JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 1989, p. 2598-2603 Vol. 27, No. 110095-1137/89/112598-06$02.00/0Copyright © 1989, American Society for Microbiology

Isolation, Acid Proteinase Secretion, and ExperimentalPathogenicity of Candida parapsilosis from

Outpatients with VaginitisFLAVIA DE BERNARDIS,' RODOLFO LORENZINI,2 RENZO VERTICCHIO,2

LUIGI AGATENSI,3 AND ANTONIO CASSONEl*Laboratories ofBacteriology 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 premeno-pausai 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 anddyspareunia). All isolates conformed morphologically, biochemically, and serologically to the standarddescription of the species. They showed high acid proteinase-secretory activity in vitro and were appreciablypathogenic for cyclophosphamide-immunodepressed mice. Some isolates were also tested for their vaginopathicpotential 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 Candidaalbicans. Periodic acid-Schiff-stained vaginal smears taken at intervals during rat experimental infectionshowed C. parapsilosis yeasts adhering to exfoliated epithelial cells of rat vagina. Overall, these resultsemphasize the proteolytic and pathogenic potential of C. parapsilosis and suggest that this fungus may be anagent of clinical vaginitis.

Vulvovaginal candidiasis is one of the most frequentdisorders in obstetrics and gynecology. It has been estimatedthat approximately three-quarters of all adult women sufferfrom at least one attack of the disease (17). Mechanismsunderlying outbreaks and recurrences of candidal vaginitisare, however, complex. A local, transient immunosuppres-sion due to prostaglandin production by macrophages hasbeen proposed as a defect in a severe recurrent form ofvaginitis in some women (21). Etiopathological interpreta-tions are also complicated by the frequent reports of asymp-tomatic women carrying the yeast in the vagina (6, 13),although some authors have argued against the assumptionof a simple vaginal carriage of Candida spp., and, mostrecently, vaginal colonization by yeasts has been found to becommonly 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 com-monly considered as true vaginopathic agents (13, 17). Forvaginitis caused by the most prevalent species, i.e., C.albicans, formation of a germ tube, adherence, and protein-ase secretion, probably interrelated to each other (5, 13, 18),have been postulated as important pathogenicity determi-nants, but it is intriguing that T. glabrata is a nongerminativeand nonproteolytic species (10, 12). In a recent survey ofoutpatients attending a gynecological center in Rome, wehave been impressed by the nonoccasional isolation ofCandida parapsilosis from the vaginas of women presentingthe clinical symptomatology of a classical vaginal candidiasis(11). C. parapsilosis is a nongerminative species whoseimplication in deep-seated diseases, mostly endocarditis, ofdebilitated hosts is well known (13). Nevertheless, an etio-logic role for this species of Candida in vaginal candidiasishas never been specifically advocated. This prompted us to

* Corresponding author.

examine more closely the pathogenic characteristics of ourC. parapsilosis isolates, including attempts to reproduce avaginal infection in ovariectomized, estrogen-administeredrats (7).

MATERIALS AND METHODS

Subjects under study. The women who participated in thisstudy were nonpregnant, nondiabetic outpatients attendingthe Center for Prevention of Female Genital Cancers atSant'Andrea Hospital in the northeastern area of Rome. Thecriteria for inclusion in the study and for the differentialclinical diagnosis of candidal vaginitis were reported else-where (3, 11, 19). In particular, the subjects enrolled had nottaken antibiotics or topical antifungal agents during the twomonths preceding clinical examination. Most women (inparticular, all but one from whom C. parapsilosis wasisolated) did not use any medication or device for birthcontrol. Each woman was examined by the same gynecolo-gist (L.A.), who was in charge of all clinical observationsand records pertaining to this study.

