Molecular characterization of Blastocystis subtypes in HIV-positive patients and evaluation of risk factors for colonization

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Studies on the prevalence of Blastocystis in HIV-positive patients are limited and dated. Methods: A cross-sectional study was carried out involving 156 HIV-positive patients to evaluate the prevalence of Blastocystis-subtypes by molecular amplification and sequencing the small subunit rRNA gene (SSU rDNA), to identify the risk factors for its transmission, to examine the relationship between the presence of the protist and gastrointestinal disorders. Furthermore, the evaluation of the faecal calprotectin by immunoassay from a sample of subjects was performed to evaluate the gut inflammation in Blastocystis-carriers. Results: Blastocystis-subtypes ST1, ST2, ST3, ST4 were identified in 39 HIV-positive patients (25%). No correlation was found between the presence of the protist and virological or epidemiological risk factors. Blastocystis was more frequently detected in homosexual subjects (p=0.037) infected by other enteric protozoa (p= 0.0001) and with flatulence (p=0.024). No significant differences in calprotectin level was found between Blastocystis-carriers and free ones. Conclusions: Blastocystis is quite common in HIV-positive patients on ART showing in examined patients 25% prevalence. Homosexual behaviour may represent a risk factor for its transmission, while CD4 count and viremia didn’t correlate with the presence of the protist. The pathogenetic role of Blastocystis remains unclear and no gut inflammation status was detected in Blastocystis-carriers. The only symptom associated with Blastocystis was the flatulence, evidencing a link between the presence of the protist and the composition and stability of gut microbiota. Keywords: Blastocystis, subtype, PCR, HIV, Italy Infectious Diseases Blastocystis subtype PCR HIV Italy Figures Figure 1 Background Blastocystis spp. is a common intestinal protist distributed worldwide infecting humans and animals, with a prevalence from 0.5%-30% and 30-76% in industrialized and developing countries, respectively [1]. This difference can be explained by poor hygiene practices and consumption of contaminated food or water [2, 3, 4, 5] since the faecal-oral route is considered to be the main mode of transmission of this protist [6]. A remarkable genetic diversity has been revealed among Blastocystis spp. isolates from humans and animals based on the comparison of the small-subunit rRNA gene (SSU rDNA) sequences [7] . Currently at least 26 subtypes of Blastocystis have been described among mammalian and avian isolates [8, 9], nine of them (ST-1 to ST-8 and ST-12) detected in human population and are potentially zoonotic [10]. Because several STs are shared between humans and animals it has been proposed that a proportion of human infections may result from zoonotic transmission. Indeed, a higher risk of Blastocystis infection was found in people with close animal contact, including zookeepers [11]. Despite several studies reported Blastocystis implicated in different intestinal diseases, potential pathogenetic factors have been described and its presence is frequently associated with symptoms in humans [12], its pathogenetic role is so far under debated and several variables, as well as the Blastocystis subtype and load, host’s immune status and dysbiosis, could affect the occurrence of the disease [13][14]. Although the protist was never considered as an opportunistic protozoon, it has also been frequently found in immunocompromised individuals presenting diarrhoea, with prevalence value ranging from 15% to 72.4% [15]. In addition, the sexual practices among men who have sex with men (MSM) have been reported to increase the transmission of Blastocystis (as for as other enteric organisms) [16] . Finally, recent data suggest that the protozoon is associated with certain gut microbiota profiles and health indices. Accordingly, a positive correlation between high bacterial richness and the presence of Blastocystis has been reported [17]. Notwithstanding it is commonly accepted that Blastocystis is a non-invasive organism, its vacuolar form is able to colonize the lamina propria, the submucosa and even the muscle layers leading to inflammation and active colitis in experimentally infected mice [18]. Currently, it is possible to perform a non-invasive evaluation of gut inflammation using biomarkers as the fecal calprotectin (FC), which is considered a surrogate marker of intestinal inflammation with good diagnostic performance for separating organic and functional intestinal disorders [19] . The increase of FC is due to faecal excretion of neutrophils and macrophages migrants from the bloodstream into the intestinal lumen, which occurs during the intestinal inflammation. The value of FC as a laboratory marker has been shown in inflammatory bowel disease [20] but its significance in other gastrointestinal diseases remains unclear. Studies evaluating the correlation between intestinal inflammatory markers and parasitic infections are scant [21, 22, 23, 24] . However, a positive correlation between the level of FC and severe infections due to intestinal parasites such as Giardia intestinalis and Schistosoma mansoni has been reported [21, 22]. Conversely, lower concentrations of FC have been reported in Blastocystis colonized individuals compared to non-colonized subjects [25]. This study aimed to evaluate the prevalence of Blastocystis subtypes in HIV-positive patients, to evaluate the potential the risk factors for its transmission and the relationship between the presence of the protozoon and gastrointestinal symptoms and, finally, to assess the level of FC in Blastocystis- colonized subjects. Methods 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 χ 2 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). Results A total of 156 consecutive HIV-positive patients were enrolled in the study between January 2016 and September 2017. Demographic and clinical data of the study population are described in Table 1. The majority of subjects were males (75%), mean age was 47.05 ± 12.38 (range 22 to 71 years), 39 participants (25%) were coming from non-EU countries and 50 ones (32%) were MSM ( men who have sex with men ). Moreover 47.4% were owner of pets and 42.9% travelled outside Europe in the last 6 months. Twenty-three patients (14.7%) were naïve and 133 (85.3%) were on ART treatment. Thirty-five subjects (26.3%) were on ART based on non-nucleoside reverse-transcriptase inhibitors (NNRTI), 34 (25.6%) on protease inhibitors (PI/r), 36 (27.1%) on integrase inhibitors and 28 (21%) on dual therapy. Three of the enrolled patients were on prophylaxis treatment with sulfamethoxazol-trimethoprim. The mean CD4 cell count was 655.04 ± 381.56 cells/mm 3 . Most of patients (62.2%) showed a CD4 count higher than 500 cells/ml, 26.3% patients had a CD4 count between 200 and 500 cells/ml and only 11.5% patients showed a CD4 count lower than 200 cells/ml. The median HIV-RNA level of naive patients was 40010 (7093-824500) -copies/mL. Among patients on ART treatment, HIV-RNA was detectable (>37 cp/mL) in 15.8% of subjects (21/133) with a median HIV-RNA level of 116 (67-991) cp/mL. Microscopic examination revealed the presence of Blastocystis spp. in 34 patients (21.8%) while PCR amplification detected the protist in 39 individuals (25%) and the expected 600 bp fragments of the SSU rRNA gene amplified were successfully sequenced. Sequences obtained showed a high identity (98–100%) to homologous sequences of Blastocystis isolates previously reported in GenBank and, according to the consensus terminology [29] and based on the phylogenetic analyses carried out, enabled the clustering of our isolates into 4 distinct Blastocystis -subtypes (Figure 1). In detail, ST3 was the most common subtype found in 51.3% of the subjects, followed by ST1 (30.8%), ST4 (10.2%) and ST2 (7.7%). As expected, ST4 was identified only in European participants. In 30.7% of the subjects, Blastocystis was found in coinfection with other enteric protozoa as Entamoeba coli (15%) , Endolimax nana (8%) , Giardia intestinalis (6.1%) and Iodamoeba butschlii (1.6%). The detection of such intestinal parasites was more frequent in Blastocystis -carriers compared to Blastocystis -free ones (p<0.0001) and in MSM compared to heterosexual subjects (p<0.0001). Neither demographic characteristics such as gender, age and nationality nor other epidemiological risk factors such as travel history or, presence of domestic animals differed significantly between the Blastocystis positive and Blastocystis negative patients (Table 2). In addition, no significant differences were found between the two groups regarding CD4+ T-cell counts, HIV-RNA undetectability and type of ART regiment. Blastocystis positive subjects reported more frequently homosexual behavior practices compared to negative ones (48.71% vs 26.49%, p=0.037). After adjusting for age, the relationship between homosexual behavior practices and the presence of Blastocystis remained significant (p=0.01). Most of the Blastocystis positive subjects were symptomatic (53.8%), while 33.6% Blastocystis -free ones referred gastrointestinal disorders (p=0.029). Among the gastrointestinal symptoms analyzed, flatulence was more frequently observed in Blastocystis carriers (30.7%) compared to Blastocystis negative ones (14.5%) (p=0.024) (Table 3). To determine whether Blastocystis colonization was associated with intestinal inflammation, we measured FC in a subset of samples divided in Blastocystis -carrier (mono-infected) (N=23) and Blastocystis- free (N=36) subjects rom the all 59 fecal samples ( Blastocystis -carrier and Blastocystis- free), 83% had a value in the standard range (lower than 50 mg/kg) and 11 (18.6%) had an increased value (median 115.7; 95% CI=71.82-303.78). However, no significant differences in calprotectin values was found between Blastocystis- carriers (median 15, 95% CI= 18.93-148.53) and non-carriers (median 13.35, 13.93-31.74) (p=0.31). Discussion Studies on the prevalence of Blastocystis spp. in HIV-positive subjects are relatively scant. In Italy the protist has so far attracted little attention, where few epidemiological surveys have been published, demonstrating a prevalence rate of about 7% and the occurrence of seven STs (ST1, ST2, ST3, ST4, ST6, ST7, ST8) in HIV-negative people [34][13]. The only study conducted on Italian HIV-positive patients date back to 1999, through the microscopic method, showed a Blastocystis prevalence of 10.3% [35]. The current study is, to our knowledge, the first to assess the prevalence of Blastocystis in HIV-positive patients through molecular methods in Europe and reported a prevalence of about 25%, in line with that reported in previous studies conducted in Mexico (30%)[36], Malaysia (19.8%) [15] and Iran (19%) [37]. The molecular characterization of Blastocystis positive isolates evidenced the circulation of 4 subtypes (ST1, ST2, ST3, ST4) already described in Italy, as previously mentioned [13, 38], being ST3 the most common one with a prevalence of 51%. In our cohort of HIV-positive patients, the presence of Blastocystis resulted no correlated to viro-immunological factors (p>0.05) confirming therefore the non-opportunistic behavior of the protist. No demographic characteristic or epidemiological drivers (presence of domestic animals, dietary habits, travels abroad) were found to be associated with Blastocystis colonization. A higher prevalence of Blastocystis was reported in MSM compared to heterosexuals as well as of the other intestinal parasites, underlying the faecal-oral contact as the main route of transmission in this group of subjects and the sexual practice and lifestyle linked to the presence of the protist more than the immunological status [16]. Despite the unresolved controversy over its pathogenicity, Blastocystis was also been frequently found in immunocompromised individuals presenting diarrhea [15]. In our study, among gastrointestinal symptoms, a positive association was found only for the flatulence. This socially disabling symptom is determined by two main factors: the diet, particularly the quantity of fermentable residues, and the composition and metabolic activity of