Candidiasis during pregnancy may result from isogenic ...downloads.hindawi.com/journals/idog/2001/941789.pdf · colonization during pregnancy has not been directly addressed. In the

Candidiasis during pregnancy may result from isogeniccommensal strains

Wayne Daniels1, Douglas D. Glover2, Michael Essmann1 and Bryan Larsen1

1Infectious Disease Research Laboratory, Des Moines University Osteopathic Medical Center,Des Moines, IA

2Consortium on Reproductive and Developmental Health, Robert C. Byrd Health Sciences Center of theWest Virginia University, Morgantown, WV

Objective: Our laboratory previously demonstrated that asymptomatic vaginal colonization during pregnancy is afactor predisposing patients to subsequentsymptomatic vulvovaginal candidiasis. It is unknown whether symptomsresult from strain replacement or a change in host relationship to the original colonizing strain. This study wasundertaken to determine whether Candida albicans isolates from asymptomatic women could be responsible forsubsequent symptomatic vaginitis.Methods: We retained isolates of C. albicans from women followed longitudinally through pregnancy, and identi-fied six pairs of cultures from women who were colonized without symptoms and who later became symptomatic(average time 14 weeks). We used a random amplification of polymorphic DNA (RAPD) analysis to determinewhether isolates from our study patients were genetically similar or dissimilar.Results: Analysis of these pairs of yeast strains by RAPD revealed that five of the six women had symptomsapparently due to the same yeast strain that was found initially as a commensal strain. To increase the power ofthese observations, we also performed RAPD analysis on six randomly selected yeast strains from other women inthis study who had not become symptomatic to determine whether any of these unrelated strains matched strainsfrom those women who became symptomatic.Conclusion: Symptomatic yeast vaginitis is usually due to strains of C. albicans already carried in the lower genitaltract, underscoring the need to understand regulation of growth and virulence of the organism in vivo.

Key words: VAGINITIS, MOLECULAR EPIDEMIOLOGY, PREGNANCY

Candida albicans is recognized both as normal florain the female genital tract and as the causative agentof vulvovaginal candidiasis. What is not clear iswhether the vaginal commensal organism increasesits virulence to cause candidiasis, the host becomesmore permissive toward proliferation of theorganism resulting in symptoms, or whetheranother, possibly more virulent, strain supplantsthe normal flora organism. The availability of

methods of DNA analysis that distinguish betweendifferent strains makes it possible to obtain insightinto this question.

Soll has recently reviewed the myriad geno-typing techniques available to raise epidemiologicinvestigations of C. albicans from the phenotypicto the genotypic level1. These techniques haveallowed the tracking of transmission of organismswithin families, from mothers to infants and within

Infect Dis Obstet Gynecol 2001;9:65–73

Correspondence to: Bryan Larsen, PhD, Des Moines University Osteopathic Medical Center, 3200 Grand Avenue, Des Moines,IA 50312. E-mail: [email protected]

Clinical study 65

hospital environments, and the association ofgenetic fingerprints with virulence and drug resist-ance. Genotyping approaches are of particularinterest to organisms such as C. albicans, which canand often do colonize individuals in an asymptom-atic fashion. One of the key questions arising fromthis observation is whether symptomatic candi-diasis results from strain replacement. While manyepidemiologic studies have been done, includingcomparison of strains carried simultaneously atdifferent body sites or sequential analysis of strainsinvolved in recurrent infections, the issue ofwhether symptomatic candidiasis can developfrom a strain associated with initially asymptomaticcolonization during pregnancy has not beendirectly addressed.

In the course of a culture-based longitudinalstudy of vaginal yeast carriage and symptomaticinfection during pregnancy, we were able to findpairs of cultures representing asymptomatic colo-nization, followed at a later time by developmentof symptoms of vulvovaginal candidiasis2. Alimited number of pairs of yeast specimens wereavailable, but the discriminating power of randomamplification of polymorphic DNA (RAPD)analysis provided sufficient data to determine therelatedness of C. albicans cultures present beforeand during symptomatic vaginitis.

