Genotyping of Cryptosporidium Species and Their Clinical Manifestations in Patients with Renal Transplantation and Human Immunodeficiency Virus Infection

In the present study we aimed to determine (i) frequency of Cryptosporidium species among patients with renal transplantation (RT) and human immunodeficiency virus (HIV) infection and (ii) relationship of the nature, severity, and duration of symptoms with different species and load of Cryptosporidium. Stool samples from 70 (42 RT and 28 HIV) and 140 immunocompromised patients with and without cryptosporidiosis by modified Kinyoun's staining were subjected to qPCR-melting curve analysis for identification of parasite species. qPCR detected one microscopically negative sample to be positive for cryptosporidiosis. C. hominis, C. parvum, and mixed infection were detected in 50/71 (70.4%), 19/71 (26.8%), and 2/71 (2.8%) patients, respectively. Patients with cryptosporidiosis had higher stool frequency (median, IQR: 4, 3–6/d versus 3, 2–4/d; P = 0.017) and watery stool (52/71 [73%] versus 64/139 [46%]; P = 0.003). Parasite load (median, IQR: Log10 6.37 (5.65–7.12), Log10 5.81 (4.26–6.65); P = 0.046) and nausea/vomiting (29/50 [58%] versus 5/19 [26%]; P = 0.032) were more frequent with C. hominis than with C. parvum infection. Thus, Cryptosporidium spp. (mainly C. hominis) is a common cause of diarrhoea in RT and HIV patients.


Introduction
The coccidian enteric parasite Cryptosporidium spp. is a common cause of gastroenteritis and diarrhoea in man [1,2]. It causes severe, voluminous watery diarrhea in immunocompromised patients such as those infected with human immunodeficiency virus (HIV) [3,4]. However, there is a paucity of studies on Cryptosporidium spp. among renal transplant (RT) recipients [5,6]. Infected patients exhibit varying degrees of clinical manifestations. Some patients have severe symptoms over long duration while others recover in 1-2 weeks after a mild attack. The reasons for such diversity, which might be attributed to different genotypes or species of Cryptosporidium spp., or their varying load or host immunity, have not been adequately investigated so far.
Human cryptosporidiosis is mainly caused by Cryptosporidium hominis and C. parvum, which are responsible for most of the outbreaks of Cryptosporidium described so far.
Other less common species are C. meleagridis, C. cuniculus, C. viatorum, C. muris, C. canis, C. felis, C. suis, and C. andersoni [7][8][9][10][11][12][13]. C. hominis may cause more severe infection than C. parvum and other zoonotic species [7,[14][15][16]. In contrast, in a study from India, HIV positive patients, persons infected with C. parvum and other zoonotic species tended to have fever more frequently than those infected with C. hominis [11]. Thus, this variation in clinical manifestations might be due to the different Cryptosporidium species. Genotyping of Cryptosporidium has mainly been studied in patients with HIV infection. Currently there is no study on genotyping of Cryptosporidium spp. among renal transplant recipients. Also, relationship between nature, severity, and duration of the symptoms and different species of Cryptosporidium, among RT recipients, is not known.
An inverse relationship between occurrence of cryptosporidiosis and CD4 T cell count has been well documented in many studies in patients with AIDS. HIV/AIDS patients RT * * recipients RT * * recipients HIV * patients HIV * patients n: 28 n: 42 n: 60 n: 80 having lower CD4 count (<200 cells/cumm) are at higher risk of cryptosporidiosis [4,17], with severe infections below 50 cells/cumm CD4 count [3,18]. Such studies are scanty among RT recipients. In fact, the involvement of cell-mediated immunity in cryptosporidiosis has not been investigated in renal transplant recipients. Microscopy and ELISA are the mainstay for diagnosis of Cryptosporidium spp. but are unable to differentiate among different species. Molecular technique like polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) is the mainstay for genotyping. However, it is time consuming, requires post-PCR processing, and is prone to cross-contamination and false-positive results [19]. Real-time quantitative PCR (qPCR) is a highly sensitive and specific molecular tool which detects Cryptosporidium spp. and quantifies its load. Being a closed tube assay it prevents post-PCR contamination and obviates the need of time consuming procedures, like gel electrophoresis. The diagnostic efficacy of qPCR needs evaluation considering lack of adequate data.
Accordingly, we undertook a prospective study to determine (i) frequency of different species of Cryptosporidium infecting immunocompromised patients (HIV and RT patients) by real-time PCR (qPCR) and (ii) relationship of the nature, severity, and duration of the symptoms with different species of Cryptosporidium and their loads.  [20]. RT recipients undergoing at least one renal transplantation were recruited. Microscopy was used for detection and qPCR for genetic characterization of Cryptosporidium spp.

