Genotyping and molecular profiling of intestinal microsporidiosis and cryptosporidiosis in HIV-infected patients in Alborz Province, Iran

Diarrhea is a major contributor to morbidity in human immunodeficiency virus (HIV)-infected patients both in developing and developed countries, affecting almost 40% of the people who die from the disease [1, 2]. Microsporidiosis and Cryptosporidiosis are opportunistic and the most prevalent parasitic infections causing chronic diarrhea in immunodeficiency conditions, especially HIV/acquired immunodeficiency syndrome (AIDS) [3, 4].

The occurrence of microsporidia and Cryptosporidium is significantly higher in HIV + patients with chronic diarrhea and CD4 + lymphocyte counts of less than 200 cells/mm³ [5]. Microsporidia parasites are obligate intracellular pathogens that infect various vertebrates and invertebrates globally [6, 7]. Currently, more than 200 genera and 1500 species of microsporidia have been described, and 17 of those species have been associated with human diseases [8].

Microsporidiosis is caused primarily by two microsporidian species, Enterocytozoon bieneusi and Encephalitozoon intestinalis [4]. Among these, E. bieneusi is the most frequently detected species in humans and was first identified in AIDS patients in 1985 [9]. Since then, E. bieneusi has been recognized as an important opportunistic pathogen in individuals with compromised immunity, including HIV/AIDS patients, cancer patients, organ transplant recipients, as well as in children, the elderly, and travelers [8]. It has been reported from a wide range of hosts—humans, livestock, birds, and wildlife and isolated from diverse environmental sources such as wastewater, drinking water, recreational waters, coastal areas, raw milk, and fresh vegetables. This wide distribution highlights its potential for both zoonotic and environmental transmission. Moreover, E. bieneusi exhibits extensive genetic diversity, with more than 500 genotypes identified so far across different host species and ecological settings, underscoring its adaptive capacity and epidemiological complexity [9].

Studies on E. bieneusi genotypes are commonly based on the analysis of the polymorphism of the internal transcribed spacer (ITS) nucleotide sequences. Presently, more than 1600 full-length E. bieneusi ITS sequences from more than 650 studies have been deposited in GenBank. The most prevalent genotypes of E. bieneusi are types IV, EbpC, A, and D. Nevertheless, Peru6, Peru8, Peru11, D, EbpC, and type IV, which are the most common genotypes in humans, have been frequently reported from domestic and wild animals, as well as various water sources, indicating the possibility of zoonotic and waterborne transmission [8].

Cryptosporidium is an intracellular protozoan that is recognized as a human and animal pathogen, given its frequency in waterborne outbreaks worldwide [10, 11]. Cryptosporidiosis causes diarrhea in both immunocompetent and immunocompromised humans and animals [12‐14]. However, it leads to serious diarrhea which may be fatal in immunocompromised individuals [3, 15]. Among at least 48 species and more than 120 recognized genotypes of Cryptosporidium reported worldwide [16], 22 species and two genotypes have been diagnosed in humans [17]. The majority of human cases of cryptosporidiosis are caused by five Cryptosporidium species: Cryptosporidium hominis, C. parvum, C. meleagridis, C. felis, and C. canis [18].

HIV/AIDS is a prominent cause of mortality and morbidity in low- and middle-income countries. Between 1990 and 2019, Iran witnessed a 108% increase in the number of HIV/AIDS-related mortalities, rising from 566 to 1,177 deaths. According to the Joint United Nations Program on HIV/AIDS (UNAIDS), the number of individuals living with HIV/AIDS in Iran in 2019 is expected to be 59,000 cases [19]. The recent increases in HIV/AIDS burden in Iran have important implications for opportunistic infections. As more people live with HIV for longer periods, gaps in care, delayed diagnosis, or incomplete antiretroviral therapy (ART) coverage can lead to increased numbers of immunosuppressed individuals who are susceptible to enteric pathogens. Opportunistic protozoa such as Cryptosporidium spp. and microsporidia exploit weakened mucosal and systemic immunity, leading to prolonged diarrheal disease, malabsorption and increased morbidity. Therefore, regional increases in HIV prevalence and HIV-related mortality necessitate strengthened surveillance of enteric opportunistic infections to guide clinical management and public-health interventions.

The aim of the current study was the molecular identification and genotyping of Enterocytozoon, Encephalitozoon, and Cryptosporidium isolates from HIV-infected patients referred to a behavioural diseases consultation centre in Alborz Province, Iran, using ribosomal RNA (rRNA) gene analysis.

