First isolations in India of Candida nivariensis, a globally emerging opportunistic pathogen

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1 Medical Mycology March 2010, 48, First isolations in India of Candida nivariensis, a globally emerging opportunistic pathogen ANURADHA CHOWDHARY*, H. S. RANDHAWA *, Z. U. KHAN, S. AHMAD, S. JUNEJA *, B. SHARMA, P. ROY*, G. SUNDAR * & L. JOSEPH *Department of Medical Mycology, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi, India, Department of Microbiology, Faculty of Medicine, Kuwait University, Safat, Kuwait, and Department of Medicine, Dr. Ram Manohar Lohia Hospital, New Delhi, India We randomly screened 363 yeast isolates during 2008 for their ability to form white colonies on CHROM agar Candida medium. Two of these isolates (0.5%) were identified as Candida nivariensis based on detailed phenotypic characterization and DNA sequencing. One was recovered from the sputum of an HIV-positive patient with a pneumonic lesion and the second from the blood of a diabetic with oropharyngeal lesions. Direct DNA sequencing of the D1/D2 region of 28S rrna gene and/or the internal transcribed spacer (ITS) regions of rdna confirmed that both of the isolates were C. nivariensis. The carbohydrate assimilation profiles with the ID 32 C and VITEK 2 yeast identification systems revealed only glucose assimilation. In vitro antifungal susceptibility profiles by broth microdilution and Etest methods revealed susceptibility of both isolates to fluconazole, itraconazole, voriconazole, amphotericin B and 5-flucytosine, with low MICs for posaconazole and caspofungin. These results document the occurrence of Candida nivariensis for the first time in India and focus on its potential as an opportunistic human pathogen. Keywords Candida nivariensis, candidiasis, emerging, opportunistic pathogen, India Introduction The incidence of invasive fungal infections has been rising in recent decades, especially among immunocompromised patients, with a mortality rate as high as 40% in cases involving candidemia [ 1 ]. More than 17 species of Candida have been reported as etiologic agents of invasive candidiasis in humans [ 2,3 ]. Although over 90% of such isolates were found to be C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, and C. krusei, the number of pathogenic species within the genus continues to grow due to changes in medical care, increasing numbers of immunocompromised patients and more frequent specieslevel identifications currently provided by mycology laboratories to optimize species specific therapy [3 5 ]. Species Received 6 April 2009; Final revision received 12 May 2009; Accepted 12 June 2009 Correspondence: H. S. Randhawa, Department of Medical Mycology, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi , India.Tel: ; fax: ; hsrandh5@ yahoo.co.in such as C. glabrata, C. lusitaniae and C. krusei are generally more resistant to antifungal agents than C. albicans. Therefore, rapid and accurate species identification is often critical in ensuring early and effective antifungal therapy. The identification of Candida and other yeast-like fungi is generally done by a combination of morphological features, ability to ferment selected sugars and assimilation of a large number of carbon and nitrogen compounds [ 6 ]. Two commercial methods for yeast identification, the API and VITEK systems require only 2 to 3 days for reading the results [ 7 ] but their databases are limited [ 8 ]. Therefore, species identification of a number of pathogenic yeast species that are difficult to identify by phenotypic methods requires the application of molecular methods. One such yeast-like pathogen is Candida nivariensis which has been recently demarcated from its closely related taxa, Candida bracarensis and Candida glabrata [9,10 ]. Currently, the available assimilation tests in the API database are not helpful for identification of C. nivariensis. Following its first isolations in 2005 from three patients in a single Spanish hospital over a 3-year period, solitary reports have 2010 ISHAM DOI: /