colonic microbiota [39]. Since we evidenced any differences in the diet between Blastocystis -carriers and Blastocystis -free subjects, we hypothesized that this symptom was linked to the presence of the protist and the gut microbiota composition. Several Authors evaluated the intestinal microbiome in HIV-positive subjects, with somewhat inconsistent or controversial results [40]. This may be because of small sample sizes, lack of appropriate controls or regional differences in dietary and environmental factors. Regardless, many authors evidenced a change in the Bacteroides:Prevotella ratio, which they suggested to be linked to the antiretroviral therapy [41, 42] or to the sexual practice and lifestyle [43]. Similarly, several studies reported Blastocystis colonization associated with higher bacterial richness and Prevotella enterotype [44]. Therefore, investigations on the gut microbiota from HIV-positive subjects Blastocystis -carriers are needed to complete and to better understand the preliminary results obtained in this study. Concerning FC, we found the median of both Blastocystis -carrier and free subjects within the normal range, supporting the non-pathogenic role of Blastocystis in inducing intestinal inflammation [25]. Conclusions Despite some critical points of this study concerning the lack of data about the faecal microbiota from enrolled subject , to our knowledge this is the first large survey to molecular characterize Blastocystis subtypes in HIV-positive patients in Europe. Our results suggest that Blastocystis is quite common in such patients (25%) and homosexual behavior resulted a risk factor for its transmission and confirm the presence of the protist not associated with pathological gut inflammation. The role of Blastocystis as intestinal pathogen remains unclear as the only symptom associated with its presence was flatulence. This result is intriguing since flatulence could be linked to the gut microbial communities, therefore the impact of the protist on the faecal microbiota and its possible role to maintain the to gut homeostasis could be interesting prospects for further studies. List of Abbreviations ART: antiretroviral therapy; CI: Confidence interval; FC: fecal calprotectin; HIV: Human Immunodeficiency Virus; MSM: men who have sex with men; NNRTI: non-nucleoside reverse-transcriptase inhibitors. Declarations Ethics approval and consent to participate The study protocol was approved by the ethics committees of Umberto I Academic Hospital (licence n. 4836) in accordance with the ethical guidelines of the Declaration of Helsinki. Written informed consents were obtained from the patients after declaring the objectives of the present study. Consent for publication Not Applicable Availability of data and materials The dataset of this article will not be available publicly, to ensure the patient’s privacy, but are available from the corresponding author on reasonable request. Competing interest The authors declare that they have no competing interests. Funding This study was financed by Sapienza University in the frame of the “Progetti di Ricerca Università Sapienza; years 2016, 2017 and 2018, grant numbers RM11615506189BE0, RM11715C81DDD2F3, RM11816432E08126”. The content is solely the responsibility of the authors and the funder was not involved in the design of the study, data collection, analysis and interpretation of the data and in writing the manuscript. Authors’ contributions LFS and SG designed the study with input from SM and GT. LFS, SG and FF conducted the data analysis, interpretation, and manuscript preparation. FF and GLM performed the laboratory analyses and molecular identification and contributed to the manuscript preparation. EB, MDA, GI, CF and MM enrolled the patients, performed the sample collection and contributed to the data analysis, data interpretation, and manuscript preparation. SG, SM and GT interpreted the results and revised the manuscript. All authors critically reviewed and approved the final version of this paper for publication. Acknowledgements Authors are grateful to the Eurospital S.p.A. for providing the Calprest immunoassay. References Alfellani MA, Stensvold CR, Vidal-Lapiedra A, Onuoha ES, Fagbenro-Beyioku AF, Clark CG. Variable geographic distribution of Blastocystis subtypes and its potential implications. Acta Trop 2013; 126:11–18. Leelayoova S, Siripattanapipong S, Thathaisong U, Naaglor T, Taamasri P, Piyaraj P, et al. Drinking water: A possible source of Blastocystis subtype 1 infection in schoolchildren of a rural community in central Thailand. Am J Trop Med Hyg 2008; 79(3):401–406. Baldursson S, Karanis P. Waterborne transmission of protozoan parasites: review of worldwide outbreaks- an update 2004-2010. Water Res 2011; 45:6603-14. Lee LI, Chye TT, Karmacharya BM, Govind SK. Blastocystis : waterborne zoonotic organism, a possibility? Parasit Vectors 2012; 28:5-130. Nagel R, Cuttell L, Stensvold CR, Mills PC, Bielefeldt-Ohmann H, Traub RJ. Blastocystis subtypes in symptomatic and asymptomatic family members and pets and response to therapy. Intern Med J 2012; 42(11):1187–1195. Yoshikawa H, Yoshida K, Nakajima A, Yamanari K, Iwatani S, Kimata I. Fecal-oral transmission of the cyst form of Blastocystis hominis in rats. Parasitol Res 2004; 94(6):391–396. Yoshikawa H, Wu Z, Nagano I, Takahashi Y. Molecular comparative studies among Blastocystis isolates obtained from humans and animals. J Parasitol. 2003;89(3):585-94. Zhao GH, Hu XF, Liu TL, Hu RS, Yu ZQ, Yang WB, et al. Molecular characterization of Blastocystis sp. in captive wild animals in Qinling Mountains. Parasitol Res. 2017; 116(8):2327-2333. Maloney JG, Lombard JE, Urie NJ, Shivley CB, Santin M. Zoonotic and genetically diverse subtypes of Blastocystis in US pre-weaned dairy heifer calves. Parasitol Res. 2019; 118(2):575-582. Stensvold CR, Clark CG. Current status of Blastocystis : A personal view. Parasitol Int 2016; 65(6):763–771. Parkar U, Traub RJ, Vitali S, Elliot A, Levecke B, Robertson I, et al. Molecular characterization of Blastocystis isolates from zoo animals and their animal-keepers. Vet Parasitol 2010; 169:8-17 Wawrzyniak I, Poirier P, Viscogliosi E, Dionigia M, Texier C, Delbac F, et al. Blastocystis , an unrecognized parasite: an overview of pathogenesis and diagnosis. Ther Adv Infect Dis 2013; 1(5):167–78. Mattiucci S, Crisafi B, Gabrielli S, Paoletti M, Cancrini G. Molecular epidemiology and genetic diversity of Blastocystis infection in humans in Italy. Epidemiol Infect 2016; 144:635-46. Tito RY, Chaffron S, Caenepeel C, Lima-Mendez G, Wang J, Vieira-Silva S, et al. Population-level analysis of Blastocystis subtype prevalence and variation in the human gut microbiota. Gut. 2019; 68(7):1180-1189. Tan TC, Ong SC, Suresh KG. Genetic variability of Blastocystis isolates obtained from cancer and HIV/AIDS patients. Parasitol Res 2009; 105(5):1283–1286. Stark D, Fotedar R, van Hal S, Beebe N, Marriott D, Ellis JT, et al. Prevalence of enteric protozoa in human immunodeficiency virus (HIV)-positive and HIV-negative men who have sex with men from Sydney, Australia. Am J Trop Med Hyg 2007; 76(3):549–552. Andersen LO, Stensvold CR. Blastocystis in Health and Disease: Are We Moving from a Clinical to a Public Health Perspective? J Clin Microbiol 2016; 54:524–528. Elwakil HS, Hewedi IH. Pathogenic potential of Blastocystis hominis in laboratory mice. Parasitol Res 2010; 107(3):685–689. Gisbert JP , McNicholl AG. Questions and answers on the role of faecal calprotectin as a biological marker in inflammatory bowel disease. Dig Liver Dis 2009; 41(1):56-66 Lehmann FS, Burri E, Beglinger C. The role and utility of faecal markers in inflammatory bowel disease. Therap Adv Gastroenterol 2015; 8(1):23–36. Bustinduy AL, Sousa-Figueiredo JC, Adriko M, Betson M, Fenwick A, Kabatereine N, et al. Fecal Occult Blood and Fecal Calprotectin as Point-of-Care Markers of Intestinal Morbidity in Ugandan Children with Schistosoma mansoni PLoS Negl Trop Dis 2013; 7:1–9. Hanevik K, Hausken T, Morken MH, Strand EA, Mørch K, Coll P, et al. Persisting symptoms and duodenal inflammation related to Giardia duodenalis J Infect. 2007; 55(6):524-30. Opintan JA, Newman MJ, Ayeh-Kumi PF, Affrim R, Gepi-Attee R, Sevilleja JE, et al. Pediatric diarrhea in southern Ghana: etiology and association with intestinal inflammation and malnutrition. Am J Trop Med Hyg. 2010; 83(4):936-43. Kohli A, Bushen OY, Pinkerton RC, Houpt E, Newman RD, Sears CL, et al. Giardia duodenalis assemblage, clinical presentation and markers of intestinal inflammation in Brazilian children. Trans R Soc Trop Med Hyg. 2008; 102(7):718-25. Nieves-Ramírez ME, Partida-Rodríguez O, Laforest-Lapointe I, Reynolds LA, Brown EM, Valdez-Salazar A, et al. Asymptomatic Intestinal Colonization with Protist Blastocystis Is Strongly Associated with Distinct Microbiome Ecological Patterns. mSystems 2018; 3(3):1–18. Falasca F, Di Carlo D, De Vito C, Bon I, d'Ettorre G, Fantauzzi A, et al. Evaluation of HIV-DNA and inflammatory markers in HIV-infected individuals with different viral load patterns. BMC Infect Dis. 2017; 17(1):581. Ridley DS, Hawgood BC. 1956. The value of formolether concentration of faecal cysts and ova. J Clin Pathol 1956; 9(1):74-6. Scicluna SM, Tawari B, Clark CG. DNA barcoding of Blastocystis . Protist 2006; 157(1):77–85. Stensvold CR, Suresh GK, Tan KS, Thompson RC, Traub RJ, Viscogliosi E, et al. Terminology for Blastocystis subtypes - a consensus. Trends Parasitol 2007; 23(3): 93–96. Mumolo MG, Bertani L, Ceccarelli L, Laino G, Di Fluri G, Albano E, et al. From bench to bedside: Fecal calprotectin in inflammatory bowel diseases clinical setting. World J Gastroenterol 2018; 24(33):3681-3694 Ronquist F, Huelsenbeck JP. MrBayes 3: Bayesian phylogenetic inference undermixed models. Bioinformatics 2003;19:1572-1574. Wilgenbusch JC, Swofford D. Inferring evolutionary trees with PAUP*. Curr Protoc Bioinformatics 2003; Chapter 6:Unit 6.4. Posada D, Buckley TR. Model selection and model averaging in phylogenetics: advantages of akaike information criterion and bayesian approaches over likelihood ratio tests. Syst Biol. 2004; 53:793-808. Masucci L, Graffeo R, Bani S, Bugli F, Boccia S, Nicolotti N, et al. Intestinal parasites isolated in a large teaching hospital, Italy, 1 May 2006 to 31 December 2008. Euro Surveill. 2006; 16:19891. Brandonisio O, Maggi P, Panaro MA, Lisi S, Andriola A, Acquafredda A, et al. Intestinal protozoa in HIV-infected patients in Apulia, South Italy. Epidemiol Infect 1999; 123:457–462. Sanchez-Aguillon F, Lopez-Escamilla E, Velez-Perez F, Martinez-Flores WA, Rodriguez-Zulueta P, Martinez-Ocaña J, et al. Parasitic infections in a Mexican HIV/AIDS cohort. J Infect Dev Ctries 2013; 7(10):763–766. Piranshahi AR, Tavalla M, Khademvatan S. Genomic analysis of Blastocystis hominis isolates in patients with HIV-positive using locus SSU-rDNA. J Parasit Dis. 2018; 42(1):28-33. Meloni D, Sanciu G, Poirier P. Molecular subtyping of Blastocystis isolates from symptomatic patients in Italy. Parasitol Res 2011; 109(3):613–619. Manichanh C, Eck A, Varela E, Roca J, Clemente JC, González A, et al. Anal gas evacuation and colonic microbiota in patients with flatulence: effect of diet. Gut 2014; 63(3):401-8. Williams B, Mirmonsef P, Boucher CA, Bushman F, Carrington-Lawrence S, Collman RG, et al. A Summary of the First HIV Microbiome Workshop 2015. AIDS Res Hum Retroviruses 2016; 32(10–11):935–941. Vesterbacka J, Rivera J, Noyan K. Richer gut microbiota with distinct metabolic profile in HIV infected Elite Controllers. Sci Rep 2017; 7(1):6269. Ling Z, Jin C, Xie T, Cheng Y, Li L, Wu N. Alterations in the Fecal Microbiota of Patients with HIV-1 Infection: An Observational Study in A Chinese Population. Sci Rep 2016; 6:30673. Noguera-Julian M, Rocafort M, Guillén Y, Rivera J, Casadellà M, Nowak P, et al. Gut Microbiota Linked to Sexual Preference and HIV Infection. EBioMedicine 2016; 5:135-46. Andersen LO, Bonde I, Nielsen HB, Stensvold CR. A retrospective metagenomics approach to studying Blastocystis . FEMS Microbiol Ecol 2015; 91:1–9. Tables Study population N (%) Gender, male 117 (75%) Age, years (mean ± SD) 47.05 ± 12.38 Foreign origin 39 (25%) MSM 50 (32.0%) Domestic animals 74 (47.4%) Travels 67 (42.9%) CD4 count, cells/ml, mean ± SD 655.04 ± 381.56 HIV-RNA (<37 copies/ml) 