Yeast colonization of Vagina in Ekpoma Nigeria 1st & best

Addresses: ¹Microbiology Department, Irrua Specialist Teaching Hospital Irrua Edo State, Nigeria. ²Department of Medical Laboratory Sciences, Faculty of Health Sciences and Technology, Nnamdi Azikiwe University, Nnewi campus Anambra state, Nigeria. ³Department of Microbiology, Kampala International University Western campus Ishaka Bushenyi, Uganda.⁴Microbiology Department, Faculty of Natural Sciences Ambrose Alli University Ekpoma, Edo State, Nigeria. ⁵Department of Medical Laboratory Sciences, Ambrose Alli University, Ekpoma Edo State, Nigeria. ^6&7Department of Obstetrics and Gynecology, Irrua Specialist Teaching Hospital, Irrua, Edo State Nigeria.

Citation: Inyang NJ, Enweani IB, Agwu E, Esumeh FI, Obiazi HAK, Eifediyi RA, Ikheloa J. Colonization And Susceptibility Profile Of Vaginal Yeast Isolates In Ekpoma, Edo State Nigeria. Special Fungal Pathogens Journal (SJ-FPJ) 2015; Vol 1, No 1: p 007-013. :

Abstract

Background: Although Candida albicans remains a persistent pathogen in humans, little is known about the relationship between commensal and infecting strains, the origins of infecting strains, the transmission of the organism between individuals, and strain specialization.

Objective: To outline the colonization profile and susceptibility to azoles of vaginal yeast isolates in Ekpoma, Nigeria.

Methods: In this study, 300 vaginal swabs were aseptically collected from women visiting Hospitals in Ekpoma. Samples were processed phenotypically using standard mycological protocols. PCR was done on representative isolates whose identity could not be phenotypically ascertained. Minimum inhibitory Concentration for azoles was tested using E- test strip.

Results: Candida albicans showed 22.3% prevalence followed by C. glabrata with 8.7% prevalence and both yeasts showed dose-dependent sensitivity to azoles. The married women aged 20-39 years (47.6% and 36.8%) were the most vulnerable followed by single asymptomatic women aged 10-19years. Nine per-cent aged 29, had no marital status.

Married women showed high prevalence in all age groups while 40-29 (62.3%) showed the highest prevalence among singles. Among the symptomatic group, 33.3% aged 30-39 with secondary education and 13.3% of symptomatic patients aged 10-19years (13.3%) were most vulnerable. Among the asymptomatic participants, 35.7% with primary education aged 40-49 were most vulnerable.

Conclusions: Yeast prevalence in VVC may result from a patient change in immune status and may not be affected by demographic and sociocultural factors. In-depth antifungal susceptibility testing is required to investigate if the routine use of antifungal drugs can lead to shifts in the colonization and susceptibility status of Candida species.

Corresponding Author: Microbiology Department, Irrua Specialist Teaching Hospital Irrua Edo State, Nigeria.

e-mail [email protected]. TeL: +2348033724973 and +2348022109358

Background

The vaginal microbiota contains a community of microbes and plays a common role in the maintenance of vaginal health. Disruption of the microbial composition can lead to increased susceptibility to various infectious diseases. Candida species infections lead to non-life-threatening mucocutaneous diseases. Among Candida species, Candida albicans is the most common infectious agent colonizing humans to cause Candidiasis in the skin, soft tissues, and genital tract. The name given to Candidiasis of the virginal tract in a healthy human is vulvovaginal candidiasis (VVC).

The presence of Candida in the vagina, in the absence of immunosuppression or damaged mucosa, may not be associated with any disease and is thus referred to as colonization. In contrast to asymptomatic colonization, VVC is defined as signs and symptoms of inflammation and an overgrowth of Candida species, particularly C. albicans, and without other infectious etiology. Uncomplicated VVC is characterized by random occurrence of a moderate disease caused by C. albicans in immunocompetent women.