Isolation and identification of yeasts. Two high (posteriorfornix) vaginal plain cotton wool swabs were taken fromeach woman. One swab was immediately smeared on a glassslide, fixed, and stained with periodic acid-Schiff stain formicroscopy, while the other swab was transported to thelaboratory (within 2 h) by using sterile saline solution as thetransport medium. Yeasts were isolated on Sabouraud glu-cose agar and presumptively identified through morphologyon cornmeal agar, the germ tube test in serum (see alsobelow), and the API 20C gallery system (Ayerst, Milan,Italy). The identification was eventually confirmed by thewhole battery of assimilation and fermentation tests, asreported by Meyer et al. (10), and by serological tests(Iatron, Tokyo, Japan) (20). The latter included a slideagglutination with a monoclonal antibody (MAbAF1) which

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recognizes an oligosaccharide epitope present on the cellsurface of C. albicans but absent on C. parapsilosis (4).

In each test of definitive identification, a representativestandard strain of the putatively isolated vaginal species was

included as a control. Growth and proteinase production invitro by the isolates of C. parapsilosis were determined on

albumin and hemoglobin as substrate, as recently describedfor vaginal isolates of C. albicans (3).

Detection of proteinase activity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. To examine the pattern ofalbumin degradation by acid proteinases of C. parapsilosisand C. albicans, the supernatant (0.1 ml) of a 4-ml culture ofeither yeast (C. parapsilosis SA-23 or C. albicans SA-40)was harvested after 40 h of growth in proteinase secretionmedium, as reported previously (3), and incubated at 37°Cfor 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 with-out enzyme addition or with addition of the proteinaseinhibitor 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 electrophoreticbuffer and subjected to unidimensional sodium dodecylsulfate-polyacrylamide gel electrophoresis, as describedelsewhere (1).

Experimental infections. For systemic mouse infections,inbred male CD2F1 mice, weighing 18 to 21 g, were obtainedfrom Charles River Breeding Laboratories (Calco, Italy).The animals were inoculated intravenously with 106 yeastcells (in a final volume of 0.2 ml) of each isolate of C.parapsilosis grown to the stationary phase in YEPD (yeastextract-peptone-dextrose) medium (4) and suspended inphosphate-buffered saline. Groups of mice were retreatedwith cyclophosphamide (Cy [Cytoxan]) (Sigma ChemicalCo., St. Louis, Mo.) given intraperitoneally at a dose of 150mg/kg (0.2 ml final volume) 2 days before microbial chal-lenge. Yeast growth in mouse organs was monitored by a

conventional microbiological assay (CFU enumeration), as

described elsewhere (2).For experimental vaginal infections, ovariectomized, fe-

male Wistar rats (80 to 100 g), obtained from Charles RiverBreeding Laboratories, were injected subcutaneously with0.5 mg of estradiol benzoate (Benzatrone; Samil, Rome,Italy) every 2 days. Six days after the first estradiol dose, theanimals were inoculated intravaginally with 107 cells (0.1-mlvolume) of each yeast isolate tested. The yeast had beengrown to stationary phase in YEPD medium at 28°C. It wasthen harvested by low-speed centrifugation, washed, andsuspended to the required number in phosphate-bufferedsaline. Yeast cells were injected into the vaginal cavitythrough a syringe equipped with a multipurpose calibratedtip (Combitip; PBI, Milan, Italy). Vaginal fluid was takenfrom each animal every 2 days, with a special-purposecalibrated (1-uul) plastic loop (Dispoinoc; PBI). Some fluidswere 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, thecontent of each loop was vigorously suspended in 0.1 ml ofphosphate-buffered saline and then portions were streakedover plates containing Sabouraud glucose agar plus chlor-amphenicol and incubated for 48 to 72 h at 28°C. At intervalsduring vaginal infections, colonies developed on the myco-logical medium were subjected to species identificationaccording to the diagnostic criteria reported above.

