Can the diagnosis of recurrent vulvovaginal candidosis be...

Can the diagnosis of recurrent vulvovaginal candidosis beimproved by use of vaginal lavage samples and cultures on

chromogenic agar?

N. Novikova1, A. Rodrigues2 and P.-A. Mårdh1

1Department of Obstetrics and Gynecology, Lund University Hospital, Lund, Sweden2Department of Microbiology, Porto Medical School, Porto, Portugal

Objective: To investigate if introital and vaginal flushing samples inoculated on chromogenic agar could increasethe recovery rate and rapid identification of Candida and non-albicans species, as compared to culture of posteriorvaginal fornix samples on Sabouraud agar and speciation of isolates by biochemical tests.Methods: Samples from the introitus and the posterior vaginal fornix and vaginal lavage samples were collectedfrom 91 women with a history suggestive of recurrent vulvovaginal candidosis (RVVC), and with a suspected newattack of the condition. The specimens were cultured on Sabouraud and CHROMagarâ. Speciation of yeastisolates was made on the chromogenic agar by API 32C® kits and by an atomized system (Vitekâ).Results: Forty-six (51%) women were positive for Candida from one or more of the samples. The introital cultureswere positive in 43 (47%) women, both on Sabouraud and chromogenic agar. From the posterior vaginal fomix,42 (46%) women were positive on the Sabouraud and 43 (47%) on chromogenic agar cultures, while the vaginallavage cultures yielded Candida on those two media in 40 (44%) and 4l (45%) cases, respectively. Candida albicanswas the most frequent species recovered, from 40 (87%) cases, followed by C. krusei in 4 (9%), C. glabrata in2 (4%), and C. parapsilosis in one case. There was only one woman who had a mixed yeast infection, by C. albicansand C. krusei. There was only one discrepancy in the speciation as demonstrated by mean of chromogenic agar andAPI 32C kit.Conclusions: Neither cultures of introital nor of vaginal lavage samples increases the detection rate of Candida inRVVC cases as compared to cultures of posterior vaginal fornix samples. Use of chromogenic agar is a convenientand reliable means to detect colonization by Candida and differentiate between C. albicans and non-albicansspecies.

Key words: VULVOVAGINAL CANDIDOSIS; CANDIDA ALBICANS; NON-ALBICANS SPECIES; VAGINAL LAVAGE;

CHROMOGENIC AGAR

The most common means to diagnose vulvo-vaginal Candida infections is to study a wet smearof vaginal secretion that has been treated bypotassium hydroxide (KOH) for Candida morpho-types, and/or to culture samples collected with acotton-tipped swab from the posterior vaginal

fornix. The specimens can either be sent to alaboratory for culture and speciation of Candidaisolates or plated on Sabouraud agar directly at theclinic1–3.

Although the vast majority of strains isolatedfrom the genital tract in recurrent vulvovaginal

Infect Dis Obstet Gynecol 2002;10:89–92

Correspondence to: Per-Anders Mårdh, MD, PhD, Department of Obstetrics and Gynecology, University Hospital, SE-221 85Lund, Sweden. E-mail: [email protected]

Clinical study 89

candidosis (RVVC) cases belongs to C. albicans4, itmay be essential to identify instances of coloniza-tion with non-albicans species. Non-albicans strainshave a natural greater resistance to the antifungaldrugs most commonly used to treat vulvovaginalcandidosis (VVC), i.e. various azole drugs5,6. Bythe use of chromogenic agar7,8 it is possible todirectly identify a number of non-albicans species ofCandida by mere inspection of the plates, i.e.C. albicans, C. tropicalis, and C. krusei, according tothe package insert7. Koehler and co-workers9

reported, however, that as many as eight Candidaspecies could be identified on chromogenic agar.The species identities on chromogenic agar corre-spond well to those obtained by fermentation andassimilation tests10. Apart from use of samplescollected by cotton-tipped swabs, analyses ofvaginal flushing samples have been proved ofvalue in the diagnosis of genital pathogens11.

