Oral candida prevalence and species specificity in leprosy

Oral candida prevalence and species specificity in leprosy

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Oral candida prevalence and species specificity in leprosy Bharti Gupta, BDS, MDS a, Shekhar Gupta, BDS, MDS b, Minal Chaudhary, BDS, MDS c, A. Thirumal Raj, BDS, MDS d, Kamran Habib Awan, BDS, PhD e, Shankargouda Patil, BDS, MDS, PhD a,∗ a

Department of Maxillofacial Surgery and Diagnostic Sciences, Division of Oral Pathology, College of Dentistry, Jazan University, Jazan, Saudi Arabia Department of Prosthetic Dental Science, College of Dentistry, Jazan University, Jazan, Saudi Arabia c Department of Oral and Maxillofacial Pathology and Microbiology, Sharad Pawar Dental College and Hospital, Datta Meghe Institute of Medical Sciences, Wardha, Maharashtra, India d Department of Oral Pathology and Microbiology, Sri Venkateswara Dental College and Hospital, Chennai, India e College of Dental Medicine, Roseman University of Health Sciences, South Jordan, Utah, United States b

a r t i c l e

i n f o

Article history: Available online xxx Keywords: Candida Leprosy Opportunistic infections Oral Prevalence



a b s t r a c t Background: Leprosy represents a chronic progressive debilitating disease. The severe morbidity associated with leprosy predisposes the patients to opportunistic infections. To assess the oral candida prevalence and species specificity in lepromatous leprosy patients. Methods: The cross-sectional study included 70 lepromatous leprosy patients under a multi-drug regimen for less than 1 year (group 1) and 70 healthy volunteers (group 2). Both group 1 and 2 were matched for potential confounding factors including age, gender, ethnicity, absence of HIV co-infection. Oral swab samples obtained from both groups were subjected to a series of conventional and molecular diagnostic modalities. Results: Yeast growth was statistically higher (0.0 0 06) in group 1 (45.7%) than in group 2 (18.5%). 28 of the 32 yeast growth in group 1 and all 13 yeast growth in group 2 were identified as candida. Among the 28 candida species in group 1, 23 (71.88%) were Candida albicans, 3 (9.37%) were Candida

Corresponding author. E-mail address: [email protected] (S. Patil).

https://doi.org/10.1016/j.disamonth.2019.100920 0011-5029/© 2019 Elsevier Inc. All rights reserved.

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parapsilosis, 1 (3.13%) was Candida lusitaniae and 1 (3.13%) was Candida nivariensis. Among group 2, 11 (84.6%) were Candida albicans, 1 (7.7%) was Candida parapsilosis and 1 was Candida tropicalis. Conclusion: Oral candida prevalence is higher in leprosy patients than in healthy individuals, indicating a predisposition towards opportunistic infections. The increasing prevalence of the non-candida albicans species in leprosy is a major concern as they have shown to possess inherent resistant towards common anti-fungal agents. © 2019 Elsevier Inc. All rights reserved.

Introduction India has the highest prevalence of leprosy in the world, followed closely by Brazil, Mozambique, Nepal, and Madagascar.1 Leprosy represents a chronic debilitating granulomatous condition caused by Mycobacterium leprae.2 Skin and peripheral nervous system are the primary targets of manifestation in leprosy. Although the introduction of multi-drug therapy has aided in effectively curbing the prevalence of leprosy, the affected individuals are predisposed to opportunistic infections, especially oral candidiasis.3 , 5 Unlike healthy individuals, oral candida infection in individuals with debilitating diseases progresses rapidly. The aggressive nature of candidiasis in these individuals can be attributed to their compromised immune system and dominance of treatment-resistant non- Candida albicans species.6–15 Reichart et al.3 , 4 conducted two studies on the oral candida prevalence and species specificity in leprosy patients. The first study3 compared the candida prevalence and species specificity between leprosy patients and healthy subjects in Thailand leprosy population. The second study compared candida prevalence and species specificity between the Thailand and Cambodian leprosy population. The 2002 study3 indicated that there was an increase in the oral candida prevalence in leprosy patients compared to the control group, but the difference was not statistically significant. A similar study conducted on the Brazilian leprosy population by de Arau jo Navas et al. inferred the same conclusion. The dominant candida species in each of these 3 studies varied. While the 2002 study by Reichart et al. found C.krusei to be the dominant species, the 2007 Reichart et al. study and 2009 de Arau jo Navas et al. study showed a predominance of C.albicans in the leprosy patients. Despite the variations in the predominant species, both Reichart et 2002 study3 and de Arau jo Navas et al. 2009 study5 showed a higher prevalence of non-candida albicans species in the leprosy population than in the control subjects. Non-candida albicans species are known for their inherent potential to resist common anti-fungal agents like fluconazole.6–12 Thus, potent anti-fungal agents like amphotericin B are required to overcome the resistance of non-candida albicans species. As the anti-fungal regimens would depend on the candida flora, it vital to first study the prevalence and species specificity of candida in a specific population before formulating treatment regimens. Thus, the present study was formulated to investigate the prevalence and the species specificity of oral candida in lepromatous leprosy patients in Central India.

