Diagnostic Microbiology and Infectious Disease 41 (2001) 29 –35
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Multicenter prospective surveillance of oral Candida dubliniensis among adult Brazilian human immunodeficiency virus-positive and AIDS patients Eveline Pı´polo Milana,b, Priscilla de Laet Sant⬘Anaa, Analy Salles de Azevedo Meloc, Derek J. Sullivand, David C. Colemand David Lewia, Arnaldo Lopes Colomboa,* a
Division of Infectious Diseases, Escola Paulista de Medicina,Universidade Federal de Sa˜o Paulo, Sa˜o Paulo, Brazil b Department of Microbiology and Parasitology, Universidade Federal do Rio Grande do Norte, Natal, Brazil c Department of Microbiology, Immunology and Parasitology, Escola Paulista de Medicina, Universidade Federal de Sa˜o Paulo, Sa˜o Paulo, Brazil d Microbiology Research Unit, Department of Oral Medicine and Oral Pathology, School of Dental Science, University of Dublin, Trinity College, Dublin 2, Republic of Ireland Received 8 March 2001; accepted 15 August 2001
Abstract The incidence of C. dubliniensis in South America has not yet been determined. In the present study, oral swab samples were taken from 108 HIV-infected/AIDS individuals attending 6 separate Brazilian HIV-treatment centers to determine the incidence of C. dubliniensis in this population. Swabs were plated onto CHROMagar Candida medium and 155 isolates, presumptively identified as C. albicans or C. dubliniensis were further investigated. In a preliminary screen for C. dubliniensis, 13 of the 155 isolates showed no or poor growth at 42°C, and all them were subjected to randomly amplified polymorphic DNA (RAPD) and polymerase chain reaction (PCR) analysis using C. dubliniensis-specific primers. We confirmed that 4 out of 13 isolates were C. dubliniensis, representing an incidence rate of 2.8% for the Brazilian HIV-infected population infected with yeasts exhibiting green colonies on CHROMagar Candida. This value is significantly lower than those reported in Ireland and the United States. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Oral candidosis; Candida dubliniensis; AIDS; Emerging pathogen
1. Introduction Candida dubliniensis was originally described by Sullivan et al. in 1995 (Sullivan et al., 1995). It is primarily associated with colonization and infection of the oral cavity of human immunodeficiency virus (HIV)-infected individuals and AIDS patients. Recently it has also been recovered from oral and non-oral sites in non-HIV-infected individuals (Brandt et al., 2000; Sullivan et al., 1997, 1999; Willis et al., 2000). Several authors have demonstrated that C. dubliniensis is closely related to C. albicans and that both species share many phenotypic characteristics (Coleman et al., 1997; Kirkpatrick et al., 1998; Sullivan et al., 1995). Consequently, accurate and rapid differentiation between these species in the clinical laboratory has proven to be
* Corresponding author. Tel./fax: ⫹1-5511-5549-6585. E-mail address:
[email protected] (A. Lopes-Colombo).
problematic. The most accurate and reliable means of discriminating between C. albicans and C. dubliniensis involves the use of molecular methods that detect genotypic differences between the two species. There are currently a wide variety of molecular techniques capable of identifying C. dubliniensis (Sullivan et al., 1999). Some of these (e.g., DNA fingerprinting) are time-consuming and expensive, while others (e.g., PCR with C. dubliniensis-specific primers) are rapid and less expensive and suitable for large sample volume throughput. Soon after the first identification of C. dubliniensis in HIV-positive and AIDS patients in Dublin, Ireland, the new species was reported from this patient group in many countries throughout the world (Jabra-Rizk et al., 1999; Odds et al., 1998; Rodriguez-Tudela et al., 1995; Sano et al., 2000; Sullivan et al., 1997). Although the prevalence of C. dubliniensis has been investigated prospectively in Europe and the United States, its prevalence in South America has not been investigated. The purpose of the present study was to
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perform the first multi-center prospective investigation of the incidence of C. dubliniensis in Brazilian HIV-positive and AIDS patients.
al., 1998; National Committee for Clinical Laboratory Standards, 1997; Rodriguez-Tudela, 1995). 2.3 RAPD analysis.