A large proportion of women suspected on thebasis of history and clinical examination provenegative for Candida on culture of vaginal posteriorfornix samples. One hypothesis is that analysis ofvaginal lavage samples would increase the chanceof detecting only a low number of Candida organ-isms in the vagina compared to the most com-monly applied sampling technique, which involvesjust touching the vaginal mucosa with a swab. Thislatter procedure may not leave enough time foradsorption of yeast organisms by the swab. Even ifrubbing the mucosa, many organisms may remainattached to the swab material after inoculation ofculture media, resulting in a negative culture.

The present study explored the possibility ofincreasing the detection rate of Candida in womenwith a history suggestive of RVVC and who didattend with symptoms and signs that were consis-tent with a new attack of the condition. Analysesof introital swab and vaginal lavage samples weremade. Furthermore, the value of chromogenicagar for culture of such specimens was investigated.

SUBJECTS AND METHODS

The 91 women recruited to the study had allattended the outpatient department of the Mater-nity Hospital 3, Kiev, because of genital symptomsand a history suggestive of RVVC. They all hadthe diagnosis of a genital Candida infection by a

physician at least four times during the previousyear on the basis of history, clinical examinationand at least one positive microscopic test forCandida morphotypes, in methylene-blue-stainedsmears prepared from posterior vaginal fornixsamples. The patients presented with symptoms,such as vulvovaginal pruritis, irritation, unpleasantodor and dyspareunia, and/or with dysuria andburning at micturition. They had signs such as oneor more of edema, erythema, fissures and caseousdischarge.

The vaginal introitus was rubbed with a sterile,cotton-tipped swab moistened with physiologicalsaline, which was plated on Sabouraud dextroseagar (Beckton Dickinson®, Baltimore, USA)3 andchromogenic agar (CHROMagar®, Paris)9, likeswab samples from the posterior vaginal fornix.The order of the inoculation on the two media waschanged for each consecutive case studied. There-after, vaginal lavage fluid was obtained by flushingthe vagina with 3 ml of sterile physiological salineand by regurgitating the fluid with the aid ofa sterile syringe. Of the mixture, 0.1 ml was inocu-lated on each of a Sabouraud and chromogenicagar plate.

Isolated strains of Candida were frozen at -70°Cin fetal calf serum for later speciation, which wasdone with the aid of API 32C® test kits and anautomized identification system, Vitek® (Bio-Mérieux, Marcy-l’Etoile, France). Chromogenicagar plates were read according to the criteriadescribed by Pfaller and co-workers10.

The study was approved by the ethic committeeof the Maternity Hospital. Written consents wereobtained from the patients.

RESULTS

Table 1 shows the results of cultures made fromswab samples from the introitus and posteriorvaginal fornix and vaginal lavage samples, inocu-lated on Sabouraud and chromogenic agar. Therecovery rate from the introitus was the same asthat from the posterior vaginal fornix. The vaginallavage samples missed two Candida-positive casesotherwise diagnosed by the swab samples.

C. albicans was the most frequent isolate,recovered in 40 (87%) cases, followed by C. kruseidetected in 4 (9%) cases, C. glabrata in two and

Use of vaginal lavage samples and cultures on chromogenic agar Novikova et al.

90 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY

C. parapsilosis in another case. There was only onecase of mixed Candida infection, i.e. by C. albicansand C. krusei. The isolation rate on chromogenicagar was almost the same as on Sabouraud agar: thedifference was 1.4% in favor of chromogenic agar.

A discrepancy in the result of speciation bymeans of chromogenic agar versus API 32C kitswas only found in two of the 46 Candida-positivecases. In these two patients, C. albicans was identi-fied by the API 32C kit, while C. glabrata was indi-cated to grow on the chromogenic agar. In anothercase, a mixed infection by C. albicans and C. kruseiwas found on the chromogenic agar, while onlygrowth of C. albicans was identified by the APIC32 and Vitek identification methods.

