Study population
A cross-sectional study was carried out from January 2016 to September 2017 involving HIV-positive patients followed at the Department of Translation and Precision Medicine, Umberto I Academic Hospital, Rome. A standardized questionnaire face-to-face interview was designed including demographic data (gender, age, profession, country of origin, recent travels) and information on clinical characteristics from each participating subject (CD4+ count and HIV-RNA load, therapy, laboratory results, comorbidity), potential risk factors for intestinal pathogens (exposure to domestic animals, diet, sexual behaviour) as well as detailed gastrointestinal symptoms (diarrhoea, abdominal pain, nausea, anorexia, weight loss, weakness, flatulence). According to World Health Organization definitions, diarrhoea was defined as the passage of three or more loose or liquid stools per day.
Written informed consent was obtained from every participant. The study was approved with respect to the Helsinki Declaration by the Ethical Committee of the Umberto I Academic Hospital (licence n. 4836).
Laboratory analyses
Blood and faecal samples were collected from each subject included in the study and submitted to the quantitative measurements of CD4+ T-lymphocytes and viral load [26], and to the microscopic observation of the wet smears stained with Lugol, directly or after Ridley concentration [27], respectively. Genomic DNA was then extracted from stool samples and submitted to PCR amplification using primers previously described [28], which target a fragment of about 600 bp from the Blastocystis-SSU rRNA gene, following PCR protocol and conditions described in Mattiucci et al., 2016 [13]. The sequences obtained were compared to those of Blastocystis spp. deposited in GenBank using the BLAST application (www.ncbi.nlm.nih.gov/BLAST). The STs were identified by determining the exact match or closest identity (99%), according to the classification given by Stensvold et al., 2007 [29].
Furthermore, the evaluation of the FC from a sample of subjects was performed using a commercial immunoassay (Calprest, EUROSPITAL, Italy), following the manufacturer’s instructions. Samples giving values above 50mg/kg were regarded as having a positive Calprest test, as reported by the manufacturer and by previous published studies [30].
Phylogenetic analyses
Bayesian inference (BI) and Neighbour-Joining (NJ) phylogenetic combined tree, based on the amplified sequences of the Blastocystis-SSU rDNA gene, was carried out by using MrBayes3.1 [31], software and PAUP4* [32] respectively; the analysis were performed using the HKY+G (G=0.134) as the best model selection for the data set, as implemented in jModeltest, with Akaike Information Criterion (AIC) [33]. For Bayesian analysis, four incrementally heated Markov Chains (using default heating values), were run for 1,000,000 generations, sampling the Markov Chains at intervals of 100 generations. For each corresponding subtype, a referring sequence by GenBank database was included in the tree. B. lapemi and B. pythoni (GenBank accession no. AY590115 and KU146575, respectively) were used as outgroups.
Statistical analysis
Continuous variables were summarized as mean ± standard deviation or median ± interquartile range (IQR) and categorical data as counts and percentages. Comparisons between groups were performed using χ2 test or Fisher’s exact test for categorical variables, and t-test or Mann-Whitney test for continuous variables. Multiple logistic regression was used to identify the predictors of Blastocystis colonization. The significance level for all analyses was set at p <0.05. Data were analysed using IBM SPSS, version 21.0 (SPSS Inc, Chicago, USA).