Complicated VVC may include severe cases, those caused by non-C. albicans species, those associated with pregnancy or other concurrent conditions like uncontrolled diabetes or immunosuppression, and recurrent VVC (RVVC) in Non-C. albicans species are emerging pathogens and can also colonize human mucocutaneous surfaces (1).

Consequently, they are also isolated in the setting of candidiasis. Mucocutaneous candidiasis can be divided into non-genital disease and genitourinary disease. Among non-genitourinary candidiasis, oropharyngeal manifestations are the most common while the most frequent manifestations of genitourinary candidiasis include vulvovaginal candidiasis (VVC) in women. In the majority of women, a diagnosis of VVC is made at least once during their childbearing years (2).

Among the many causes of vaginitis, VVC is the second most common after bacterial vaginosis and is diagnosed in up to 40% of women with vaginal complaints in the primary care setting (3). Long-term suppressive antifungal therapy is commonly required to control RVVC, and recurrence rates of up to 40% to 50% occur after discontinuation of suppressive therapy (4). Compared to the case for women with other chronic vaginal symptoms, RVVC is reported to have the greatest negative impact on work and social life (5).

Reliable diagnosis of VVC requires a correlation of clinical features with mycological evidence. VVC is not traditionally considered a sexually transmitted disease. Sometimes sexual transmission of Candida can occur during vaginal intercourse. Particularly Candida transmission and sexual behaviors are linked to RVVC. Although the yeast Candida albicans remains a persistent pathogen in humans, relatively little is known about the relationship between commensal and infecting strains, the origins of infecting strains, the transmission of the organism between individuals, and strain specialization.

Although considerable information has accumulated in the last decade regarding rates of both vaginal colonization and vulvovaginal candidiasis (VVC) in HIV-positive women, gaps in knowledge remain, particularly about the pathophysiology of clinical disease. It is unclear, whether cell-mediated immunity in women with RVVC are systemically derived or are seized or separated in the vaginal mucosa and whether additional host, organism, or exogenous factors affect normal vaginal defenses.

Analysis of vaginal yeast isolated from women with recurrent candidal vaginitis uncommonly reveals a higher percentage of non-albicans Candida species. There is no indication that resistance to azoles is a causal factor of vaginal yeast colonization and no other fungal virulence factors have been identified to explain the repeated attacks. It is thought that widespread use of ‘over-the-counter’ azoles may increase the incidence of resistant Candida glabrata. Infections with species other than Candida albicans frequently do not respond to standard azole treatments.

Objective

Based on the above gaps, this study was designed to outline the colonization profile and susceptibility status of vaginal yeast isolates in Ekpoma, Nigeria.

Materials and Methods

In this descriptive cross-sectional laboratory-based study, three hundred high vaginal swab samples were aseptically collected from 300 women aged 18-55years visiting Hospitals and Medical laboratories in Ekpoma and its environs. One hundred samples each were collected from: women clinically diagnosed with vaginal Candidosis, apparently healthy non-pregnant females, and pregnant females with symptoms of Vaginal Candidosis.

Informed consent was sought and obtained from the participants. In the Microbiology laboratory of Irrua Teaching Hospital, Edo State, Nigeria, samples were cultured on Sabouraud dextrose agar (SDA) and incubated at 37⁰C for 48hrs. Direct Gram for yeast cells was also done to eliminate contamination during the plate reading. Germ tube test was done on all Candida albicans isolates; they were also tested for heat tolerance at 45⁰C, osmotic resistance in broth containing 6.5% NaCl.

All presumptively identified yeast isolates were taken to Molecular Biology Laboratory, Department of Veterinary Microbiology, College of Veterinary Medicine, Makerere University, Kampala Uganda for further analysis. Suspect yeast isolates were first grown on CHROMagar Candida medium at 37+ 2⁰C for 24-48 hours (6) and later grown on: Pal’s agar (7) and CHROMagar Candida medium supplemented with Pal’s agar (8). The protocol and choice of sera including incubation atmosphere during germ tube testing as suggested by Isibor et al. (9) were adopted.

