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Alterations in the Genital Microbiota in Women With Spinal Cord Injury

Pires, Cristhiane V.G., PT; Linhares, Iara M., MD, PhD; Serzedello, Felipe, MD; Fukazawa, Eiko I., MD; Baracat, Edmund C., MD, PhD; Witkin, Steven S., PhD

doi: 10.1097/AOG.0000000000001257
Contents: Original Research
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OBJECTIVE: To evaluate the vaginal and cervical microbiota in women with spinal cord injury compared with mobile women.

METHODS: Fifty-two women with spinal cord injury (study group) and 57 mobile women (control group) were evaluated in a case–control study. All answered a structured questionnaire and were submitted to the following microbiological tests: microscopic examination of vaginal secretions for Trichomonas vaginalis and yeasts, Nugent score by Gram stain, bacterial culture, yeast culture, and endocervical sampling for Chlamydia trachomatis, Neisseria gonorrhoeae, and Mycoplasma species.

RESULTS: Candida species detected by direct microscopic examination of vaginal fluid was more common in women with spinal cord injuries than in control women: 17.3% (9/52) compared with 3.5% (2/57), respectively (P=.017). However, the frequency of yeast-positive cultures was similar in both groups (21.2% [10/52] compared with 15.8% [14/57]). Women with spinal cord injury were more likely to have positive vaginal cultures for Escherichia coli (15.4% [8/52] compared with 0% [0/57], P=.002) and Corynebacterium species (25.0% [13/52] compared with 8.8% [5/57], P=.037) and less likely for Lactobacillus species (63.5% [33/52] compared with 94.7% [54/57], P<.001). Women with spinal cord injury were more likely to have intermediate flora by Gram stain (Nugent score 4–6) than did the women in the control group (13.5% [7/52] compared with 1.8% [1/57], P=.033). The frequency of Mycoplasma species detection was similar in both groups (36.9% [18/52] compared with 34.6% [21/57]). No woman in either group was positive for T vaginalis, C trachomatis, or N gonorrhoeae.

CONCLUSION: Women with spinal cord injury have an alteration in their vaginal microbiota away from a Lactobacillus species–dominated flora and a higher concentration of vaginal Candida species than do mobile women.

Women with spinal cord injuries have lower vaginal levels of Lactobacilli spp. and higher levels of Candida spp. Corynebacterium spp., and Escherichia coli.

Discipline of Gynecology, Department of Obstetrics and Gynecology, College of Medicine, University of São Paulo, São Paulo, Brazil; and the Department of Obstetrics and Gynecology, Weill Cornell Medical College, New York, New York.

Corresponding author: Cristhiane V.G. Pires, PT, Praça Capitão Alberto Mendes Júnior Centro, 71, Torre 3, Apto 132, Guarulhos, São Paulo 07012-031, Brazil; e-mail: c.garabellopires@gmail.com.

Financial Disclosure The authors did not report any potential conflicts of interest.

The authors thank Fleury Laboratories, São Paulo, Brazil, for providing the vaginal cultures for our study participants and performing the laboratory tests.

Spinal cord injury is a major concern for the public health system. The majority of patients are young and at the height of their productive lives. The injuries in this serious disability syndrome encompass various degrees of difficulty in mobility, lack of sensitivity, and loss of sphincter control.1,2 Because approximately 80% of spinal cord injuries affect men, studies have rarely focused on the needs of women with spinal cord injury.3

A major effect on the quality of life in women with spinal cord injury is decreased or absent genitourinary sensitivity. A second major concern is the loss of urinary sphincter control, identified as a neurogenic bladder. Most of the time these women need to use diapers and absorbent pads for their continuous urinary loss, regardless of their use of intermittent urinary catheterization. All of these conditions can increase the likelihood of genital tract infections and an altered vaginal microbiota in women with spinal cord injury.4,5

The aim of the present investigation was to evaluate the genital tract microbiota in women with spinal cord injury and to determine the likelihood of susceptibility to urinary tract infections. Little is known about the genital tract microbiota in women with spinal cord injuries.

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MATERIALS AND METHODS

Fifty-two women with spinal cord injury (study group) and 57 mobile women (control group) were evaluated in a case–control study. This spinal cord injury population was obtained from The Association of Assistance to Children with Disability Ibirapuera Unit—São Paulo. Fifty-two women registered in The Service of Medicals Archive from April 2010 until December 2014 were invited to participate and all agreed. The control group consisted of women seen for an annual health checkup as well as relatives of women with spinal cord injury.

