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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.

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.

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., Highlights • The incidence of taeniosis cases in Cuba is rising, with 2020 being a peak year • Although assumed to be Taenia saginata, this has not been confirmed • We investigated 37 samples from 2001 until 2020 that had been stored in formalin • Duplex real-time PCR indicated T. saginata; 3 cases were confirmed by sequencing

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.

Giardia duodenalis is a worldwide protozoan parasite that infects humans and other mammals including dogs. Due to the risk of zoonotic transmission between dogs and humans, we aimed in this study to determine the prevalence of the intestinal parasites and the distribution of assemblages of G. duodenalis among dogs analysed. A descriptive cross-sectional study was carried out in La Habana from June 2014 to March 2015 in the Zoonosis Unit of La Lisa municipality. A total of 98 dogs were analysed by three different techniques (microscopy with faecal concentration, Enzyme-Linked ImmunoSorbent Assay, and Polymerase Chain Reaction) in order to detect Giardia in stool samples. Out of 98 dogs studied, 43 (43.9%) were infected with intestinal parasites. The zoonotic parasites Ancylostoma caninum (21.4%), Trichuris vulpis (16.3%) and the protozoan Giardia duodenalis (11.2%) were the most prevalent parasites. In regards to the G. duodenalis, seven dogs were positive by microscopy after faecal concentration, nine by NOVITEC® Giardia Microplate Assay, and ten and eleven samples were amplified by the β-giardin and SSU-RNA PCRs, respectively. After PCR sequence analysis of both genes only zoonotic assemblages (A and B) were detected. The SSU-RNA sequence results revealed a distribution of 8 assemblage A and 4 assemblage B, whereas only assemblage A were identified by the β-giardin analysis. Among subassemblage classification by β-giardin phylogenetic tree, four isolates showed an AI pattern and one isolate displayed an AII distribution. Mixed infections were detected in three isolates. These findings highlight the risk of zoonotic transmission of Giardia duodenalis between dogs and humans.

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.

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.

Giardia duodenalis is one of the most frequent intestinal parasitic infections in children worldwide. To date, eight main assemblages of G. duodenalis have been described, but only A and B genetic groups are known to infect humans. A cross-sectional study was conducted to determine the prevalence and risk factors of Giardia duodenalis infection in 417 preschool children from the Fomento municipality in the central region of Cuba between January and June 2013. The overall prevalence of Giardia infection was 10.79 %. Assemblage identification was carried out by the amplification of a fragment of the triose phosphate isomerase (tpi) gene. DNA from 36 of 45 (80 %) samples was successfully amplified by PCR-tpi. Assemblage B and mixed assemblages A + B represented 52.78 and 36.11 % respectively, of genotyped samples. Assemblage A accounts for only 11.11 %. Children who were cared for at home were associated with diarrhea caused by assemblage B. No associations were found between other clinical variables with infecting assemblage of Giardia. Univariate analysis identified the use of unsafe water resources (OR 2.9; CI 1.2–6.8) and—even more interestingly—keeping dogs indoor (OR 2.5; CI 1.2–5.4) were significant risk factors associated with Giardia infection among children. Multivariate analysis using introduction test logistic regression ratified the association of these two risk factors: kept dogs indoor (OR 2.8, CI 1.1–5.3), and untreated water (OR 1.4, CI 1.4–4.9) with Giardia infection. This information may be useful for an effective prevention and control programme of giardiasis in this population.

Giardia duodenalis is one of the most prevalent intestinal parasites worldwide that infect humans, especially children. This study was aimed to determine the prevalence of assemblages of Giardia among a group of children. A descriptive cross-sectional study was performed in the Pediatric Academic Hospital “William Soler” from January 2014 to September 2014 in 286 children as part of a surveillance program of intestinal parasitic infections in scholar children. Out of 286 samples examined, 27 were positive for G. duodenalis for an infection rate of 9.34%. All Giardia positive samples were successfully amplified by a polymerase chain reaction (PCR) assay targeting the β-giardin gene. Restriction fragment length polymorphism analysis of these PCR products revealed that children infected with assemblage B were more prevalent (19/27; 70.4%) than those who harbored assemblage A (8/27; 29.6%) of this parasite. Abdominal pain was statistically associated with the infecting assemblage B of Giardia. The sequence of PCR products of β-giardin gene from symptomatic children infected with assemblage A revealed a distribution of four subassemblages AII divided in two subtypes A3 and two A2. Cases infected with subassemblage AII-A3 had a more abrupt onset of symptomatology. These findings highlighted the use of novel molecular tools for a better discrimination of assemblages at the subassemblages and genotypes levels are needed to verify possible correlations between Giardia genotypes and symptomatology of giardiasis.

