Indian J Sex Transm Dis Indian J Sex Transm Dis
Official Publication of the Indian Association for the Study of Sexually Transmitted Diseases
Indian J Sex Transm Dis
The Journal | Search | Ahead Of Print | Current Issue | Archives | Instructions | Subscribe | Login    Users online: 611   Home Email this page Print this page Bookmark this page Decrease font size Default font size Increase font size


 
  Table of Contents  
ORIGINAL ARTICLE
Year : 2015  |  Volume : 36  |  Issue : 1  |  Page : 53-58
 

Diversity and antifungal resistance patterns of prevalent opportunistic pathogenic yeasts colonizing the oral cavities of asymptomatic human immunodeficiency virus-infected individuals, and their relation to CD4 + counts


1 Department of Biosciences, Jamia Millia Islamia, New Delhi, India
2 Department of Regional STD Teaching Training and Research Centre, VMMC and Safdarjung Hospital, New Delhi, India
3 Department of Microbiology, AIIMS, New Delhi, India
4 Department of Laboratory Medicine, JPNA Trauma Centre - AIIMS, New Delhi, India

Date of Web Publication8-May-2015

Correspondence Address:
Dr. Luqman Ahmad Khan
Department of Biosciences, Jamia Millia Islamia, New Delhi - 110 025
India
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0253-7184.156724

Rights and Permissions

 

   Abstract 

Background: Yeasts are important opportunistic pathogens, in individuals infected with human immunodeficiency virus (HIV). Yeast species inhabiting the oral mucosa of HIV-infected persons can act as source of oral lesions, especially as the individual progresses towards immunocompromised state. Present study was conducted to evaluate the diversity of yeasts in oral cavities of asymptomatic HIV-infected persons and their association with CD4 + cell counts. Materials and Methods: 100 HIV seropositive subjects and 100 healthy controls were screened for oral yeast carriage using standard procedures. Results: Of the 100 HIV-seropositive persons screened, 48 were colonized by different yeasts, either alone or in association with another species. Candida albicans was the most common species (56.90%) while non C. albicans Candida (NCAC) accounted for 39.65%. Among NCAC, Candida tropicalis and Candida krusei were most common. One isolate each of rare opportunistic pathogenic yeasts, Geotrichum candidum and Saccharomyces cereviseae, was recovered. The control group had an oral candidal carriage rate of 23%; C. albicans was the predominant species, followed by Candida glabrata, C. tropicalis and Candida parapsilosis. Antifungal susceptibility testing revealed no resistance in C. albicans, to the commonly used antifungal agents, whereas resistance or dose dependent susceptibility to fluconazole was observed in some of the NCAC species. Conclusion: Oral carriage of opportunistic pathogenic yeasts was greater in HIV-seropositive persons heading towards immunocompromised state, as evidenced by their CD4 + cell count. The predominant yeast isolated in this study (C. albicans), was found to be susceptible to commonly used antifungals.


Keywords: Antifungal susceptibility test, CD4 + count, human immunodeficiency virus, seropositive, opportunistic infections, oral candidiasis, oral yeast carriage


How to cite this article:
Kumar DA, Muralidhar S, Banerjee U, Basir SF, Mathur P, Khan LA. Diversity and antifungal resistance patterns of prevalent opportunistic pathogenic yeasts colonizing the oral cavities of asymptomatic human immunodeficiency virus-infected individuals, and their relation to CD4 + counts. Indian J Sex Transm Dis 2015;36:53-8

How to cite this URL:
Kumar DA, Muralidhar S, Banerjee U, Basir SF, Mathur P, Khan LA. Diversity and antifungal resistance patterns of prevalent opportunistic pathogenic yeasts colonizing the oral cavities of asymptomatic human immunodeficiency virus-infected individuals, and their relation to CD4 + counts. Indian J Sex Transm Dis [serial online] 2015 [cited 2019 Nov 22];36:53-8. Available from: http://www.ijstd.org/text.asp?2015/36/1/53/156724