Materials and Methods
Oocyst load was also determined by qPCR. Clinical and laboratory parameters of patients with cryptosporidiosis were compared with 140 immunocompromised controls (60 HIV and 80 RT recipients) without cryptosporidiosis ( Figure 1). Data on demographic, clinical, and laboratory parameters were recorded in a standard questionnaire in each patient. The study protocol was approved by Institutional Ethics Committee (Ref. number PGI/PhD/IEC/57/21.10.2011) and it was in accordance with the Helsinki Declaration [21].

Sample Collection.
Three consecutive stool samples from each patient and control were subjected to microscopy and culture. A part of it was stored at −40 ∘ C in normal saline for DNA extraction. Blood in EDTA vials was collected from the patients for determining the CD4 count of the patients.

Sample
Processing. Stool samples were subjected to routine microscopy using saline and iodine direct wet mount technique for detection of cysts and trophozoites of parasites. After stool concentration by formal ether concentration technique, acid fast staining (modified Kinyoun's) was used for detection of oocysts of Cryptosporidium spp. Briefly, the smear was incubated in modified Kinyoun's stain for 20 minutes and malachite green was used as counter stain [22]. The stained smears were subjected to light microscopy. Stool samples were also subjected to culture on MacConkey, deoxycholate citrate agar (DCA), and dextrose sorbitol rhamnose agar (DSRA) to exclude other common bacterial agents causing diarrhoea such as Salmonella and Shigella spp. [23]. the manufacturer's protocol with some modifications; these included initial washing in 2% polyvinyl polypyrrolidone (PVPP) dissolved in 1x phosphate buffered saline (PBS) and incubation with stool lysis buffer (Buffer ASL) at 80 ∘ C for 10 minutes, with occasional stirring in between. DNA was purified and eluted as per manufacturer's instructions.

Polymerase Chain Reaction (PCR).
Polymerase chain reaction (PCR) was performed using four primers for the amplification of dihydrofolate reductase (DHFR) gene, one sense and three antisense primers (CINF and CINR, 1R, 2R), aiming to detect Cryptosporidium spp., C. hominis, and C. parvum, respectively. The primers used have been described previously [24]. Samples, positive for Cryptosporidium spp. but negative for the two genotypes, were sequenced. Primerpairs CINF and CINR amplified a 575 bp region of the DHFR gene. Primer-pair CINF and 1R amplified a 357 bp region and primer-pair CINF and 2R, a 190 bp region of the DHFR gene. PCR was performed in a total volume of 20 L, consisting of 10 L of Green Taq Master Mix (2x), 10 pmoles of forward and reverse primers, and 1 L of template DNA. 100 ng/ L of template DNA from each sample (extracted DNA) was used in PCR reactions. Three sets of reactions were performed with each sample; one with primer set CINF and CINR (for detection of Cryptosporidium spp.), second with primer set CINF and 1R (for detection of C. hominis); third with primer set CINF and 2R (for detection of C. parvum). PCR conditions were initial denaturation at 95 ∘ C for 5 min, followed by 40 cycles of denaturation at 94 ∘ C for 40 sec, annealing at 54 ∘ C for 30 sec, and elongation at 72 ∘ C for 45 sec. This was followed by a final elongation step of 72 ∘ C for 5 min.
The PCR products were confirmed by sequencing. Amplified products were subjected to electrophoresis in 2% agarose gel. Sequencing of the amplicons was performed using respective species-specific PCR primers. The sequences were compared with those available in the Gen-Bank databases with the BLAST N program.

