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 Table of Contents  
ORIGINAL ARTICLE
Year : 2022  |  Volume : 34  |  Issue : 1  |  Page : 38-44

Identification & correlation of candida strains with CD4+ count among HIV patients on HAART and evaluation of in-vitro susceptibility to fluconazole and voriconazole


1 Department of Oral Medicine and Radiology, Lenora Institute of Dental Sciences, Rajahmundry, Andhra Pradesh, India
2 GSL Dental College and Hospital, Andhra Pradesh, India

Date of Submission25-Mar-2021
Date of Decision18-Oct-2021
Date of Acceptance26-Jan-2022
Date of Web Publication25-Mar-2022

Correspondence Address:
Dr. Naidu Harika
Department of Oral Medicine and Radiology, Lenora Institute of Dental Sciences, Rajahmundry, Andhra Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0019-5049.340768

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   Abstract 


Background: HIV is a life-threatening disease that increases morbidity and mortality due to the alterations of the body's immune system, leading to several opportunistic infections due to the progressive loss of CD4+ T lymphocytes. Aim: To identify and correlate the Candida strains with CD4+ count in Human Immunodeficiency Virus (HIV) patients on Highly active antiretroviral therapy (HAART) and evaluate the in-vitro susceptibility to fluconazole and voriconazole. Materials and Methods: A total of 100 HIV-positive patients who were not under HAART therapy were examined at three different intervals for the presence of Candida strains along with their CD4+ count and evaluated for in-vitro susceptibility to fluconazole and voriconazole. The obtained values were analyzed using the Spearman correlation test and Chi-square tests with a significant P value of 0.005 and SPSS version 20.0 software. Results: C. albicans was the most common species isolated, followed by C. krusei, C. glabrata, and C. tropicalis. C. glabrata showed a positive correlation with CD4+ count at 6 months of HAART therapy. The variation in CD4+ count prior to HAART therapy and at 3 and 6 months of HAART therapy showed a significant increase in the CD4+ count. Antifungal susceptibility testing showed that all Candida strains were resistant to fluconazole and susceptible to voriconazole. Conclusion: The correlation of CD4+ count with Candida strains and in-vitro susceptibility of voriconazole to all the identified strains of Candida as observations from the present study suggests the need for more periodic studies among different sample populations with larger sample size.

Keywords: Antifungal susceptibility, CD4+ count, CHROMagar Chromogenic media, highly active antiretroviral therapy, human immunodeficiency virus


How to cite this article:
Harika N, Sridevi K, Krishnaveni B, Kumar NP, Mounika G, Naga Deepika AD. Identification & correlation of candida strains with CD4+ count among HIV patients on HAART and evaluation of in-vitro susceptibility to fluconazole and voriconazole. J Indian Acad Oral Med Radiol 2022;34:38-44

How to cite this URL:
Harika N, Sridevi K, Krishnaveni B, Kumar NP, Mounika G, Naga Deepika AD. Identification & correlation of candida strains with CD4+ count among HIV patients on HAART and evaluation of in-vitro susceptibility to fluconazole and voriconazole. J Indian Acad Oral Med Radiol [serial online] 2022 [cited 2022 May 27];34:38-44. Available from: https://www.jiaomr.in/text.asp?2022/34/1/38/340768




   Introduction Top


Human immunodeficiency virus (HIV) is an RNA retrovirus that affects the human immune system, resulting in loss of normal defense mechanisms and life-threatening infections. Its detection for the first time in India was in April 1986 in the state of Tamil Nadu.[1] HIV infects and kills CD4+ T cells; therefore, during HIV infection, infected and healthy CD4+ T cells act in opposition to each other, reproducing virus particles and resulting in progressive failure of the immune system.[2]

Opportunistic infections are the common and early complications and are predictors for the progression of the HIV disease, resulting in shortening the life span.[3] Oral candidiasis is the most common opportunistic fungal infection in HIV patients caused by C. albicans. Although C. albicans is the most common strain, other non-albicans strains are also predominantly seen in HIV patients. The high incidence of candidiasis in HIV patients and increased use of antifungal drugs eventually results in increased resistance to the conventional antifungal agents. Moreover, the prevalence of non-albicans strains that do not respond to conventional antifungal therapy is on the rise.[4]

Moreover, oral physicians get to encounter HIV patients with candidiasis on a daily basis. As various strains of Candida are known to be resistant to conventionally used drugs such as fluconazole, we also aimed to evaluate the in-vitro susceptibility of Candida strains to fluconazole and voriconazole.