   Introduction Top


The oral cavity of human beings is rich in microbial flora of extremely diverse nature, with opportunistic pathogenic yeasts being important components. [1] These yeasts are often a source of local infection, especially in immunocompromised patients, as in human immunodeficiency virus (HIV)/AIDS disease. [2] India is home to a large number of people living with HIV/AIDS, where many do not have access to antiretroviral therapy (ART), and oropharyngeal candidiasis (OPC) has been reported as the most common HIV-associated opportunistic infection. [2] Among the yeasts inhabiting the oral cavity, genus Candida, specifically the most virulent species, Candida albicans, is found to be commonly distributed either as colonizer or as pathogen. [3],[4],[5],[6] Furthermore, there are other yeasts, previously thought to be nonpathogenic, now gaining importance, especially in the era of HIV/AIDS. [7],[8] In the Indian scenario, only a few reports are available on asymptomatic carriage of yeasts in the oral cavity of HIV seropositive individuals. [3],[4],[5],[9],[10] Most of these studies are, however, from the southern states of the country. [3],[4],[9] Of these, still fewer studies have looked for the association of yeast carriage in relation to CD4 + count. [5],[9]

Present study was planned to determine the current status of opportunistic yeasts, especially Candida, including non C. albicans Candida (NCAC), in the oral cavities of asymptomatic HIV seropositive persons. The study also explores the antifungal susceptibility patterns of the prevalent isolates.


   Materials and Methods Top


Oral swabs were collected from consecutive HIV seropositive individuals, attending ART center of Safdarjung Hospital, New Delhi, on the particular day of sample collection. Control group included 100 healthy volunteers from staff and student population of Safdarjung hospital, who were age and sex matched. None of these subjects had any underlying predisposing conditions, as explored and ruled out by a detailed questionnaire. Subjects excluded were those with a history of treatment with antifungals during previous 6 months, with dentures, with pregnancy or on oral contraceptives. Samples from the oral mucosa were collected from both male and female HIV-infected persons (68 + 32 = 100) (Confirmed by 3 rapid tests), attending the ART center. None of the subjects had any clinical signs of oral lesions at the time of sample collection. A detailed history-taking preceded the collection of samples. CD4 + lymphocyte count was performed on the same day as collection of oral samples. Informed consent was obtained from each subject. The study was approved by Institutional Ethics Committee (No. 26-11-EC [21/31]).

Two swabs were collected by depressing the tongue and gently rubbing the surface of gum, tooth, tonsils and tongue using sterile cotton swabs. The first swab was inoculated on Sabouraud's Dextrose Agar (SDA) and incubated at 37°C for up to 7 days, and observed daily for growth, while the second swab was used to prepare a smear for Gram staining. All isolates were identified by their morphology on Gram's smear, cultural characteristics on SDA, Corn Meal Agar with 1% Tween 80, Chrom Agar (color, texture, etc.), germ tube test and carbohydrate assimilation/fermentation tests. [11] When conventional methods failed to identify unusual isolates, they were verified by automated Vitek-2 YST system. To differentiate C. albicans from Candida dubliniensis, all C. albicans isolates (confirmed by conventional methods) were subjected to growth at 45°C and Tween 80 hydrolysis test, along with the ATCC control strains. [12] Subsequent to species identification, antifungal susceptibility testing was performed, using fluconazole (FL), ketoconazole, voriconazole and amphotericin B, by E-test strip method. This was performed by suspending a portion of the isolated colony in normal saline and adjusting its turbidity to 0.5 McFarland standard. The suspension was then spread over the surface of a predried RPMI-1640 +2% glucose agar media, using a sterile cotton swab. E-test strips were applied using a sterile forceps and minimum inhibitory concentrations (MICs) were determined after 24 h and 48 h of incubation at 37°C. [13] In the case of doubt in the results by E-test, MIC was confirmed by micro broth dilution method, to determine the discordance if any. MIC for each antifungal agent was interpreted as per Clinical and Laboratory Standards Institute criteria. [14] CD4 + cells were analyzed and enumerated on a FACS count flow cytometer (Becton Dickinson, California, USA), using two color monoclonal antibody panel.

Data analysis

Data analyses were performed by Chi-square test using GraphPad prism software version 5.00 developed by GraphPad Software, Inc., La Jolla, CA, USA. A P < 0.05 was considered significant.


