Clinical significance of isolation of Haemophilus no ducreyi in genital samples. Systematic review

Abstract

Introduction and objectives

Currently, the microbiological diagnosis of genital infections is carried out with molecular methods, which allow the detection of less frequent etiological agents but with potential pathogenic importance, such as Haemophilus spp. The objective of this review is to analyse and highlight the clinical importance of the isolation of Haemophilus spp. in genital and rectal infections, excluding Haemophilus ducreyi.

Material and methods

A systematic review was carried out based on an exhaustive search of the publications included in the MEDLINE database up to August 5, 2021, on the presence of Haemophilus spp. in genital and rectal infections, excluding H. ducreyi.

Results

After reviewing what was described in the literature, Haemophilus spp. (excluding H. ducreyi: HSNOD) was detected in 2397 episodes of genital infection, the most frequently isolated species being H. influenzae and H. parainfluenzae. Most of the episodes (87,6%) are constituted by single isolation. There is a slight predominance in women (48,3%) where it can cause vaginitis, salpingitis, endometritis or complications during pregnancy. In men, the clinical picture usually corresponds to urethritis. Most of the samples correspond to vaginal and urethral exudates, with a minority representation at the rectal level (2.3%).

Conclusion

HSNOD plays a relevant pathogenic role in episodes of genital infection, so microbiological diagnostic protocols must include methods that allow their detection, as well as include them in the etiological spectrum of this type of clinical picture.

Resumen

Introducción y objetivos

Actualmente el diagnóstico microbiológico de las infecciones genitales se realiza con métodos moleculares, los cuales permiten detectar agentes etiológicos menos frecuentes, pero con potencial importancia patogénica, como Haemophilus spp. El objetivo de esta revisión es analizar y resaltar la importancia clínica del aislamiento de Haemophilus spp. en infecciones genitales y rectales, excluyendo H. ducreyi.

Material y métodos

Se ha realizado una revisión sistemática en base a una búsqueda exhaustiva de las publicaciones incluidas en la base de datos MEDLINE hasta el 5 de agosto de 2021, sobre la presencia de Haemophilus spp. en infecciones genitales y rectales, excluyendo H. ducreyi.

Resultados

Tras revisar lo descrito en la literatura, las especies de Haemophilus (excluyendo H. ducreyi: HSNOD) se detectaron en 2397 episodios de infección genital, siendo las especies más frecuentemente aisladas H. influenzae y H. parainfluenzae. La mayoría de los episodios (87,6%) están constituidos por aislamiento único. Existe un ligero predominio en mujeres (48,3%) donde puede producir cuadros de vaginitis, salpingitis, endometritis o complicaciones durante el embarazo. En hombres, el cuadro clínico suele corresponder a una uretritis. La mayoría de las muestras corresponde a exudados vaginales y uretrales, con una representación minoritaria a nivel rectal (2,3%).

Conclusión

HSNOD desempeña un papel patogénico relevante en episodios de infección genital, por lo que los protocolos de diagnóstico microbiológico deben incluir métodos que permitan su detección, así como incluirlos en el espectro etiológico de este tipo de cuadros clínicos.

Introduction

Genital tract infections are common entities that share a clinical presentation but can be caused by myriad aetiological agents apart from the microorganisms commonly regarded as pathogens¹. In addition, there is a large diversity of microbiota and significant inter-individual variability that makes it even more difficult to distinguish between a possible pathogenic role or not². These less common agents that play a possibly pathogenic role include the species of the genus Haemophilus, excluding Haemophilus ducreyi (HSNOTD). The genus Haemophilus, belonging to the Pasteurallaceae family, is made up of small non-sporulating, pleomorphic, non-motile, facultative anaerobic gram-negative bacilli. It is characterised by having demanding nutritional requirements and requires chocolate agar medium for proper growth and identification. The species of HSNOTD most frequently involved are Haemophilus influenzae (H. influenzae), Haemophilus parainfluenzae (H. parainfluenzae) and Haemophilus haemolyticus (H. haemolyticus). The first one is well-known, it is a colonising agent of the upper respiratory and genital tract and can behave as an opportunistic pathogen. In recent years, more and more cases of urethritis associated with H. influenzae, and more recently its association with non-gonococcal urethritis, have been described both in men who have sex with men (MSM) and women³. In addition, it is the second most common cause of vulvovaginitis in girls not linked to sexual transmission⁴. H. parainfluenzae predominantly colonises the oropharynx and can lead to infections through contiguity, bacteraemia, endocarditis and genital infections⁵. H. haemolyticus is difficult to distinguish from the non-encapsulated species of H. influenzae due to its morphological, biochemical and genetic similarities, and it is sometimes difficult to distinguish between them by conventional microbiological methods, hence some authors advocate the combination of conventional methods and polymerase chain reaction (PCR) analysis; ⁶ mass spectrometry (MALDI-TOF) has also shown good results in differentiation capacity⁷. It rarely has pathogenic potential.

In addition, there are the biotype IV cryptic genospecies, genetically related to H. haemolyticus, but with a different phenotype and location, such as "Haemophilus quentini", whose presence has been described in the genitourinary tract and can cause genital infections in pregnant women, chorioamnionitis and preterm delivery, and even pneumonic or septic conditions in newborns⁸.

The objective of this study is to analyse the presence of HSNOTD species in the development of genital infections through a systematic literature review.

Materials and methods

A search was made in the MEDLINE database, through PubMed, for studies that describe the presence of Haemophilus spp. in genital (vaginal, endocervical and urethral) and rectal exudates (in MSM). The following search terms were used: "Haemophilus and urethritis", "Haemophilus and proctitis", "Haemophilus and vaginitis", "Haemophilus and cervicitis", "Haemophilus and salpingitis", "Haemophilus and endometritis", "Haemophilus and bartholinitis", "Haemophilus and tubo-ovarian abscess", "Haemophilus and septic abortion", "Haemophilus and chorionamnionitis". The inclusion criteria were: studies published up until 1 September 2021; and studies published in English or Spanish. The exclusion criteria were: studies referring to the H. ducreyi or Haemophilus vaginalis species; and studies that analysed samples from sources other than genital or rectal. The references listed in the studies were also reviewed to reduce the number of losses.

