Detection of pathogens causing genital ulcer disease by multiplex polymerase chain reaction / 中国医学科学杂志(英文版)

<p><b>OBJECTIVE</b>To establish a multiplex polymerase chain reaction (M-PCR) assay for simultaneous detection of pathogens causing genital ulcer disease (GUD).</p><p><b>METHODS</b>Based on the gene-specific region of the following pathogens: Chlamydia trachomatis omp1/ompb, herpes simplex virus (HSV) DNA polymerase, Treponema pallidum tpp47, Haemophilus ducreyi 16s rRNA, four sets of primers were designed and an M-PCR assay was developed to detect four pathogens in one test. The assay was evaluated with diagnostic result of golden standard for each pathogen.</p><p><b>RESULTS</b>Of the 51 clinical samples, M-PCR showed slightly higher positive rate (47.1%) of HSV than cell culture (23.6%). Meanwhile, the positive rate of T. pallidum detected by M-PCR and dark-field microscopy was 19.6% (10/51) and 15.7% (8/51), respectively. Only one sample was positive for H. ducreyi and no sample was positive for C. trachomatis detected by both M-PCR assay and culture.</p><p><b>CONCLUSION</b>This primary study indicated that M-PCR assay can simultaneously and rapidly detect the four etiologic pathogens causing GUD.</p>

Episome profiles and mobilizable beta-lactamase plasmid in Haemophilus ducreyi.

Chancroid caused by Haemophilus ducreyi has been described as a significantly predisposing factor of HIV heterosexual transmission in an endemic region of both diseases. The fastidious, H. ducreyi has been reported world wide with various antimicrobial susceptibility patterns. A high tendency of drug resistances has generally been found among isolates derived in Thailand. In this study, the plasmids of H. ducreyi were isolated and analysed from 63 clinically derived organisms. Twenty-nine out of 63 isolates (46%) revealed the same plasmid profiles. Plasmid DNA was further cloned into Escherichia coli and transformants were selected. A 3.6 kb plasmid (pCb) carrying ampicillin resistance was subsequently identified. The pCb conferred resistance to various beta-lactam antibiotics including penicillin G, carbenicillin, piperacillin, cefazolin, cefoperazone, ampicillin-sulbactam, and amoxicillin-clavulanate but not to cefoxitin. Co-resistance to streptomycin, chloramphenicol and tetracycline was not detected. Beta-lactamase gene was located on the major pCb fragment of EcoRI and AatII cutting.

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

Drug resistance of Haemophilus ducreyi.

Chancroid, the disease caused by H. ducreyi is one of the common sexually transmitted diseases (STD) in Thailand and other tropical countries. In Thailand, the diagnosis of chancroid is still based on clinical appearance which may be confused with other STD manifested by genital ulcers. In recent years the increasing resistance strains of H. ducreyi to these antimicrobial agents has been reported so that cultivation and antimicrobial susceptibility tests of this organism have become more important. This study showed that MBV is the best medium for isolation with a success rate of 48%. All strains tested from isolates of this study were resistant to ampicillin, due to production of beta-lactamase. Approximately 99% of the strains were resistant to tetracycline 92% of strains were resistant to sulfamethoxazole and 32% were resistant to trimethoprim. All isolates were susceptible to chloramphenicol, ceftriaxone, erythromycin and the fluorinated quinolones ciprofloxacin, norfloxacin, ofloxacin and pefloxacin. Beta-lactamase enzymes produced by 37 strains of H. ducreyi were determined for their isoelectric point (pI). All had pI of 5.4, indicative of plasmid-mediated beta-lactamase type TEM-1.

Chancroid: Report of Three Case / 대한피부과학회지

Chancroid is an acute localized, autoinoculable venereaI disease caused by Haemophilus ducreyi, characterized clinicaIly by painful ulceration at the site of incubation, and frequently accompanied by regional lymphadenopathy, and short incubation period. Three cases of chancroid were seen in Won Ju city, Kangwondo, Korea. All of the patients were male, and painful ulcers developed on genital region in 2 to 3 days after sexual contacts. Direct smear showed the characteristic "school-of-fish" arrangement of Haemophilus ducreyi by Gram stain. There were no regional lymphadenopathies and VDRL test were negative. Treatments were done with sulfisoxazole (Gantrisin) in case I, with sulfamethoxydiazine(Bayrena) and streptomycin in case 2, and with sulfamethoxydiazine and tetracycline in case 3 with exccllent results.