Pathogenicity of Candida parapsilosis from Outpatients with Vaginitis

Vol. 27, No. 11

JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 1989, p. 2598-2603 0095-1137/89/112598-06$02.00/0 Copyright © 1989, American Society for Microbiology

Isolation, Acid Proteinase Secretion, and Experimental Pathogenicity of Candida parapsilosis from Outpatients with Vaginitis FLAVIA DE BERNARDIS,' RODOLFO LORENZINI,2 RENZO VERTICCHIO,2 LUIGI AGATENSI,3 AND ANTONIO CASSONEl* Laboratories of Bacteriology and Medical Mycologyl and Veterinary Medicine,2 Istituto Superiore di Sanità, and Gynecology Department, Sant'Andrea Hospital,3 Rome, Italy Received 8 May 1989/Accepted 9 August 1989 Candida parapsilosis was isolated from the vaginas of several nonpregnant, nondiabetic, mostly premenopausai outpatients who presented the characteristic signs and symptoms of a frank vulvovaginal candidiasis (heavy discharge with cottage cheese appearance and intense itching, with or without vulvar erythema and dyspareunia). All isolates conformed morphologically, biochemically, and serologically to the standard description of the species. They showed high acid proteinase-secretory activity in vitro and were appreciably pathogenic for cyclophosphamide-immunodepressed mice. Some isolates were also tested for their vaginopathic potential in ovariectomized rats under estradiol administration. In all cases, the rat vagina was colonized by C. parapsilosis to an extent and duration not different from those caused by a vaginopathic isolate of Candida albicans. Periodic acid-Schiff-stained vaginal smears taken at intervals during rat experimental infection showed C. parapsilosis yeasts adhering to exfoliated epithelial cells of rat vagina. Overall, these results emphasize the proteolytic and pathogenic potential of C. parapsilosis and suggest that this fungus may be an agent of clinical vaginitis.

Vulvovaginal candidiasis is one of the most frequent disorders in obstetrics and gynecology. It has been estimated that approximately three-quarters of all adult women suffer from at least one attack of the disease (17). Mechanisms underlying outbreaks and recurrences of candidal vaginitis are, however, complex. A local, transient immunosuppression due to prostaglandin production by macrophages has been proposed as a defect in a severe recurrent form of vaginitis in some women (21). Etiopathological interpretations are also complicated by the frequent reports of asymptomatic women carrying the yeast in the vagina (6, 13), although some authors have argued against the assumption of a simple vaginal carriage of Candida spp., and, most recently, vaginal colonization by yeasts has been found to be commonly associated with vulvovaginal symptoms (9). Although several Candida species can be isolated from the vaginas of diseased or healthy subjects, only two species, i.e., Candida albicans and Torulopsis glabrata, are commonly considered as true vaginopathic agents (13, 17). For vaginitis caused by the most prevalent species, i.e., C. albicans, formation of a germ tube, adherence, and proteinase secretion, probably interrelated to each other (5, 13, 18), have been postulated as important pathogenicity determinants, but it is intriguing that T. glabrata is a nongerminative and nonproteolytic species (10, 12). In a recent survey of outpatients attending a gynecological center in Rome, we have been impressed by the nonoccasional isolation of Candida parapsilosis from the vaginas of women presenting the clinical symptomatology of a classical vaginal candidiasis (11). C. parapsilosis is a nongerminative species whose implication in deep-seated diseases, mostly endocarditis, of debilitated hosts is well known (13). Nevertheless, an etiologic role for this species of Candida in vaginal candidiasis has never been specifically advocated. This prompted us to *

examine more closely the pathogenic characteristics of our C. parapsilosis isolates, including attempts to reproduce a vaginal infection in ovariectomized, estrogen-administered rats (7). MATERIALS AND METHODS

