Vulvovaginitis in prepubertal children is a common complained problem in clinical practice. The anatomy of vulva and the thin uncornified vulval epithelium at prepubertal age makes it susceptible to infection[1]. Because few hospitals provide specialist paediatric gynaecological outpatient services, children with vulvovaginitis are managed mainly in primary care[3]. Therefore, there were few studies comprehensively explored the prevalence of H. influenzae vulvovaginitis in prepubertal girls. This study was performed at a tertiary university hospital, which provides specialist paediatric gynaecological outpatient services.
Vulvovaginitis caused by upper respiratory flora is generally considered to be the most common gynecological problem in prepubertal girls. A multicenter study showed that paediatric inflammatory vulvovaginitis is mainly caused by pathogens of the upper respiratory tract and the most common risk factor for this infection is to have suffered an upper respiratory tract infection in the previous month[6]. A study of case report provided the direct evidence of the nose-hand-vagina method of transmission[7]. It has been assumed that respiratory bacteria were transmitted to the perineal area via the hands[8]. Therefore, advice on hygiene and behavior may be an important strategy to prevent vulvovaginitis in prepubertal girls.
Several previous studies have indicated that vulvovaginitis in girls is mostly caused by the bacteria from the upper respiratory tract, S. pyogenes and H. influenzae[1]. In a large study from Liverpool, H. influenzae was a more common cause of this complaint than β haemolytic streptococci[9]. However, H. influenzae is fastidious in its growth requirements and laboratories may not isolate it unless they include appropriate culture medium for genital swabs received for young girls[10]. In the present study, all the specimens were inoculated onto Haemophilus selective chocolate agar for the isolation of H. influenzae. Published studies have described a large number of Gram-negative and Gram-positive bacteria as the possible causes of vulvovaginitis in girls. However, the pure or predominant growth of a possible pathogenic microorganism, associated with the signs of inflammation, is certainly of diagnostic relevance[1]. In this study, the finding of a large number of polymorphonuclear leukocytes on Gram-stained smears was used to indicate the presence of inflammatory reaction, which ensured that the H. influenzae isolated from vulval swabs was a possible pathogenic microorganism. There were 649 (15.7%) of 4142 vulval swabs from children yielded H. influenzae in this study, which was in agreement with the opinions described above that vulvovaginitis in girls is mostly caused by the bacteria from the upper respiratory tract, S. pyogenes and H. influenzae, suggesting that H. influenzae was a common pathogen of vulvovaginal infection in prepubertal girls in Zhejiang, China. There were peaks of isolates from April to July seen in the vulval isolates, which was consistent with that from the respiratory tract specimens, suggesting that vaginal H. influenzae strains might from the respiratory tract[6, 11]. The age range of children carrying the H. influenzae isolates was from 0.5y to 13y but 477 (75%) were between the ages of 3 and 7 years, which were in agreement with the results of previous studies[11, 12].
Ampicillin became the drug of choice for the treatment of H. influenzae infections since the 1970s[13]. In recent years, with the extensive use of antibiotics, the drug resistance of H. influenzae strains to ampicillin has gradually increased. The ampicillin resistance rate of H. influenzae strains in China was increased from12% in 2000–2002[14] to 58.1% in 2016[15]. In this study, the ampicillin resistance rate was 39.1%, which was higher than that in genital strains (26.4%) but lower than that in respiratory strains (58.4%)in 2015 reported by our study team[15]. In different years, the ampicillin resistance of H. influenzae strains isolated from vulval specimens had gradually increased, from 33.1% in 2016 to 42.8% in 2018.Therefore, it’s worth noting of ampicillin resistance of H. influenzae in clinical management. However, the production of β-lactamase is still the main mechanism of ampicillin resistance of H. influenzae strains in this study, which was consistent with the results in other reports[12, 16, 17], but different from the mechanism in Japan (BLNAR accounted for > 50% after 2014)[18], while BLNAR accounted for only 6.6% in our study, suggesting great differences in antibiotic resistance and drug-resistant mechanisms of H. influenzae strains around the world. There have been studies comparing the H. influenzae resistance profiles between respiratory tract and urinary tract[19], respiratory isolates and vaginal isolates [11], the resistance profiles of H. influenzae vary greatly depending on the infection site, which indicated that the optimal antibiotic treatment for H. influenzae might vary depending on the region and the site of infection. For BLNAR strains, these drugs should be avoided to use with the reason that BLNAR H. influenzae strains were all resistant to amoxycillin-clavulanic acid, ampicillin-sulbactam and cefuroxime. The resistance rates of the H. influenzae isolates to amoxycillin-clavulanic acid, and ampicillin-sulbactam were 26.4%, 21.8% in this study, which might be attributed to BLNAR strains and β-lactamase-producing clavulanic acid/amoxicillin-resistant (BLPACR) strains of H. influenzae. The mechanisms of BLPACR strains might be β-lactamase and PBP amino acid substitutions[20].
Generally, H. influenzae strains are highly susceptible to third-generation cephalosporins. The non-susceptibility rate of H. influenzae to third-generation cephalosporins was under 2% in the present study, which was much lower than that in Iran (33.1%)[21] and Japan (49.4%) [22], but similar to the rate of genital strains in china in 2015(5.5%), a reason for the differences might be explained by the different sites of infection. Typically, H. influenzae is sensitive to carbapenem; however, carbapenem-non-susceptible H. influenzae has been reported previously [23]. In this study, we found one H. influenzae strain non-susceptible to meropenem; its mechanism is worthy to research in further study.
A high prevalence of sulfamethoxazole-trimethoprim resistance (47.7%) among H. influenzae isolates was found in this study. However, no significant difference was found between the current results (47.7%, 306/641) in 2016–2019 and the previous results in 2015 (51.8%, 57/110)[11], which might be because of fewer applications of sulfamethoxazole-trimethoprim. There were 10.2% of the H. influenzae isolates resistant to azithromycin in this study, and a significantly increased resistance was seen between 2018 and 2019, which might because the extensive use of azithromycin in respiratory infections in China. H. influenzae strains were all sensitive to levofloxacin in the study; and 1.1% of H. influenzae strains were resistant to chloramphenicol, which might because of that these antibiotics are not used in children in China. However, ofloxacin or levofloxacin gel is still widely used as topical antibiotics in treating local infections in China, including prepubertal vulvovaginitis. MDR was present in 41 (6.4%) of the 642 H. influenzae isolates. The most prevalent resistance phenotype was ampicillin-sulfamethoxazole-trimethoprim-azithromycin resistance, which was in accordance with the previous findings[15].