112 (71.8%) Naïve subjects 23 (14.74%) 2NRTI + NNRTI 35 (26.3%) 2NRTI + IP/r 34 (25.6%) 2NRTI + INI 36 (27.1%) Dual therapy 28 (21%) Table 1. Epidemiological, demographic, immunologic and virological characteristics of the enrolled patients (N=156). A total of N=23 were n aïve and N=133 on ART. Blastocystis -carriers N(%) Blastocystis- free N(%) p-value Gender, male 33 (82.0%) 84 (72.6%) 0.289 Age, years (mean ± SD) 44.71 ± 11.33 47.05 ± 12.37 0.06 Foreign origin 15 (38.46%) 25 (21.36%) 0.138 MSM Domestic animals Travels 19 (48.71%) 18 (46.15%) 20 (51.28%) 31 (26.49%) 56 (47.86%) 47 (40.17%) 0.037* 0.721 0.296 CD4 count, cells/ml, mean ± SD 637.43± 263.32 660.90± 414.35 0.823 CD4 count > 500 cells/ml CD4 count 200– 500 cells/ml CD4 count <200 cells/ml 28 (71.79%) 8 (20.51%) 3 (7.69%) 69 (58.97%) 33 (28.20%) 15 (12.82%) 0.484 HIV-RNA (<37 copies/ml) 32 (82.05%) 80 (68.37%) 0.257 Naïve subjects 2NRTI + NNRTI 2NRTI + IP/r 2NRTI + INI Dual therapy 5 (12.82%) 9 (23.07%) 7 (17.94%) 12 (30.76%) 6 (15.38%) 18 (15.38%) 26 (22.22%) 27 (23.07%) 24 (20.51%) 22 (18.80%) 0.765 Table 2. Comparison of epidemiological, demographic, immunologic and virological characteristics between Blastocystis - carrier (N=39) and Blastocystis -free (N=117) patients. Significant results were marked with *. Symptoms Blastocystis- carriers N(%) Blastocystis -free N(%) p-value Abdominal pain 7 (17.94%) 26 (22.22%) 0.836 Diarrhoea 10 (25.64%) 20 (17.09%) 0.283 F latulence 12 (30.76%) 17 (14.52%) 0.024* Nausea 3 (7.69%) 6 (5.12%) 0.887 Poor appetite 5 (12.82%) 10 (8.54%) 0.180 Weight loss 3 (7.69%) 10 (8.54%) 0.658 Table 3. Gastrointestinal symptoms observed in Blastocystis - carrier (N=39) and Blastocystis -free (N=117) patients. Significant results were marked with *. Cite Share Download PDF Status: Published Journal Publication published 22 Oct, 2019 Read the published version in BMC Infectious Diseases → Version 3 posted Submission checks completed at journal 06 Nov, 2019 Editorial decision: Accept 08 Oct, 2019 Editor assigned by journal 08 Oct, 2019 Editor invited by journal 07 Oct, 2019 You are reading this latest preprint version Show more versions Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3113","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research article","associatedPublications":[],"authors":[{"id":181972,"identity":"37499d5a-764c-4915-9018-44e02a7ac9ca","order_by":1,"name":"Lucia Fontanelli Sulekova","email":"","orcid":"","institution":"Universita degli Studi di Roma La Sapienza","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Lucia","middleName":"Fontanelli","lastName":"Sulekova","suffix":""},{"id":181973,"identity":"3fc91021-dd54-48a2-9e69-4641fae976ca","order_by":2,"name":"Simona Gabrielli","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0002-1596-8036","institution":"Universita degli Studi di Roma La Sapienza Facolta di Farmacia e Medicina","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Simona","middleName":"","lastName":"Gabrielli","suffix":""},{"id":181974,"identity":"49f7d83f-2c24-49a4-ace6-791513c53587","order_by":3,"name":"Federica Furzi","email":"","orcid":"","institution":"Universita degli Studi di Roma 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13:14:24","currentVersionCode":3,"declarations":"","doi":"10.21203/rs.2.12345/v3","doiUrl":"https://doi.org/10.21203/rs.2.12345/v3","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12879-019-4537-7","type":"published","date":"2019-10-22T12:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":116840,"identity":"f3905e10-f6d2-49de-8ce8-0709c58bbd35","added_by":"auto","created_at":"2019-10-09 16:34:01","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":31844,"visible":true,"origin":"","legend":"Bayesian inference (BI) and Neighbour-Joining (NJ) phylogenetic combined tree showing the relationships among Blastocystis-SSU rRNA gene sequences from this study (Bh) and published Blastocystis-subtypes ST1, ST2, ST3, ST4 (GenBank accession numbers indicated in the tree), carried out by using MrBayes3.1 and PAUP4 respectively, as above described. The number of sequences from each isolate (Bh) was reported in the brackets. Bootstrap values and posterior probabilities, respectively, are reported at the nodes; B. lapemi and B. pythoni (accession no. AY590115 and KU146575, respectively) were used as outgroups.","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/a97b588f-f2d4-4cb7-b111-0284a7a07153/v3/1.png"},{"id":13475607,"identity":"a1fc3881-222b-483f-ae99-b6034556f0f9","added_by":"auto","created_at":"2021-09-16 21:25:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":363683,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3113/v3/91fcc958-2ecb-4fdd-87e5-a474538900cd.pdf"}],"financialInterests":"","formattedTitle":"Molecular characterization of Blastocystis subtypes in HIV-positive patients and evaluation of risk factors for colonization","fulltext":[{"header":"Background","content":"\u003cp\u003e\u003cem\u003eBlastocystis\u003c/em\u003e spp. is a common intestinal protist distributed worldwide infecting humans and animals, with a prevalence from 0.5%-30% and 30-76% in industrialized and developing countries, respectively [1].\u0026nbsp; This difference can be explained by poor hygiene practices and consumption of contaminated food or water [2, 3, 4, 5] since the faecal-oral route is considered to be the main mode of transmission of this protist [6]. A remarkable genetic diversity has been revealed among \u003cem\u003eBlastocystis\u003c/em\u003e spp. isolates from humans and animals based on the comparison of the small-subunit rRNA gene (SSU rDNA) sequences [7]\u003cem\u003e.\u003c/em\u003e Currently at least 26 subtypes of \u003cem\u003eBlastocystis\u003c/em\u003e have been described among mammalian and avian isolates [8, 9], nine of them (ST-1 to ST-8 and ST-12) detected in human population and are potentially zoonotic [10]. Because several STs are shared between humans and animals it has been proposed that a proportion of human infections may result from zoonotic transmission. Indeed, a higher risk of \u003cem\u003eBlastocystis\u003c/em\u003e infection was found in people with close animal contact, including zookeepers [11]. Despite several studies reported \u003cem\u003eBlastocystis\u003c/em\u003e implicated in different intestinal diseases, potential pathogenetic factors have been described and its presence is frequently associated with symptoms in humans [12], its pathogenetic role is so far under debated and several variables, as well as the \u003cem\u003eBlastocystis\u003c/em\u003e subtype and load, host\u0026rsquo;s immune status and dysbiosis, could affect the occurrence of the disease [13][14].\u003c/p\u003e\n\u003cp\u003eAlthough the protist was never considered as an opportunistic protozoon, it has also been frequently found in immunocompromised individuals presenting diarrhoea, with prevalence value ranging from 15% to 72.4% [15]. In addition, the sexual practices among men who have sex with men (MSM) have been reported to increase the transmission of \u003cem\u003eBlastocystis\u003c/em\u003e (as for as other enteric organisms) [16]\u003cem\u003e.\u003c/em\u003e Finally, recent data suggest that the protozoon is associated with certain gut microbiota profiles and health indices. Accordingly, a positive correlation between high bacterial richness and the presence of \u003cem\u003eBlastocystis\u003c/em\u003e has been reported [17].\u003c/p\u003e\n\u003cp\u003eNotwithstanding it is commonly accepted that \u003cem\u003eBlastocystis \u003c/em\u003eis a non-invasive organism, its vacuolar form is able to colonize the lamina propria, the submucosa and even the muscle layers leading to inflammation and active colitis in experimentally infected mice [18]. Currently, it is possible to perform a non-invasive evaluation of gut inflammation using biomarkers as the fecal calprotectin (FC), which is considered a surrogate marker of intestinal inflammation with good diagnostic performance for separating organic and functional intestinal disorders [19]\u003cem\u003e. \u003c/em\u003eThe increase of FC is due to faecal excretion of neutrophils and macrophages migrants from the bloodstream into the intestinal lumen, which occurs during the intestinal inflammation. The value of FC as a laboratory marker has been shown in inflammatory bowel disease [20] but its significance in other gastrointestinal diseases remains unclear. Studies evaluating the correlation between intestinal inflammatory markers and parasitic infections are scant [21, 22, 23, 24]\u003cem\u003e. \u003c/em\u003eHowever, a positive correlation between the level of FC and severe infections due to intestinal parasites such as \u003cem\u003eGiardia intestinalis \u003c/em\u003eand \u003cem\u003eSchistosoma mansoni\u003c/em\u003e has been reported [21, 22]. Conversely, lower concentrations of FC have been reported in \u003cem\u003eBlastocystis \u003c/em\u003ecolonized individuals compared to non-colonized subjects [25].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis study aimed to evaluate the prevalence of \u003cem\u003eBlastocystis\u003c/em\u003e subtypes in HIV-positive patients, to evaluate the potential the risk factors for its transmission and the relationship between the presence of the protozoon and gastrointestinal symptoms and, finally, to assess the level of FC in \u003cem\u003eBlastocystis-\u003c/em\u003ecolonized subjects.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eStudy population\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA 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\u003cem\u003e.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eWritten 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).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLaboratory analyses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBlood 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 \u003cem\u003eBlastocystis\u003c/em\u003e-SSU rRNA gene, following PCR protocol and conditions described in Mattiucci et al., 2016 [13]. The sequences obtained were compared to those of \u003cem\u003eBlastocystis\u003c/em\u003e 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].\u003c/p\u003e\n\u003cp\u003eFurthermore, the evaluation of the FC from a sample of subjects was performed using a commercial immunoassay (Calprest, EUROSPITAL, Italy), following the manufacturer\u0026rsquo;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].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhylogenetic analyses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBayesian inference (BI) and Neighbour-Joining (NJ) phylogenetic combined tree, based on the amplified sequences of the \u003cem\u003eBlastocystis\u003c/em\u003e-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. \u003cem\u003eB. lapemi \u003c/em\u003eand \u003cem\u003eB. pythoni \u003c/em\u003e(GenBank accession no. AY590115 and KU146575, respectively\u003cem\u003e) \u003c/em\u003ewere used as outgroups.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eContinuous variables were summarized as mean \u0026plusmn; standard deviation or median \u0026plusmn; interquartile range (IQR) and categorical data as counts and percentages. Comparisons between groups were performed using \u0026chi;\u003csup\u003e2\u003c/sup\u003e test or Fisher\u0026rsquo;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 \u003cem\u003eBlastocystis\u003c/em\u003e colonization. The significance level for all analyses was set at p \u0026lt;0.05. Data were analysed using IBM SPSS, version 21.0 (SPSS Inc, Chicago, USA).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eA total of 156 consecutive HIV-positive patients were enrolled in the study between January 2016 and September 2017. Demographic and clinical data of the study population are described in Table 1. The majority of subjects were males (75%), mean age was 47.05 \u0026plusmn; 12.38 (range 22 to 71 years), 39 participants (25%) were coming from non-EU countries and 50 ones (32%) were MSM (\u003cem\u003emen\u003c/em\u003e\u0026nbsp;who have\u0026nbsp;\u003cem\u003esex\u003c/em\u003e\u0026nbsp;with\u0026nbsp;\u003cem\u003emen\u003c/em\u003e). Moreover 47.4% were owner of pets and 42.9% travelled outside Europe in the last 6 months.