2 Candida nivariensis in India 417 appeared on the isolation of the yeast from Japan, Indonesia, United Kingdom and Australia [ 9,11 14 ]. In this communication, we report for the first time the isolation of C. nivariensis in India. The yeast-like organism is a globally emerging opportunistic pathogen. Material and methods Clinical specimens and C. nivariensis isolation During 2008, 363 yeast isolates recovered from miscellaneous clinical specimens referred to the Diagnostic Mycology Laboratory of V. P. Chest Institute were randomly screened for their ability to form white yeast-like colonies on CHROM agar Candida medium (Difco Becton Dickinson, Sparks, MD, USA). Two of these isolates that developed white colonies on CHROM agar Candida medium were identified as C. nivariensis. Brief case histories of the two patients from whom C. nivariensis were recovered are as follows: Case 1 A 45-year-old male, resident of Delhi, India, was diagnosed as HIV-positive two years prior to his admission to a medical ward at Dr. Ram Manohar Lohia Hospital, New Delhi, in May He complained of fever, cough, dyspnoea, occasional bouts of hemoptysis and chest pain for the past 15 days and had diarrhoea and weight-loss over the last few months. At the time of admission, his general physical examination revealed a thin, cachectic, pale, anicteric patient with pulse rate 82 per minute, respiration rate 20 breaths per minute and pyrexia of F. He further showed crepitations in interscapular and right infrascapular area of the chest with no hepatosplenomegaly or lymphadenopathy. His hemoglobin was 10 g/dl, total leukocyte count 10,500/ μl, platelet count 210,000/mm 3 and CD4 count 408 cells/mm 3. He was not on fluconazole prophylaxis. Chest X-ray showed right middle zone pneumonia and sputum examination yielded negative results for acidfast bacilli, Pneumocystis jiroveci cysts and trophozites, and pyogenic organisms. The patient was treated with parenteral antibiotics augmentin, levofloxacin and amikacin for 14 days without alleviation of symptoms or changes in chest X-ray findings. Three consecutive early morning sputum samples were collected from the patient after he thoroughly rinsed his oropharyngeal cavity with sterile distilled water and were used for direct microscopy (KOH wet mounts) and culture for fungus. The material was inoculated on routine mycological media, including Sabouraud dextrose agar (SDA) supplemented with chloramphenicol (0.005%) and on the same medium supplemented with cycloheximide (0.05%). One set of inoculated Petri-plates was incubated at 28 C and the second at 37 C. Direct microscopic examination of KOH wet mounts of all three sputum specimens showed mainly budding yeast cells. After 2 days of incubation, numerous yeast-like colonies appeared in culture on SDA with chloramphenicol but not on SDA with cycloheximide. The isolate was assigned accession no. VPCI 32/08 for a detailed study and species identification. Thereafter, the patient was not available for further laboratory and clinical evaluation as he left the hospital against medical advice. Case 2 A 70-year-old male who had a 12 year history of diabetes, was admitted to Hindu Rao Hospital, Delhi, with a fever of 5 6 days duration and pain in swallowing for the last two weeks. At the time of admission, his general physical examination revealed clubbing but the respiratory and other systems were unremarkable. Examination of his oral cavity revealed diffuse erythematous lesions with white plaques on the edge of the tongue. His total leukocyte count was 7,500/mm 3, platelet count-220,000/mm 3 and serum glucose level (fasting) on admission 425 mg/ dl. A blood sample collected on biphasic brain-heart-infusion (BHI) broth was received in the Diagnostic Mycology Laboratory of V. P. Chest Institute for isolation of a possible fungal pathogen. The patient was not available for subsequent follow up. The biphasic BHI broth blood culture bottle was incubated at 37 C and vented for the first 2 3 days. After 48 h, the bottle was tilted for min so that the broth fully covered the agar slope after which the bottle was again incubated upright for further observations. Two days later, the BHI slope cultures showed numerous yeast-like colonies which were subcultured. One of the isolates obtained in pure culture on SDA was assigned the accession number VPCI 1293/08 for detailed study and species identification. Identifi cation of C. nivariensis Morphological and physiological characterization. Both of the C. nivariensis isolates, VPCI 32/08 and VPCI 1293/08, were studied for their morphological and physiological characteristics including fermentation on appropriate media, using standard mycological techniques [ 6 ]. Briefly, after initial germ tube tests, the isolates were cultured on CHROMagar Candida medium, tween 80 rice agar medium (in-house preparation) and tested for pellicle formation and turbidity in yeast peptone dextrose broth (Difco). Candida nivariensis, CBS 9983, C. bracarensis, CBS 10154, and Candida glabrata, ATCC 15545, were included for comparison purposes in the phenotypic characterization