   Results Top


Of the 100 HIV-seropositive persons investigated, 48 (48.00%) were colonized with different species of yeasts, while 52 (52.00%) had a negative culture. While 39 specimens yielded single species, nine yielded more than one species, giving a total of 58 isolates. Of the 58 isolates, from HIV seropositive individuals, C. albicans was the most frequently isolated species, 33 (56.90%), followed by an equal distribution (15.52%) of Candida tropicalis and Candida krusei [Table 1]. Of the nine specimens, yielding multiple isolates, six had a combination of C. albicans with various NCAC, while three had a combination of two NCAC species (C. tropicalis and C. krusei, C. tropicalis and Candida glabrata and C. glabrata and C. krusei). Of the 100 samples from healthy controls, only 23 yielded a growth of Candida, with a predominance of C. albicans (18), followed by C. glabrata (3), Candida parapsilosis (1) and C. tropicalis (1) [Table 1].
Table 1: Species distribution of various yeasts colonizing the oral cavities of HIV seropositive individuals and healthy controls


Click here to view


Relationship between yeast carriage and the CD4 + count

According to CD4 + count, HIV seropositive subjects were classified into three groups: Group A: CD4 + count >400 cells/µl; Group B: CD4 + count between 201 and 400 cells/µl; Group C: CD4 + count <200 cells/µl.

Yeasts isolated from Group A

A total of 30 samples were collected from the subjects belonging to Group A. Of these, eight (26.66%) yielded different yeast species [Table 2], six of which grew only single species, two yielded multiple species, giving a total of 11 isolates [Table 1]. A rare opportunistic pathogenic yeast Saccharomyces cereviseae was isolated in combination with C. albicans and C. krusei from this group. Besides biological activities (seen by Vitek-2 YST system), identification of S. cereviseae was also confirmed by genetic study (from a reference center, PGIMER, Chandigarh).
Table 2: Oral yeast carriage in relation with CD4+ cell count


Click here to view


Yeasts isolated from Group B

Of 42 samples collected from the subjects belonging to Group B, 20 (47.62%) yielded different Candida species [Table 2]. While 16 of these grew only one single species, four yielded multiple species, giving a total of 24 isolates [Table 1].

Yeasts isolated from Group C

A total of 28 samples were collected from subjects belonging to Group C. Of these; 20 (71.43%) yielded yeast isolates [Table 2]. 17 of which grew only single species, including an unusual yeast Geotrichum candidum. Three specimens yielded multiple species, giving a total of 23 isolates [Table 1].

Antifungal resistance patterns of the isolates

In vitro antifungal susceptibility testing of the isolates revealed that all C. albicans isolated from both the control and the HIV-seropositive group did not show resistance to any of the antifungal agents tested. NCAC isolated from the control group also yielded similar findings. Among the HIV-seropositive group, FL resistance was observed in 100% of C. krusei isolates and dose dependent susceptibility in two of the four isolates of C. glabrata [Table 3].
Table 3: Antifungal resistance patterns of the yeast isolates


Click here to view



   Discussion Top


Various species of yeasts inhabit the oral cavity of humans as harmless commensals. [1] However, in recent past, especially in the era of HIV promoted diseases, these yeasts have gained importance as opportunistic pathogens, causing oral lesions. [1] It is also shown that OPC, is the most common opportunistic infection in HIV-seropositive individuals, as the disease progresses. [2],[3],[9] Often, the yeast routinely seen as normal flora are the sources of OPC, as the individual progress toward an immunocompromised state. [1],[2]

Earlier studies conducted in India and elsewhere revealed that asymptomatic oropharyngeal colonization with yeast is known to be significantly higher among HIV-infected persons. Published reports shows carriage rates to vary between 27.9% and 68% in this group. [4],[9],[10] Present study recorded an overall yeast carriage rate of 48%, which included different Candida species along with other yeasts. This figure is comparable to those reported by Pavithra et al.,[4] from Southern India and Hung et al.,[15] from Taiwan, but lesser than that of a similar study carried out a decade ago by Gugnani et al.[10] (68% - included 65.35% Candida and 2.7% other yeasts) from New Delhi. A recent study by Maurya et al.,[5] reported a carriage rate of 33.1% in a similar group of patients. [5] This variance in the isolation rates may be due to the difference in distribution of Candida in several geographical areas, time of sampling or the use of different collection methods for yeast recovery. [5],[16] Among the control group, candidal carriage was observed in 23% of subjects, which is in agreement with other Indian studies. [4],[5]