Results

145, 3, 583, 595, 21, 73, 14, 6, 16 and 30 publications were obtained for the criteria "Haemophilusand urethritis", "Haemophilusand proctitis", "Haemophilusand vaginitis", "Haemophilusand cervicitis", "Haemophilusand salpingitis", "Haemophilusand endometritis", "Haemophilusand bartholinitis", "Haemophilusand tubo-ovarian abscess", "Haemophilusand septic abortion", "Haemophilus and chorionamnionitis", respectively, which were subsequently submitted to the inclusion and exclusion criteria, yielding 117 studies. Of these, 13 could not be located (Northwest Med. 1955;54(9):992−3, J Pathol Bacteriol. 1967;93(1):109−18, Am J Vet Res. 1971;32(12):2067−9, Am J Clin Pathol. 1980;73(2):285−7, Med J Aust. 1985;143(5):223, Med J Aust. 1985;142(9):531, J Infect Dis. 1986;153(1):165−7, Acta Paediatr Scand. 1987;76(2):363−4, Med Clin (Barc). 1989;92(9):335−7, Med Clin (Barc). 1989;93(19):758−9, Pediatr Infect Dis J. 1994;13(3):243, Am J Obstet Gynecol. 1994;170(4):1008−17, J Am Board Fam Pract. 1994;7(4):335−41). Table 1 shows 104 works, which group together a total of 2397 episodes of genital infection caused by Haemophilus spp. The most frequently isolated species were H. influenzae (57.7%; 1383/2397) and H. parainfluenzae (35.7%; 855/2397). No species was identified in 6.7% (159/2397) of the episodes. In most episodes (87.6%; 2099/2397), Haemophilus was isolated in monomicrobial culture. Otherwise, it was often detected along with Chlamydia trachomatis, Neisseria gonorrhoeae, Ureaplasma spp. or Mycoplasma spp.

Table 1.

Studies reporting the isolation of Haemophilus spp. in genital samples.

