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The occurrence of vulvovaginal Candida species and their antifungal susceptibility pattern in HIV seropositive women in Ahvaz, Southwest Iran

Published:March 07, 2020DOI:https://doi.org/10.1016/j.cegh.2020.02.021

      Abstract

      Background

      Vulvovaginal candidiasis (VVC) is a prevalent infection among HIV-seropositive women that still needs to be subjected to more researches. This study is aimed at determining the occurrence of vulvovaginal Candida species and their antifungal susceptibility pattern in HIV-seropositive women from the Behavioral Disease Counseling Center in Ahvaz, Iran.

      Method

      In this analytical cross-sectional study, 85 HIV-seropositive women with the mean age of 26.15 years (SD. 9.60) were evaluated. Vaginal swabs were collected from women to identify various Candida species through conventional methods and API 20 C AUX system. Antifungal susceptibility testing was done on specimens against Amphotericin B and Azole antifungals. Eventually, all data were analyzed by SPSS.

      Results

      The occurrence of vulvovaginal Candida species was 34.11% (29) among 85 HIV-seropositive women. Out of 29 Candida spp., 18 (62.06%) C. albicans, 6 (20.68%) C. glabrata, 4 (13.79%) C. dubliniensis, and 1 (3.44%) C. krusei were identified. There was no significant association between VVC and some medical variables such as antiretroviral therapy, birth control methods, underlying diseases, and CD4 count (PV > 0.05). However, there was a significant association between VVC and age and pregnancy (PV < 0.05). The most Candida spp. showed considerably resistant to Amphotericin B and Itraconazole while all species were susceptible to Caspofungin.

      Conclusion

      The high occurrence of Candida spp. (34.11%) among HIV-seropositive women is notable, with the highest frequency of C. albicans, C. glabrata and C. dubliniensis, respectively. Also, their notable resistance to Amphotericin B and Azole antifungals necessitates more effective treatments for VVC, particularly in pregnant women.