Interestingly, among the yeasts isolated, the predominant species was C. albicans (56.90%), which is in concordance with other reports. [3],[4],[5] NCAC species accounted for 39.65%, of which C. tropicalis and C. krusei head the list. Though the isolation of C. tropicalis, as the second most common species, is in agreement with most other Indian reports on asymptomatic carriage or in OPC, [3],[4] the scenario is different as far as C. krusei isolation is concerned. Only one report from southern India recorded C. krusei (26.31%) as the predominant NCAC species isolated from a similar patient group, as in the present study. [4] This spectrum is quite different from the results of a study conducted a decade ago, by one of the authors (UB) on OPC in HIV patients, where not a single isolate was C. tropicalis. [2] Another important observation in the present study is the isolation of potentially less opportunistic pathogenic yeasts like S. cereviseae and G. candidum. S. cereviseae was isolated in combination with C. albicans and C. krusei from one subject. S. cereviseae, also known as brewer's yeast, is commonly used in baking and fermentation of alcoholic drinks. [17] It is often considered as a commensal organism of the oral cavity and has been isolated from immunocompromised patients, as a colonizer. [7] Sporadic reports are available in the literature, revealing its presence as a pathogen causing oral lesion and mimicking candidiasis. [18] From India, there is a single report available from New Delhi, on pan gastrointestinal-mucosal involvement, by S. cereviseae in an AIDS patient. [19]

Geotrichum candidum was present as a single isolate from another subject. Though rare, various Geotrichum species are found to affect the respiratory tract, skin, oral cavity and also invasive infections, in the immunocompromised host. [8] Probably, this is the first report on the isolation of G. candidum from Indian HIV seropositive individuals.

The present study has brought forth an important finding, that the yield of potentially pathogenic yeast isolates was highest in the subjects belonging to Group C, with CD4 + counts below 200 cells/µl. The isolation was lesser in Group B subjects with CD4 + counts between 200 and 400 cells/µl, and least in subjects belonging to Group A, with CD4 + counts >400 cells/µl [Table 2]. This clearly indicates that the carriage of potentially pathogenic yeasts in the oral cavities is greater in patients, as they head toward immunosuppressive state. Though this finding is in corroboration with Maurya et al.,[5] there is a difference in observation as reported by Costa et al.,[6] and Ananthalakshmi et al. [9]

There are reports on emerging FL, a common antifungal used in therapy, resistant strains of C. albicans both in HIV seropositive [20] and seronegative persons. [21] Hence, an in vitro antifungal susceptibility test of Candida species was performed to determine their resistance patterns, and highlight an emerging trend if any, for future reference. Besides as therapy, in HIV seropositive individuals, FL is also recommended in lifelong prophylaxis to prevent complicated severe opportunistic infections in AIDS. [2] Though most Indian studies on in vitro antifungal resistance patterns of Candida isolates, revealed either nil or very low percentage of FL resistance in C. albicans isolated from various clinical specimens, [2] a higher percentage of C. albicans, isolated from Intensive Care Unit patients, were found to be resistant to FL. [21]

Interestingly, none of the C. albicans isolated from either HIV seropositive or control groups, showed resistance to FL and other antifungals. Absence of drug resistance in these isolates can be attributed to several factors including, degree of prior exposure to a particular drug, acquisition of resistance gene, and the intrinsic lack of resistance of Candida species. [2] Although a few HIV seropositive patients were on ART, none of them, was on FL prophylaxis, which probably explains the outcome of in vitro susceptibility test results.