Author (year of publication)	No. of episodes	Microorganisms	Age/sex	Possible predisposing factors	Clinical manifestations	Clinical sample	Microbiological diagnosis reflected	Treatment
Skirrow MB (1970)^9	1	H. influenzae	48/F	NP	Tubo-ovarian abscess	Abscess exudate	Culture	Surgery and ampicillin
Hurley R (1970)^10	1	H. influenzae	29/F	Intrauterine device	Endometritis	Vaginal exudate	Culture	Removal of device
Farrand RJ (1971)^11	1	H. influenzae	4/ F	NP	Vaginal dischargeand irritation Inflamed vagina with purulent exudate	Vaginal exudate	Culture	Ampicillin
	1	H. influenzae	35/ F	Tubal ligation surgery 7 years previously.	Right tubal abscess	Puncture-aspiration of the abscess	Culture	Ampicillin + cloxacillin
Berczy J (1973)^12	1	H. influenzae	27/F	NP	Septic abortion	Vaginal exudate	Culture	NP
Khuri-Bulos N (1975)^13	8	H. influenzae	NP/F	NP	Neonatal sepsis	Vaginal and/or cervical exudate	NP	NP
Herva E (1975)^14	1	H. influenzae	NP/F	NP	Salpingitis	Tubal exudate	Culture	NP
Nicholls S (1975)^15	1	H. influenzae	31/ F	NP	Neonatal sepsis	Vaginal exudatePlacental biopsy	Culture	Penicillin + kanamycin (NR)Ampicillin
	1	H. influenzae	18/ F	NP	Antepartum haemorrhage and vaginal discharge.Neonatal sepsis and death	Vaginal exudate and respiratory samples from the newborn	Culture	Penicillin + kanamycin
Bowie WR (1977)^16	10	7 H. parainfluenzae4 + C. trachomatis3 H. influenzae	NP/M	NP	4 urethritis and3 asymptomatic3 asymptomatic	Urethral exudate	Culture (Virginia Polytechnic Institute)	NP
Albritton WL (1978)^17	5	H. influenzae	18 F, 20 F, 22 F 30 F,33/F	2 Pregnancy	1 vulvovaginitis, 2 septic abortion, 1 acute salpingitis	Vaginal exudate, blood cultures	Culture	Ampicillin, kanamycin
Gibson M (1978)^18	1	H. influenzae	NP/F	28 weeks pregnant. Premature rupture of membranes	Amnionitis	Amniotic fluid	Culture	NP
Ogden E (1979)^19	1	H. influenzae	NP/F	16 weeks pregnant	Amnionitis	Amniotic fluid	Culture	NP
Simon HB (1980)^20	1	H. influenzae	36/F	NP	Salpingitis/endometritis	Vaginal exudate	Culture	NP
Arias JW (1981)^21	1	H. parainfluenzae	18/F	29 weeks pregnant	Chorioamnionitis	Placenta	Culture	NP
De Pass EE (1982)^22	1	H. influenzae	22/F	NP	Bilateral salpingitis and abscess	Tubal exudate	Culture	Bilateral salpingo-oophorectomy, hysterectomy and ampicillin
Pastorek J (1982)^23	2	H. influenzae	26 F and 17F	Pregnant (37 and 40 weeks)	ChorioamnionitisEndometritis	Placenta and cervical exudate	Culture	Ampicillin, gentamicin
Chowdhury MN (1983)^24	1	H. parainfluenzae	NP/M	NP	Urethritis, urethral discharge	Urethral exudate	NP	Amoxicillin
Hall GD (1983)^25	11	11 H. influenzae	NP/F2 M	NP	4 vaginitis, 2 IUD-associated endometritis, 1 incomplete septic abortion, 1 urethral syndrome and 1NP2 urethritis	Vaginal, cervical or urethral exudate	Biochemical tests	NP
Messing M (1983)^26	1	H. parainfluenzae	NP/M	NP	Nongonococcal urethritis: genital erythema and urethral discharge	Urethral exudate	Culture and biochemical tests	NP
	1	H. parainfluenzae		NP	Ulcerative lesions on the genitals
	1	H. parainfluenzae		Secondary syphilis	Genital erythema and inguinal adenopathy
	1	H. parainfluenzae		Pubic folliculitis	Erythematous genital lesions
	1	H. parainfluenzae		Gonorrhoea and condyloma acuminata	Urethral discharge and genital lesion
	1	H. influenzae		NP	Dysuria and urethral discharge
	1	H. influenzae		Primary syphilis	Ulcerative lesions on the genitals
	1	H. influenzae		NP	Nongonococcal urethritis: dysuria and urethral discharge
Tebbutt GM (1984)^27	6	H. influenzae1 + N. gonorrhoeae	NP/F	NP	3 girls with vaginal discharge2 postnatal infection1 pelvic inflammatory disease	Vaginal exudate	Culture and biochemical tests	NP
Nakamura KT (1984)^28	1	H. parainfluenzae	24/F	28 weeks pregnant.	Premature rupture of membranes	Placenta and cervical exudate	Culture	Ampicillin and gentamicin
Paavonen J (1985)^29	2	H. influenzae	22/F, 29/F	1 Previous risky sexual relationship	1 pyosalpinx1 tubo-ovarian abscess	Tubal and abscess exudate	Culture	Doxycycline and metronidazolePenicillin G and metronidazole
Campognone P (1986)^30	17	H. influenzae	17 F	17 pregnant. 1 diabetic	Chorioamnionitis	Placenta and maternal blood	Culture	NP
Crowe HM (1987)^31	2	H. influenzae	NP/F	1 Crohn's disease	2 tubo-ovarian abscesses	Abscess exudate	Culture	Chloramphenicol and tobramycin
LW Davis (1987) ^32	1	H. influenzae	NP/F	NP	Tubo-ovarian abscess	NP	NP	NP
Winn HN (1987)^33	1	H. influenzae	25/F	22 weeks pregnant.	Chorioamnionitis	Maternal blood and amniotic fluid	Culture	Ampicillin
Casin I (1988)^34	60	60 H. influenzae52 solitarily3 + C. albicans3 + T. vaginalis1 + N. gonorrhoeae1 + C. trachomatis	NP/37 M, 20 F and 3 NP	NP	NP	37 Urethral exudate19 Vaginal and/or cervical exudate3 urine1 Bartholin's gland	Culture and biochemical tests	NP
Andreu A (1989)^35	10	7 H. parainfluenzae3 H. influenzae7 solitarily3 + other*	NP/M	NP	7 urethritis	Urethral exudate	NP	NP
	20	15 H. influenzae5 H. parainfluenzae + others*	NP/F	5 IUD carriers	8 vaginitis, 3 salpingitis and 9 NP	Vaginal or endocervical exudate	NP	NP
Drouet EB (1989)^36	244	216 H. parainfluenzae28 H. influenzae	NP	NP	NP	NP	NP	NP
Houang E (1989)^37	73	H. parainfluenzae	NP/71 M and 2 F	NP	NP	71 urethral exudate2 vaginal exudate	MicroScan	NP
Martel AY (1989)^38	103	Haemophilus spp.	NP/56 M, 33 F and 14 NP	NP	NP	55 genital exudate48 rectal exudate	Biochemical tests	NP
Leiberman JR (1989)^39	1	H. influenzae	35/F	16 weeks pregnant.	Amnionitis	Amniotic fluid	Culture	NP
van Bosterhaut B (1990)^40	3	H. influenzae	NP/F	NP	3 bartholinitis	Gland exudate	Culture	NP
Christensen JJ (1990)^41	17	Haemophilus spp.	NP/F	NP	Bartholinitis, salpingitis and/or vaginal discharge	NP	NP	NP
Quentin R (1990)^42	8	H. influenzae	NP/F	Bartholin's gland cyst or abscess surgery	Bartholinitis	Aspirated cyst or abscess	NP	NP
