CLINICAL STUDY

Mycotic VulvoVaginitis: Epidemiology, Pathogenesisand Profile of Antifungal Agents

AI-Zahraa Karam EI-Din1 Ph. D, Fawzia Habib2 M.D,Najwa Abd-Allah! Ph. D, Omayma Khorshid3 M.D

Departments ofBiology-Faculty of Science', Obstetrics and Gynecology2, and of Pharmacologu',College ofMedicine, Taibah University, Al Madinah Al Munawarah, Kingdom ofSaudi Arabia

AbstractObjectiveVulvoVaginitis is the most common gynecological problem affecting millions of womenworld wide. At some point in their life time, nearly 75% of all women experience an attack ofCandida Vulvovaginitis. Little is known about the prevalence of different causes ofVulvovaginitis and risk factors for this entity in Saudi Arabia. This survey was conducted tostudy the etiologic agents associated with mycotic Vulvovaginitis and review somepredisposing factors correlated with this type of infection in Al -Madina AI- Munawarah,Saudi Arabia.MethodsHigh vaginal swabs (HVS) specimens were collected from 1000 patients attendinggynecological out patients clinics of two general hospitals in AI-Madinah AI-Munawarah .Specimens were cultured on specific medium for yeasts and identification of the positiveisolates was carried out using "API" kit. Antifungal sensitivity pattern of the isolates wastested out using "Candifast" kit. Proteolytic enzyme activity of the isolates was also detected.ResultsThree hundred and forty nine positive cases of yeast infection out of 1000 diagnosed casesrepresenting 34.9% were recorded in this study. These positive cases were classified on thebasis of the risk factor as; diabetic (28.9%); pregnant, (32.1%); pregnant and diabetic (7.5%);Menopause (5.5%); oral contraceptive users (10.6%); post hysterectomy (1.4%) and noobserved factor in 14% of the cases. Twenty one different species belonging to 7 genera wererecovered in this study. The genus Candida was the most common (93.4%) and included 14different species. Candida albicans was the highest in prevalence (51.3%). The non-Candidaspecies were Saccharomyces cervisiae (2.9%); Rhodotorula rubra (1.1%); R. minuta (0.9%)Debaryomyces hansenii (0.6%); Trichosporon mucoides (0.6%); Cryptococcus neoformans (0.3%) andPichia ohmeri (0.3%). Most of the Candida species were sensitive towards nystatin,amphoterian B and fluconazole and resistant to other azole drugs. Saccharomyces cervisiae andDebaryomyces hansenii were sensitive to almost all the tested antifungal drugs.The rest of thespecies were variable in their pattern. Proteolytic activity of C. albicans reached its maximumvalue after 72 hours. Neutral proteases was the highest at pH 6.5 followed by alkalineproteases at pH 8.2 and the least amount was acid protease at pH 3.5.ConclusionThe results of this survey which is the first study done in this region threw some light on theprevalence, etiology, sensitivity profile of the etiologic agents and the risk factors ofVulvovaginitis in AI-Madina AI-Munawarah Saudi Arabia. Awareness towards the increaseincidence of Vulvovaginits needs more attention to be paid for fungal infection andantifungal sensitivity.

Key words: Fungal infection, Vulvovaginitis, epidemiology, antifungal, enzyme activity

Journal of Taibah University Medical Sciences 2009; 4(2):123 - 136

123

Correspondence to:Prof. AI-Zahraa Karam EI-DinDepartment of BiologyFaculty of Sciences, Taibah University~ 344 Al Madinah Al Munawarah, Saudi Arabiatil +966 4 8460008 Ext. 3197~ zahraakaram@yahoo.com

Introduction

VUlvo~agi~tis i~ a gene~al term used todescribe infection or Inflammation of

the Vulva and vagina. Inflammation ofvagina due to infectious agents is verycommon, both as an overgrowth of normalor common colonizers, or as a frankinfection. The most common causes ofVulvovaginitis are yeast, bacteria, protozoa,viruses and parasites.'. Egan- had reportedthat Vulvovaginitis is the most frequentlygynecological diagnosis encountered byphysicians who provide primary care towomen.There are number of vaginal candidiasis riskfactors have been identified. The increasedsusceptibility of women to develop vaginalcandidiasis is known to be correlated withthe high levels of reproductive hormonesand increase in the glycogen content in thevaginal environment. The incidence in theoverall population is found to be increasedsignificantly during pregnancy; in thoseusing high dose estrogen contraceptive pills,after an antibacterial treatment regime, andwas also more frequently reported indiabetic women 3.