Materials and methods Group 1 70 lepromatous leprosy patients admitted to the Maharogi Kushta Seva Samiti Wardha and Varora (Anandwan) and Department of Dermatology, JNMC, Wardha (Free leprosy treatment center for central India) were included. The diagnosis of lepromatous leprosy was confirmed by Please cite this article as: B. Gupta, S. Gupta and M. Chaudhary et al., Oral candida prevalence and species specificity in leprosy, Disease-a-Month, https://doi.org/10.1016/j.disamonth.2019.100920

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clinical examination (six or more skin lesions) and microbial examination (all lesions positive smear for AFB) as recommended by the WHO Committee on Global Strategy for Further Reducing the Leprosy Burden and Sustaining Leprosy Control Activities 2006–2010.1 All included patients were on the WHO recommended leprosy multidrug treatment regimen [rifampicin, clofazimine, and dapsone] for less than 1 year. None of the patients were on any anti-fungal regimens. ELISA, Immunocomb, and Tri-dot were used to exclude the possibility of HIV-co-infection in the included cases.

Group 2 70 healthy individuals visiting the Sharad Pawar Dental College were included. To confirm the absence of leprosy and/or HIV infections, all the control subjects were subjected to WHO diagnostic tests for leprosy and serological tests (ELISA, Immunocomb, and Tri-dot) for HIV.

Matching To avoid confounding factors, both the groups were matched for age, gender and ethnicity. The average age of group 1 and 2 were 45 years and 44 years respectively. Group 1 consisted of 44 males (62.85%) and 26 females (37.15%) while group 2 consisted of 40 males (57.14%) & 30 females (57.14%).

Ethics and consent Institutional (Datta Meghe Institute of Medical Sciences) ethics committee approval (reference number-DMIMSU/IEC/09) was obtained. The study was explained and consent was obtained from all included participants.

Sample collection From each patient, two oral swabs (from the buccal mucosa and hard palate) were collected using a sterile fungi quick swab (Himedia, India). Swabs were immediately transported in charcoal alginate transport media. Among the two swabs, one was subjected to Gram staining and other was used for culture.

Investigations used A combination of gram staining, sabouraud’s dextrose agar culture, germ tube test, chlamydospore formation on cornmeal agar, sugar assimilation test, CHROMagar culture were used to assess the candida flora of the samples. The findings were confirmed using polymerase chain reaction.

Gram staining Swabs collected from patients were rolled on slides to prepare smears for gram staining. Smears were allowed to air dry. They were heat-fixed by rapidly passing them 2–3 times over a flame of a bunsen burner. Staining included crystal violet as the basic stain and safranin as the counterstain. Gram-positive dark purple yeast cells approximately 4–8 μm with budding were considered positive for candida. Please cite this article as: B. Gupta, S. Gupta and M. Chaudhary et al., Oral candida prevalence and species specificity in leprosy, Disease-a-Month, https://doi.org/10.1016/j.disamonth.2019.100920

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Sabouraud’s dextrose agar culture (SDA) The culture media (SDA, Himedia-India) was dispensed in test tubes and cooled at the slanted position and stored in the refrigerator. Samples were inoculated on the media. For each patient, 2 SDA were inoculated. One medium was incubated at 37 °C, the other at 42–45 °C (as C. albicans grows at this temperature and some strains of C. dubliniensis do not grow at 42–45 °C.). The colony morphology interpretation was as follows: (a) cream colored, pasty and smooth were C. albicans; (b) cream colored to off-white, glistening to dull, soft, smooth or wrinkled with mycelial fringe were C.tropicalis; (c) cream to yellow, flat, dull and dry were C. krusei; (d) cream colored, glistening, smooth were C. glabrata (e) cream colored smooth and pasty were C. dubliniensis. Following culture, direct microscopy was used to confirm the diagnosis. Gram-positive budding yeast cells approximately 4–8 μm with pseudohyphae were confirmed as candida.