Silverberg K (1990)^43	1	H. influenzae	17/F	29 weeks pregnant.	Chorioamnionitis	Amniotic fluid and placenta	Culture	Ampicillin, gentamicin, clindamycin
Pinhas-Hamiel O (1991)^44	1	H. influenzae	28/F	13 weeks pregnantPrevious risky sexual relationship	Septic abortion	Blood cultures	Culture	NP
Facinelli B (1991)^45	1	H. parainfluenzae	NP	Risky sexual relationship	Urethritis	NP	NP	NP
Bendig JW (1991)^46	2	H. influenzae	35/F, 29/F	1 IUD	2 purulent salpingitis	Peritoneal and tubal exudate	Culture	Drainage, ceftriaxone, metronidazole
Lefevre JC (1991)^47	21	H. parainfluenzae	NP/M	NP	Urethritis	Urethral exudate	NP	NP
Bosch J (1991)^48	9	H. influenzae	NP/F	NP	7 Bartholin's gland abscesses1 post-caesarean section endometritis1 chorioamnionitis	Vaginal and gland exudate	NP	Ampicillin
Rusin P (1991)^49	13	H. influenzae	NP/F	NP	13 endometritis/chorioamnionitis	NP	NP	NP
Mazor M (1991)^50	1	H. influenzae	28/F	29 weeks pregnant.Premature rupture of membranes	Amnionitis	Amniotic fluid and neonatal blood	Culture	Ampicillin and gentamicin
Kragsberg P (1993)^51	6	H. influenzae	NP/F	6 pregnancies1 Recurrent endometritis	3 tubo-ovarian abscesses2 septic abortions1 postpartum sepsis	Abscess exudateVaginal exudate	Culture	Cephalosporin, ampicillin, metronidazole, doxycycline
Ault KA (1993)^52	3	H. influenzae	18 F, 22 F, 29F	3 Pregnancies (29, 31 and 31 weeks)	Amnionitis	Placenta and maternal blood	Culture	Vancomycin and gentamicinAmpicillin and gentamicinCeftizoxime
Kinney JS (1993)^53	6	H. influenzae	NP/F	Pregnant 24–40 weeks	Chorioamnionitis	Placenta and neonatal blood	Culture	1 Ampicillin, 1 cephalosporin
Gill MV (1995)^54	1	H. influenzae	36/F	35 weeks pregnant, placenta percreta	Chorioamnionitis	Blood cultures and cervical exudate	Culture	Ceftazidime and aztreonam
Llanes Caballero R (1996)^55	1	H. influenzae	NP/M	Previous schistosomiasis	Discomfort while urinating and purulent urethral discharge	Urethral exudate	NP	Norfloxacin
Pena MJ (1996)^56	20	H. influenzae	NP/F	Prepubescent girls	Vulvovaginitis	Vaginal exudate	NP	NP
Vázquez F (1996)^57	155	100 H. parainfluenzae45 H. influenzae10 Haemophilus spp.21 solitarily134 + others*	2 M, 11 F and 142 NP	Female sex workers and men with urethritis	8 urethritis2 epididymo-orchitis9 cervicitis and/or vaginitis2 Bartholin's gland abscesses134 NP	NP	Culture	NP
Carmeci C (1997)^58	1	H. influenzae	48/F	NP	Acute salpingitis and septic shock	Peritoneal exudate and blood cultures	Culture	Salpingo-oophorectomy and ceftriaxone
Cox RA (1997)^4	11	H. influenzae	<14/ F	NP	Vulvovaginitis	Vaginal and vulvar exudate	Culture	Amoxicillin
García E (1997)^59	30	18 H. influenzae	NP/F	Pregnant, parturient, puerperal	1 abortion2 antepartum deaths1 postpartum death14 live newborns (5 confirmed infection and 5 suspected infection and 4 healthy)	Vaginal exudate taken intrapartum or in the immediate postpartum	NP	NP
		12 H. parainfluenzae			1 antepartum death12 live newborns (10 suspected infection)
Aydin MD (1998)^60	19	11 H. parainfluenzae8 H. influenzae	NP/M	NP	Urethritis	Urethral exudate	Biochemical tests	NP
Rodríguez-Guardado (2000)^61	1	H. influenzae	36/F	NP	Bilateral salpingitis	Peritoneal exudate	Culture	Salpingectomy, clindamycin, gentamicin
Cherpes TL (2002)^62	1	H. influenzae	39/F	8 weeks pregnant	Septic abortion. Acute chorioamnionitisLow-grade fever, vaginal bleeding, and headache	Blood culturePlacental biopsy	NP	Ampicillin + gentamicin + clindamycinCeftriaxoneLevofloxacin
Cox RA (2002)^63	39	38 H. influenzae1 H. parainfluenzae8 + mixed anaerobic flora	18 months -11 years/ F	14 recurrent vulvovaginitis5 two previous episodes of vulvovaginitis due to H. influenzae, 1 three previous episodes	Vulvovaginitis: vaginal discharge and irritation	Vaginal and vulvar exudate	Culture	19 amoxicillin9 amoxicillin/clavulanic acid6 trimethoprim6 miconazole2 metronidazole2 clarithromycin, erythromycin
Campos J (2003)^64	2	H. influenzae serotype e	NP/F	NP	Vaginitis	NP	NP	NP
Cuadros J (2004)^65	12	H. influenzae	2−12/ F	NP	Vulvovaginitis	Vaginal exudate	NP	NP
Mikamo H (2005)^66	1	H. influenzae	NP/F	NP	Bartholinitis	Gland exudate	Culture	Cefteram pivoxil
Tanaka K (2005)^67	8	H. influenzae	NP/F	NP	Bartholin's gland abscess	Gland exudate	Culture and PCR	NP
Iser P (2005)^68	2	H. influenzae	NP/M	Heterosexual	Nongonococcal urethritis	Urethral exudate	Culture	NP
Tsai HY (2006)^69	1	H. influenzae	NP/F	Prepubescent girl. No sexual relations, no abuse.	Vulvovaginitis, profuse vaginal discharge	Vaginal exudate	NP	NP
Varela JA (2006)^70	12	Haemophilus spp.	NP/M	HeterosexualCouple with cervical intraepithelial neoplasia (CIN)	Asymptomatic	Urethral exudate	Culture	NP
Kohlberger P (2007)^71	11	H. influenzae	NP/F	Girls with a history of sexual abuse	NP	Vaginal exudate	NP	NP
Santiago JL (2008)^72	1	H. parainfluenzae	41/M	HeterosexualUnprotected oral sex with a sex worker	Dysuria, urethral discharge and inguinal adenopathy.Conjunctival hyperemia with purulent exudate, tarsal follicular reaction, blepharitis and palpebral oedema	Urethral and conjunctival exudate	Culture and biochemical tests	Ceftriaxone + doxycycline
Orellana MA (2009)^73	36	22 H. parainfluenzae14 H. influenzae	NP/M	NP	NP	Urethral exudate	API NH® test	NP
Sikanić-Dugić N (2009)^74	5	H. influenzae	2−8/ F	NP	Vulvovaginitis	Vaginal exudate	Culture	NP
Kakisi (2010)^75	1	H. influenzae	42F	NP	Tubo-ovarian abscess	Abscess exudate	Culture	Surgery and cefoxitin/metronidazole
Glover WA (2011)^8	1	H. quentini	28/M	Previous episodes of urethritis and prostatitis.Unprotected sex	Scarce urethral discharge, painful ejaculation	Urethral exudate	16S rRNA sequencing	Ceftriaxone + azithromycin
	1	H. quentini	30/M	NP	NP			NP
	1	H. quentini +N. gonorrhoeae	32/M	NP	NP			NP
Mc Kechnie ML (2011)^76	11	H. influenzae	2 < 25,5 25−29,4 > 35/ F	NP	7 symptomatic4 asymptomatic	Vaginal exudate Endocervical exudateUrine	PCR	NP