      Keywords

      1. Introduction

      Vulvovaginal candidiasis (VVC) is the most prevalent gynecological infections among childbearing women.
      • Khan M.
      • Ahmed J.
      • Gul A.
      • Ikram A.
      • Lalani F.K.
      Antifungal susceptibility testing of vulvovaginal Candida species among women attending antenatal clinic in tertiary care hospitals of Peshawar.
      The frequency of vaginal Candida spp. was significantly higher among HIV-seropositive women than HIV-seronegative women.
      • Ohmit S.-E.
      • Sobel J.-D.
      • Schuman P.
      • et al.
      Longitudinal study of mucosal Candida species colonization and candidiasis among human immunodeficiency virus (HIV)-Seropositive and at-risk HIV-seronegative women.
      Mucosal candidiasis (vaginal, oropharyngeal, and esophageal) is one of the most common infections among HIV-infected patients.
      • Sangeorzan J.A.
      • Bradley S.F.
      • He X.
      • et al.
      Epidemiology of oralcandidiasis in HIV-infected patients: colonization, infection, treatment,and emergence of fluconazole resistance.
      • Fichtenbaum C.J.
      • Powderly W.G.
      Refractory mucosal candidiasis in patients with human immunodeficiency virus infection.
      • Moore R.D.
      • Chaisson R.E.
      Natural history of opportunistic disease in anHIV-infected urban clinical cohort.
      Various behavioral and biological risk factors may predispose individuals to mucosal candidiasis.
      • Reed B.D.
      • Gorenflo D.W.
      • Gillespie B.W.
      • Pierson C.L.
      • Zazove P.
      Sexual behaviors and other risk factors for Candida vulvovaginitis.
      For example, risk factors including high sexual activity, pregnancy, diabetes, and or using antibiotics can arise the risk of vaginal candidiasis.
      • Lifson A.R.
      • Hilton J.F.
      • Westenhouse J.L.
      • et al.
      Time from HIV seroconversion to oral candidiasis or hairy leukoplakia amonghomosexual and bisexual men enrolled in three prospective cohorts.
      ,
      • Schuman P.
      • Sobel J.D.
      • Ohmit S.E.
      • et al.
      Mucosal candidal colonization and candidiasis in women with or at risk for human immunodeficiency virus infection. HIV Epidemiology Research Study (HERS) Group.
      Moreover, cell-mediated immunodeficiency increases the risk of mucosal Candida colonization and candidiasis.
      • Schuman P.
      • Sobel J.D.
      • Ohmit S.E.
      • et al.
      Mucosal candidal colonization and candidiasis in women with or at risk for human immunodeficiency virus infection. HIV Epidemiology Research Study (HERS) Group.
      ,
      • Schuman P.
      • Sobel J.D.
      • Ohmit S.E.
      • et al.
      HIV Epidemiology Research Study (HERS) Group. Mucosal candidal colonization and candidiasis in women with or at risk for human immunodeficiency virus infection.
      The spectrum of Candidiasis is varied from asymptomatic colonization to symptomatic form. The low CD4 T-lymphocyte count has previously been shown as the main risk factor for the development of mucosal candidiasis.
      • Anwar K.P.
      • Malik A.
      • Subhan K.H.
      Profile of candidiasis in HIV infected patients.
      Despite the therapeutic effects of antiretroviral therapy (ART) on the AIDS-related infectious,
      • Michelet C.
      • Arvieux C.
      • François C.
      • et al.
      Opportunistic infections occurring during highly active antiretroviral treatment.
      Candidiasis still remains a common opportunistic infection in HIV seropositive patients,
      • Morgan J.
      Global trends in candidemia: review of reports from 1995–2005.
      mostly because of increasing antibiotic resistance. The high prevalence of candidiasis (≥50%) was reported among HIV positive patients in different regions,
      • Pruthvi B.C.
      • Vikram S.
      • Suman S.K.
      • Jayaprakash B.
      • Rau N.R.
      13th International Congress on Infectious Diseases. Spectrum of Clinical Presentation and Opportunistic Infections in HIV.
      • Nagalingeswaran K.
      • Solomon S.
      • Madhivanan P.
      • et al.
      Correlation between plasma viral load and CD4+T cell count to opportunistic infections in persons with HIV in South India.
      • Singh A.
      • Bairy I.
      • Shivananda P.G.
      Spectrum of opportunistic infections in AIDS cases.
      • Pandey S.
      • Sundar S.
      • Hasan H.
      • Shankar R.
      • Singh S.P.
      Clinical profile and opportunistic infection in HIV/AIDS patients attending SS Hospital, Varanasi.
      and C. albicans as the most common species.
      • Anwar K.P.
      • Malik A.
      • Subhan K.H.
      Profile of candidiasis in HIV infected patients.
      ,
      • Pruthvi B.C.
      • Vikram S.
      • Suman S.K.
      • Jayaprakash B.
      • Rau N.R.
      13th International Congress on Infectious Diseases. Spectrum of Clinical Presentation and Opportunistic Infections in HIV.
      ,
      • Pandey S.
      • Sundar S.
      • Hasan H.
      • Shankar R.
      • Singh S.P.
      Clinical profile and opportunistic infection in HIV/AIDS patients attending SS Hospital, Varanasi.
      ,
      • Nurat A.A.
      • Babalola G.O.
      • Shittu M.O.
      • Tijani M.A.
      • Adekola S.A.
      Detection and epidemiology of vulvovaginal candidiasis among asymptomatic pregnant women attending a tertiary hospital in Ogbomoso, Nigeria.
      Based on the researches in different regions of Iran, the prevalence of VVC and Candida spp. have been reported, for example Kermanshah (20%), Mazandaran (28.2%), Esfahan (54.6%), and Tehran (87.5%), respectively.
      • Faraji R.
      • Rahimi M.A.
      • Rezvanmadani F.
      • Hashemi M.
      Prevalence of vaginal candidiasis infection in diabetic women.
      • Hedayati M.T.
      • Taheri Z.
      • Galinimoghadam T.
      • Aghili S.R.
      • Yazdani Cherati J.
      • Mosayebi E.
      Isolation of different species of Candida in patients with vulvovaginal candidiasis from sari, Iran.
      • Pouladian S.
      • Movahedi M.
      • Mohammadi R.
      Clinical and mycological study of vulvovaginal candidiasis (VVC); identification of clinical isolates by polymerase chain reaction-fragment size polymorphyim (PCR-FSP) technique.
      • Mahmoudi Rad M.
      • Zafarghandi A.
      • Amel Zabihi M.
      • Tavallaee M.
      • Mirdamadi Y.
      Identification of Candida species associated with vulvovaginal candidiasis by multiplex PCR.
      However, few data are available regarding the rates of vulvovaginal candidiasis and or Candida species among HIV-infected patients, especially from Iran. Furthermore, the increasing global risk of antibiotic-resistant infections particularly azole-resistant Candida species
      • Sobel J.D.
      • Ohmit S.E.
      • Schuman P.
      • et al.
      The evolution of Candida species and fluconazole susceptibility among oral and vaginal isolates recovered from human immunodeficiency virus (HIV)-seropositive and at-risk HIV-seronegative women.
      ,
      • Cassone A.
      • Cauda R.
      Candida and candidiasis in HIV-infected patients: where commensalism, opportunistic behavior and frank pathogenicity lose their borders.
      necessitate assessing their antifungal susceptibility pattern in HIV-infected patients. So, the present study report the occurrence of vulvovaginal Candida species and their antifungal susceptibility pattern in HIV-seropositive women from the Behavioral Disease Counseling Center in Ahvaz, Iran.