Real-Time PCR.
The amplification was carried out in real-time PCR machine Corbett Research 6000 Q-PCR instrument (Rotor-Gene 6000 software, Sydney, Australia) using the same sets of species-specific primers used in the traditional PCR [24]. The amplification round was carried out in a total volume of 20 L containing 10 L of KAPA-SYBR FAST Master Mix (Universal qPCR kit; Kapa Biosystems), containing KAPA-SYBR DNA Polymerase (an engineered version of Taq DNA Polymerase designed specifically for realtime PCR, SYBR Green I dye and optimized magnesium chloride concentration). Template DNA (having a concentration of 100 ng/mL per sample) was added. PCR conditions were as follows: initial denaturation at 95 ∘ C for 10 min, 40 cycles at 95 ∘ C for 45 sec, then annealing at 54 ∘ C for 60 sec, and extension at 72 ∘ C for 40 sec. Fluorescence data were collected in the extension step. For meting curve analysis (MCA), after completion of last PCR cycle, a quick denaturation was performed at 95 ∘ C for 1 sec, followed by a 30 sec annealing step at 45 ∘ C with a slow ramp (0.2 ∘ C/sec) up to 80 ∘ C. Reaction mixture without template DNA was used as negative control.

Standard Graph.
Standard graph was plotted with DNA extracted from purified Cryptosporidium parvum oocyst, commercially purchased from The University of Arizona, USA. Oocysts (concentration of 5 × 10 7 oocysts/mL in antibiotic solution) were subjected to DNA extraction by previously mentioned protocol using the QIAamp DNA Mini Kit (QIAGEN Inc., Valencia, CA, USA) [25,26]. Standard graph was generated via serial dilution of oocyst stock (5 × 10 7 oocysts/mL) to 5-fold (corresponding to approximately 10 7 to 10 3 oocysts/mL/dilution). The DHFR gene copies/ L corresponded approximately to four times the oocysts/dilution due to the presence of single-copy of this gene/sporozoite or 4 copies/oocyst, as one oocyst consists of 4 sporozoites. DNA was extracted from each serial dilution and 100 ng/ L of the extracted DNA was subjected to qPCR using primers specific for C. parvum to estimate the DHFR gene copy number of each serially diluted DNA and thus to construct the standard graph ( Figure 2).

Data Analysis.
Nonparametric and parametric continuous variables were analysed by Mann-Whitney test and unpaired -test, respectively. Categorical variables were analysed by Chi-square test. The values of equivalent oocyst concentrations (parasite load) were transformed into Log 10 and presented as median and interquartile range (IQR). values <0.05 were considered significant. All the statistical analyses were performed using Statistical Package for the Social Sciences (SPSS 15, Inc., Chicago, IL, USA).