Using the method reported by Cheesbrough (10), yeast colonies that were germ-tube positive were tested for chlamydospores production using standard methods. Meanwhile, yeast colonies that were germ-tube positive and produced chlamydospores were sub-cultured onto SDA plates to obtain fresh isolates and incubated at a temperature of 45^oC for 4-5 days. Those yeast isolates that did not grow at 45^oC became suspect colonies of Candida dubliniensis while germ-tube positive and chlamydospores producing yeast isolates that grew at 45^oC were presumptively identified as C. albicans colonies (11).

Besides, suspected colonies of yeast strains were further identified and differentiated by a biochemical test using sugar assimilation test pattern (ID32 C) for Candida species provided by BioMereux^(R) Paris, France. Standard methods recommended by the manufacturers were adopted in setting up the experiment, reading, and interpretation of results (BioMereux^(R), Paris, France).

Molecular PCR Assay of yeast

Suspected colonies of C. albicans and C. dubliniensis were subjected to further analysis using polymerase chain reaction (PCR) test method because they are closely related. Candida template DNA was prepared using the method of Donnelly et al. (12). Briefly, fresh three yeast colonies growing on PDA were sub-cultured onto Yeast Peptone Dextrose Agar (YPDA), incubated at 37^oC for 48hours and a single yeast colony from the YPDA culture was suspended in 50µl sterile distilled water.

Cell suspensions were boiled for 10 min and then lysed cells were subjected to a clearing spin for 5 min at 20,000 g. Template DNA contained in 25 µl supernatant was used for PCR amplification. The PCR test for the differentiation of Candida albicans and Candida dubliniensis colonies was carried out in a 50 µl final volume (master mix) containing: 25.0µl (variable) of sample Candida species DNA template; 10.0 µl of GoTaq^(R) Flexi buffer; 0.25 µl GoTaq^(R) polymerase; 5.0 µl of Magnesium chloride (variable); 1.0 µl of each Deoxy-Nucleotide Triphosphates (dNTPs: A, T, C, G); 2.5 µl (variable) of each Candida dubliniensis-specific primers:

DUBF: 5″-GTATTTGTCGTTCCCCTTTC-3″; DUBR: 5″GTGTTGTGT G C ACTAACGTC-3″; RNAF: 5″-GCATA TCAAT AAGCG GA GGAAAAG-3″ and RNAR, 5″-GGTCCGTGTTTCAAGACG-3″ (12). The Candida dubliniensis specific primers were designed to amplify a DNA fragment of 288 base pair from C. dubliniensis template DNA while the universal fungal primers which also served as an internal positive control, was synthesized by Fell (13). It was designed to amplify a DNA fragment of 610 base pairs from fungal template DNA. The internal negative control used constituted 50 µl final volume of the master mix and Nuclease free water without template DNA.

The reaction mixture was placed in a PCR machine (Thermo Electron Corporation, Milford, MA, USA) preheated to 95^oC and ran according to pre-set PCR program of an initial denaturation step at 95^oC for two minutes (to ensure the entire DNA samples were single-stranded); final denaturation step at 95^oC for thirty seconds (to completely denature the double-stranded template DNA); annealing step at 58^oC for thirty seconds (to enable primers to anneal to template DNA); extension step at 72^oC for one minute /kilobase pair (to synthesize DNA from the primers).

Denaturation, annealing, and extension steps described above were set to repeat for thirty-five times or cycles after which, a final extension step at 72^oC for ten minutes was added (to ensure all of the extensions were completed). The program held the tubes at 4^oC until the instrument was turned off. Using the guideline of Sambrook and Russell (14), two percent (w/v) agarose gel containing 0.5 µg ethidium bromide ml^-1 was prepared, and 15 µl each of the DNA amplification products and DNA Molecular Size Marker (MSM; Standard) were loaded onto the 2% gel, ran at 90V for about 2 hours to ensure adequate separation.