2. Materials and methods 2.1 HIV-infected patients and oral Candida isolates A prospective study was conducted over a 24-month period, from March 1998 to February 2000, aimed at determining the incidence of C. dubliniensis in the oral cavities of HIV-positive and AIDS patients in Brazil. Six tertiary care medical centers with active HIV patient treatment clinics joined the project at different stages during the two year study period. These included the Universidade Federal de Sa˜ o Paulo-UNIFESP, Universidade Federal do Rio Grande do Norte (Natal), Universidade de Sa˜ o Paulo (Ribeira˜ o Preto), Universidade Federal do Parana´ (Curitiba), Universidade Federal da Bahia (Salvador) and Faculdade de Medicina de Uberlaˆ ndia. Samples were taken from HIV-positive and AIDS patients attending the HIV treatment clinics at each participating center by swabbing the oral mucosa of the subjects with sterile cotton wool swabs. These specimens were forwarded immediately to the microbiology laboratory in each center and subsequently plated onto CHROMagar Candida agar medium (CHROMagar, Paris, France) and incubated at 30°C for up to 5 days. Plates were examined for light green colonies (typical of C. albicans) and dark green colonies (typical of C. dubliniensis) (Kirkpatrick et al., 1998; Schoofs et al., 1997; Sullivan & Coleman, 1997) following 48 h incubation and thereafter daily up to five days. When present on primary isolation plates, representative light green and/or dark green colonies were chosen at random, purified by subculture on Sabouraud dextrose agar (SDA, Difco Laboratories, Detroit, Mich.) and forwarded to the reference laboratory, at UNIFESP, together with case report forms containing clinical and epidemiologic information. Cultures which presented other colors than green were excluded from this study, but selected for any other.
Template DNA for Randomly Amplified Polymorphic DNA (RAPD) analysis was prepared from yeast cells grown in 2 mL of YPD medium for 16 h at 30°C with agitation (220 r.p.m.) or until a density of 2 ⫻ 108 cells/mL was achieved. DNA was extracted using a rapid, small-scale isolation protocol described previously (Wash et al., 1994). RNA was removed by treatment with RNase A (Amersham Pharmacia Biotech, Piscataway-NJ, USA) for 1 h at 37°C. DNA concentration and purity were determined by optical density at 260 nm and ratio O.D. 260 nm/280 nm determinations, respectively. RAPD tests were also performed with template DNA from reference strains of C. albicans (ATCC 76615) and C. dubliniensis (CBS 7987). RAPD analysis was performed as described previously with the oligonucleotide primer CDU (5⬘ GCG ATC CCC A 3⬘) (Sullivan et al., 1995) and the oligonucleotide primer B-14 (5⬘ GAT CAA GTC C 3⬘), the latter previously reported by Bauer et al. (1993). Usually 40 ng of total DNA were added to a 25 L reaction containing 2.5 L of a 10⫻ PCR buffer (100 mM Tris-HCl, pH 8.3, 500 mM KCl, 3.5 mM MgCl2), 4 L of dNTP mix (1.25 mM each dNTP), 0.4 M of primer, 0.5% (vol/vol) Tween 20 and 1.0 unit of Taq DNA polymerase (Amersham Pharmacia Biotech). Cycle conditions were 94°C for 1 min, 40°C for 1 min, 72°C for 2 min, for 45 cycles and a final extension at 72°C for 10 min, in a Perkin-Elmer DNA thermocycler (Gene Amp PCR System 9600). PCR products were separated by electrophoresis for 3 h at 100 V in 1.5% (wt/vol) agarose gels using TrisAcetate-EDTA (TAE) buffer. Following electrophoresis, gels were stained with a solution containing 0.5 g/mL ethidium bromide for 10 min and destained twice for 15 min in 300 mL of distilled water. RAPD profiles were then visualized on a UV transilluminator and photographed.