The spectrum of results was different in NCAC isolates. Among the isolates from HIV group, resistance to FL was observed in all isolates of C. krusei while two isolates of C. glabrata showed dose-dependent susceptibility. However, NCAC species from healthy controls did not show resistance to any of the antifungal agents tested [Table 3]. The resistant isolates can be described as primarily or intrinsically resistant strains. Moreover, like C. albicans, none of the isolates showed resistance to other antifungal agents tested. However, this is only a 1 time testing; the study should ideally be repeated with isolates from the same patients at different time intervals to observe any changes in the patterns of carriage and susceptibility to various antifungal agents. [2]

To conclude, periodic surveys of oropharyngeal yeast colonization in a high-risk population, such as HIV-infected subjects, are useful in the clinical arena to bring out changes in spectrum if any. This study showcases the present scenario of the yeast distribution and resistance patterns of predominant species, colonizing the oral cavities of HIV seropositive individuals. This will be of help in understanding the current status of distribution of yeast species, in this high-risk patient population, as oral carriage may predispose to the development of clinically overt oral lesions especially in individuals heading toward immunocompromised state. Furthermore, our findings clearly indicate that the carriage of potentially pathogenic yeasts in the oral cavities is associated with reduced CD4 + count.


   Acknowledgments Top


The research group acknowledges, with thanks, the support rendered by the faculty and staff of the Department of Medicine, VMMC and Safdarjung Hospital, New Delhi, Dr. Malini Capoor, Department of Microbiology, VMMC and Safdarjung Hospital, New Delhi, in performing CLSI micro broth dilution test, Dr. Arunaloke Chakrabarti, Professor and Head, Department of Medical Microbiology, PGIMER, Chandigarh, in the genetic confirmation of S. cereviseae isolate and Dr. Rahul Saxena, Assistant Professor, Department of Biochemistry, School of Medical Sciences, Sharada University, Uttar Pradesh, in statistical analysis.

 
   References Top

1.
Sharifzadeh A, Khosravi AR, Shokri H, Asadi Jamnani F, Hajiabdolbaghi M, Ashrafi Tamami I. Oral microflora and their relation to risk factors in HIV+ patients with oropharyngeal candidiasis. J Mycol Med 2013;23:105-12.  Back to cited text no. 1
    
2.
Lattif AA, Banerjee U, Prasad R, Biswas A, Wig N, Sharma N, et al. Susceptibility pattern and molecular type of species-specific Candida in oropharyngeal lesions of Indian human immunodeficiency virus-positive patients. J Clin Microbiol 2004;42:1260-2.  Back to cited text no. 2
    
3.
Mane A, Panchvalli S, Bembalkar S, Risbud A. Species distribution and antifungal susceptibility of oral Candida colonising or infecting HIV infected individuals. Indian J Med Res 2010;131:836-8.  Back to cited text no. 3
[PUBMED]  Medknow Journal  
4.
Pavithra AJ, Kulkarni RD, Ajantha DS, Shubhada CA. Comparative evaluation of oral Candida carriage in HIV-infected individuals and HIV seronegative healthy individuals in North Karnataka. J Biosci Technol 2011;2:232-7.  Back to cited text no. 4
    
5.
Maurya V, Srivastava A, Mishra J, Gaind R, Marak RS, Tripathi AK, et al. Oropharyngeal candidiasis and Candida colonization in HIV positive patients in northern India. J Infect Dev Ctries 2013;7:608-13.  Back to cited text no. 5
    
6.
Costa CR, Cohen AJ, Fernandes OF, Miranda KC, Passos XS, Souza LK, et al. Asymptomatic oral carriage of Candida species in HIV-infected patients in the highly active antiretroviral therapy era. Rev Inst Med Trop Sao Paulo 2006;48:257-61.  Back to cited text no. 6
    
7.
Meurman JH, Siikala E, Richardson M, Rautemaa R. Non-Candida albicans Candida yeast of the oral cavity. In: Mendez-Vilas A, editor. Communicating Current Research and Educational Topics and Trends in Applied Microbiology, Formatex, Spain; 2007. p. 719-31.  Back to cited text no. 7
    
8.
Bonifaz A, Vázquez-González D, Macías B, Paredes-Farrera F, Hernández MA, Araiza J, et al. Oral geotrichosis: Report of 12 cases. J Oral Sci 2010;52:477-83.  Back to cited text no. 8
    
