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Epidemiological Study of Some Candida albicans Strains in Patients

Undergoing Chemotherapy by Using PCR Technique in Whole Blood

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Second Scientific Conference – Science College – Tikrit University 2012

Epidemiological Study of Some Candida albicans Strains in Patients

Undergoing Chemotherapy by Using PCR Technique in Whole Blood
Ahmad Salih Helal11 , Adnan F. AL-azawy2 , Thekraa Ahmad Hamadaa3
1
B. Med. Tech./Med. Lab. Sc. North oil company .
2
College of Science Dep. Of Biology , Tikrit university, Tikrit, Iraq .
3
College of medicine , Tikrit university, Tikrit, Iraq .

Abstract:
Candida species were common human commensals that can cause a wide spectrum of disease, major concern
was ahematogenously disseminated infection which was occurring with increased prevalence in
immunecompromised patients, although Candida albicans remains the most frequent cause of fungemia and
hematogenously disseminated candidiasis. In the presented study was 102 patient undergoing chemotherapy and
73 control outpatients returning Tikrit teaching hospital, were examined to determine the prevalence of
candidemia by using blood culture method from isolaltion, cultural identification, were 17.6% from cancer
patients undergoing chemotherapy positive for candidemia, while all control patient negative. Were83.4% from
rural area for province of Tikrit , in addition, were 61.2% from south direction Tikrit city and DNA extraction
from whole blood samples dependening on distribution cancer patients and candidemia to detected direct
diagnosis and universal strains in our epidemiological study, were 80% Candida albicans strains of ATCC
10261, 3153, A72, MEN, Ci002, CiOll, Ci012,CiO35,CiO60, and CiO61 by using PCR technique.
Key: Candidemia, Chemotherapy, Blood Culture, PCR.
Introduction
Opportunistic fungal infections have increased reproductive structures useful for identifying isolated
dramatically in recent years. This had often been as a fungi may take days to weeks to develop in culture,
result of advanced medical treatments, such as more and evaluation of these characteristics requires
intensive regimens of cancer therapy, complications expertise in mycology. Commercial methods used to
of abdominal or cardiothoracic surgery, prolonged identify yeast including API system require 2 to 3
broad spectrum antibiotic therapy, invasive devices days before biochemical reactions can be
such as indwelling catheters and prolonged hospital interpreted(5). In addition, their databases sometimes
stays(1,10). Under these conditions an antibiotic- are ambiguous. Molecular techniques utilizing
resistant replacement flora, including Candida amplification of target DNA, provide alternative
species, can proliferate in the gut and invade deep methods for diagnosis and identification of some
tissues from mucosal foci. This is especially the case organisms(6). PCR based detection of fungal DNA
when mucosal integrity has been disrupted due to sequences can be rapid, sensitive and specific (7).
chemotherapy or surgery. At least 200 fungal species The positions of the primers used for PCRs within the
have been identified as human pathogens but most C. albicans rDNA region are shown in Figure 1,
opportunistic fungal infections are caused by yeasts, synthetic oligonucleotide primers (from DNA
and the most important yeasts are Candida species(3). Express, Colorado State University) used for
Rapid identification of Candida isolates to the species amplification of the C. albicans NTS fragment were
level in the clinical laboratory has become more 5'-TAG CGA TGA GGT AGT GCA AGT (PSpA1)
important because the incidence of candidiasis and 5'-GCT GCA GCT ACG AAT GTT AG (PSpA2)
continues to rise in proportion to an increasing (8). Using purification of genomic DNA from fresh
number of patients at risk for infection withCadinda whole blood collected in EDTA, heparin anticoa-
albicans and recently, with innately azole-resistant gulant tubes and detected no adverse effects upon
non-albicans Candida species. Some Candida species subsequent manipulations of the DNA, including
including C. glabrata and C. krusei are emerging PCR(9). Anticoagulant blood samples may be stored
possibly because they are innately less susceptible to at 2–8°C for up to two months, but DNA yield will be
azole drugs (4) . Consequently, rapid identification to reduced with increasing length of storage(9).
the species level is necessary for more rapid and The yeast strains used in our study are listed in
effective antifungal therapy, to facilitate hospital Table 1. The identities and serotypes of the Candida
infection control measures and generally because strains were confirmed with an immune agglutination
epidemiological studies. Identification of this identification system (Iatron Laboratories, Tokyo,
increasing diversity of pathogen by conventional Japan). C. albicans A72, MEN, and CiO35 were
methods is often difficult and sometimes serotype B; all other C albicans strains were serotype
impossible(5). Morphological features and A(8).

