- Email: [email protected]
Study of fungal isolates in patients with chronic diarrhea at a tertiary care hospital in north India
Journal de Mycologie Médicale (2013) 23, 21—26
Available online at
www.sciencedirect.com
ORIGINAL ARTICLE/ARTICLE ORIGINAL
Study of fungal isolates in patients with chronic diarrhea at a tertiary care hospital in north India ´Etude des levures isole ´ es chez des patients souffrant de diarrhe ´ e chronique ˆ pital de soins tertiaires du nord de l’Inde dans un ho P. Banerjee a,*, R. Kaur b, B. Uppal b a b
Department of Laboratory, Indian Spinal Injuries Centre, Sector-C, Vasant Kunj, 110070 New Delhi, India Department of Microbiology, Maulana azad medical college, Bahadurshah Zafar marg, 110002 New Delhi, India
Received 10 June 2012; received in revised form 29 November 2012; accepted 2 December 2012 Available online 30 January 2013
KEYWORDS Yeast; Chronic diarrhea; Antifungal drugs
Summary Objective. — The aim of this study was to evaluate the rate of isolation and antifungal susceptibility profile of various opportunistic yeasts in faeces of patients with chronic diarrhea. Patients. — The study included 120 patients who received the consultation and treatment for the complaints of chronic diarrhea for a period of more than 15 days. Materials and methods. — The microscopy of samples of stool was followed by the culture on Sabourauds-dextrose agar. Isolate were tried for their sensitivity to fluconazole, flucytosine, amphotericin B and voriconazole. Results. — The significant growth of fungal organism was seen in 32 (26.7%) stool specimen. These fungal isolates include five (15.6%) of C. albicans, 14 (43.8%) of C. tropicalis, five (15.6%) of C. krusei, two (6.3%) of C. famata, two (6.3%) of C. parapsilosis isolates and one isolate each of C. lusitaniae, C. guilliermondii, Stephanoascus ciferri and Trichosporon asahii (3.1% each). Most of the yeast isolates (65.6%) were found to be sensitive to all four antifungal agents i.e., fluconazole, flucytosine, amphotericin B and voriconazole. The highest resistance was seen against fluconazole in 10 (31.3%) isolates followed by flucytosine in eight (25%) isolates. No resistance to voriconazole was observed in any of the isolates. The resistance to the antifungal agents was higher among the other species of Candida compared to C. albicans. Conclusion. — The study could establish the relation between the opportunistic yeasts and chronic diarrhea and gives information on the antifungal sensitivity profile of the isolated yeasts. # 2012 Elsevier Masson SAS. All rights reserved.
* Corresponding author. E-mail address: [email protected] (P. Banerjee). 1156-5233/$ — see front matter # 2012 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.mycmed.2012.12.002
22
MOTS CLÉS Levure ; Diarrhée chronique ; Antifongiques
P. Banerjee et al. Re ´sume ´ Objectif. — Le but de cette étude est d’évaluer le taux d’isolement et le profil de susceptibilité à divers antifongiques des levures opportunistes isolées des matières fécales de malades avec diarrhée chronique. Malades. — L’étude a inclus 120 malades reçus en consultation et traités pour diarrhée chronique pour une période de plus de 15 jours. Mate´riels et me´thodes. — L’examen microscopique des prélèvements a été suivi par la culture sur Sabouraud-dextrose agar. Les levures isolées ont été testées pour leur sensibilité au fluconazole, à la flucytosine, à l’amphotericine B et au voriconazole. Re ´sultats. — La croissance significative d’organisme fongique a été vue dans 32 (26,7 %) prélèvements. Ces isolats fongiques correspondent à cinq (15,6 %) C. albicans, 14 (43,8 %) C. tropicalis, cinq (15,6 %) C. krusei, deux (6,3 %) C. famata, deux (6,3 %) C. parapsilosis et une souche de C. lusitaniae, C. guilliermondii, Stephanoascus ciferri et Trichosporon asahii (3,1 % chacun). La plupart des levures isolées (65,6 %) ont été trouvés sensibles à quatre agents antifongiques i.e., fluconazole, flucytosine, amphotericine B et voriconazole. La plus haute résistance a été observée contre le fluconazole dans dix (31,3 %) isolats suivi par la flucytosine dans huit (25 %) isolats. Aucune résistance au voriconazole a été observée. La résistance aux agents antifongiques était plus élevée parmi les espèces de Candida comparativement à C. albicans. Conclusion. — L’étude pourrait permettre d’établir une relation entre les levure opportunistes et la diarrhée chronique et donner des informations sur le profil de sensibilité aux antifongiques des levures isolées. # 2012 Elsevier Masson SAS. Tous droits réservés.
Introduction Diarrhea is a common cause of morbidity and mortality among all age groups worldwide. The infectious agents causing diarrhea can be enteric bacteria, parasites, viruses and in some cases, fungi. Yeasts/yeast-like fungi are usually found in the gastrointestinal tract in small numbers since their attachment and colonization to the mucosal surface is prevented by the anaerobic micro-flora. Prolonged administration of antibiotics can cause imbalance in protective microbial flora in gastrointestinal tract, leading to antibiotic associated diarrhea (AAD) [14,13]. Increased rates of isolation of yeasts/yeast-like organisms especially Candida species have been reported in faecal culture of patients following long-term antibiotic use. Isolation of these yeasts has been reported in increased frequency and quantity in faeces of the patients, which can actually result from antibiotic treatment of diarrhea instead of being the cause of diarrhea [13]. Although not commonly suspected clinically, such pathogenic yeasts/yeast-like fungi can increase the severity of diarrhea causing severe dehydration, malnutrition and mortality in already debilitated patients especially, immunecompromised individuals, young children and elderly patients. Discontinuing the antibiotic use and if required, administration of specific antifungal therapy can help reduce morbidity and mortality in such patients. Many of these opportunistic yeasts or yeast-like fungi are even resistant to commonly used antifungal agents [14,13,17]. Identification of the underlying fungal pathogen and information on their antifungal susceptibility profile can help the clinicians to decide upon the appropriate management of the patient. Not many studies have provided such detailed information in Indian scenario. Hence, the present study was aimed to evaluate the rate of isolation and antifungal susceptibility
profile of various opportunistic pathogenic yeasts/yeast-like fungi in faeces of patients with chronic diarrhea.