   Materials and Methods Top


Source of data

The study was conducted in the Department of Oral Medicine and Radiology, Lenora Institute of Dental Sciences and Government Hospital February 2019 to July 2020, Rajahmundry, East Godavari district, Andhra Pradesh for a period of 1 year and 6months (February 2019 to July 2020). The study protocol was approved by the institutional ethical committee (102/IEC/LIDS/2018) and by the District Medical and Health Officer, following the Helsinki rule of declaration,and informed consent was obtained from the participants. A total of 100 HIV-positive patients were included in the study. The sample size was calculated based on the pilot study conducted on 30 patients, of which 60% showed the presence of Candida strains. The results were substituted in the formula n = 4 pq/d2 or n = Z2 p(1-p)/d2, where n = sample size, P = expected prevalence (60% = 0.6), d = precision (10% = 0.1), and Z2 = 3.84 (constant value) at 95% confidence interval and 80% power.

n = 3.84 × 0.6 × 0.4 ÷ 0.1 × 0.1 = 92.16

The obtained sample size of 92.16 was rounded off to a total sample of 100 and was included in the study.


   Aim Top


To identify the Candida strains in HIV patients on HAART, and correlate with the CD4+ count and to evaluate the in-vitro susceptibility to fluconazole and voriconazole.


   Objectives Top


  1. To identify Candida strains, record CD4+ cell count, and evaluate the in-vitro susceptibility of Candida strains to fluconazole and voriconazole at baseline prior to initiation of HAART.
  2. To identify Candida strains, record CD4+ cell count, and evaluate the in-vitro susceptibility of Candida strains to fluconazole and voriconazole after 3 months of HAART.
  3. To identify Candida strains, record CD4+ cell count, and evaluate the in-vitro susceptibility of Candida strains to fluconazole and voriconazole after 6 months of HAART.


Inclusion criteria

Patients diagnosed as HIV positive by TRI-DOT test and prior to initiation of HAART.

Exclusion criteria

Patients under HAART therapy by the time of enrolment. Patients with diseases that lead to immunosuppression other than HIV infection.


   Methodology Top


All the participants of the study were patients visiting the HIV ward of Government Hospital, Rajahmundry immediately after have been tested positive with TRI-DOT and prior to the initiation of HAART therapy.

The details of demographic data, which also included the CD4+ counts, were recorded in a specially designed case history proforma from all the 100 HIV-positive patients.

The baseline buccal swabs were obtained on the first day of visit to the hospital after being tested positive for HIV prior to the initiation of HAART therapy. The collected swabs were diluted in 1 mL of PBS solution in test tubes. Next, 10 μL of the obtained sample was transferred to a Petri dish containing CHROMagar medium by using a 10-μL pipette. Similarly, 10 μL of the sample was transferred to the remaining two Petri dishes containing CHROMagar with 8 μg/mL dilutions of fluconazole and voriconazole each. All the Petri dishes were then incubated at 37°C for 48 h.

Again, the buccal swabs were collected at the intervals of 3 and 6 months after the commencement of HAART therapy (tenofovir disoproxil fumarate- 300 mg + lamivudine- 300 mg + doultegravir- 50 mg- fixed-dose regimen) along with their CD4+ counts. The Petri dishes containing CHROMagar with fluconazole and voriconazole were observed and investigated for in-vitro susceptibility of different strains of candidiasis to voriconazole and fluconazole.