\u003c/p\u003e\n\u003cp\u003eTwenty-three patients (14.7%) were na\u0026iuml;ve and 133 (85.3%) were on ART treatment. Thirty-five subjects (26.3%) were on ART based on non-nucleoside reverse-transcriptase inhibitors (NNRTI), 34 (25.6%) on protease inhibitors (PI/r), 36 (27.1%) on integrase inhibitors and 28 (21%) on dual therapy. Three of the enrolled patients were on prophylaxis treatment with sulfamethoxazol-trimethoprim. The mean CD4 cell count was 655.04 \u0026plusmn; 381.56 cells/mm\u003csup\u003e3\u003c/sup\u003e. Most of patients (62.2%) showed a CD4 count higher than 500 cells/ml, 26.3% patients had a CD4 count between 200 and 500 cells/ml and only 11.5% patients showed a CD4 count lower than 200 cells/ml. The median HIV-RNA level of naive patients was 40010 (7093-824500) -copies/mL. Among patients on ART treatment, HIV-RNA was detectable (\u0026gt;37 cp/mL) in 15.8% of subjects (21/133) with a median HIV-RNA level of 116 (67-991) cp/mL.\u003c/p\u003e\n\u003cp\u003eMicroscopic examination revealed the presence of \u003cem\u003eBlastocystis \u003c/em\u003espp. in 34 patients (21.8%) while PCR amplification detected the protist in 39 individuals (25%) and the expected 600 bp fragments of the SSU rRNA gene amplified were successfully sequenced. Sequences obtained showed a high identity (98\u0026ndash;100%) to homologous sequences of \u003cem\u003eBlastocystis\u003c/em\u003e isolates previously reported in GenBank and, according to the consensus terminology [29] and based on the phylogenetic analyses carried out, enabled the clustering of our isolates into 4 distinct \u003cem\u003eBlastocystis\u003c/em\u003e-subtypes (Figure 1).\u003c/p\u003e\n\u003cp\u003eIn detail, ST3 was the most common subtype found in 51.3% of the subjects, followed by ST1 (30.8%), ST4 (10.2%) and ST2 (7.7%). As expected, ST4 was identified only in European participants. In 30.7% of the subjects, \u003cem\u003eBlastocystis\u003c/em\u003e was found in coinfection with other enteric protozoa as \u003cem\u003eEntamoeba coli \u003c/em\u003e(15%)\u003cem\u003e, Endolimax nana \u003c/em\u003e(8%)\u003cem\u003e, Giardia intestinalis \u003c/em\u003e(6.1%) and\u003cem\u003e Iodamoeba butschlii \u003c/em\u003e(1.6%). The detection of such intestinal parasites was more frequent in \u003cem\u003eBlastocystis\u003c/em\u003e-carriers compared to \u003cem\u003eBlastocystis\u003c/em\u003e-free ones (p\u0026lt;0.0001) and in MSM compared to heterosexual subjects (p\u0026lt;0.0001).\u003c/p\u003e\n\u003cp\u003eNeither demographic characteristics such as gender, age and nationality nor other epidemiological risk factors such as travel history or, presence of domestic animals differed significantly between the\u003cem\u003e Blastocystis \u003c/em\u003epositive and \u003cem\u003eBlastocystis\u003c/em\u003e negative patients (Table 2). In addition, no significant differences were found between the two groups regarding CD4+ T-cell counts, HIV-RNA undetectability and type of ART regiment. \u003cem\u003eBlastocystis\u003c/em\u003e positive subjects reported more frequently homosexual behavior practices compared to negative ones (48.71% vs 26.49%, p=0.037).\u0026nbsp; After adjusting for age, the relationship between homosexual behavior practices and the presence of \u003cem\u003eBlastocystis \u003c/em\u003eremained significant (p=0.01).\u003c/p\u003e\n\u003cp\u003eMost of the \u003cem\u003eBlastocystis\u003c/em\u003e positive subjects were symptomatic (53.8%), while 33.6% \u003cem\u003eBlastocystis\u003c/em\u003e-free ones referred gastrointestinal disorders (p=0.029). Among the gastrointestinal symptoms analyzed, flatulence was more frequently observed in \u003cem\u003eBlastocystis\u003c/em\u003e carriers (30.7%) compared to \u003cem\u003eBlastocystis\u003c/em\u003e negative ones (14.5%) (p=0.024) (Table 3).\u003c/p\u003e\n\u003cp\u003eTo determine whether \u003cem\u003eBlastocystis\u003c/em\u003e colonization was associated with intestinal inflammation, we measured FC in a subset of samples divided in \u003cem\u003eBlastocystis\u003c/em\u003e-carrier (mono-infected) (N=23) and \u003cem\u003eBlastocystis-\u003c/em\u003efree (N=36) subjects rom the all 59 fecal samples (\u003cem\u003eBlastocystis\u003c/em\u003e-carrier and \u003cem\u003eBlastocystis-\u003c/em\u003efree), 83% had a value in the standard range (lower than 50 mg/kg) and 11 (18.6%) had an increased value (median 115.7; 95% CI=71.82-303.78). However, no significant differences in calprotectin values was found between \u003cem\u003eBlastocystis-\u003c/em\u003ecarriers (median 15, 95% CI= 18.93-148.53) and non-carriers (median 13.35, 13.93-31.74) (p=0.31).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eStudies on the prevalence of \u003cem\u003eBlastocystis\u003c/em\u003e spp. in HIV-positive subjects are relatively scant. In Italy the protist has so far attracted little attention, where few epidemiological surveys have been published, demonstrating a prevalence rate of about 7% and the occurrence of seven STs (ST1, ST2, ST3, ST4, ST6, ST7, ST8) in HIV-negative people [34][13]. \u003c/p\u003e\n\u003cp\u003eThe only study conducted on Italian HIV-positive patients date back to 1999, through the microscopic method, showed a \u003cem\u003eBlastocystis\u003c/em\u003e prevalence of 10.3% [35]. The current study is, to our knowledge, the first to assess the prevalence of \u003cem\u003eBlastocystis\u003c/em\u003e in HIV-positive patients through molecular methods in Europe and reported a prevalence of about 25%, in line with that reported in previous studies conducted in Mexico (30%)[36], Malaysia (19.8%) [15] and Iran (19%) [37]. The molecular characterization of \u003cem\u003eBlastocystis\u003c/em\u003e positive isolates evidenced the circulation of 4 subtypes (ST1, ST2, ST3, ST4) already described in Italy, as previously mentioned [13, 38], being ST3 the most common one with a prevalence of 51%. In our cohort of HIV-positive patients, the presence of \u003cem\u003eBlastocystis\u003c/em\u003e resulted no correlated to viro-immunological factors (p\u0026gt;0.05) confirming therefore the non-opportunistic behavior of the protist. No demographic characteristic or epidemiological drivers (presence of domestic animals, dietary habits, travels abroad) were found to be associated with \u003cem\u003eBlastocystis \u003c/em\u003ecolonization. A higher prevalence of \u003cem\u003eBlastocystis\u003c/em\u003e was reported in MSM compared to heterosexuals as well as of the other intestinal parasites, underlying the faecal-oral contact as the main route of transmission in this group of subjects and the sexual practice and lifestyle linked to the presence of the protist more than the immunological status [16].\u003c/p\u003e\n\u003cp\u003eDespite the unresolved controversy over its pathogenicity, \u003cem\u003eBlastocystis\u003c/em\u003e was also been frequently found in immunocompromised individuals presenting diarrhea [15]. \u0026nbsp;In our study, among gastrointestinal symptoms, a positive association was found only for the flatulence. This socially disabling symptom is determined by two main factors: the diet, particularly the quantity of fermentable residues, and the composition and metabolic activity of colonic microbiota [39]. Since we evidenced any differences in the diet between \u003cem\u003eBlastocystis\u003c/em\u003e-carriers and \u003cem\u003eBlastocystis\u003c/em\u003e-free subjects, we hypothesized that this symptom was linked to the presence of the protist and the gut microbiota composition. Several Authors evaluated the intestinal microbiome in HIV-positive subjects, with somewhat inconsistent or controversial results [40]. This may be because of small sample sizes, lack of appropriate controls or regional differences in dietary and environmental factors. Regardless, many authors evidenced a change in the Bacteroides:Prevotella ratio, which they suggested to be linked to the antiretroviral therapy [41, 42] or to the sexual practice and lifestyle [43]. Similarly, several studies reported \u003cem\u003eBlastocystis\u003c/em\u003e colonization associated with higher bacterial richness and Prevotella enterotype [44]. Therefore, investigations on the gut microbiota from HIV-positive subjects \u003cem\u003eBlastocystis\u003c/em\u003e-carriers are needed to complete and to better understand the preliminary results obtained in this study.\u003c/p\u003e\n\u003cp\u003eConcerning FC, we found the median of both \u003cem\u003eBlastocystis\u003c/em\u003e-carrier and free subjects within the normal range, supporting the non-pathogenic role of \u003cem\u003eBlastocystis\u003c/em\u003e in inducing intestinal inflammation [25].\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eDespite some critical points of this study concerning the lack of data about the faecal microbiota from enrolled subject\u003cem\u003e, \u003c/em\u003eto our knowledge this is the first large survey to molecular characterize \u003cem\u003eBlastocystis\u003c/em\u003e subtypes in HIV-positive patients in Europe. Our results suggest that \u003cem\u003eBlastocystis \u003c/em\u003eis quite common in such patients (25%) and homosexual behavior resulted a risk factor for its transmission and confirm the presence of the protist not associated with pathological gut inflammation. The role of \u003cem\u003eBlastocystis\u003c/em\u003e as intestinal pathogen remains unclear as the only symptom associated with its presence was flatulence. This result is intriguing since flatulence could be linked to the gut microbial communities, therefore the impact of the protist on the faecal microbiota and its possible role to maintain the to gut homeostasis could be interesting prospects for further studies.\u003c/p\u003e"},{"header":"List of Abbreviations","content":"\u003cp\u003eART: antiretroviral therapy; CI: Confidence interval; FC: fecal calprotectin; HIV: Human Immunodeficiency Virus; MSM: men who have sex with men; NNRTI: non-nucleoside reverse-transcriptase inhibitors.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study protocol was approved by the ethics committees of Umberto I Academic Hospital (licence n. 4836) in accordance with the ethical guidelines of the Declaration of Helsinki. Written informed consents were obtained from the patients after declaring the objectives of the present study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot Applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe dataset of this article will not be available publicly, to ensure the patient\u0026rsquo;s privacy, but are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was financed by Sapienza University in the frame of the \u0026ldquo;Progetti di Ricerca Universit\u0026agrave; Sapienza; years 2016, 2017 and 2018, grant numbers RM11615506189BE0, RM11715C81DDD2F3, RM11816432E08126\u0026rdquo;. The content is solely the responsibility of the authors and the funder was not involved in the design of the study, data collection, analysis and interpretation of the data and in writing the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLFS and SG designed the study with input from SM and GT. LFS, SG and FF conducted the data analysis, interpretation, and manuscript preparation. FF and GLM performed the laboratory analyses and molecular identification and contributed to the manuscript preparation. EB, MDA, GI, CF and MM enrolled the patients, performed the sample collection and contributed to the data analysis, data interpretation, and manuscript preparation. SG, SM and GT interpreted the results and revised the manuscript. All authors critically reviewed and approved the final version of this paper for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors are grateful to the Eurospital S.p.A. for providing the Calprest immunoassay.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAlfellani MA, Stensvold CR, Vidal-Lapiedra A, Onuoha ES, Fagbenro-Beyioku AF, Clark CG. Variable geographic distribution of \u003cem\u003eBlastocystis\u003c/em\u003e subtypes and its potential implications. Acta Trop\u0026nbsp; 2013; 126:11\u0026ndash;18.\u003c/li\u003e\n\u003cli\u003eLeelayoova S, Siripattanapipong S, Thathaisong U, Naaglor T, Taamasri P, Piyaraj P, et al. Drinking water: A possible source of \u003cem\u003eBlastocystis\u003c/em\u003e subtype 1 infection in schoolchildren of a rural community in central Thailand. Am J Trop Med Hyg 2008; 79(3):401\u0026ndash;406.