Orellana MA (2011)^77	77	45 H. parainfluenzae32 H. influenzae4 + C. trachomatis2 + U. urealyticum	>15/M	NP	Urethritis	Urethral exudate	API NH® test	NP
Calner PA (2012)^78	1	H. influenzae	36/F	16 weeks pregnant	Septic abortion	Blood cultures	Culture	Curettage, ampicillin, gentamicin, clindamycin
Ranđelović G (2012)^79	2	H. influenzae	2−12/ F	NP	Vulvovaginitis	Vaginal exudate	NP	NP
Martin D (2013)^80	1	H. influenzae	48/ F	Uterine leiomyoma. No IUD	Acute endometritisLower abdominal pain and fever	Endometrial biopsy	PCR	NP
Tinguely R (2013)^81	1	H. parainfluenzae +N. gonorrhoeae	NP/M	Homosexual.Recently treated syphilis	Urethritis	Urethral exudate	16S rRNA sequencing	Ciprofloxacin
	1			Homosexual			PCR	Ciprofloxacin + doxycycline (NR)Ceftriaxone + azithromycin
Mendz GL (2014)^82	1	H. parainfluenzae +C. curvus	29/ F	Pregnant (25 weeks)	Chorioamnionitis, premature rupture of membranes, neonatal sepsis	Vaginal exudate	16S rRNA sequencing	Penicillin + gentamicin + metronidazole
Cardines R (2015)^83	46	37 H. parainfluenzae7 H. pittmaniae2 H. parahaemolyticus	32.5/ F	Pregnant (25−38 weeks)	Asymptomatic	Vaginal exudate	16S rRNA sequencing	NP
Hsu MS (2015)^84	1	H. parainfluenzae + Enterococcus spp.	29/M	Homosexual, unprotected sex	Dysuria, purulent urethral discharge	Urethral exudate	16S rRNA sequencing	Ceftriaxone + doxycycline
	1	H. parainfluenzae +N. gonorrhoeae +M. morganii	32/M		Dysuria, frequency, purulent urethral discharge			Ceftriaxone + doxycycline
	1	H. parainfluenzae + Enterococcus spp.	27/M		Dysuria, purulent urethral discharge, inguinal adenopathy			Clarithromycin
Deza G (2016)^85	52	45 H. parainfluenzae7 H. influenzae24 + other microbes10 + N. gonorrhoeae8 + C. trachomatis3 + C. albicans2 + G. vaginalis1 + U. urealyticum	31.8/M	31 (60%) homosexual and 21 (40%) heterosexual4 (8%) HIV9 (17%) previous STI52 (100%) unprotected oral sex29 (55%) anal sex, 44% unprotected19 (36%) vaginal sex, 31% unprotected	37 (71%) purulent urethral discharge13 (25%) non-purulent urethral discharge2 (4%) dysuria without urethral discharge	Urethral exudate	API20E	48 (92%) ceftriaxone + azithromycin/doxycycline2 (4%) doxycycline1 (2%) azithromycin1 (2%) ciprofloxacin
		28 solitarily23 H. parainfluenzae5 H. influenzae	33.7/M	15 (54%) homosexual and 13 (46%) heterosexual.3 (11%) HIV5 (18%) previous STI28 (100%) unprotected oral sex (5 (18%) only sexual practice)13 (46%) anal sex, 39% unprotected11 (39%) vaginal sex, 32% unprotected	18 (64%) purulent urethral discharge9 (32%) non-purulent urethral discharge1 (4%) dysuria without urethral discharge			28 (100%) ceftriaxone + azithromycin/ doxycycline
Frølund M (2016)^86	9	6 H. influenzae solitarily2 + adenovirus1 + M. genitalium	NP/M	NP	Acute urethritisDysuria and urethral discharge	First morning urine	PCR	NP
Ito S (2016)^87	22	21 H. influenzae solitarily1 + U. urealyticum	7 < 31 and14 > 31/M	11 married and 10 single.12 prior urethritis.21 unprotected oral sex(12 exclusively).9 unprotected oral and vaginal sex.13 sex worker partner	21 urethral discharge (13 moderate and 8 scant) in 10 serous,6 mucoid and 5 mucopurulent15 urethral irritation12 moderate dysuria3 meatitis and/or balanitis3 pharyngeal discomfortIncubation < 7 days	Urethral exudate	NP	NP
Kim H (2016)^88	1	H. influenzae	0−9/ F	Prepubescent girl	Vulvovaginitis	Vaginal or vulvar exudate	Culture	NP
You C (2016)^89	8	H. influenzae	NP/M	NP	Urethritis	First morning urine	16S RNA sequencing	NP
Porter M (2016)^90	31	H. influenzae	21−29/F	31 pregnancies	31 Chorioamnionitis	Placenta	Culture	NP
Baba H (2017)^91	1	H. influenzae	31/ F	17 weeks pregnant.History of upper respiratory infection with cough.	Septic abortion.High fever, vaginal bleeding, and abdominal pain	Vaginal exudateBlood culture	Culture, MALDI-TOF, PCR.	Piperacillin/tazobactamCefotaxime + metronidazole
Chen X (2017)^92	1	H. influenzae	6/ F	History of a foreign body in the vagina.Recurrent and chronic rhinitis. In nasal exudate, H. influenzae is isolated, derived from the same clone as the one detected in the vagina	Vulvovaginitis associated with rhinitis	Vaginal exudate	NP	NP
Deguchi T (2017)^93	73	H. influenzae	NP/M	NP	67 acute urethritis4 acute epididymitis2 urethritis and epididymitis	Urethral exudate	NP	NP
Ito S (2017)^1	68	54 H. influenzae solitarily5 + U. urealyticum4 + U. urealyticum + M. hominis2 + U. parvum1 + C. trachomatis1 + N. gonorrhoeae	35/M		Urethritis	Urethral exudate	HN-20 Rapid system identification test	43 azithromycin (5 NR)20 sitafloxacin3 levofloxacin2 ceftriaxone
	6	H. influenzae	NP/M	NP	Epididymitis			NP
	1				Acute prostatitis
Li JP (2017)^94	110	H. influenzae	NP/F	Prepubescent girls	Vulvovaginitis	Vaginal exudate	Culture	55 ofloxacin49 β-lactams6 NP
Caballero Mateos AM (2018)^95	1	H. parainfluenzae + C. trachomatis L2 (lymphogranuloma venereum)	34/M	Homosexual, unprotected sexHepatitis B previously treated. Secondary syphilis.Drug addict.	Proctalgia, constipation, rectal bleeding, fever.Rectal ulcer	Rectal biopsy	NP	Doxycycline
Horie K (2018)^96	1	H. quentini + Streptococcus spp. +M. hominis +U. urealyticum	34/M	Heterosexual. SinglePrevious urethritis (no other previous STIs)Casual partners	Moderate mucoid urethral dischargeIncubation 12 days	Urethral exudate	16S rRNA sequencing	Azithromycin
	1	H. quentini	24/M	Heterosexual SingleNo previous STIsHIV negativeUnprotected oral sex with a sex worker	Scant mucoid urethral discharge, dysuriaIncubation 4 days			Sitafloxacin
	1	H. quentini + Streptococcus spp. +	30/M	Heterosexual SingleNo previous STIsHIV negativeUnprotected oral and vaginal sex with a stable partner and other partners	Condyloma acuminatum on the glansScant serous urethral discharge, urethral irritation, dysuria			Levofloxacin
Kondo H (2018)^97	1	H. influenzae + Streptococcus spp. +U. urealyticum	32/M	Heterosexual Married4 previous episodes of urethritis (no other STIs)Multiple sexual partners	Urethral irritation, mucopurulent urethral discharge	Urethral exudate		AzithromycinND