After 48 h, the clear to slightly opalescent gel form of CHROMagar was observed for areas of color change to identify the four different strains of Candida as follows:

  1. Light green - C. albicans [Figure 1]
  2. Metallic blue - C. tropicalis [Figure 2]
  3. Rose pink – C. krusei [Figure 3]
  4. White – C. glabrata [Figure 4].
Figure 1: CHROMagar candida medium showing the presence of C. albicans

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Figure 2: CHROMagar candida medium showing the presence of C. albicans and C. tropicalis

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Figure 3: CHROMagar candida medium showing the presence of C. krusei

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Figure 4: CHROMagar candida medium showing the presence of C. glabrata

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The Petri dishes containing 8 μL dilutions of voriconazole and fluconazole were intercepted as follows.

The absence of color change indicates the fungicidal activity of the drug and thus states that all the strains were susceptible to that particular drug, whereas any change in color depicts the fungistatic activity of the drugs.

Statistical analysis

The obtained values were tabulated and statistically analyzed using SPSS version 20.0 software. Chisquare test and ANOVA tests were used for the analysis and Spearman correlation test for correlation between CD4+ counts and Candida strains, and a significance level of ≤0.005 was considered for correlation between three different intervals of HAART therapy.


   Results Top


The sample included 100 subjects with ages ranging from 20 years to 60 years, among which 43% were males with a mean age of 36.84 years and 57% were females with a mean age of 36.72 years, which reflected a slight female predilection for HIV [Table 1].
Table 1: Depiction of demographic details of the study population (HIV patients)

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Of the four cultured strains, the most common species isolated at baseline was C. albicans (81%), followed by C. krusei (26%), C. glabrata (19%), and C. tropicalis (9%). Upon taking buccal swabs at an interval of 3 months, the common species isolated was C. albicans (83%), followed by C. krusei (38%), C. tropicalis (16%), and C. glabrata (15%), while the most common species isolated after 6 months of HAART therapy was C. albicans (82%), followed by C. krusei (40%), C. glabrata (31%), and C. tropicalis (16%) [Table 2].
Table 2: Presence of Candida strains at baseline, 3 months, and 6 months' intervals of HAART therapy

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A change in the CD4 + count has been observed using ANOVA test at different intervals of HAART therapy with a mean CD4+ count of 275.41 prior to HAART therapy, followed by 325.80 CD4+ count at 3 months after HAART therapy and 380.78 CD4+ count at 6 months after initiation of HAART therapy. This increase in the CD4+ count was statistically significant with a P value of 0.001 [Table 3].
Table 3: Distribution of CD4 count prior to HAART THERAPY, 3 months, and 6 months after initiation of HAART therapy

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Out of all the Candida strains, C. albicans was found to decrease after the initiation of HAART therapy, with a descending order of frequency of about 57.8% at baseline, 54.6% after 3 months of HAART therapy, and 48.5% after 6 months of HAART therapy, with a positive correlation value of −0.272 among the other strains. Only C. glabarata was found to have a very high positive correlation of about 0.420 [Table 4].
Table 4: Correlation of CD4 count and Candida strains

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Of the total sample, 33.6% and 76.5% of the Candida strains were susceptible to fluconazole and voriconazole, respectively. However, 66.3% were resistant to fluconazole, whereas only 23.4% of the Candida strains were resistant to voriconazole.

Of all the Candida strains, 37.8% were susceptible to fluconazole, whereas 72.8% were susceptible to voriconazole at baseline.

After 3 months of HAART therapy, 32.2% & 76.8% of Candida strains were susceptible to fluconazole and voriconazole, respectively.

Further, 31.3% and 73.3% of Candida strains were susceptible to fluconazole and voriconazole, respectively, after 6 months of HAART therapy [Table 5].
Table 5: Distribution of candida strains according to susceptibility to fluconazole and voriconazole at baseline, 3 months, and 6 months' intervals of HAART therapy

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   Discussion Top


HIV infection is a serious disorder of the immune system in which the body's normal defenses against infection breaks down, leaving it vulnerable to a host of life-threatening infections.[1]

HIV is a retrovirus that impairs the body's immune system and leaves the patient susceptible to life-threatening opportunistic infections. HIV enters the body through mucosal tissues and blood, infecting the T cells, dendritic cells, and macrophages.[5]

