The prevalence and of Haemophilus infuenzae in prepubertal girls with vulvovaginitis

Mingming Zhou The Children's Hospital, Zhejiang University School of Medicine Liying Sun The Children's Hospital, Zhejiang University School of Medicine Xuejun Chen (  [email protected] ) Chao Fang The Children's Hospital, Zhejiang University School of Medicine Jianping Li The Children's Hospital, Zhejiang University School of Medicine Chunzhen Hua The Children's Hospital, Zhejiang University School of Medicne

Research article

Keywords: Haemophilus infuenzae, Vulvovaginitis, Prepubertal, Antimicrobial resistance, Vulval

Posted Date: July 16th, 2020

DOI: https://doi.org/10.21203/rs.3.rs-41705/v1

License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License

Page 1/16 Abstract

Background: To determine the prevalence of Haemophilus infuenzae vulvovaginitis in prepubertal girls and the antimicrobial resistance of H.infuenzae strains isolated from vulval specimens.

Methods: Isolates of H.infuenzae from vulval swabs of prepubertal girls with vulvovaginitis received at The Children's Hospital, Zhejiang University School of Medicine during 2016-2019 were studied. Vulval specimens were inoculated on Haemophilus selective chocolate agar. Antimicrobial susceptibility tests were performed using the disk diffusion method. A cefnase disk was used to detect β-lactamase.

Results: A total of 4142 vulval specimens were received during the 4 years, 649 isolates of H. infuenzae were isolated from 642 girls aged 6 months to 13 years, with a median of 5y. There were peaks of isolates from April to July seen in the vulval isolates. In total, the resistance rate was 39.1% (250/640); 33.2% strains (211/636) were for β-lactamase-positive isolates, 6.6% strains (42/635) were β- lactamase-negative and ampicillin-resistant (BLNAR) isolates. The resistance rates of H. infuenzae isolates to amoxycillin-clavulanic acid, ampicillin-, , , , , levofoxacin, sulfamethoxazole-trimethoprim, azithromycin, and chloramphenicol were 26.4%, 21.8%, 24.8%, 1.7%, 1.0%, 0.2%, 0%, 47.7%, 10.2%, and 1.1%, respectively. MDR was present in 41 (6.4%) of the 642 H. infuenzae isolates, with the most prevalent MDR phenotype of ampicillin- sulfamethoxazole-trimethoprim-azithromycin resistance.

Conclusions: H. infuenzae is a common cause of vulvovaginitis in prepubertal girls. Laboratories should ensure that they include media appropriate for the isolation of H. infuenzae. It’s worth noting of ampicillin resistance of H. infuenzae in clinical management.

Background

Vulvovaginitis is a common problem in prepubertal girls, which is most commonly attributed to poor hygiene or nonspecifc irritants, but can occur as a part of bacterial infection[1]. Several studies have reported that Streptococcus pyogenes and Haemophilus infuenzae (H. infuenzae) are the most common bacterial causes of juvenile vulvovaginitis[1–4]. The association between H.infuenzae and prepubertal vulvovaginitis was frst highlighted by MacFarlane in 1987[5]. But Cox RA’s study in 1997 showed that H. infuenzaewas an underrated cause of vulvovaginitis in young girls[4]. However, until now, few studies have comprehensively explored the prevalence of H. infuenzae vulvovaginitis in prepubertal girls. Furthermore, published studies that included a large sample size are also scarce. Therefore, we described a four-year study, undertaken in the laboratory of a tertiary university hospital, to determine the prevalence of H. infuenzae vulvovaginitis in prepubertal girls and the antimicrobial resistance of H.infuenzae strains isolated from vulval specimens.

Methods

Page 2/16 Strain Collection and Identifcation

This was a retrospective analysis of data from prepubertal girls who presented to the outpatient clinic of pediatric and adolescent gynecology at The Children’s Hospital, Zhejiang University School of Medicine between January 2016 and December 2019. Two vulval swabs were taken from each girl, one for microscopic examination and the other for cultivation. The fnding of a large number of polymorphonuclear leukocytes on Gram-stained smears indicated the presence of infammatory reaction. The specimens were inoculated directly onto Columbia blood agar, Chocolate agar (Haemophilus and

Gonorrhoeae selective chocolate agar), and Sabouraud’s agar plates in air or 5% CO2, as appropriate, at 35 °C for 24–48 h. Suspected pathogens on Haemophilus selective chocolate agar were identifed by standard methods using Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS, Bruker).

