, 2010-2018

Introdución: Los cestodos son helmintos parásitos del humano y de animales, con complejos ciclos de vida. En las infecciones por los cestodos no existen programas de desparasitación masiva implementados para disminuir la carga parasitaria de estos a nivel mundial, dada la baja prevalencia que se informa en estudios epidemiológicos. Existen pocos trabajos sobre la epidemiología y la detección de estas infecciones en la literatura internacional. Objetivo: Describir la frecuencia de infección de cestodos y sus características epidemiológicas. Métodos: Estudio descriptivo y retrospectivo, realizado entre el 3 de enero de 2010 y 28 de diciembre de 2018. Se analizaron todas las muestras de heces, parásitos adultos y metacestodos enviadas de la red de Salud al Laboratorio Nacional de Referencia de Parasitismo Intestinal-IPK. El universo de estudio estuvo constituido por 9833 muestras, que fueron enviadas mayoritariamente de la provincia La Habana. Resultados: La infección por Inermicapsifer madagascariensis predominó en niños menores de 5 años (69,6 por ciento; IC 95 por ciento: 47,1-86,8). La infección intestinal por Taenia spp. se diagnosticó prinipalmente en pacientes entre 15 y 65 años de edad (88,2 por ciento; IC95 por ciento: 63,6-98,5). De los 47 casos positivos a la infección por cestodos, 24 pertenecieron al sexo femenino (51,1 por ciento; IC95 por ciento: 35,7-66,4) y 23 al sexo masculino (48,9 por ciento; IC95 por ciento: 33,6-64,3). No hubo diferencias significativas entre la infección por Inermicapsifer madagascariensis y Taenia spp. y el sexo de los pacientes (p˃ 0,05). Conclusiones: Aunque la frecuencia de infección de las cestodiosis detectadas es baja, estos resultados pudieran ser útiles para un control integrado de las principales cestodioisis en los diferentes grupos etarios(AU)

Introduction: Cestodes are helminths of complex life cycles which may infect animals and humans. No mass deworming programs are in place to reduce the worldwide parasite load of cestodes, given the low prevalence reported by epidemiological research. Few studies are available in the international literature about the epidemiology and detection of these infections. Objective: Describe the frequency of cestode infection and its epidemiological characteristics. Methods: A retrospective descriptive study was conducted from 3 January 2010 to 28 December 2018. Evaluation was performed of all the samples of fecal matter, adult parasites and metacestodes submitted by the health care network to the Intestinal Parasitic Disease National Reference Laboratory at Pedro Kourí Institute. The study universe was 9 833 samples, mostly received from the province of Havana. Results: Inermicapsifer madagascariensis infection prevailed among children aged under 5 years (69.6 percent; CI 95 percent: 47.1-86.8). Taenia sp. intestinal infection was mainly diagnosed in patients aged 15-65 years (88.2 percent; CI 95 percent: 63.6-98.5). Of the 47 cestode infection positive cases, 24 were female (51.1 percent; CI 95 percent: 35.7-66.4) and 23 were male (48.9 percent; CI 95 percent: 33.6-64.3). No significant differences were found between Inermicapsifer madagascariensis and Taenia sp. infection and the sex of patients (p˃ 0.05). Conclusions: Although the frequency of cestode infection detected is low, these results could be useful for an integrated control of the main cestode infections affecting the different age groups(AU)

Diagnosis of intestinal protozoan infections in patients in Cuba by microscopy and molecular methods: advantages and disadvantages.

Microscopy is the gold standard for diagnosis of intestinal parasitic diseases in many countries, including Cuba, although molecular approaches often have higher sensitivity as well as other advantages. Fecal samples from 133 patients were analyzed by light microscopy and also real-time multiplex qPCR targeting Giardia duodenalis, Cryptosporidium spp., and Entamoeba histolytica, and, separately, Dientamoeba fragilis. Microscopy revealed G. duodenalis occurred most commonly (17 patients), followed by Blastocystis spp. (12 patients). In a few patients, Entamoeba histolytica/E. dispar, Cryptosporidium spp., and Cyclospora cayetanensis were identified. Molecular analysis identified 4 more G. duodenalis infections and 2 more Cryptosporidium spp. infections; concordance between microscopy and PCR showed almost perfect agreement for G. duodenalis (κ = 0.88) and substantial agreement for Cryptosporidium (κ = 0.74). PCR indicated that E. dispar, rather than E. histolytica, had been identified by microscopy. Additionally, 16 D. fragilis infections were detected using molecular methods. Although both microscopy and molecular techniques have a place in parasitology diagnostics, for parasites such as D. fragilis, where microscopy can underestimate occurrence, molecular techniques may be preferable, and also essential for distinguishing between morphologically similar microorganisms such as E. histolytica and E. dispar. Although in resource-constrained countries such as Cuba, microscopy is extremely important as a diagnostic tool for intestinal parasites, inclusion of molecular techniques could be invaluable for selected protozoa.