Subjects under study. The women who participated in this study were nonpregnant, nondiabetic outpatients attending the Center for Prevention of Female Genital Cancers at Sant'Andrea Hospital in the northeastern area of Rome. The criteria for inclusion in the study and for the differential clinical diagnosis of candidal vaginitis were reported elsewhere (3, 11, 19). In particular, the subjects enrolled had not taken antibiotics or topical antifungal agents during the two months preceding clinical examination. Most women (in particular, all but one from whom C. parapsilosis was isolated) did not use any medication or device for birth control. Each woman was examined by the same gynecologist (L.A.), who was in charge of all clinical observations and records pertaining to this study. Isolation and identification of yeasts. Two high (posterior fornix) vaginal plain cotton wool swabs were taken from each woman. One swab was immediately smeared on a glass slide, fixed, and stained with periodic acid-Schiff stain for microscopy, while the other swab was transported to the laboratory (within 2 h) by using sterile saline solution as the transport medium. Yeasts were isolated on Sabouraud glucose agar and presumptively identified through morphology on cornmeal agar, the germ tube test in serum (see also below), and the API 20C gallery system (Ayerst, Milan, Italy). The identification was eventually confirmed by the whole battery of assimilation and fermentation tests, as reported by Meyer et al. (10), and by serological tests (Iatron, Tokyo, Japan) (20). The latter included a slide agglutination with a monoclonal antibody (MAbAF1) which

Corresponding author. 2598

C. PARAPSILOSIS AND VAGINITIS

VOL. 27, 1989

recognizes an oligosaccharide epitope present on the cell surface of C. albicans but absent on C. parapsilosis (4). In each test of definitive identification, a representative standard strain of the putatively isolated vaginal species was included as a control. Growth and proteinase production in vitro by the isolates of C. parapsilosis were determined on albumin and hemoglobin as substrate, as recently described for vaginal isolates of C. albicans (3). Detection of proteinase activity by sodium dodecyl sulfatepolyacrylamide gel electrophoresis. To examine the pattern of albumin degradation by acid proteinases of C. parapsilosis and C. albicans, the supernatant (0.1 ml) of a 4-ml culture of either yeast (C. parapsilosis SA-23 or C. albicans SA-40) was harvested after 40 h of growth in proteinase secretion medium, as reported previously (3), and incubated at 37°C for 15 min with bovine serum albumin (1%) in citrate buffer, pH 3.2, 0.1 M (0.4 ml). Control experiments consisted of the same mixture without enzyme addition or with addition of the proteinase inhibitor pepstatin, at a final concentration of 50 ,ug/ml (4). After incubation, a 40-jl portion of each mixture was added with 10 ,ul of sodium dodecyl sulfate electrophoretic buffer and subjected to unidimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, as described elsewhere (1). Experimental infections. For systemic mouse infections, inbred male CD2F1 mice, weighing 18 to 21 g, were obtained from Charles River Breeding Laboratories (Calco, Italy). The animals were inoculated intravenously with 106 yeast cells (in a final volume of 0.2 ml) of each isolate of C. parapsilosis grown to the stationary phase in YEPD (yeast extract-peptone-dextrose) medium (4) and suspended in phosphate-buffered saline. Groups of mice were retreated with cyclophosphamide (Cy [Cytoxan]) (Sigma Chemical Co., St. Louis, Mo.) given intraperitoneally at a dose of 150 mg/kg (0.2 ml final volume) 2 days before microbial challenge. Yeast growth in mouse organs was monitored by a conventional microbiological assay (CFU enumeration), as described elsewhere (2). For experimental vaginal infections, ovariectomized, female Wistar rats (80 to 100 g), obtained from Charles River Breeding Laboratories, were injected subcutaneously with 0.5 mg of estradiol benzoate (Benzatrone; Samil, Rome, Italy) every 2 days. Six days after the first estradiol dose, the animals were inoculated intravaginally with 107 cells (0.1-ml volume) of each yeast isolate tested. The yeast had been grown to stationary phase in YEPD medium at 28°C. It was then harvested by low-speed centrifugation, washed, and suspended to the required number in phosphate-buffered saline. Yeast cells were injected into the vaginal cavity through a syringe equipped with a multipurpose calibrated tip (Combitip; PBI, Milan, Italy). Vaginal fluid was taken from each animal every 2 days, with a special-purpose calibrated (1-uul) plastic loop (Dispoinoc; PBI). Some fluids were used for microscopical examination, while other fluids (one vaginal sample per rat per culture) were used for a measurement of vaginal colonization. For this purpose, the content of each loop was vigorously suspended in 0.1 ml of phosphate-buffered saline and then portions were streaked over plates containing Sabouraud glucose agar plus chloramphenicol and incubated for 48 to 72 h at 28°C. At intervals during vaginal infections, colonies developed on the mycological medium were subjected to species identification according to the diagnostic criteria reported above. Statistical evaluation. Both parametric (Student's t test) and nonparametric (x2, Fisher exact test, and Mann-Whitney