β -Lactamase Detection and Antimicrobial Susceptibility Test

The antimicrobial susceptibility test by disk diffusion was performed and interpreted according to the Clinical Laboratory Standards Institute (CLSI) guidelines M100-S29with ampicillin (10 µg), amoxycillin-clavulanic acid (20 µg/10 µg), ampicillin-sulbactam (10 µg/10 µg), cefuroxime (30 µg), ceftriaxone (30 µg), cefotaxime (30 µg), meropenem (10 µg), levofoxacin (5 µg), sulfamethoxazole- trimethoprim (1.25/23.75 µg), azithromycin (15 µg), and chloramphenicol (30 µg) (Oxoid, UK). β- lactamase was measured using the cefnase disc method (BioMérieux, France). H. infuenzae strain ATCC49247 was used for quality control throughout the test. Multi- (MDR) was defned as resistant to three or more than three different types of antibiotics. Statistical Analysis

Antibiotic resistant rates were analyzed with WHONET 5.6. Comparisons of resistant rate between groups were performed with the X2 test. Age data, which were non-normally distributed, were described as medians (IQR). P values < 0.05 were considered statistically signifcant.

Results Sources of Isolates

From January 2016 to December 2019 a total of 4142vulval swabs from children were received, increasing from 803 in 2016 to 1488 in 2019. In total, 649 swabs (15.7%) from 642 patients yielded H. infuenzae. Table 1 shows the yearly totals with an increase in numbers received but a decrease in proportion of positives for H. infuenzae. The monthly totals for positive isolates of H. infuenzae show some peaks and troughs throughout the year (Fig. 1). The numbers of isolates per month ranged from 2 in October 2017 to a maximum of 27 in July 2019. There were peaks of isolates from April to July seen in the vulval isolates. The age range of children carrying the H. infuenzae isolates was from 0.5y to 13y but 477 (75%) were between the ages of 3 and 7 years, with a median of 5y (IQR: 3)(Fig. 2).

Page 3/16 Table 1 Yearly distribution of H. infuenzae strains isolated from vulval specimens, 2016–2019 Year Total received H. infuenzae positives

N %

2016 803 149 18.6

2017 931 144 15.5

2018 920 155 16.8

2019 1488 194 13.0

Total 4142 642 15.5

β -Lactamase Detection and Antimicrobial Susceptibility Test

In total, the ampicillin resistance rate was 39.1% (250/640); 33.2% strains (211/636) were for β- lactamase-positive isolates, 6.6% strains (42/635) were β-lactamase-negative and ampicillin-resistant (BLNAR) isolates. The resistance rates of the H. infuenzae isolates toamoxycillin-clavulanic acid, ampicillin-sulbactam, cefuroxime, ceftriaxone, cefotaxime, meropenem, levofoxacin, sulfamethoxazole- trimethoprim, azithromycin, and chloramphenicol were 26.4%, 21.8%, 24.8%, 1.7%, 1.0%, 0.2%, 0%, 47.7%, 10.2%, and 1.1%, respectively (Table 2). In different years, the resistance rates of H. infuenzae strains to cefuroxime and azithromycin had a signifcantly statistical difference (P < 0.05; Table 3). β-lactamase- positive H. infuenzae strains had signifcantly higher resistance rates to ampicillin, amoxycillin-clavulanic acid, cefuroxime, sulfamethoxazole-trimethoprim, azithromycin, and chloramphenicol than β-lactamase- negative strains did (P < 0.01; Table 4). BLNAR H. infuenzae strains were all resistant to amoxycillin- clavulanic acid, ampicillin-sulbactam and cefuroxime while all susceptible to levofoxacin, azithromycin and chloramphenicol (Table 5).