\u003c/li\u003e\n\u003cli\u003eBaldursson S, Karanis P. Waterborne transmission of protozoan parasites: review of worldwide outbreaks- an update 2004-2010. Water Res 2011; 45:6603-14.\u003c/li\u003e\n\u003cli\u003eLee LI, Chye TT, Karmacharya BM, Govind SK. \u003cem\u003eBlastocystis\u003c/em\u003e: waterborne zoonotic organism, a possibility? Parasit Vectors 2012; 28:5-130.\u003c/li\u003e\n\u003cli\u003eNagel R, Cuttell L, Stensvold CR, Mills PC, Bielefeldt-Ohmann H, Traub RJ. \u003cem\u003eBlastocystis\u003c/em\u003e subtypes in symptomatic and asymptomatic family members and pets and response to therapy. Intern Med J 2012; 42(11):1187\u0026ndash;1195.\u003c/li\u003e\n\u003cli\u003eYoshikawa H, Yoshida K, Nakajima A, Yamanari K, Iwatani S, Kimata I. Fecal-oral transmission of the cyst form of \u003cem\u003eBlastocystis\u003c/em\u003e hominis in rats. Parasitol Res 2004; 94(6):391\u0026ndash;396.\u003c/li\u003e\n\u003cli\u003eYoshikawa H, Wu Z, Nagano I, Takahashi Y. Molecular comparative studies among \u003cem\u003eBlastocystis \u003c/em\u003eisolates obtained from humans and animals. J Parasitol. 2003;89(3):585-94.\u003c/li\u003e\n\u003cli\u003eZhao GH, Hu XF, Liu TL, Hu RS, Yu ZQ, Yang WB, et al. Molecular characterization of Blastocystis sp. in captive wild animals in Qinling Mountains. Parasitol Res. 2017; 116(8):2327-2333.\u003c/li\u003e\n\u003cli\u003eMaloney JG, Lombard JE, Urie NJ, Shivley CB, Santin M. Zoonotic and genetically diverse subtypes of Blastocystis in US pre-weaned dairy heifer calves. Parasitol Res. 2019; 118(2):575-582.\u003c/li\u003e\n\u003cli\u003eStensvold CR, Clark CG. Current status of \u003cem\u003eBlastocystis\u003c/em\u003e: A personal view. \u003cem\u003eParasitol Int \u003c/em\u003e2016; 65(6):763\u0026ndash;771.\u003c/li\u003e\n\u003cli\u003eParkar U, Traub RJ, Vitali S, Elliot A, Levecke B, Robertson I, et al. Molecular characterization of \u003cem\u003eBlastocystis\u003c/em\u003e isolates from zoo animals and their animal-keepers. Vet Parasitol 2010; 169:8-17\u003c/li\u003e\n\u003cli\u003eWawrzyniak I, Poirier P, Viscogliosi E, Dionigia M, Texier C, Delbac F, et al. \u003cem\u003eBlastocystis\u003c/em\u003e, an unrecognized parasite: an overview of pathogenesis and diagnosis. Ther Adv Infect Dis 2013; 1(5):167\u0026ndash;78.\u003c/li\u003e\n\u003cli\u003eMattiucci S, Crisafi B, Gabrielli S, Paoletti M, Cancrini G. Molecular epidemiology and genetic diversity of \u003cem\u003eBlastocystis\u003c/em\u003e infection in humans in Italy. Epidemiol Infect 2016; 144:635-46.\u003c/li\u003e\n\u003cli\u003eTito RY, Chaffron S, Caenepeel C, Lima-Mendez G, Wang J, Vieira-Silva S, et al. Population-level analysis of Blastocystis subtype prevalence and variation in the human gut microbiota. Gut. 2019; 68(7):1180-1189.\u003c/li\u003e\n\u003cli\u003eTan TC, Ong SC, Suresh KG. Genetic variability of \u003cem\u003eBlastocystis\u003c/em\u003e isolates obtained from cancer and HIV/AIDS patients. Parasitol Res 2009; 105(5):1283\u0026ndash;1286.\u003c/li\u003e\n\u003cli\u003eStark D, Fotedar R, van Hal S, Beebe N, Marriott D, Ellis JT, et al. Prevalence of enteric protozoa in human immunodeficiency virus (HIV)-positive and HIV-negative men who have sex with men from Sydney, Australia. Am J Trop Med Hyg 2007; 76(3):549\u0026ndash;552.\u003c/li\u003e\n\u003cli\u003eAndersen LO, Stensvold CR. \u003cem\u003eBlastocystis\u003c/em\u003e in Health and Disease: Are We Moving from a Clinical to a Public Health Perspective? J Clin Microbiol 2016; 54:524\u0026ndash;528.\u003c/li\u003e\n\u003cli\u003eElwakil HS, Hewedi IH. Pathogenic potential of \u003cem\u003eBlastocystis\u003c/em\u003e hominis in laboratory mice. Parasitol Res 2010; 107(3):685\u0026ndash;689.\u003c/li\u003e\n\u003cli\u003eGisbert JP , McNicholl AG. Questions and answers on the role of faecal calprotectin as a biological marker in inflammatory bowel disease. Dig Liver Dis 2009; 41(1):56-66\u003c/li\u003e\n\u003cli\u003eLehmann FS, Burri E, Beglinger C. The role and utility of faecal markers in inflammatory bowel disease. Therap Adv Gastroenterol 2015; 8(1):23\u0026ndash;36.\u003c/li\u003e\n\u003cli\u003eBustinduy AL, Sousa-Figueiredo JC, Adriko M, Betson M, Fenwick A, Kabatereine N, et al. Fecal Occult Blood and Fecal Calprotectin as Point-of-Care Markers of Intestinal Morbidity in Ugandan Children with \u003cem\u003eSchistosoma mansoni\u003c/em\u003e PLoS Negl Trop Dis 2013; 7:1\u0026ndash;9.\u003c/li\u003e\n\u003cli\u003eHanevik K, Hausken T, Morken MH, Strand EA, M\u0026oslash;rch K, Coll P, et al. Persisting symptoms and duodenal inflammation related to \u003cem\u003eGiardia duodenalis \u003c/em\u003e J Infect. 2007; 55(6):524-30.\u003c/li\u003e\n\u003cli\u003eOpintan JA, Newman MJ, Ayeh-Kumi PF, Affrim R, Gepi-Attee R, Sevilleja JE, et al. Pediatric diarrhea in southern Ghana: etiology and association with intestinal inflammation and malnutrition. Am J Trop Med Hyg. 2010; 83(4):936-43.\u003c/li\u003e\n\u003cli\u003eKohli A, Bushen OY, Pinkerton RC, Houpt E, Newman RD, Sears CL, et al. Giardia duodenalis assemblage, clinical presentation and markers of intestinal inflammation in Brazilian children. Trans R Soc Trop Med Hyg. 2008; 102(7):718-25.\u003c/li\u003e\n\u003cli\u003eNieves-Ram\u0026iacute;rez ME, Partida-Rodr\u0026iacute;guez O, Laforest-Lapointe I, Reynolds LA, Brown EM, Valdez-Salazar A, et al. Asymptomatic Intestinal Colonization with Protist Blastocystis Is Strongly Associated with Distinct Microbiome Ecological Patterns. mSystems 2018; 3(3):1\u0026ndash;18.\u003c/li\u003e\n\u003cli\u003eFalasca F, Di Carlo D, De Vito C, Bon I, d'Ettorre G, Fantauzzi A, et al. Evaluation of HIV-DNA and inflammatory markers in HIV-infected individuals with different viral load patterns. BMC Infect Dis. 2017; 17(1):581.\u003c/li\u003e\n\u003cli\u003eRidley DS, Hawgood BC. 1956. The value of formolether concentration of faecal cysts and ova. J Clin Pathol 1956; 9(1):74-6.\u003c/li\u003e\n\u003cli\u003eScicluna SM, Tawari B, Clark CG. DNA barcoding of \u003cem\u003eBlastocystis\u003c/em\u003e. Protist 2006; 157(1):77\u0026ndash;85.\u003c/li\u003e\n\u003cli\u003eStensvold CR, Suresh GK, Tan KS, Thompson RC, Traub RJ, Viscogliosi E, et al. Terminology for \u003cem\u003eBlastocystis\u003c/em\u003e subtypes - a consensus. Trends Parasitol 2007; 23(3): 93\u0026ndash;96.\u003c/li\u003e\n\u003cli\u003eMumolo MG, Bertani L, Ceccarelli L, Laino G, Di Fluri G, Albano E, et al. From bench to bedside: Fecal calprotectin in inflammatory bowel diseases clinical setting. World J Gastroenterol 2018; 24(33):3681-3694\u003c/li\u003e\n\u003cli\u003eRonquist F, Huelsenbeck JP. MrBayes 3: Bayesian phylogenetic inference undermixed models. Bioinformatics 2003;19:1572-1574.\u003c/li\u003e\n\u003cli\u003eWilgenbusch JC, Swofford D. Inferring evolutionary trees with PAUP*. Curr Protoc Bioinformatics 2003; Chapter 6:Unit 6.4.\u003c/li\u003e\n\u003cli\u003ePosada D, Buckley TR. Model selection and model averaging in phylogenetics: advantages of akaike information criterion and bayesian approaches over likelihood ratio tests. Syst Biol. 2004; 53:793-808.\u003c/li\u003e\n\u003cli\u003eMasucci L, Graffeo R, Bani S, Bugli F, Boccia S, Nicolotti N, et al. Intestinal parasites isolated in a large teaching hospital, Italy, 1 May 2006 to 31 December 2008. Euro Surveill. 2006; 16:19891.\u003c/li\u003e\n\u003cli\u003eBrandonisio O, Maggi P, Panaro MA, Lisi S, Andriola A, Acquafredda A, et al. Intestinal protozoa in HIV-infected patients in Apulia, South Italy. Epidemiol Infect 1999; 123:457\u0026ndash;462.\u003c/li\u003e\n\u003cli\u003eSanchez-Aguillon F, Lopez-Escamilla E, Velez-Perez F, Martinez-Flores WA, Rodriguez-Zulueta P, Martinez-Oca\u0026ntilde;a J, et al. Parasitic infections in a Mexican HIV/AIDS cohort. J Infect Dev Ctries 2013; 7(10):763\u0026ndash;766.\u003c/li\u003e\n\u003cli\u003ePiranshahi AR, Tavalla M, Khademvatan S. Genomic analysis of \u003cem\u003eBlastocystis hominis\u003c/em\u003e isolates in patients with HIV-positive using locus SSU-rDNA. J Parasit Dis. 2018; 42(1):28-33.\u003c/li\u003e\n\u003cli\u003eMeloni D, Sanciu G, Poirier P. Molecular subtyping of \u003cem\u003eBlastocystis\u003c/em\u003e isolates from symptomatic patients in Italy. Parasitol Res 2011; 109(3):613\u0026ndash;619.\u003c/li\u003e\n\u003cli\u003eManichanh C, Eck A, Varela E, Roca J, Clemente JC, Gonz\u0026aacute;lez A, et al. Anal gas evacuation and colonic microbiota in patients with flatulence: effect of diet. Gut 2014; 63(3):401-8.\u003c/li\u003e\n\u003cli\u003eWilliams B, Mirmonsef P, Boucher CA, Bushman F, Carrington-Lawrence S, Collman RG, et al. A Summary of the First HIV Microbiome Workshop 2015. AIDS Res Hum Retroviruses 2016; 32(10\u0026ndash;11):935\u0026ndash;941.\u003c/li\u003e\n\u003cli\u003eVesterbacka J, Rivera J, Noyan K. Richer gut microbiota with distinct metabolic profile in HIV infected Elite Controllers. Sci Rep 2017; 7(1):6269.\u003c/li\u003e\n\u003cli\u003eLing Z, Jin C, Xie T, Cheng Y, Li L, Wu N. Alterations in the Fecal Microbiota of Patients with HIV-1 Infection: An Observational Study in A Chinese Population. Sci Rep 2016; 6:30673.\u003c/li\u003e\n\u003cli\u003eNoguera-Julian M, Rocafort M, Guill\u0026eacute;n Y, Rivera J, Casadell\u0026agrave; M, Nowak P, et al. Gut Microbiota Linked to Sexual Preference and HIV Infection. EBioMedicine 2016; 5:135-46.\u003c/li\u003e\n\u003cli\u003eAndersen LO, Bonde I, Nielsen HB, Stensvold CR. A retrospective metagenomics approach to studying \u003cem\u003eBlastocystis\u003c/em\u003e. FEMS Microbiol Ecol 2015; 91:1\u0026ndash;9.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003ctable style=\"border-collapse: collapse; border: none;\"\u003e\n\u003ctbody\u003e\n\u003ctr style=\"height: 1.0pt;\"\u003e\n\u003ctd style=\"width: 155.7pt; border-top: windowtext; border-left: #BFBFBF; border-bottom: windowtext; border-right: #BFBFBF; border-style: solid; border-width: 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 1.0pt;\" width=\"208\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; line-height: 200%;\"\u003e\u003cstrong\u003e\u003cspan style=\"color: #212121;\"\u003eStudy population\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 85.05pt; border-top: solid windowtext 1.0pt; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid #BFBFBF 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 1.0pt;\" width=\"113\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; line-height: 200%;\"\u003e\u003cstrong\u003e\u003cspan style=\"color: #212121;\"\u003eN (%)\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 1.0pt;\"\u003e\n\u003ctd style=\"width: 155.7pt; 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SD)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 85.05pt; border-top: none; border-left: none; border-bottom: solid #BFBFBF 1.0pt; border-right: solid #BFBFBF 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 1.0pt;\" width=\"113\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; line-height: 200%;\"\u003e\u003cspan style=\"color: #212121;\"\u003e47.05 \u0026plusmn; 12.38\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 1.0pt;\"\u003e\n\u003ctd style=\"width: 155.7pt; border: solid #BFBFBF 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt; height: 1.0pt;\" width=\"208\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; line-height: 200%;\"\u003e\u003cspan style=\"color: #212121;\"\u003eForeign origin\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 85.05pt; border-top: none; border-left: none; border-bottom: solid #BFBFBF 1.0pt; border-right: solid #BFBFBF 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 1.0pt;\" width=\"113\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; line-height: 200%;\"\u003e\u003cspan style=\"color: #212121;\"\u003e39 (25%)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 1.0pt;\"\u003e\n\u003ctd style=\"width: 155.7pt; border: solid #BFBFBF 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt; height: 1.0pt;\" width=\"208\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; line-height: 200%;\"\u003e\u003cspan style=\"color: #212121;\"\u003eMSM\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 85.05pt; border-top: none; border-left: none; border-bottom: solid #BFBFBF 1.0pt; border-right: solid #BFBFBF 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 