Patients The prospective study was carried out between June and November 2010 at the Department of Microbiology, Maulana azad medical college and associated Lok Nayak (LN) hospital, New Delhi, India. Faecal specimen were collected and included in the study from 120 patients receiving treatment from wards or outpatient departments, with complaints of chronic diarrhea, i.e., passage of stool greater than 200 g/day or of soft and watery stool more than 3 times/day with or without blood or mucus in the stool, for a period of more than 15 days.
Materials and methods Stool samples were collected in screw-capped sterile plastic containers. Microscopic examination of the samples was done after preparing wet mount, Gram’s stained and modified acid fast stained smears and observations were recorded. Inoculation of faeces was done on Sabouraud’s dextrose agar (HI media, Mumbai, India) for fungal pathogen. Culture was also done directly on alkaline bile salt agar, xylose lysine deoxycholate agar and MacConkey agar (HI Media, Mumbai, India) to isolate any bacterial pathogen. The plated culture media were incubated at 37 8C till 24 h. Bacterial isolates were identified using standard diagnostic procedures. Yeast and yeast-like organisms were identified and characterized by standard phenotypic methods including morphology and biochemical properties [9,15]. An overgrowth of these fungi was identified if greater or equal to 105 colony forming units/ ml were obtained after culture [14].
Study of fungal isolates in patients with chronic diarrhea Antifungal susceptibility of the isolates was tested against fluconazole, flucytosine, amphotericin B and voriconazole using YST-AST cards in Vitek1 2 compact system (Biomerieux, France). Interpretation of susceptibility patterns was made by taking minimum inhibitory concentration (MIC) breakpoints according to the manufacturer’s instructional manual. The data tabulation and statistical analysis of the variables was done using statistical software SPSS version 20.00 (IBM, USA).
Results Among the 120 stool samples studied, significant growth of yeasts/yeast-like fungi i.e., greater or equal to 105 colony forming units/ml was seen in 32 (26.7%) samples. In another five samples, insignificant growth of yeast-like organisms i.e., less than 104 colony forming units/ml was seen. In 83 samples, no fungal growth was seen on culture. Among the 32 patients with significant growth of fungal organism in stool specimen, a majority (n = 27, 84.4%) were admitted and receiving treatment in various wards of the hospital (Table 1). Paediatric age group was predominant (n = 21, 65.6%) among these cases. Two cases were HIV infected adults on anti-retroviral therapy. Enteropathogenic Escherichia coli were isolated in two stool samples. Microscopic examination of faeces revealed the presence of budding yeasts cells in 16 (50%) of these 32 samples thus, correlating with the significant growth of yeast-like organisms. Pus cells were found in 20 samples. Of these, 12 were also positive for yeast cells. Red blood cells were found in two samples. No protozoal cysts or helminthic eggs were found on microscopy. In all the stool samples, single yeast/yeast-like species were isolated except in one, in which colonies of both C. albicans and C. guilliermondii were isolated in equal numbers. Overall, the yeasts/yeast-like fungi found with significant growth in the diarrhea cases included five (15.6%) of C. albicans, 14 (43.8%) of C. tropicalis, five (15.6%) of C. krusei, two (6.3%) of C. famata, two (6.3%) of C. parapsilosis isolates and one isolate each of C. lusitaniae, C. guilliermondii, Stephanoascus ciferri and Trichosporon asahii (3.1% each).
23 Table 1 The analysis of results for faecal samples with significant growth of yeast and yeast-like isolates (n = 32). ´ sultats pour des ´echantillons fe ´ caux avec Analyse des re croissance significative de levure (n = 32). Variable
Result
Male: Female sex ratio among patients Adult: Paediatric age group among patients Inpatient (admitted): Outpatient ratio HIV positive patients Faecal samples with microscopic findings of yeast/yeast-like fungi Faecal samples with both bacterial and fungal isolates Faecal sample with more than one fungal isolates Faecal samples with single fungal isolate
1:1.1
Percentage (%)
1:1.9 1:5.4 2/32 16/32
6.3 50
2/32
6.3
1/32
3.1
31/32
96.9
Upon testing antifungal susceptibility, most yeast isolates (65.6%) were found sensitive to all four antifungal agents i.e., fluconazole, flucytosine, amphotericin B and voriconazole. Maximum resistance was seen against fluconazole in 10 (31.3%) isolates followed by flucytosine in eight (25%) isolates. No resistance to voriconazole was observed in any of the strains. Antifungal resistance in C. albicans isolates (Table 2) was seen against fluconazole and flucytosine in one (20%) and two (40%) strains respectively while among the non-albicans Candida species, seven (28%) isolates were resistant to fluconazole and six (24%) strains were resistant to flucytosine. All C. krusei isolates were resistant to fluconazole and flucytosine.
Discussion Diarrhea is a leading cause of morbidity and mortality among children and adults worldwide. The pathogenic causes of
Table 2 Resistance to individual antifungal agents among the Candida isolates. ´ sistance aux antifongiques des Candida isole ´ s. Re Candida species
Isolates resistant to fluconazole (n = 8)
Isolates resistant to flucytosine (n = 8)
Isolates resistant to amphotericin B (n = 0)
Isolates resistant to voriconazole (n = 0)
C. C. C. C. C. C. C.
1 1 5 0 1 0 0
2 (40) 1 (7.1) 5 (100) 0 0 0 0
0 0 0 0 0 0 0
0 0 0 0 0 0 0
albicans (n = 5) tropicalis (n = 14) krusei (n = 5) famata (n = 2) parapsilosis (n = 2) lusitaniae (n = 1) guilliermondii (n = 1)
(20) (7.1) (100) (50)
Figures in parenthesis represent percentage.