Page 4/16 Table 2 Antibiotic resistances of H. infuenzae strains isolated from vulval specimens, 2016–2019 Antibiotic N %R %I %S

β-lactamase 636 33.2 66.8

Ampicillin 640 39.1 6.6 54.4

Amoxycillin-clavulanic acid 349 26.4 0 73.6

Ampicillin-sulbactam 641 21.8 0 78.2

Cefuroxime 640 24.8 2.5 72.7

Ceftriaxone 350 1.7༊ 0 98.3

Cefotaxime 288 1༊ 0 99

Meropenem 632 0.2༊ 0 99.8

Levofoxacin 640 0 0 100

Sulfamethoxazole-trimethoprim 641 47.7 1.4 50.9

Azithromycin 469 10.2༊ 0 89.8

Chloramphenicol 641 1.1 0 98.9

Note: S, susceptible; I, intermediate; R, resistant; * rate ofnon-susceptible.

Page 5/16 Table 3 Antibiotic resistances of H. infuenzae strains isolated from vulval specimens in different years Antibiotic 2016 2017 2018 2019

N %R N %R N %R N %R

β-lactamase 148 31.8 142 26.8 156 35.9 192 37

Ampicillin 148 33.1 145 37.2 155 41.9 194 42.8

Amoxycillin-clavulanic acid / / / / 154 23.4 194 28.9

Ampicillin-sulbactam 149 16.1 145 26.9 155 21.3 194 22.7

Cefuroxime# 149 17.4 145 26.2 154 23.4 194 30.4

Ceftriaxone / / / / 155 1.3 194 2.1

Cefotaxime 146 1.4 143 0.7 / / / /

Meropenem 148 0 137 0.7 155 0 194 0

Levofoxacin 148 0 145 0 155 0 194 0

Sulfamethoxazole-trimethoprim 149 49 145 45.5 155 47.7 194 48.5

Azithromycin# / / / / 155 7.1 191 14.7

Chloramphenicol 149 1.3 145 1.4 155 0 194 1.5

Note: R, resistant; * rate of non-susceptible.

Page 6/16 Table 4 Comparison of antibiotic resistance between β-lactamase-positive and β-lactamase-negative H. infuenzae strains isolated from vulval specimens, 2016–2019 Antibiotic β-lactamase (+) β-lactamase (-)

N %R %I %S N %R %I %S

Ampicillin1 211 98.6 0.5 0.9 424 9.9 9.4 80.7

Amoxycillin-clavulanic acid1 126 38.9 0 61.1 222 18.9 0 81.1

Ampicillin-sulbactam2 212 26.9 0 73.1 424 19.6 0 80.4

Cefuroxime1 212 33 6.6 60.4 423 21 0.5 78.5

Ceftriaxone 127 3.1* 0 96.9 222 0.9* 0 99.1

Cefotaxime 83 1.2* 0 98.8 201 1* 0 99

Meropenem 210 0 0 100 417 0.2* 0 99.8

Levofoxacin 211 0 0 100 424 0 0 100

Sulfamethoxazole-trimethoprim1 212 59.4 0.5 40.1 424 42 1.9 56.1

Azithromycin1 161 28* 0 72 307 1* 0 99

Chloramphenicol1 212 3.3 0 96.7 424 0 0 100

Note: S, susceptible; I, intermediate; R, resistant; * rate ofnon-susceptible; 1P<0.01; 2P<0.05.

Page 7/16 Table 5 Antibiotic resistances of BLNAR H. infuenzae strains isolated from vulval specimens, 2016–2019 Antibiotic N %R %I %S

Amoxycillin-clavulanic acid 22 100 0 0

Ampicillin-sulbactam 42 100 0 0

Cefuroxime 42 100 0 0

Ceftriaxone 22 9.1* 0 90.9

Cefotaxime 20 10* 0 90

Meropenem 36 2.8* 0 97.2

Levofoxacin 42 0 0 100

Sulfamethoxazole-trimethoprim 42 47.6 9.5 42.9

Azithromycin 24 0 0 100

Chloramphenicol 42 0 0 100

Note: S, susceptible; I, intermediate; R, resistant; * rate ofnon-susceptible. MDR Pattern

MDR was present in 41 (6.4%) of the 642 H. infuenzae isolates. The most prevalent resistance phenotype was ampicillin-sulfamethoxazole-trimethoprim-azithromycin resistance, which was detected in 16 isolates, representing 39% of MDR strains (Table 6).