Vulvovaginitis caused by Candida speciesrepresents 20-30% of the overall infection. Itis a common cause of morbidity, in women.Some women have infrequent occasionalepisodes of varying severity that respond toantifungal treatment, while others sufferfrom recurrent, often chronic candidiasis 4.

Candida albicans was the most common typeof yeast infection (91.8%). Odds" had foundthat C. glabrata to be common yeast otherthan C. albicans to be isolated. Certain yeastspecies are commonly associated withantifungal resistance. Resistance toamphotericin B has been demonstrated inCandida lusitaniae 6, other Candida spp. Such

124

as, C.guilliermondii C. inconspicua, C. kefyr, C.krusei, C. rugosa', and Trichosporon Sp.8.Additionally, azole (e.g fluconazole)resistance has been demonstratedrepeatedly in C.glabrata and C.krusei 9.

The invasion of host tissues by microbialcells possesses constitutive or induciblehydrolytic enzymes which destroy ordegrade constituents of cell membranesleading to membrane dysfunction and orphysical disruption. Since host cellmembranes are made up from lipids andproteins, it is obvious that these biochemicalprocesses include the largest part of enzymeattack'",The production of hydrolytic enzymes,especially secreted aspartic proteinases, askey virulence to determinants has beencomprehensively studied 11. Proteinaseproduction by C. albicans is associated withpathogenicityt-.P Proteinase enzyme islocated as a mannoprotein, functions as aligand for attachment to host cells and wasidentified as virulence determinantl-.

Materials and Methods

Selection ofSubiectsOne thousand patients attendinggynecological outpatients clinics of twogeneral hospitals (Ohod and the Maternityhospitals in Al-Madinah AI-Munawarah),complaining of vaginaldischarge and itching, over a period of 8months ( October ,2007 to May ,2008 ) weretested for the presence of Vulvovaginalfungal infection.

Collection of the clinical samplesThis study protocol was approved by theDeanship of Scientific Research of TaibahUniversity, Al-Madinah AI-Munawarah,Saudi Arabia.

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High vaginal swabs (HVS) specimens weretaken from each patients, Each HVS wascultured on a Sabouraud dextrose agar(SDA) plate (supplemented with 500mg/liter of chloramphenicol .The plateswere incubated at 37 C for 5 to 7 days beforediscarding as negative. Only patients whoyielded heavy growth of yeasts in culturewere selected for the study. Other Bacterial,Parasitic infections were not tested in thisstudy.The following tests were carried out for eachculture",

• Purification of the culture.• Yeast morphology on corn & rice-meal

tween 80 agar media• Germ tube test

API 20 C AUX for identification of the yeastisolateIdentification of the isolates was carried outusing API 20 CUX kit (Bio Merieux, SAMarcy- L' Etoile, France)16.

The pathogenic potentialities of the isolateswere tested for• Production of proteolytic enzymes.• Production of lipolytic enzymes• And Blood Haemolysis: 17

Sensitivity of the isolated strains to someantifungal agents using "Candifast ES TwinKit".Antifungal sensitivity testing of pathogenicCandida species were carried out using"CANDIFAST ES Twin kit" (ELITECH,France SAS)18. It provides a sensitivityprofile against 7 antifungal agents(amphotericin B, nystatin, flucytosine,econazole, ketoconazole, miconazole andfluconazole).

Quantitative determination of proteolyticenzymes activity19,2o.Enzyme assay0.1 M sodium citrate buffer containing 2 gmBSA/litre and the pH adjusted at 3.5, 6.5and 8.5.• Culture supernatant, assay medium were

kept on ice.• Reaction starts by adding 0.1 ml

supernatant + 0.9 ml assay medium.

