Putative vaccine candidates and drug targets identified by reverse vaccinology and subtractive genomics approaches to control Haemophilus ducreyi, the causative agent of chancroid.

Chancroid is a sexually transmitted infection (STI) caused by the Gram-negative bacterium Haemophilus ducreyi The control of chancroid is difficult and the only current available treatment is antibiotic therapy; however, antibiotic resistance has been reported in endemic areas. Owing to recent outbreaks of STIs worldwide, it is important to keep searching for new treatment strategies and preventive measures. Here, we applied reverse vaccinology and subtractive genomic approaches for the in silico prediction of potential vaccine and drug targets against 28 strains of H. ducreyi We identified 847 non-host homologous proteins, being 332 exposed/secreted/membrane and 515 cytoplasmic proteins. We also checked their essentiality, functionality and virulence. Altogether, we predicted 13 candidate vaccine targets and three drug targets, where two vaccines (A01_1275, ABC transporter substrate-binding protein; and A01_0690, Probable transmembrane protein) and three drug targets (A01_0698, Purine nucleoside phosphorylase; A01_0702, Transcription termination factor; and A01_0677, Fructose-bisphosphate aldolase class II) are harboured by pathogenicity islands. Finally, we applied a molecular docking approach to analyse each drug target and selected ZINC77257029, ZINC43552589 and ZINC67912117 as promising molecules with favourable interactions with the target active site residues. Altogether, the targets identified here may be used in future strategies to control chancroid worldwide.

Macrolides for treatment of Haemophilus ducreyi infection in sexually active adults.

BACKGROUND: Chancroid is a genital ulcerative disease caused by Haemophilus ducreyi. This microorganism is endemic in Africa, where it can cause up to 10% of genital ulcers. Macrolides may be an effective alternative to treat chancroid and, based on their oral administration and duration of therapy, could be considered as first line therapy. OBJECTIVES: To assess the effectiveness and safety of macrolides for treatment of H ducreyi infection in sexually active adults. SEARCH METHODS: We searched the Cochrane STI Group Specialized Register, CENTRAL, MEDLINE, Embase, LILACS, WHO ICTRP, ClinicalTrials.gov and Web of Science to 30 October 2017. We also handsearched conference proceedings and reference lists of retrieved studies. SELECTION CRITERIA: Randomized controlled trials (RCTs) comparing macrolides in different regimens or with other therapeutic alternatives for chancroid. DATA COLLECTION AND ANALYSIS: Two review authors independently assessed trials for inclusion, extracted data and assessed risk of bias. We resolved disagreements through consensus. We used the GRADE approach to assess the quality of the evidence. MAIN RESULTS: Seven RCTs (875 participants) met our inclusion criteria, of which four were funded by industry. Five studies (664 participants) compared macrolides with ceftriaxone, ciprofloxacin, spectinomycin or thiamphenicol. Low quality evidence suggested there was no difference between the groups after treatment in terms of clinical cure (risk ratio (RR) 1.09, 95% confidence interval (CI) 0.97 to 1.21; 2 studies, 340 participants with syndromic approach and RR 1.06, 95% CI 0.98 to 1.15; 5 studies, 348 participants with aetiological diagnosis) or improvement (RR 0.89, 95% CI 0.52 to 1.52; 2 studies, 340 participants with syndromic approach and RR 0.80, 95% CI 0.42 to 1.51; 3 studies, 187 participants with aetiological diagnosis). Based on low and very low quality evidence, there was no difference between macrolides and any other antibiotic treatments for microbiological cure (RR 0.93, 95% CI 0.74 to 1.16; 1 study, 45 participants) and minor adverse effects (RR 1.34, 95% CI 0.24 to 7.51; 3 studies, 412 participants).Two trials (269 participants) compared erythromycin with any other macrolide type. Low quality evidence suggested that, compared with azithromycin or rosaramicin, long courses of erythromycin did not increase clinical cure (RR 1.00, 95% CI 0.91 to 1.10; 2 studies, 269 participants with syndromic approach and RR 1.04, 95% CI 0.93 to 1.16; 2 studies, 211 participants with aetiological diagnosis), with a similar frequency of minor adverse effects between the groups (RR 1.14, 95% CI 0.63 to 2.06; 1 trial, 101 participants). For this comparison, subgroup analysis found no difference between HIV-positive participants (RR 1.02, 95% CI 0.73 to 1.43; 1 study, 38 participants) and HIV-negative participants (RR 1.04, 95% CI 0.94 to 1.14; 1 study, 89 participants). We downgraded the quality of evidence to low, because of imprecision, some limitations on risk of bias and heterogeneity.None of the trials reported serious adverse events, cost effectiveness and participant satisfaction. AUTHORS' CONCLUSIONS: At present, the quality of the evidence on the effectiveness and safety of macrolides for treatment of H ducreyi infection in sexually active adults is low, implying that we are uncertain about the estimated treatment effect. There is no statistically significant difference between the available therapeutic alternatives for the treatment of sexually active adults with genital ulcers compatible with chancroid. Low quality evidence suggests that azithromycin could be considered as the first therapeutic alternative, based on their mono-dose oral administration, with a similar safety and effectiveness profile, when it is compared with long-term erythromycin use.Due to sparse available evidence about the safety and effectiveness of macrolides to treat H ducreyi infection in people with HIV, these results should be taken with caution.