3 418 Chowdhary et al. of the strains. Carbohydrate assimilation profiles of the test isolates were determined with ID 32C kit, read by the miniapi (biomérieux, Marcy l Etoile, France), and by the VITEK 2 (biomérieux) system according to the manufacturer s instructions. In addition, the assimilation profiles were verified by the auxanographic method with yeast nitrogen base (Difco). PCR amplifi cation and direct DNA sequencing of C. nivariensis VPCI 32/08 and VPCI 1293/08 isolates. The molecular identification of both the C. nivariensis isolates was done by PCR amplification and DNA sequencing of internally transcribed spacer regions ITS-1 and ITS-2 of rdna. Additionally, sequencing of the D1/D2 region of 28S rrna gene was done for isolate VPCI 32/08. The DNA of the isolates was extracted as described previously [ 15, 16 ]. The ITS region of rdna containing the ITS-1, 5.8S rrna and ITS-2 was amplified by using panfungal primers ITS1 and ITS4 and both strands of the amplified DNA were sequenced as described previously [ 17 ]. Apart from the ampli fication primers, sequencing primers included ITS1FS, ITS2, ITS3 and ITS4RS [ 18 ]. The D1/ D2 region of 28S rrna was amplified by using panfungal primers NL-1 and NL-4 and both strands of amplified DNA were sequenced by using NL-1, NL-2A, NL-3A and NL4RS primers as described previously [ 19 ]. Reverse complement was generated by using the Bioinformatics site ( ) and aligned with forward sequences, using ClustalW ( ). GenBank basic local alignment search tool (BLAST) searches ( ) were performed for species identification. Antifungal susceptibility testing In vitro antifungal susceptibility testing of both yeast isolates was determined by the broth microdilution method (CLSI, formerly NCCLS, M27-A2) [ 20 ] and Etest (AB BIODISK, Solna, Sweden). The antifungals included amphotericin B (Sigma, St. Louis, Mo, USA), fluconazole, and voriconazole (Pfizer, Groton, CT) and 5-flucytosine (Sigma). Additionally, posaconazole and caspofungin were tested by Etest and itraconazole (Lee Pharma, Hyderabad, India) by the microbroth dilution method. For the broth microdilution test, RPMI 1640 medium with glutamine without bicarbonate (Sigma) buffered to ph 7 with M 3-N-morpholinepropanesulfonic acid (Sigma) was used with a final yeast inoculum concentration of cells/ml as measured by spectrophotometer. The final concentrations of the drugs were μg/ ml for fluconazole and μg/ml for amphotericin B, itraconazole and voriconazole. Drug-free and yeast-free controls were included and microplates were incubated at 35 C for h. CLSI recommended quality control strains, Candida krusei, ATCC 6258, and Candida parapsilosis, ATCC 22019, were used with each test. The MIC end points were read visually and defined for fluconazole, voriconazole and itraconazole as the lowest drug concentration that caused a prominent decrease in growth (50%) vis-à-vis the controls. For amphotericin B, the MIC was defined as the lowest concentration at which there was 100% inhibition of growth compared with the drug-free control wells. Etest was done in accordance with the manufacturer s instructions on RPMI-1640 medium supplemented with 2% glucose and buffered to ph 7.0 with 0.165M 3-N-morpholinopropane-sulphonic acid. MIC endpoints were read visually after 48 h incubation at 35 C. Reference strains of Candida albicans, ATCC 90028, and Candida parapsilosis, ATCC 22019, were used for quality control of susceptibility testing. Results Neither of the C. nivariensis isolates formed pseudohyphae or true hyphae after 48 h at 28 C on Dalmau plate cultures on rice agar with tween 80. They were germ tubenegative and inhibited by 0.1% cycloheximide. The isolates grew well on SDA at 28 C and 37 C and showed smooth, convex, cream-coloured colonies. However, they failed to grow at 45 C, and contrary to C. glabrata, they didn t show turbidity in YPD broth and neither C. nivariensis nor C. glabrata showed pellicle formation in the same medium. On CHROMagar Candida medium they developed white colonies, whereas those of C. glabrata reference strain were typically pink. The carbohydrate assimilation profile with the ID 32 C yeast identification system after 48 h incubation at 30 C demonstrated that only glucose was assimilated, yielding the ID 32 C profile This code provides a low discrimination between Z ygosaccharomyces species, Candida valida and C. glabrata. Furthermore, no species identification was possible with YST cards, VITEK 2 system. However, it may be pointed out that the VITEK and miniapi system do not claim to identify C. nivariensis. We repeated the aforementioned assimilation tests by auxanographic method which confirmed that only glucose was assimilated. Candida glabrata, ATCC 15545, revealed the unique profile , corresponding to the excellent identification grade. Isolates VPCI 32/08 and VPCI 1293/08 of C. nivariensis fermented trehalose after the 3rd and 4th day, respectively, whereas C. glabrata, ATCC 15545, fermented trehalose on the 2nd day. Candida nivariensis,