9.
Ananthalakshmi R, Murali S, Sekar B. Association of asymptomatic oral candidal carriage, oral candidiasis with CD4 lymphocyte count in HIV/AIDS patients - A comparative study. J Indian Acad Dent Spec 2011;2:6-10.  Back to cited text no. 9
    
10.
Gugnani HC, Becker K, Fegeler W, Basu S, Chattopadhya D, Baveja U, et al. Oropharyngeal carriage of Candida species in HIV-infected patients in India. Mycoses 2003;46:299-306.  Back to cited text no. 10
    
11.
Milne LJ. Fungi. In: Collee JG, Duguid JP, Fraser AG, Marmion BP, editors. Mackie and McCartney: Practical Medical Microbiology. 13 th ed. Chichester: Churchill Livingstone; 1989. p. 675-99.  Back to cited text no. 11
    
12.
Slifkin M. Tween 80 opacity test responses of various Candida species. J Clin Microbiol 2000;38:4626-8.  Back to cited text no. 12
    
13.
Maxwell MJ, Messer SA, Hollis RJ, Boyken L, Tendolkar S, Diekema DJ, et al. Evaluation of E test method for determining fluconazole and voriconazole MICs for 279 clinical isolates of Candida species infrequently isolated from blood. J Clin Microbiol 2003;41:1087-90.  Back to cited text no. 13
    
14.
Clinical and Laboratory Standards Institute. Reference method for broth dilution antifungal susceptibility testing of yeasts, approved standard. 3 rd ed. Wayne, PA: CLSI Document M27-A3, Clinical and Laboratory Standards Institute (CLSI); 2008.  Back to cited text no. 14
    
15.
Hung CC, Yang YL, Lauderdale TL, McDonald LC, Hsiao CF, Cheng HH, et al. Colonization of human immunodeficiency virus-infected outpatients in Taiwan with Candida species. J Clin Microbiol 2005;43:1600-3.  Back to cited text no. 15
    
16.
Xu J, Mitchell TG. Geographical differences in human oral yeast flora. Clin Infect Dis 2003;36:221-4.  Back to cited text no. 16
    
17.
Premanathan M, Shakurfow FA, Ismail AA, Berfad MA, Ebrahim AT, Awaj MM. Treatment of oral candidiasis (thrush) by Saccharomyces cerevisiae. Int J Med Med Sci 2011;3:83-6.  Back to cited text no. 17
    
18.
Kwamin F, Nartey NO, Codjoe FS, Newman MJ. Distribution of Candida species among HIV-positive patients with oropharyngeal candidiasis in Accra, Ghana. J Infect Dev Ctries 2013;7:41-5.  Back to cited text no. 18
    
19.
Wadhwa A, Kaur R, Bhalla P. Saccharomyces cerevisiae as a cause of oral thrush and diarrhoea in an HIV/AIDS patient. Trop Gastroenterol 2010;31:227-8.  Back to cited text no. 19
    
20.
Jeddy N, Ranganathan K, Devi U, Joshua E. A study of antifungal drug sensitivity of Candida isolated from human immunodeficiency virus infected patients in Chennai, South India. J Oral Maxillofac Pathol 2011;15:182-6.  Back to cited text no. 20
[PUBMED]  Medknow Journal  
21.
Kaur R, Goyal R, Dhakad MS, Bhalla P, Kumar R. Epidemiology and virulence determinants including biofilm profile of Candida infections in an ICU in a tertiary hospital in India. J Mycol 2014; ID 359630.  Back to cited text no. 21
    



 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

Top
Print this article  Email this article
 

    

 
  Search
 
  
 
    Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
    Article in PDF (580 KB)
    Citation Manager
    Access Statistics
    Reader Comments
    Email Alert *
    Add to My List *
* Registration required (free)  


    Abstract
   Introduction
    Materials and Me...
   Results
   Discussion
   Acknowledgments
    References
    Article Tables

 Article Access Statistics
    Viewed1364    
    Printed37    
    Emailed0    
    PDF Downloaded72    
    Comments [Add]    

Recommend this journal