Statistical evaluation. Both parametric (Student's t test)and nonparametric (x2, Fisher exact test, and Mann-Whitney

TABLE 1. Clinical and microbiological data of vaginalisolates of C. parapsilosisa

Proteinase

Isolate Patient code; Symptomsb activity on':age (yr) BSA HB

(score) (U/ml)

SA-17 BMM-42; 30 CD + PR + DY + 1.36SA-19 BMM-64; 45 CD + PR + ER + 0.99SA-23 BMM-79; 48 CD + PR + ER ++ 1.89

+ DYSA-25 BMM-103; 45 CD + PR ++ 0.70/1.22SA-36 BMM-127; 38 CD + PR + ER ++ 0.85/0.95

+ DYSA-139 BMM-360; 37 CD + PR + ER + 0.63/1.02

+ DYSA-327 BMM-451; 53 CD + PR + ER ++ 1.60

+ DYSA-365 BMM-487; 46 CD + PR + ER ++ 1.29

+ DYSA-381 BMM-493; 46 CD + PR + ER ++ NDdSA-383 BMM-501; 55 CD + PR ND NDSA-393 BMM-627; 27 CD + PR ++ ND

a All isolates conformed microbiologically and serologically to the standarddescription of C. parapsilosis (see reference 10).

b CD, Clumpy discharge with cottage cheese appearance; PR, vulvarpruritus; ER, vulvar erythema; DY, dyspareunia.

c BSA, Bovine serum albumin; U/ml, units of activity. For technical detailson proteinase assays, see reference 3.

d ND, Not done.

U test) tests were used, as appropriate, and indicated insingle experiments (see below).

RESULTS

Clinical and microbiological data. During the period thisstudy was performed (January 1985 to December 1988), atotal of 155 yeast isolates from women with active, sympto-matic vaginitis were identified. The large majority (around65%) of the isolates belonged, as expected, to C. albicansand T. glabrata. C. parapsilosis was isolated from 13 sub-jects with signs and symptoms characteristic of candidatevaginitis. In two cases, C. parapsilosis was isolated in mixedculture with C. albicans; these two isolates of C. parapsilo-sis were excluded from subsequent studies.The main clinical features of the 11 vaginitis patients who

harbored C. parapsilosis as single yeast species in the vaginaare shown in Table 1.

All patients had the two characteristic signs and symptomsof candidate vaginitis, i.e., the clumpy discharge with cottagecheese aspect and an intense pruritus, but most also showederythema or painful sexual intercourse or both.

All putative C. parapsilosis isolates were repeatedlytested to exclude the possession of properties typical of C.albicans, e.g., germ tube and chlamydospore formation invitro; no isolate formed these morphological elements in anymedium. Agglutination tests based on the serological schemeof Tsuchiya et al. (20) by Iatron's reagents confirmed theidentification of C. parapsilosis. Finally, an immunoglobulinM 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 ofthe vaginal isolates of C. parapsilosis were also assayed fortheir proteolytic activity in vitro; Table 1 shows that theyextensively hydrolyzed both hemoglobin and albumin. Theacid proteinase activity of C. parapsilosis was confirmed bythe analysis of albumin degradation products by sodium

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2600 DE BERNARDIS ET AL.

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

Mortality of:Isolate of Untreated mice Cy-treated mice

C. parapsilosisMSTa DITb MST D/T

SA-17 >30 0/6 >30 2/6SA-19 >30 0/6 >30 4/15SA-23 >30 1/7 >30 3/6SA-25 >30 1/7 3C 7/1d

a MST, Median survival time (days).b D/T, Dead/total (after 30 days of observation).Pp < 0.01, as evaluated by Mann-Whitney U test, comparing the value with

the MSTs of C. parapsilosis-injected, Cy-untreated mice and with the MSTsof SA-17, SA-19, or SA-23-injected, Cy-treated mice.

d P < 0.05, comparing SA-25 and SA-19 (Fisher exact method).

dodecyl sulfate-polyacrylamide gel electrophoresis (Fig. 1);the pattern of hydrolysis by the enzyme in the supernatant ofC. parapsilosis (isolate SA-23) was nearly identical to thatshown by the enzyme of a vaginopathic isolate of C. albicans(SA-40). In both cases, a major hydrolytic product of thereaction was a protein fragment of about 20 kilodaltons.

