TNF alpha and NRAMP1 polymorphisms in leprosy.

OBJECTIVE: To determine the association of TNF alpha and NRAMP1 polymorphisms in leprosy. MATERIAL AND METHOD: The polymorphisms of TNF alpha at -238, -308, and NRAMP1 at INT4, D543N, and3' UTR were examined in 37 patients with leprosy (24 multibacillary and 13 paucibacillary) and 140 healthy controls. PCR-SSP and PCR-SSO method were used to type TNF and NRAMP1 polymorphisms. RESULTS: The genotype frequency of TNF-308 G/A was significantly increased in all leprosy patients compared to the controls (p = 0.04, OR = 2.69). When leprosy types were divided, the allele frequency of TNF-308A was significantly increased in multibacillary leprosy compared to the normal controls (p = 0.04, OR = 2.93). There was no significant difference in the distribution of the genotypes and allele frequencies of TNF -238 and NRAMP1 polymorphisms between the patients and controls. CONCLUSION: TNF-308A was associated with susceptibility to multibacillary leprosy.

Detection and identification of Mycobacterium species by polymerase chain reaction (PCR) from paraffin-embedded tissue compare to AFB staining in pathological sections.

BACKGROUND: Polymerase chain reaction (PCR) is a recent, rapid and reliable method in the detection of causative organism. The authors tried to determine the possibility of using PCR technique as an alternative way to detect mycobacterial DNA from paraffin-embedded tissue to avoid repeated biopsy from the patient. MATERIAL AND METHOD: Paraffin-embedded tissue blocks, the corresponding histopathologic slides, and cultural results were retrospectively searched for according to the patient's records, the granuloma clinic, Department of Dermatology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand from 1994-2000. One hundred and thirty-one tissue blocks and slides were found but only 120 cultural results were retrieved Histologic sections were reviewed for AFB findings and PCR was done using 16S rRNA sequences to detect M. tuberculosis by one-tube nested technique and multiplex PCR for M. marinum and M. fortuitum complex. RESULTS: The causative organisms were identified by AFB staining in pathologic sections 31.29%, by PCR 35.87%, and by culture 30.00% of tested samples. The sensitivity of PCR when compared to AFB result was 29.26%, specificity 61.11% but when compared to cultural results, the sensitivity of PCR was 66.67% and AFB sensitivity was 41.66% with specificity 76.19% and 72.61% respectively. CONCLUSION: The low sensitivity of the PCR method may be due to formalin fixation, deparaffinization process, DNA extraction method, the use of 16S rRNA-based primers and the length of the expected product, and the tissue type that may have Taq polymerase inhibitor. Therefore, PCR should be used to augment the information of the conventional method in the diagnosis of mycobacterial infection.

Gender difference in socio-epidemiological factors for leprosy in the most hyper-endemic district of Nepal.

Leprosy which has caused stigma and social ostracism for millennium is nearing elimination worldwide as a public health problem, but the leprosy burden in Nepal is still 4.4 times greater than WHO's target level of less than one case per 10,000 population. Although leprosy affects both the sexes, in most parts of the world males are affected more than females at a ratio of 2:1. The general objective of the study was to investigate the gender difference in socio-epidemiological factors for leprosy. The analytic cross-sectional study was carried out in one of the most hyper endemic district- Dhanusa district of Nepal. Stratified random sampling method was applied for the selection of the patients. Chi-square/Fisher's exact test was applied to assess statistically significant differences in values between males and females. There were 580 leprosy patients (385 male and 195 female) aged above 15 years registered for multi drug therapy between April 1, 2001 to March 31,2002 in the 16 main health centers of the district. Out of 580 patients, 273 patients (183 males and 90 females) were included in the study in order to collect the data on socio-demographics, patient's knowledge on leprosy, treatment seeking behaviour, and social problems faced by the patients. Data were collected using a structured interview schedule. The mean age of the male patients was 45.1 years (range 15-77 years) and female patients were 40.3 years (range 15-75 years). Among male patients 93.4% were married while among female patients 70.0% were married. Among male patients 51.9% were illiterate whereas 71.1% were illiterate among female patients. Most of the patients (69.6%) lived in joint family and the rest in nuclear family. Among male patients, 86.9% had good knowledge about the disease compared to 73.3% among females. This study showed that among the female patients 12.2% were facing high level of social problems, while among male patients only 4.4% were facing the same. About 15% patients had poor treatment seeking behaviors (8.2% among males and 27.8% among females). A significant gender differences among leprosy patients have been found in age distribution, educational status, marital status, caste types, family members, and overall knowledge on the general aspect of leprosy, social problems faced by the patients and treatment seeking behaviour.

Mycobacterial skin infections: comparison between histopathologic features and detection of acid fast bacilli in pathologic section.