Germ tube test (Raynaulds–Braude phenomenon) This rapid screening procedure is used to differentiate C. dubliniensis and C. albicans from other Candida species. The cultured samples were incubated with 1 ml of mammalian sterile serum (fetal bovine, sheep or normal human) for 2–4 h at 37 °C. Following the incubation, the presence of the germ tube was confirmed by examining a drop of the suspension on a glass slide under a microscope. Germ tubes appear as a distal extension from the yeast cell. The characteristic feature of these germ tubes is the lack of constriction at point of attachment to the yeast cell. Production of germ tube is only the presumptive identification of C. albicans and C. dubliniensis.

Cornmeal Agar Culture Cornmeal Agar Culture was used to demonstrate the formation of chlamydospores. Cornmeal and agar was added to 10 0 0 ml of water. The medium was autoclaved\121 °C for 15 min and poured in plates. Slide culture technique was used for inoculation of cornmeal agar. Blocks of corn meal agar of the volume 1936 mm3 (22 × 22 × 4) were used. A block was placed on a sterile slide and a sterile coverslip was placed over it. A part of the colony was taken with the help of a sterile straight wire and was inoculated at the corners of the block, 1–2 mm below the surface. The slide was placed in a moist chamber and incubated at room temperature for 48 h. The slides were observed under the microscope for in situ morphology. The growth pattern, morphology of hyphae, arrangement of blastospores and production of chlamydospores were observed. Candida albicans and Candida dubliniensis are capable of producing chlamydospores on cornmeal agar.

Sugar assimilation test The ability of Candida organisms to assimilate different sugars was assessed for tentative identification of subtypes. Himedia sugar discs used for sugar assimilation tests included: Lactose (La), Maltose (Ma), Raffinose (Rf), Dextrose (De), Sucrose (Su), Cellobiose (Ce), Galactose (Go), Trehalose (Te), Melibiose (Mb), Xylose (Xy), Inositol (Is), Dulcitol (Du). Each disc had 25 mg of respective sugar. All sugar discs were inoculated on Yeast Nitrogen Base Agar. A twin pack was used to prepare Yeast Nitrogen Base Agar. (HiMedia Laboratories Limited). Yeast nitrogen base agar medium was used to perform a sugar assimilation test. A single isolate was swabbed on the whole culture plate to obtain lawn culture. Five different carbohydrate differentiation discs were placed on the surface of this medium at a distance of 3 cm from each other. The ability to assimilate five sugars by an isolate was tested at a time. These Please cite this article as: B. Gupta, S. Gupta and M. Chaudhary et al., Oral candida prevalence and species specificity in leprosy, Disease-a-Month, https://doi.org/10.1016/j.disamonth.2019.100920

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plates were incubated at 37 °C for 24–48 h. At the end of 48 h, the plates were observed for any growth around the sugar discs. The positive test was indicated by growth around the disc. The growth detects the ability of the sugar deprived organism to assimilate particular carbohydrate present in the disc. No growth around the disc denoted that the organism could not use the sugar for its growth. Table 1 summarizes the sugar assimilation characteristics for Candida species

Table 1 Sugar assimilation characteristics for candida species. Species

Su

Ga

Ma

Xy

Mb

Te

Ce

Rf

Du

In

La

C.albicans C.dubliniensis C.lusitaniae C.parapsilosis C.krusei C.glabrata C.tropicalis

+ve +ve +ve +ve –ve –ve +ve

+ve +ve +ve +ve –ve –ve +ve

+ve +ve +ve +ve –ve –ve +ve

+ve –ve +ve +ve –ve –ve +ve

–ve –ve –ve –ve –ve –ve -ve

+ve +ve +ve +ve –ve +ve +ve

–ve –ve +ve –ve –ve –ve +ve

–ve –ve –ve –ve –ve –ve –ve

–ve –ve –ve –ve –ve –ve –ve

–ve –ve –ve –ve –ve –ve –ve

–ve –ve –ve –ve –ve –ve –ve

CHROMagar It is a chromogenic media which distinguished the candida species based on colors resulting from biochemical reactions between specific enzymes and proprietary chromogenic substrate. It enables the identification of several species in each sample. Thus patients with mixed candida species can be assessed through CHROMagar. The samples were incubated in the CHROMagar at 37 °C. The cultures were examined at regular intervals (1st, 2nd, and 3rd day) after inoculation. The observations recorded include colony growth, colors, and forms. Color code for each Candida species in the CHROMagar are as follows: C. albicans-Light green; C. parapsilosis-Cream colored; C. tropicalis- Blue with pink halo; C. krusei-Pink; C. glabrata- Purple; C. dubliniensis-Dark green; C.lusitaniae- Pink; C. nivariensisCream to white

Urease test It is used to test the ability of the Candida species to dissimilate urea. The test was performed using pure growth from selective agar. The inoculated slope was incubated at 35–37 °C in the incubator. Slopes were examined after 4 h and after overnight incubation. A positive result was observed as a purple/pink color. There was a need to apply urease test for those samples which did not show any growth on chromagar for further confirmation.