All personal and clinical information was obtained from electronic records, including the extent of disease as characterized by the International Statistical Classification of Diseases and Related Health Problems and the American Spine Injury Association. American Spine Injury Association A is defined as a total lesion without any motor function and sensation below the level of the lesion, American Spine Injury Association B is the loss of motor function with a preservation of sensitivity below the lesion, American Spine Injury Association C is preservation of motor function below the lesion with diminished muscle strength, and American Spine Injury Association D is preservation of motor function below the neurologic level with moderate muscle strength.

Inclusion criteria for the spinal cord injury women were: living in the metropolitan region of Sao Paulo; premenopausal age; sexually active (current or previous); spinal cord injury diagnosis by clinic investigation and radiography, tomography, or magnetic resonance evaluation; tetraplegic or paraplegic; no cognitive disturbance; and time of injury more than 6 months. Exclusion criteria were: clinical instability; pregnancy; use of antibiotics, corticosteroids, or vaginal creams in the previous 30 days; intercourse in the last 72 hours; and associated diseases (autoimmune diseases, diabetes, lupus erythematosus, arthritis rheumatoid, hepatitis, malignancies, and human immunodeficiency virus-positive).

The control group was composed of sexually active women of reproductive age without physical disabilities with no signs or symptoms of a genital tract disturbance. The exclusion criteria were the same as for the spinal cord injury group.

The project was approved by the Research Ethics Committee of the Department of Gynecology and Obstetrics from Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo and the Ethics Committee for Research Project Analysis protocol no. 229/12, receiving number of Certificate of Presentation to Ethics Assessment 02216612.0.0000.00.65 by the Brazil Plataform. All patients provided informed written consent.

Personal data and aspects of gynecologic health were collected by interview and a structured questionnaire. After this, patients meeting the inclusion criteria were sent to a commercial laboratory for direct microscopic examination of vaginal secretions for Trichomonas vaginalis and yeasts, Nugent score analysis by Gram stain,6 bacterial culture on agar blood medium, yeast culture on Sabouraud medium, and endocervical sampling for Chlamydia trachomatis and Neisseria gonorrhoeae by polymerase chain reaction and Mycoplasma culture on U9, A7 medium.

Statistical analysis included comparing the absolute and relative frequencies of individual microorganisms in spinal cord-injured and control women by Fisher exact test.7 The level of significance was set at 5%. The analyses were conduced using Microsoft Excel 2003 and SPSS 20.0.

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RESULTS

Demographic information for women in both groups is described in Table 1. The age distribution and mean age of women in the spinal cord injury group, 33.9 years (range 20–49 years), was similar to the women in the control group, 33.1 years (range 20–49 years). A comparable percentage of women in the spinal cord injury (36.5%) and control (31.6%) groups were married. The majority of women in the spinal cord injury and control groups were white (69.2% compared with 71.9%) or of mixed race (26.9% compared with 17.5%), respectively, and these differences were not significant. Women with spinal cord injury had a lower level of education (P<.001) and a higher frequency of being unemployed (P<.001) compared with the women in the control group.

Table 1

Table 1

The microbiological analyses are shown in Table 2. On microscopic examination of fresh vaginal secretions, 9 of 52 (17.3%) women with spinal cord injury compared with 2 of 57 (3.5%) women in the control group were positive for yeast (P=.024, 95% confidence interval [CI] 1.181–28.047). However, when the vaginal secretions were cultured 11 of 52 (21.2%) of the women with spinal cord injury and 9 of 57 (15.8%) of the women in the control group were positive for yeast species. This difference was not significant. Candida albicans was identified by culture in seven women in each group, and non-albicans Candida yeast was present in four women with spinal cord injury and in two women in the control group. By culture, Lactobacillus species was identified in 63.5% of patients with spinal cord injury (33/52) as opposed to 94.7% (54/57) of the women in the control group (P<.001, 95% CI 2.845–37.750). Conversely, Escherichia coli was cultured from 8 of 52 women (15.4%) with spinal cord injury and in none (0/57) of the women in the control group (P=.002, 95% CI 1.234–391.18) and Corynebacterium species was present in 13/52 (25.0%) of the patients with spinal cord injury as opposed to 5 of 57 (8.8%) of the women in the control group (P=.037, 95% CI 1.140–10.541). There were no differences in isolation rates for Gardnerella, Staphylococcus, Streptococcus, or Enterococcus species. The frequency of detection of Mycoplasma hominis (5.8% compared with 5.3%) and Ureaplasma urealyticum or parvum (28.8% compared with 31.6%) was similar in the spinal cord injury and control groups. No woman in either group was positive for T vaginalis, C trachomatis, or N gonorrhoeae. Women with spinal cord injury had an 83% less chance of being positive for Lactobacillus species as compared with women in the control group (P=.018).