A total of 275 HIV-positive patients were examined for Cryptosporidium and Microsporidia infections. Molecular analysis identified Cryptosporidium spp. in 7.6% (21/275) and Microsporidia spp. in 9.1% (25/275) of participants. Among them, C. parvum was detected in 5.8% (16/275), C. hominis in 1.8% (5/275), E. bieneusi in 6.5% (18/275), and E. intestinalis in 2.5% (7/275). Mixed infections involving more than one parasite were observed in 1.8% (5/275) of patients.

This study highlights the clinical and epidemiological significance of microsporidiosis and cryptosporidiosis in HIV-infected individuals in Alborz, Iran, revealing valuable insights into local transmission dynamics, genotype distribution, and potential risk factors. Despite advancements in antiretroviral therapy (ART), enteric protozoan infections continue to pose a substantial health risk to immunocompromised individuals, particularly in resource-limited settings where diagnostic challenges persist [33].

Our findings confirmed the presence of E. bieneusi and E. intestinalis in HIV-positive patients, with a higher prevalence of E. bieneusi. The predominance of genotype D (n = 10) is consistent with previous reports identifying this genotype as the most common in both immunocompetent and immunocompromised individuals globally [34, 35]. The detection of zoonotic genotypes Peru6 (n = 5) and J (n = 3) further indicates the significance of animal reservoirs in the local transmission cycle [36]. The identification of genotype J in this cohort represents the first report of this genotype in humans in Iran. Although genotype J has previously been detected in cattle within the country [37‐39], its presence in human patients provides the first molecular evidence of a potential zoonotic spillover [8]. The Bayesian phylogenetic analysis (BEAST, TN93 + G) showed genotype J forming a distinct, well-supported clade (posterior probability = 0.95), reinforcing its novelty and epidemiological importance in the Iranian context and suggesting a possible recent cross-species transmission event or previously unrecognized local circulation.

The presence of C. parvum subtypes IIdA20G1 (n = 8), IIdA19G1 (n = 4), and IIaA15G2R1 (n = 3) reflects a subtype distribution that differs from patterns observed in Western populations, where C. hominis is more prevalent [40]. The dominance of IId subtypes aligns with other Middle Eastern and Asian studies that have linked these subtypes to infections from small ruminants such as sheep and goats [41, 42]. The strong association between these subtypes and animal contact or environmental exposure further underscores the zoonotic nature of cryptosporidiosis in this region. Bayesian phylogenetic analysis in BEAST confirmed the predominance of zoonotic IId subtypes, which clustered closely with sequences from both animals and humans in China, India, and Australia, with strong posterior support (0.82–1.00), supporting the hypothesis of cross-species transmission. Meanwhile, C. hominis isolates all belonged to the IdA15G1 subtype and formed a tightly clustered clade with reference strains from Europe and Asia (posterior >0.9), suggesting anthroponotic transmission routes.

While zoonotic transmission is strongly supported by the predominance of C. parvum IId subtypes and the detection of zoonotic E. bieneusi genotypes such as Peru6 and J, other transmission routes should also be considered. The identification of C. hominis subtype IdA15G1 in several cases—together with its well-established anthroponotic nature—suggests that human-to-human transmission may occur within households, healthcare facilities, or communities with inadequate sanitation. Moreover, the observed association between well-water use and C. hominis infection in this cohort indicates a waterborne pathway, as contaminated groundwater can serve as a vehicle for oocyst dissemination in areas lacking effective water treatment. Consequently, infection-control programs should integrate WASH-based interventions (water, sanitation, and hygiene) alongside zoonotic-control strategies to limit both environmental and interpersonal spread of these opportunistic pathogens.

The identification of E. bieneusi genotype J in this cohort represents the first report of this genotype in humans in Iran. Although genotype J has previously been detected in cattle within the country [20‐22], its presence in human patients provides the first molecular evidence of a potential zoonotic spillover. The phylogenetic placement of genotype J in a distinct and well-supported clade further reinforces its novelty and epidemiological importance in the Iranian context. From a public health perspective, this emergence underscores the need for targeted One Health investigations to (i) identify animal reservoirs, (ii) assess environmental sources such as drinking water and agricultural runoff, and (iii) conduct clinical surveillance to determine possible genotype-specific outcomes. We recommend expanded molecular monitoring across human, animal, and environmental samples, coupled with comparative genomic analyses to explore virulence markers and public-awareness programs to reduce zoonotic exposure.