2.2 Phenotypic characterization of clinical isolates.
2.4 PCR identification of C. dubliniensis
Following receipt, all clinical isolates were purified on SDA agar and tested for their ability to grow at 42°C on SDA and for their ability to produce chlamydoconidia on cornmeal Tween 80 Agar (Difco laboratories, Detroit, Mich.), both as described previously (Sullivan et al., 1995; Warren & Hazen, 1995). All C. dubliniensis isolates were also tested for susceptibility to amphotericin B, fluconazole, itraconazole and ketoconazole by broth microdilution, in accordance with the NCCLS M27-A standard (National Committee for Clinical Laboratory Standards, 1997). Breakpoint definitions for fluconazole and itraconazole MICs were those proposed by the NCCLS (National Committee for Clinical Laboratory Standards, 1997). Due to the lack of consensual definitions of breakpoints for ketoconazole and amphotericin B MICs, arbitrary values were established, following those suggested in previous studies (Milan et
The identity of chlamydospore-positive isolates identified as C. dubliniensis on the basis of RAPD profiles and ID 32C assimilation profiles and by lack of growth or poor growth at 42°C was confirmed using PCR with C. dubliniensis-specific primer pair DUBF and DUBR as described previously (Donnelly et al., 1999). This primer pair is complimentary to sequences within the ACT1-associated intron sequence of C. dubliniensis and yields an amplimer of 288 bp. Each reaction mixture also contained the universal fungal primers RNAF and RNAR (Fell, 1993), which amplify a fragment of approximately 610 bp from all fungal large-subunit rRNA genes, as an internal positive control. Template DNA extracted from the C. dubliniensis type strain CBS 7987 and the C. albicans reference strains 132A and 179B (Gallagher et al., 1992) were used as positive and negative controls, respectively.
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Fig. 1. Screening for C. dubliniensis among isolates exhibiting green colonies on CHROMagar Candida medium.
3. Results
fected individuals included in the study showed clinical signs of oral candidiasis at sampling.
3.1 Clinical isolates 3.3 Screening for C. dubliniensis. The centers sent a total of 175 Candida isolates to the reference laboratory. Of note, 155 isolates were identified by CHROMagar as C. albicans or C. dubliniensis, according to the green color of the colonies. Otherwise, 20 isolates exhibited colonies color others than green, which were further identified as non-albicans species, mainly represented by C. tropicalis and C. glabrata. These non-albicans isolates were excluded from this study. We evaluated the 155 oral yeast isolates which exhibited green color on CHROMagar plates that were recovered from 108 HIV-infected individuals attending HIV treatment clinics in 6 Brazilian medical centers. The origin of the 155 strains was: 10 isolates from Uberlaˆ ndia, 13 isolates from Curitiba, 9 isolates from Salvador, 24 isolates from Ribeira˜ o Preto, 46 isolates from Sa˜ o Paulo and 53 isolates from Natal. All them were subjected to further analysis. 3.2 Clinical and demographic data of enrolled patients. The 108 HIV-infected persons enrolled in this study included 21 HIV-seropositive individuals and 87 individuals with AIDS. Sixty-eight of the HIV-infected individuals were male and 40 were female with a median age of 35 years (range 16 – 65 years). The mean CD4 lymphocyte count of the HIV-seropositive individuals was 413 cells/mL (range 200 –902 cells/mL) and that of the AIDS patients was 57 cells/mL (range 2.9 –199 cells/mL). Forty-one of the 108 patients were sampled on two separate occasions. All of the AIDS patients were receiving Highly Active Anti-Retroviral Therapy (HAART) at the time of sampling. None of the HIV-positive patients and 50/87 AIDS patients had been treated previously with antifungal drugs. The remaining 37 AIDS patients had been exposed once or twice to antifungal drug therapy, including topical or systemic drugs prior to sampling. The majority (89/108, 82.4%) of the HIV-in-
The screening for C. dubliniensis isolates was performed according to the Fig. 1. 3.4 RAPD results All 13 isolates with weak or no growth at 42°C were further evaluated by RAPD profile analysis. The RAPD banding patterns of the 13 clinical isolates obtained using the 2 primers tested are illustrated in Fig. 1. Using the primer CDU, 4/13 clinical isolates yielded RAPD profiles very similar to the corresponding profile obtained with the C. dubliniensis type strain CBS 7987. These profiles consisted of 12 strong bands, with molecular sizes ranging from 100 to 2000 bp. (Fig. 2A). In contrast, the remaining isolates yielded RAPD profiles similar to that obtained with the C. albicans reference strain ATCC 76615 (Fig. 2A). These results were confirmed following RAPD analysis of the isolates with the B-14 primer (Fig. 2B). The same 4 isolates which yielded RAPD profiles similar to the C. dubliniensis type strain with the CDU primer, yielded very similar banding profiles to C. dubliniensis CD36 with the B-14 primer, with profiles consisting of 11 strong bands, ranging in size from 200 to 1,300 bp (Fig. 2B). These findings strongly suggested that these 4 isolates were C. dubliniensis and that the remaining 9 were C. albicans. 3.5 PCR results Definitive identification of the 4 presumptive C. dubliniensis isolates was made by PCR analysis using PCR primers specific for the ACT1 intron of C. dubliniensis. All 4 isolates yielded C. dubliniensis-specific amplimers (Fig. 3).