	1	H. influenzae	27/M	Heterosexual MarriedNo previous STIsStable sexual partner	Urethral irritation, scant mucoid urethral dischargeIncubation 16 days
	1	H. influenzae +S. agalactiae	30/M	Heterosexual SingleNo previous STIsUnprotected oral sex with a sex worker	Urethral irritation, scant mucoid urethral dischargeIncubation 5 days
	1	H. influenzae	29/M	Heterosexual Married1 previous episode of urethritis (no other STIs)Unprotected oral sex with a sex worker	Dysuria, scanty mucoid urethral dischargeIncubation 12 days
	1	H. influenzae	21/M	Heterosexual Married1 previous episode of urethritis (no other STIs)Unprotected oral and vaginal sex with a sex worker	Urethral irritation, mucopurulent urethral dischargeIncubation 5 days
	1	H. influenzae	25/M	Heterosexual MarriedNo previous STIsUnprotected oral sex with a sex worker	Dysuria, urethral irritation, mucopurulent urethral dischargeIncubation 9 days
Saxena A (2018)^98	1	H. parainfluenzae	33/M	No previous STIsHIV negativeUnprotected oral sex with a sex worker	Urethral pruritus, urethral discharge	Urethral exudate	MALDI-TOF	Norfloxacin + amoxic-clav + fluconazole (NR)Azithromycin + ciprofloxacin
Cevik M (2019)^99	1	H. influenzae	35/ F	14 weeks pregnant	Septic abortion. Acute chorioamnionitisAbdominal pain, vaginal discharge/bleeding, fever.	Placental biopsyBlood cultureCervical exudate	NP	β-lactams
	1		22/ F	14 weeks pregnant
	1		33/ F	20 weeks pregnant.Premature rupture of membranesPrevious abortion. History of chorioamnionitis
Magdaleno Tapial J (2019)^100	38	32 H. parainfluenzae5 H. influenzae1 H. haemolyticus15 Haemophilus spp. solitarily9 + M. hominis/ Ureaplasma spp7 + Chlamydia6 + Neisseria2 + M. genitalium	30.5/35 (92%) M3 (8%) F	21 (55%) M homosexual13 (34%) M heterosexual3 (8%) F heterosexual1 (3%) NP5 (13%) HIV20 (57%) previous STI38 (100%) unprotected oral sex	22 (58%) purulent urethral discharge6 (16%) dysuria10 (26%) asymptomatic, risky sexual contact	Urethral exudate	NP	17 (45%) ceftriaxone + azithromycin12 (31%) ceftriaxone + doxycycline5 azithromycin4 doxycycline6NR
Wang HJ (2019)^101	230	H. influenzae	NP/F	Prepubescent girls	Vulvovaginitis	Vaginal exudate	Vitek culturesystem NH	NP
Alsuhaibani MA (2019)^102	1	H. parainfluenzae	26/F	Pregnant	Chorioamnionitis	Placenta and neonatal blood	Culture	Cefotaxime + gentamicin
Ducours M (2020)^103	1	H. parainfluenzae +S. hominis + E. faecalis + S. anginosus + p. harei	20/M	HeterosexualUnprotected vaginal sex	Urethral discharge	Urethral exudate	MALDI-TOF	Ceftriaxone + azithromycin
	1	H. parainfluenzae +N. gonorrhoeae +S. epidermidis +S. anginosus + S. mitis	31/M	HomosexualUnprotected anal sex	Dysuria, urethral discharge			Ceftriaxone + azithromycin
	1	H. parainfluenzae +M. genitalium +E. faecalis + S. mitis	37/M	Heterosexual	Dysuria, urethral discharge			Minocycline + pristinamycin
	1	H. parainfluenzae +N. gonorrhoeae +S. haemolyticus + Corynebacterium sp.	43/M	HomosexualHIVUnprotected oral and anal sex	Dysuria, urethral discharge			Ceftriaxone + doxycycline
	1	H. parainfluenzae +S. haemolyticus +E. faecalis	30/M	Homosexual. PrEPUnprotected oral and anal sex	Dysuria, urethral discharge			Cotrimoxazole (NR)Gentamicin +ciprofloxacin
	1	H. parainfluenzae +S. haemolyticus + S. mitis	28/M	HomosexualUnprotected anal sex	Dysuria, urethral discharge			Ceftriaxone + doxycycline
	1	H. parainfluenzae +S. haemolyticus	27/M	NP	NP			NP
	1	H. influenzae	55/M	Homosexual. PrEPUnprotected oral and anal sex	Urethral discharge			Ceftriaxone + azithromycin
	1	H. parainfluenzae +N. gonorrhoeae + E. coli	33/M	Homosexual. PrEPUnprotected oral and anal sex	Dysuria, urethral discharge			Ceftriaxone
Nishimura Y (2020)^104	1	H. influenzae	51/ F	Polypoid adenomyomaEarly stage endometrioid adenocarcinoma	Microabscesses in adenomyoma.	Vaginal exudateBlood culture	MALDI-TOF	Ceftriaxone + hysterectomy
Sierra Y (2020)^105	175	H. parainfluenzae ± NP125 NOT multiresistant50 multiresistant30 H. parainfluenzae +C. trachomatis/N. gonorrhoeae/T. pallidum10 NP10 H. parainfluenzae solitarily	37.8/97 (55%) M78 (45%) FNPNPNPNP42/M	NPNPNPNPNP4 homosexuals, 4 heterosexuals and 2 NP.4 HIV6 previous STI8 unprotected sex with a stranger, 1 unprotected sex with a stable partner and 1 NP	NPNP26 urethritis and 24 NP16 urethritis and 14 NP5 asymptomatic and 5 NP10 (100%) urethritis	79 (45%) urethral exudate62 (35%) vaginal13 (7%) preputial9 (5%) cervical6 (3%) rectal3 (2%) genital ulcer1 (1%) pharyngeal1(1%) semen1 (1%) urineNPNPNPNP10 urethral exudate	MALDI-TOF	NPNPNPNPNP5 ceftriaxone + azithromycin2 ceftriaxone + doxycycline1 ceftriaxone + azithromycin + doxycycline1 amox-clav
Snirivasan S (2020)^3	40	H. influenzae	NP/M	23 heterosexual17 homosexual	16 urethritis and 7 asymptomatic16 urethritis and 1 asymptomatic	First morning urine	16S rRNA sequencing	NP
Vives A (2020)^106	30	19 H. parainfluenzae10 H. influenzae1 Haemophilus sp.25 solitarily5 + other microbes4 + C. trachomatis1 + N. gonorrhoeae	36.6/M	17 (57%) heterosexual, 8 (27%) homosexual, 2 (7%) bisexual and 3 (10%) NP3 (10%) HIV with no detectable viral load13 (43%) previous STI21 (75%) oral sex, 15 (54%) vaginal sex, and 10 (36%) anal sex.N.o sexual partners 3.5 [1–20]	13 (43%) urethral discharge7 (23%) dysuria5 (17%) testicular pain2 (7%) genital ulcer1 (4%) hematospermia1 (4%) painful ejaculation1 (4%) lower abdominal pain	Urethral exudate	API NH® test	Ceftriaxone + azithromycin/doxycyclineAzithromycin ±ciprofloxacinCiprofloxacinDoxycycline8 NR
Hu BF (2021)^107	140	H. influenzae	5.8/F	NP	Symptomatic vulvovaginitis	Vaginal exudate	Vitek	Ampicillin
Bruins MJ (2021)^108	127	H. influenzae	37 (<12 y)/F2(12−17 y)/F45(18−51 y)/F43(>51)/F	NP	Symptomatic vulvovaginitis	Vaginal exudate	Culture	NP