DISCUSSION

The diagnosis of genital Candida infections, incases with a history of RVVC who attend withsymptoms and signs suggestive of a new attack, failsmore often than generally realized – even if bothmicroscopic and culture studies are made. Thusthe presence of Candida cannot be confirmed.Specialists in gynecology often misdiagnose thecondition if not based on patient-close or centrallaboratory test results. In a previous study byLedger12, the presence of Candida in the genitaltract could only be confirmed in half of cases giventhe diagnoses of VVC or RVVC based on history,clinical examination and patient-close microscopyof vaginal secretion. In our study, half the women

also proved to be culture-negative for Candida inspite of a ‘typical’ RVVC case history and findingsat the clinical examination. In another study,Candida was identified by vaginal cultures in only28% of 554 women with symptoms consistentwith ‘candidal vaginitis’13. Thus, there are reasonsto believe that a current genital Candida infection iscommonly overdiagnosed in women diagnosedwith RVVC. In fact, those women may sufferfrom other vaginal flora changes, such as those seenin bacterial vaginosis (BV)14. Women may switchbetween BV and VVC15.

At least 10–15% of all women of reproductiveage are colonized in the vagina by Candida, i.e. arecarriers without any occurrence of either VVC orRVVC4. This colonization rate adds to the diffi-culty in diagnosing RVVC exclusively on the basisof genital yeast cultures, i.e. to differentiate carriersfrom cases with a true vaginal mucosal and/orintroital invasion by yeast.

In our study, the sensitivity of chromogenic agarcompared to Sabouraud dextrose agar for detec-tion of Candida did not differ (p = 1). This is inaccordance with the findings by Beighton andco-workers8. Nor did we find the use of vaginallavage samples to positively influence the recoveryrate of Candida.

The use of chromogenic agar has one disadvan-tage compared to Sabouraud agar, namely its ratherhigh cost. On the other hand, chromogenic agarcan identify most of the species occurring in thefemale genital tract. Consequently other expensiveidentification tests may be avoided. The use of anincubation temperature below 30°C does notallow speciation of Candida strains9,16 on chromo-genic agar. Thus, chromogenic agar cannot beused at doctors’ offices for speciation if no incuba-tor is available.

C. albicans, C. krusei and C. tropicalis have beenindicated to represent more than 99% of all yeastisolates from the human vagina7. In an Italian studyof symptomatic women with a positive Candidaculture, C. glabrata was the next most commonspecies found, identified in 1207 of 3351 cases.C. krusei and C. tropicalis were found in 404 and290 cases, respectively17. In our study, C. glabratawas found in only two cases.

The possibility of rapidly identifying the speciesof Candida by which a women is colonized


INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 91

Number of positive Candidacultures

Sample typeSabouraud agar

(n = 91)Chromogenic agar

(n = 91) p-value

Introital swabPosterior vaginal

fornix swabVaginal lavage

samples

43 (47%)

42 (46%)

40 (44%)

43 (47%)

43 (47%)

41 (45%)

1

1

1

Table 1 Results of swab sample cultures from theintroitus and the posterior vaginal fornix and of vaginallavage samples from 91 patients with a history andcurrent symptoms of recurrent vulvovaginal candidosisgrown on Sabouraud and chromogenic agar

increases the possibility of obtaining successfulcure. Women infected with non-albicans speciesmay not respond to azole drugs, to which such

species are naturally resistant or have a low sensiti-vity18. In that respect chromogenic agar serves adiagnostic purpose in RVVC cases.

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15. Mårdh P-A, Rodrigues A, Pina-Vaz C. Can in vitroobservation explain interactions in vivo between alactobacilli-dominated vaginal flora and vulvo-vaginal candidosis? Ital J Obstet Gynecol 2001;13:89–93

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18. Mårdh P-A, Rodrigues A, Genc M, et al. Facts andmyths on recurrent vulvovaginal candidosis – areview on epidemiology, clinical manifestations,diagnosis, pathogenesis and therapy. Int J STDAIDS, in press

RECEIVED 10/10/01; ACCEPTED 01/04/02


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