The amplification products were visualized on a 302 nm UV ultra-illuminator. Pictures of the ethidium-bromide-stained gels containing the separated amplification DNA product were taken and printed out. All the isolates were tested for minimum inhibitory concentration using an E. test strip (Biomerieux, France). Reading of plate for fungal MIC was made when growth was seen clearly after overnight incubation or 48 hours incubation depending on when visible growth was seen. The guideline provided by CLSI, (15) was strictly followed in the interpretation of the result.

Besides, a visual aid (chart) provided by the manufacturer (AB BIODISK, Solna, Sweden) was used as a guide during reading and interpretation of results. The CLSI (15) guideline in brief states that MIC for antifungals should be read as follows: Fluconazole: MIC<8µg/ml =sensitive; MIC>8µg/ml and ≤32µg/ml = sensitive dose-dependent (SDD) and MIC>32µg/ml = resistant. For Itraconazole: MIC ≤ 0.125µg/ml = sensitive; MIC> 0.125µg/ml and ≤ 0.5 µg/ml = sensitive dose dependent and MIC > 0.5 µg/ml = Resistant.

Results:

In this study, Candida albicans had an overall prevalence of 22.3% while C. glabrata was 8.7%. In table 1, the marital status of participants concerning age is displayed for 58 symptomatic and 68 asymptomatic women who participated in this study. Among the symptomatic married women, the age groups 20-29 and 30 -39years (36.8% and 47.6%) were the most vulnerable while the 10-19years age group were the most vulnerable among the symptomatic women that are single followed by 40-49 years (14.3%) of age.

There was no report of divorced or separated participants giving a zero prevalence in this population. Worthy of note is the 9.2% registered among the age group 20-29 years who could not disclose their marital status. On the other hand, among the 68 asymptomatic women surveyed, married women clearly showed high prevalence in all age groups while the age group 40-29 (62.3%) showed the highest prevalence, followed by age group 30-39years with the prevalence of 42.9% (Table 1). The divorced and others showed the same trend of little (6.7%) or no disclosure (0.0%) as in most age groups.

Table 2 shows the education status of the participants. Among the symptomatic group, the vulnerable population is those aged 30-39years (33.3%) with secondary education followed by those aged 10-19 years (26.7%) with secondary education. Besides, among the symptomatic patients with no education, the age group 10-19years (13.3%) was the highest followed by 10.5% seen among the age group 20-29 years.

Symptomatic participants with tertiary education aged 40-49 years showed a 14.3% prevalence. On the other hand among the asymptomatic participants the decreasing order of prevalence were: 35.7% among those aged 40-49years with primary education, 28.6% among those aged 40-49years with no education, 19.7% among those aged 20-29years with primary education, and 19.0% among those aged 30-39years with no education. Other results are shown in Table 2.

The occupational status of participants showed in Table 3 indicates that yeast prevalence was most common among asymptomatic students aged 20-29years with 32.9% prevalence followed by asymptomatic traders, aged 40-49years with 28.6% prevalence, and symptomatic peasants aged 10-19years with 26.7% prevalence and symptomatic students aged 10-19 years with 23.8% prevalence. Other results include 20% asymptomatic peasants, 19% asymptomatic students, 18.4% asymptomatic peasants, 14.3% symptomatic civil servants, and others whose status was not disclosed.

Candida species demonstrated a high overall yeast prevalence of 42% (126 out of 300 samples) in this investigation. Table 4 gives details of this yeast distribution with age. Candida albicans maintained this prevalence across all age groups ranging from 73.3% in age group 10-19years followed by 57.1% among age group 30-39years and 42.9% among age group 40-49years. Candida glabrata was the second most prevalent yeast among the studied population with 28.6% prevalence among the age group 30-49 years old and 19.7% among age group 20-29years.