4 Candida nivariensis in India 419 CBS 9983, and C. bracarensis, CBS 10154, fermented trehalose on the 4th and 5th day, respectively. Both of our C. nivariensis isolates and the species reference strain, CBS 9983, were susceptible to fluconazole, itraconazole, voriconazole, amphotericin B and 5-flucytosine ( Table 1 ). In addition, the test C. nivariensis isolates revealed low MICs for posaconazole and caspofungin ( Table 1 ). Amplicons of ~750 bp obtained for the complete ITS region from both the isolates were sequenced. The BLAST searches showed that the ITS region sequence from isolate 1293/08 was completely identical, while the sequence from isolate 32/08 exhibited two nucleotide differences with the corresponding sequence available in the data bank for C. nivariensis reference strain, CBS10161 (GenBank accession nos. EF056322). The BLAST searches also revealed nearly complete ( 99%) identity with the ITS region sequences available in databank from three other clinical isolates of C. nivariensis. An amplicon of ~600 bp obtained for the D1/D2 regions from isolate 32/08 was also sequenced and DNA sequences when subjected to the BLAST search revealed only three nucleotide differences with the corresponding sequences available in the databank for the C. nivariensis reference strain, CBS10161 (Gen- Bank accession no. EF056323). Based on the observed DNA sequence identity and previous observations that strains belonging to the same species exhibit 1% differences in D1/D2 and/or ITS region sequences, the molecular identity of our isolates was determined as C. nivariensis. The DNA sequence data of our isolates have been deposited in the EMBL under the accession numbers FM955316, FM and FM Table 1 Antifungal susceptibility of Candida nivariensis isolates, VPCI 32/08 and VPCI 1293/08 Species Antifungal agents MIC value μg/ml Broth Microdilution method Etest C. nivariensis VPCI 32/ 08 Amphotericin B Fluconazole 2 2 Itraconazole 0.25 ND* Voriconazole Flucytosine Posaconazole ND 0.38 Caspofungin ND 0.25 C. nivariensis VPCI 1293/08 Amphotericin B Fluconazole 1 6 Itraconazole 0.25 ND Voriconazole Flucytosine Posaconazole ND 0.38 Caspofungin ND ND* not done. Discussion The results document the occurrence of C. nivariensis for the first time in India and focus on its potential as an opportunistic human pathogen. Evidence is provided in support of its etiologic role in a diabetic patient with candidemia. Concerning its repeated isolation from sputum samples of an HIV-positive patient with pneumonic lesion and signs of systemic infection, its significance remains uncertain due to the lack of histopathologic investigations and inadequate clinical follow up. In the present study, C. nivariensis represented 0.5% of the 363 randomly collected yeast isolates identified in our diagnostic laboratory during a period of one year. Lockhart et al. recently examined 1,595 globally collected C. glabrata isolates for the presence of the cryptic species, C. nivariensis and C. bracarensis, and reported the isolation of a solitary C. nivariensis isolate originating from Australia [ 14 ]. Although C. nivariensis has so far been reported only from a few countries in Europe, Asia and Australia ( Table 2 ), further studies are likely to reveal its wider global distribution than apparent from the current literature [ 9, ]. As pointed out by earlier investigators, C. nivariensis is likely to be misidentified as C. glabrata by commercial identification products such as the ID 32 C and VITEK 2 systems [ 9, 11 ]. Furthermore, the development of white colonies on CHROMagar Candida medium by both of our isolates, as also noted in published reports [9,11,13,14 ] does not permit its morphological differentiation from C. bracarensis, C. norvegensis and C. inconspicua [21 ]. A definitive identification of C. nivariensis, therefore, requires the application of molecular methods but facilities for these types of studies are generally lacking in most of the diagnostic laboratories, especially in developing countries. As awareness about this potential opportunistic pathogen grows, it is likely to be encountered more frequently in clinical specimens than at present. Both of our C. nivariensis isolates were susceptible to the antifungals tested Table 2 Global distribution of reported Candida nivariensis clinical isolates Country Source No. of isolates Year reported References Spain BAL, blood and urine [9] Japan Blood [11] Indonesia Oral rinse [12] UK Blood, oral cavity, pelvic [13] abscess, ascitic fluid, peritoneal fluid, lung biopsy, etc Australia Pleural fluid [14] India Sputum and Blood 2 Present study