24 diarrhea include enteric bacteria, parasites and viruses. Few fungi too have been reported as causative agents of diarrhea. Candida and Trichosporon species are colonizers of gastrointestinal tract. Indigenous flora of gut forms a dense layer of mucus gel, which outcompetes these fungi for adhesion sites and produces inhibitory substances, which decreases their adhesion to mucosa [14,13]. In the presence of previous mucosal injury, depressed immune system, hormonal abnormalities, increased sugar levels, stress and broad-spectrum antibiotic intake, these fungi may become pathogenic and cause diarrhea [17]. In the present study, in 32 (%) out of 120 patients, yeasts/ yeast-like organisms could be associated with diarrhea considering their overgrowth i.e., greater or equal to 105 colony forming units/ml in faecal culture. Another five samples had growth less than 104 colony forming units/ml and hence could be considered part of normal micro-flora [14,13]. A high isolation rate (65.6%) was seen in age group of 0 to 12 years. This may be due to relatively lower immune resistance in paediatric population leading to the colonizing yeasts becoming pathogenic in them. A previous study has reported 48.28% prevalence of Candida-associated diarrhea in 203 paediatric cases [7] while Forbes et al. [8], in 2001 reported the prevalence of Candida in diarrhoeic cases among children to be 39%. Although, children have been found to have Candida spp in their stool, the rate of carriage is found higher amount among hospitalized children and the proportion increases with the duration of hospitalization. In the present study, 27 (84.4%) cases with yeast-associated diarrhea were admitted patients receiving treatment in the various inpatient wards at the hospital. Their general health was poor and in many of them, broad-spectrum antibiotics, corticosteroids etc. were included in the treatment therapies. Two patients were diagnosed with HIV and were receiving anti-retroviral therapy. The condition of all these patients indicates a depressed immune system. Studies by Danna et al. [6] and Gupta et al. [11] reported higher rates of isolation of faecal Candida spp. in critically ill, malnourished patients with diarrhea in absence of any other possible infective, inflammatory or chemical cause for the symptoms which subsided with administration of antifungal therapy. A co-existing bacterial pathogen could be isolated in only two (6.3%) of the 32 faecal samples with significant colonies of yeasts/yeast-like fungi. It can thus be considered that these yeasts/yeast-like organisms could primarily be the agents causing diarrhea. Among these fungal isolates with significant growth in faeces of the patients included in the present study, five (15.6%) were C. albicans strains, while 25 (78.1%) were nonalbicans Candida species. C. albicans has been reported as a causative agent in diarrhea esp., associated with intake of broad-spectrum antibiotics and immune-compromised state. A study from Cameroon, Africa (Nkuo-Akenji et al.) in 2002 reported a 35.9% prevalence of C. albicans in 184 diarrhoeic stool cultures [17]. Forbes et al. also reported the isolation rates of C. albicans as 35% and non-albicans Candida species like C. parapsilosis, C.guillermondii and C.glabrata as 1.9% each in culture of faecal specimen [8]. C. albicans was previously reported as the predominant pathogen among yeasts and yeast-like fungi. However, in
P. Banerjee et al. recent times, rate of isolation of non-albicans Candida species has even surpassed that of C. albicans. Most common among these are C. tropicalis, C. parapsilosis, C. glabrata and C. krusei. A study from Japan (Tashiro et al.) in 2010 reported the prevalence of C. albicans as 54% in 782 stool samples [23]. Compared to it, the present study shows a lower prevalence (15.6%) of C. albicans showing the increasing importance and emergence of non-albicans Candida species as pathogens. Similarly, Vaishnavi et al. in 2008 reported a higher prevalence of C. tropicalis (50%) compared to C. albicans (43.8%) in stool samples of patients receiving antibiotics [24]. In our study too, C. tropicalis isolation rate (43.8%) was highest among all the Candida species. The study by Tashiro et al. reported a prevalence of C. tropicalis to be 4% while the predominant non-albicans Candida species was C. glabrata (13%). Among the non-albicans Candida isolated in the present study, five (15.6%) isolates were of C. krusei, two (6.3%) each of C. famata and C. parapsilosis and one (3.1%) each of C. lusitaniae and C. guilliermondii. Non-albicans Candida species are replacing C. albicans as the opportunistic yeast-like fungi causing systemic and invasive infections in immune-compromised and debilitated individuals [1,18,21]. Antifungal susceptibility testing of the yeast isolates indicated a higher resistance pattern against fluconazole (in 31.3%) and flucytosine (in 25%) as compared to amphotericin B (in 6.3%). Fluconazole is an azole agent used commonly in the treatment of yeast infections. Primary resistance to fluconazole is seen in C. krusei [10,16], which was seen in our study too. Resistance to fluconazole has been on the rise among other Candida species as well [16,20,2,12,5,25,3]. Besides C. krusei, resistance to fluconazole was seen in one isolate each of non-albicans Candida species including C. tropicalis and C. parapsilosis. All the isolates of C. famata, C. lusitaniae and C. guilliermondii were sensitive to fluconazole. No resistance to voriconazole was seen in any of the yeast isolates. Voriconazole is a relatively newer antifungal agent which is more potent than other azoles. Even C. krusei isolates remain susceptible to voriconazole, despite inherent fluconazole resistance. This is probably due to stronger binding to target enzyme [10,16]. Fluconazole susceptibility is considered an indirect marker for susceptibility of Candida isolates to voriconazole [10]. However, this relation could not be established in the present study among the five isolates of C. krusei and one isolate each of C. albicans, C. tropicalis and C. parapsilosis. There is a paucity of Indian studies reporting antifungal susceptibility of faecal yeast isolates against voriconazole. Fungi like Trichosporon asahii and Stephanoascus ciferri were isolated in significant amounts from stool sample of one patient each. Trichosporon is a soil inhabitant and commensal flora of the gastrointestinal tract and skin in humans. It is rarely reported as a pathogen causing invasive systemic infections in immune-compromised individuals including cancer patients and non-cancer patients following chronic illness or break in skin and mucous membranes [19,4]. In our study, it was isolated as the sole probable cause of diarrhea in a 1-month-old child who had not developed sufficient immunity to the pathogen. Amphotericin B, flucytosine and azoles have been found to be more potent than
Study of fungal isolates in patients with chronic diarrhea echinocandins for treating infections caused by Trichosporon. However, the strain isolated in present study was resistant to both amphotericin B and fluconazole. Stephanoascus ciferri (or Candida ciferri) is a fungus that has emerged as an opportunistic pathogen causing superficial infection in ear canal or nose and systemic invasive mycoses in immune-deficient individuals eg, patients of acute myeloid leukemia. Strains have been found resistant to common antifungal agents like fluconazole, flucytosine and itraconazole [22]. The isolate in our study was found resistant to amphotericin B and fluconazole. Both the above relatively uncommon opportunistic fungi were found susceptible to voriconazole, thus emphasizing the importance of voriconazole as the antifungal of choice in infections caused by these fungi.
Conclusion There is paucity of studies giving information on epidemiological distribution of pathogenic yeast/yeast-like species among patients with chronic diarrhea. Since these are generally opportunistic pathogen in immune-compromised individuals and are usually resistant to the commonly used antifungal agents, the clinicians face the problem of identifying the cause of diarrhea in these patients and choosing the right antifungal agent for these fungi resulting in delay in treatment thus causing high morbidity and mortality. Prompt identification of the causative fungal agent and information on its susceptibility pattern against various antifungal agents helps in starting the specific therapy early and quicker recovery of the patient. The present study has been able to establish the relationship between opportunistic yeast/yeast-like fungi and chronic diarrhea. It has also provided information on antifungal susceptibility pattern of the isolated fungal strains within the limitations of the relatively smaller number of isolates and lack of information available from previous studies regarding fungi like Trichosporon and Stephanoascus species. More studies are suggested with inclusion of larger study group, which can provide relevant and significant data to evaluate pathogenesis and susceptibility patterns of the opportunistic fungi causing antibiotic associated diarrhea, thus helping in developing therapeutic regimes in such patients.