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Second Scientific Conference – Science College – Tikrit University 2012

FIG. 1. (a) Physical map of the C. albicans rDNA region showing the positions of rRNA genes, the NTS
region, and the direction of transcription of rRNAs (from reference 11). (b) Expanded map of the region
showing restriction enzyme sites (from reference 2), the positions of primers used in PCR, and the
predicted sizes of the amplified products.

Materials and Methods:- stained and subcultured onto appropriate media for
Patients and control groups :102 patient aged 23-67 identification. Sabouraud’s agar for 48 h at 30°C.
years old were undergoing chemotherapy for different DNA extraction by used Wizard® Genomic DNA
types of cancer, and 73 outpatients control group Purification Kit (promega) (for 3ml blood sample
returning in Tikrit Teching Hospital from January Volume)(9) and a modification of the
2012 to May 2012. hexadecyltrimethyl-ammonium bromide (CTAB)
Specimens : 5ml of venous blood collected from buffer method(12,13).
patient and control group; 2.5 ml into EDTA (K3) Procedure.
storage at -32 C0 for DNA extraction in using for 1- Add 7.5ml of Cell Lysis Solution to a sterile 15ml
PCR technique and 2.5 ml for yeast blood culture to centrifuge tube.
determine the yeast infection in these two group. 2- Gently rock the tube of blood until thoroughly
Yeast isolation : slant of 5 ml of brain heart infusion mixed; then transfer blood to the tube containing the
agar overlaid with 20 ml of brain heart infusion broth Cell Lysis Solution. Invert the tube 5–6 times to mix.
(Difco) with chloramphincol provided by the 3- Incubate the mixture for 10 minutes at room
Bacteriology Department of the Teaching Tikrit temperature (invert 2–3 times once during the
Hospital. were processed for each contained. After incubation) to lyse the red blood cells Centrifuge at
inoculation of 2.5 ml of blood into the culture , they 2,000 × g for 10 minutes at room temperature
were vented and incubated in an upright position for 4- Remove and discard as much supernatant as
10 days at 300 C. Cultures were examined daily for possible without disturbing the visible white pellet.
visual evidence of growth, and each examination was Approximately 50–100μl of residual liquid will
followed by a gentle mixing of the blood-broth remain in the 15ml tube .
mixture over the agar slants. Visual growth was 5-repeat Steps 1–4 until pellet is white. There may be
some loss of DNA from frozen samples.

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Second Scientific Conference – Science College – Tikrit University 2012

6-Vortex the tube vigorously until the white blood 8-. Add 50μl of DNA Rehydration Solution.
cells are resuspended (10–15seconds). 9-. Add 1.5μl of RNase Solution to the purified DNA
7- Add700 µl of 2x CTAB buffer (100 mM Tris-HCl sample. Vortex the sample for 1second. Centrifuge
[pH 8], 1.4 M aCl, 25 mM EDTA, 2% CTAB),vortex briefly in a microcentrifuge for 5 seconds to collect
,transfer to in 1.5-ml microcentrifuge Tubes the liquid and incubate at 37°C for 15 minutes.
incubate 60 minutes at 65C0 . and 700 µl of phenol- 10-. Rehydrate the DNA by incubating at 65°C for 1
chloroform was added. The mixture was Vortexed 5 hour. Periodically mix the solution by gently tapping
minute again and centrifuged at 12,000 X g for 5 min. the tube. Alternatively, rehydrate the DNA by
The upper aqueous phase was transferred to a new incubating the solution overnight at room temperature
microcentrifuge tube, and the DNA was precipitated or at 4°C.
by adding an equal volume of isopropanol and 11-. Store the DNA at 2–8°C.
centrifuging at 12,000 X g for 10 min. The DNA
pellet was washed once in 70% ethanol (12).
The Used Primers in This Study (14).
No Code 5ˉ to 3ˉ
1. PConl AGT TTC GCG TAT GGT CTC CC
2. PCon2 GTT GCG GCC ATA TCT AGC AG
3. PSpA1 TAG CGA TGA GGT AGT GCA AGT
4. PSpA2 GCT GCA GCT ACG AAT GTT AG
The reaction mixture 20µL(2µL AccuPower® PCR by nanodroup (Thermo®) and 15µL deionizer
Pre Mix, 2µL(F,R)primer (1.25 ,uM each), water). 30 cycles 1:-94ċ-3m. 2:-94ċ-30sc,60ċ-45sc,
1µLtemplate 50ng purification and concentration 72ċ-1m. 3:-72ċ-5m.
Results and discussion showed in Table 2. While control group was
All 102 patients undergoing chemotherapy and negative results.
control group were tested blood culture classified
according to positive and negative blood culture as
Table 2. prevalence of Candidemia in patients undergoing chemotherapy.
Blood culture results for candidemia Patients Controls
No. % No. %
Positive 18 17.6 0 0
Negative 84 82.4 73 100
Total 102 100 73 100
2
X =5.77 P = 0.008 P˂ 0.01 High significant

