The etiology and treatment of the two most common infection-related vulvovaginal disorders, vulvovaginal candidiasis and bacterial vaginosis, remain unresolved. Vulvovaginal candidiasis is primarily caused by Candida albicans, a dimorphic yeast that is present as a commensal microorganism in up to 30% of reproductive-aged women.1 The mechanisms involved in conversion of this microbe into a vaginal pathogen are debatable and incompletely understood.2,3 In addition, about 5% of women with a first symptomatic episode will subsequently develop recurrent vulvovaginal candidiasis, defined as at least four culture-verifiable occurrences within a 1-year period.4 Bacterial vaginosis is characterized by greatly reduced levels of vaginal Lactobacillus species while concentrations of Gardnerella vaginalis, Mycoplasma hominis, and anaerobic Prevotella, Bacteroides, Porphyromonas, and Mobiluncus species drastically increase.5 There is a concomitant elevation in the vaginal pH and, in some women, a malodorous discharge. In a recent trial of antibiotic maintenance therapy, half the women with bacterial vaginosis experienced a recurrent episode within 3 months of cessation of treatment.6 The mechanism(s) responsible for alterations of the vaginal flora that are characteristic of bacterial vaginosis remain a matter of speculation.
The influence of the innate immune system on the development and recurrence of vaginal disorders has only recently begun to be explored. Mannose-binding lectin (MBL) is a protein present in the circulation and body secretions, which binds to mannose, glucosamine, and fucose residues present on microbial surfaces. As a consequence of this interaction, the microorganism reacts with receptors on phagocytic cells and is subject to opsonization. In addition, MBL activates the complement cascade resulting in direct microbial killing as well as promoting opsonization via complement receptors.7 Several studies have demonstrated that MBL binds to C albicans8 and participates in anti-Candida innate immunity.9–11
The gene coding for MBL, gene symbol MBL2, has three functional polymorphisms in exon I. The polymorphism at codon 54 is most common in white populations. The codon 57 polymorphism is primarily present in blacks, while the codon 52 polymorphism is present at a low frequency (less than 5%) in both populations.12 We have demonstrated that carriage of a single nucleotide guanine-adenine polymorphism in codon 54 of the MBL gene was associated with lowered vaginal concentrations of MBL protein and recurrent vulvovaginal candidiasis in Latvian women.13 This substitution results in the replacement of guanine with aspartic acid and synthesis of an MBL protein with greatly reduced stability.12,14 In this communication we estimated the association between the MBL codon 54 and 57 gene polymorphisms and acute or recurrent vulvovaginal candidiasis and bacterial vaginosis in Brazilian women.
MATERIALS AND METHODS
A cross-sectional study to assess the association between MBL codon 54 and 57 gene polymorphisms and acute and recurrent vulvovaginal candidiasis and bacterial vaginosis was conducted at two outpatient infectious diseases clinics in the gynecology departments at the State University of Campinas and the Federal University of Rio Grande do Norte in Brazil between August 2004 and February 2006. All subjects were sexually active, and no one refused to take part in the study. Women with recurrent problems were seen at least every 3 months over a 1-year period; women with a single acute episode were seen during the episode and followed over a 1-year period to document the absence of recurrences. Controls were randomly selected women seen for routine yearly examinations with no history of vulvovaginal candidiasis or bacterial vaginosis and had a Nugent Gram stain score15 of 1–3. Each participant underwent a speculum-based gynecological examination and filled out a detailed questionnaire in consultation with her physician. A history of allergy was defined, in consultation between patient and physician, as symptoms of sneezing, congestion, irritation, and/or itching in response to specific environmental exposures. Vulvovaginal candidiasis was diagnosed by the presence of Candida sp on a Sabouraud agar culture, a white vaginal discharge, and clinical symptoms of pruritus and/or erythema. Bacterial vaginosis was diagnosed by Nugent's criteria as a score between 7 and 10.15 Recurrent vulvovaginal candidiasis and bacterial vaginosis were defined as at least four separate microscopically confirmed symptomatic episodes within a 1-year period. Exclusion criteria were pregnancy, systemic disease, use of vaginal cream, treatment for a vaginal discharge within the previous month, lack of sexual intercourse for more than 3 months, and inability to give informed consent. The final study population was composed of 13 women with acute bacterial vaginosis, 20 with recurrent bacterial vaginosis, 28 with acute vulvovaginal candidiasis, 50 with recurrent vulvovaginal candidiasis, and 66 controls. None of the subjects were positive for both vulvovaginal candidiasis and bacterial vaginosis. Written informed consent was obtained from all subjects, and the study was approved by the institutional review board at each medical center.