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TABLE 1. Clinical and microbiological data of vaginal isolates of C. parapsilosisa Proteinase Isolate

Patient code; age (yr)

SA-17 SA-19 SA-23

BMM-42; 30 BMM-64; 45 BMM-79; 48

SA-25 SA-36

BMM-103; 45 BMM-127; 38

SA-139

BMM-360; 37

SA-327

BMM-451; 53

SA-365

BMM-487; 46

SA-381 SA-383 SA-393

BMM-493; 46 BMM-501; 55 BMM-627; 27

Symptomsb

CD + PR + CD + PR + CD + PR + + DY CD + PR CD + PR + + DY CD + PR + + DY CD + PR + + DY CD + PR + + DY CD + PR + CD + PR CD + PR

on':

activity BSA HB (score) (U/ml)

DY ER ER

+ + ++

1.36 0.99 1.89

ER

++ ++

0.70/1.22 0.85/0.95

ER

+

0.63/1.02

ER

++

1.60

ER

++

1.29

ER

++ ND ++

NDd ND ND

a All isolates conformed microbiologically and serologically to the standard description of C. parapsilosis (see reference 10). b CD, Clumpy discharge with cottage cheese appearance; PR, vulvar pruritus; ER, vulvar erythema; DY, dyspareunia. c BSA, Bovine serum albumin; U/ml, units of activity. For technical details on proteinase assays, see reference 3. d ND, Not done.

U test) tests were used, as appropriate, and indicated in single experiments (see below). RESULTS Clinical and microbiological data. During the period this study was performed (January 1985 to December 1988), a total of 155 yeast isolates from women with active, symptomatic vaginitis were identified. The large majority (around 65%) of the isolates belonged, as expected, to C. albicans and T. glabrata. C. parapsilosis was isolated from 13 subjects with signs and symptoms characteristic of candidate vaginitis. In two cases, C. parapsilosis was isolated in mixed culture with C. albicans; these two isolates of C. parapsilosis were excluded from subsequent studies. The main clinical features of the 11 vaginitis patients who harbored C. parapsilosis as single yeast species in the vagina are shown in Table 1. All patients had the two characteristic signs and symptoms of candidate vaginitis, i.e., the clumpy discharge with cottage cheese aspect and an intense pruritus, but most also showed erythema or painful sexual intercourse or both. All putative C. parapsilosis isolates were repeatedly tested to exclude the possession of properties typical of C. albicans, e.g., germ tube and chlamydospore formation in vitro; no isolate formed these morphological elements in any medium. Agglutination tests based on the serological scheme of Tsuchiya et al. (20) by Iatron's reagents confirmed the identification of C. parapsilosis. Finally, an immunoglobulin M monoclonal antibody which agglutinated all isolates tested (64) of C. albicans did not agglutinate any isolate of C. parapsilosis (data not shown; see also reference 4). Most of the vaginal isolates of C. parapsilosis were also assayed for their proteolytic activity in vitro; Table 1 shows that they extensively hydrolyzed both hemoglobin and albumin. The acid proteinase activity of C. parapsilosis was confirmed by the analysis of albumin degradation products by sodium

J. CLIN. MICROBIOL.

DE BERNARDIS ET AL.

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TABLE 2. Mortality of mice challenged intravenously with 106 cells of vaginal isolates of C. parapsilosis Mortality of: Isolate of C. parapsilosis

SA-17 SA-19 SA-23 SA-25

Untreated mice

Cy-treated mice

MSTa

DITb

MST

D/T

>30 >30 >30 >30

0/6 0/6 1/7 1/7

>30 >30 >30 3C

2/6 4/15 3/6

7/1d

MST, Median survival time (days). D/T, Dead/total (after 30 days of observation). P < 0.01, as evaluated by Mann-Whitney U test, comparing the value with p the MSTs of C. parapsilosis-injected, Cy-untreated mice and with the MSTs of SA-17, SA-19, or SA-23-injected, Cy-treated mice. d P < 0.05, comparing SA-25 and SA-19 (Fisher exact method). a