DNA extraction from yeast colonies 500 μl suspension of the fungal samples were centrifuged for 5 min at 15,300 g using a refrigerated centrifuge. The resulting pellet was mixed with a 600 μl solution of 10% Emercaptoethanol, 10 mM EDTA, 50 mM Tris, 250 U/ml lyticase (L2524, Sigma). To promote cellwall digestion, the samples were kept for 2 h 30 min at 37 °C. For digesting the proteins and lyzing the nucleus, 200 μg/ml proteinase K and 0.2% SDS (Banglore Genie) were added and the samples were maintained for 45 min at 65 °C, followed by 5 min centrifugation at 15,300 g. Isopropanol (Merck) (v/v) was added to the resulting supernatant and the sample was maintained overnight at −20 °C. Following another round of centrifugation (at 15,300 g for 5 min), 500 μl of 70% ethanol (Merck) was added to the DNA containing pellet for rehydration. 1 M Tris-EDTA Please cite this article as: B. Gupta, S. Gupta and M. Chaudhary et al., Oral candida prevalence and species specificity in leprosy, Disease-a-Month, https://doi.org/10.1016/j.disamonth.2019.100920

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buffer was used to rehydrate the final DNA pellet which was then maintained at −20 °C until further procedures.

Polymerase chain reaction The ribosomal DNA’s ITS region was amplified using ITS1 5 -TCCGTAGGTGAACCTGCGG-3 was used as the forward primer and ITS4 5 -TCCTCCGCTTATTGATATGC-3 as the reverse primer. The ITS region contains several regions of relatively well-conserved sequences useful to obtain proper sequence alignment and yet the ITS region also contains sufficient sequence variability. Amplifications were carried out in a final volume of 50 μl. Each reaction contained 27.5 μl distilled water, 6 μl of template DNA, 5 μl of 10× PCR buffer, 3 μl each primer at 0.5 μM, 4 μl of deoxynucleoside triphosphate (dNTP) at 0.25 mM, 0.25 μl Taq DNA Polymerase (5 U/μL). The thermal cycler program consisted of 5-min denaturation at 94 °C, followed by 35 consequent cycles, each of 30-second duration at 94 °C. The denaturation was followed by 45-s annealing at 56 °C. The last phase of the cycle consisted of the final extension for 1 min at 72 °C and a final extension at 72 °C for 10 min.

Agarose gel electrophoresis 2 g of agarose was dissolved in 100 ml of 0.5× TBE by heating to prepare 2% Agarose gel. Then, 2 μL of ethidium bromide (10 mg/ml) was added to the agarose solution. The comb was placed in the sealed gel-casting tray. After it cooled to 50–55 °C the solution was allowed to set by pouring without any bubbles into the gel platform. Wells was created in the agarose gel, which was loaded with a mixture of gel loading dye (1 μL) and the PCR product (8 μL). The electrophoresis was performed at 100 V for 40 min.

PCR product recovery After getting the visible bands under UV light the bands were cut using a scalpel. GeNei Quick PCR purification kit was used to purify the species-specific PCR products. The purified products were sent for sequencing.

Analysis of sequence data Samples were sequenced in 1st BASE DNA sequencing lab, Singapore. Both the strands were sequenced separately for all samples. Species identification was done with nucleotide sequence provided by 1st BASE using Basic Local Alignment Search Tool (BLAST) facility in NCBI database.

Results Candida growth on SDA 32 (45.7%) samples in group 1 and 13 (18.5%) samples in group 2 exhibited yeast growth in SDA. The difference between group 1 and 2 was statistically significant (0.0 0 06). 28 out of the 32 yeast samples in group 1 were identified to be candida based on their cream-colored glistening, soft and smooth appearance (Fig. 1(A)). Rest of the 4 samples in group 1 have different colony characteristics and were designated as non-candida species (Fig. 1(B)). Among the candida positive samples, 23 from group 1 (71.88%) and 11 (78.57%) from group 2 were identified as C. albicans. Please cite this article as: B. Gupta, S. Gupta and M. Chaudhary et al., Oral candida prevalence and species specificity in leprosy, Disease-a-Month, https://doi.org/10.1016/j.disamonth.2019.100920