Finally, all isolates of C. parapsilosis were susceptible invitro to common antimycotics (nystatin, flucytosine, andimidazole derivatives; data not shown).The growth potential of each vaginal isolate was measured

by using the YEPD medium (3). No statistically significantdifferences in growth rate and yield after 24 h of incubationin the above medium were found among the various isolatesof 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 experimentalpathogenicity after a systemic (intravenous) challenge inmice. The animals were either unmodified or had loweredanti-infectious defenses by administration of the strongimmunodepressant drug Cy (2). None of the vaginal isolatescaused more than a marginal mortality in normal mice (Table2). However, they were appreciably pathogenic for Cy-immunodepressed mice, with one isolate (SA-25) killing 7out of 10 infected, Cy-immunodepressed mice in less than 1week. Collectively, the isolates killed 16 out of 37 infectedanimals of the Cy group, whereas they killed only 2 out of 26normal mice, the difference being statistically highly signif-icant (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 3

FIG. 1. The electrophoretic pattern of albumin hydrolysis by

secretory acid proteinase of C. parapsilosis (lane 2) and C. albicans

(lane 3). Controls are: buffered bovine serum albumin only, without

enzyme (lane 1), and C. parapsilosis enzyme activity blocked by

acid proteinase inhibitor pepstatin (lane 4). The arrows point to

molecular weight standards.

infecting celis in two target organs for invasive candidiasis,e.g., the kidney and the heart, coupled with histopathologi-cal observations. Both organs were heavily parasitized(CFU greater than 106 per organ) by each isolate during thefirst 3 to 4 days after challenge, followed (in surviving mice)by a dramatic drop (2 or more orders of magnitude) in thenumber of microbial units by day 7 (data not shown).

Experimental vaginitis infection with C. parapsilosis. Ova-riectomized, estradiol-treated rats were used in attempts toreproduce an experimental vaginal infection with C. para-psilosis. Three isolates of C. parapsilosis were used for thisstudy, and the experimental design included rats challengedintravaginally with an isolate of C. albicans from an activevaginitis (SA-40) and a strain of Saccharomyces cerevisiae(SA-264) isolated from the vagina of a healthy, fully asymp-tomatic woman, as positive and negative controls, respec-tively. Table 3 shows the viable counts of each isolatedetected in the vaginal material and the number of ratsinfected (>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

CFU/ml ± SE (103) (no. of rats infected/total)Day afterchallenge C. parapsilosis C. parapsilosis C. parapsilosis C. albicans S. cerevisiae

SA-25 SA-23 SA-19 SA-40 SA-264

1 >100 (5/5) >100 (4/4) >100 (4/4) >100 (5/5) 75 ± 24.8 (2/2)3 69.0 ± 7.5 (5/5) 82.5 ± 10.3 (4/4) 58.7 ± 23.5 (4/4) 72.5 ± 1.9 (5/5) 6.5 ± 1.48c (2/2)7 47.5 ± 21.2 (4/5) >100 (4/4) 75.0 ± 14.4 (4/4) 50.2 ± 8.6 (5/5) <1 (0/2)d14 18.7 ± 2.3 (3/5) 25.0 ± 6.1 (4/4) 41.5 ± 20.7 (4/4) 14.0 ± 9.2 (2/5) <1 (0/2)22 3.6 ± 1.4 (2/5) 1.5 ± 0.7 (2/4) 11.0 ± 6.0 (2/4) 2.0 (1/5) <1 (0/2)29 1.0 (1/5) 1.0 ± 0.25 (2/4) 2.0 ± 0.7 (2/4) 1.0 (1/5) <1 (0/2)

a A 1-,u calibrated loop was used for a semiquantitative assessment of vaginal Candida counts. The value reported is the mean plus or minus the standard errorof all independent counts for each vaginal sample obtained from each rat. Only infected rats (>103 cells per ml of vaginal fluid) were counted.

b Each isolate of C. parapsilosis and control yeasts was injected intravaginally with 107 CFU suspended in 0.1 ml of phosphate-buffered saline.C p < 0.01, as evaluated by Student's t test, comparing the value of CFU for S. cerevisiae to CFU of each isolate of C. albicans on day 3.d P < 0.01, comparing the number of rats infected over total between S. cerevisiae and the three isolates of C. parapsilosis cumulatively (Fisher exact method).