MATERIALS AND METHODSMore than 300 women who sought obstetric careat the Robert C. Byrd Health Sciences Center andits outreach clinics were the source of vaginalspecimens, which were plated onto BIGGY(bismuth sulfite–glucose–glycine–yeast) agar in aneffort to identify colonization by C. albicans. Eachwoman was told of our interest in obtainingepidemiologic information on yeast vaginitis inpregnancy and signed a consent form approved bythe West Virginia University Institutional ReviewBoard. The demographic composition of thiscohort has been described in an earlier paper2. Allsix patients in this study were pregnant at the timeof their first culture (range 8–35 weeks’ gestation;average gestational age at initial booking was 16weeks). Two patients were primigravidas and theremaining four patients ranged from gravida 2 to

gravida 4; two patients had aborted previously.Three patients had experienced a prior yeast infec-tion and a fourth had experienced recurrent infec-tions. None was symptomatic for yeast or othertype of vaginitis (abnormal discharge, itching,burning or malodor) at the time of their initial pre-natal examination and all had wet mount andKOH examination to evaluate signs of bacterialvaginosis, trichomoniasis, and candidiasis. All werenegative at the initial examination. Each patientwas asked to call the clinic in the event of vaginalsymptoms. In addition, at each subsequent clinicvisit throughout the pregnancy, participants werequestioned about symptoms consistent withvulvovaginal candidiasis. Vaginal cultures wereobtained from women who developed symptomsso that matched pairs of C. albicans isolates could beassembled for this study. Symptomatic individualswere those who complained of or had symptoms ofpruritus, inflammation, white curd-like discharge,positive microscopic evidence of vaginal yeast, andpositive culture for C. albicans.

Cultures obtained from study participants wereheld at room temperature for at least 72 hoursbefore being discarded as negative. Cultures thatdeveloped characteristic brown colonies were heldat 4°C until needed. All isolates, in addition to pro-ducing brown colonies on BIGGY agar, wereshown to have characteristic microscopicmorphology and produced germ tubes in humanserum, which presumptively classified them asC. albicans3. A total of seven pairs of cultures repre-senting asymptomatic colonization followed bysymptomatic vulvovaginal candidiasis were avail-able. One of these pairs could not be used becausethe ‘asymptomatic’ culture became nonviable instorage, leaving six for analysis. These sampleswere identified by a patient study number(assigned at West Virginia University) and addedto the culture collection maintained at Des MoinesUniversity.

The six women whose yeast isolates were ulti-mately included in this study were all white, aged17–26 years and ranged from 8 to 35 weeks’ gesta-tion at their first clinic visit. Two patients wereprimigravidas and none were private-pay patients,nor did they have alternate sources for medicalcare. Because of this, all were followed consistentlyby the study physician.

Candidiasis during pregnancy Daniels et al.

66 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY

Just before polymerase chain reaction (PCR)analysis, the paired cultures were again streaked forisolation on BIGGY agar plates, and an individualcolony was picked and maximally propagated on aSabouraud’s dextrose agar plate. The growth fromthis plate was recovered with a sterile inoculatingloop and suspended in 293 ml of 50 mmol/lEDTA. LyticaseÒ (Sigma Chemical Co., St. Louis,MO), 7.5 ml of 20 mg/ml solution, was added andthe cell suspension mixed by gentle pipetting.The samples were incubated at 37°C for 1 hourto digest the cell walls. The spheroplasts werebrought to room temperature and centrifuged at13 000 g, and the supernatant fluid was discarded.

DNA isolation employed a commercial kit(Wizard KitÒ, Promega, Madison, WI), and themanufacturer’s directions were followed. TheDNA was rehydrated in Tris-EDTA buffer and theabsorbance at 260 and 280 nm determined to allowdilution to a final concentration of 10 ng/ml,which served as template for subsequent RAPDanalysis.

Our RAPD protocol employed two of theprimers from the Ready-To-Go-RAPDÒ system(Amersham Pharmacia Biotech, Piscataway, NJ):Primer 1 (5¢-d[GGTGCG GGAA]-3¢) and primer4 (5¢-d[AAGAGCC CGT]-3¢). For amplification,15 ml of sterile distilled water, 5 ml primer, 5 ml oftemplate DNA (50 mg) and one RAPD bead wereplaced in a reaction tube and reacted in an MJMinicycler (MJ Research Inc., Watertown, MA).Because the instrument has a heated lid, no mineraloil was added to the reaction mix. The cycling pro-file was: one cycle at 95°C for 5 min, 40-45 cyclesat 95°C for 5 min, 36°C for 1 min, 72°C for 2 min.