CD4+ T lymphocytes are the primary target of HIV infection because of the affinity of the virus to the CD4+ cell surface markers. HIV infection causes a gradual decline of CD4+ T lymphocyte levels in peripheral bloodstream, leading to compromised immunity. The pathogenesis of HIV infection is mostly attributed to the decrease in the number of T-cells with CD4+ cell surface receptors.[5]

Owing to a weakened immune system, HIV/AIDS infected persons are at an increased risk of a wide variety of opportunistic infections.[6] The most common opportunistic pathogens isolated from the oral cavity are Candida species. There are many species of Candida, but the most prevalent one, which is recovered from the oral cavity, in both commensal state and in cases of oral candidiasis, is C. albicans.[7]

The infection caused by Candida may be superficial and affect the mucous membrane or may attack the bloodstream and spread into internal organs if left unattended.[8] Thus, identification of specific strains of Candida is important because isolates of Candida species differ widely, both in their ability to cause infection and in their susceptibility to antifungal agents.

Early initiation of antifungal therapy along with controlling the risk factors is crucial to improve the prognosis. One of the most commonly prescribed antifungal drugs for Candida infections is fluconazole, a triazole antifungal. The increased use of these antifungal agents can lead to an increase in opportunity for antifungal resistance.[9]

The present study comprised 100 HIV-positive patients with an age range of 20–60 years and a mean of 36.7 years, with a slight female predilection comprising 57% females and 43% males. The minor variation in the gender predominance could be due to the variation in sampling with respect to the lifestyle and geographic area of the sample population. The demographic details of our study with female predominance were in accordance with other reported studies conducted by Annapurna (58%)[1] [Table 1].

Prior to the initiation of HAART therapy, the most common species in the present study was found to be C. albicans (81%), followed by C. krusei (26%), C. glabrata (19%), and C. tropicalis (9%), whereas the most common species isolated after 3 months of HAART therapy was C. albicans (83%), followed by C. krusei (38%), C. tropicalis (16%), and C. glabrata (15%). The most common species isolated after 6 months of HAART therapy was C. albicans (82%), followed by C. krusei (40%), C. glabrata (31%), and C. tropicalis (16%) [Table 2].

As C. albicans is the most common species, this was in accordance with various other reported studies conducted by Ribeiro et al.,[4] and other studies.[10],[11],[12],[13],[14],[15],[16],[17],[18],[19],[20]

In the present study, the incidence of C. glabarata was found to be increased despite the initiation of HAART therapy from 3 months to 6 months with a significant P value of <0.0023. This shows that there is no significant association between Candida species and HAART therapy, which was in accordance with other studies conducted by Khedri et al.[14] and Luque et al.[18]

According to WHO, the CD4+ count in HIV patients was characterized as stage I count ≥500 cell/cc.mm, stage II between 200 and 499 cells/cc.mm, and stage III <200 cells/cc.mm. However, the present study showed the mean CD4+ count at baseline, that is, prior to HAART therapy, 3 months, and 6 months after initiation of HAART therapy were 275.41, 325.8, and 380.78, respectively, reflecting a significant increase in the CD4 count with a P value of 0.001 after the initiation of HAART [Table 3].

Out of all the Candida strains, C. albicans was found to decrease after the initiation of HAART therapy, with a descending order of frequency of about 57.8% at baseline, 54.6% after 3 months of HAART therapy, and 48.5% after 6 months of HAART therapy, with a positive correlation value of −0.272. Among the other strains, only C. glabarata was found to have a very high positive correlation of about 0.420 [Table 4]. To the best of our knowledge there were no reports in the literature correlating the CD4+ counts with Candida strains. Further the studies regarding the evaluation of susceptibility of Candida strains in HIV patients on HAART to voriconazole were very few [12].Interestingly the candida strains in the present study showed susceptibility to voriconazole.

Various topical and systemic antifungal drugs are available for treating oral candidiasis (OC). They interfere with the biosynthesis or integrity of ergosterol, the major sterol in the fungal cell membrane, and cause disruption of the fungal cell wall. Triazoles and imidazoles are the most widely used antifungal drugs and act primarily by inhibiting the fungal cytochrome P450 enzyme, 14 α-demethylase.[7]

Of the total sample, 33.6% and 76.5% of the Candida strains were susceptible to fluconazole and voriconazole, respectively. However, 66.3% were resistant to fluconazole, whereas only 23.4% of the Candida strains were resistant to voriconazole.