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C. PARAPSILOSIS AND VAGINITIS 2601

a

b

eb P.

4

e~~~~ls %*g~~~1ke G~~

Ja-~4

s

7s'*4

FIG. 2. Periodic acid-Schiff-stained vaginal smears from a rat infected with C. albicans (a; strain SA-40; day 9) and one infected with C.parapsilosis (b; strain SA-19; day 16). Magnifications, x400 (a) and x 100 (b). For other details, see Materials and Methods.

number in a 4-week period, after a vaginal challenge of 107cells.The infection by the nonpathogenic isolate of S. cerevisiae

was eliminated from the vagina of the infected rats within thefirst week after the intravaginal challenge, whereas thepathogenic isolate of C. albicans persisted at high, althoughprogressively descending counts, all over the experimentalperiod, with all rats being heavily infected after 1 week andone rat out of five still infected on day 29. All three isolatesof C. parapsilosis tested gave a vaginal infection in all rats,with a sustained and prolonged high number of viable cellsfor 14 days, followed by a declining period to the end of theexperiment. On day 29, 5 of 13 rats vaginally challenged withC. parapsilosis isolates were still infected. With one isolate(SA-23), the experiment of vaginal infection was repeatedafter 6 months, during which time it had undergone frequentpassages in artificial culture medium (yeast extract malt

agar). The results were comparable in terms of mean CFUand ratio of infected over total rats to those obtained in thefirst experiment. Collectively, the results of rat vaginalinfection with C. parapsilosis did not significantly differ fromthose with C. albicans nor was any statistically significantdifference noted in the vaginal CFU and proportion ofinfected rats over the total among the three different isolatesof C. parapsilosis.

Vaginal scrapings (taken on day 16) showed, in the case ofC. parapsilosis-infected rats, numerous ungerminated yeastsadhering to exfoliated cells, without any coarse indication ofinflammatory cells (Fig. 2b). C. albicans showed typicalhyphal growth in the vaginal smear (Fig. 2a).

Estradiol-untreated, ovariectomized rats eliminated bothC. albicans and C. parapsilosis from the vagina in 5 to 7days. Moreover, no yeast could be isolated from the vaginasof five ovariectomized control rats maintained for 1 month

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2602 DE BERNARDIS ET AL.

under estradiol but not challenged with any fungus (data notshown).

DISCUSSION

Although C. parapsilosis has long been known as anopportunistic yeast, its pathogenicity has always been re-garded as occasional or limited to some deep-seated infec-tions in severely debilitated hosts. Large surveys (reviewedin reference 13) of yeast isolation from diseased or healthywomen documented spotted recovery of C. parapsilosisfrom the vagina, but a causal association of this yeast withvaginitis has not, to our knowledge, been found or specifi-cally described. The experimental pathogenicity of C. para-psilosis is considered to be much lower than that of C.albicans, often being undetectable both in immunodepressedmice (2) and in phagocyte infection in vitro (15). Finally, weare unaware of any successful attempt to obtain an experi-mental vaginal infection with C. parapsilosis. Nonetheless,the clinical and experimental evidence reported in this paperseems to favor the idea of C. parapsilosis as a true humanvaginopathic agent.