1.0pt;\" width=\"113\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; line-height: 200%;\"\u003e\u003cspan style=\"color: #212121;\"\u003e50 (32.0%)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 1.0pt;\"\u003e\n\u003ctd style=\"width: 155.7pt; border: solid #BFBFBF 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt; height: 1.0pt;\" width=\"208\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; line-height: 200%;\"\u003e\u003cspan style=\"color: #212121;\"\u003eDomestic animals\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 85.05pt; border-top: none; border-left: none; border-bottom: solid #BFBFBF 1.0pt; border-right: solid #BFBFBF 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 1.0pt;\" width=\"113\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; line-height: 200%;\"\u003e\u003cspan style=\"color: #212121;\"\u003e74 (47.4%)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 1.0pt;\"\u003e\n\u003ctd style=\"width: 155.7pt; border: solid #BFBFBF 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt; height: 1.0pt;\" width=\"208\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; line-height: 200%;\"\u003e\u003cspan style=\"color: #212121;\"\u003eTravels\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 85.05pt; border-top: none; border-left: none; border-bottom: solid #BFBFBF 1.0pt; border-right: solid #BFBFBF 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 1.0pt;\" width=\"113\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; line-height: 200%;\"\u003e\u003cspan style=\"color: #212121;\"\u003e67\u003c/span\u003e\u003cspan style=\"color: #212121;\"\u003e (42.9%)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 1.0pt;\"\u003e\n\u003ctd style=\"width: 155.7pt; border: solid #BFBFBF 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt; height: 1.0pt;\" width=\"208\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; line-height: 200%;\"\u003e\u003cspan style=\"color: #212121;\"\u003eCD4 count, cells/ml, mean \u0026plusmn; SD\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 85.05pt; border-top: none; border-left: none; border-bottom: solid #BFBFBF 1.0pt; border-right: solid #BFBFBF 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 1.0pt;\" width=\"113\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; line-height: 200%;\"\u003e\u003cspan style=\"color: #212121;\"\u003e655.04 \u0026plusmn; 381.56\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 1.0pt;\"\u003e\n\u003ctd style=\"width: 155.7pt; border: solid #BFBFBF 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt; height: 1.0pt;\" width=\"208\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; line-height: 200%;\"\u003e\u003cspan style=\"color: #212121;\"\u003eHIV-RNA (\u0026lt;37 copies/ml)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 85.05pt; border-top: none; border-left: none; border-bottom: solid #BFBFBF 1.0pt; border-right: solid #BFBFBF 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 1.0pt;\" width=\"113\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; line-height: 200%;\"\u003e\u003cspan style=\"color: #212121;\"\u003e112 (71.8%)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 1.0pt;\"\u003e\n\u003ctd style=\"width: 155.7pt; border: solid #BFBFBF 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt; height: 1.0pt;\" width=\"208\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; line-height: 200%;\"\u003e\u003cspan style=\"color: #212121;\"\u003eNa\u0026iuml;ve subjects\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 85.05pt; border-top: none; border-left: none; border-bottom: solid #BFBFBF 1.0pt; border-right: solid #BFBFBF 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 1.0pt;\" width=\"113\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; line-height: 200%;\"\u003e\u003cspan style=\"color: #212121;\"\u003e23 (14.74%)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 1.0pt;\"\u003e\n\u003ctd style=\"width: 155.7pt; border: solid #BFBFBF 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt; height: 1.0pt;\" width=\"208\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; line-height: 200%;\"\u003e\u003cspan style=\"color: #212121;\"\u003e2NRTI + NNRTI\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 85.05pt; border-top: none; border-left: none; border-bottom: solid #BFBFBF 1.0pt; border-right: solid #BFBFBF 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 1.0pt;\" width=\"113\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; line-height: 200%;\"\u003e\u003cspan style=\"color: #212121;\"\u003e35 (26.3%)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 1.0pt;\"\u003e\n\u003ctd style=\"width: 155.7pt; border: solid #BFBFBF 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt; height: 1.0pt;\" width=\"208\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; line-height: 200%;\"\u003e\u003cspan style=\"color: #212121;\"\u003e2NRTI + IP/r\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 85.05pt; border-top: none; border-left: none; border-bottom: solid #BFBFBF 1.0pt; border-right: solid #BFBFBF 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 1.0pt;\" width=\"113\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; line-height: 200%;\"\u003e\u003cspan style=\"color: #212121;\"\u003e34 (25.6%)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 1.0pt;\"\u003e\n\u003ctd style=\"width: 155.7pt; border: solid #BFBFBF 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt; height: 1.0pt;\" width=\"208\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; line-height: 200%;\"\u003e\u003cspan style=\"color: #212121;\"\u003e2NRTI + INI\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 85.05pt; border-top: none; border-left: none; border-bottom: solid #BFBFBF 1.0pt; border-right: solid #BFBFBF 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 1.0pt;\" width=\"113\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; line-height: 200%;\"\u003e\u003cspan style=\"color: #212121;\"\u003e36 (27.1%)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 1.0pt;\"\u003e\n\u003ctd style=\"width: 155.7pt; border: solid #BFBFBF 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt; height: 1.0pt;\" width=\"208\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; line-height: 200%;\"\u003e\u003cspan style=\"color: #212121;\"\u003eDual therapy\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 85.05pt; border-top: none; border-left: none; border-bottom: solid #BFBFBF 1.0pt; border-right: solid #BFBFBF 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 1.0pt;\" width=\"113\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; line-height: 200%;\"\u003e\u003cspan style=\"color: #212121;\"\u003e28 (21%)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eTable 1. Epidemiological, demographic, immunologic and virological characteristics of the enrolled patients (N=156). A total of N=23 were \u003cspan style=\"color: #212121;\"\u003en\u003c/span\u003e\u003cspan style=\"color: #212121;\"\u003ea\u0026iuml;ve and\u003c/span\u003e N=133 on ART.\u003c/p\u003e\u003c/br\u003e\u003c/br\u003e\n\u003ctable style=\"border-collapse: collapse; border: none;\"\u003e\n\u003ctbody\u003e\n\u003ctr style=\"height: 30.9pt;\"\u003e\n\u003ctd style=\"width: 165.3pt; border-top: windowtext; border-left: #BFBFBF; border-bottom: windowtext; border-right: #BFBFBF; border-style: solid; border-width: 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 30.9pt;\" width=\"220\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal;\"\u003e\u003cspan style=\"color: #212121;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 118.9pt; border-top: solid windowtext 1.0pt; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid #BFBFBF 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 30.9pt;\" width=\"159\"\u003e\n\u003cp style=\"margin-bottom: 10.0pt; text-align: center; line-height: normal;\"\u003e\u003cstrong\u003e\u003cem\u003eBlastocystis\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e-carriers\u003c/strong\u003e\u003c/p\u003e\n\u003cp style=\"margin-bottom: 10.0pt; text-align: center; line-height: normal;\"\u003e\u003cstrong\u003eN(%)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 105.2pt; border-top: solid windowtext 1.0pt; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid #BFBFBF 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 30.9pt;\" width=\"140\"\u003e\n\u003cp style=\"margin-bottom: 10.0pt; text-align: center; line-height: normal;\"\u003e\u003cstrong\u003e\u003cem\u003eBlastocystis-\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003efree\u003c/strong\u003e\u003c/p\u003e\n\u003cp style=\"margin-bottom: 10.0pt; text-align: center; line-height: normal;\"\u003e\u003cstrong\u003eN(%)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 92.0pt; border-top: solid windowtext 1.0pt; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid #BFBFBF 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 30.9pt;\" width=\"123\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: normal;\"\u003e\u003cstrong\u003ep-value\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 165.3pt; border: solid #BFBFBF 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"220\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; line-height: 200%;\"\u003e\u003cspan style=\"color: black;\"\u003eGender, male\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; \u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 118.9pt; border-top: none; border-left: none; border-bottom: solid #BFBFBF 1.0pt; border-right: solid #BFBFBF 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"159\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e33 (82.0%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 105.2pt; border-top: none; border-left: none; border-bottom: solid #BFBFBF 1.0pt; border-right: solid #BFBFBF 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"140\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e84 (72.6%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 92.0pt; border-top: none; border-left: none; border-bottom: solid #BFBFBF 1.0pt; border-right: solid #BFBFBF 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"123\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e0.289\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 165.3pt; border: solid #BFBFBF 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"220\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; line-height: 200%;\"\u003eAge, years (mean \u0026plusmn; SD)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 118.9pt; border-top: none; border-left: none; border-bottom: solid #BFBFBF 1.0pt; border-right: solid #BFBFBF 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"159\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e44.71 \u0026plusmn; 11.33\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 105.2pt; border-top: none; border-left: none; border-bottom: solid #BFBFBF 1.0pt; border-right: solid #BFBFBF 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"140\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e47.05 \u0026plusmn; 12.37\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 92.0pt; border-top: none; border-left: none; border-bottom: solid #BFBFBF 1.0pt; border-right: solid #BFBFBF 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"123\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e0.06\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 165.3pt; border: solid #BFBFBF 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"220\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; line-height: 200%;\"\u003eForeign origin\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 118.9pt; border-top: none; border-left: none; border-bottom: solid #BFBFBF 1.0pt; border-right: solid #BFBFBF 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"159\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e15 (38.46%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 105.2pt; border-top: none; border-left: none; border-bottom: solid #BFBFBF 1.0pt; border-right: solid #BFBFBF 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"140\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e25 (21.36%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 92.0pt; border-top: none; border-left: none; border-bottom: solid #BFBFBF 