Vulvovaginitis is recognized as a common complaint of the genital system in prepubertal and pubescent girls that causes anxiety in both parents and girls. Different physical, chemical or infectious agents have been implicated as causes of this clinical entity.1 Although the suggestion of sexual abuse should always be investigated and ruled out, previous data suggest that, mostly in prepubertal girls, vulvovaginitis is usually nonspecific, caused by irritants, allergic reactions or dermatological conditions.2
Before puberty, the lack of estrogen and the generally neutral or alkaline vaginal pH with few or no lactobacilli create a favorable environment for infections.1, 3
On the contrary, puberty represents an important transition period from childhood to adulthood. With its onset, lactobacilli increase and become the predominant part of the vaginal microbiome, and the pH becomes acidic.4 In addition to maintaining an acidic vaginal pH, lactobacilli play an important role in minimizing the risk of vaginal infections.
Any disruption to the healthy vaginal environment may predispose the patient to vulvovaginitis. In the pubertal patient, infectious vulvovaginitis is more common and sexually transmitted infections should also be considered and investigated.5
The female anatomy, with the urethra and anal region in close proximity to the vagina, puts the genital tract at risk of infection, particularly when local hygiene is poor or inadequate. Furthermore, oropharyngeal pathogens can easily reach the genital area through autoinoculation. Such infection, if not responsive to hygienic measures, needs further investigation as treatment must be tailored to each specific patient.
Microbiological diagnosis, through microscopic evaluation and cultures of vulvovaginal samples, can be an important tool to exclude or identify pathogens involved in these infections. However, the significance of pathogens isolated from vaginal cultures should be evaluated only after taking into account clinical information and potential risk factors, if any.
The authors conducted this study to investigate the pathogens isolated in prepubertal and pubertal girls who presented to the hospital with signs and symptoms of vulvovaginitis.
| Materials and methods |
This was a retrospective analysis of data from 2314 girls aged 2 to 16 years, who attended the Pediatric and Adolescent Gynecology Outpatient Clinic of the Aretaieio University Hospital, with symptoms suspicious of vulvovaginitis (vaginal discharge, genital erythema, pruritus, pelvic pain, vulvodynia , bad odor or bleeding), between January 2009 and December 2020. All cases referring to recurrent infections, vaginal foreign body or sexual abuse were excluded, as well as all girls who were receiving antibiotic treatment at that time or in the month former.
Vaginal samples were collected by pediatric and adolescent gynecologists using a sterile newborn suction catheter carefully inserted into the vagina and immediately sent to the microbiology laboratory. Wet mounts and Gram stains were examined microscopically for the presence of leukocytes, epithelial cells, trichomonads, clue cells, hyphae, pseudohyphae, or budding yeast.
For the diagnosis of bacterial vaginosis (BV), the Nugent score was applied to Gram-stained vaginal smears and the number of lactobacilli, Gardnerella vaginalis, and variable small and/or curved Gram morphotypes were evaluated.6 A score > 6 was indicative. from VB. Additionally, vaginal samples were directly inoculated onto 5% sheep blood agar, MacConkey agar, mannitol salt agar, chocolate agar, and Wilkins Chalgren agar, as well as Sabouraud dextrose agar and Gardnerella agar, followed by incubation in an aerobic, anaerobic, or CO2 at 37 °C for 24 or 48 h, as appropriate.
All microorganisms classified as pathogens were grown as pure isolates or as clearly dominant bacteria in the culture plates and were subsequently identified by conventional methods, using morphological and biochemical characteristics, Gram stain, while the definitive identification was performed using the automated system. VITEK. 2
In girls with genital pruritus or anal itching, a Graham test for oxyurius was performed in the early morning for 3 consecutive days, by pressing a strip of adhesive tape to the skin adjacent to the anus which was then removed and placed on a slide for examination. the presence of Enterobius vermicularis eggs by microscopy. Statistical analysis was performed with SPSS for Windows version 25.0. Categorical variables were analyzed using the chi-square test for comparison of data between groups. Statistical significance was set at a p value of 0.05.
| Results |
Over a 12-year period , 2,314 pediatric patients presenting with symptomatic vulvovaginitis were included in this study. The cases were divided into 2 groups: 1,094 girls in a prepubertal state, Tanner stage 1, with absence of secondary sexual characteristics, and 1,220 pubertal girls in Tanner stage ≥2. The most common clinical symptoms at presentation were vaginal discharge, genital erythema, and pruritus.
