4.6.1. Direct Microscopy

The presence of *Cryptosporidium* oocysts in stools was first established using the modified Ziehl–Neelsen (mZN) staining method as described by the World Health Organization (WHO) [35]. Briefly, thin smears of fresh samples and concentrated pellet from formolether concentration technique were prepared on the same glass side and air-dried before mZN staining. Samples were read using a microscope; the results were recorded as a positive for those where oocysts of the parasite were visualized under 100X magnification. All stool samples were collected, labeled, processed and stored following the laboratory standard procedures, including Good Laboratory Practice (GLP) recommendations. For the parasitological assay, each internal quality control was made, and all sample readings were double-checked for concordance by two technicians. In case of discordant results, a third observation was required.

#### 4.6.2. DNA Extraction

DNA was extracted from frozen stool sample using a commercial QIAmp stool Mini Kit (Qiagen, Hilden, Germany) following the manufacturer's protocol with the following modification: the lysis temperature was raised to 95 ◦C and the DNA was eluted in 100 μL. The pre-treating of the sample and usage of the QIAmp stool Mini Kit was done in a standard procedure as in similar studies, and with this there is no need to freeze or apply thawing cycles [32]. DNA extracts were stored at −20 ◦C until use.

#### 4.6.3. Molecular Detection by Conventional Polymerase Chain Reaction (PCR)

The 18S rRNA gene was amplified for all samples (positive and negative by mZN staining) by conventional polymerase chain reaction. The forward primer 5'-AACCTGGTTGATC CTGCCAGTAGTC-3' and reverse primer 5'-TGATCCTTCTGCAGGTTCACCTACG-3' as described by Xiao et al. [36] were used. Briefly, the PCR contained 10X PCR buffer (MgCl2) at a final concentration of 1X, 5mM MgCl2, 200 mM (each) deoxynucleotides triphosphate (dNTP), 1.0 U of Taq polymerase (Invitrogen Life Technologies, São Paulo, SP-Brasil), 100 nM (each) primer (Extend, SP-Brasil) and 2.5 μL of DNA template in a total 25 μL reaction mixture. The following parameter was adjusted in our study: MgCl2 concentration, Taq polymerase quantity when compared with the study that was used as reference. Each PCR had small adjustments; annealing was set to 61 ◦C for 45 s and extension to 72 ◦C for 7 min. The PCR efficacy and the identification of the *Cryptosporidium* genetic material from samples were verified through electrophoresis in agarose gel (1.2%).

Stool samples were considered positive if oocysts with typical characteristics (approximately 4 μm and 6 μm in diameter; stained bright pink within a clear halo under green field) or *Cryptosporidium* DNA with expected base pair (bp) were detected by conventional PCR.

#### 4.6.4. Characterization of *Cryptosporidium* spp. Isolates by Nested PCR and Restriction Fragment Polymorphism (RFLP) Analysis

All samples that were previously positive for 18S rRNA gene amplification were genotyped by a PCR-RFLP technique using genomic DNA as template. Firstly, a PCR product of approximately 1325 bp of the SSU rRNA gene was amplified by a nested PCR using the following primers 5'-TTCTAGAGCTAATACATGCG-3' and 5'-CCCTAATCCTTCGAAACAG GA-3' [11,12]. The first PCR contained 1X PCR buffer (MgCl2), 6 mM of MgCl2, 2.5 U of Taq polymerase, 200 μM of each primer concentration (500 nM each) and 2 μL of DNA template in a total volume of the reaction mixture (50 μL). The initial denaturation was 94 ◦C for 3 min, followed by the amplification performed in 35 total cycles: 94 ◦C for 45 s for denaturation, 58 ◦C for 45 s for annealing and 72 ◦C for 60 s for extension. The final extension was 72 ◦C for 7 min. For secondary PCR, for a product of 826 to 864 bp, it was done by using the following primers 5'-GGAAGGGTTGTATTTATTAGATAAAG-3' and 5'-AAGGAGTAAGGAACAACCTC CA-3' [11]. A total volume of the reaction mixture (50 μL), Taq DNA amount (1 U) and the primary PCR product was optimized (0.5 μL diluted at 1:20 or 1 μL non-diluted product). Amplification condition for secondary PCR was as follows: 94 ◦C for 45 s, 59 ◦C for 30 s and 72 ◦C for 45 s in 25 cycles with an initial hot start at 94 ◦C for 3 min and a final extension at 72 ◦C for 7 min. The PCR efficacy and the identification of the *Cryptosporidium* genetic material from samples were verified through electrophoresis in agarose gel (1.2%).

Genotype identification was made through the analysis of pattern of the secondary product after restriction digestion with the enzymes SspI and AseI (New England BioLabs Inc., Beverly, MA, USA) as described by Xiao et al. [11]. The AseI enzyme has the same digestion function and pattern as the enzyme VspI. Each set of experiments included a negative PCR control (laboratory-grade distilled water). For the restriction, 20 μL of the second product of the nested PCR, 10 U of SspI or AseI, 5 μL pf specific enzyme buffer was digested in a 50 μL of the reaction by 37 ◦C for one hour under conditions recommended by the supplier manufacturer. Aliquots of amplified and digested fragments were separated and visualized under Ultra-Violet light translucent (Bio-Rad, Milan, Italy) after separation

in 1.5% to 2% agarose gel (Invitrogen, Aukland, New Zealand) by electrophoresis stained with 3X GelRed (Biotium, San Francisco, CA, USA). The expected band for each enzyme varied according to the species detected were the most observed highlighted in bold (Table 3).


**Table 3.** Expected band of the restriction digestion using SspI and AseI enzymes.