Candida parapsilosis showed 11.8% prevalence among the age group 20-29years and 7.1% among the age group 40-49years. While found no Saccharamycess cerevecie in any sample. Other unclassified yeasts which we could not identify due to limited resources had 14.3% prevalence among the age group 40-49 and 13.3% among age group 10-19years respectively.

Table 5 shows yeast distribution according to symptoms, age, and pregnancy status. Yeast prevalence of 42.9% among pregnant asymptomatic women aged 40-49years was the highest followed by 38.1% prevalence seen among pregnant symptomatic women and pregnant asymptomatic women aged 30-39years; 33.3% prevalence seen in pregnant symptomatic women aged 10-19years, 31.6% seen in pregnant asymptomatic women aged 20-29years and 27.6% prevalence seen in pregnant symptomatic women aged 20-29years respectively. Other results are shown in Table 5 below.

Table 6 reveals the mean representative minimal inhibitory concentration of the 4 yeast strains isolated in this study when challenged with fluconazole and Itraconazole. Candida glabrata isolates were completely (MIC=>32.0) resisted Itraconazole and its sensitivity to fluconazole (MIC= ≤12.0) was dependent on the dosage according to the table (6). Candida parapsilosis (MIC≤0.38 and 0.01), C. tropicalis (MIC≤1.0 and 0.5), and C albicans (MIC≤0.5) all showed sensitivity to fluconazole and Itraconazole respectively as shown in Table 6.

Fig 1 shows the amplified DNA products of yeast isolates suspected to be C. dubliniensis. Lanes 1 and 13 (100bp) are Molecular Size DNA Markers. Universal fungal primers were included as an internal positive control for all yeast strains (610bps). Lane 2 is a negative control containing all the constituents of the PCR master mix but no DNA added. Lanes 3 and 4 are internal positive control for C. dubliniensis first showing a band of about 610bp to show it is yeast followed by another band of about 288bp specific for C. dubliniensis. Lanes 5-12 are C. albicans suspected to be C. dubliniensis.

Table 1: Marital Status of study participants in Ekpoma

Age No Symptomatic (n=58) Asymptomatic (n=68)

Yrs Ex Single Married Divorced Others Single Married Divorced Others

<10 0 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0)

10-19 15 5(33.3) 3(20.0) 0(0.0) 0(0.0) 2(13.3) 4(26.7) 0(0.0) 1(6.7)

20-29 76 1(1.3) 28(36.8) 0(0.0) 7(9.2) 16(21.1) 20(23.3) 0(0.0) 4(5.3)

30-39 21 0(0.0) 10(47.6) 0(0.0) 0(0.0) 2(9.5) 9(42.9) 0(0.0) 0(0.0)

40-49 14 2(14.3) 2(14.3) 0(0.0) 0(0.0) 1(7.1) 9(62.3) 0(0.0) 0(0.0)

50-59 0 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0)

≥60 0 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0)

n=number sampled, No=Number, Preg=pregnant, %=percentage

Table 2: Education status of study participants in Ekpoma

Age No Symptomatic (n=?) Asymptomatic (n=?)

Years Exam Non Primary 2⁰ 3⁰ Non Primary 2⁰ 3⁰

<10 0 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0)

10-19 15 2(13.3) 2(13.3) 4(26.7) 0(0.0) 2(13.3) 1(6.7) 3(20) 1(1.7)

20-29 76 8(10.5) 6(7.9) 12(15.8) 10(13.2) 10(13.2) 15(19.7) 9(11.8) 6(7.9)

30-39 21 0(0.0) 1(4.7) 7(33.3) 2(9.5) 4(19.0) 3(14.3) 2(9.5) 2(9.5)

40-49 14 0(0.0) 0(0.0) 2(14.3) 2(14.3) 4(28.6) 5(35.7) 1(7.1) 0(0.0)

50-59 0 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0)

≥60 0 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0)