Demographic, Clinical, and Laboratory Parameters of
Patients and Controls. 70 immunocompromised patients were positive for Cryptosporidium spp. by both microscopy and qPCR and one by qPCR alone. Though the oocyst load in the stool of this microscopically negative RT recipient, initially included in the immunocompromised control group, was comparatively lower, being log 10 6.65 equivalent oocyst concentration, he was considered as infected. Patients with other parasitic infestation and enteropathogenic bacteria were excluded from analysis. The demographic and clinical parameters of the 71 patients with cryptosporidiosis and 139 immunocompromised controls are shown in Table 1. Cryptosporidiosis was associated with diarrhoea, increased stool frequency, and watery stool (Table 1). Immunocompromised (both HIV and RT) patients with cryptosporidiosis had lower CD4 count than noninfected ones (mean ± SD: 214 ± 162 cells/cumm versus 260 ± 144 cells/cumm, = 0.041). CD4 count of patients with HIV infection was lower than patients with RT (145 ± 10.6 cells/cumm versus 313 ± 12.7 cells/cumm, = 0.004). CD4 count of HIV-infected patients with cryptosporidiosis was lower than those without (95 ± 11.3 cells/cumm versus 169 ± 13.7 cells/cumm, = 0.002) (Figure 3). In contrast, CD4 counts of RT patients, with and without cryptosporidiosis, were comparable (290 ± 23.6 cells/cumm versus 326 ± 15 cells/cumm, = 0.269) (Figure 3). Moreover, CD4 count of HIV-infected patients with cryptosporidiosis was lower than that of Cryptosporidium infected RT patients (223 ± 23 cells/cumm versus 260 ± 12.3 cells/cumm, = 0.09); neutrophil count in RT patients with cryptosporidiosis tended to be lower than that in Cryptosporidium infected HIV patients (67 ± 10.5 cells/cumm versus 72 ± 14 cells/cumm, = 0.08).

Relationship between the Nature, Severity, and Duration of the Symptoms and Different
Species. The demographic, laboratory, and clinical parameters of the patients infected with Cryptosporidium hominis and C. parvum are shown in Table 2. Patients with C. hominis infection more often had nausea and/or vomiting and increased oocyst load (denoted by equivalent concentration of oocysts in the stool samples) compared to patients infected with C. parvum ( < 0.05; Table 2). However, fever, abdominal pain, frequency, and duration of diarrhoea were comparable between patients with C. hominis and C. parvum infection ( Table 2).