5 420 Chowdhary et al. which is in conformity with the susceptibility profile of a solitary isolate reported from Indonesia [ 12 ]. Likewise, the C. nivariensis strains originating from the pleural fluid in Australia was found to be susceptible to azoles, as well as echinocandins [ 14 ]. On the other hand, the C. nivariensis isolates reported from Japan and UK were found to be resistant to azoles [ 11, 13 ]. The antifungal susceptibility of C. nivariensis may thus vary from strain to strain or by different prophylactic and/or therapeutic regimens. Acknowledgements The work was carried out with financial assistance from the Indian Council of Medical Research, New Delhi, and Kuwait University Research Grant MI01/08. H.S.R acknowledges the award of an Honorary Scientist position to the Indian National Science Academy, New Delhi. Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. References 1 Pfaller MA, Diekema DJ. Epidemiology of invasive candidiasis: a persistent public health problem. Clin Microbiol Rev 2007; 20: Hazen KC. New and emerging yeast pathogens. Clin Microbiol Rev 1995; 8: Pfaller MA, Diekema DJ. Rare and emerging opportunistic fungal pathogens: concern for resistance beyond Candida albicans and Aspergillus fumigatus. J Clin Microbiol 2004; 42: Pappas PG, Rex JH, Sobel JD, et al. Guidelines for treatment of candidiasis. Clin Infect Dis 2004; 38: Spellberg BJ, Filler SG, Edwards JE, Jr. Current treatment strategies for disseminated candidiasis. Clin Infect Dis 2006; 42: Kurtzman CP, Fell JW (eds). The Yeasts: A Taxonomic Study. Amsterdam : Elsevier, Fenn JP, Segal H, Barland B, et al. Comparison of updated Vitek Yeast Biochemical Card and API 20C yeast identification systems. J Clin Microbiol 1994; 32: Ramani R, Gromadzki S, Pincus DH, Salkin IF, Chaturvedi V. Efficacy of API 20C and ID 32C systems for identification of common and rare clinical yeast isolates. J Clin Microbiol 1998; 36: Alcoba-Flórez J, Méndez-Alvarez S, Cano J, et al. Phenotypic and molecular characterization of Candida nivariensis sp. nov., a possible new opportunistic fungus. J Clin Microbiol 2005; 43: Correia A, Sampaio P, James S, Pais C. Candida bracarensis sp. nov., a novel anamorphic yeast species phenotypically similar to Candida glabrata. Int J Syst Evol Microbiol 2006; 56: Fujita S, Senda Y, Okusi T, et al. Catheter-related fungemia due to fluconazole-resistant Candida nivariensis. J Clin Microbiol 2007; 45: Wahyuningsih R, SahBandar IN, Theelen B, et al. Candida nivariensis isolated from an Indonesian human immunodeficiency virus-infected patient suffering from oropharyngeal candidiasis. J Clin Microbiol 2008; 46: Borman AM, Petch R, Linton CJ, et al. Candida nivariensis, an emerging pathogenic fungus with multidrug resistance to antifungal agents. J Clin Microbiol 2008; 46: Lockhart SR, Messer SA, Gherna M, et al. Identification of Candida nivariensis and Candida bracarensis in a large global collection of Candida glabrata isolates: Comparison to the literature. J Clin Microbiol 2009; 47: Al-Sweih N, Ahmad S, Khan ZU, Khan S, Chandy R. Prevalence of Candida dubliniensis among germ tube-positive Candida isolates in a maternity hospital in Kuwait. Mycoses 2005; 48: Ahmad S, Khan Z, Mustafa AS, Khan ZU. Semi-nested PCR for diagnosis of candidemia: comparison with culture, antigen detection, and biochemical methods for species identification. J Clin Microbiol 2002; 40: Ahmad S, Al-Mahmeed M, Khan ZU. Characterization of Trichosporon species isolated from clinical specimens in Kuwait. J Med Microbiol 2005; 54: Al-Obaid I, Ahmad S, Khan ZU, Dinesh B, Hejab HM. Catheterassociated fungemia due to Exophiala oligosperma in a leukemic child and review of fungemia cases caused by Exophiala species. Eur J Clin Microbiol Infect Dis 2006; 25: Khan ZU, Ahmad S, Mokaddas E, et al. Actinomucor elegans var. kuwaitiensis isolated from the wound of a diabetic patient. Antonie van Leeuwenhoek 2008; 94: Clinical and Laboratory Standards Institute (formerly National Committee of Clinical Laboratory Standards) Reference method for broth microdilution antifungal susceptibility testing of yeast, 2nd ed. Approved standard M27-A2. CLSI Standards, Wayne PA. 21 Bishop JA, Chase N, Lee R, Kurtzman CP, Merz WG. Production of white colonies on CHROMagar Candida medium by members of the Candida glabrata clade and other species with overlapping phenotypic traits. J Clin Microbiol 2008; 46: This paper was first published online on Early Online on 01 February 2010.