125

• Rapidly shaking at 37°C for 10 min.• Add equal volume of 5% trichloroacetic

acid.• After and additional 10 min, the reaction

mixture was centrifuged.• The supernatant was decanted, and the

absorbance at 280 nm was read againstblank containing distilled water.

Enzyme units are expressed as the amountof tyrosine in micromoles released perminute per milliliter of culture supernatant(108cells of Candida species).At the same time of enzyme assay, thegrowth rate was determined (absorbance at660nm).

Results

Out of 1000 female patients (ranged from 16to 65 years) attending the out patientsgynecological clinics with Vulvovaginalitching and discharge, Candida species andother non- Candida were isolated from 349out of the 1000 tested case. Positive casesrepresented 34.9% (Table 1). Positive caseswere classified according to the suspectedpredisposing factor for fungal infection: 101diabetic (28.9%), 112 pregnant (32.1%), 26pregnant and diabetic (7.5%), 19 Menopause(5.5%), 37 oral contraceptive (10.6%), 5 post­hysterectomy (1.4%) and 49 with noobserved factors (14%) as shown in Table 2.Patients were classified into 5 groupsaccording to their age. The maximumpositive cases were recorded in the secondgroup (age range 26-35) followed by the firstgroup (age range 15-25) shown in Table 3.Data of Table 6 revealed that 21 yeastspecies were identified belonging to 7genera; Candida, Cryptococcus, Debaryomyces,Pichia, Rhodotorula, Saccharomyces andtrichospron.The prevalence of non-Candida species was6.6% (23/349) (Table 6). The non-Candidaisolates included; Saccharomyces cervisiae 10(2.9%); Rhodotorula rubra 4 (1.1%)Rhodotonula minuta 3 (0.9%), 2 case of each:Debaryomyces hansenii, and Trichosporonmucoides each representing 0.6% and onecase of each of Cryptococcus neoformans andpichia ohmeri (representing 0.3% in Table 6).

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In this study, the genus Candida recordedthe highest prevalence 326 caes representing93.4% of the total positive. Candida albicanswas the most prevalent species recovered inthis study. It was isolated from 179 caseconstituting 51.3% of the positive cases.Candida glabrata came next in rank; it wasisolated from 78 case constituting 22.3% ofthe positive cases. Candida lusitaniae, C.tropicalis C. pseudotrepicalis and C. krusei wereof low occurrence representing 5.4%, 4.3%,3.4% and 2.6 respectively. The rest of theisolated Candida spp. were of rareoccurrence (Table 6).Candida albicans recorded the highestisolation rate in all studied groups. It wasrecovered as follows: 54 cases out of 101 in

Table 1: Prevalence rate of the test sample

diabetic group, 62 out of 112 in the pregnantgroup, 14 out of 26 of the diabetic - pregnantgroup; 9 out 19 in Menopause group, 21 outof 37 in oral contraceptive group, 2 out of 5in post hysterectomy group and 17 out of 49in the unobserved factor group (Table 5).The tested species proves their ability tohydrolyze casein and fat which indictedtheir potentiality and their implication in thepathogenesis process. Also almost all(except Saccharomyces and Debaryomyces spp.)isolates were positive in the bloodhaemolysis test which proves their invasiveand disseminated form.

Samples

Patients

Total rested samples

1000

Positive cases

349

Percentage 0/0

34.9

Table 2: Distribution of yeast positive cases according to suspected predisposing factors

Predisposing Factor

Diabetic

Pregnant

Pregnant and Diabetic

Menopause

Oral contraceptive

Post-hysterectomy

No observed factor

No. of Cases

101

112

26

19

37

5

63

Percentage0/0

28.9

32.1

7.5

5.5

10.6

1.4

14

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Mycotic Vulva Vaginitis

Table 3: Distribution of yeast positive cases according to patient's age

Groups of patients

15-25

26-35

36-45

46-55

56-65

No. of cases

118

135

64

28

4

Percentage 0/0

33.8

38.7

18.3

8.1

1.4

Total

Table 4: Frequency and percentage of isolated genera and species of yeasts from test sample