The products were applied to a 2% agarose gelcontaining 1 mg/ml ethidium bromide. A mini-submarine gel format was found to be adequate forseparating bands generated from C. albicans. ADNA ladder (BioRad, Hercules, CA) with a rangeof 50–2000 bp was applied to the first lane. Thesecond and third lanes contained the products fromprimer 1 (the asymptomatic strain in the secondlane and the symptomatic strain in the third lane),and the fourth and fifth lanes contained theproducts from primer 4 (asymptomatic followedby symptomatic).

After electrophoresis, the resulting bandpatterns were visualized and recorded using the

FluorS-Multi-ImagerÒ (BioRad). Analytic den-sitometry was performed using the Multi-AnalystÒ

software (BioRad). Digitized densitometry scanswere exported to a Microsoft ExcelÒ spreadsheetfor further analysis.

The densitometry scans of two electrophoreticpatterns from the same gel were compared bycross-correlation analysis using a custom programwritten with LabViewÒ programming software(National Instruments, Austin, TX). The nearercross-correlation coefficients were to 1.0, themore similar were the two banding patterns. Crosscorrelation analysis proceeded as follows.

Initially all comparisons were made on adjacentlanes on the same electrophoresis gel, but to com-pare unrelated strains it was necessary to compareRAPD patterns between gels. Because the numberof data points differed between gel images, twodata manipulations were performed. First, bothelectrophoretic patterns were normalized withrespect to landmarks on the x axis: that is, the xco-ordinate for each data point was reported as apercentage of the total length between the twotracking dyes. Second, one of the electrophoreticpatterns was interpolated so that the data pointswere matched with respect to the normalized xco-ordinate of the other normalized electro-phoretic pattern. Electrophoretic patterns wereanalyzed using baseline shifts of ±100 lags (lags aredata points added by the computer to adjust thelength of one x axis).

RESULTSSix pairs of cultures representing the vaginalorganisms present before symptoms developed andwhile the patient was symptomatic were analyzedby RAPD analysis. A seventh pair of organisms wasidentified, but in this case the earliest isolate did notsurvive in storage. The average length of timebetween the first C. albicans isolation and develop-ment of symptoms was 14 weeks (range 9–20weeks).

Figure 1 shows a composite image of all thePCR products elicited from RAPD analysis. Evenwithout the aid of densitometric analysis it is clearthat the banding pattern from the two isolates wasidentical for both primer 1 and primer 4 in allbut one of the six pairs of cultures. The interval


INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 67

between cultures for the non-matching pair was18 weeks.

Analysis of the PCR products from the 12 yeastisolates in this study generated data on 125 bandswith each isolate generating between 11 and 16bands from the two primers used for the compari-son. In order to evaluate this quantity of data, amathematical method for comparing the bandingpatterns from the pairs of organisms was employed.Video densitometry provided a more objective

method for capturing the RAPD data in a usefulform. Figure 2 shows the alignment of twodensitometric scans plotted in the LabView soft-ware package. This figure consists of the densito-metric scan from the two organisms beingcompared. In this example the two scans appearvisually similar when overlaid. The lower plotshows the result of cross-correlation in which thedegree of correlation (values ranging from 0–1with 1 representing perfect correlation) on the



Figure 1 Electrophoretic pattern of random amplification of polymorphic DNA polymerase chain reaction(RAPD-PCR) products from 12 strains of Candida albicans from six patients. Each panel represents the isolate recov-ered before symptoms and the organism isolated while the patient was symptomatic. The data are from patientsGT054 (A), GT151 (B), GT397 (C), GT150 (D), GT377 (E), and GT387 (F). The lanes in each panel are as follows: lane1: standards (2000 bp and less); lane 2: primer 1 (see Materials and Methods) with target DNA from the asymptomaticpatient isolate; lane 3: primer 1 with target DNA from the same patient while symptomatic; lane 4: primer 4 with targetDNA from asymptomatic isolate from the same patient; and lane 5: primer 4 with symptomatic isolate

vertical axis and the number of lags (extra datapoints used to ‘stretch’ one of the traces to maxi-mize the alignment). In the example, there was ahigh degree of correlation. The ‘tightness’ of thecorrelation diminished when more than a few lagswere inserted.