Symptoms were significantly more prevalent in pubertal girls, except vulvodynia , which was more common among prepubertal girls. A vaginal sample was collected from each patient; Thus, 2314 samples were taken in total.
Symptoms were significantly more (P < 0.01) positive cultures recorded in girls at puberty (926/1220, 75.9%) compared to the results obtained in the group of girls before puberty (587/1094, 53 .7%). A greater number of pathogens was detected in the vagina in samples from pubescent girls compared to prepubertal girls (984 vs. 613, P < 0.01)
Among the Gram-positive cocci and Gram-negative bacilli isolated in this study, fecal pathogens were the most prevalent.
An unexpected finding was the increased prevalence of BV in both groups, although it increased significantly in pubertal girls (P < 0.01). Finally, Candida species were mainly isolated from pubertal girls.
| Discussion |
Vulvovaginitis is one of the most common gynecological complaints in the pediatric population. The microbial flora in girls with clinical signs and symptoms of vulvovaginitis is variable. What is considered normal vaginal flora in different age periods is still a matter of controversy, and different data can be found in the literature. The vaginal microbiome is complex and the presence of potential pathogens does not necessarily imply that they are responsible for the infection.7-9 Therefore, vaginal cultures obtained from cases of vulvovaginitis should be evaluated cautiously before choosing a specific antibacterial treatment.
The common causes of vulvovaginitis in the prepubertal patient are different from those considered in the pubertal patient. When girls present with vulvar itching, burning, or pain, the most common etiology is nonspecific vulvovaginitis; no pathogen can be isolated and hygiene measures are recommended.5 Furthermore, younger girls cannot accurately express their symptoms; Therefore, the clinical examination must make the distinction between nonspecific and infectious vulvovaginitis.1
Much attention is required in girls with symptoms secondary to intravaginal foreign bodies or sexually transmitted infections, where the concern of specialists should focus on the possibility of sexual abuse.1, 2
In this study, although a specific pathogen was not isolated in all cases, the symptoms of all our patients were characteristic of vulvovaginitis. As previously reported, the most common complaints detected in patients were vaginal discharge, vulvar erythema, and pruritus.10-12 Pathogens can reach the genital area from the surrounding skin, as well as the rectal and urethral area.1
The results of vaginal cultures obtained from prepubertal girls differed from those sampled from pubescent girls. In prepubertal girls, Gram-positive cocci and Enterobacteriaceae were mainly isolated as demonstrated in previous studies.11, 13 In contrast, vulvovaginal candidiasis and BV were frequently detected in adolescents, as previously published.2, 14–16 To the best of our knowledge, there are very few reports in the literature that comprise series as large as those presented in this study.
The most frequently isolated pathogens in symptomatic pediatric patients with vulvovaginitis include respiratory tract and enteric microorganisms.3
Haemophilus influenzae is a common respiratory tract pathogen. Young children, in particular, tend to have a higher incidence of upper respiratory tract infections and, by touching their nose and mouth with their hands, can easily spread pathogens to other anatomical regions, such as the genital area. Although previous studies indicated that H. influenzae type b was one of the most common pathogens isolated in girls with vulvovaginitis, the introduction of the respective vaccine against this pathogen resulted in a significant decrease in its prevalence, thus protecting against respiratory tract infections and as result, against vulvovaginitis.1, 8, 17, 18
Cases that are still reported, even in vaccinated populations, are probably mostly caused by nonencapsulated or nontypeable strains of H. influenzae. The low rate detected in this study can be attributed to the implementation of the vaccine starting in 1995 in Greece, and this result is in agreement with reports from different countries where vaccination is available.13, 19, 20 On the contrary, a recent report identified H. influenzae as the second most common pathogen in preschool girls with a prevalence of 27.2%, while McGreal and Wood reported it as the third most common pathogen with a prevalence of 10% in prepubertal girls. 12, 21
Streptococcus pyogenes (group A beta-hemolytic streptococcus) is an important human pathogen responsible for different infections in the human body. It has often been reported to be the most common or one of the most common causes of vulvovaginitis in girls.8, 12, 13, 19, 20, 22 This is a common upper respiratory tract pathogen and therefore, Children who are colonized with S. pyogenes are at increased risk for streptococcal vulvovaginitis, as this pathogen can be easily transmitted to the genital area.23 Although not the most common pathogen in the study population, the prevalence of S. pyogenes was similar to recent reports.12, 20 Staphylococcus aureus is an important human pathogen.