F: woman; M: male; NP: not provided; NR: no response to treatment.

*

+ others: these studies do not describe which other microorganisms are isolated concomitantly.

Most of the studies adequately described the method used in the laboratory for the isolation of Haemophilus. Until 2011, mainly culture techniques and biochemical tests were used. From 2011 onwards, molecular biology techniques, such as PCR or the sequencing of the 16S subunit of rRNA, became more prominent. More recently, mass spectrophotometry has been used (MALDI -TOF).

Regarding patient gender, 34.8% (835/2397) were men; 48.3% were women (1158/2397) and 16.9% (404/2397) were not reported. Only half of the studies provided data on patient age. The men were all adults with a mean age of 30 years^20–55. The women were divided into two groups, one of children under 14 years of age (53.9%; 624/1158) and another of adults (14 years of age or older, 28.4%; 329/1158). The age was not recorded in 17.7% of women (205/1158).

In the group of men, H. parainfluenzae (44.1%; 367/835) and H. influenzae (42.6%; 356/835) were detected with almost equal frequency. The vast majority were diagnosed with urethritis. The most frequent clinical manifestations were urethral discharge, purulent or not, dysuria and urethral pruritus or irritation. Regarding sample type, in the vast majority of cases it was urethral exudates. The most frequently administered treatment was ceftriaxone with azithromycin and/or doxycycline, although the treatment was only specified in 40% (15/37) of the publications referring to men.

We subdivided the group of adult women according to whether the infection occurred during pregnancy (52.9%; 174/329) or not (47.1%; 155/329). In the latter, there were episodes of vaginitis, bartholinitis, salpingitis, endometritis or tubal abscesses. The absence of a significant portion of data prevents us from drawing conclusions about the prevalence of a specific species of Haemophilus. In pregnant women, almost all the studies refer to septic abortion, acute chorioamnionitis, premature rupture of membranes and neonatal sepsis. H. influenzae was detected more frequently (64.9%; 113/174) than H. parainfluenzae (30.5%; 53/174). H. influenzae was isolated in all the samples from girls (vaginal or vulvar exudates). Most of them presented vaginal discharge, vulvar irritation or pruritus and did not have predisposing factors. Antibiotic treatment included β-lactams or quinolones.

Samples of rectal origin only represented 2.3% (55/2397). The clinical and epidemiological information available on these patients was very scant, with the exception of one reported case⁷.

Table 2 summarises the information on the susceptibility ofHaemophilus to different groups of antibiotics (penicillin, carbapenems, tetracycline, quinolones, first-, second- and third-generation cephalosporins) contributed by 21 of the 67 studies. Four of these were excluded because they did not disaggregate the results by sample origin, as were those with fewer than five reported cases to avoid bias. Overall susceptibility of Haemophilus spp. to penicillins was 78.9%, and the overall rate of β-lactamase production was 26.4%. It should be remembered that although the production of β-lactamases is the main mechanism of resistance in this genus, there are also strains that do not produce β-lactamases but have alterations in the penicillin-binding proteins with a low affinity for β-lactams and even a combination of both mechanisms¹⁰⁹.

Table 2.

Antibiotic susceptibility (%) of Haemophilus spp. isolates.

Author (year of publication)	Species	No. cases	Prod. βL	Pen	CFZ	Ceph 2nd	Ceph 3rd	Ceph 4th	Carba	Macr	Quin	TC	RIF	FOS
Campognone P (1986)^30	Hi	17	–	100	100					100		100
Casin I (1988)^34	Hi	60	16.7	83.3	–	–	–	–	–	100	100	85.8	–	–
Houang E (1989)^37*	Hp + Hi	73
Bosch J (1991)	Hi	9		100
Vázquez F (1996)^57	HpHiH	1004510	2926.7–	7173.3–	–––	–––	–––	–––	–––	–––	–––	–––	–––	–––
Kragsberg P (1993)^51	Hi	6	0	100	100							100
Cox RA (1997)^4	Hi	11	–	100	–	–	–	–	–	–	–	–	–	–
García E (1997)^59	Hi	18	27	–	–	–	–	–	–	–	–	–	–	–
Cox RA (2002)^63	Hi + Hp	39	10	95	–	100	–	–	–	95	–	–	–	–
Tanaka K (2005)^67	Hi	8	37.5	37.5
Orellana MA (2009)^73	Hp + Hi	36	–	76.5	–	94.1	100	–	–	64.7	91.1	–	–	–
Orellana MA (2011)^77	Hp + Hi	77	34.2	75.9	–	92.4	100	–	–	73	87.8	76.8	–	–
Cardines R (2015)^83	HpH. pittmaniaeH. haemolyticus	3772	13.5––	85.1100100	–––	–––	97.3100100	–––	100100100	–––	91.9100100	–––	–––	–––
Deguchi T (2017)^93	Hi	73	27.4	67.1	–	58.9	82.6	–	99.5	95.2	100	98.6	–	–
Li JP (2017)^94*	Hi	110
Kondo H (2018)^97	Hi	6	16.7	16.7	–	–	–	–	–	–	33.3	–	–	–
Magdaleno Tapial J (2019)^100	Hp + Hi +H. haemolyticus	38	–	79	–	92.1	94.8	–	–	65.8	90.8	73.7	–	–
Wang HJ (2019)^101*	Hi	230
Ducours M (2020)^103	Hp + Hi	5	–	10	–	–	25	–	100	–	22.2	0	–	–
Sierra Y (2020)^105	Hp	175	–	87.1	–	92.3	99.4	–	–	88.2	78.6	53.7	–	–
Hu BF (2021)^107	Hi	136	35	–	–	81.6	98.5	–	100	91.9	100