When cross-correlation was used to determinethe relatedness of the RAPD products generatedfrom the remaining C. albicans isolates obtainedbefore and after development of symptoms it con-firmed that in only one of six cases was there anapparent change in yeast strain from asymptomaticto symptomatic colonization (Table 1).

To assess the meaningfulness of this level ofagreement, we selected an additional six yeast iso-lates randomly from the women in our study whohad been found to be colonized without symp-toms. Table 2 uses the six isolates from asymptom-atic women who later became symptomatic asindex cases. These are paired with randomlychosen isolates of C. albicans obtained during the

course of the longitudinal study previously pub-lished with comparisons of the level of discordanceby means of cross-correlation analysis2. None ofthe random strains matched the six index isolates,whereas five of six pairs from the initial pairingsappeared to be the same (Yates c2 p = 0.019). Thisanalysis confirms that five of the six women whodeveloped yeast vaginitis symptoms did so as aresult of the same organism that colonized themasymptomatically earlier in their pregnancies.

DISCUSSIONIn the past decade, a variety of molecular tech-niques have emerged which allow a high degree ofreliability in epidemiologic tracking of variousstrains of pathogenic yeasts. These techniquesinclude pulsed field gel electrophoresis4–6, restric-tion fragment length polymorphism (RFLP)6–9,Southern hybridization of specific probes10,11,RAPD11–19, and combinations of various methods.



Figure 2 Graphic results of cross-correlation analysis for isolate GT151. (a) Original densitometric data (x axis is dis-tance (relative units); y axis is relative optical density units); (b) superimposed traces of asymptomatic and symptomaticisolates; (c) change in cross-correlation coefficient with varying numbers of lags, from –100 to +100 lags (x axis is thenumber of lags; y axis is the correlation coefficient)

Recently, Soll published an extensive review ofthe methods and pitfalls associated with DNAfingerprinting of fungal organisms1. Strain mainte-nance over long periods of time appears to be com-mon, but studies that have used very sensitivegenotyping methods have revealed the possibilityof microevolution among colonizing strains20. Inaddition, the work of Lockhart and colleagues,who investigated recurrent vaginitis, indicated thatthese women had a repertoire of similar strainsthat became intermittently predominant strainsthrough a process they termed ‘substrainshuffling’21.

Several reports on C. albicans typing have com-pared different methods4,22,23, and found RAPD tobe a useful method of epidemiologic analysis.Following these organisms within populations isbecoming increasingly important because of theprevalence of fungal infections among compro-mised hosts and other individuals. In addition tounderstanding the pathogenesis of yeast vaginitis

during pregnancy, there is concern about infants,especially premature infants, acquiring C. albicansinfections. Molecular typing techniques haveindicated that one-third of premature infants werecolonized and that mother-to-infant transmissionof the identical strain occurred in 14% of infants24.In another study, there were six cases among 103newly parturient women in which both motherand infant were colonized by identical strains ofC. albicans5.

C. albicans and other related yeast are frequentlycommensal flora on mucosal sites, and it is possiblefor individuals to carry more than one strain ofyeast at a time25. It is important to understand ifsymptoms arise after acquisition of a virulent strainof yeast or if the endogenous organisms canincrease their virulence toward the host. Magee’sgroup used an RFLP method to determine thatdifferent strains are carried in the vagina andrectum and that recurrent vaginitis is usually due torelapse with the same vaginal strain rather thanreinfection from the rectum9. Despite this infor-mation, it is not clear whether yeast strains presentin asymptomatic women are the same ones that areinvolved in symptomatic infection. Certainly,there is ample evidence that individuals sometimeschange strains of yeast and other organisms.Concordance of strains among family membersis common26, as is person-to-person transmissionof organisms as documented by Muller andcolleagues in an HIV-infected family27, implyingthat the development of symptomatic infectioncould be the result of acquiring a new yeast strain.