Disclosure of interest The authors declare that they have no conflicts of interest concerning this article.
References [1] Adhikary R, Joshi S. Species distribution and anti-fungal susceptibility of Candidaemia at a multi super-speciality center in southern India. Indian J Med Microbiol 2011;29:309—11. [2] Bourgeois N, Dehandschoewercker L, Bertout S, Bousquet PJ, Rispail P, Lachaud L. Antifungal susceptibility of 205 Candida spp. isolated primarily during invasive Candidiasis and comparison of the Vitek2 system with the CLSI broth microdilution and Etest methods. J Clin Microbiol 2010;48:154—61.
25 [3] Bruder-Nascimento A, Camargo CH, Sugizaki MF, Sadatsune T, Montelli AC, Mondelli AL, et al. Species distribution and susceptibility profile of Candida species in a Brazilian public tertiary hospital. BMC Res Notes 2010;3:1—5. [4] Chagas-Neto TC, Chaves GM, Melo ASA, Colombo AL. Bloodstream infections due to Trichosporon spp: Species distribution, Trichosporon asahii genotypes determined on the basis of ribosomal DNA intergenic spacer 1 sequencing, and antifungal susceptibility testing. J Clin Microbiol 2009;47:1074—81. [5] Cordoba S, Vivot W, Bosco-Borgeat ME, Taverna C, Szusz W, Murisengo O, et al. Species distribution and susceptibility profile of yeasts isolated from blood cultures: results of a multicenter active laboratory-based surveillance study in Argentina. Rev Argent Microbiol 2011;43:176—8. [6] Danna PL, Urban C, Bellin E, Rahal JJ. Role of Candida in pathogenesis of antibiotic-associated diarrhoea in elderly patients. Lancet 1991;337:511—4. [7] Enweani B, Obi CL, Jokpeyibo M. Prevalence of Candida species in Nigerian children with diarrhoea. J Diarrhoeal Dis Res 1994;12:133—5. [8] Forbes D, Ee L, Camer-Pesci P, Ward PB. Faecal Candida and diarrhoea. Arch Dis Child 2001;84:328—31. [9] Forbes BA, Sahm DF, Weissfeld AS. Laboratory methods in basic mycology. In: Forbes BA, Sahm DF, Weissfeld AS, editors. Bailey & Scott’s Diagnostic Microbiology. . 11th ed, St. Louis, Missouri: Mosby; 2002. p. 711—89. [10] Fukuoka T, Johnston DA, Winslow CA, et al. Genetic basis for differential activities of fluconazole and voriconazole against Candida krusei. Antimicrob Agents Chemother 2003;47: 1213—9. [11] Gupta TP, Ehrinpreis MN. Candida-associated diarrhea in hospitalized patients. Gastroenterology 1990;98:780—5. [12] Hazen KC, Baron EJ, Colombo AL, Girmenia C, Sanchez- Sousa A, Palacio AD, et al. Comparison of the susceptibilities of Candida spp. to fluconazole and voriconazole in a 4-year global evaluation using disk diffusion. J Clin Microbiol 2003;41:5623— 32. [13] Krause R, Schwab E, Bachhiesl D, Daxbock F, Wenisch C, Krejs GJ, et al. Role of Candida in antibiotic-associated diarrhea. J Infect Dis 2001;184:1065—9. [14] Krause R, Krejs GJ, Wenisch C, Reisinger EC. Elevated fecal Candida counts in patients with antibiotic-associated diarrhea: role of soluble fecal substances. Clin Diagn Lab Immunol 2003;10:167—8. [15] Larone DH. Yeasts and yeastlike organisms. In: Larone DH, editor. Medically important fungi: a guide to identification. . 2nd ed., New York: Elsevier Science Publishing Co; 1987 . p. 53—75. [16] Lat A, Thompson GR. Update on the optimal use of voriconazole for invasive fungal infections. Review. Infect Drug Resist 2011;4:443—53. [17] Nkuo-Akenji TK, Ndip RN, Ntoko A. The prevalence of Candida albicans-associated diarrhoea in Buea, South West Cameroon. Afr J Health Sci 2002;9:153—7. [18] Rangel-Frausto MS, Wiblin T, Blumberg HM, et al. National Epidemiology of Mycosis Survey (NEMIS): variations in rates of candidal bloodstream infections in seven surgical ICUs and six neonatal ICUs. Clin Infect Dis 1999;29:253—8. [19] Ruan SY, Chien JY, Hsueh PR. Invasive Trichosporonosis caused by Trichosporon asahii and other unusual Trichosporon species at a medical center in Taiwan. Clin Infect Dis 2009;49:e11—7. [20] Slavin MA, Sorrell TC, Marriott D, Thursky KA, Nguyen Q, Ellis DH, et al. Candidaemia in adult cancer patients: risks for fluconazole-resistant isolates and death. J Antimicrob Chemother 2010;65:1042—51. [21] Snydman DR. Shifting patterns in the epidemiology of nosocomial Candida infections. Chest 2003;123:500S—3S.
26 [22] Soki H, Nagase Y, Yamazaki K, Oda T, Kikuchi K. Isolation of the yeast like fungus Stephanoascus ciferri by culturing the aural discharge of a patient with intractable otitis media. Case report. Kansenshogaku Zasshi 2010;84:210—2. [23] Tashiro M, Murakami H, Yoshizawa, Tateda K, Yamaguchi K. Isolation rate and susceptibilities of Candida species from blood, vascular catheter, urine and stool. Kansenshogaku Zasshi 2010;84:187—92.
P. Banerjee et al. [24] Vaishnavi C, Kaur S, Prakash S. Speciation of fecal Candida isolates in antibiotic-associated diarrhea in non-HIV patients. Jpn J Infect Dis 2008;61:1—4. [25] Zepelin MBV, Kunz L, Ruchel R, Reichard U, Weig M, Grob U. Epidemiology and antifungal susceptibilities of Candida spp. to six antifungal agents: results from a surveillance study on fungaemia in Germany from July 2004 to August 2005. J Antimicrob Chemother 2007;60:424—8.