Of all the Candida strains, 37.8% were susceptible to fluconazole, whereas 72.8% were susceptible to voriconazole at baseline.

After 3 months of HAART therapy, 32.2% and 76.8% of Candida strains were susceptible to fluconazole and voriconazole, respectively.

Further, 31.3% and 73.3% of Candida strains were susceptible to fluconazole and voriconazole, respectively, after 6 months of HAART therapy [Table 5] and [Figure 5] and [Figure 6]. This shows that Candida strains are more susceptible to voriconazole when compared to fluconazole in HIV-positive patients. This is similar to the study done by Badiee et al.[12] This greater susceptibility of voriconazole was highly significant with a P value of <0.001. The increased resistance for fluconazole is due to increased drug efflux, alterations or increase in the drug target, and the development of compensatory pathways for producing the target sterol, ergosterol. However, voriconazole showed more susceptibility to the Candida strains compared to fluconazole.[9]
Figure 5: Sample cultured on CHROMagar candida without an antifungal agent (fluconazole, voriconazole) showing the presence of C. albicans. (a) Sample containing C. albicans cultured on CHROMagar media containing fluconazole showing susceptibility to fluconazole. (b) Sample containing C. albicans cultured on CHROMagar media containing voriconazole showing susceptibility to voriconazole

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Figure 6: Sample cultured on CHROMagar candida without an antifungal agent (fluconazole, voriconazole) showing the presence of C. albicans and C. krusei. (a) Sample containing C. albicans and C. krusei cultured on CHROMagar media containing fluconazole showing the susceptibility of C. albicans to fluconazole and C. krusei resistant to fluconazole. (b) Sample containing C. albicans and C. krusei cultured on CHROMagar media containing voriconazole showing the susceptibility of C. albicans to voriconazole and C. krusei resistant to voriconazole

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Limitations and future study prospects

  • More periodic studies among different sample populations with larger sample sizes may further continue and validate the observations of the present study.
  • More sensitive methods that quantify the Candida species may further define the susceptibility/sensitivity to fluconazole and voriconazole.
  • In-vivo studies may be taken forward.



   Conclusion Top


The present study gives an insight into the presence of Candida species in HIV-positive patients as the increased and prolonged use of fluconazole can lead to the development of resistant Candida strains, including the non-albicans strains, which necessitates the identification of different strains of Candida. Therefore, the quest for an alternative drug to make the resistant strains to susceptible is under progress.

Declaration of patient consent

The authors certify that they have obtained all appropriate patient consent forms. In the form, the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

1.
Annapurna CS, Prince CN, Sivaraj S, Ali IM. Oral manifestations of HIV patients in South Indian population. J Pharm Bioall Sci 2012;4:364-8.  Back to cited text no. 1
[PUBMED]  [Full text]  
2.
Aavani P, Allen LJS. The role of CD4 T cells in immune system activation and viral reproduction in a simple model for HIV infection. Appl Math Model 2019;75:210-22.  Back to cited text no. 2
    
3.
Srirangaraj S, Venkatesha D. Opportunistic infections in relation to antiretroviral status among AIDS patients from south India. Indian J Med Microbiol 2011;29:395-400.  Back to cited text no. 3
[PUBMED]  [Full text]  
4.
Ribeiro Ribeiro AL, de Alencar Menezes TO, de Melo Alves-Junior S, de Menezes SA, Marques-da-Silva SH, Rosário Vallinoto AC. Oral carriage of Candida species in HIV-infected patients during highly active antiretroviral therapy (HAART) in Belém, Brazil. Oral Surg Oral Med Oral Pathol Oral Radiol. 2015 Jul; 120 (1):29-33.  Back to cited text no. 4
    
5.
Sridevi K, Malathi S, Chalapathi KV, Chowdary GN, Gayathri M, Eswar Chand G, et al. CD4 cell counts, lipid profile, and oral manifestations in HIVInfected and AIDS patients. Front Dent 2019;16:436-49.  Back to cited text no. 5
    