14 326179 51.378 22.319 5.415 4.312 3.49 2.64 1.13 0.92 0.61 0.31 0.31 0.31 0.31 0.3

110 2.8

24 1.13 0.9

11 0.6

1 0.62

11 0.3

11 0.3

General and species ofyeast

Candida(1) C. albicans(2) C. glabrata(3) C. lusitaniae(4) C. tropicalis(5) C. pseudotropicalis(6) C. Krusei(7) C. famata(8) C. guilliermondii(9) C. parapsilosis(10) C. ciferii(11) C. dubliniensis(12) C. pelliculosa(13) C. rugosa(14) C. zylanoides

Saccaromyces(15) S. cervisiaeRhodutorula(16) R. ruburu(17) R. minutaDebaryomyces(18) D. hanseniiTrichosporon(19) T. mucoidesCryptococcus(20) C. neoformansPichia(21) P. ohmeri

No. of species No. of cases

Total

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Al-Zahraa Karam EI-Din et al

Table 5 : Distribution of the isolated yeast species among the different groups of patients

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Table 6: Percentage of Sensitivity and Resistance of the isolated pathogenic yeast towards the common antifungals

Organism

C. albicans

C. glabrata

C. tropicalis

C. lusitaniaeC. pseudotropicalis

C. Krusei

Ci famaiaC. guilliermondii

C. Parapsilosis

C.Ciferii

C. dubiliensis

C. pelliculosa

C. rugosa

C. ZylanoidesSaccharomycescervisiaeRhodotorularubraR. minutaDebaryomyceshanseniiTrichosporonmucoidesPichia ohmariCryptococcusneo ormaus

11 ••_ ••••••••179 67.8 32.2 82 18 78 22 78.5 21.5 70.6 29.4 49.7 50.3 53.7 46.3

78 64.1 35.4 84.6 15.4 62.8 37.2 65.4 34.6 52.6 47.4 30.8 69.2 37.2 62.8

19 84.2 15.8 89.5 10.5 100 a 89.5 10.5 73.7 26.3 47.4 52.6 31.6 68.4

15 66.7 33.3 66.7 33.3 80 20 66.7 33.3 80 20 53.3 46.7 46.7 53.3

12 75 25 91.7 8.3 50 50 50 50 58.3 41.7 41.7 58.3 83.3 16.7

12 33.3 66.7 83.3 16.7 25 75 33.3 66.7 33.3 66.7 16.7 83.3 58.3 41.7

4 25 75 50 50 50 50 25 75 25 75 25 75 25 75

3 a 100 66.7 33.3 66.7 33.3 33.3 66.7 33.3 66.7 33.3 66.7 33.3 66.7

2 a 100 100 a a 100 a 100 a 100 a 100 100 a1 100 a 100 a 100 a 10 a 100 a 100 a 100 a1 100 a 100 a 100 a a 100 a 100 a 100 a 100

1 100 a 100 a 100 a 100 a 100 a 100 a a 100

1 100 a 100 a 100 a 100 a 100 a 100 a 100 a100 a 100 a 100 a a 100 a 100 a 100 a 100

10 80 20 90 10 90 10 80 20 80 20 70 30 70 30

4 75 25 25 75 50 50 50 50 50 50 75 25 75 25

3 66.7 33.3 66.7 33.3 66.7 33.3 66.7 33.3 66.7 33.3 66.7 33.3 66.7 33.32 100 a 100 a 100 a 100 a 100 a 50 50 100 a

2 50 50 50 50 50 50 25 75 50 50 a 100 75 25

1 100 a 100 a 100 a a 100 a 100 a 100 100 a1 100 a 100 a 100 a 100 a 100 a a 100 100 a

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Anti fungal sensitivity testing of theisolated speciesSensitivity testing of the isolated species wascarried out using candi fast kit. It provides asensitivity profile against 7 anti fungalagents (Ampoteriin B, Nystatin, 5­flurocytosine, Econazole, Ketonazole,Miconazole and Fluconazole).The test showed that most Candida isolateswere sensitive to Nystatin. Most of theisolates of Candida albicans (80%), the mostcommon species in this investigation wasalso sensitive to Nystatin, Amphotericin Band some Azole compounds especiallyFluconzole.Almost all the isolates of Saccharomycescervisiae and the two isolates of Debaryomyceshansenii were sensitive to all antifungaldrugs (Table 6).Data in Table 6 showed the variableresponse of the other isolated speciestowards the tested antifungal drugs used inthis study.