Before discussing studies comparing different sensitivity or specificity, the fungal culture were the
techniques for culturing Candida spp. from blood, gold standard for diagnosis (16,15).
two points regarding their limitations must be made. The present study revealed that the rate of candidemia
First, none of these studies describe in detail in patients undergoing chemotherapy (as shown in
characteristics of the patients from whom positive table 2) through the isolation of Candida spp. from
cultures were obtained. Thus, it was impossible to the blood of these patients this finding less than that
determine whether blood culturing was more recorded in other studies; 32.1%(17) in Australia, 32%
effective in detecting fungemia in one population of in USA (18,19), these different results may be due to
patients compared with another. Frequency of the difference of techniques for blood culture as the
culturing and total volume of blood cultured during sample size to the volume of media and daily
an admission might influence the likelihood of inverting inoculated broth with blood sample. In
detecting fungemia more than the particular blood addition, the method used in our study by BHI
culture technique used (13,15). Although there was no broth/agar, that insider as a good significant to
similar study in Iraq, particularly in province of Tikrit screening by visional growth of candid..
for comparing this results to other study, this finding The relation between residence group and candidemia
give the importance of candidemia in patients revealed that there were 16.6% of infection in urban
undergoing chemotherapy and the significant from total group in South direction 61.2%, but 22.2%
mortality and morbidity of candidemia associated in urban from total group 83.4% as shown in table 3.
with this patients that have resulted in considerable Selected 50% of sampling from each rural, south and
efforts to use develop rapid, reliable diagnostic tests, other direction to detected (rural 2sample,south
in addition, to use blood culture to facilitate clinical 5sample and other direction 3sample) Candida
treatment decisions. Despite continued efforts to albicans strains as showed in table 1, by PCR
develop such tests, most tests developed to date lack technique and the prevalence that to region
genotyping.

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Second Scientific Conference – Science College – Tikrit University 2012

Table 3. prevalence of Candidemia in Residence.

Subjects Residences
Total
Urban Rural
South direction Other directions
No. % No. % No. % No. %
Patients 18 17.6 70 68.6 14 13.7 102 100
Positive blood 3 16.6 11 61.2 4 22.2 18 100
culture
X2 =19.8 P=0.003 P ˂ 0.01 High significant

In the presented study the highest rate of increasing Although the increased rates of candidemia in rural
candidemia in rural area was 82.4% . Table.3. This area there is variable of rate within different
may be due to highly contacting with animals that distribution of locations were the high rates recorded
normally found or infected with Candida species, In within rural areas of south direction from Tikrit
the current other study 75% isolation Candida spp. provinces, this may due to environmental factors or
from cow milk(20), poor sanitation and personal type of animal domestication that harbors Candida
hygiene’s with low knowledge’s about the species that affecting the spreading the infection.
transmission of Candida from the animals to human. PCR technique results:-

Table 4. Nanodruop Results.

data No. of DNA purfication
sample concentration A260/A280
ng/µL
14/05/12 1 81.3 1.27
14/05/12 2 120.5 1.88
14/05/12 3 91.9 2
14/05/12 4 66.1 1.9
14/05/12 5 158.9 1.58
14/05/12 6 61.3 2
14/05/12 7 33.7 1.09
14/05/12 8 85.4 1.91
14/05/12 9 50.2 1.87
14/05/12 10 68.3 1.9