6.
Nugraha AP, Ernawati DS, Parmadiati AE, Soebadi B, Triyono E. Prevalence of Candida species in oral candidiasis and correlation with CD4+ count in HIV/AIDS patients at Surabaya, Indonesia. 2018;11:81-5.  Back to cited text no. 6
    
7.
Byadarahally Raju S, Rajappa S. Isolation and identification of Candida from the oral cavity. ISRN Dent 2011;487921.  Back to cited text no. 7
    
8.
Maheronnaghsh M, Tolouei S, Dehghan P, Chadeganipour M, Yazdi M. Identification of Candida species in patients with oral lesion undergoing chemotherapy along with minimum inhibitory concentration to fluconazole. Adv Biomed Res 2016;5:132.  Back to cited text no. 8
[PUBMED]  [Full text]  
9.
Berkow EL, Lockhart SR. Fluconazole resistance in Candida species: a current perspective. Infect Drug Resist. 2017;10:237-245.  Back to cited text no. 9
    
10.
Anwar KP, Malik A, Subhan KH. Profile of candidiasis in HIV infected patients. Iran J Microbiol 2012;4:204-9.  Back to cited text no. 10
    
11.
Kaur R, Mehra B, Dhakad MS, Goyal R, Bhalla P, Dewan R. Clinico-mycological analysis and antifungal resistance pattern in human immunodeficiency virusassociated candidiasis: An Indian perspective. Indian J Sex Transm Dis 2018;39:111-9.  Back to cited text no. 11
[PUBMED]  [Full text]  
12.
Badiee P, Alborzi A, Davarpanah MA, Shakiba E. Distributions and antifungal susceptibility of Candida species from mucosal sites in HIV positive patients. Arch Iran Med 2010;13:282-87.  Back to cited text no. 12
    
13.
Pomarico L, Cerqueira DF, de Araujo Soares RM, de Souza IPR, de Araujo Castro GFB, Socransky S, et al. Associations among the use of highly active antiretroviral therapy, oral candidiasis, oral Candida species and salivary immunoglobulin A in HIV-infected children. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2009;108:203-210.  Back to cited text no. 13
    
14.
Khedri S, Santos ALS, Roudbary M, Hadighi R, Falahati M, Farahyar S, et al. Iranian HIV/AIDS patients with oropharyngeal candidiasis: Identification, prevalence and antifungal susceptibility of Candida species. Lett Appl Microbiol 2018;67:392-9.  Back to cited text no. 14
    
15.
Aboualigalehdari E, Birgani MT, Fatahinia M, Hosseinzadeh M, et al. Candida spp. in oral mucosa of HIV-infected individuals. Epidemiol Health 2020;42:e2020033.  Back to cited text no. 15
    
16.
Costa CR, Cohen AJ, Fernandes OFL, Miranda KC, Passos XS, Souza LKH, et al. Asymptomatic oral carriage of Candida species in HIV-infected patients in the highly active antiretroviral therapy era. Rev Inst Med trop S Paulo 2006;48:257-61.  Back to cited text no. 16
    
17.
Campisi G, Pizzo G, Milici ME, Mancuso S, Margiotta V. Candida carriage in the oral cavity of human immunodeficiency virus-infected subjects. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2002;93:281-6.  Back to cited text no. 17
    
18.
Luque AG, Biasoli MS, Tosello ME, Binolfi A, Lupo S, Magaró HM. Oral yeast carriage in HIV-infected and non-infected populations in Rosario, Argentina. Mycoses 2009;52:53-9.  Back to cited text no. 18
    
19.
Kalpana Devi V, Geetha Lakshmi S. Species distribution and in-vitro antifungal susceptibility pattern of oral candida isolates in HIV patients and correlation with CD4 count. Journal of Evolution of Medical and Dental Sciences 2013;2:9231-8.  Back to cited text no. 19
    
20.
Banu A, Khan KA, Manasa S, Krishnam SP. Study of candida colonization and speciation in HIVpositive patients in a tertiary care hospital. Int J Curr Res 2015;7:12935-39.  Back to cited text no. 20
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

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