In children, it represents the main cause of skin and soft tissue infections and can subsequently cause various and even invasive infections with significant morbidity and mortality. Although nasal and skin colonization by S. aureus is associated with an increased risk of infection, its importance in vulvovaginitis remains controversial.20 However, many reports in the literature have shown that S. aureus is an important pathogen in symptomatic patients with vulvovaginitis.2, 3, 5, 8, 19, 24
On the contrary, Jariene and collaborators isolated S. aureus in both study (9.6%) and control (7.1%) groups, in mixed culture and did not consider it as the main pathogen.20 However, Kim and collaborators isolated S. aureus in 15% of prepubertal girls with vulvovaginitis, while others, according to our data, reported a prevalence of 5.8%.8, 12, 19
Streptococcus agalactiae or group B beta-hemolytic Streptococcus frequently colonizes the genitourinary and gastrointestinal tract, as well as the oropharynx.25 However, the implication of this pathogen during pregnancy for the health of the newborn is of utmost importance and is always a reason of concern in women of reproductive age. S. agalactiae has been included as a common pathogen in symptomatic patients presenting with vulvovaginitis.5 The authors isolated S. agalactiae from 6.7% and 2.2% of prepubertal and pubertal girls, respectively.
Similar to these data, Randelovic et al detected this pathogen in 7% of prepubertal patients, while others reported a much higher isolation rate of 12%, with it being the second most common cause of vulvovaginitis in their group of prepubertal girls. 8, 21 There was an increase in the prevalence of enteric uropathogens.
The role of fecal pathogens in the pathogenesis of vulvovaginitis is still unclear and is a matter of controversy among specialists, since these pathogens are also considered contaminants in healthy control groups.10, 13, 20, 24 Interestingly, Gorbachinsky et al reported a significant increase in uropathogenic bacteria (79%) in periurethral samples from girls with vulvovaginitis compared to girls without vulvovaginitis (18%).26
Randelovic and collaborators reported bacteria of fecal origin in 33.8% of symptomatic girls, in pure culture, mostly Proteus mirabilis (14.4%), Enterococcus faecalis (12.2%) and Escherichia coli (7%).8 Others have also isolated fecal pathogens in significant quantities.2, 19, 22 As in previous reports, pathogens found in fecal flora were an important cause of vulvovaginitis in prepubertal and pubertal girls in the present study, probably due to the lack of hygiene regarding urination and cleaning habits.
In prepubertal girls, the vaginal pH is alkaline with a hypoestrogenic environment that allows the excessive growth of different microorganisms, often of enteric or oropharyngeal origin.2, 3, 9 The frequent isolation of enteric pathogens in vaginal samples could be explained by the anatomical proximity of the vulva and anus, poor hygiene or hygiene habits that favor colonization.
Children often have poor hygiene in the anogenital region, do not wash their hands regularly, and can easily transfer pathogens from the oropharynx to the genital area.
On the other hand, pubertal girls, although more resistant to infections compared to prepubertal boys, due to a higher microbial flora, may still be prone to infections by enteric pathogens.
Many cases of vulvovaginitis will improve and resolve by implementing careful hygienic measures and improved perineal hygiene.24 However, it has been suggested that symptomatic pediatric patients should be treated accordingly, as gastrointestinal microorganisms are characterized by greater pathogenicity.5
BV was found in significant percentages in both groups studied. BV has rarely been reported in the literature as a cause of vulvovaginitis in girls and was not associated with sexual activity.13, 27
In a normal prepubertal vagina, lactobacilli are missing and, along with different aerobic species, anaerobic bacteria can also be found.2, 7, 9, 15 McGreal and Wood reported an increase in anaerobes (51%) in prepubertal girls.21 However, In pubertal girls, the most common causes of infectious vulvovaginitis are BV, Candida species, and Trichomonas vaginalis.2, 15 Unfortunately, their exact prevalence is unknown as these infections are not reportable. In these girls in whom the vaginal flora is enriched with lactobacilli, G. vaginalis and different anaerobes can be isolated even without evidence of sexual transmission.1, 14, 21
Huppert et al reported a prevalence of BV of 23% in adolescents aged 14 to 19 years while Xu et al reported an incidence of BV and intermediate type of BV of 25.7% and 19.3%, respectively, in girls in the late puberty, stating that these girls are more susceptible to BV and that more attention should be paid to menstrual hygiene.16, 28
Vaca et al reported a BV prevalence of 31.5% in adolescent girls in Ecuador, suggesting genetic, behavioral, and environmental causes as possible factors for the increased prevalence.14 However, due to a higher activity rate sexually transmitted infections in adolescents, sexually transmitted infections should also be considered.5 In fact, it has been documented that BV facilitates the acquisition of sexually transmitted infections.14 BV differs from other vaginal infections because, generally, does not present inflammation.