1.0pt; border-right: solid #BFBFBF 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"123\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e0.138\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 165.3pt; border: solid #BFBFBF 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"220\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; line-height: 200%;\"\u003e\u003cspan style=\"color: black;\"\u003eMSM\u0026nbsp;\u0026nbsp;\u0026nbsp; \u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"margin-bottom: .0001pt; line-height: 200%;\"\u003e\u003cspan style=\"color: black;\"\u003eDomestic animals \u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; \u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"margin-bottom: .0001pt; line-height: 200%;\"\u003e\u003cspan style=\"color: black;\"\u003eTravels \u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 118.9pt; border-top: none; border-left: none; border-bottom: solid #BFBFBF 1.0pt; border-right: solid #BFBFBF 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"159\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e19 (48.71%)\u003c/p\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e18 (46.15%)\u003c/p\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e20 (51.28%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 105.2pt; border-top: none; border-left: none; border-bottom: solid #BFBFBF 1.0pt; border-right: solid #BFBFBF 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"140\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e31 (26.49%)\u003c/p\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e56 (47.86%)\u003c/p\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e47 (40.17%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 92.0pt; border-top: none; border-left: none; border-bottom: solid #BFBFBF 1.0pt; border-right: solid #BFBFBF 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"123\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e0.037*\u003c/p\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e0.721\u003c/p\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e0.296\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 165.3pt; border: solid #BFBFBF 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"220\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; line-height: 200%;\"\u003eCD4 count, cells/ml, mean \u0026plusmn; SD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 118.9pt; border-top: none; border-left: none; border-bottom: solid #BFBFBF 1.0pt; border-right: solid #BFBFBF 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"159\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u003cspan style=\"color: #212121;\"\u003e637.43\u0026plusmn; 263.32\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 105.2pt; border-top: none; border-left: none; border-bottom: solid #BFBFBF 1.0pt; border-right: solid #BFBFBF 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"140\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u003cspan style=\"color: #212121;\"\u003e660.90\u0026plusmn; 414.35\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 92.0pt; border-top: none; border-left: none; border-bottom: solid #BFBFBF 1.0pt; border-right: solid #BFBFBF 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"123\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e0.823\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 165.3pt; border: solid #BFBFBF 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"220\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; line-height: 200%;\"\u003eCD4 count \u0026gt; 500 cells/ml\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp style=\"margin-bottom: .0001pt; line-height: 200%;\"\u003eCD4 count 200\u0026ndash; 500 cells/ml\u0026nbsp;\u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp style=\"margin-bottom: .0001pt; line-height: 200%;\"\u003eCD4 count \u0026lt;200 cells/ml\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 118.9pt; border-top: none; border-left: none; border-bottom: solid #BFBFBF 1.0pt; border-right: solid #BFBFBF 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"159\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e28 (71.79%)\u003c/p\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e8 (20.51%)\u003c/p\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e3 (7.69%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 105.2pt; border-top: none; border-left: none; border-bottom: solid #BFBFBF 1.0pt; border-right: solid #BFBFBF 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"140\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e69 (58.97%)\u003c/p\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e33 (28.20%)\u003c/p\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e15 (12.82%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 92.0pt; border-top: none; border-left: none; border-bottom: solid #BFBFBF 1.0pt; border-right: solid #BFBFBF 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"123\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e0.484\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 165.3pt; border: solid #BFBFBF 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"220\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; line-height: 200%;\"\u003eHIV-RNA (\u0026lt;37 copies/ml)\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 118.9pt; border-top: none; border-left: none; border-bottom: solid #BFBFBF 1.0pt; border-right: solid #BFBFBF 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"159\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e32 (82.05%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 105.2pt; border-top: none; border-left: none; border-bottom: solid #BFBFBF 1.0pt; border-right: solid #BFBFBF 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"140\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e80 (68.37%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 92.0pt; border-top: none; border-left: none; border-bottom: solid #BFBFBF 1.0pt; border-right: solid #BFBFBF 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"123\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e0.257\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 165.3pt; border-top: none; border-left: solid #BFBFBF 1.0pt; border-bottom: solid windowtext 1.0pt; border-right: solid #BFBFBF 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"220\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; line-height: 200%;\"\u003eNa\u0026iuml;ve subjects\u0026nbsp;\u003c/p\u003e\n\u003cp style=\"margin-bottom: .0001pt; line-height: 200%;\"\u003e2NRTI + NNRTI\u003c/p\u003e\n\u003cp style=\"margin-bottom: .0001pt; line-height: 200%;\"\u003e2NRTI + IP/r\u0026nbsp;\u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp style=\"margin-bottom: .0001pt; line-height: 200%;\"\u003e2NRTI + INI\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp style=\"margin-bottom: .0001pt; line-height: 200%;\"\u003eDual therapy\u0026nbsp;\u0026nbsp;\u0026nbsp; \u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 118.9pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid #BFBFBF 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"159\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e5 (12.82%)\u003c/p\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e9 (23.07%)\u003c/p\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e7 (17.94%)\u003c/p\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e12 (30.76%)\u003c/p\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e6 (15.38%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 105.2pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid #BFBFBF 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"140\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e18 (15.38%)\u003c/p\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e26 (22.22%)\u003c/p\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e27 (23.07%)\u003c/p\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e24 (20.51%)\u003c/p\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e22 (18.80%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 92.0pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid #BFBFBF 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"123\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e0.765\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eTable 2. Comparison of epidemiological, demographic, immunologic and virological characteristics between \u003cem\u003eBlastocystis\u003c/em\u003e- carrier (N=39) and \u003cem\u003eBlastocystis\u003c/em\u003e-free (N=117) patients. \u003cspan style=\"color: black; background: white;\"\u003eSignificant results were marked with *.\u003c/span\u003e\u003c/p\u003e\u003c/br\u003e\u003c/br\u003e\n\u003ctable style=\"border-collapse: collapse; border: none;\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 91.9pt; border-top: windowtext; border-left: #BFBFBF; border-bottom: windowtext; border-right: #BFBFBF; border-style: solid; border-width: 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"123\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 10.0pt; line-height: 200%; color: black;\"\u003eSymptoms\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 106.3pt; border-top: solid windowtext 1.0pt; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid #BFBFBF 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"142\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u003cstrong\u003e\u003cem\u003e\u003cspan style=\"font-size: 10.0pt; line-height: 200%;\"\u003eBlastocystis-\u003c/span\u003e\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 10.0pt; line-height: 200%;\"\u003ecarriers\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 10.0pt; line-height: 200%;\"\u003eN(%)\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 92.15pt; border-top: solid windowtext 1.0pt; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid #BFBFBF 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"123\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u003cstrong\u003e\u003cem\u003e\u003cspan style=\"font-size: 10.0pt; line-height: 200%;\"\u003eBlastocystis\u003c/span\u003e\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 10.0pt; line-height: 200%;\"\u003e-free\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 10.0pt; line-height: 200%;\"\u003eN(%)\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 49.6pt; border-top: solid windowtext 1.0pt; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid #BFBFBF 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"66\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; line-height: 200%;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 10.0pt; line-height: 200%;\"\u003ep-value\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 91.9pt; border: solid #BFBFBF 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"123\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 10.0pt; line-height: 200%;\"\u003eAbdominal pain\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 106.3pt; border-top: none; border-left: none; border-bottom: solid #BFBFBF 1.0pt; border-right: solid #BFBFBF 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"142\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 10.0pt; line-height: 200%;\"\u003e7 (17.94%)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 92.15pt; border-top: none; border-left: none; border-bottom: solid #BFBFBF 1.0pt; border-right: solid #BFBFBF 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"123\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 10.0pt; line-height: 200%;\"\u003e26 (22.22%)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 49.6pt; border-top: none; border-left: none; border-bottom: solid #BFBFBF 1.0pt; border-right: solid #BFBFBF 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"66\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 10.0pt; line-height: 200%;\"\u003e0.836\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 91.9pt; border: solid #BFBFBF 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"123\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 10.0pt; line-height: 200%;\"\u003eDiarrhoea\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 106.3pt; border-top: none; border-left: none; border-bottom: solid #BFBFBF 1.0pt; border-right: solid #BFBFBF 