Production of extracellular proteinases byCandida albicansIn this study the determination of the acid,neutral and alkaline proteases activities wascarried out at the following pH values (3.5,6.5 and 8.5). The growth rate and enzymeactivity were determined every 12 hours fora total incubation time of 96 hours. Table 7and Figure 1 showed that the proteolyticactivity reach its maximum values after 72hours. This result was relevant to maximumgrowth rate of the tested strain, C. albicans.The proteolytic activity of C. albicans thatmeasured at pH 3.5 reached its maximumvalue of 685 umol / ml. Also the proteolyticactivity of C. albicans reach to the value of643 umol / ml at pH 8.2. From the aboveresults, it was found that the neutralproteases (pH 6.5) were released inappreciable amounts in C. ablicans followedby alkaline proteases (pH 8.2) while the leastamount recorded was of acid proteases (pH3.5) Table 8.

Protease enzyme productivity [umol] tyrosinefml

Inc. PeriodpH value

Table 7: Protease enzyme productivity at different incubation periods by C. albicans atpH value 3.5, 6.5 and 8.2

Ea:...._ ........_

pH 3.5

pH 6.5

pH 8.2

335

455

575

395

543

758

383

528

478

427

595

557

461

608

580

493

685

643

345

486

413

409

592

535

BOO~

:~ JOO'-.J

600:Jn0 5000.(,.' 400ctI: JOO.;J

.200-,/I

r;...,100~

0....0-::a..

~=- ~;i. h ~.

IJ" .~-,~

l1:h 14h 3bll 48h tOh 7211 84h 96h

inc uba Lion period (hourt

Figure1: Protease enzyme productivity at different incubation periods by C. albicans at pH values 3.5, 6.5and 8.2

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Mycotic Vulva Vaginitis

Table 8: Protease standard values using different tyrosine concentrations at pH values3.5, 6.5 and 8.2pH valueTyrosine conc. H3.5

O.D.

H6.5 H8.2

diabetic (7.5%), menopause (3.4%), post­hysterectomy 1.4% and (14.1%) with noobserved factors.It is well documented that pregnancy anddiabetes mellitus increases the rate ofvaginal colonization and infection withCandida24,22,25 . Potential risk factors forVulvovaginal candidiasis have beenidentified, including women of childbearing age, post menopausal women whohave underlying risk factors such ashormone replacement therapy orimmunosuppression caused by medicationsor diseases , using high estrogen containingcombined oral contraceptive pills, vaginaldouching, some sexual behaviors,contraception devices, (diaphragm,intrauterine device etc.) and antibiotics26,27,28,25 .Concerning the etiology in thepresent study, the genus Candida(represented with 14 species) was the mostcommon etiologic agent documented byculture, microscopy and the results of theAPI Kit. This genus constituted 93.4% of thetotal positive cases, while the prevalence ofthe non-Candida genera was 6.6% of thetotal positive cases. This result is similar tothat of Saporiti et a[29. Candida albicans wasby far the most common pathogen detectedin this study (51.3% of the positive cases).Vasquez et al.3D reported that, the majoropportunistic pathogen has been Candidaalbicans. The proportion of genital C. albicansin symptomatic women ranges fromapproximately 90% in Australian samples31to approximately 65% in Belgium", Turkey'"and Saudi Arabia's, The non-albicansCandida species isolated during this studyrepresented 42.1% including C. glabrata(22.3%), C. tropiclis (5.4%), C. lusitaniae