Page 8/16 Table 6 The main MDR patterns of H. infuenzae strains isolated from vulval specimens, 2016–2019 MDR pattern Number of isolates Percent (%)

β-lactams-SXT-AZM

AMP-SXT-AZM 16 39.0

AMP-CXM-SXT-AZM 4 9.8

AMP-CXM-AMC-SXT-AZM 3 7.3

AMP-CXM-SAM-AMC-SXT-AZM 3 7.3

AMP-AMC-SXT-AZM 2 4.9

AMC-SXT-AZM 1 2.4

AMP-CXM-SAM-SXT-AZM 1 2.4

SAM-SXT-AZM 1 2.4

CXM-SXT-AZM 1 2.4

AMP-SAM-AMC-SXT-AZM 1 2.4

AMP-CXM-SAM-AMC-CRO-SXT-AZM 1 2.4

β-lactams-SXT-CHL

AMP-SXT-CHL 3 7.3

AMP-AMC-SXT-CHL 1 2.4

AMP-SAM-SXT-CHL 1 2.4

AMP-CXM-SXT-CHL 1 2.4

β-lactams-SXT-AZM-CHL

AMP-SXT-AZM-CHL 1 2.4

Note: AMP, Ampicillin; CXM, Cefuroxime; SAM, Ampicillin-sulbactam; AMC, Amoxycillin-clavulanic acid; CRO, Ceftriaxone; SXT, Sulfamethoxazole-trimethoprim; AZM, Azithromycin; CHL, Chloramphenicol.

Discussion

Vulvovaginitis in prepubertal children is a common complained problem in clinical practice. The anatomy of vulva and the thin uncornifed 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

Page 9/16 explored the prevalence of H. infuenzae 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 fora is generally considered to be the most common gynecological problem in prepubertal girls. A multicenter study showed that paediatric infammatory 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 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. infuenzae[1]. In a large study from Liverpool, H. infuenzae was a more common cause of this complaint than β haemolytic streptococci[9]. However, H. infuenzae 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. infuenzae. 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 infammation, is certainly of diagnostic relevance[1]. In this study, the fnding of a large number of polymorphonuclear leukocytes on Gram-stained smears was used to indicate the presence of infammatory reaction, which ensured that the H. infuenzae isolated from vulval swabs was a possible pathogenic microorganism. There were 649 (15.7%) of 4142 vulval swabs from children yielded H. infuenzae 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. infuenzae, suggesting that H. infuenzae 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. infuenzae strains might from the respiratory tract[6, 11]. The age range of children carrying the H. infuenzae 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. infuenzae infections since the 1970s[13]. In recent years, with the extensive use of antibiotics, the drug resistance of H. infuenzae strains to ampicillin has gradually increased. The ampicillin resistance rate of H. infuenzae 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. infuenzae 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. infuenzae in clinical management.

Page 10/16 However, the production of β-lactamase is still the main mechanism of ampicillin resistance of H. infuenzae 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. infuenzae strains around the world. There have been studies comparing the H. infuenzae resistance profles between respiratory tract and urinary tract[19], respiratory isolates and vaginal isolates [11], the resistance profles of H. infuenzae vary greatly depending on the infection site, which indicated that the optimal antibiotic treatment for H. infuenzae 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. infuenzae strains were all resistant to amoxycillin-clavulanic acid, ampicillin-sulbactam and cefuroxime. The resistance rates of the H. infuenzae 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/-resistant (BLPACR) strains of H. infuenzae. The mechanisms of BLPACR strains might be β-lactamase and PBP amino acid substitutions[20].

Generally, H. infuenzae strains are highly susceptible to third-generation . The non- susceptibility rate of H. infuenzae 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. infuenzae is sensitive to ; however, carbapenem-non- susceptible H. infuenzae has been reported previously [23]. In this study, we found one H. infuenzae 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. infuenzae isolates was found in this study. However, no signifcant 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. infuenzae isolates resistant to azithromycin in this study, and a signifcantly increased resistance was seen between 2018 and 2019, which might because the extensive use of azithromycin in respiratory infections in China. H. infuenzae strains were all sensitive to levofoxacin in the study; and 1.1% of H. infuenzae strains were resistant to chloramphenicol, which might because of that these antibiotics are not used in children in China. However, ofoxacin or levofoxacin 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. infuenzae isolates. The most prevalent resistance phenotype was ampicillin-sulfamethoxazole- trimethoprim-azithromycin resistance, which was in accordance with the previous fndings[15].