To analyze for the MBL codon 54 and 57 gene polymorphisms, buccal cells were obtained with a cotton swab after rinsing of the mouth with water, and samples were stored at 4°C before shipping to New York at ambient temperature. The swabs were assigned a number and tested without knowledge of any clinical data. The cells on the swab were suspended by shaking in a 1% solution of the nonionic detergent, Brij 35 in Tris buffer containing 5 mg/mL proteinase K. Lysis was induced by incubation at 56°C for 60 minutes, and the proteinase K was subsequently inactivated by incubation at 95°C for 10 minutes. The extracts were diluted 1:5 in 10 mmol/L Tris–hydrogen chloride (HCl) that contained 1.5 mmol/L magnesium chloride (MgCl2), 50 mmol/L potassium chloride (KCl), 0.2 mmol/L each of deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), and thymidine triphosphate (TTP), 1.25 units of Taq DNA polymerase, and 30 pmol of oligonucleotide primers that amplified the codon 54 and 57 polymorphic regions of the MBL gene.16,17 The final volume was 0.05 mL. Samples were incubated in a thermal cycler for 2 minutes at 94°C, followed by 35 cycles of 94°C for 50 seconds, 58°C for 1.5 minutes, and 72°C for 15 seconds, and then by a final incubation at 72°C for 5 minutes. For the codon 54 polymorphism, the final polymerase chain reaction (PCR) products were digested at 37°C for 18 hours with Ban I endonuclease (New England BioLabs, Beverly, MA), and the resulting amplicon fragments were analyzed on 2% agarose gels after staining with ethidium bromide. The wild-type allele (allele A) was seen as two 260 and 89 base pair fractions, while the variant (allele B) yielded a single band of 349 base pairs. For the codon 57 polymorphism, the PCR product was incubated with Mbo II endonuclease (New England BioLabs). The wild-type allele is uncut (349 base pairs), and the variant (allele C) is cleaved into two 279 and 70 base pair fragments. Duplicate analysis of a subset of samples always yielded identical results.
Associations between MBL genotype or alleles and clinical variables were assessed by Fisher exact test. Genotype and allele frequencies were determined by direct counting and then divided by the number of chromosomes to obtain allele frequency and by the number of women to obtain genotype frequency. Goodness of fit to Hardy-Weinberg equilibrium was determined by comparing the expected genotype frequencies with the observed values, using the χ2 test. P<.05 was considered significant.
The demographics and history of the study subjects is shown in Table 1. There was a higher frequency of vulvovaginal candidiasis among whites (P=.007) and a trend toward an increased frequency of bacterial vaginosis in nonwhites that did not reach statistical significance (P=.05). Both vulvovaginal candidiasis and bacterial vaginosis were associated with a history of allergy (P<.02), vulvovaginal candidiasis was strongly associated with oral contraceptive use (P<.001), and both vulvovaginal candidiasis and bacterial vaginosis were associated with having sexual intercourse more than three times a week (P<.001). There were no differences in age, education, or smoking history among the three groups. All results were consistent between the two individual clinics.
The MBL genotype distribution from the control women was in Hardy-Weinberg equilibrium (P=.691), indicating that these subjects were representative of the overall population. The association between clinical diagnosis and MBL genotype and alleles is shown in Table 2. In women with recurrent vulvovaginal candidiasis, but not in those with a single acute episode of vulvovaginal candidiasis, the frequency of the variant MBL allele B was about 2½ times that of the control population (P=.004). Similarly, women with recurrent bacterial vaginosis, but not those with a single acute bacterial vaginosis occurrence, had an allele B frequency twice that of the controls; this did not quite reach statistical significance (P=.06). However, allele B was more prevalent in women with recurrent bacterial vaginosis (22.5%) than in those with acute bacterial vaginosis (0%) (P=.009). There were no associations between MBL genotype and race or history of allergy (Table 3).
Only eight women (4.5%) were positive for the polymorphism at codon 57. Three patients with recurrent bacterial vaginosis, two with recurrent vulvovaginal candidiasis, and two controls were heterozygotes; one control woman was homozygous for this polymorphism. Thus, there was no association between codon 57 polymorphism carriage and either vulvovaginal candidiasis or bacterial vaginosis. Five (62.5%) of the women with the polymorphism were nonwhite.