Available online at
www.sciencedirect.com
ORIGINAL ARTICLE/ARTICLE ORIGINAL
Study of fungal isolates in patients with chronic diarrhea at a tertiary care hospital in north India ´Etude des levures isole ´ es chez des patients souffrant de diarrhe ´ e chronique ˆ pital de soins tertiaires du nord de l’Inde dans un ho P. Banerjee a,*, R. Kaur b, B. Uppal b a b
Department of Laboratory, Indian Spinal Injuries Centre, Sector-C, Vasant Kunj, 110070 New Delhi, India Department of Microbiology, Maulana azad medical college, Bahadurshah Zafar marg, 110002 New Delhi, India
Received 10 June 2012; received in revised form 29 November 2012; accepted 2 December 2012 Available online 30 January 2013
KEYWORDS Yeast; Chronic diarrhea; Antifungal drugs
Summary Objective. — The aim of this study was to evaluate the rate of isolation and antifungal susceptibility profile of various opportunistic yeasts in faeces of patients with chronic diarrhea. Patients. — The study included 120 patients who received the consultation and treatment for the complaints of chronic diarrhea for a period of more than 15 days. Materials and methods. — The microscopy of samples of stool was followed by the culture on Sabourauds-dextrose agar. Isolate were tried for their sensitivity to fluconazole, flucytosine, amphotericin B and voriconazole. Results. — The significant growth of fungal organism was seen in 32 (26.7%) stool specimen. These fungal isolates include five (15.6%) of C. albicans, 14 (43.8%) of C. tropicalis, five (15.6%) of C. krusei, two (6.3%) of C. famata, two (6.3%) of C. parapsilosis isolates and one isolate each of C. lusitaniae, C. guilliermondii, Stephanoascus ciferri and Trichosporon asahii (3.1% each). Most of the yeast isolates (65.6%) were found to be sensitive to all four antifungal agents i.e., fluconazole, flucytosine, amphotericin B and voriconazole. The highest resistance was seen against fluconazole in 10 (31.3%) isolates followed by flucytosine in eight (25%) isolates. No resistance to voriconazole was observed in any of the isolates. The resistance to the antifungal agents was higher among the other species of Candida compared to C. albicans. Conclusion. — The study could establish the relation between the opportunistic yeasts and chronic diarrhea and gives information on the antifungal sensitivity profile of the isolated yeasts. # 2012 Elsevier Masson SAS. All rights reserved.
* Corresponding author. E-mail address: [email protected] (P. Banerjee). 1156-5233/$ — see front matter # 2012 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.mycmed.2012.12.002
22
MOTS CLÉS Levure ; Diarrhée chronique ; Antifongiques
P. Banerjee et al. Re ´sume ´ Objectif. — Le but de cette étude est d’évaluer le taux d’isolement et le profil de susceptibilité à divers antifongiques des levures opportunistes isolées des matières fécales de malades avec diarrhée chronique. Malades. — L’étude a inclus 120 malades reçus en consultation et traités pour diarrhée chronique pour une période de plus de 15 jours. Mate´riels et me´thodes. — L’examen microscopique des prélèvements a été suivi par la culture sur Sabouraud-dextrose agar. Les levures isolées ont été testées pour leur sensibilité au fluconazole, à la flucytosine, à l’amphotericine B et au voriconazole. Re ´sultats. — La croissance significative d’organisme fongique a été vue dans 32 (26,7 %) prélèvements. Ces isolats fongiques correspondent à cinq (15,6 %) C. albicans, 14 (43,8 %) C. tropicalis, cinq (15,6 %) C. krusei, deux (6,3 %) C. famata, deux (6,3 %) C. parapsilosis et une souche de C. lusitaniae, C. guilliermondii, Stephanoascus ciferri et Trichosporon asahii (3,1 % chacun). La plupart des levures isolées (65,6 %) ont été trouvés sensibles à quatre agents antifongiques i.e., fluconazole, flucytosine, amphotericine B et voriconazole. La plus haute résistance a été observée contre le fluconazole dans dix (31,3 %) isolats suivi par la flucytosine dans huit (25 %) isolats. Aucune résistance au voriconazole a été observée. La résistance aux agents antifongiques était plus élevée parmi les espèces de Candida comparativement à C. albicans. Conclusion. — L’étude pourrait permettre d’établir une relation entre les levure opportunistes et la diarrhée chronique et donner des informations sur le profil de sensibilité aux antifongiques des levures isolées. # 2012 Elsevier Masson SAS. Tous droits réservés.
Introduction Diarrhea is a common cause of morbidity and mortality among all age groups worldwide. The infectious agents causing diarrhea can be enteric bacteria, parasites, viruses and in some cases, fungi. Yeasts/yeast-like fungi are usually found in the gastrointestinal tract in small numbers since their attachment and colonization to the mucosal surface is prevented by the anaerobic micro-flora. Prolonged administration of antibiotics can cause imbalance in protective microbial flora in gastrointestinal tract, leading to antibiotic associated diarrhea (AAD) [14,13]. Increased rates of isolation of yeasts/yeast-like organisms especially Candida species have been reported in faecal culture of patients following long-term antibiotic use. Isolation of these yeasts has been reported in increased frequency and quantity in faeces of the patients, which can actually result from antibiotic treatment of diarrhea instead of being the cause of diarrhea [13]. Although not commonly suspected clinically, such pathogenic yeasts/yeast-like fungi can increase the severity of diarrhea causing severe dehydration, malnutrition and mortality in already debilitated patients especially, immunecompromised individuals, young children and elderly patients. Discontinuing the antibiotic use and if required, administration of specific antifungal therapy can help reduce morbidity and mortality in such patients. Many of these opportunistic yeasts or yeast-like fungi are even resistant to commonly used antifungal agents [14,13,17]. Identification of the underlying fungal pathogen and information on their antifungal susceptibility profile can help the clinicians to decide upon the appropriate management of the patient. Not many studies have provided such detailed information in Indian scenario. Hence, the present study was aimed to evaluate the rate of isolation and antifungal susceptibility
profile of various opportunistic pathogenic yeasts/yeast-like fungi in faeces of patients with chronic diarrhea.
Patients The prospective study was carried out between June and November 2010 at the Department of Microbiology, Maulana azad medical college and associated Lok Nayak (LN) hospital, New Delhi, India. Faecal specimen were collected and included in the study from 120 patients receiving treatment from wards or outpatient departments, with complaints of chronic diarrhea, i.e., passage of stool greater than 200 g/day or of soft and watery stool more than 3 times/day with or without blood or mucus in the stool, for a period of more than 15 days.