Conclusions

To our knowledge, this study currently represents the largest population-based study of H. infuenzae vulvovaginitis in prepubertal girls in China. H. infuenzae is a common cause of vulvovaginitis in prepubertal girls in Zhejiang, China. Laboratories should ensure that they include media appropriate for

Page 11/16 the isolation of H. infuenzae. It’s worth noting of ampicillin resistance of H. infuenzae in clinical management. Advice on hygiene and behavior may be an important strategy to prevent vulvovaginitis in prepubertal girls.

Abbreviations

H.infuenzae Haemophilus infuenzae; CLSI:Clinical and laboratory standards institute; MDR:Multi-Drug Resistance; BLNAR:β-lactamase-negative and ampicillin-resistant;

Declarations

Ethics approval and consent to participate

The study obtained approval from the Research and Ethics committee of The Children's Hospital, Zhejiang University School of Medicine (2019-IRB-049). The Ethics Committee waived the requirement for informed consent as the investigated isolates were obtained from clinical specimens referred to the diagnostic laboratory as part of routine care.

Consent for publication

Not applicable.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors declare no conficts of interest.

Funding

Not applicable

Authors' contributions

Liying Sun, Xuejun Chen and Chunzhen Hua guided in the study design; Mingming Zhou and Xuejun Chen performed analysis and interpretation of data and drafted the manuscript; Mingming Zhou, Chao Fang and Jianping Li participated in strain identifcation and antimicrobial susceptibility test. All authors have read and approved the fnal manuscript.

Acknowledgements

Page 12/16 Not applicable

References

1. Randelovic G, Mladenovic V, Ristic L, Otasevic S, Brankovic S, Mladenovic-Antic S, Bogdanovic M, Bogdanovic D. Microbiological aspects of vulvovaginitis in prepubertal girls. Eur J Pediatr. 2012;171(8):1203–8. 2. Sikanic-Dugic N, Pustisek N, Hirsl-Hecej V, Lukic-Grlic A. Microbiological fndings in prepubertal girls with vulvovaginitis. Acta Dermatovenerol Croat. 2009;17(4):267–72. 3. Cox RA, Slack MP. Clinical and microbiological features of Haemophilus infuenzae vulvovaginitis in young girls. J Clin Pathol. 2002;55(12):961–4. 4. Cox RA. Haemophilus infuenzae: an underrated cause of vulvovaginitis in young girls. J Clin Pathol. 1997;50(9):765–8. 5. Macfarlane DE, Sharma DP. Haemophilus infuenzae and genital tract infections in children. Acta Paediatr Scand. 1987;76(2):363–4. 6. Cuadros J, Mazon A, Martinez R, Gonzalez P, Gil-Setas A, Flores U, Orden B, Gomez-Herruz P, Millan R. Spanish Study Group for Primary Care I. The aetiology of paediatric infammatory vulvovaginitis. Eur J Pediatr. 2004;163(2):105–7. 7. Chen X, Chen L, Zeng W, Zhao X. Haemophilus infuenzae vulvovaginitis associated with rhinitis caused by the same clone in a prepubertal girl. J Obstet Gynaecol Res. 2017;43(6):1080–3. 8. Hansen MT, Sanchez VT, Eyster K, Hansen KA. Streptococcus pyogenes pharyngeal colonization resulting in recurrent, prepubertal vulvovaginitis. J Pediatr Adolesc Gynecol. 2007;20(5):315–7. 9. Pierce AM, Hart CA. Vulvovaginitis: causes and management. Arch Dis Child. 1992;67(4):509–12. 10. Macsween KF, Ridgway GL. The laboratory investigation of vaginal discharge. J Clin Pathol. 1998;51(8):564–7. 11. Li JP, Hua CZ, Sun LY, Wang HJ, Chen ZM, Shang SQ. Epidemiological Features and Antibiotic Resistance Patterns of Haemophilus infuenzae Originating from Respiratory Tract and Vaginal Specimens in Pediatric Patients. J Pediatr Adolesc Gynecol. 2017;30(6):626–31. 12. Kim H, Chai SM, Ahn EH, Lee MH. Clinical and microbiologic characteristics of vulvovaginitis in Korean prepubertal girls, 2009–2014: a single center experience. Obstet Gynecol Sci. 2016;59(2):130–6. 13. Tristram S, Jacobs MR, Appelbaum PC. Antimicrobial resistance in Haemophilus infuenzae. Clin Microbiol Rev. 2007;20(2):368–89. 14. Shen XZ, Lu Q, Deng L, Yu S, Zhang H, Deng Q, Jiang M, Hu Y, Yao KH, Yang YH. Resistance of Haemophilus infuenzae isolates in children under 5 years old with acute respiratory infections in China between 2000 and 2002. J Int Med Res. 2007;35(4):554–63. 15. Wang HJ, Wang CQ, Hua CZ, Yu H, Zhang T, Zhang H, Wang SF, Lin AW, Cao Q, Huang WC, et al. Antibiotic Resistance Profles of Haemophilus infuenzae Isolates from Children in 2016: A