Table 3: Occupational status of study participants in Ekpoma n= 126

Age No Peasants Traders Students Civil servants others

Yrs Ex Sym Asy Sym Asy Sym Asym Sym Asym Sym Asym

<10 0 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0 (0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0)

10-19 15 4(26.7) 3(20.0) 2 (13.3) 1(6.7) 2(13.3) 2(13.3) 0(0.0) 0(0.0) 1(6.7) 0(0.0)

20-29 76 14 (18.4) 7(9.2) 5 (6.6) 6 (7.9) 7 (9.2) 25 (32.9) 5(6.6) 4(5.3) 0(0.0) 3(3.9)

30-39 21 1 (4.8) 2(9.5) 2 (9.5) 5 (23.8) 5 (23.8) 4 (19.0) 2(9.5) 0(0.0) 0(0.0) 0(0.0)

40-49 14 0(0.0) 3 (21.4) 2(14.3) 4(28.6) 0 (0.0) 0 (0.0) 2(14.3) 1(7.1) 0(0.0) 2(14.3)

50-59 0 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0 (0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0)

≥60 0 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0 (0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0)

n=number sampled, Sym=symptomatic, Asy=Asymptomatic

Table 4: Age-specific prevalence of Yeast species isolated from 300 samples of consenting participants

Age No n=126 (42%) positive

Yr Exam C albi C. glab C. trop C. parap Sacch Sp others

<10 0 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0)

10-19 15 11(73.3) 1(6.7) 0(0.0) 1(6.7) 0(0.0) 2(13.3)

20-29 76 38(50.0) 15(19.7) 13(17.1) 9(11.8) 0(0.0) 1(1.3)

30-39 21 12(57.1) 6(28.6) 1(4.8) 0(0.0) 0(0.0) 2(9.5)

40-49 14 6(42.9) 4(28.6) 1(7.1) 1(7.1) 0(0.0) 2(14.3)

50-59 0 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0)

≥60 0 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0)

n=number sampled, C=Candida, Sacch=Saccharomyces Spp=species, No=number albi=albicans glab=glabrata top=tropicalis parap=parapsilosis

Table 5: Distribution of virginal yeasts from samples of symptomatic (Candidiasis) and asymptomatic (Candidosis) participants in Ekpoma

Age No Symptomatic (n=58) Asymptomatic (n=68)

Years Exam A B C D

<10 0 0(0.0) 0(0.0) 0(0.0) 0(0.0)

10-19 15 5(33.3) 4(26.7) 3(20.0) 3(20.0)

20-29 76 21(27.6) 14(18.4) 24(31.6) 17(23.4)

30-39 21 8(38.1) 2(9.5) 8(38.1) 3(14.3)

40-49 14 3(21.4) 1(7.1) 6(42.9) 4(28.6)

50-59 0 0(0.0) 0(0.0) 0(0.0) 0(0.0)

≥60 0 0(0.0) 0(0.0) 0(0.0) 0(0.0)

n=number sampled, No=Number, Preg=pregnant, %=percentage A=symptomatic Preg n=37(%), B=Symptomatic non-preg n=21(%), C=Asymptomatic Pregn n=41(%), D=Asymptomatic Non-preg n=27 (%)

Table 6: Resistance profile of yeasts to azoles among participants in Ekpoma

Yeasts No exa Flu MIC Itra MI

1. C. albicans 67 0.50^S 0.14^SDD

2. C. glabrata 26 12.00^SDD >32.00^R

3. C. tropicalis 15 1.00^S 0.50^S

C. parapsilosis 11 0.38^S 0.01^S

n=number sampled, Itra=Itraconazole, Flu=Fluconazole

1 2 3 4 5 6 7 8 9 10 11 12

Plate 1: Agarose gel electrophoresis photograph of UV ultra-illuminated DNA amplified products of suspect Candida dubliniensis separated.