Concordance of Giardia duodenalis assemblages determined by different PCR methodologies in three observational studies in Cuba.

Giardia duodenalis is one of the most important intestinal parasites globally, especially in children, and in Cuba is the leading cause of chronic paediatric diarrhoea in this population. G. duodenalis is composed of eight genetic groups (or assemblages), two of which (A and B) are apparently zoonotic, occurring in both humans and other animals. However, consensus on the most appropriate genotyping scheme for optimal characterization of G. duodenalis isolates is lacking. In this article we present the results of three descriptive observational studies conducted in Havana, Cuba between 2010 and 2013, with the aim of comparing the results from molecular (PCR) approaches targeting different genes in order to assign with confidence 224 isolates of G. duodenalis to the correct assemblages. In each sub-study, following DNA isolation by the phenol/chloroform/isoamyl alcohol extraction method, PCR targeting the triose phosphate isomerase (tpi) gene was used for molecular characterization, as well as one additional PCR-method targeting another gene or pair of genes. DNA amplification was obtained in 87%, 83%, and 80% in the three sub-studies. Although excellent agreement (kappa index = 1) was recorded between results from some pairs of genes, for other combinations only moderate or substantial agreement was achieved. These results highlight the importance of interpretation of genotyping data, especially when single genetic markers are used. From the results of our studies, PCR targeting a combination of the tpi gene and the intergenic spacer region of rDNA may be a useful approach for the molecular characterization of G. duodenalis isolates.

Molecular analysis of Giardia duodenalis isolates from symptomatic and asymptomatic children from La Habana, Cuba.

Giardiasis is considered the most common intestinal parasitic disease in humans worldwide. In Cuba, this infection has particularly a strong clinical impact on the child population. Giardia duodenalis is a highly diverse protozoan, which comprises a complex of eight morphologically identical genetic assemblages, further divided into sub-assemblages. The present study used triose phosphate isomerase (tpi) and small-subunit ribosomal RNA (SSU rRNA) genes as genetic markers for the identification of G. duodenalis assemblages and sub-assemblages in correlation with clinical and epidemiological data in children attended at the Paediatric Hospital "William Soler" and at Pedro Kouri Institute, between 2015 and 2016. A prevalence of 8% of G. duodenalis infection was recorded in stool samples after concentration techniques from 68 children out of 847 analysed. A 100% detection of Giardia DNA was achieved by a SSU-rRNA PCR, whereas DNA from 63 of 68 (92.6%) was successfully amplified by tpi-PCR. By this assemblage-specific tpi-PCR 32 (50.8%) assemblage B, 17 (27.0%) assemblage A and 14 (22.2%) mixed infection (A + B) were identified. Assemblage B was significantly (P < 0.02) more frequently found in children with diarrhoea. Sequence analysis of the tpi gene of Giardia isolates from symptomatic children showed that assemblage A belonged to the sub-assemblage AII, and 4 sub assemblages BIV and 1 sub assemblage BIII were also recorded. Only 2 discordant genotyping results were observed by phylogenetic comparison of SSU-rRNA and tpi sequences. Further studies with novel molecular tools for a better discrimination at the sub-assemblage level are needed to identify the dynamics of spread of giardiasis and to verify possible correlations between Giardia genetic diversity and clinical manifestation.

A dual PCR-based sequencing approach for the identification and discrimination of Echinococcus and Taenia taxa.

Reliable and rapid molecular tools for the genetic identification and differentiation of Echinococcus species and/or genotypes are crucial for studying spatial and temporal transmission dynamics. Here, we describe a novel dual PCR targeting regions in the small (rrnS) and large (rrnL) subunits of mitochondrial ribosomal RNA (rRNA) genes, which enables (i) the specific identification of species and genotypes of Echinococcus (rrnS + L-PCR) and/or (ii) the identification of a range of taeniid cestodes, including different species of Echinococcus, Taenia and some others (17 species of diphyllidean helminths). This dual PCR approach was highly sensitive, with an analytical detection limit of 1 pg for genomic DNA of Echinococcus. Using concatenated sequence data derived from the two gene markers (1225 bp), we identified five unique and geographically informative single nucleotide polymorphisms (SNPs) that allowed genotypes (G1 and G3) of Echinococcus granulosus sensu stricto to be distinguished, and 25 SNPs that allowed differentiation within Echinococcus canadensis (G6/7/8/10). In conclusion, we propose that this dual PCR-based sequencing approach can be used for molecular epidemiological studies of Echinococcus and other taeniid cestodes.