Materials and methods Stool samples were collected in screw-capped sterile plastic containers. Microscopic examination of the samples was done after preparing wet mount, Gram’s stained and modified acid fast stained smears and observations were recorded. Inoculation of faeces was done on Sabouraud’s dextrose agar (HI media, Mumbai, India) for fungal pathogen. Culture was also done directly on alkaline bile salt agar, xylose lysine deoxycholate agar and MacConkey agar (HI Media, Mumbai, India) to isolate any bacterial pathogen. The plated culture media were incubated at 37 8C till 24 h. Bacterial isolates were identified using standard diagnostic procedures. Yeast and yeast-like organisms were identified and characterized by standard phenotypic methods including morphology and biochemical properties [9,15]. An overgrowth of these fungi was identified if greater or equal to 105 colony forming units/ ml were obtained after culture [14].
Study of fungal isolates in patients with chronic diarrhea Antifungal susceptibility of the isolates was tested against fluconazole, flucytosine, amphotericin B and voriconazole using YST-AST cards in Vitek1 2 compact system (Biomerieux, France). Interpretation of susceptibility patterns was made by taking minimum inhibitory concentration (MIC) breakpoints according to the manufacturer’s instructional manual. The data tabulation and statistical analysis of the variables was done using statistical software SPSS version 20.00 (IBM, USA).
Results Among the 120 stool samples studied, significant growth of yeasts/yeast-like fungi i.e., greater or equal to 105 colony forming units/ml was seen in 32 (26.7%) samples. In another five samples, insignificant growth of yeast-like organisms i.e., less than 104 colony forming units/ml was seen. In 83 samples, no fungal growth was seen on culture. Among the 32 patients with significant growth of fungal organism in stool specimen, a majority (n = 27, 84.4%) were admitted and receiving treatment in various wards of the hospital (Table 1). Paediatric age group was predominant (n = 21, 65.6%) among these cases. Two cases were HIV infected adults on anti-retroviral therapy. Enteropathogenic Escherichia coli were isolated in two stool samples. Microscopic examination of faeces revealed the presence of budding yeasts cells in 16 (50%) of these 32 samples thus, correlating with the significant growth of yeast-like organisms. Pus cells were found in 20 samples. Of these, 12 were also positive for yeast cells. Red blood cells were found in two samples. No protozoal cysts or helminthic eggs were found on microscopy. In all the stool samples, single yeast/yeast-like species were isolated except in one, in which colonies of both C. albicans and C. guilliermondii were isolated in equal numbers. Overall, the yeasts/yeast-like fungi found with significant growth in the diarrhea cases included five (15.6%) of C. albicans, 14 (43.8%) of C. tropicalis, five (15.6%) of C. krusei, two (6.3%) of C. famata, two (6.3%) of C. parapsilosis isolates and one isolate each of C. lusitaniae, C. guilliermondii, Stephanoascus ciferri and Trichosporon asahii (3.1% each).
23 Table 1 The analysis of results for faecal samples with significant growth of yeast and yeast-like isolates (n = 32). ´ sultats pour des ´echantillons fe ´ caux avec Analyse des re croissance significative de levure (n = 32). Variable
Result
Male: Female sex ratio among patients Adult: Paediatric age group among patients Inpatient (admitted): Outpatient ratio HIV positive patients Faecal samples with microscopic findings of yeast/yeast-like fungi Faecal samples with both bacterial and fungal isolates Faecal sample with more than one fungal isolates Faecal samples with single fungal isolate
1:1.1
Percentage (%)
1:1.9 1:5.4 2/32 16/32
6.3 50
2/32
6.3
1/32
3.1
31/32
96.9
Upon testing antifungal susceptibility, most yeast isolates (65.6%) were found sensitive to all four antifungal agents i.e., fluconazole, flucytosine, amphotericin B and voriconazole. Maximum resistance was seen against fluconazole in 10 (31.3%) isolates followed by flucytosine in eight (25%) isolates. No resistance to voriconazole was observed in any of the strains. Antifungal resistance in C. albicans isolates (Table 2) was seen against fluconazole and flucytosine in one (20%) and two (40%) strains respectively while among the non-albicans Candida species, seven (28%) isolates were resistant to fluconazole and six (24%) strains were resistant to flucytosine. All C. krusei isolates were resistant to fluconazole and flucytosine.
Discussion Diarrhea is a leading cause of morbidity and mortality among children and adults worldwide. The pathogenic causes of
Table 2 Resistance to individual antifungal agents among the Candida isolates. ´ sistance aux antifongiques des Candida isole ´ s. Re Candida species
Isolates resistant to fluconazole (n = 8)
Isolates resistant to flucytosine (n = 8)
Isolates resistant to amphotericin B (n = 0)
Isolates resistant to voriconazole (n = 0)
C. C. C. C. C. C. C.
1 1 5 0 1 0 0
2 (40) 1 (7.1) 5 (100) 0 0 0 0
0 0 0 0 0 0 0
0 0 0 0 0 0 0
albicans (n = 5) tropicalis (n = 14) krusei (n = 5) famata (n = 2) parapsilosis (n = 2) lusitaniae (n = 1) guilliermondii (n = 1)
(20) (7.1) (100) (50)
Figures in parenthesis represent percentage.