First of all, C. parapsilosis was isolated, as single yeastspecies, from cases of vaginitis clinically similar to thosecaused by the classical candidal vaginitis agent C. albicans.Since C. parapsilosis can occasionally be isolated from both

healthy and diseased vaginas in conjunction with C. albicansor T. glabrata (13), it could be argued that the clinicaldisease was indeed caused by either or both of these twoclassical vaginopathic yeasts, whose isolation was masked

by that of concomitant C. parapsilosis. The isolation andidentification procedures used in this study allowed a cleardistinction of each agent. As recently emphasized by Odds etal. (14), yeast numbers in the vagina appear to correlate withobjective clinical signs and symptoms. Therefore, if thosesevere symptoms noted in our patients were due to C.albicans infection, there should have been a sufficient num-ber of this fungus to allow its isolation, at least concomi-tantly with that of C. parapsilosis. When present, C. albi-cans was easily coisolated with C. parapsilosis. If theclinical disease was due to C. albicans (or another vagino-pathic yeast) and C. parapsilosis was only a safe, althoughcopious, vaginal bystander, it would not be expected that alltested isolates of C. parapsilosis from vaginitis patientswould be appreciably pathogenic for Cy-immunodepressedmice and would give experimental vaginitis in rats. Duringthe course of the experimental infections, the yeast wassubjected to multiple reisolations and identifications frommouse organs and rat vagina; all the assays confirmed theidentification of the species C. parapsilosis.

All vaginal isolates of C. parapsilosis reported here werestrong producers of secretory acid proteinase in vitro, whencompared with the proteolytic activity expressed under thesame conditions by classically proteolytic isolates of C.albicans from vaginitis-affected patients (3). There is nowconsiderable evidence for a role of acid proteinase as avirulence factor of C. albicans (8, 12, 16). The existence of astrong correlation between proteinase secretion and clini-cally active vaginitis has also been reported (3). Clinical andenvironmental isolates of C. parapsilosis vary in their pro-teolytic potential, but, in general, C. parapsilosis is notregarded as a high producer of secretory acid proteinase (12,16). This enzyme has recently been studied by Ruchel et al.(15), who concluded, however, that C. parapsilosis wasunable to secrete acid proteinase in vivo. Although ourresults do not permit us to draw a definite conclusion on this

specific aspect, they do indicate the need for investigation ofa possible involvement of acid proteinase in the vaginitis dueto C. parapsilosis.We have also shown that C. parapsilosis is capable of

producing an experimental infection in mice and is also ableto infect the vagina of pseudoestrous rats. The course of theexperimental vaginal infection by C. parapsilosis did notsubstantially differ from that of a vaginopathic isolate of C.albicans, and estrogen administration was, as for C. albicans(7), essential for the maintenance of C. parapsilosis infec-tion. The histological picture suggested pronounced abilityof C. parapsilosis to adhere to exfoliated epithelial cells inthe absence of significant inflammatory reaction. Adherenceor ability to form germ tubes invading the cornified vaginalepithelium have been proposed as essential factors forestablishment of vaginal infection by C. albicans (12, 18). C.parapsilosis does not form germ tubes and true hyphae, andthis inability has been confirmed in our vaginal scrapings.The data shown in this paper suggest an etiological in-

volvement of C. parapsilosis in human vulvovaginal candi-diasis and should also alert the clinical microbiologist not tosimply regard C. parapsilosis as a safe component of humanvaginal mycoflora.

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2. Bistoni, F., A. Vecchiarelli, E. Cenci, G. Sbaraglia, S. Perito, andA. Cassone. 1984. A comparison of experimental pathogenicityof Candida species in cyclophosphamide-immunodepressedmice. Sabouraudia 22:409-418.

3. Cassone, A., F. De Bernardis, F. Mondello, T. Ceddia, and L.Agatensi. 1987. Evidence for a correlation between proteinasesecretion and vulvovaginal candidosis. J. Infect. Dis. 156:777-783.

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6. Hurley, R., V. C. Stanley, B. G. S. Leask, and J. De Louvois.1974. Microflora of the vagina during pregnancy, p. 155-185. InF. A. Skinner and J. G. Carr (ed.), The normal microbial flora ofman. Academic Press, Inc. (London), Ltd., London.

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