Table 2

Table 2

The Nugent score is a method to classify the bacteria present on a Gram stain of vaginal secretions based on bacterial morphotypes. A score of 1–3 indicates a predominance of Lactobacilli species, a score of 4–6 indicates a decrease in Lactobacilli species and an increased proportion of other bacteria, and a score of 7–10 indicates a substantial loss of Lactobacilli species accompanied by a very large increase in the numbers of other bacteria and indicates a diagnosis of bacterial vaginosis. A Nugent score of 1–3 was identified in 78.8% (41/52) of women with spinal cord injury and in 93.0% (53/57) of women in the control group (P=.049). Conversely, a Nugent score of 4–6 was present in vaginal secretions from 13.5% (7/52) of women with spinal cord injury compared with 1.8% (1/57) of women in the control group (P=.033, 95% CI 1.003–69.095). Secretions from very few women, 7.7% (4/52) with spinal cord injury and 5.3% (3/57) in women in the control group, had a Nugent score 7–10 and this difference was not significant.

Relating the bacterial findings to the degree of spinal cord injury (Table 3), women classified as American Spine Injury Association A had a much higher frequency of Corynebacterium species isolation (12/30) as compared with women with American Spine Injury Association B, C, and D (1/22, P=.011, 95% CI 1.655–118.45). There were no other differences in microbial detection by American Spine Injury Association classification in women with spinal cord injury. Women in the spinal cord injury group who had poor bladder control (spontaneous unintentional loss of urine) had a lower isolation frequency of Lactobacillus species (17/34 [50.0%]) than did women with good urine control (16/18 [88.9%]) (P=.007, 95% CI 1.588–40.296). Conversely, Corynebacterium species were detected in 38.2% (13/34) of women with urine loss as opposed to no women with good urine control (P=.002, 95% CI 1.290–418.45) (Table 4). Women with spinal cord injury who continually used diapers or pads (n=34) had a higher isolation frequency only of Corynebacterium species as compared with the other (n=18) patients with spinal cord injury (P=.019, 95% CI 1.247–88.789). There was no association between intermittent catheterization in the study group and the isolation of specific bacteria or an altered Nugent score (data not shown). Patients with spinal cord injury who currently engaged in sexual intercourse (n=27) did not differ in bacterial analysis from women who were not now sexually active (n=25).

Table 3

Table 3

Table 4

Table 4

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DISCUSSION

Women with spinal cord injury had an increased occurrence of Candida species, E coli, and Corynebacteria species and a lower detection rate of Lactobacilli species in their vaginas than did a matched group of fully mobile women. An absence or reduction in vaginal sensation leading to alterations in hygiene coupled with involuntary urination and prolonged use of absorbent pads likely contribute to these differences.

Vulvovaginal complaints are among the most common reasons that women seek gynecologic care.8,9 However, in women with spinal cord injury, variations in the vaginal microbiota that typically result in clinical symptoms may go unnoticed as a result of the loss of sensitivity in the genital area. Nevertheless, these alterations can indicate the presence of local immune system alterations that predispose to development of more serious conditions such as urinary tract or bladder infections or acquisition of sexually transmitted diseases.

In the present study it was not surprising that Candida species was cultured from a similar number of women with spinal cord injury and women in a control group. Candida species, especially C albicans, are frequent low-level components of the vaginal microbiota in healthy women.10,11 However, the detection of Candida species by direct microscopic examination of vaginal secretions was more frequent in women with spinal cord injury than in women in a control group (P=.017). This clearly indicates that the vaginal concentration of Candida species was higher in the spinal cord injury group. Women with spinal cord injury may not notice the burning and itching typically associated with an elevated concentration of vaginal Candida species.12,13 Nevertheless, its occurrence can be an indication of altered vaginal antimicrobial immunity and an increased likelihood of atypical bacterial growth.

