Ethical statement
The present study was approved by the Ethics Committee of the Shiraz University of Medical Sciences, Fars Province, Iran (Approval No: IR.IUMS.REC.1398.266) and was conducted from October 2021 to May 2023.
Fecal samples collection and microscopic examination
200 fecal samples (5-10 g each) were collected from children and adolescents under 18 years old with malignancies at Amir, Nemazee, and Saadi hospitals in association with Shiraz University of Medical Sciences. Direct microscopy using saline and iodine wet mount was applied, and fecal preparations were stained with Wheatley’s Trichrome stain. Three general groups of cancers were found among examined patients: hematologic, non-hematologic (cranial), and non-hematologic (extra-cranial).
DNA extraction
Genomic DNA was extracted from collected fecal samples using the QIAamp Fast DNA Stool Mini Kit (Qiagen, Hilden, Germany) based on the manufacturer’s instructions. Next, the quantity and quality of the purified DNA samples were determined using a nano-spectrophotometer (NanoDrop ND-1000, Biocompare, San Francisco, USA). The final DNA was eluted in 50 µL of elution buffer and kept at −20 °C for further use.
Polymerase chain reaction (PCR)
All Fecal samples underwent further examination using molecular techniques for G. duodenalis. First, a nested PCR protocol was used to validate the presence of the parasite through amplification of a ~130-bp fragment of the SSU-rRNA gene. The outer primer pair RH11 and RH4, and the inner primer set GiarF and GiarR were employed in both primary and secondary reactions. The reaction mixture had 2 μl of template DNA for both primary and secondary PCR reactions. Cycling conditions were identical for the first and second steps of PCR. The primary denaturation was at 95°C for 3 min, followed by 35 cycles of amplification (denaturation at 95°C for 30 s, annealing at 55°C for 30 s), with a final extension of 7 min at 72 °C.
For the amplification of the G. duodenalis DNA fragment in tpi gene (530 bp), two primer pairs were used, described by Sulaiman and colleagues [16]. The first PCR was done using AL3543 and AL3546 (0.5 μM each) primer pair, which amplify a 605 bp fragment, whereas the second PCR was performed using AL3544 and AL3545 (0.5 μM each) primer pair. All PCR reactions included 2 mM MgCl2, 200 μM dNTPs, 0.5 U of GoTaq DNA polymerase (Promega), and 5 μL of the extracted DNA (for primary PCR) or 1 μL of the primary PCR product (for secondary PCR). Of note, the extracted DNA from G. duodenalis assemblage A was used as a positive control. The following cycling conditions were applied to the thermal cycler device: initial denaturation (96◦C for 4 min), followed by 35 cycles of denaturation (96◦C for 30 s), annealing (55◦C [primary PCR] or 59◦C [secondary PCR] for 30 s) and extension (72 ◦C for 45 s), as well as an excessive step of extension at 72◦C for 4 min. Finally, the secondary PCR products were visualized by electrophoresis on 1.5% agarose gel stained with Safe Stain (SinaClon, Tehran, Iran).
DNA sequencing and phylogenetic analysis
Purified PCR products of the G. duodenalis tpi gene were sequenced bi-directionally using MACROGEN, (Korea), the chromatograms were analyzed and edited and the obtained sequences were aligned using MEGA Version X. The maximum likelihood algorithm was used for the phylogenetic analysis of the isolates from current study and reference ones. The phylogenetic tree was drawn using a neighbor-joining parameter model with the highest likelihood algorithm value. The reliability of clusters was evaluated using a bootstrap of 1000 iterations. Giardia muris (AF069565) was placed as an outgroup in the phylogenetic tree.
Statistical analysis
Collected data were analyzed by SPSS software (version 20, IBM Inc., USA). To determine the correlation between Giardia infection and subject variables such as age, sex, residence, symptomatology, number of chemotherapy cycles and type of cancers, the Chi-square test was used. P<0.05 was considered statistically significant.