From a clinical standpoint, the study observed that infections were not limited to severely immunocompromised individuals. Microsporidial infections occurred even among patients with relatively stable CD4 + T-cell counts and those receiving ART, suggesting persistent exposure and possible gaps in immune protection or reinfection. Importantly, specific genotypes such as E. bieneusi genotype D were associated with more severe gastrointestinal symptoms, particularly in HAART-naïve individuals. This observation is supported by recent studies highlighting genotype-dependent virulence and cytokine modulation [43‐45].

The overall prevalence of Cryptosporidium spp. (7.64% by PCR) and E. bieneusi (6.5%) observed in our study falls within the range reported across various regions of Iran. For Cryptosporidium, the prevalence among Iranian individuals ranges widely from 0.36% to 12%, with the lowest rates reported in Kurdistan and Hamedan, and the highest in Bandar Abbas and Yazd [46‐49]. Similarly, E. bieneusi prevalence has been reported from as low as 1.3% to as high as 20%, with lower rates in Tehran (1.3%) and Rasht (2.3%), while higher prevalence has been observed in Tabriz (10.6%), Kerman (13.88%), and Mazandaran (20%) [6, 34, 50‐52]. These variations in prevalence may reflect differences in regional environmental conditions, water sources, sanitation, host immunity, and diagnostic methods [53, 54]. Notably, the higher detection rates in our study may be attributed to the use of sensitive molecular techniques, such as nested PCR, which provided more accurate results compared to conventional microscopy, which detected a lower prevalence of Cryptosporidium (2.2%).

Our Bayesian phylogenetic analysis based on ITS sequencing revealed that genotype D strains clustered closely with isolates from both humans and animals, including cattle, cat, beaver, chinchilla, primate and dog, from geographically diverse regions such as China, Bangladesh, Thailand, Argentina, Poland, Slovak Republic, Brazil, and Turkey [44, 55‐61]. This global clustering highlights the broad host range and high zoonotic potential of genotype D, supporting the One Health perspective [62, 63]. The clear Bayesian phylogenetic separation of Cryptosporidium species based on 18 S rRNA sequencing provided additional validation of molecular identification and demonstrated close evolutionary relationships among human-infecting species. The C. parvum subtypes identified in this study have been widely reported in livestock in Iran and neighbouring countries, reinforcing the risk of zoonotic transmission in agricultural communities [20, 47, 64‐69].

The findings of this study have several public health implications [70]. First, the detection of zoonotic genotypes in patients using well water indicates vulnerabilities in local water treatment infrastructure [71, 72]. Notably, C. hominis subtype IdA15G1, known for its resistance to chlorine-based disinfection, was detected in association with well water consumption, echoing previous global reports [73, 74]. These insights call for enhanced surveillance and improved water safety measures, particularly in high-risk areas. Second, the continued presence of these infections in patients on ART suggests that immune reconstitution alone may not be sufficient to prevent infection or that reinfection is occurring. Routine screening of stool samples in HIV patients, especially those with CD4 + counts below 100 cells/µl—could facilitate early diagnosis and intervention. Our data also suggest the presence of asymptomatic carriers who may contribute to ongoing transmission within communities [75, 76].Finally, this study supports the integration of a One Health framework in the control and prevention of protozoan infections, particularly those with zoonotic transmission potential [77]. Coordinated efforts involving veterinary, environmental, and human health sectors are essential to address shared risk factors and implement effective interventions, such as livestock vaccination, environmental sanitation, and public health education [78, 79].

This study provides molecular and epidemiological evidence for the occurrence of Cryptosporidium and Microsporidia infections among HIV-positive individuals in Alborz, Iran. The identification of zoonotic genotypes and subtypes—particularly C. parvum IIdA20G1 and E. bieneusi genotype J—highlights the ongoing risk of cross-species transmission in this vulnerable population. Bayesian phylogenetic analyses (BEAST, TN93 + G model) confirmed the genetic relatedness of Iranian isolates to strains from both human and animal sources worldwide, underscoring the importance of a One Health approach in surveillance and control strategies. The associations between infections and environmental exposure, immunosuppression, and lack of antiretroviral therapy further emphasize the multifactorial nature of infection risk. These findings call for integrated public health interventions including improved water safety, targeted screening in immunocompromised patients, and strengthened collaboration across human, veterinary, and environmental health sectors to mitigate the burden of these opportunistic infections in endemic settings.