      2. Materials and methods

      In this descriptive-analytic and cross-sectional study, 85 HIV-seropositive women with the age range between 16 and 60 years (26.15 ± 9.60) were evaluated, who were referred to the Behavioral Disease Counseling Center in Ahvaz. This study was approved by the ethics committee of Ahvaz Jundishapur University of Medical Sciences with Ethical Code: ajums.REC.1396.410. The informed consent forms were signed by all participants after explaining its contents.
      Vaginal swab specimens were collected from women to identify various Candida species through conventional methods including culturing on the mediums of CHROMagar Candida or Cornmeal agar plus %1 Tween 80, Germ tube test and API 20 C AUX system.
      • 1.
        Vulvovaginal Candida species
      Three vaginal swabs were taken from the posterior vaginal fornix, and one of them was used for direct smear examination, and the others were aerobically inoculated on sabouraud dextrose agar (SDA; Zist Kavosh Iranian Company, IRAN) at 37 °C. In order to examination of vaginal candidiasis swabs, 10% Potassium hydroxide preparation (KOH preparation; Zist Kavosh Iranian Company, IRAN), Gram staining as well as microscopic evaluation were used. The second swab was cultured into SDA (Zist Kavosh Iranian Company, IRAN) and SDA with chloramphenicol (Zist Kavosh Iranian Company, IRAN), and then incubated for 48 h at 27 °C. The pure colonies were isolated and confirmed on Gram staining. For detecting Candida spp., isolated Candida colonies were subcultured onto differential CHROM agar Candida medium for 24 h at 37 °C (based on colony color of growing Candida spp.).
      • 1.1.
        Candida Strains
      The API 20 C AUX system (Bio merieux, France) was used for identification and confirmation of Candida species (based on manufacturer guidelines of kit). The strips were read after incubation at 30 °C for 48–72 h. Based on this method, C. albicans (green colonies), C. dublinensis (dark green colonies), Candida glabrata (purple colonies), Candida krusei (pink colonies) were detected.
      • 2.
        The association between medical variables and vulvovaginal candidiasis
      The characteristics and some medical variables of women were evaluated, including the usage history of antibiotics, antiretroviral therapy, history or presence of underlying diseases such as diabetes, CD4 count (less or greater than 200 cells/mm3) and duration of AIDS.
      • 3.
        Antifungal susceptibility test
      Antifungal susceptibility testing was done on specimens against various antifungals by broth microdilution assay and according to M27 A3 CLSI protocol.
      An overnight culture of isolates was prepared in Sabouraud dextrose agar (Merck, Germany). A suspension of each isolate prepared and was adjusted to 0.5 McFarland standard. A 100 μL of each serial dilution of antifungals including, amphotericin B (Sigma - Aldrich, Germany) (16–0.062 μg/mL), caspofungin (Sigma - Aldrich, Germany) (4–0.031 μg/mL), itraconazole (Sigma - Aldrich, Germany) (16–0.125 μg/mL) and fluconazole (Serva, USA) (64–0.5 μg/mL) was inoculated into each well in 96 wells microplate. Then, 100 μL of diluted suspension was added into each well and incubated at 35 °C for 24–48 h. Minimum inhibitory concentration (MIC) rage, MIC50, MIC90 and MICGM were calculated.
      • 4.
        Data analysis
      All data were analyzed using SPSS ver. 22. The data related to qualitative and quantitative variables were respectively reported as frequency or percentage, and mean ± standard deviation.
      The primary assessment normal distribution of variables by Kolmogorov-Smirnov Test showed that none of them follows a normal distribution. So, the results and assumptions were eventually analyzed by Mann-Whitney U test and Chi-Square test with considering PV < 0.05 as statistically significant.