b

dodecyl sulfate-polyacrylamide gel electrophoresis (Fig. 1); the pattern of hydrolysis by the enzyme in the supernatant of C. parapsilosis (isolate SA-23) was nearly identical to that shown by the enzyme of a vaginopathic isolate of C. albicans (SA-40). In both cases, a major hydrolytic product of the reaction was a protein fragment of about 20 kilodaltons. Finally, all isolates of C. parapsilosis were susceptible in vitro to common antimycotics (nystatin, flucytosine, and imidazole derivatives; data not shown). The growth potential of each vaginal isolate was measured by using the YEPD medium (3). No statistically significant differences in growth rate and yield after 24 h of incubation in the above medium were found among the various isolates of C. parapsilosis. Experimental pathogenicity in a systemic infection of mice. The first four vaginal isolates of C. parapsilosis (SA-17, SA-19, SA-23, and SA-25) were tested for their experimental pathogenicity after a systemic (intravenous) challenge in mice. The animals were either unmodified or had lowered anti-infectious defenses by administration of the strong immunodepressant drug Cy (2). None of the vaginal isolates caused more than a marginal mortality in normal mice (Table 2). However, they were appreciably pathogenic for Cyimmunodepressed mice, with one isolate (SA-25) killing 7 out of 10 infected, Cy-immunodepressed mice in less than 1 week. Collectively, the isolates killed 16 out of 37 infected animals of the Cy group, whereas they killed only 2 out of 26 normal mice, the difference being statistically highly significant (P < 0.01; x2 test). The infections caused by the isolates SA-19 and SA-23 in Cy-treated mice were also monitored for the number of the

2

FIG.

1.

The

3

electrophoretic pattern of albumin hydrolysis by

secretory acid proteinase of C. parapsilosis (lane 2) and C. albicans are: buffered bovine serum albumin only, without (lane 1), and C. parapsilosis enzyme activity blocked by acid proteinase inhibitor pepstatin (lane 4). The arrows point to

(lane 3). Controls

enzyme

molecular

weight standards.

infecting celis in two target organs for invasive candidiasis, e.g., the kidney and the heart, coupled with histopathological observations. Both organs were heavily parasitized

(CFU greater than 106 per organ) by each isolate during the first 3 to 4 days after challenge, followed (in surviving mice) by a dramatic drop (2 or more orders of magnitude) in the number of microbial units by day 7 (data not shown). Experimental vaginitis infection with C. parapsilosis. Ovariectomized, estradiol-treated rats were used in attempts to reproduce an experimental vaginal infection with C. parapsilosis. Three isolates of C. parapsilosis were used for this study, and the experimental design included rats challenged intravaginally with an isolate of C. albicans from an active vaginitis (SA-40) and a strain of Saccharomyces cerevisiae (SA-264) isolated from the vagina of a healthy, fully asymptomatic woman, as positive and negative controls, respectively. Table 3 shows the viable counts of each isolate detected in the vaginal material and the number of rats infected (>103 cells per ml of vaginal fluid) over the total

TABLE 3. Quantitationa of vaginal colonization in pseudoestrous rats infected' with isolates of C. parapsilosis and controls Day after challenge

1 3 7 14 22 29

CFU/ml ± SE (103) (no. of rats infected/total) C. parapsilosis SA-25

C. parapsilosis

SA-23

C. parapsilosis SA-19

C. albicans SA-40

S. cerevisiae SA-264

>100 (5/5) 69.0 ± 7.5 (5/5) 47.5 ± 21.2 (4/5) 18.7 ± 2.3 (3/5) 3.6 ± 1.4 (2/5) 1.0 (1/5)

>100 (4/4) 82.5 ± 10.3 (4/4) >100 (4/4) 25.0 ± 6.1 (4/4) 1.5 ± 0.7 (2/4) 1.0 ± 0.25 (2/4)

>100 (4/4) 58.7 ± 23.5 (4/4) 75.0 ± 14.4 (4/4) 41.5 ± 20.7 (4/4) 11.0 ± 6.0 (2/4) 2.0 ± 0.7 (2/4)

>100 (5/5) 72.5 ± 1.9 (5/5) 50.2 ± 8.6 (5/5) 14.0 ± 9.2 (2/5) 2.0 (1/5) 1.0 (1/5)

75 ± 24.8 (2/2) 6.5 ± 1.48c (2/2)