0.032

0.056

0.070

0.087

0.118

0.178

Discussion

Over the last several decades, medicaladvances have become available that makehuman life more safe. Factors such astransplant surgery and concomitantimmunosuppressive therapies, anti-cancertherapies, medical devices that traverse theprotective skin barrier (e.g. central venouslines, catheters, etc.), broad spectrumantibacterial therapies, corticosteroidtherapies, certain disease states (e.g.malignancy, human immunodeficiencyvirus infection, etc.), and others havecontributed to increased numbers ofimmunocompromised individuals. Theseimmune-deficient individuals are at higherrisk for yeast infections and the spectrum ofoffending species is ever increasing. Speciesthat were considered to be saprophytic arebecoming opportunists causing humandiseases?'.In the present study, 349 (34.9%) positivecases were recovered out of 1000 testedpatients.This result is in agreement with that ofMargriti-' , who reported that mycoticVulvo vaginitis is the most common clinicalmanifestation of fungal infections causinghuman mycoses; the incidence occurs in10% of women, while, during pregnancy theincidence achieves 30% of cases. Candidaspecies was the most common pathogen in35.5% of symptomatic women and 15% ofasymptomatic controls-" . Data of this studyrevealed that yeast vaginal infection amongthe classified groups of patients was;pregnant (32.1%) diabetic (28.9%), oralcontraceptive users (10.6%), pregnant and

131

0.033

0.065

0.079

0.086

0.108

0.166

0.026

0.036

0.062

0.087

0.098

0.178

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AI-Zahraa Karam EI-Din et al

(4.3%), C. pseudotropiclis (3.4%), C. Krusei(2.6%), C. famata (1.1%), C. guilliermondii(0.9%), C. parapsilosis (0.6%) and thefollowing species C. ciferii, C. dubliniensis, C.pelliculosa, C. rugosa and C. zylanoides were ofrare occurrence and each represented 0.3%of the total positive cases. This studyconfirmed that Candida glabrata currentlyranks second as causative agent of vaginalcandidal infection and are common inimmunocompromised persons or those withdiabetes mellitus as reported by Geiger etal28 and Paul et al14 Two specialized clinicshad reported rates of 10% to 20% of non­albicans Vulvovaginal candidiasis, andCandida glabrata had consistently been thedominant species 27, 35. It is worthmentioning that there is a variation in theyield of the species of Candida, where 14species were recovered belonging to thisgenus, some of them may be reported ascausal agent of Vulvovaginitis rarely or forthe first time. Saccharomyces cervisive (2.9%);Rhodotorula rubra and minuta (2%); each ofDebaryomyces hansenii and Trichosporonmucoides represented (0.6%) and 0.3% foreach of Cryptococcus neoformans and Pichiaohmeri. This list was also reported as causalagents of vaginitis by many authors2,36,37

while other species were recorded as causalagents of other clinical cases 38,39 • It wassurprising to report one case caused byCryptococcus neoformans. Cryptococcalinfection is opportunistic and occurs mostcommonly in immunocompromisedpatients. Cryptococcal infection usuallypresents as meningoencephalitis orpulmonary infection. Skin, bone and genitalinfections are very rare 41. Cryptococcalinfection of the vagina was reported beforeby Chen et a1.42 and Ranganathan et a1.43•

The case recorded in the present study isone of the rare cases to report cryptococcalvaginitis. Drug resistance is a major problemin treating yeast infectionsw, At present,yeast infections are usually treated as ageneral fungal infection and agents such asthe polyene, amphotericin B or the newerazole drugs, which are intended to control abroad array of fungi, are usedv, The Candidaspecies collected during this study with highfrequency rate (C.albicans, 179 strain and