The present study was possible because we wereengaged in following women longitudinallyduring pregnancy with a culture for C. albicanswhen they booked for obstetric care and then againif they developed symptoms consistent with yeastvaginitis. Clearly, a larger number of pairs of strainswould have been desirable for evaluation, butbecause of the prospective nature of the culturecollection and our inability to predict whichwomen would become symptomatic, we believethat we would need to follow an additional 50women through pregnancy to elicit just one morepair of cultures. Based on RAPD analysis, themajority of vulvovaginitis cases were apparentlydue to the same strain of organism that was presentmany weeks before the symptoms arose. These



Patient number

Primer 1cross-correlationcoefficient (r2)

Primer 4cross-correlation

coefficient (r2)

GT054GT150GT151GT377GT387GT397

0.980.950.980.290.960.96

1.000.880.970.480.980.97

Table 1 Correlation of electrophoretic patterns ofrandom amplification of polymorphic DNA (RAPD)products from Candida albicans isolates before and afterdevelopment of symptoms

Patient number(index case/unrelated case)



GT054/GT196GT150/GT275GT151/GT158GT377/GT157GT387/GT188GT397/GT199

0.250.120.240.100.180.13

0.420.070.290.370.280.11

Table 2 Correlation of presumably unrelated Candidaalbicans strains from asymptomatic women

findings suggest that the changing relationshipbetween the host and microorganism during theprogression of the pregnancy is probably moreimportant in the development of symptoms than isstrain replacement or even a genetic change in thecommensal organism. We believe this study, andpotentially others like it, can benefit from themathematical method of expressing the compari-son of two gel patterns as a single correlationcoefficient. Additional work will be required toascertain the level of sensitivity to generic differ-ences represented by the cross-correlation tech-nique, but clearly it distinguishes paired samplesfrom random samples.

One limitation of our approach was that onlyone colony was isolated from each of theC. albicans-positive cultures, which was done toensure purity of the isolates we tested. Although inthe 12 positive cultures forming the core of thisstudy the morphotypes of the colonies on primaryplating media appeared identical, a small possibilityexists that we could have missed isolating a secondstrain that might have been present in the same cul-ture. Because the occurrence of two concomitantstrains of yeast in the same vaginal culture is rela-tively uncommon, and because yeast causingsymptoms are generally thought to be in numericalabundance, the likelihood of having missed asecond yeast strain in this cohort of patients is con-sidered very low. On the other hand, the one dis-cordant pair of yeast we discovered could havebeen the result of having two strains simulta-neously in the same patient and our havinghappened to isolate the nonidentical strain in the‘symptomatic’ culture.

Until obtaining DNA sequence data becomesthe standard method for genotyping there is apossibility that less sensitive analytical techniquessuch as RAPD will miss mutations and micro-evolution occurring in organisms carried for along period of time. A study by Metzgar andco-workers16 found small changes in nucleotidesequence data during the course of fluconazoletreatment in some HIV-infected patients, raising

the possibility of genetic changes in the short timefrom isolation of a commensal organism to thetime that symptoms develop. The study of Metzgarand co-workers involved a much more in-depthanalysis of the genotypic characteristics of theirorganisms than our RAPD analysis. Thus, smallmutational events could have been more readilyapparent in their work. In fact, their work suggeststhat during therapy the patients in their studyprobably retained the same organisms, but thatthese organisms were undergoing microevolution.

Despite the fact that there are more sensitivemethods of genetic characterization of micro-organisms, the evidence gained from the use ofRAPD analysis provides a good basis for believingthat in most cases vaginal symptoms may resultfrom the same organism present prior to thedevelopment of symptoms. This raises a furtherquestion of whether a change in host physiology orin regulation of virulence attributes of the micro-organism (or a combination of both) is responsiblefor genesis of symptoms in these patients. Whilethe occurrence of mucocutaneous candidiasis inHIV patients emphasizes the importance ofchanging immune status in fungal pathogenesis,there is also a growing recognition thatmicroorganisms have the ability to alter theiraggressiveness toward the host in response tomicroenvironmental cues. Environmental factorssuch as glucose concentration28, iron29, orhormonal factors30 may play a role in modulatingC. albicans virulence. This fact, coupled with therecognition that the same organism which colo-nizes the vaginal epithelium may subsequentlycause vaginal irritation, underscores the impor-tance of understanding the regulation of microbialphysiology and the role of host factors in microbialregulation.

ACKNOWLEDGEMENTThe assistance of Joseph Weir, PhD, in analyzingdensitometry patterns aided materially in the pre-sentation of this work.



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RECEIVED 08/11/00; ACCEPTED 01/18/01



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Candidiasis during pregnancy may result from isogenic ...downloads.hindawi.com/journals/idog/2001/941789.pdf · colonization during pregnancy has not been directly addressed. In the