Responses of different individuals to any given microorganism exhibit considerable variability. The recognition and elucidation of factors that determine susceptibility to specific outcomes are critical for development of more focused treatments with improved efficacy for individual women.18,19 The present investigation highlights the influence of a specific polymorphism in codon 54 in the MBL2 gene on recurrent vulvovaginal candidiasis and bacterial vaginosis in Brazilian women. Clearly, variations in a single gene are inadequate to fully explain the etiology of complex disease entities. In addition, the possibility remains that the MBL polymorphism is not directly involved in development of recurrent vulvovaginal candidiasis or recurrent bacterial vaginosis but rather is in linkage disequilibrium with an as yet unidentified causative marker. Further studies of independent populations are needed to confirm our observations. We have previously reported very similar findings in women with recurrent vulvovaginal candidiasis from eastern Europe.13 The two combined studies emphasize that this polymorphism may be one determinant of susceptibility to recurrent vulvovaginal candidiasis. The present investigation also extends these findings to include susceptibility to recurrent bacterial vaginosis. Bacterial vaginosis is characterized by large increases in vaginal concentrations of a variable number of bacterial species.4,6 Many of these bacteria have been shown to be recognized by MBL.8,20,21 Although the factors involved in conversion of a healthy vaginal flora to one characteristic of bacterial vaginosis remain unknown, the present observations implicate MBL as one component of the vaginal milieu that protects against the repeated proliferation of an atypical vaginal microflora. The finding that the MBL polymorphism was not associated with acquisition of a single incident of bacterial vaginosis suggests that multiple factors besides local MBL concentrations are capable of triggering a single episode of altered vaginal bacteria.
Because the population of Brazil is a heterogeneous mixture of whites, blacks, and native Indians, we assessed whether the MBL2 codon 57 polymorphism was also associated with recurrent vulvovaginal disorders. This polymorphism is almost exclusively present in black individuals.12 However, identification of this polymorphism was infrequent in our study and control populations, and further studies on much larger numbers of subjects will be required to determine the possible influence of codon 57 carriage on vulvovaginal candidiasis and bacterial vaginosis.
The MBL codon 54 polymorphism is common in the general population, and most individual female carriers do not have recurrent vaginal disorders. In the absence of a specific trigger, such as a transient deficit in cell-mediated immunity in women positive for commensal carriage of C albicans in their vagina, or exposure to unknown events that alter the vaginal microbial milieu, women positive for the MBL polymorphism will probably not be at a heightened risk for developing recurrent vulvovaginal candidiasis or bacterial vaginosis. In fact, an MBL deficiency appears to be beneficial against infection under certain conditions. Mycobacterium tuberculosis penetrates into host cells by an MBL-mediated mechanism, and therefore, decreased MBL levels reduce the likelihood of infection by this microorganism.22
Vulvovaginal candidiasis was more common in white than in nonwhite Brazilian women, and there was an opposite trend of bacterial vaginosis being more prevalent in the nonwhite patients. In addition, a history of allergy was associated with both vulvovaginal candidiasis and bacterial vaginosis. However, the MBL codon 54 gene polymorphism was not associated with either ethnicity or allergic history. The not unexpected conclusion from these observations is that susceptibility to recurrent vaginal complaints is multifactorial and that an MBL genetic polymorphism is just one pathway leading to development of these disorders.
Women with a history of recurrent vulvovaginal candidiasis or bacterial vaginosis might benefit from testing for the MBL codon 54 gene polymorphism. If they belong to the subset whose disorder is associated with allele B carriage, a more prolonged course of antibiotic treatment might compensate for their relative deficiency in this innate defense mechanism. It also remains to be determined whether administration of purified or recombinant MBL as a preventive or therapeutic measure will decrease the incidence of vulvovaginal candidiasis and bacterial vaginosis recurrences in allele B–positive women with this history. Studies of MBL-mediated infusions in humans have already been reported, with no adverse consequences and preliminary indications of success.23
1. Giraldo P, von Nowaskonski A, Gomes FA, Linhares I, Neves NA, Witkin SS. Vaginal colonization by Candida
in asymptomatic women with and without a history or recurrent vulvovaginal candidiasis. Obstet Gynecol 2000;95:413–6.
2. Witkin SS, Giraldo PC, Linhares I. New insights into the immune pathogenesis of recurrent vulvovaginal candidiasis. It J Gynecol Obstet 2000;12:114–8.