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Fig. 1. (A) C. albicans exhibiting cream-colored glistening, soft and smooth colonies; (B) cream colored highly wrinkled or radially folded colonies identified as non-candida species. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2. (A) Gram-positive dark purple yeast cells; (B) chlamydospore formation in cornmeal agar. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Direct microscopy Gram-positive dark purple yeast cells approximately 4–8 μm (10 0 0×) with budding were considered positive for candida. All the cases from both groups which showed cream colored glistening, soft and smooth colonies in SDA exhibited gram-positive dark purple yeast cells (Fig. 2(A)). Chlamydospore formation test Based on the morphology of hyphae, the arrangement of blastospores and production of chlamydospores, C.albicans was identified in 23 samples of group 1 (23 out of 32, 71.88%) and 11 samples (78.57%) of group 2 (Fig. 2(B)). Please cite this article as: B. Gupta, S. Gupta and M. Chaudhary et al., Oral candida prevalence and species specificity in leprosy, Disease-a-Month, https://doi.org/10.1016/j.disamonth.2019.100920

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Fig. 3. (A) Showing positive sugar assimilation (growth around all disc) for xylose, maltose trehalose, galactose, and sucrose; (B) showing positive sugar assimilation (growth around all disc) for cellobiose and negative (no growth around the disc) for raffinose, melibiose, inositol, and dulcitol.

Germ tube test 23 samples of group I (71.88%) and 11 samples (78.57%) in group 2 demonstrated germ tube formation, which could be considered positive for either Candida albicans or Candida dubliniensis or both.

Sugar assimilation test The ability of Candida organisms to assimilate different sugars was assessed for identification of subtypes. Himedia sugar discs were used for sugar assimilation tests. All the 23 samples which had germ tube formation and showed growth at 42–45 °C, were able to assimilate all sugars [Dextrose (De), Maltose (Ma), Sucrose (Su), Galactose (Go), Trehalose (Te), Xylose (Xy), except Raffinose (Rf), Melibiose (Mb), Inositol (Is), Dulcitol (Du), Lactose (La), Cellobiose (Ce)] indicating C.albicans (Fig. 3(A)). 4 samples which had different colony morphology did not assimilate any sugar. Two samples were positive for cellobiose which confirms them to be either candida lucitaniae or candida parapsilosis (Fig. 3(B)). Collective results of sugar assimilation test are tabulated in Table 2.

CHROMagar culture Group 1 23 samples (71.88%) showed light green colored colonies which were considered to be positive for C. albicans (Fig. 4(A)). One sample showed a distinct cream-colored colony which is suggestive of C. nivareniesis (Fig. 4(B)). One sample (3.2%) showed pink colored colony which is suggestive of C. lusitaniae (Fig. 4(C)) and 3 samples (9.37%) showed white colored colony which is considered to be positive for C. parapsilosis (Fig. 4(D)). Other four samples (12.50%) did not show any kind of growth on chromagar. Group 2 11 (84.6%) showed light green colored colonies which were considered to be positive for C.albicans. 2 samples (15.4%) showed a white colored colony which was considered to be positive for C. parapsilosis. The results of the candida growth on CHROMagar are summarized in Table 3. Please cite this article as: B. Gupta, S. Gupta and M. Chaudhary et al., Oral candida prevalence and species specificity in leprosy, Disease-a-Month, https://doi.org/10.1016/j.disamonth.2019.100920

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Table 2 Results of sugar assimilation tests. Sap.no.

Su

Ga

Ma

Xy

Mb

Te

Ce

Rf

Du

In

La

3 5 8 12 14 15 24 30 40 45 51 52 55 60 62 64 65 66 68 71 73 76 78 79 82 83 84 85 88 90 95 98

+ve +ve +ve +ve +ve +ve +ve +ve +ve +ve +ve +ve –ve +ve +ve +ve +ve +ve +ve +ve +ve –ve +ve +ve +ve +ve –ve +ve +ve +ve –ve +ve

+ve +ve +ve +ve +ve +ve +ve +ve +ve +ve +ve +ve –ve +ve +ve +ve +ve +ve +ve +ve +ve –ve +ve +ve +ve +ve –ve +ve +ve +ve –ve +ve

+ve +ve +ve +ve +ve +ve +ve +ve +ve +ve +ve +ve –ve +ve +ve +ve +ve +ve +ve +ve +ve –ve +ve +ve +ve +ve –ve +ve +ve +ve –ve +ve

+ve +ve +ve +ve +ve +ve +ve +ve +ve +ve +ve +ve –ve +ve +ve +ve +ve +ve +ve +ve +ve –ve +ve +ve +ve +ve –ve +ve +ve +ve –ve +ve

–ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve

+ve +ve +ve +ve +ve +ve +ve +ve +ve +ve +ve +ve –ve +ve +ve +ve +ve +ve +ve +ve +ve –ve +ve +ve +ve +ve –ve +ve +ve +ve –ve +ve

+ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve

–ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve

–ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve

–ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve

–ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve –ve

Probable diagnosis sugar assimilation C. lusitaniae C. albicans C. albicans C. albicans C. Parapsilosis C. albicans C. albicans C. Parapsilosis C. albicans C. Parapsilosis C. albicans C. albicans Negative C. albicans C. albicans C. albicans C. albicans C. albicans C. albicans C. albicans C. albicans Negative C. albicans C. Parapsilosis C. albicans C. albicans Negative C. albicans C. albicans C. albicans Negative C. albicans

Table 3 Percentage-wise distribution of growth on chromagar in both groups. Species

Group I

Group II

Candida albicans. Candida parapsilosis. Candida lusitaniae Candida nivariensis Noncandida species which do not show any growth on chromagar Total

23 (71.88%) 3 (9.37%) 1 (3.13%) 1(3.13%) 4(12.50%)

11 (84.6%) 2 (15.4%) –

32(10 0.0 0%)

13(10 0.0 0%)



Candida flora in group 1 and 2 as identified by combining the results of the SDA culture, germ tube test, chlamydospore formation, sugar assimilation test, and CHROMagar Group 1 23 out of 32 samples (71.88%) were positive for C. albicans in group 1. 3 samples (9.37%) were positive for C.parapsilosis. 1 sample (3.13%) was identified as Candida lusitaniae and 1 sample as C. nivariensis. Four samples did not show any growth on chromagar and could not be identified by any other conventional diagnostic procedure. Different colony morphology on SDA of these samples was suggestive of non-candida species like Trichosporon, Blastocystomysis, and Geotricum. Further differentiation was done by urease Test. Out of four samples, one sample Please cite this article as: B. Gupta, S. Gupta and M. Chaudhary et al., Oral candida prevalence and species specificity in leprosy, Disease-a-Month, https://doi.org/10.1016/j.disamonth.2019.100920

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Fig. 4. (A) Light green colored colonies of C. albicans; (B) cream colored colonies of C. nivariensis; (C) pink colored colonies of lusitaniae; (D) white to cream colored colonies of C. parapsilosis. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) Table 4 Candida flora in group 1 and 2 as identified using the SDA culture, germ tube test, chlamydospore formation, sugar assimilation test, and CHROMagar. Species

Group I

Group II

χ 2 -value

C. albicans C. parapsilosis C. nivareniesis C. lusitaniae Noncandida species Total

23(71.88%) 3(9.37%) 1(3.13%) 1(3.13%) 4(12.50%) 32(10 0.0 0%)

11(84.64%) 2(15.36%) – – – 13(10 0.0 0%)

2.93 p-value=0.56 Non-significant, p > 0.05

showed positivity which is probably suggestive of Trichosporon species. No C. dublineinsis or C.krusei was found in group 1 or group 2 patients. Group 2 11 (84.6%) samples were considered positive for Candida albicans and two samples were (15.45) considered positive for C. parapsilosis. The overall candida prevalence was higher in group 1 than group 2. The predominant species found in both the groups was C. albicans. The summary of the findings is tabulated in Table 4.

Confirmation of the candida species through PCR The Candida species identified using the conventional methods (SDA culture, germ tube test, chlamydospore formation, sugar assimilation test, and CHROMagar were confirmed using PCR (Fig. 5(A) and (B)). Please cite this article as: B. Gupta, S. Gupta and M. Chaudhary et al., Oral candida prevalence and species specificity in leprosy, Disease-a-Month, https://doi.org/10.1016/j.disamonth.2019.100920

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Fig. 5. Agarose gel of samples post-DNA amplification. (A) Lane 1- NC, Lane 2- C. lusitaniae, Lane 3,4,5,6,7,9,10 and 11- C. albicans, Lane 12- C. parapsilosis, Lane 8- 100bp ladder; (B) Lane C4,5,18,23,30,36,41,60,64,71,8,78,79,82,83,88,90Candida albicans; Lane C45-Candida nivariensis, Lane 76, 84, 85-Trichosporon asahii, the center- 100bp ladder. Table 5 Candida flora in group 1 and 2 as identified by PCR. Species

Group I

Group II

χ 2 -value

C. albicans C. parapsilosis C. nivariensis C. lusitaniae C. tropicalis Trichosporon asahii Total

23(71.88%) 3(9.38%) 1(3.13%) 1(3.13%) – 4(12.50%) 32(100%)