Amin: aminoglycosides; ATM: aztreonam (monobactam); Carba: carbapenems; Ceph 2nd: 2nd-generation cephalosporins; Ceph 3rd: 3rd-generation cephalosporins; Ceph 4th: 4th-generation cephalosporins; CFZ: cefazolin (1st-generation cefalosporin); CHL: chloramphenicol; FOS: fosfomycin; Hi: Haemophilus influenzae; Hp: Haemophilus parainfluenzae; Macr: macrolides; Pen: penicillins; Prod. βL: β-lactamase production; Quin: quinolones; RIF: rifampicin; SXT: trimethoprim-sulfamethoxazole; TC: tetracyclines.

*

The data provided by these studies are not included because they include samples of a different origin to the genital apparatus.

In addition, susceptibility to these antibiotics was determined separately for each species. These data are shown in Table 3.

Table 3.

Rates of antibiotic susceptibility and presence of β-lactamases separated by species (%).

Species	Prod. βL	Pen	CFZ	Ceph 2nd	Ceph 3rd	Carba	Macr	Quin	TC
H. influenzae	27.97	76.14	100	73.7	92.9	99.7	94.9	98.5	93.9
H. parainfluenzae	24.81	81.66		92.3	99	100	88.2	80.9	53.7

Carba: carbapenems; Ceph 2nd: 2nd-generation cephalosporins; Ceph 3rd: 3rd-generation cephalosporins; CFZ: cefazolin (1st-generation cephalosporin); Macr: macrolides; Pen: penicillins; Prod. βL: β-lactamase production; Quin: quinolones; TC: tetracyclines.

Discussion

It is difficult to demonstrate the pathogenic role of microorganisms that can be colonisers and present together with other species, as in the case of HSNOTD and in particular Haemophilus influenzae. There are few studies with a control group on the prevalence of the latter in men with urethritis, although in one of them³, an association is demonstrated with non-gonococcal urethritis in both MSM and heterosexuals, which would support its aetiological and pathogenic role in urethritis. Doubts remain as to the pathogenic role of the rest of the HSNOTD species since there are no rigorous studies with valid methodology, and most of them are case series.

The acquisition mechanism varies depending on the characteristics of the patient. For example, in girls with no history of sexual intercourse or abuse, there could be an auto-inoculation mechanism from the nasal location to the vaginal area. In contrast, in adults, the transmission route is predominantly sexual. One of the most relevant data items we can extract from this bibliographical review is the high frequency with which urethritis caused by HSNOTD is associated with the practice of unprotected oral sex, which in many cases is the only form of exposure reported. Genital infection by HSNOTD seems to be associated with sexual contact with multiple sexual partners and with the domain of prostitution. However, it is important to be cautious when drawing conclusions about possible predisposing factors to genital infection by HSNOTD, since the information provided by the different publications is scant. Regarding the sexual orientation of male patients, 53.3% were heterosexual and 46.7% homosexual, but the information available is very limited and does not allow us to conclude that the risk of infection is related to sexual orientation. The same occurs with previous having had sexually transmitted infections (urethritis, HIV, syphilis, etc.) as a risk factor. We found the same number of patients with a history of STIs as without them, although once again we cannot draw definitive conclusions due to the general lack of information. The low number of publications in which HSNOTD is considered a pathogenic agent causing proctitis is striking.

The susceptibility rates of Haemophilus against cephalosporins, especially third-generation, are very high, which together with amoxicillin/clavulanic acid are the treatments of choice. Taking into account the antibiotic susceptibility profile of all the species described above, we can deduce that the empiric treatment administered for STIs could be more effective against H. influenzae than against H. parainfluenzae, since the latter presents lower rates of susceptibility to macrolides, tetracyclines and quinolones.

Traditionally, vulvovaginitis in girls has been treated with penicillins, and a great tendency to a recurrence of these episodes has been described³, which could be due to the ineffectiveness of empiric treatment, since the susceptibility rate of H. influenzae to these antibiotics is only 72.3%. This hypothesis is reinforced by the fact that the recurrence rate is higher in girls who had received penicillins in the preceding months to treat infections in other locations. One alternative to penicillins are cephalosporins, which show good susceptibility rates and do not have the possible side effects of other antibiotics, such as quinolones, in children. Finally, one of the works reviewed⁴⁶ demonstrates the frequency with which Haemophilus spp. is detected in the vaginal exudate of asymptomatic pregnant women, with percentages close to 10%. This could entail a greater risk of vertical transmission and infectious complications both at maternal level and regarding neonatal sepsis, and prophylactic eradication may be considered as a strategy. However, a deeper analysis of the risk-benefit ratio of this intervention, which could increase antibiotic pressure, is necessary.

This review has several limitations. An extensive search was carried out. However, articles in Spanish and English were included and we did not have access to a series of published studies, listed at the beginning of the article, meaning that not all published studies may not have been included. In addition, the wide variability of data published between articles regarding epidemiological, microbiological and antibiotic susceptibility data makes it difficult to analyse the data homogeneously and therefore draw conclusions. Furthermore, the methodology of most of the included studies is outmoded and the aetiological role of these microorganisms cannot therefore be firmly established.

Following the review, we can highlight, in HSNOTD, the role ofHaemophilus influenzae as an aetiological agent in cases of non-gonococcal urethritis in men, both MSM and heterosexuals, and therefore the usefulness of systematically searching for this microorganism in this entity, as it can explain a significant percentage of urethritis without microbiological isolation. It proved impossible to find consistently described epidemiological risk factors and/or an acquisition mechanism, hence more studies are needed. On the other hand, taking the antibiotic resistance profile into account, third-generation cephalosporins, amoxicillin-clavulanic acid and quinolones are postulated as the most successful eradication options. With regard to azithromycin and doxycycline, which are widely used in the empiric treatment of STIs, we did not find significant resistance rates in the review, although we are seeing a growing trend of strains with a higher resistance profile and higher minimum inhibitory concentrations for doxycycline¹¹⁰.

Conflicts of interest

The authors declare that they have no conflicts of interest.