It is characterized by a change in the vaginal microflora with a significant decrease or even disappearance of lactobacilli and an increase in G. vaginalis, Atopobium vaginae, different anaerobic bacteria and a plethora of other microorganisms that cannot be cultured and, as a result, Identification can only be achieved through molecular methods.29, 30 Furthermore, it has been shown that BV is not only a polymicrobial infection, but a more complex clinical entity, since it is characterized by the formation of biofilms,30, 31 which could offer a possible explanation for the frequent recurrences observed.15
The increased prevalence of microorganisms associated with BV in both groups, although significantly higher in the pubertal group, is a cause for concern for future implications on their reproductive potential, since BV has been associated with pelvic inflammatory disease ( EPI) and, therefore, with subfertility.32 Generally, vulvovaginitis due to Candida species, as an estrogen-dependent condition, is more common in pubertal girls and quite unusual in prepuberty.3, 4, 8, 10 , twenty
Higher levels of estrogen during puberty along with higher levels of glucose in the vaginal environment and poor menstrual hygiene are predisposing factors for vaginal candidiasis.13, 16 Without a doubt, in the presence of risk factors such as systemic antibiotic treatment recent history, immunosuppression, diabetes mellitus or sexual abuse, the incidence may increase in both groups.33
Candida species are members of the normal skin or intestinal flora. Most fungal infections are caused by Candida albicans, while non-albicans species show reduced susceptibility to antifungals, specifically topical or oral azoles currently used as first-line treatment.2
In this study, Candida vulvovaginitis was defined significantly more frequently in pubertal girls than in prepubertal girls, the result of which is in accordance with previous works.13, 14, 16 On the contrary, Hu et al12 reported an increased prevalence 22.3% in a large group of prepubertal girls, while Xu et al.16 isolated Candida in 29.4% of girls in late puberty. There are some limitations in the present study.
They did not include a healthy asymptomatic control group to compare the results. However, this would have been very difficult to attempt since it is not easy to obtain permission from parents or guardians to collect vaginal samples from healthy, asymptomatic virginal girls. Furthermore, nasopharyngeal cultures were not performed in parallel nor did we study the patients’ gastrointestinal flora. These cultures would have provided important information about the pathogens that colonized the patients. Finally, retrospective record review limited the ability to collect and analyze treatment outcomes.
| Conclusions |
Although vaginal cultures represent an important tool for the correct identification of the responsible pathogens, their results should be evaluated with caution when deciding the cause of vulvovaginitis in girls. In prepubertal girls, the most common isolated pathogens were opportunistic bacteria of fecal origin, while girls in late puberty were more susceptible to BV and vulvovaginal candidiasis.
The combination of a complete history with an appropriate physical examination and methodical laboratory evaluation should lead the physician to a correct diagnosis and specific treatment. However, general personal hygiene recommendations should always be provided to symptomatic girls of all ages.
Vulvovaginitis is the most common reason for referral to pediatric and adolescent gynecology services and, to ensure reproductive health later in life, knowledge of the pathogens often involved is essential.
| Comment |
Vulvovaginitis is a relatively common cause of concern and consultation in prepubertal and pubescent girls and is usually due to various etiologies. This study focused on investigating the pathogens isolated in prepubertal and pubertal girls with signs and symptoms of vulvovaginitis.
Bacterial vaginosis was diagnosed in 22.8% of prepubertal girls and 37.9% of pubertal girls. Candida was isolated mainly in pubescent girls.
According to what was evaluated in this study, culture results should be evaluated with caution in girls with vulvovaginitis. In prepubertal girls, the most commonly isolated pathogens were opportunistic bacteria of fecal origin, while girls in late puberty were more susceptible to bacterial vaginosis and candidiasis.