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Fig. 2. Agarose gels showing amplified DNA products generated using either (A) the CDU primer or (B) the B-14 primer in RAPD analysis of Candida isolates. The profiles shown in lanes 2 and 3 correspond to the C. dubliniensis type strain CD36 (CBS 7987) and the C. albicans reference strain ATCC 76615, respectively. The remaining profiles correspond to the 13 clinical isolates which grew poorly or not at all at 42°C on SDA agar. Profiles shown in lanes 4 –7 correspond to the 4 C. dubliniensis isolates identified in this study, whereas profiles shown in lanes 8 –16 correspond to C. albicans isolates. Lane 1, molecular weight size reference markers (100 bp ladder)
3.6 Susceptibility profile of C. dubliniensis The 4 C. dubliniensis oral isolates exhibited MIC values ranging from 0.125 to 0.5 for amphotericin B, 0.125 for
fluconazole, 0.03 to 0.06 for itraconazole and 0.015 to 0.03 for ketoconazole. These data indicated that all of the C. dubliniensis isolates were susceptible to the 4 antifungal drugs tested.
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Fig. 3. Agarose gel showing ethidium bromide-stained amplimers from PCRs with the C. dubliniensis -specific primers DUBF and DUBR (288 bp product) and the fungal universal primers RNAF and RNAR (610 bp product). The amplimers shown in the lanes were obtained with template DNA from Candida isolates and strains as follows: lanes 2–5, 4 Brazilian clinical C. dubliniensis isolates; lanes 6 – 8, C. dubliniensis reference strains (including the type strain CD36 (lane 6)); lanes 9 and 10, C. albicans reference strains 132A and 179B (9). Lane 1, molecular weight size markers (100 bp ladder).
3.7 Clinical data of patients harboring C. dubliniensis The 4 isolates of C. dubliniensis were recovered from 3/108 (2.8%) patients recruited from the cities of Sa˜ o Paulo and Natal. The patients included two females (a 32 and a 39 year old with CD4⫹ counts of 160 and 315, respectively), and one male (32 years old with a CD4⫹ count of 339). Based on the CDC classification system for HIV infection and expanded AIDS surveillance case definition, the clinical category of the two females was A2 and the male was classified as A3. Laboratory tests revealed plasma HIV-1 RNA levels of 3,394, 2,900 and 61,000 copies/mL, respectively. None of the three patients showed any oral signs or symptoms indicative of oral candidiasis at the time C. dubliniensis was recovered and none had received prior treatment with antifungal drugs. C. dubliniensis was the only species isolated from two of the HIV-infected subjects, while in the third it was isolated together with C. albicans. In one of the colonized individuals C. dubliniensis was recovered on two separate occasions 6 months apart, suggesting that colonization was persistent.
4. Discussion Candida dubliniensis is an emerging yeast pathogen that was originally associated with oral colonization and infection in HIV-infected and AIDS patientes (Sullivan & Coleman, 1997; Sullivan et al., 1995). However, recent reports have also described cases of superfical and systemic candidiasis in HIV-infected and non-HIV-infected individuals (Brandt et al., 2000; Sullivan et al., 1997; Willis et al., 2000). As the number of reports of infections caused by C.