11(84.62%) 1(7.69%) – – 1(7.69%) – 10 0.0 0

1.27 p-value=0.87 NS, p> 0.05

Group 1 The majority of the yeasts in group 1 were candida albicans (23 of 32, 71.88%). The second most common Candida species was candida parapsilosis (3 of 32, 9.38%). The remaining species were candida lusitaniae (1 of 32, 3.13%) and candida nivariensis (1 of 32, 3.13%). Interestingly we found the second largest yeast group as that of trichosporon species. 4 samples out of 32 were positive for Trichosporon asahii. Literature has shown that Trichosporon asahii has been isolated from various types of clinical specimens from immunocompromised patients. Trichosporon asahii can be found as commensals in the human gut or skin flora and in the environment. Candida dubliniensis and candida krusei were absent. Group 2 11 (84.62%) out of 13 samples were positive for candida albicans, one sample (7.69%) was candida parapsilosis and one (7.69%) was candida tropicalis. The summary of the Candida flora as identified by PCR is tabulated in Table 5 Please cite this article as: B. Gupta, S. Gupta and M. Chaudhary et al., Oral candida prevalence and species specificity in leprosy, Disease-a-Month, https://doi.org/10.1016/j.disamonth.2019.100920

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Table 6 Comparison of diagnostic potential between PCR and conventional microbiological diagnostic methods (SDA culture, germ tube test, chlamydospore formation, sugar assimilation test, and CHROMagar). Yeast growth Number of samples identified Number of samples unidentified Total Sensitivity Specificity Positive predictive value Negative predictive value

Group 1 (Lepromatous leprosy patients) Conventional Diagnostic method

DNA sequencing

28 4 32 Sensitivity Analysis 87.50% 0.00% 46.67% 0.00%

32 0 32 95% confidence interval 71–96.49% 0–10.89% 33.67–60.00% 0.00–60.24%

Comparison of the conventional and molecular diagnostic methods are tabular in Table 6. The sensitivity of conventional techniques was 87.50% while PCR had 100% sensitivity. The 4 cases in group 1 which we unidentified in conventional methods and termed as non-candida species, were identified as Trichosporon asahii through PCR.

Discussion Immuno-compromised state predisposes to opportunistic infections. Oral candidiasis is one of the most common opportunistic infection seen in debilitating chronic diseases such as AIDS, cancer, and diabetes.6–15 Unlike healthy individuals, the candida infections in debilitated patient’s progress rapidly, which can be attributed to both the suppressed immune system and the emergence of non-candida albicans species. The clinical significance of the floral shift is due to the inherent resistance shown by non-candida albicans species to common anti-fungal agents.6–12 Leprosy represents a chronic progressive granulomatous disease. Although the introduction of multidrug regimens has aided in curbing the progression of leprosy, the irreversible damage induced by the disease predisposes the patients towards opportunistic infection.1–5 Unlike, AIDS, cancer and diabetes, the oral candida flora in leprosy are relatively less explored. PubMed search revealed only 3 studies in the English language exploring the oral candida prevalence and flora in leprosy patients.3–5 The first study was conducted by Reichart et al. in 20023 on Thailand population. They did not find any statistically significant difference in the candida prevalence between leprosy (80% prevalence) and the control group (65% prevalence). Non-candida albicans C. krusei was the predominant candida species in leprosy as opposed to C. albicans in healthy individuals. C. albicans was the second most common in leprosy patients. The other non-candida albicans species isolated from both groups included C. tropicalis, C. parapsilosis. The non-candida albicans present only in the control group were C. cerevisiae and H.polymorpha. The non-candida species present only in the leprosy group included C. famata, C. rugosa, C. guilliermondii, and Pichia ohmeri. The growing resistance of the non-candida albicans was confirmed by C. krusei resistance towards common anti-fungals (fluconazole and ketoconazole) while showing sensitivity to amphotericin B.3 In order to confirm their earlier findings, Reichart et al. in 20074 investigated the candida prevalence and flora in Thailand population and compared it to the Cambodian population. There was no difference in the candidal prevalence between the two population. Unlike their 2002 study,3 the predominant candida species in both Thailand and Cambodian population was C.albicans. The third study on candida flora in leprosy patients was conducted in a Brazilian population by de Arau jo Navas et al. in 2009.5 The study included leprosy patients who were under the multidrug regimen for at least 45 days. Similar to Reichart et al. 2002 and 2006 findings,3 , 4 there was no statistically significant difference (p = =0.099) in the candida prevalence between leprosy patients (65.8%) and healthy individuals (47.4%). The C. albicans, C. tropicalis, and C. kefyr Please cite this article as: B. Gupta, S. Gupta and M. Chaudhary et al., Oral candida prevalence and species specificity in leprosy, Disease-a-Month, https://doi.org/10.1016/j.disamonth.2019.100920