Figure 3. Electrophoresis 2% agarose jel within 1X TBE at stage 1

V.45,mA 60,T.15 and stage 2 V.60 ,mA 90,T.120

The idea of epidemiological strain study beyond the pathogen were selected samples of whole blood of a
diagnosis of the colony identification to reduce the disease in our study, there were 5 samples isolated
cost and to demonstrate the level of strains in the from rural south direction, 3 rural other direction and

138
Second Scientific Conference – Science College – Tikrit University 2012

2 urban residence as shown in table 3, completed 10 detection and identification of the infecting species
sample whole blood from all residence patients in blood would facilitate prompt, appropriate
cancer undergoing chemotherapy and camdidemia treatment.
was detected by PCR Technique, were 80% Candida In conclusion, the PCR assay designed and tested as
albicans results and 20% non-albicans as show in described here provides a high sensitivity and
Figures 3. specificity for the detection of fungal DNA in blood
The epidemiological strain study by rapid diagnosis samples and rapidly identifies most Candida species.
of Candida species through DNA extraction of Thus, the vast majority of clinically relevant fungal
Candida from fresh blood sample of patients pathogens in the immunocompromised patient were
undergoing chemotherapy were 80% for Candida detected by this assay, distribution of Candida
albicans while 20% for non albicans candida. This albicans in patients undergoing chemotherapy was a
agree with geographic distribution of candida(21). high rate and Nan-albicans Candida as a significant
Identification of candidemia takes a minimum of 2 rate. BHI agar with broth for isolation was increased
days with an optimal blood culture system(9), so there visual growth screening, using IgG and IgM antibody
was a need for a rapid, sensitive, and specific test to by ELISA technique increased incidence of early
aid in diagnosis of disseminated yeast infection. detection candidemia, CTAB buffer as instead
DNA- diagnostic methods not sensitive and specific lyticase enzyme in promega extraction DNA kit, in
only but also, have the potential decrease time taken additional, incubation the buffer at 650C in 60
for the laboratory identification of pathogens that minutes was good methods, PCR technique were very
were slowly growing or difficult to culture. Such tests good methods for diagnosis candidemia in patients
may detect nonviable and non cultureable cells as undergoing chemotherapy, The strains of Candida
well as viable cells. Drug susceptibilities of Candida albicans detection by PCR was same strains in USA,
species vary; for example, most C. krusei strains are Italy, UK and New Zealand.
resistant to fluconazole(22). Therefore, earlier
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‫‪3‬‬
‫اﺣﻣد ﺻﺎﻟﺢ ﻫﻼل‪ ، 1‬ﻋدﻧﺎن ﻓﺎﺿل اﻟﻌزاوي‪ ، 2‬ذﻛرى اﺣﻣد ﺣﻣﺎدة‬
‫ﺷرﻛﺔ ﻧﻔط اﻟﺷﻣﺎل ‪ ،‬اﻟﻌراق‬ ‫‪1‬‬