Discussion

Vulvovaginal candidiasis remains one of the most frequently diagnosed inflammatory diseases of the vagina, which affects most sexually active women. In most patients, it is manifested as acute inflammation which is easy to diagnose and treat. However, in the susceptible population, it may be characterized by recurrent episodes, usually with an unknown cause or exacerbating moment. In resource-limited settings, VVC is not a reportable disease and is often diagnosed by attending clinicians without confirmatory tests and treated with over-the-counter (OTC) medications, and thus the exact incidence remains unknown.

Although VVC affects women globally, we are not aware of any population-based studies from different countries or regions of the world nor have we seen large studies from ethnically diverse regions to confirm whether incidence rates of VVC vary according to ethnic origin to warrant racial discrimination of the disease incidence especially in most African sub-regions with poor resources to confirm disease etiology before commencing treatment.

Our result of 47% to 68% range of virginal yeast carriage among symptomatic and asymptomatic women (Table 1) in this study is similar to the 55% prevalence reported from University female students in the United States (16, 17). It is also similar to 42% reported by Enweani et al in (17) and four years later (18) in this region. It is similar to the 40% reported by Ikit and Guzel (19). This range is higher than 20% to 11% reported in another setting by Spielberg et al, (20) and Richards et al, (21).

It is interesting to note that our result is in line with what other researchers in both the developing and developed world have established confirming the already known fact that VVC distribution is similar in most settings despite the demographic, racial, and geographic divide among the studied population as shown in Table 2 and 3. Host-related risk factors that have been significantly associated with VVC and RVVC include antibiotic use, uncontrolled diabetes, conditions with high reproductive hormone levels, and genetic predispositions to VVC (16, 17, 22).

It is clear that antibiotics alter the bacterial microflora of the vaginal and gastrointestinal tracts and thus allow for overgrowth of Candida spp. After antibiotic use, the increase in vaginal colonization with Candida spp., mostly C. albicans, is estimated to range from 10 to 30%, and VVC occurs in 28 to 33% of cases (22). It is commonly postulated that the reduction of lactobacilli in the vaginal tract predisposes women to VVC. Lactobacilli play a key role in the vaginal flora through the production of hydrogen peroxide, bacteriocins, and lactic acid, which protect against overgrowth of pathogenic species yeasts.

It has already been reported that diabetes is responsible for delayed wound healing which may harbor a mixed population of microorganisms including bacteria and fungi (23, 24) Diabetes-related infections is among the most common soft tissue infection associated with diabetes mellitus, with disease-related peripheral neuropathy and peripheral vascular disease playing major roles in this complication of diabetes.

More serious complications include failure of ulcers to heal and gangrene which may lead to osteomyelitis, amputation, and death. However, the exact importance of microorganisms in non-healing wounds whether internal or external, that do not show clinical signs of infection is presently being questioned concerning whether the density of microorganisms, the presence of specific pathogens, or total absence of microorganisms could be the critical factor determining whether a wound is likely to heal or experience delayed healing.

Finding an answer to this question is likely to outline the conditions which are conducive for the observed pattern of VVC in this study. In Nigeria and indeed many other African countries, little has been documented about diabetes care in most resource-limited settings. Fewer data exist for known cases and a high prevalence of unknown cases in where people only discover they are diabetic when they can no longer contain the associated complications makes management and control even worst. Lack of diabetes clinics in major hospitals and at the grass-root could explain the poor education of diabetic patients on what to do and how to manage the situation.

While Candida albicans showed a prevalence of 22.3% C glabrata showed 8.7% prevalence (Table 4). This is also very different from 16% virginal Candida infection among Ekpoma women reported eight years ago in Ekpoma by Agwu et al, (25). The 8.7% prevalence of C glabrata is similar to the 8% prevalence of re-current VVC in US women by (16, 17). From the foregoing, it appears that Candida distribution and resistance to surface-active agents is relatively similar in most populations with or without symptoms (Table 5) including the present study where the isolated C albicans and C glabrata had similar dose-dependent sensitivity to the two azoles tested (Table 6).

