Molecular Identification
The DNA was extracted from the isolated yeasts using the genomic DNA extraction mini kit (Favor PrepTM Fungi/Yeast Genomic DNA-Canada). The extracted DNA was migrated using agarose gel electrophoresis. The PCR amplification was performed in a final volume of 25 µl. Each reaction consists of 12.5 µl of Green Master Mix, 0.5 µl of MgCl2, 0.5 µl of each forward (ITS1,F´5-TCC GTA GGT GAA CCT GCG G-´3) and reverse (ITS4, R-5´ TCC GCT TAT TGA TAT GC-´3) primer, and 1.5 µl of the template DNA. The reaction volume was completed to the final volume with 9.5 µl. The thermal cycles were programmed as follows: an initial denaturation step at 94ºC for 5 min, a second step of 25 denaturation cycles at 94ºC for 30 s of annealing at 56ºC for 45 s, and extraction at 72ºC for 1 min with a last step at 72ºC for 7 min. The amplified products were visualised using 0.8% agarose gel electrophoresis in the TBE buffer and then stained with 0.2 µl of ethidium bromide and photographed [40]. The PCR products of the isolated strains were sent to Macrogen Company (hhtt://dna. Macrogen. Com, South Korea) for sequencing.

Results and Discussion

Out of the 29 Candida strains that were recovered from the oral cavity of male and female children with leukaemia before and after chemotherapy, three isolates (10.3%) were identified as C. africana based on morphological, biochemical, and molecular features.
As shown in Table 1, the ICL21 isolate was obtained from the oral cavity of a 5.5-year-old female with acute lymphoblastic leukaemia (ALL) after receiving chemotherapy. Before induction, there was no signs of infection with candidiasis, as the culture of the concerned sample was negative. The ICL26 was isolated from oral swabs taken from a 10-year-old male afflicted with acute myeloid leukaemia (AML) after chemotherapy. The patient showed the typical oral signs and symptoms, including pseudomembranous candidiasis on the tongue and diffused painful erythematous mucositis. Unfortunately, this patient died after 9 months of chemotherapy. The third isolate ICL27 was recovered from the oral Candida growth of a 2.8-year-old male suffering from ALL before having any course of chemotherapy.
All patients were examined thoroughly by the physicians and subjected to a specific protocol of chemotherapy including methotrexate (MTX), 6-mercaptopurine (6MP), and vincristine (VCR).
Developing such diverse and frequent oral yeast infections and mucositis is attributed to the chemotherapy courses, which increases the number and diversity of such infections [41, 42]. Various prophylactic schedules, including oral hygiene, Mycostatin droplets, and chlorohexidine, may be considered to reduce oral opportunistic pathogens [43-45].
To our knowledge, the present findings represent the first recoveredC. africana isolates from the oral cavity of immunocompromised patients with leukaemia, and the first report of the species in Iraq. To date, the majority of C. africana isolates have been registered from female genital samples. However, it has also been reported in other sites of the human body [2, 10, 21, 12].
Similar to Romeo and Criseo [46], our data showed that C. albicans was the most frequent species that comprised 89.6% of the strains, followed by C. africana at 10.3%. This study did not recover any C. dubliniensis strains, which is a clear contradiction with the findings by Romeo and Criseo [46].
The present isolates of C. africana possess several diagnostic features that distinguish them from other typical strains of C. albicans . These phenotypic characteristics are shown in Table 2, including negative results for chlamydospore on corn-meal agar with 1% Tween 80, no growth at 42ºC, the inability to assimilate the amino sugars N-acetylglucosamine and glucosamine as well as trehalose and DL-lactate, and small, bluish-green colonies on the CHROMagar (Fig. 1A).
These results agree with those obtained by previous studies [2, 3, 5, 7-9]. However, our C. africana isolates are associated withC. albicans in the positive germ-tube formation, growing well at 30ºC and 37ºC. These features are identical to those described by Tietz et al. [2], Romeo and Criseo [8, 47], and Borman et al. [9]. Colonies of C. africana grow well on Sabouraud dextrose agar at 28ºC, as small cream-coloured (Fig. 1B). pseudohyphae are rare.
Specific universal primers for Candida strains were employed to amplify the ITS1-5.8S-ITS2 regions of rDNA genes, giving the PCR product a molecular size of 572 bp (Fig. 2). Nonetheless, comparing all sequences that were obtained in this study with the database of GenBank and CBS revealed that three (10.34%) out of 29 isolates were identified as C. africana, and they showed >99% sequence similarity with that of the C. africana strain (CBS 8781), while the rest of the isolates (89.65%) belonged to C. albicans, which were mostly analogous to the strain C. albicans (ATCC 18804).
In addition to the phynotypic similarities between C. africanaand other species of C. albicans complex, there was a high percentage of molecular proximity among them. Therefore, based on several reports and the sequencing of the D1-D2 regions, the C. africana was considered a variant and was not discriminated from theC. albicans species complex because the level of dissimilarity was too small to be a distinct species [7, 8, 47, 49]. However, several investigators showed that molecular diagnostic methods, such as sequencing the internal transcribed spacer region 2 or the PCR amplicon length of the HWP1 could be used for definitive identification ofC. africana [9, 12, 14, 16, 48, 50], while Rodriguez-Leguizmon et al. [15] indicated that the identified isolates based on sequencing the D1-D2 region of the rDNA and the HWP1 gene were in agreement with those for C. albicans . Therefore, their study recommended using the matrix-assisted laser desorption-ionisation time of flight mass spectrophotometry (MALDI-TOF MS) and phenotypic and molecular identification to separate the three species of C. albicans, C. africana, and C. dubliniensis.
In our study, the isolation of C. africana in the oral cavity of the patients with leukaemia was not expected because it has been documented that the species is common in human genital organs, particularly in vaginal samples [1-3,7,9,10,18,20, 21,47,49]. However, it was possible to use genes and more to separate this species from C. albicans. In contrast to local and international studies regarding C. dubliniensis, which primarily occurs in the oral cavity [8, 51, 52], the techniques employed in this work did not show the presence of this species. Abdul-Raheem [32] and Aldossary et al. [36] recovered this species from the oral cavity of diabetic and cancer patients, respectively. Therefore, isolating C. africana from the oral cavity is a surprising finding, as it is the first study reporting the presence of C. africana in the oral cavities of immunocompromised patients.Candida africana has been reported from Saudi Arabia (west border of Iraq) [3] and recently from Iran (east border of Iraq) [4, 5] as an etiological agent of vulvovaginal candidiasis, while it was absent in all re-examined vaginal specimens in Turkey [22]. Hence, the present findings represent the first report of C. africana from the oral cavity of immunocompromised patients with leukaemia receiving chemotherapy and the first record of the species in Iraq. However, further epidemiological, clinical, phenotypical, and molecular inspections based on a larger sample size are strongly recommended to uncover the actual recrudescence of C. africana in clinical specimens.
Disclosure statement: The authors declare that they have no conflicts of interest
Acknowledgements The authors wish to thank all the children and their parents for their generous cooperation with us during the sample collection. We extend a sincere thanks to the staff of Basrah Children’s Specialty Hospital for their kind help and facilities