‫ﻛﻠﯾﺔ اﻟﻌﻠوم‪ ،‬ﺟﺎﻣﻌﺔ ﺗﻛرﯾت ‪ ،‬ﺗﻛرﯾت ‪ ،‬اﻟﻌراق‬ ‫‪2‬‬

‫ﻛﻠﯾﺔ اﻟطب ‪ ،‬ﺟﺎﻣﻌﺔ ﺗﻛرﯾت ‪ ،‬اﻟﻌراق‬ ‫‪3‬‬

‫اﻟﻣﻠﺧص‬
‫أﺟﻧﺎس ﺧﻣﺎﺋر اﻟﻣﺑﯾﺿﺔ اﻻﻛﺛر ﺗﻌﺎﯾﺷﺎ ﻟﻸﻧﺳﺎن واﻟﺗﻲ ﺗﺳﺑب اﻟﻌدﯾد ﻣن أﻷﻣراض اﻟواﺳﻌﺔ اﻻﻧﺗﺷﺎر وﺧﺎﺻﺔ اﻟﻣرﺿﻰ ﻏﯾر اﻟﺳوي ﻣﻧﺎﻋﯾﺎ ‪.‬واﻟﻘﻠق اﻟﺑﺎﻟﻎ‬
‫ﻣن ﺗداﺧل واﻧﺗﺷﺎر اﻷﺻﺎﺑﺔ واﻟﺗﻲ ﺗظﻬر ﺿراوة ﻋﺎﻟﯾﺔ ﻓﻲ ﻣرﺿﻰ ﻏﯾر اﻟﺳوي ﻣﻧﺎﻋﯾﺎ‪.‬وان اﻟﻣﺑﯾﺿﺔ اﻟﺑﯾﺿﺎء اﻻﻛﺛر اﺻﺎﺑﺔ ﻣن ﺑﯾن اﺻﻧﺎف‬
‫اﻟﻣﺑﯾﺿﺎت‪ .‬اﺟرﯾت اﻟدراﺳﺔ ﻋﻠﻰ ‪ 102‬ﻣرﯾﺿﺎ ﺧﺎﺿﻌﯾن ﻟﻠﻌﻼج اﻟﻛﯾﻣﯾﺎﺋﻲ واﻟﻣﺻﺎﺑﯾن ﺑﻣﺧﺗﻠف اﻧواع اﻟﺳرطﺎن‪ ،‬و‪ 73‬ﻣرﯾﺿﺎ ﻣراﺟﻊ ﻟﻠﻣﺳﺗﺷﻔﻰ‬
‫ﻛﻣﺟﻣوﻋﺔ ﺳﯾطرة واﻟﻠذﯾن ارﺗﺎدو اﻟﻰ ﻣﺳﺗﺷﻔﻰ ﺗﻛرﯾت اﻟﺗﻌﻠﯾﻣﻲ ﺧﻼل اﻟﻔﺗرة ﻣن ﻛﺎﻧون اﻟﺛﺎﻧﻲ ‪ 2012‬اﻟﻰ اﯾﺎر ‪. 2012‬اظﻬرت اﻟدراﺳﺔ واﻟﺗﻲ اﺟرﯾت‬
‫ﻋﻠﻰ ﻧﻣﺎذج اﻟدم ﻟﻠﻣرﺿﻰ ﻟﻌزل اﻟﺧﻣﺎﺋر اﻟﻣﺑﯾﺿﺔ ﺑﺎﺳﺗﺧدام ﺑﻌض اﻻوﺳﺎط اﻟزرﻋﯾﺔ اﻟﺧﺎﺻﺔ و ﺗﺷﺧﯾﺻﻬﺎ ‪ ،‬وﺟد ‪ %17.6‬ﻣن اﻟﻣﺑﯾﺿﺎت ﻓﻲ اﻟدم‬
‫وﻟم ﺗﺟد أي أﺻﺎﺑﺔ ﻓﻲ ﻣﺟﻣوﻋﺔ اﻟﺳﯾطرة‪ ،‬وأن ‪ %83.4‬ﻣن اﻷﺻﺎﺑﺔ ﻫم ﻣن اﻟﻣﻧﺎطق اﻟرﯾﻔﯾﺔ ﻟﻠﻣﺣﺎﻓظﺔ وأﺿﺎﻓﺗﺎ ﻟذاﻟك ‪ %61.2‬ﻣن اﻷﺗﺟﺎﻩ اﻟﺟﻧوﺑﻲ‬
‫ﻟﻠﻣدﯾﻧﺔ ﺗﻛرﯾت واﺳﺗﺧﻼص اﻟﺣﻣض اﻟﻧووي ﻣن ﻧﻣﺎذج اﻟدم اﻟﻌﺷرة أﻋﺗﻣﺎدا ﻋﻠﻰ اﻟﺗوزﯾﻊ وأﻧﺗﺷﺎر ﻣرﺿﻰ اﻟﺳرطﺎن اﻟﻣﺻﺎﺑﯾن ﻟﻠﺗﺄﻛﯾد اﻟﺗﺷﺧﯾص اﻟﺳرﯾﻊ‬
‫و ﻟﻠﺗﻌرف ﺳﻼﻻت اﻟﻣﺑﯾﺿﺔ اﻟﺑﯾﺿﺎء ﻛﺎﻧت ﻣن ﺿﻣن اﻟﺳﻼﻻت اﻟﺷﺎﺋﻌﺔ ﻋﺎﻟﻣﯾﺎ ﺑﺎﺳﺗﺧدام ﺗﻘﻧﯾﺔ ﺑﻠﻣرة اﻟﺗﻔﺎﻋل اﻟﻣﺗﺳﻠﺳل‪.‬‬

‫‪140‬‬

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