Consistent with this observation, the presence of E coli and Corynebacterium species was more frequently detected by culture in women with spinal cord injury. Conversely, Lactobacilli species were less frequently isolated. Also, Gram stain secretions from women with spinal cord injury were less likely to have a Nugent score indicative of a Lactobacillus-dominated vaginal microbiota and more likely to have a score consistent with an altered microbiota. Thus, women with spinal cord injury are more likely than other women to have an altered bacterial and fungal vaginal microbiota.

The immunologic changes leading to this altered vaginal microbiota in women with spinal cord injury and the possible medical consequences remain to be determined. Changes in the vaginal flora, especially the reduction of Lactobacillus species, have been shown to predispose to colonization of the vagina by E coli in women without spinal cord disability.14E coli is a frequent urinary tract pathogen.15Corynebacterium species are frequent components of the bacterial vaginal microbiota in healthy asymptomatic women and do not typically cause symptoms.16 However, when vaginal immunity is impaired, Corynebacterium species has been associated with the occurrence of urinary tract infections.17 In women with spinal cord injury classified as American Spine Injury Association A, who have no genital sensation and are totally dependent on wheelchairs, physical alterations in the vulvovaginal area resulting from their sedentary life may increase the likelihood of Corynebacterium species prolifereration.18,19 In support of increased Corynebacterium species growth under an altered vaginal milieu, the continuous use of pads and diapers by women in our study was associated with isolation of Corynebacterium species (P<.023). There are no prior studies related to preferential Corynebacterium species proliferation in women with spinal cord injury. Studies in populations without spinal disability have reported that the presence of Corynebacterium species is related to the production of lithiasis kidney.20

There are no reports in the literature citing a reduction of Lactobacillus species in women with spinal cord injury. However, there are studies that the overuse of antibiotics complicates the resolution of a neurogenic bladder in people with spinal cord injury21,22 as well as the frequent use of antibiotics for prophylaxis against the occurrence of urinary tract infections.23,24 There is a link between a decrease of Lactobacillus species in the vaginal flora and antibiotic use.25,26 The history of antibiotic use in women with spinal cord injury and its relation to vaginal Lactobacilli species remains to be determined.

Lactobacillus species are constituents of normal bacterial flora of the vagina and play an important role in maintaining the balance between commensal microorganisms and pathogenesis.18,19 In the present study, a lower frequency of Lactobacillus species and increased Corynebacterium species in the population with spinal cord injury and urinary loss could be associated with an increased prevalence of some other microorganism that was not isolated by the culture medium used for this study. There are no studies in the literature that related urinary loss with the presence of microorganisms such as Corynebacterium species and a lower frequency of Lactobacillus species in vaginal culture in women with spinal cord injury. A recent prospective study in the United States of 161 male and female patients with spinal cord injury showed that 82.7% of individuals with involuntary urinary loss evolved into urologic complications such as renal atrophy andhydronephrosis.27 Therefore, vaginal bacterial alterations in women with spinal cord injury need further assessment for possible predisposing factors for urinary, bladder, and kidney problems.

Several authors described the technique of intermittent catheterization as a protective factor against development of pathogenic microorganisms in the urogenital system.28,29 Rabadi and Aston evaluated the risks and complications resulting from a neurogenic bladder in people with spinal cord injury and found that patients who performed intermittent catheterization were less frequently positive for E coli in urine culture tests.27,29 However, in the present study we did not find any association between intermittent catheterization and E coli detection. This may perhaps be the result of the low number of women in our study who used this procedure and further investigations are indicated.

The strength of the study was our relationship with the spinal cord-injured women, allowing us to easily recruit all participants who were approached and their willingness to have samples obtained for microbiological testing in a clinical laboratory. Having all testing performed in the same laboratory under identical conditions also insured uniformity of results. A limitation of the study is that the small sample size precluded analyzing differences between women who were paraplegic and tetraplegic. In addition, we did not perform a power analysis for this study because there were no prior data for guidance. The relatively small sample size indicates that our study is hypothesis-testing and primarily descriptive and is underpowered to detect meaningful differences between groups. Further research is needed to validate and extend our observations.

The study reinforces the need for regular periodic gynecologic care, including evaluation of vaginal microorganisms, in women with spinal cord injury. They appear to have an increased occurrence of an atypical vaginal microbiota that may elevate their susceptibility to urinary and genital tract infections.

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