dubliniensis continues to grow, there is an increasing need for an in-depth epidemiologic investigation into the prevalence of this species. Currently there are no substantial data available on the incidence of C. dubliniensis in South American populations. The purpose of the present study was to prospectively determine the oral incidence of C. dubliniensis in Brazilian HIV-infected and AIDS patients attending HIV treatment clinics at 6 different centers in Brazil. Of the 155 Candida isolates tested, 13 (8.4%) failed to grow or grew poorly at 42°C suggesting that they may have been C. dubliniensis isolates. Clearly phenotypic tests, such as colony color on CHROMagar medium or growth at 42°C, are helpful in preliminary screens of isolate collections for the presence of C. dubliniensis. However, it is evident from the present and previous studies (Kirkpatrick et al., 1998; Odds et al., 1998; Schoofs et al., 1997) that these tests alone are insufficient for the definitive identification of C. dubliniensis; e.g., in the present Study 9/151(6%) C. albicans isolates failed to grow or grew poorly at 42°C. Although, inhability to grow at 42°C or 45°C is considered the best phenotypical triage test, definitive identification of C. dubliniensis can only be determined unequivocally using appropriate molecular tests. The 4 C. dubliniensis isolates identified in this study were recovered from three HIV-infected patients with clinical category A2 and A3, according to the CDC classification system. Two of the isolates were recovered from the same individual at separate clinical evaluations, six-months apart. According to Sullivan et al. (1999), many of the HIVinfected individuals colonized by C. dubliniensis have a history of recurrent oral candidiasis and have received protracted treatments with azole drugs. C. dubliniensis had
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already been recovered from the oral cavity of HIV-infected individuals or AIDS patients with or without clinical symptoms of oral candidiasis. The risk factors for acquiring C. dubliniensis infections remains unclear (Schoofs et al., 1997; Kirkpatrick et al., 1998). There is no available data about correlation among C. dubliniensis and AIDS clinical category, CD4 count and HIV-1 RNA level. Our C. dubliniensis isolates were isolated from assymptomatic patients in a initial phase of disease, that have not been received antifungal drugs in the last 10 months. This finding suggests that C. dubliniensis can colonize oral cavity of AIDS patients without previous use of azole drugs. The overall incidence of C. dubliniensis in the Brazilian HIV-infected population with thrush due to yeasts exhibiting green colonies on CHROMagar Candida was very low (3/108 (2.8%)) compared to the reported incidence levels in other populations of HIV-infected individuals. Currently, the most extensive reported data on the incidence of C. dubliniensis in HIV-infected individuals comes from Ireland. In a study of Irish HIV-infected individuals the incidence ranged from 18 –32%, depending on whether the individuals studied were HIV-seropositive or had AIDS, and on the presence or absence of oral candidiasis. In the same study, an oral incidence level of 3% was reported for Irish HIV-negative healthy, dentate individuals (Coleman et al., 1997; Sullivan et al., 1999). Studies conducted in the USA have reported oral incidence rates for C. dubliniensis in HIV-infected individuals ranging from 11.1 to 17.5% (Kirkpatrick et al., 1998; Meiller et al., 1999). It is difficult to explain the observed low C. dubliniensis incidence rate in Brazilian HIV-infected individuals relative to these other HIV-infected populations. It is possible that absence of prior treatment with antifungal drugs may have contributed to the low incidence of C. dubliniensis in the Brazilian HIVinfected population, since in other studies the majority of patients had previously received antifungal therapy (Coleman et al., 1997; Kirkpatrick et al., 1998; Meiller et al., 1999; Sullivan et al., 1999). Alternatively, the low incidence of C. dubliniensis in the Brazilian HIV-infected population may reflect a genuine difference in the subject population. All of the Brazilian AIDS patients were receiving HAART at the time of sampling for oral Candida. Whether HAART therapy can influence the incidence of oral carriage of particular Candida species awaits further study. One could suggest that the low incidence of C. dubliniensis we found in the present study could be related to the fact that some C. dubliniensis isolates may not have been identified following primary culture from the oral samples. However, this is unlikely as primary isolation was performed on CHROMagar medium which supports very well the growth of Candida species. Actually CHROMagar Candida has been successfully used by investigators in Europe and the USA to investigate oral colonization by C. dubliniensis (Kirkpatrick et al., 1998; Schoofs et al., 1997; Sullivan & Coleman, 1997). As a conclusion, C. dubliniensis is an emergent pathogen
also present in oral cavity of brazilian HIV-infected or AIDS patients. This species may colonize patients with initial phase of AIDS disease and low previous exposition to antifungal drugs. The prevalence rate of C. dubliniensis among Brazilian HIV-infected/AIDS patients seems to be lower than in the USA or Europe.
Acknowledgments We thank Dr. Roberto Martinez, Dr. Marcelo Sima˜ o, Dr. Flavio Telles and Dr. Ana Paula Alcaˆ ntara for sending some of the oral Candida albicans isolates used in this study. Work performed in Brasil was supported by Fundac¸ a˜ o de Amparo a` Pesquisa do Estado de Sa˜ o Paulo grant 1999/ 07134 –3. Work performed in Dublin was supported by Irish Health Research Board grant 05/97.
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