Page 13/16 Multicenter Study in China. Can J Infect Dis Med Microbiol. 2019; 2019:6456321. 16. Fluit AC, Florijn A, Verhoef J, Milatovic D. Susceptibility of European beta-lactamase-positive and - negative Haemophilus infuenzae isolates from the periods 1997/1998 and 2002/2003. J Antimicrob Chemother. 2005;56(1):133–8. 17. Wang CY, Xu HM, Deng JK, Yu H, Chen YP, Lin AW, Cao Q, Hao JH, Zhang T, Deng HL, et al. A multicentric clinical study on clinical characteristics and drug sensitivity of children with pneumococcal meningitis in China. Zhonghua Er Ke Za Zhi. 2019;57(5):355–62. 18. Yamada S, Seyama S, Wajima T, Yuzawa Y, Saito M, Tanaka E, Noguchi N. beta-Lactamase-non- producing ampicillin-resistant Haemophilus infuenzae is acquiring multidrug resistance. J Infect Public Health. 2019. 19. Deguchi T, Ito S, Hatazaki K, Horie K, Yasuda M, Nakane K, Mizutani K, Tsuchiya T, Yokoi S, Hanaoka N, et al. Antimicrobial susceptibility of Haemophilus infuenzae strains isolated from the urethra of men with acute urethritis and/or epididymitis. J Infect Chemother. 2017;23(11):804–7. 20. Matic V, Bozdogan B, Jacobs MR, Ubukata K, Appelbaum PC. Contribution of beta-lactamase and PBP amino acid substitutions to amoxicillin/clavulanate resistance in beta-lactamase-positive, amoxicillin/clavulanate-resistant Haemophilus infuenzae. J Antimicrob Chemother. 2003;52(6):1018–21. 21. Vaez H, Sahebkar A, Pourfarzi F, Yousef-Avarvand A, Khademi F. Prevalence of Antibiotic Resistance of Haemophilus Infuenzae in Iran- A Meta-Analysis. Iran J Otorhinolaryngol. 2019;31(107):349–57. 22. Nagai K, Kimura O, Domon H, Maekawa T, Yonezawa D, Terao Y. Antimicrobial susceptibility of Streptococcus pneumoniae, Haemophilus infuenzae, and Moraxella catarrhalis clinical isolates from children with acute otitis media in Japan from 2014 to 2017. J Infect Chemother. 2019; 25(3):229 – 32. 23. Kitaoka K, Kimura K, Kitanaka H, Banno H, Jin W, Wachino JI, Arakawa Y. Carbapenem- Nonsusceptible Haemophilus infuenzae with -Binding Protein 3 Containing an Amino Acid Insertion. Antimicrob Agents Chemother. 2018; 62(8).

Figures

Page 14/16 Figure 1

Seasonal distribution of H. infuenzae strains isolated from vulval specimens, 2016-2019

Figure 2

Page 15/16 Distribution of ages in children carrying the H. infuenzae strains isolated from vulval specimens, 2016- 2019

Page 16/16