This observation is slightly different from what is known about the two yeast strains regarding the inherent resistance of C. glabrata to conventional antimicrobial agents compared to C. albicans which is hardly implicated in any refractile diseases. Thus one would have expected resistance in the mean MIC of C. glabrata resistance while that of C. albicans shows sensitivity.

A brief comparative analogy of the pathogenicity of the two yeasts may highlight the reason why we are surprised at this unusual similar activity of both yeasts in this study. Candida glabrata, formerly known as Torulopsis glabrata, contrasts with other Candida species in its non-dimorphic blastoconidial morphology and haploid genome (16). Although C. glabrata has few virulence attributes, the high mortality rate and the rapidity of the spread of disease would argue to the contrary (26, 17).

If C. glabrata is low in virulence, the lack of hypha formation may be a contributing factor because hypha formation is a recognized means of increased adherence, tissue invasion by C. albicans, a means of increasing proteolytic enzyme elaboration and antigen modulation with little, known proteinase production(17, 27).

Adherence is known to be an extremely important virulence factor, although the actual adherence property may be enhanced by other virulence properties. C. glabrata is not as sensitive or as influenced by environmental factors compared to C. albicans (28). C. glabrata may not express some specific adhesins found in C. albicans and thus would have a disadvantage in adherence (17). Extracellular membrane-damaging phospholipases are known virulence factors for C. albicans and phospholipase activity has not been studied in C. glabrata.

Although phenotypic switching was studied largely as an in vitro phenomenon, there is some evidence of in vivo phenotype switching and an association of switched phenotypes with virulence. Recently, it was determined that phenotype switching does occur in C. glabrata (17). Interestingly, such a phenomenon would occur in non-dimorphic organisms as well as in haploid organisms. Although the relationship of this C. glabrata phenotype switching to virulence is unknown, it may enhance virulence and play a role in causing symptomatic infection.

Little is known about host defense against C. glabrata infections. Since C. glabrata is a commensal organism similar to C. albicans, there are likely to be normal host mechanisms that effectively control C. glabrata, holding it in check and suppressing the expression of its pathogenic properties, thereby preventing infection. Unfortunately, C. glabrata does not induce endothelial-cell phagocytosis (17), suggesting that this endothelial-cell activity may be species specific or restricted to C. albicans alone.

While T cells may be important for the protection of some tissues against C. glabrata infection, antibodies are not critical to protection against C. glabrata infections (17). The use of PCR, carbohydrate assimilation system and chromogenic agar in characterizing the yeast isolates should have confirmed the identity of all isolates in a carefully designed experimental study like this (plate 1). However, the appearance of some yeast strains which could not be characterized and hence grouped under others shows the complexity in designing an investigation to isolate an organism where there is a mixed microbial population.

The unknown unclassified group of yeast showing some resistance may mean it may be any of the resistance yeast strains frequently isolated from human infection sites. Changing disease epidemiology makes it difficult to predict the transmission dynamics of microbial agents of infection at any one time and the emergence and re-emergence of disease pathogens.

Conclusion

Yeast prevalence in cases of V.V.C. may not be affected by the demographic and socio-cultural disposition of patients. We found that asymptomatic women like their symptomatic counterparts harbor different Candida strains and that there was cohabitation within the same individual. It was also concluded that symptomatic vaginal Candidiasis may result from a patient change in immune status which will allow the proliferation of the Candida strains that were kept in check.

Conflict of interest: This yeast study was supported in part by CHROMagar Corporation through their donation of the media for which presumptive Identification was made.

Acknowledgment:

Ann Natinza is acknowledged for assisting in the molecular identification protocol for the yeast isolates. Professor Victoria Pazos of Kampala International University for her Assistance in providing the enabling environment for yeast purification and quantification before the molecular analysis. Management of Irrua Specialist Hospital for granting the permission to travel to Uganda for the molecular aspect of the work.

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Colonization and susceptibility profile of vaginal yeast isolates in Ekpoma, Edo state Nigeria.

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