132

C.glabrata, 78 strain),gave meaningful datawith the sensitivity test. The overall ofC.allbicans isolates showed resistance to "AB(32.2%); NY (18%); FCT (22%); ECZ (21.5%);KTZ (29.4%); MCZ (50.3%) and FCZ (46.3%).While C. glabrata showed the followingresistance pattern "AB (35.4%); NY (15.4%);FCT (37.2%); ECZ (34.6%); KTZ (47.4%);MCZ (69.2%) and FCZ (62.8%). Othercollected species with low or rare frequencygave meaningless data with the antifungalsensitivity test. Generally the incidence ofamphotericin B- resistant candida species inour study was 35.5%; Nystatin 17.5%Fluctosine 27.9%; Econazole, 29.4%;ketokonazalo 36.5%; Miconazole, 57.4% andFluconazole, 51.2%. This incidence ofantifungal resistant candida species in ourresults is higher than that reported by otherstudies 45,46,47,48. Sanglard and Odds 50

concluded that Candida albicans and relatedspecies pathogenic to man become moreresistant to antifungal agents, in particulartriazole compounds, by expression of effuxpumps that reduce drug accumulation,alteration of membrane sterol compositionresistance towards most of the testedantifungal drugs. In the case ofVulvovaginal candidiasis, an analysis ofclinical isolates indicates that resistance isdue to not only to resistant strains of C.albicans but also to an increasing number ofnon-albicans Candida strains. Various Candidaspecies appear to develop resistance to thecommonly used drugs at frequencies muchhigher than that for C. albicans 50. In thepresent study, the Candida species other thanalbicans and glabrata showed variablepatterns of resistance and sensitivity andmost of them were sensitive to Nystatinexcept some isolates of C. krusei and C.guilliermondii. Mashburn and Facumvsuggested that uncomplicated vaginalcandidiasis is easily treated with topicalazole antifungal medications in single orshort-term doses. This class of drugs isusually more effective than the oldernystatin class of drugs. Capoor et al.46

reported that the spectrum of candidiasishas changed with the emergence of non­candida species and acquired antifungalresistance. Other non-Candida species such

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Mycotic Vulva Vaginitis

as Saccharomyces cervisiae and Debaryomyceshansenii were sensitive to most of the testedantifungal drugs. While Rhodotorula rubra,Rh. Minuta Trichosporon mucoides, Pichiaohmeri and Cryptococcus neoformans werevariable in their resistance and sensitivitypattern towards the tested antifungal drugs.. Many yeasts and molds are known tosecrete extracellular proteases'f For Candidaspecies, some investigators have reportedthe determination of proteolytic activity'<whereas others have detected no activity's,This discrepancy may be attributed to theconditions used for eliciting and measuringprotease activity. In this study, it was foundthat the level of proteolytic enzyme was lowduring the earlier exponential growth phaseand reached its maximum value after 72hours. This clearly shows a directcorrelation between the growth rate of yeastcells and the proteolytic activities of Candidaisolates. This correlation was also reportedby56 who connected between theproteolytic production and the growth rateof yeast cells. Many workers agreed thatCandida species need 72 hours, incubationtime for the maximum proteaseproductionv-", In this study, the neutralproteases (pH 6.5) were released inappreciable amount; 685 umol/ml. in case ofC. albicans. This followed by alkalineproteases 643 umol/ml. These resultscoincide with that of Dostal et al.( 2003) whoreported that the pH value was the criticalfactor in proteolytic activity becauserelatively small pH shifts can cause changesin extracellular proteolytic activity. Also,these results were supported by Taylor etal.53 who found that the human vaginalinfections were accompanied by elevation ofpH values (neutral to alkaline) andsubsequently the protease activity reachedto the maximum levels. Odds" and Fidel etal.59 reported that Candida albicans producesa higher amount of proteases in comparisonwith other Candida species.

ConclusionThe incidence of fungal infection and vulvovaginal candidiasis is increasing rapidly, inrelation to the growing number of diabetic

133

patients and immunocompromisedindividuals in the population.Candida albicans is the most common causeof Mycotic Vulvovaginitis . Other non­albicans species such as Candida glabrata andtropicalis , which are increasing in frequencyare also involved . Topical and oralantifungal therapies are effective.Resistance of the Candida spp. towards thecommonly used antifungal drugs isincreasing.

AcknowledgementThis project was funded by the Deanship ofScientific Research of Taiba University AI­Madinah AI-Munawarah, Kingdom of SaudiArabia.Sincere thanks to all who took part in thestudy.

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