Etiology of Genital Ulcer Disease in Male Patients Attending a Sexually Transmitted Diseases Clinic: First Assessment in Cuba.

BACKGROUND: Sexually transmitted diseases (STDs) and in particular genital ulcer disease (GUD) have a major impact on morbidity and mortality in developing countries. The World Health Organization recommends the use of syndromic guidelines for the treatment of sexually transmitted infections (STIs) in resource-constrained countries. Surveillance of autochthonous etiologies provides epidemiological information contributing to the prevention and treatment of STIs. We investigated the etiology and factors associated with GUD among male patients attending a STD clinic in Havana, Cuba. METHODS: Swabs from genital ulcers of 113 male patients, collected from May 2012 to June 2015, were analyzed using PCR for herpes simplex virus types 1 and 2, Treponema pallidum, Haemophilus ducreyi, and Chlamydia trachomatis. We also investigated the clinical and epidemiological characteristics associated with the presence of these pathogens in GUD. RESULTS: At least one of the pathogens was detected in 70% of patients. The occurrence of the pathogens was herpes simplex virus type 2 (HSV-2) (51.3%), T. pallidum (29.2%), and C. trachomatis (1.8%). Co-infections occurred as follows: T. pallidum-HSV-2 (10.6%), C. trachomatis-HSV-2 (0.9%) and C. trachomatis-T. pallidum (0.9%). Herpes simplex virus type 1 and H. ducreyi were not detected. Ages 15 to 40 years, HIV-positive serostatus, and no condom use were significant risk factors for the presence of HSV-2 in genital ulcers. CONCLUSIONS: Our preliminary results highlight the predominance of HSV-2 and T. pallidum as the leading GUD etiologies in the study population and identified risk factors associated with HSV-2. This information should help to inform guidelines for better management of GUD in Havana, Cuba.

Haemophilus ducreyi detection by polymerase chain reaction in oesophageal lesions of HIV patients.

HIV patients frequently have opportunistic oesophageal infections. We report Haemophilus ducreyi genetic material detected by polymerase chain reaction in biopsies of oesophageal lesions in three HIV-1-infected patients. This finding may be an indication of its aetiopathological role in oesophageal lesions of HIV patients.

Epidemic Lymphogranuloma venereum during epidemics of crack cocaine use and HIV infection in the Bahamas.

BACKGROUND: Since the early 1980s, the Bahamas has experienced sequential epidemics of freebase/crack cocaine use, genital ulcer-inguinal adenopathy disease (GUD), and heterosexual HIV infection. GOAL: To prospectively define the etiology of GUD in patients at the Princess Margaret Hospital during outbreaks of crack cocaine use, GUD, and HIV infection in the Bahamas. STUDY DESIGN: In Nassau, 47 consecutive patients with GUD underwent serologic testing for syphilis and for infections with HIV, herpes simplex virus type 2 (HSV-2), and Chlamydia trachomatis. Genital ulcer specimens were tested by culture and/or polymerase chain reaction (PCR) assay for Haemophilus ducreyi; by PCR and/or antigen assay for HSV; and by PCR for C trachomatis. Lymph node aspirates were tested by PCR for C trachomatis and H ducreyi. RESULTS: Twenty patients (43%) had HIV infection; eight had lymphogranuloma venereum (LGV), confirmed by PCR detection of C trachomatis sequences consistent with the L2 serovar; and nine others had possible LGV, on the basis of serum microimmunofluorescent C trachomatis antibody titers > or =256. Inguinal lymphadenopathy or bubo was present in 15 of 17 patients, who thus met the laboratory criteria for definite or possible LGV, and in 7 of 30 who did not meet such laboratory criteria (P < 0.001). Thirteen patients had confirmed genital herpes, seven had confirmed chancroid, and four had probable or possible primary syphilis. CONCLUSIONS: The epidemics in the Bahamas of crack use, heterosexual HIV infection, and GUD apparently included epidemic transmission of LGV.