      3. Results

      The occurrence of vulvovaginal Candida species was 34.11% (29) among 85 HIV seropositive women. Out of 29 Candida spp., 18 (62.06%) C. albicans, 6 (20.68%) C. glabrata, 4 (13.79%) C. dubliniensis, and 1 (3.44%) C. krusei were identified. There was no significant association between vulvovaginal candidiasis and some medical variables, including the usage history of antibiotics, antiretroviral therapy, birth control methods, history or presence of underlying diseases such as diabetes, CD4 count (less or greater than 200 cells/mm3) and duration of AIDS [Table 1].
      Table 1The statistically association between vulvovaginal candidiasis and some medical variables.
      Medical VariablesHIV-infected women with candidiasis (n = 29)HIV-infected women without candidiasis (n = 29, randomly selected)P Value
      Duration of AIDS (month)29.17 ± 17.5931.34 ± 16.790.5646 MWW
      AGE (year)24.68 ± 8.9933.96 ± 15.590.0298 MWW*
      CD4 > 200 cells/mm3430.68 ± 364.69455.86 ± 377.150.9566 MWW
      CD4 ≤200 cells/mm3424.65 ± 359.39433.96 ± 364.490.8825 MWW
      The usage history of
       _ Antibiotics17 (58.62%)22 (75.86%)0.1619 c
       _ Birth control pills2 (6.89%)4 (13.79%)0.3885 c
       _ Antiretroviral therapy20 (68.96%)25 (86.20%)0.1154 c
       _ Contraception (Birth control methods)16 (55.17%)17 (58.62%)0.7909 c
      History or presence of
       _Underlying diseases5 (17.24%)9 (31.03%)0.2197 c
       _Pregnancy17 (55.17%)1 (3.44%)<0.0001 c ***
       _Diabetes4 (13.79%)2 (6.89%)0.3885 c
      MWW: Mann-Whitney U test.
      C: Chi-square test.
      *PV < 0.05 is considered as statistically significant value.
      However, there was a significant association between vulvovaginal candidiasis and age and pregnancy (PV < 0.05). In this regard, the mean age of HIV-infected women with candidiasis (24.68 ± 8.99) was considerably less than women without candidiasis (33.96 ± 15.59). The most Candida spp. showed considerably resistant to Amphotericin B and Itraconazole while all species showed antifungal susceptibility to Caspofungin. Although, C. dubliniensis showed dose-dependent resistance to Itraconazole after 48 h growing, it was absolutely resistance after 72 h growing (Fig. 1).
      Fig. 1
      Fig. 1Antifungal susceptibility of Candida specimens against various antifungals.