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were isolated from both the leprosy patients and the control subjects, with C. albicans being the predominant species in both the groups. C. parapsilosis was noted only in the leprosy patient while C. glabrata was found only in the control subject.5 Overall the results of previous studies have shown that there was a mild increase in the prevalence of candida in leprosy patients than in control subject, although the difference was not statistically significant. The constant findings in the previous studies were the significant increase in the non-candida albicans population in leprosy patients compared to the control subjects. The predominant non-candida albican species and the non-candida albicans flora varied with each study which in turn could have been due to differences in the population. To the best of our knowledge, the present study is the first to explore the candida prevalence and flora on leprosy population in India. The study participants were from Maharogi Kushta Seva Samiti Wardha and Varora (Anandwan) and Department of Dermatology, JNMC, Wardha. All the included patients were under multi-drug therapy for at least 6 months. As HIV prevalence is high in India, all included patients were checked for potential HIV co-infection. Three serological tests including the Immunocomb, ELISA and Tri-dot were used to confirm the absence of HIV in the included patients. Unlike, the findings of Reichart et al. 2002, Reichart et al. 2006 and de Arau jo Navas et al. 2009,3–5 there was a statistically significant difference in the yeast prevalence between the healthy population and the leprosy patients. The significant difference in the present study could have been a result of the relatively larger sample size. C. albicans was the predominant species identified in both the groups. SDA media showed growth of yeast in 32 samples from leprosy patients and 13 from the control group. Although it was possible to identify all the 13 yeast growth in the control group using conventional methods (SDA, chlamydospore formation, gram staining, germ tube test, sugar assimilation tests, CHROMagar), only 28 of the 32 yeast growth was recognized as candida in the leprosy group. The remaining 4 samples were designated as non-candida species. In order to confirm the findings and to identify the 4 non-candida species, all the samples were subjected to PCR. All 11 C. albicans (84.6%) samples in the control group were confirmed by PCR, but among the 2 samples identified as C. parapsilosis, only one was confirmed while the other was identified as C. tropicalis. Among the leprosy patients, PCR confirmed all the species identified in the conventional methods as C. albicans (71.88%), C. parapsilosis (9.37%), C. lusitaniae (3.13%), and C. nivariensis (3.13%). C. albicans and C. parapsilosis were isolated from both leprosy and control group. C. lusitaniae (3.13%), and C. nivariensis were present only in the leprosy patients while C. tropicalis was detected only in the control subjects. In addition to these, there were 4 samples with yeast growth which were broadly classified by conventional methods as non-candida species. These 4 samples were identified by PCR as Trichosporon asahii. Similar to candida, T. asahii is an opportunistic microbe. It can be present as a commensal on the skin, stool, and urine. T. asahii infections are noted mostly in immunocompromised states including AIDS, post-transplant cases, hematogenous cancers, especially acute myelogenous leukemia.16 In addition to an immunocompromised state, the use of central vein catheter is considered as a predisposing factor for T. asahii infections, as the organism has the ability to adhere and form biofilms on catheters causing deep invasive infections. Like non-candida albicans species, T. asahii, have known to show resistance to common anti-fungal agents and requires potent antifungal agents like amphotericin B to curb its infection.16–18 The presence of T. asahii in leprosy patients under multi-drug therapy is an indicator that despite the treatment regimens, leprosy patients have a severely debilitating condition predisposing them to opportunistic infections. To summarize, although the oral candida flora of leprosy patients varied among different populations, a common finding in all the published data and the present study was the increasing prevalence of non-candida albicans species.

Please cite this article as: B. Gupta, S. Gupta and M. Chaudhary et al., Oral candida prevalence and species specificity in leprosy, Disease-a-Month, https://doi.org/10.1016/j.disamonth.2019.100920

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Conclusion The increasing prevalence of non-candida albicans species in leprosy could be detrimental to the oral health status of the patients. In addition to being treatment resistant, these noncandida albicans species have shown to be a potential risk factor a number of progressive oral diseases including oral cancer.19–22 Thus, it is vital that in addition to receiving multidrug regimen, the leprosy patients are also closely monitored for potential oral infections, especially oral candidiasis. As mentioned earlier, common anti-fungals including fluconazole and itraconazole may not be effective against oral candidiasis in leprosy patients due to the presence of noncandida albicans species. Thus, it may be necessary to use potent anti-fungal like amphotericin B to overcome the inherent resistance of the candida flora in leprosy patients.

Declaration of Competing Interest None declared.

Disclaimer None.

Funding None.

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