Discussion
In the current study, we found that (a) C. hominis and C. parvum were the only two Cryptosporidium spp. detected in immunocompromised patients, (b) cryptosporidiosis was more commonly associated with diarrhoea, increased stool frequency, and watery stool, (c) C. hominis infection was more frequently associated with nausea and/or vomiting and increased oocyst load than C. parvum, and (d) patients with diarrhoea had higher copy number (or equivalent oocyst concentration) of Cryptosporidium spp. than those without.
In the present study, C. hominis and C. parvum were the only two Cryptosporidium spp. detected in immunocompromised patients. To date, genotyping of Cryptosporidium spp. has been done mostly on HIV-infected patients. There are scanty data on genotyping of Cryptosporidium spp. among RT recipients. In studies on HIV-infected patients from South Africa [27], North America [28,29], South America [7,30], and Europe [31], C. hominis was the most prevalent species. However, in a few other studies, C. parvum was found to be more prevalent than C. hominis [32][33][34][35]. Studies from India also revealed C. hominis to be the most prevalent species infecting HIV-infected patients [36,37]. A recent study from northern India reported two of three Cryptosporidium isolates from RT recipients to be C. hominis and one C. parvum by PCR-RFLP for small subunit (SSU) rRNA gene [38]. This indicates that human is the major source of transmission of the infection. To the best of our knowledge, the present study is perhaps the only one from India, which used real-time PCR for detection and genetic characterization of Cryptosporidium spp.
In this study qPCR detected Cryptosporidium in one sample that was missed by microscopy. The load of oocysts in this sample was comparatively lower than the rest. Hence, stool microscopy done by an expert is as good as qPCR for detection of Cryptosporidium. However, qPCR is useful when oocyst load is less. In addition, species identification, which may have clinical implications, can be done by qPCR and is not possible by microscopy [36,39]. qPCR has the advantage of real-time detection of PCR amplicons omitting the requirement of restriction digestion or sequence analysis for genotyping.
The current study shows that CD4 count was less in HIV patients with Cryptosporidium infection than without. It has been well documented in previous studies that patients having lower CD4 count are at higher risk of cryptosporidiosis. Chronic cryptosporidiosis generally occurs in patients with CD4 counts below 200 cells/cumm [18], and patients with CD4 count below 50 cells/cumm suffer from severe infections [3]. In fact, several studies have documented a correlation between CD4 count <200 cells/cumm and symptomatic cryptosporidiosis, in HIV patients [11,17]. However, such studies are scanty in RT recipients. In our study, CD4 count among RT patients was not as low as that in HIV positive patients with cryptosporidiosis, suggesting that depletion of immune cells and factors other than CD4 count is responsible for susceptibility to cryptosporidiosis in RT patients. Subgroup analysis revealed neutrophil count tended to be lower in Cryptosporidium infected RT patients than in HIV patients. Low neutrophil count of RT patients might be attributed to some immune-suppressive drugs as well. Thus, not only immune restoration in the form of HAART therapy, but also management of immune-suppressive therapy may help in clearance of the parasite, in immunocompromised hosts.
In this study, the RT recipients with cryptosporidiosis were given mostly triple immunosuppressive drugs than double and single immunosuppressants. The combination of mycophenolate mofetil (MMF), Wysolone, and Tacrolimus drugs was mostly used in both patient and control groups. In a study from Pakistan, the immunosuppressive drugs administered to RT recipients having cryptosporidiosis comprised of cyclosporine/FK506, azathioprine/mycophenolate mofetil (MMF), and steroids (Prednisolone/Wysolone) [40]. In another study cyclosporin A has been used in infected RT recipients [41]. Till date no association of Cryptosporidium infection has been shown with any immunosuppressive drug. In fact, there are scanty data on cryptosporidiosis in RT recipients. Large-scale follow-up studies, explaining the type and dose of immunosuppressive agents and their management in Cryptosporidium infected RT recipients, could clarify this issue further.
In the present study, C. hominis was associated with frequent nausea and/or vomiting and increased oocyst load compared to C. parvum. Thus, infection caused by C. hominis was more severe than C. parvum infection. This is in accordance with the previous studies from Peru, which showed that C. hominis was associated with diarrhea, nausea, vomiting, general malaise, and increased oocyst shedding intensity and duration. In contrast, zoonotic isolates like C. parvum were associated with diarrhea only [7]. In a study from United Kingdom, duration of C. parvum infection was significantly lower than that of C. hominis ( = 8.312, = 0.005) [14]. Also, C. hominis was reported to cause more severe infection than C. parvum [14]. It has been mentioned in this study that C. hominis being a "specialist," particularly infecting the human host, show more virulent characteristics than a "generalist" like C. parvum. From Brazil, children with C. hominis infection have been reported to shed significantly more oocysts in stool [42]. Similarly in a study on children from India, C. hominis infection was associated with a greater severity of diarrhea [16]. In contrast, in a study from India on HIV positive patients, persons infected with C. parvum and other zoonotic species tended to have fever more frequently than those infected with C. hominis [11]. It is suggested that immunodeficiency is responsible for alteration of host susceptibility to the Cryptosporidium species that are normally noninfectious to humans [11].
The present study has a few limitations. The study does not involve follow-up of the patients. Follow-up studies on the same will determine the actual epidemiology of the infection and heterogeneity of clinical manifestations. Our findings, however, add some new information to the existing knowledge.
In conclusion, Cryptosporidium spp. is a common cause of diarrhoea in renal transplant recipients, as in HIV positive patients. Hence, clinicians should not overlook the possibility of occurrence of cryptosporidiosis in these patients. C. hominis is the most common Cryptosporidium spp. detected in both HIV and RT patients, suggesting that anthroponotic mode of transmission of the parasite is dominant in immunocompromised patients, particularly in northern India. Real-time PCR is an important and time-effective tool for diagnosis of Cryptosporidium spp. as well as its genetic characterization.