ORIGINAL RESEARCH ARTICLE

Haemophilus ducreyi detection by polymerase chain reaction in oesophageal lesions of HIV patients
M C Borges
MD PhD*,

J K B Colares

MD PhD*,

D M Lima

PhD*

and B A L Fonseca

MD PhD*

o Paulo Medical School at Ribeira o Preto; Department of Medicine, University of Sa o Carlos, *Department of Internal Medicine, University of Sa SP, Brazil

Summary: 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 nding may be an indication of its aetiopathological role in oesophageal lesions of HIV patients. Keywords: chancroid, microbiology, sexually transmitted diseases, diagnosis, DNA bacterial

INTRODUCTION
AIDS encompasses several clinical manifestations, ranging from asymptomatic infection to development of severe and potentially lethal opportunistic infections. Oesophageal disease occurs in as many as 40% HIV patients at some point of the HIV infection,1 infection being the most important aetiology. Candida albicans is the most prevalent infection, followed by cytomegalovirus and herpes virus infections.2,3 Other unusual agents have also been implicated in the aetiology of oesophageal disease. A considerable part of oesophageal lesions does not have an identied agent, and are considered idiopathic.2,4 These idiopathic oesophageal lesions can be due to HIV infection, apoptosis or another agent not yet identied by traditional diagnostic methods.5,6 Considering the efcacy of currently available treatment for most of the pathogens, a denitive diagnosis has an important prognostic implication. Therefore, the use of novel diagnostic methods should be considered in these patients. Owing to the diverse sexual activity described by our patients, we hypothesized that Haemophilus ducreyi could have an aetiopathologic role in oesophageal lesions of HIV-1-infected patients, and polymerase chain reaction (PCR) could improve its detection. Therefore, we studied the role of PCR to detect H. ducreyi in biopsies obtained from oesophageal lesions of HIV-1-infected patients.

METHODS
Seventy-nine HIV-1-infected patients presenting with gastrointestinal symptoms and who were attended at the Clinical o Paulo Medical School at Hospital of the University of Sa o Preto were included in the study. Seventy patients Ribeira
rio de Correspondence to: Dr B A L Fonseca, Laborato o Paulo University Medical School at Virologia Molecular, Sa o Preto, Av. Bandeirantes, 3900, Ribeira o Preto, SP Ribeira 14048.900, Brazil Email: baldfons@fmrp.usp.br

were submitted to one upper endoscopy, eight patients to two upper endoscopies and one patient to three upper endoscopies, with a total of 89 procedures. Ninety-six oesophageal biopsies were collected, kept either in liquid nitrogen or at 2 708C, and processed for PCR amplication. The clinical characteristics of these HIV-1-infected patients are listed in Table 1. Biopsies were also sent to routine histopathological studies according to our hospital protocol. The need for a biopsy was dened by the medical team, without any interference by the authors. The study was approved by the local Ethics Committee. All biopsies were thawed at room temperature in a laminar ow cabinet and grounded with a sterile scalpel. The material was transferred to a sterile tube, and tissue lysis was performed by adding 500 mL of lysis solution (10 mmol/L Tris-HCl, pH 8.0; 10 mmol/L EDTA, pH 8.0; 100 mmol/L NaCl; 1% SDS) and 1 mL of proteinase K (20 mg/mL), followed by incubation at 558C for two hours. The DNA extraction was carried out by adding 500 mL of phenol equilibrated with TE buffer (10 mmol/L Tris-HCl, pH 8.0; 1 mmol/L EDTA). The mix was agitated and centrifuged at 13,000 g for two minutes at room temperature. The aqueous phase was carefully removed to a new tube and 500 mL of chloroform was added. Similarly, fresh agitation and centrifugation were performed and the aqueous phase was transferred to a new tube. Then, 1000 mL of 100% ethanol and 50 mL of 3 mol/L sodium acetate ( pH 7.08.0) were added and incubated at 2 208C overnight. After that, the mix was centrifuged at 13,000 g for 30 minutes at 48C. The pellet was washed twice with 70% ethanol and dried in the speed-vac for 5 minutes. The pellet was resuspended in 200 mL of TE buffer (10 mmol/L Tris-HCl, pH 8.0; 1 mmol/L EDTA) and incubated at 688C for 20 minutes. The extracted DNA sample was conserved in a 2 208C freezer. We used specic primers to H. ducreyi, designed to amplify the 16S rRNA gene.7 The amplicon for H. ducreyi has an expected size of 799 base pairs. In all reactions, negative and positive controls were added. As a positive control, extracted DNA from a reference strain of H. ducreyi obtained from o Paulo was used in all PCR reactions. Adolfo Lutz Institute, Sa

International Journal of STD & AIDS 2009; 20: 238 240. DOI: 10.1258/ijsa.2008.008317

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Table 1

Characteristics of HIV-1-infected patients 79 33.94 (20 65) 49 (62%) 103.59 (1 795.2) 52 24 19 7 14 13 5 4 3 11

Patients (n) Age (years) Gender (male) CD4 T-cell count (cells/mL) CD4 T-cell , 200 (cells/mL) Primary symptom, n Vomiting Epygastralgia Odynophagia Dysphagia Abdominal pain Sternal pain Diarrhoea Gastrointestinal Bleeding Other or unknown
These results are expressed in mean and limits Twenty patients had more than one primary symptom