Intestinal parasitic infections in dogs represent a major concern in developing countries as they are important definitive or reservoir hosts for several zoonotic parasites. The present study was conducted to determine the prevalence of intestinal parasitic infections in stray dogs and domestic dogs in the municipality of la Lisa, in La Havana, between June 2014 to March 2015. A total of 97 faecal samples from 56 stray dogs and 41 household dogs were examined by parasitological concentration techniques. The overall prevalence of gastrointestinal parasites was 44.3% (95% CI: 33.9-54.7). Ancylostoma caninum (21.6%), Trichuris vulpis (16.5) and Giardia duodenlis (7.2%) were the most prevalent intestinal parasites identified in canine faecal samples, indicating an important faecal contamination of the analyzed area. There were no significant differences between the prevalence of gastrointestinal parasites and the kind of dog analyzed. However stray dogs were associated with a higher risk of infection as well as those older than 1 year old. This information may be useful in the implementation of effective prevention and control programmes by the National Group of Zoonosis in our country. Appropriate public health education for dog’s owners is necessary to reduce the risks of zoonotic infections

Blastocystis sp. is one of the most common intestinal parasites diagnosed worldwide, but its pathogenic role is still regarded by some authors as controversial. The frequency of Blastocystis sp. was studied during a two year period of time and its association with some clinical symptoms. An observational descriptive study was carried out from January 2012 to December 2013. A total of 3140 stool samples were investigated for the presence of parasites by various parasitological techniques. Among 3140 analyzed stool samples the prevalence of Blastocystis infection observed was 3.54%, the fourth most prevalent. A total of 111 stool samples were positive for Blastocystis sp., and 71 samples showed co-infection with one or more other intestinal parasites. The group of 5 to 14 years age group showed a higher percentage of infections in the group of symptomatic persons than in asymptomatic ones and it was statistically significant, while in persons with ages more than 40 years the frequency of infection with this parasite was higher in asymptomatic persons. Blastocystis infection is one of the most frequent parasitic infections diagnosed in our setting. The relationship with symptomatology found in individuals infected with Blastocystis was significant and this association was higher in younger children.

Giardia duodenalis is one of the most frequent intestinal parasitic infections in children worldwide. To date, eight main assemblages of G. duodenalis have been described, but only A and B genetic groups are known to infect humans. In Cuba, this parasite has most clinical impact on children. The aim of this investigation was genetic characterization of G. duodenalis isolated from children with giardiasis diagnosed at the Paediatric Hospital "William Soler" between 2010 and 2011, and to compare the genetic results with clinical and epidemiological data. A total of 103 stool samples from 452 children were positive for G. duodenalis and co-infections with other parasites were noted in 5 cases. Assemblage identification was carried out by the amplification of a fragment of the triosephosphate isomerase (tpi) gene. Sub-assemblages of assemblage A (AI and AII) were identified by a nested PCR using the intergenic spacer (IGS) region of ribosomal deoxyribonucleic acid gene as a target. DNA from 90 of 103 (87.4%) samples was successfully amplified by PCR-tpi. The prevalence of assemblages A and B was 40% and 42%, respectively. Infections with both assemblages were reported in 16 cases. No associations between epidemiological information and assemblage was detected, but assemblage B was significantly (P0.01) more frequently found in children with diarrhea, flatulence or abdominal pain than assemblage A. Sub-assemblage AII accounted for the majority of cases (86.5%).

Fungal infections, also called mycoses, are important causes of morbidity and mortality in humans. Some fungal infections are endemic, and these infections are usually caused by fungi that are present in the environment and whose spores enter humans. Other fungal infections are said to be opportunistic because the causative agents cause mild or no disease in healthy individuals but may infect and cause severe disease in immunodeficient persons. The human airway is continuously open to the nonsterile environment where fungal spores have the potential to reach lung tissue and produce disease. In the immunocompromised host, many fungi, including species of fungi typically considered nonpathogenic, have the potential to cause serious morbidity and mortality. Over the last several decades the advent of the human immunodeficiency virus (HIV) epidemic and the increasing use of immunosuppressive drugs for serious medical conditions have dramatically increased the number of persons who are severely immunocompromised. In addition, the range and diversity of fungi that cause disease have broadened. Although Candida and Aspergillus species continue to be the fungal pathogens that most frequently cause invasive fungal disease in immunocompromised persons overall, infections due to previously uncommon hyaline and dematiaceous filamentous fungi are being reported with increasing frequency. This is significant because, despite marked advances in antifungal therapy, infections caused by opportunistic fungal infections (rare and emerging) continue to be associated with high morbidity, high mortality, and poor patient outcomes. This results from a combination of drug-resistant strains, lack of robust clinical studies evaluating treatments, and severe underlying diseases in the patient [2].