      4. Discussion

      Many studies have reported the prevalence of candidiasis among women from different regions. Nurat et al.'s study reported that the prevalence of VVC was 25% among asymptomatic pregnant women in Nigeria. In this regard, the prevalence of candidiasis in women in the age range of 20–29 years (33.8%) was more than those in the age range of 30–39 years (24.3%).
      • Nurat A.A.
      • Babalola G.O.
      • Shittu M.O.
      • Tijani M.A.
      • Adekola S.A.
      Detection and epidemiology of vulvovaginal candidiasis among asymptomatic pregnant women attending a tertiary hospital in Ogbomoso, Nigeria.
      In another study, the prevalence of VVC was reported 30% in Nigeria
      • Okonkwo N.
      • Umeanaeto P.
      Prevalence of vaginal candidiasis among pregnant women in Nnewi Town of Anambra State, Nigeria.
      and 30.7% in Jamaica.
      • Kamara P.
      • Hylton-Kong T.
      • Brathwaite A.
      • et al.
      Vaginal infections in pregnant women in Jamaica: prevalence and risk factors.
      Beigi et al.'s study revealed that asymptomatic VVC was about 5–30% in Ohio, USA.
      • Beigi R.H.
      • Meyn L.A.
      • Moore D.M.
      • Krohn M.A.
      • Hillier S.L.
      Vaginal yeast colonization in nonpregnant women: a longitudinal study.
      A high rate of VVC was reported in women in the age range of the 20–29 years in many studies,
      • Nurat A.A.
      • Babalola G.O.
      • Shittu M.O.
      • Tijani M.A.
      • Adekola S.A.
      Detection and epidemiology of vulvovaginal candidiasis among asymptomatic pregnant women attending a tertiary hospital in Ogbomoso, Nigeria.
      ,
      • Nelson M.
      • Wanjiru W.
      • Margaret M.W.
      Prevalence of vaginal candidiasis and determination of the occurrence of Candida species in pregnant women attending the antenatal clinic of Thika District Hospital, Kenya.
      which was consistent with our findings (24.68 ± 8.99 years), probably due to drug usage and or contraceptives. Spinillo et al. reported a high rate of Candidiasis among married women between 30 and 45 years with frequent sexual activity, reproductive history, and taking contraceptives.
      • Spinillo A.
      • Capuzzo E.
      • Nicola S.
      • Baltaro F.
      • Ferrari A.
      • Monaco A.
      The impact of oral contraception on vulvovaginal candidiasis.
      Our findings showed that the rate of pregnancy is significantly more frequent among women with VVC. In this regard, Dias et al.'s study was reported the frequency distribution of 92.3% C. albicans, 3.3% C. krusei, 2.2% C. glabrata, 1.1% C. parapsilosis and 1.1% C. tropicalis among pregnant women with VVC in Brazil.
      • Dias L.B.
      • Melhem MdSC.
      • Szeszs M.W.
      • Meirelles Filho J.
      • Hahn R.C.
      Vulvovaginal candidiasis in Mato Grosso, Brazil: pregnancy status, causative species and drugs tests.
      Moreover, other studies were reported C. albicans as the most prevalent isolate from VVC in HIV-infected and uninfected women (80–90%).
      • Sobel J.D.
      Vulvovaginal candidosis.
      ,
      • Merenstein D.
      • Hu H.
      • Wang C.
      • et al.
      Colonization by Candida species of the oral and vaginal mucosa in HIV-infected and noninfected women.
      Oliveira et al. reported a higher frequency of Candida spp. among HIV-infected women (29.7%) than controls (14.5%) in Brazil. Based on their findings, C. albicans was the most frequent isolated species in both HIV-infected (52.3%) and uninfected women (85.7%), followed by C. parapsolis in 17.6% and 14.3%, respectively. However, the frequency of Candida spp. colonization and VVC were similar in both groups. Only the isolate of C. glabrata has shown resistant to fluconazole while others were susceptible to Amphotericin B, Voriconazole, Flucytosine, and Caspofungin
      • Oliveira P.M.
      • Mascarenhas R.E.
      • Lacroix C.
      • et al.
      Candida species isolated from the vaginal mucosa of HIV-infected women in Salvador, Bahia, Brazil.
      .
      In regard to the frequency of C. albicans, our findings were in consistent with the mentioned studies. Our study reported the frequency distribution of 18 (62.06%) C. albicans, 6 (20.68%) C. glabrata, 4 (13.79%) C. dubliniensis, and 1 (3.44%) C. krusei among HIV seropositive women with VVC. The most Candida spp. showed considerable resistant to Amphotericin B and Itraconazole but antifungal susceptibility to Caspofungin. Although, there are similar evidences of Azole antifungal resistance in Candida spp in other studies too,
      • Oliveira P.M.
      • Mascarenhas R.E.
      • Lacroix C.
      • et al.
      Candida species isolated from the vaginal mucosa of HIV-infected women in Salvador, Bahia, Brazil.
      • Richter S.S.
      • Galask R.P.
      • Messer S.A.
      • Hollis R.J.
      • Diekema D.J.
      • Pfaller M.A.
      Antifungal susceptibilities of Candida species causing vulvovaginitis and epidemiology of recurrent cases.
      • Yesudhason B.L.
      • Mohanra M.K.
      Candida tropicalis as a predominant isolate from clinical specimens and its antifungal susceptibility pattern in a tertiary care hospital in Southern India.
      a considerable resistant to Amphotericin B was rarely reported. Hence, our findings emphasize on the use of non- Amphotericin B therapy in addition to non-azole therapy.
      According to Badiee et al.'s study in Shiraz, C. albicans (50%) followed by C. glabrata (21.4%), C. dubliniensis (13.3%), C. krusei (9.8%), C. kefyr (3.1%), C. parapsilosis (1.6%), and C. tropicalis (0.8%) were the most prevalent isolated species from mucosal sites in HIV positive patients. All species showed notable sensitivity to amphotericin B, nystatin, voriconazole, ketoconazole and caspofungin while some isolates were resistance to fluconazole and itraconazole.
      • Badiee P.
      • Alborzi A.
      • Davarpanah M.A.
      • Shakiba E.
      Distributions and antifungal susceptibility of Candida species from mucosal sites in HIV positive patients.
      Their findings were significantly similar to our findings. However, the notable resistant to Amphotericin B was a rare issue in our study.
      The frequency of Candida spp colonization in HIV-infected women from United States and Italy was in a higher range (81%–92%),
      • Sobel J.D.
      • Ohmit S.E.
      • Schuman P.
      • et al.
      The evolution of Candida spp. and fluconazole susceptibility among oral and vaginal isolates recovered from human immunodeficiency virus (HIV)-seropositive and at-risk HIV-seronegative women.
      ,
      • Beltrame A.
      • Matteelli A.
      • Carvalho A.C.
      • et al.
      Vaginal colonization with Candida spp. in human immunodeficiency virus-infected women: a cohort study.
      compared to our finding (34.11%). Such variation could be due to the variation in the immune status of people in various regions or other Candidiasis-related-risk factors. So, in the present study we also assessed the association between vulvovaginal candidiasis and some medical variables. In this regard, our findings showed a significant association between VVC and age and pregnancy (PV < 0.05) while there was no significant association between CD4 count, the usage history of antibiotics, antiretroviral therapy, birth control methods, history or presence of underlying diseases, and duration of AIDS (PV > 0.05).