Molecular Characterization and Risk Factors of Giardia duodenalis among School Children from La Habana, Cuba.

Giardia duodenalis is considered the most common protozoan infecting humans worldwide. Molecular characterization of G. duodenalis isolates has revealed the existence of eight groups (assemblages A to H) which differ in their host distribution. A cross-sectional study was conducted in 639 children from La Habana between January and December 2013. Two assemblage-specific PCRs were carried out for the molecular characterization. The overall prevalence of Giardia infection was 11.9%. DNA from 63 of 76 (82.9%) samples was successfully amplified by PCR-tpi, while 58 from 76 (76.3%) were detected by PCRE1-HF. Similar results by both PCRs were obtained in 54 from 76 samples (71%). According to these analyses, assemblage B and mixed assemblages A + B account for most of the Giardia infections in the cohort of children tested. Our current study identified assemblage B as predominant genotype in children infected with Giardia. Univariate analysis indicated that omission of washing hands before eating and keeping dogs at home were significant risk factors for a Giardia infection. In the future, novel molecular tools for a better discrimination of assemblages at the subassemblages level are needed to verify possible correlations between Giardia genotypes and symptomatology of giardiasis.

Aplicación de herramientas serológicas y moleculares para el diagnóstico de coriorretinitis por Toxoplasma gondii / Application of serological and molecular tools for diagnosis of chorioretinitis caused by Toxoplasma gondii

Introducción: Toxoplasma gondii, agente causal de la toxoplasmosis, es un protozoario intracelular obligado que puede afectar al globo ocular, siendo la causa más común de uveítis posterior. Objetivo: determinar la utilidad de las técnicas serológicas y moleculares para el diagnóstico de la toxoplasmosis ocular en pacientes con uveítis. Métodos: se diseñó un estudio de corte transversal para comparar un grupo de pacientes afectados por coriorretinitis toxoplásmica y otro con signos sugestivos de coriorretinitis no toxoplásmica. En ambos grupos se utilizaron métodos serológicos como la inmunofluorescencia indirecta y la prueba de inmunoensayo enzimático (ELISA), y la reacción en cadena de la polimerasa para el diagnóstico de la toxoplasmosis ocular. Resultados: las técnicas serológicas permitieron detectar los anticuerpos IgG anti-Toxoplasma en 100 % de los pacientes con coriorretinitis toxoplásmica, de ellos fueron positivos a los anticuerpos IgM solo 3 pacientes y 1 tuvo un resultado débil a la avidez IgG. La reacción en cadena de la polimerasa detectó el ADN de Toxoplasma gondii en 15 de los 47 pacientes para una sensibilidad de 31,9 %. Conclusiones: se reporta la detección de anticuerpos anti-Toxoplasma, en todos los casos de coriorretinitis toxoplásmica a través de los métodos serológicos, aunque la técnica más sensible fue la inmunofluorescencia indirecta. Se empleó la detección molecular del ADN de Toxoplasma gondii en pacientes con toxoplasmosis ocular, por primera vez en Cuba. Los resultados permiten sugerir el uso de técnicas serológicas y moleculares que ayuden a confirmar el diagnóstico de infección por Toxoplasma en pacientes con coriorretinitis.

Introduction: Toxoplasma gondii, the causative agent of toxoplasmosis, is an intracellular protozoan that can affect the eye, and be the most common cause of posterior uveitis. Objective: to determine the usefulness of serological and molecular techniques for the diagnosis of ocular toxoplasmosis in patients with uveitis. Methods: a cross-sectional study was designed in order to compare a group of patients with toxoplasmic retinochoroiditis and another affected by non-toxoplasmic retinochoroiditis. In both groups serological methods such as indirect immunofluorescence assay, immunoassay (ELISA), and the polymerase chain reaction were used for the diagnosis of ocular toxoplasmosis. Results: serological techniques allowed detecting anti-Toxoplasma IgG antibodies in 100 % of patients with toxoplasmic retinochoroiditis, and only 3 patients were positive to IgM antibodies. Finally, only one had a weak result for IgG avidity. The polymerase chain reaction detected DNA from Toxoplasma gondii in 15 out of 47 patients for a sensitivity of 31.9 %. Conclusions: this paper reports the detection of anti-Toxoplasma antibodies in all toxoplasmic retinochoroiditis cases by serological methods; but the most sensible method was immunofluorescence assay. The molecular detection of Toxoplasma gondii`s DNA in patients with ocular toxoplasmosis was employed for the first time in Cuba. The results suggest that serological and molecular techniques can be applied to confirm the diagnosis of Toxoplasma infection in patients with toxoplasmic retinochoroiditis.

