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