24 diarrhea include enteric bacteria, parasites and viruses. Few fungi too have been reported as causative agents of diarrhea. Candida and Trichosporon species are colonizers of gastrointestinal tract. Indigenous flora of gut forms a dense layer of mucus gel, which outcompetes these fungi for adhesion sites and produces inhibitory substances, which decreases their adhesion to mucosa [14,13]. In the presence of previous mucosal injury, depressed immune system, hormonal abnormalities, increased sugar levels, stress and broad-spectrum antibiotic intake, these fungi may become pathogenic and cause diarrhea [17]. In the present study, in 32 (%) out of 120 patients, yeasts/ yeast-like organisms could be associated with diarrhea considering their overgrowth i.e., greater or equal to 105 colony forming units/ml in faecal culture. Another five samples had growth less than 104 colony forming units/ml and hence could be considered part of normal micro-flora [14,13]. A high isolation rate (65.6%) was seen in age group of 0 to 12 years. This may be due to relatively lower immune resistance in paediatric population leading to the colonizing yeasts becoming pathogenic in them. A previous study has reported 48.28% prevalence of Candida-associated diarrhea in 203 paediatric cases [7] while Forbes et al. [8], in 2001 reported the prevalence of Candida in diarrhoeic cases among children to be 39%. Although, children have been found to have Candida spp in their stool, the rate of carriage is found higher amount among hospitalized children and the proportion increases with the duration of hospitalization. In the present study, 27 (84.4%) cases with yeast-associated diarrhea were admitted patients receiving treatment in the various inpatient wards at the hospital. Their general health was poor and in many of them, broad-spectrum antibiotics, corticosteroids etc. were included in the treatment therapies. Two patients were diagnosed with HIV and were receiving anti-retroviral therapy. The condition of all these patients indicates a depressed immune system. Studies by Danna et al. [6] and Gupta et al. [11] reported higher rates of isolation of faecal Candida spp. in critically ill, malnourished patients with diarrhea in absence of any other possible infective, inflammatory or chemical cause for the symptoms which subsided with administration of antifungal therapy. A co-existing bacterial pathogen could be isolated in only two (6.3%) of the 32 faecal samples with significant colonies of yeasts/yeast-like fungi. It can thus be considered that these yeasts/yeast-like organisms could primarily be the agents causing diarrhea. Among these fungal isolates with significant growth in faeces of the patients included in the present study, five (15.6%) were C. albicans strains, while 25 (78.1%) were nonalbicans Candida species. C. albicans has been reported as a causative agent in diarrhea esp., associated with intake of broad-spectrum antibiotics and immune-compromised state. A study from Cameroon, Africa (Nkuo-Akenji et al.) in 2002 reported a 35.9% prevalence of C. albicans in 184 diarrhoeic stool cultures [17]. Forbes et al. also reported the isolation rates of C. albicans as 35% and non-albicans Candida species like C. parapsilosis, C.guillermondii and C.glabrata as 1.9% each in culture of faecal specimen [8]. C. albicans was previously reported as the predominant pathogen among yeasts and yeast-like fungi. However, in
P. Banerjee et al. recent times, rate of isolation of non-albicans Candida species has even surpassed that of C. albicans. Most common among these are C. tropicalis, C. parapsilosis, C. glabrata and C. krusei. A study from Japan (Tashiro et al.) in 2010 reported the prevalence of C. albicans as 54% in 782 stool samples [23]. Compared to it, the present study shows a lower prevalence (15.6%) of C. albicans showing the increasing importance and emergence of non-albicans Candida species as pathogens. Similarly, Vaishnavi et al. in 2008 reported a higher prevalence of C. tropicalis (50%) compared to C. albicans (43.8%) in stool samples of patients receiving antibiotics [24]. In our study too, C. tropicalis isolation rate (43.8%) was highest among all the Candida species. The study by Tashiro et al. reported a prevalence of C. tropicalis to be 4% while the predominant non-albicans Candida species was C. glabrata (13%). Among the non-albicans Candida isolated in the present study, five (15.6%) isolates were of C. krusei, two (6.3%) each of C. famata and C. parapsilosis and one (3.1%) each of C. lusitaniae and C. guilliermondii. Non-albicans Candida species are replacing C. albicans as the opportunistic yeast-like fungi causing systemic and invasive infections in immune-compromised and debilitated individuals [1,18,21]. Antifungal susceptibility testing of the yeast isolates indicated a higher resistance pattern against fluconazole (in 31.3%) and flucytosine (in 25%) as compared to amphotericin B (in 6.3%). Fluconazole is an azole agent used commonly in the treatment of yeast infections. Primary resistance to fluconazole is seen in C. krusei [10,16], which was seen in our study too. Resistance to fluconazole has been on the rise among other Candida species as well [16,20,2,12,5,25,3]. Besides C. krusei, resistance to fluconazole was seen in one isolate each of non-albicans Candida species including C. tropicalis and C. parapsilosis. All the isolates of C. famata, C. lusitaniae and C. guilliermondii were sensitive to fluconazole. No resistance to voriconazole was seen in any of the yeast isolates. Voriconazole is a relatively newer antifungal agent which is more potent than other azoles. Even C. krusei isolates remain susceptible to voriconazole, despite inherent fluconazole resistance. This is probably due to stronger binding to target enzyme [10,16]. Fluconazole susceptibility is considered an indirect marker for susceptibility of Candida isolates to voriconazole [10]. However, this relation could not be established in the present study among the five isolates of C. krusei and one isolate each of C. albicans, C. tropicalis and C. parapsilosis. There is a paucity of Indian studies reporting antifungal susceptibility of faecal yeast isolates against voriconazole. Fungi like Trichosporon asahii and Stephanoascus ciferri were isolated in significant amounts from stool sample of one patient each. Trichosporon is a soil inhabitant and commensal flora of the gastrointestinal tract and skin in humans. It is rarely reported as a pathogen causing invasive systemic infections in immune-compromised individuals including cancer patients and non-cancer patients following chronic illness or break in skin and mucous membranes [19,4]. In our study, it was isolated as the sole probable cause of diarrhea in a 1-month-old child who had not developed sufficient immunity to the pathogen. Amphotericin B, flucytosine and azoles have been found to be more potent than
Study of fungal isolates in patients with chronic diarrhea echinocandins for treating infections caused by Trichosporon. However, the strain isolated in present study was resistant to both amphotericin B and fluconazole. Stephanoascus ciferri (or Candida ciferri) is a fungus that has emerged as an opportunistic pathogen causing superficial infection in ear canal or nose and systemic invasive mycoses in immune-deficient individuals eg, patients of acute myeloid leukemia. Strains have been found resistant to common antifungal agents like fluconazole, flucytosine and itraconazole [22]. The isolate in our study was found resistant to amphotericin B and fluconazole. Both the above relatively uncommon opportunistic fungi were found susceptible to voriconazole, thus emphasizing the importance of voriconazole as the antifungal of choice in infections caused by these fungi.
Conclusion There is paucity of studies giving information on epidemiological distribution of pathogenic yeast/yeast-like species among patients with chronic diarrhea. Since these are generally opportunistic pathogen in immune-compromised individuals and are usually resistant to the commonly used antifungal agents, the clinicians face the problem of identifying the cause of diarrhea in these patients and choosing the right antifungal agent for these fungi resulting in delay in treatment thus causing high morbidity and mortality. Prompt identification of the causative fungal agent and information on its susceptibility pattern against various antifungal agents helps in starting the specific therapy early and quicker recovery of the patient. The present study has been able to establish the relationship between opportunistic yeast/yeast-like fungi and chronic diarrhea. It has also provided information on antifungal susceptibility pattern of the isolated fungal strains within the limitations of the relatively smaller number of isolates and lack of information available from previous studies regarding fungi like Trichosporon and Stephanoascus species. More studies are suggested with inclusion of larger study group, which can provide relevant and significant data to evaluate pathogenesis and susceptibility patterns of the opportunistic fungi causing antibiotic associated diarrhea, thus helping in developing therapeutic regimes in such patients.