Estudio comparativo de medición entre lecturas visuales y espectrofotométricas en pruebas de susceptibilidad in vitro de aislamientos de candida / Comparative study of measure visual and spectrophotometer lectures in a in vitro antifungal susceptibility testing in candida isolates

Se realizó un estudio de susceptibilidad in vitro frente a itraconazol, ketoconazol y clotrimazol de 144 cepas de Candida, conservadas y previamente identificadas, aisladas de la cavidad oral de pacientes infectados por el virus de inmunodeficiencia humana (VIH) con cuadros clínicos de candidiasis orofaríngea (COF). El estudio se llevó a cabo mediante dos metodologías; la primera, utilizando los requerimientos del Clinical and Laboratory Standard Institute (CLSI), en el cual está establecida la lectura visual para determinar los patrones de susceptibilidad; y la segunda, mediante la propuesta de la European Committee on Antibiotic Susceptibility Testing, el cual tiene fijado la lectura espectrofotométrica para eliminar las posibles subjetividades de la metodología del CLSI. Los resultados obtenidos mediante ambas lecturas no mostraron diferencias mayores a dos diluciones en los valores de concentración mínima inhibitoria, y demostraron que ambos métodos se correlacionan y es importante para aquellos laboratorios de pocos recursos económicos.

A study of in vitro susceptibility was realized opposite to itraconazol, ketoconazol and clotrimazol of 144 strains of Candida, conserved and previously identified, isolated of the oral cavity of patients infected by the virus of human immunodeficiency (VIH) with clinical pictures of oropharingeal candidiasis (COF). The study was realized by means of two methodologies; the first one, using the requests of the Clinical and Laboratory Standard Institute (CLSI), in which the visual reading is established to determine the patterns of susceptibility; and the second one, by means of the proposal of the European Committee on Antibiotic Susceptibility Testing, which has fixed the spectrophotometric to eliminate the possible subjectivities of the methodology of the CLSI. The results obtained by means of both readings did not show differences bigger than two dilutions in the values of minimal inhibitory concentration, demonstrating that both methods are correlated and it is important for those laboratories of few economic resources.

Estudio comparativo de medición entre lecturas visuales y espectrofotométricas en pruebas de susceptibilidad in vitro de aislamientos de candida / Comparative study of measure visual and spectrophotometer lectures in a in vitro antifungal susceptibility testing in candida isolates

Se realizó un estudio de susceptibilidad in vitro frente a itraconazol, ketoconazol y clotrimazol de 144 cepas de Candida, conservadas y previamente identificadas, aisladas de la cavidad oral de pacientes infectados por el virus de inmunodeficiencia humana (VIH) con cuadros clínicos de candidiasis orofaríngea (COF). El estudio se llevó a cabo mediante dos metodologías; la primera, utilizando los requerimientos del Clinical and Laboratory Standard Institute (CLSI), en el cual está establecida la lectura visual para determinar los patrones de susceptibilidad; y la segunda, mediante la propuesta de la European Committee on Antibiotic Susceptibility Testing, el cual tiene fijado la lectura espectrofotométrica para eliminar las posibles subjetividades de la metodología del CLSI. Los resultados obtenidos mediante ambas lecturas no mostraron diferencias mayores a dos diluciones en los valores de concentración mínima inhibitoria, y demostraron que ambos métodos se correlacionan y es importante para aquellos laboratorios de pocos recursos económicos(AU)

A study of in vitro susceptibility was realized opposite to itraconazol, ketoconazol and clotrimazol of 144 strains of Candida, conserved and previously identified, isolated of the oral cavity of patients infected by the virus of human immunodeficiency (VIH) with clinical pictures of oropharingeal candidiasis (COF). The study was realized by means of two methodologies; the first one, using the requests of the Clinical and Laboratory Standard Institute (CLSI), in which the visual reading is established to determine the patterns of susceptibility; and the second one, by means of the proposal of the European Committee on Antibiotic Susceptibility Testing, which has fixed the spectrophotometric to eliminate the possible subjectivities of the methodology of the CLSI. The results obtained by means of both readings did not show differences bigger than two dilutions in the values of minimal inhibitory concentration, demonstrating that both methods are correlated and it is important for those laboratories of few economic resources(AU)