Genital ulcers: etiology, clinical diagnosis, and associated human immunodeficiency virus infection in Kingston, Jamaica.

Individuals presenting consecutively with genital ulcers in Kingston, Jamaica, underwent serological testing for human immunodeficiency virus (HIV) infection, chlamydial infection, and syphilis. Ulcer material was analyzed by multiplex polymerase chain reaction (M-PCR) analysis. DNA from herpes simplex virus (HSV), Haemophilus ducreyi, and Treponema pallidum was detected in 158 (52.0%), 72 (23.7%), and 31 (10.2%) of 304 ulcer specimens. Of the 304 subjects, 67 (22%) were HIV-seropositive and 64 (21%) were T. pallidum-seroreactive. Granuloma inguinale was clinically diagnosed in nine (13.4%) of 67 ulcers negative by M-PCR analysis and in 12 (5.1%) of 237 ulcers positive by M-PCR analysis (P = .03). Lymphogranuloma venereum was clinically diagnosed in eight patients. Compared with M-PCR analysis, the sensitivity and specificity of a clinical diagnosis of syphilis, herpes, and chancroid were 67.7%, 53.8%, and 75% and 91.2%, 83.6%, and 75.4%, respectively. Reactive syphilis serology was 74% sensitive and 85% specific compared with M-PCR analysis. Reported contact with a prostitute in the preceding 3 months was associated with chancroid (P = .009), reactive syphilis serology (P = .011), and HIV infection (P = .007). The relatively poor accuracy of clinical and locally available laboratory diagnoses pleads for syndromic management of genital ulcers in Jamaica. Prevention efforts should be intensified.

Genital ulcers: etiology, clinical diagnosis, and associated human immunodeficiency virus infection in Kingston, Jamaica

Individuals presenting consecutively with genital ulcers in Kingston, Jamaica, underwent serological testing for human immunodeficiency virus (HIV) infection , chlamydial infection, and syphilis. Ulcer material was analyzed by the multiplex polymerase chain reaction (M-PCR) analysis DNA from herpes simplex virus (HSV), Haemophilus ducreyi, and Treponema pallidum was detected in 158 (52.0 percent), 72 (23.7 percent), and 31 (10.2 percent) of 304 ulcer specimens. Of the 304 subjects, 67 (22 percent) were HIV-seropositive and 64 (21 percent) were T. pallidum-seroactive. Granuloma inguinale was clinically diagnosed in nine (13.4 percent) of 67 ulcers negative by M-PCR analysis and in 12 (5.1 percent) of 237 ulcers positive by M-PCR analysis (P = .03). Lymphogranuloma venereum was clinically diagnosed in eight patients. Compared with M-PCR analysis, the sensitivity and specificity of a clinical diagnosis of syphilis, herpes, and chancroid were 67.7 percent, 53.8 percent, and 75 percent and 91.2 percent, 83.6 percent, and 75.4 percent, respectively. Reactive syphilis serology was 74 percent sensitive and 85 percent specific compared with M-PCR analysis. Reported contact with a prostitute in the preceding 3 months was associated with chancroid (P = .009), reactive syphilis serology (P = .011), and HIV infection (P = .007). The relatively poor pleads for syndromic management of genital ulcers in Jamaica. Prevention efforts should be intensified.(Au)

Frecuencia de Haemophilus ducreyi en la etiología de la úlcera genital y papel de la coloración de Gram en el diagnóstico de chancroide