3. Fidel PL Jr. History and new insights into host defense against vaginal candidiasis. Trends Microbiol 2004;12:220–7.
4. Sobel JD. Epidemiology and pathogenesis of recurrent vulvovaginal candidiasis. Am J Obstet Gynecol 1985;152:924–35.
5. Amsel R, Totten PA, Spiegel CA, Chen KC, Eschenbach D, Holmes KK. Nonspecific vaginitis: diagnostic criteria and microbial and epidemiologic associations. Am J Med 1983;74:14–22.
6. Sobel JD, Ferris D, Schwebke J, Nyirjesy P, Wiesenfeld HC, Peipert J, et al. Suppressive antibacterial therapy with 0.75% metronidazole vaginal gel to prevent recurrent bacterial vaginosis. Am J Obstet Gynecol 2006;194:1283–9.
7. Eisen DP, Minchinton RM. Impact of mannose-binding lectin on susceptibility to infectious diseases. Clin Infect Dis 2003;37:1496–505.
8. Neth O, Jack DL, Dobbs AW, Holzel H, Klein NJ, Turner MW. Mannose-binding lectin binds to a range of clinically relevant microorganisms and promotes complement deposition. Infect Immun 2000;68:688–93.
9. Ip WK, Lau YL. Role of mannose-binding lectin in the innate defense against Candida albicans
: enhancement of complement activation, but lack of opsonic function, in phagocytosis by human dendritic cells. J Infect Dis 2004;190:632–40.
10. Pellis V, De Seta F, Crovella S, Bossi F, Bulla R, Guaschino S, et al. Mannose binding lectin and C3 act as recognition molecules for infectious agents in the vagina. Clin Exp Immunol 2005;139:120–6.
11. Lillegard JB, Sim RB, Thorkildson P, Gates MA, Kozel TR. Recognition of Candida albicans
by mannan-binding lectin in vitro and in vivo. J Infect Dis 2006;193:1589–97.
12. Babovic-Vuksanovic D, Snow K, Ten RM. Mannose-binding lectin (MBL) deficiency. Variant alleles in a Midwestern population of the United States. Ann Allergy Asthma Immunol 1999;82:134–43.
13. Babula O, Lazdane G, Kroica J, Ledger WJ, Witkin SS. Relation between recurrent vulvovaginal candidiasis, vaginal concentrations of mannose-binding lectin, and a mannose-binding lectin gene polymorphism in Latvian women. Clin Infect Dis 2003;37:733–7.
14. Lipscombe RJ, Sumiya M, Summerfield JA, Turner MW. Distinct physiochemical characteristics of human mannose binding protein expression by individuals of differing genotype. Immunology 1995;85:660–7.
15. Nugent RP, Krohn MA, Hillier SL. Reliability of diagnosing bacterial vaginosis is improved by a standardized method of gram stain interpretation. J Clin Microbiol 1991;29:297–301.
16. Madsen HO, Garred P, Kurtzhals JA, Lamm LU, Ryder LP, Thiel S, et al. A new frequent allele is the missing link in the structural polymorphism of the human mannan-binding protein. Immungenetics 1994;40:37–44.
17. Babula O, Danielsson I, Sjoberg I, Ledger WJ, Witkin SS. Altered distribution of mannose-binding lectin alleles at exon I codon 54 in women with vulvar vestibulitis syndrome. Am J Obstet Gynecol 2004;191:762–6.
18. Witkin SS, Linhares I, Giraldo P, Jeremias J, Ledger WJ. Individual immunity and susceptibility to female genital tract infection. Am J Obstet Gynecol 2000;183:252–6.
19. Ledger WJ, Witkin SS. Are you a lumper or a splitter? Am J Obstet Gynecol 2006;195:1205–9.
20. Townsend R, Read RC, Turner MW, Klein NJ, Jack DL. Differential recognition of obligate anaerobic bacteria by human mannose-binding lectin. Clin Exp Immunol 2001;124:223–8.
21. Hamvas RM, Johnson M, Vlieger AM, Ling C, Sherriff A, Wade A, et al. Role for mannose binding lectin in the prevention of mycoplasma infection. Infect Immun 2005;73:5238–40.
22. Soborg C, Madsen HO, Andersen AB, Lillebaek T, Kok-Jensen A, Garred P. Mannose-binding lectin polymorphisms in clinical tuberculosis. J Infect Dis 2003;188:777–82.
© 2007 by The American College of Obstetricians and Gynecologists. Published by Wolters Kluwer Health, Inc. All rights reserved.
23. Jensenius JC, Jensen PH, McGuire K, Larsen JL, Thiel S. Recombinant mannan-binding lectin (MBL) for therapy. Biochem Soc Trans 2003;31:763–7.