Table 2 Characteristics of HIV-1-infected patients with oesophageal lesions whose polymerase chain reactions were positive to H. ducreyi Patient 1 Age (years) Gender CD4 T-cell count (cells/mL) Symptom leading to upper endoscopy Upper endoscopy 40 F 28.9 Sternal pain Patient 2 36 M 15 Dysphagia Patient 3 46 M 194.7 Epygastralgia

Oesophageal candidiasis

Oesophageal ulcers

Histopathological ndings of endoscopy biopsies

RESULTS
The PCR was performed in all oesophageal biopsies. In three biopsies from three different patients, the PCR for H. ducreyi was positive (Figure 1). The characteristics of these HIV-1-infected patients with a positive PCR for H. ducreyi, and the endoscopic and histopathological ndings are given in Table 2. Only one of these patients, patient 3, had a history of a sexually transmitted disease, i.e. syphilis. Patient 2 had a history of drug abuse and patient 1 reported unprotected sexual intercourse. The PCR was repeated with the same biopsies and the positive result for H. ducreyi was conrmed. Because these biopsies were retrospectively analysed, it was not possible to ascertain whether these patients were treated or not for H. ducreyi infection, and they were no longer available for medical evaluation.

Mild to moderate chronic oesophagitis associated with an ulcerative lesion and Candida albicans infection

Oesophageal candidiasis and an oesophageal ulcer Mild chronic Exsudative inammatory chronic histiocytic oesophagitis process probably associated associated with with Candida mycobacterium sp. infection infection. Moderate chronic, active and ulcerative oesophagitis associated with Candida sp. and herpes virus

DISCUSSION
This study is the rst to demonstrate the presence of H. ducreyi genetic material in oesophageal biopsies of HIV-1-infected

Figure 1 Detection of H. ducreyi genome on 1% agarose gel electrophoresis, stained with ethidium bromide. From left to right: molecular weight marker (M) (100 pb DNA ladder), positive control (C) and three biopsies positive to H. ducreyi. The amplicon for H. ducreyi has 799 base pairs. As a positive control, extracted DNA from a reference strain of H. ducreyi obtained o Paulo was used from Adolfo Lutz Institute, Sa

patients. This nding raises the need for a better understanding of the aetiology of oesophageal lesions in HIV patients, especially in patients with a severe immunodeciency. Owing to the diverse sexual activity described by our patients and considering the different modes of acquiring sexually transmitted diseases, the rationale of this study was to evaluate the role of sexually transmitted infections in oesophageal ulcers of HIV patients. Therefore, we also performed PCR with respect to syphilis in all samples, but the results were all negative. The H. ducreyi genetic material was detected by PCR in three of the 79 patients studied. Two of these patients had a severe immunodeciency, demonstrated by a CD4 T-cell count of , 100 cells/mL. This nding has been indicated as a risk factor for the development of oesophageal lesions by atypical pathogens.1 H. ducreyi is a strict human pathogen, and there is no known animal or environmental reservoir. Human-to-human transmission of H. ducreyi is primarily by sexual means alone.8 The route of infection of the patients in our study is unclear since it was not possible to obtain this information either from the charts, due to the retrospective design of the study, or in person, due to the death of these patients. Chancroid has a worldwide distribution, typically associated with low socioeconomic status and precarious hygiene. It is a common cause of genital ulcers in developing countries, predominantly in men with a history of promiscuity.9 The prevalence of chancroid in Brazil is not known. Non-genital chancroid, such as of the mouth, ngers, thighs, abdomen, feet and limbs, is rarely described.10,11 Oesophageal lesions caused by H. ducreyi have not been previously reported. The diagnosis of H. ducreyi based on morphology by direct microscopy, with the nding of Gram-negative cocobacilli, has a low accuracy (30 50%).12 14 The culture was considered the gold standard, but is a difcult method, requires special cultural media and has a sensitivity of only about 75% at

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best.15 17 In an attempt to improve the sensitivity of the diagnosis, DNA amplication techniques have been developed.18 According to Chui et al.,7 the PCR used in this study has a sensitivity of 83 98% and a specicity of 51 67%.7 The low specicity of PCR in some studies probably reects the poor sensitivity of culture for diagnosing chancroid. In all three patients, the histopathological evaluation demonstrated another agent (candida, mycobacterium and herpes virus) that could be the aetiology of the oesophageal lesions. However, we believe that the nding of another pathogen in these patients does not rule out the aetiopathological role of the H. ducreyi, since several simultaneous pathogens have been described in HIV patients with oesophageal symptoms. In the majority of patients, candida is one of the co-infectious agents.1,2 The nding of H. ducreyi genetic material in oesophageal lesions of HIV patients raises the possibility of a new cause of oesophageal lesions and creates the necessity for further studies for a better understanding of its role in oesophageal ulcer causation in HIV patients. Epidemiological studies should be undertaken to determine the likely mode of transmission and reservoir for H. ducreyi. In conclusion, the nding of H. ducreyi genetic material in oesophageal lesions is an indication that a new pathogen may be playing a role in the causation of gastrointestinal lesions of HIV patients, and clinicians involved in the care of HIV-infected patients should consider H. ducreyi in the aetiology of oesophageal ulcers.

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