Objetivo: determinar la frecuencia de H. ducreyi en la úlcera genital (UG) y definir la importancia de la coloración de Gram en el diagnóstico de chancroide. Material y métodos: se trata de un estudio prospectivo, longitudinal. Con todos los pacientes se siguió un protocolo de laboratorio para el estudio de la UG que incluyó: coloración de Gram, prueba de Tzanck, campo oscuro, VDRL, FTA-ABS, aislamiento en línea celular para Herpes simplex y cultivo de H. ducreyi utilizando un medio bifásico. Resultados: se studiaron 79 pacientes con predominio del sexo masculino. En 35 pacientes (44.3 por ciento) se encontró uno o más agentes de transmisión sexual y en 44 (55.6 por ciento) no se encontró agente etiológico. En los 35 pacientes con UG los agentes encontrados fueron: H. ducreyi en 11 (13.9 por ciento), Herpes simplex en 10 (12.7 por ciento), Treponema pallidum en tres (3.8 por ciento), Calymatabacterium granulomatis en dos (2.5 por ciento) y en nueve (11.4 por ciento) la etiología fue mixta. Al comparar el cultivo de H. ducreyi con la coloración de Gram se encontró en esta última una sensibilidad de 87.5 por ciento, especificidad de 94.2 por ciento y valores predictivos positivos y negativos de 82.3 por ciento y 96.0 por ciento respectivamente, con un intervalo de confianza de 95 por ciento. Conclusiones: H. ducreyi es la primera causa de UG en este estudio; el encontrar etiologías mixtas en la UG hace necesario establecer protocolos que permitan buscar la mayoría de los agentes etiológicos de la UG. La coloración de Gram continúa siendo de mucha utilidad para sostener el diagnóstico de H. ducreyi

Propuesta y ensayo de un medio de cultivo para mejorar la recuperación de haemophilus ducreyi y detección de antígenos específicos de dos especies de haemophilus: ducreyi e influenza / Proposal and trial of cultives medio to Haemophilus ducreyi: and detection of specific antigens of two species of Haemophilus: ducrey and influenza

El diagnóstico de laboratorio mediante, cultivo de Haemophilus ducreyi, causante del chancro blando resulta conflictivo, por ser bacterianutricionalmente exigente. La serología no es eficaz por la existencia de antígenos comunes con otras bacterias, por lo que, el diagnóstico muchas veces es soloclínico. En el presente trabajo se abordó la posibilidad de mejorar y aumentar el crecimiento de Haemophilus ducreyi e influenzae considerando sus requerimientos nutricionales y condiciones de cultivo, introduciendose modificaciones que optimizaron el procedimiento, lo cual, permitió obtener cantidades importantes de antígenos para estudios inmunoquímicos, caracterizar fracciones antígenicas que presenten específicidad para cada una de las especies analizadas, con aplicación futura para desarrollar pruebas serológicas eficaces. Se obtuvieron antisueros policlonales en conejos contra ambas especies de Haemophilus, se determinó el título precipitante y se evidenció la existencia de antígenos comunes y específicosque, mostraron diferencias antigénicas en pruebas de inmunodifusión doble, tal diferencia se comprobó en electroforesis en gel de poliacrilamida (PAGE-SDS). Se observaron proteínas de peso molecular aparentemente específico para cada uno delos Haemophilus: para el ducreyi una proteína de peso molecular aproximado de 24Kda. y, en el influenzae otra que fluctúa entre 70 y 80 Kda. Estos estudios aún continúan en proceso, en una segunda fase se intenta establecer si esta diferencia antigénica es detectada por el suero de pacientes infectados con estos microorganismos mediante el Western Blott

Treatment of chancroid with a single dose of spectinomycin.

Fifty patients with lesions characteristic of chancroid were enrolled in an open-label prospective study to examine the efficacy of a single 2-gm dose of spectinomycin for treatment of chancroid. Only those patients (41 men; aged 18 to 49 years) with positive culture results for Haemophilus ducreyi were included in the analysis. Patients each received a single 2-gm dose of spectinomycin intramuscularly. The recovery process began on the third day of follow-up, as evidenced by the occurrence of epithelialization and a decrease in inflammation. By the seventh day after treatment, only one patient had ulcers; 40 patients experienced eradication of all ulcers (P less than 0.0001). The condition of nodes affected by infection also indicated efficacy of treatment (P less than 0.01); only one patient still had a swollen node by the fourteenth day after treatment. Of the 41 patients, 37 (90%) had negative culture results for H. ducreyi on the third day after treatment. Only 4 patients (10%) required a second dose of spectinomycin on the seventh day to affect a cure. Treatment with spectinomycin resulted in a 98% cure rate 14 days after treatment. The minimum inhibitory concentration (MIC) of spectinomycin was 1 microgram/mL to 3 micrograms/mL in the 15 strains studied. The drug was well tolerated and no adverse reactions were reported. It is concluded that a single 2-gm dose of spectinomycin is a safe and effective alternative drug for treatment of chancroid.