Disclosure of interest The authors declare that they have no conflicts of interest concerning this article.
References [1] Adhikary R, Joshi S. Species distribution and anti-fungal susceptibility of Candidaemia at a multi super-speciality center in southern India. Indian J Med Microbiol 2011;29:309—11. [2] Bourgeois N, Dehandschoewercker L, Bertout S, Bousquet PJ, Rispail P, Lachaud L. Antifungal susceptibility of 205 Candida spp. isolated primarily during invasive Candidiasis and comparison of the Vitek2 system with the CLSI broth microdilution and Etest methods. J Clin Microbiol 2010;48:154—61.
25 [3] Bruder-Nascimento A, Camargo CH, Sugizaki MF, Sadatsune T, Montelli AC, Mondelli AL, et al. Species distribution and susceptibility profile of Candida species in a Brazilian public tertiary hospital. BMC Res Notes 2010;3:1—5. [4] Chagas-Neto TC, Chaves GM, Melo ASA, Colombo AL. Bloodstream infections due to Trichosporon spp: Species distribution, Trichosporon asahii genotypes determined on the basis of ribosomal DNA intergenic spacer 1 sequencing, and antifungal susceptibility testing. J Clin Microbiol 2009;47:1074—81. [5] Cordoba S, Vivot W, Bosco-Borgeat ME, Taverna C, Szusz W, Murisengo O, et al. Species distribution and susceptibility profile of yeasts isolated from blood cultures: results of a multicenter active laboratory-based surveillance study in Argentina. Rev Argent Microbiol 2011;43:176—8. [6] Danna PL, Urban C, Bellin E, Rahal JJ. Role of Candida in pathogenesis of antibiotic-associated diarrhoea in elderly patients. Lancet 1991;337:511—4. [7] Enweani B, Obi CL, Jokpeyibo M. Prevalence of Candida species in Nigerian children with diarrhoea. J Diarrhoeal Dis Res 1994;12:133—5. [8] Forbes D, Ee L, Camer-Pesci P, Ward PB. Faecal Candida and diarrhoea. Arch Dis Child 2001;84:328—31. [9] Forbes BA, Sahm DF, Weissfeld AS. Laboratory methods in basic mycology. In: Forbes BA, Sahm DF, Weissfeld AS, editors. Bailey & Scott’s Diagnostic Microbiology. . 11th ed, St. Louis, Missouri: Mosby; 2002. p. 711—89. [10] Fukuoka T, Johnston DA, Winslow CA, et al. Genetic basis for differential activities of fluconazole and voriconazole against Candida krusei. Antimicrob Agents Chemother 2003;47: 1213—9. [11] Gupta TP, Ehrinpreis MN. Candida-associated diarrhea in hospitalized patients. Gastroenterology 1990;98:780—5. [12] Hazen KC, Baron EJ, Colombo AL, Girmenia C, Sanchez- Sousa A, Palacio AD, et al. Comparison of the susceptibilities of Candida spp. to fluconazole and voriconazole in a 4-year global evaluation using disk diffusion. J Clin Microbiol 2003;41:5623— 32. [13] Krause R, Schwab E, Bachhiesl D, Daxbock F, Wenisch C, Krejs GJ, et al. Role of Candida in antibiotic-associated diarrhea. J Infect Dis 2001;184:1065—9. [14] Krause R, Krejs GJ, Wenisch C, Reisinger EC. Elevated fecal Candida counts in patients with antibiotic-associated diarrhea: role of soluble fecal substances. Clin Diagn Lab Immunol 2003;10:167—8. [15] Larone DH. Yeasts and yeastlike organisms. In: Larone DH, editor. Medically important fungi: a guide to identification. . 2nd ed., New York: Elsevier Science Publishing Co; 1987 . p. 53—75. [16] Lat A, Thompson GR. Update on the optimal use of voriconazole for invasive fungal infections. Review. Infect Drug Resist 2011;4:443—53. [17] Nkuo-Akenji TK, Ndip RN, Ntoko A. The prevalence of Candida albicans-associated diarrhoea in Buea, South West Cameroon. Afr J Health Sci 2002;9:153—7. [18] Rangel-Frausto MS, Wiblin T, Blumberg HM, et al. National Epidemiology of Mycosis Survey (NEMIS): variations in rates of candidal bloodstream infections in seven surgical ICUs and six neonatal ICUs. Clin Infect Dis 1999;29:253—8. [19] Ruan SY, Chien JY, Hsueh PR. Invasive Trichosporonosis caused by Trichosporon asahii and other unusual Trichosporon species at a medical center in Taiwan. Clin Infect Dis 2009;49:e11—7. [20] Slavin MA, Sorrell TC, Marriott D, Thursky KA, Nguyen Q, Ellis DH, et al. Candidaemia in adult cancer patients: risks for fluconazole-resistant isolates and death. J Antimicrob Chemother 2010;65:1042—51. [21] Snydman DR. Shifting patterns in the epidemiology of nosocomial Candida infections. Chest 2003;123:500S—3S.
26 [22] Soki H, Nagase Y, Yamazaki K, Oda T, Kikuchi K. Isolation of the yeast like fungus Stephanoascus ciferri by culturing the aural discharge of a patient with intractable otitis media. Case report. Kansenshogaku Zasshi 2010;84:210—2. [23] Tashiro M, Murakami H, Yoshizawa, Tateda K, Yamaguchi K. Isolation rate and susceptibilities of Candida species from blood, vascular catheter, urine and stool. Kansenshogaku Zasshi 2010;84:187—92.
P. Banerjee et al. [24] Vaishnavi C, Kaur S, Prakash S. Speciation of fecal Candida isolates in antibiotic-associated diarrhea in non-HIV patients. Jpn J Infect Dis 2008;61:1—4. [25] Zepelin MBV, Kunz L, Ruchel R, Reichard U, Weig M, Grob U. Epidemiology and antifungal susceptibilities of Candida spp. to six antifungal agents: results from a surveillance study on fungaemia in Germany from July 2004 to August 2005. J Antimicrob Chemother 2007;60:424—8.