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"142\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 10.0pt; line-height: 200%;\"\u003e10 (25.64%)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 92.15pt; border-top: none; border-left: none; border-bottom: solid #BFBFBF 1.0pt; border-right: solid #BFBFBF 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"123\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 10.0pt; line-height: 200%;\"\u003e20 (17.09%)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 49.6pt; border-top: none; border-left: none; border-bottom: solid #BFBFBF 1.0pt; border-right: solid #BFBFBF 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"66\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 10.0pt; line-height: 200%;\"\u003e0.283\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 91.9pt; border: solid #BFBFBF 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"123\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 10.0pt; line-height: 200%;\"\u003eF\u003c/span\u003e\u003cspan style=\"font-size: 10.0pt; line-height: 200%;\"\u003elatulence\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 106.3pt; border-top: none; border-left: none; border-bottom: solid #BFBFBF 1.0pt; border-right: solid #BFBFBF 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"142\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 10.0pt; line-height: 200%;\"\u003e12 (30.76%)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 92.15pt; border-top: none; border-left: none; border-bottom: solid #BFBFBF 1.0pt; border-right: solid #BFBFBF 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"123\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 10.0pt; line-height: 200%;\"\u003e17 (14.52%)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 49.6pt; border-top: none; border-left: none; border-bottom: solid #BFBFBF 1.0pt; border-right: solid #BFBFBF 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"66\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 10.0pt; line-height: 200%;\"\u003e0.024*\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 91.9pt; border: solid #BFBFBF 1.0pt; border-top: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"123\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 10.0pt; line-height: 200%;\"\u003eNausea\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 106.3pt; border-top: none; border-left: none; border-bottom: solid #BFBFBF 1.0pt; border-right: solid #BFBFBF 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"142\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 10.0pt; line-height: 200%;\"\u003e3 (7.69%)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 92.15pt; border-top: none; border-left: none; border-bottom: solid #BFBFBF 1.0pt; border-right: solid #BFBFBF 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"123\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 10.0pt; line-height: 200%;\"\u003e6 (5.12%)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 49.6pt; border-top: none; border-left: none; border-bottom: solid #BFBFBF 1.0pt; border-right: solid #BFBFBF 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"66\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 10.0pt; 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padding: 0in 5.4pt 0in 5.4pt;\" width=\"123\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 10.0pt; line-height: 200%;\"\u003e10 (8.54%)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 49.6pt; border-top: none; border-left: none; border-bottom: solid #BFBFBF 1.0pt; border-right: solid #BFBFBF 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"66\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 10.0pt; line-height: 200%;\"\u003e0.180\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 91.9pt; border-top: none; border-left: solid #BFBFBF 1.0pt; border-bottom: solid windowtext 1.0pt; border-right: solid #BFBFBF 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"123\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 10.0pt; line-height: 200%;\"\u003eWeight loss\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 106.3pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid #BFBFBF 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"142\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 10.0pt; line-height: 200%;\"\u003e3 (7.69%)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 92.15pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid #BFBFBF 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"123\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 10.0pt; line-height: 200%;\"\u003e10 (8.54%)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 49.6pt; border-top: none; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: solid #BFBFBF 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"66\"\u003e\n\u003cp style=\"margin-bottom: .0001pt; text-align: center; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 10.0pt; line-height: 200%;\"\u003e0.658\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eTable 3. Gastrointestinal symptoms observed in \u003cem\u003eBlastocystis\u003c/em\u003e- carrier (N=39) and \u003cem\u003eBlastocystis\u003c/em\u003e-free (N=117) patients. \u003cspan style=\"color: black; background: white;\"\u003eSignificant results were marked with *.\u003c/span\u003e\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-infectious-diseases","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"infd","sideBox":"Learn more about [BMC Infectious Diseases](http://bmcinfectdis.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/infd","title":"BMC Infectious Diseases","twitterHandle":"#bmcinfectdis","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Blastocystis, subtype, PCR, HIV, Italy","lastPublishedDoi":"10.21203/rs.2.12345/v3","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.2.12345/v3","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Background: Blastocystis is one of the most common intestinal protozoa in human faecal samples with uncertain impact on public health. Studies on the prevalence of Blastocystis in HIV-positive patients are limited and dated.\nMethods: A cross-sectional study was carried out involving 156 HIV-positive patients to evaluate the prevalence of Blastocystis-subtypes by molecular amplification and sequencing the small subunit rRNA gene (SSU rDNA), to identify the risk factors for its transmission, to examine the relationship between the presence of the protist and gastrointestinal disorders. Furthermore, the evaluation of the faecal calprotectin by immunoassay from a sample of subjects was performed to evaluate the gut inflammation in Blastocystis-carriers.\nResults: Blastocystis-subtypes ST1, ST2, ST3, ST4 were identified in 39 HIV-positive patients (25%). No correlation was found between the presence of the protist and virological or epidemiological risk factors. Blastocystis was more frequently detected in homosexual subjects (p=0.037) infected by other enteric protozoa (p= 0.0001) and with flatulence (p=0.024). No significant differences in calprotectin level was found between Blastocystis-carriers and free ones.\nConclusions: Blastocystis is quite common in HIV-positive patients on ART showing in examined patients 25% prevalence. Homosexual behaviour may represent a risk factor for its transmission, while CD4 count and viremia didn’t correlate with the presence of the protist. The pathogenetic role of Blastocystis remains unclear and no gut inflammation status was detected in Blastocystis-carriers. The only symptom associated with Blastocystis was the flatulence, evidencing a link between the presence of the protist and the composition and stability of gut microbiota.\nKeywords: Blastocystis, subtype, PCR, HIV, Italy","manuscriptTitle":"Molecular characterization of Blastocystis subtypes in HIV-positive patients and evaluation of risk factors for colonization","msid":"","msnumber":"","nonDraftVersions":[{"code":3,"date":"2019-10-09 16:34:01","doi":"10.21203/rs.2.12345/v3","editorialEvents":[{"type":"communityComments","content":0},{"type":"checksComplete","content":"","date":"2019-11-06T12:00:00+00:00","index":"","fulltext":""},{"type":"decision","content":"Accept","date":"2019-10-08T12:00:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2019-10-08T12:00:00+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2019-10-07T12:00:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-infectious-diseases","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"infd","sideBox":"Learn more about [BMC Infectious Diseases](http://bmcinfectdis.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/infd","title":"BMC Infectious Diseases","twitterHandle":"#bmcinfectdis","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}},{"code":2,"date":"2019-10-07 21:16:36","doi":"10.21203/rs.2.12345/v2","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorAssigned","content":"","date":"2019-10-04T12:00:00+00:00","index":"","fulltext":""},{"type":"decision","content":"Minor revision","date":"2019-10-04T12:00:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2019-10-03T12:00:00+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2019-10-03T12:00:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-infectious-diseases","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"infd","sideBox":"Learn more about [BMC Infectious Diseases](http://bmcinfectdis.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/infd","title":"BMC Infectious Diseases","twitterHandle":"#bmcinfectdis","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}},{"code":1,"date":"2019-08-05 10:53:30","doi":"10.21203/rs.2.12345/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2019-09-09T12:00:00+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2019-09-08T12:00:00+00:00","index":2,"fulltext":"Form responses:\n---\n* Does the work include the necessary controls?\nIf not, please specify which controls are required in your comments to the authors.\tYes: **Are the conclusions drawn adequately supported by the data shown?\nIf not, please explain in your comments to the authors.\tNo**\n"},{"type":"reviewerAgreed","content":"","date":"2019-08-22T12:00:00+00:00","index":2,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2019-08-06T12:00:00+00:00","index":1,"fulltext":"Form responses:\n---\n* Does the work include the necessary controls?\nIf not, please specify which controls are required in your comments to the authors.\tYes: **Are the conclusions drawn adequately supported by the data shown?\nIf not, please explain in your comments to the authors.\tYes**\n"},{"type":"reviewerAgreed","content":"","date":"2019-08-04T12:00:00+00:00","index":1,"fulltext":""},{"type":"editorAssigned","content":"","date":"2019-08-02T12:00:00+00:00","index":"","fulltext":""},{"type":"reviewersInvited","content":"","date":"2019-08-02T12:00:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2019-07-29T12:00:00+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2019-07-29T12:00:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"","date":"2019-07-25T12:00:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-infectious-diseases","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"infd","sideBox":"Learn more about [BMC Infectious Diseases](http://bmcinfectdis.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/infd","title":"BMC Infectious Diseases","twitterHandle":"#bmcinfectdis","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"19b646cb-6d5a-40ff-b5c0-03eb22c85898","owner":[],"postedDate":"October 9th, 2019","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":30372,"name":"Infectious Diseases"}],"tags":[],"updatedAt":"","versionOfRecord":{"articleIdentity":"rs-3113","link":"https://doi.org/10.1186/s12879-019-4537-7","journal":{"identity":"bmc-infectious-diseases","isVorOnly":false,"title":"BMC Infectious Diseases"},"publishedOn":"2019-10-22 12:00:00","publishedOnDateReadable":"October 22nd, 2019"},"versionCreatedAt":"2019-10-09 16:34:01","video":"","vorDoi":"10.1186/s12879-019-4537-7","vorDoiUrl":"https://doi.org/10.1186/s12879-019-4537-7","workflowStages":[]},"version":"v3","identity":"rs-3113","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"identity":"rs-3113","version":["v3"]},"buildId":"ApUGefWb6u5IBVtyqm6d5","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}