      5. Conclusion

      The high occurrence of vulvovaginal Candida spp. (34.11%) among HIV seropositive women in Ahvaz (Southwest Iran) is notable, with the highest frequency of C. albicans (62.06%), followed by C. glabrata (20.68%) and C. dubliniensis (13.79%). The considerable resistance of all Candida spp to Amphotericin B and Azole antifungals necessitates more effective treatment programs to reduce the rate of this infection in HIV-infected women, particularly in pregnant women.

      Funding

      This study was funded by Ahvaz Jundishapur University of Medical Sciences (No: OG-94170 ).

      Ethical approval

      All procedures performed in studies involving human participants were in accordance with the ethical standards of the national research committee and with the 2008 Helsinki declaration and its later amendments or comparable ethical standards. This study was approved by the ethics committee of Ahvaz Jundishapur University of Medical Sciences with Ethical Code: ajums.REC.1396.417.

      Authors’ contributions

      Study concept and design: Mehran Varnasiri, Shokrolah Salmanzadeh, Ali Zarei Mahmoudabadi.
      Sampling and conducting the experiments: Ali Zarei Mahmoudabadi, Marzieh Halvaeezadeh, Simin Taghipour, Shahla Molavi.
      Data analysis and interpretation of the results: Mehran Varnasiri, Seyed Mohammad Alavi*, Khojasteh Hoseiny Nezhad, Parastoo Moradi Choghakabodi.
      Drafting of the manuscript: Seyed Mohammad Alavi, Khojasteh H. Nezhad, Parastoo Moradi Choghakabodi.
      Critical revision of the manuscript: Mehran Varnasiri, Seyed Mohammad Alavi, Khojasteh H. Nezhad, and Parastoo Moradi Choghakabodi.

      Declaration of competing interest

      Dr. Mehran Varnaseri and Dr. Seyed Mohammad Alavi has received research grants from Ahvaz Jundishapur University of Medical Sciences . Other coauthors report no conflicts of interest relevant to this article.

      Acknowledgments

      We would like to thank the Health Research Institute, Infectious and Tropical Diseases Research Center , Ahvaz Jundishapur University of Medical Sciences for their support. This study was a MD thesis (Mehran Varnasiri) supported by a grant (No: OG-94170 ) from the Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran .

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