BACKGROUND: Detection of acid fast bacilli (AFB) in chronic granulomatous inflammation is an important clue for mycobacterial infection. DESIGN: A retrospective review of 104 pathologic sections (from 1994 to 2001) of suspected cases of mycobacterial (tuberculous and nontuberculous) skin infections to study histopathologic features and the correlation with the presence of AFB in the section was performed. RESULTS: All cases showed granulomatous inflammations that can be categorized into 4 types: mixed cell, suppurative, tuberculoid and palisading granuloma. AFB was found in 32 sections (30.77%). Ninety five specimens from 104 specimens were simultaneously cultured. AFB positive cases yielded higher positive cultural results, 17 from 29 cases (58.62%) compared to the AFB negative group, 23 from 66 cases, (34.85%). Mixed cell granuloma was the most common histologic feature, but suppurative granuloma was the most common histological feature (56.25%) in which AFB could be found, which was statistically significantly different from other types of granuloma. Tuberculoid granuloma was more common in the AFB negative group (20.83%) compared to the AFB positive group (9.37%) but the difference was not statistically significant. In cases that AFB could not be found, the inflammation tended to be located in the upper half of the dermis. CONCLUSION: AFB can be more frequently detected in suppurative granuloma that might be located in any portion of the dermis. This finding was not species specific.

Gender differences in epidemiological factors associated with treatment completion status of leprosy patients in the most hyperendemic district of Nepal.

The introduction of multidrug therapy (MDT), recommended by WHO, has been a major advance in the treatment of leprosy because of its relatively short treatment course and low rate of relapse. Although leprosy treatment is provided to both sexes equally, in most parts of the world significant differences have been found in treatment status. The main objective of the study was to investigate gender differences in epidemiological factors associated with treatment status of leprosy patients. An analytic cross-sectional study was carried out in the most hyperendemic Dhanusa District, Nepal. Stratified random sampling was applied for selection of the patients. Statistical analysis of the differences in treatment status, between males and females, and among other epidemiological factors of interest was carried out using multiple logistic regression. Chi-square/Fisher's exact test were also used to assess significant differences in values between males and females. There were 580 leprosy patients (385 male and 195 female) aged >15 years registered for MDT between April 1, 2001 to March 31, 2002 in the 16 main health centers of the district. Of the 580 patients, a total of 273 (183 male and 90 female) were included in the study, to collect data on clinical type of leprosy, patterns of physical deformity/disability, site of skin lesions, and socio-demographic information. There were 183 male (68.3% on MB-MDT) and 90 female (61.1% MB-MDT) leprosy patients. We found that 79.2% of male patients completed treatment, while 34.4% female patients did not complete within the given time frame. Significant gender differences among leprosy patients were found in the distribution of disability grades and treatment completion status. However, there was no significant gender difference in the distribution of leprosy types and skin lesion sites. The study also found significant associations between treatment completion status and gender (adjusted OR 2.05, 95% CI: 1.07-3.94), educational status (adjusted OR 2.37, 95% CI: 1.12-4.99), disability grade I (adjusted OR 3.14, 95% CI: 1.23-8.04), and disability grade 0 (adjusted OR 2.92, 95% CI: 1.14-7.47) after adjustment for all other leprosy/demographic factors.

Cutaneous leukocytoclastic vasculitis: the yield of direct immunofluorescence study.

BACKGROUND: Leukocytoclastic vasculitis (LCV) is a clinico-pathological entity. Previous direct immunofluorescence study (DIF) studies of vasculitis showed positive findings mainly in the early stage of the disease. OBJECTIVE: To study the positive yield and patterns of DIF in patients with various stages of LCV. DESIGN: One hundred patients with LCV who attended the Department of Dermatology, Siriraj Hospital from 1997 to 2000 were enrolled in the study. RESULTS: The study showed immunoreactive deposits in blood vessel walls in 76 cases (76%). Forty seven per cent of patients showed immunoreactant deposit only in superficial blood vessel walls, 3% had deposits only in deep blood vessel walls. Superficial and deep blood vessel wall deposits were seen in 26%. Dermo-epidermal deposit in addition to blood vessel wall deposit was found in 39%. The most common immunoreactive deposit was C3 (71%), followed by IgM (35%), IgA (12%) and IgG (8%) respectively. The age of the skin lesions at the time of biopsy ranged from 1 to 7 days. 82% of patients with one day old lesions showed immunoreactive deposits in the blood vessel walls and 74% of the group with lesions aged 2-7 days at the time of biopsy showed immunoreactive deposits in the blood vessel walls. CONCLUSION: The present study showed a 76% positive yield for DIF study in patients with LCV when biopsies were performed within one week of onset. There was a tendency for the percentage of positive DIF results to decline when the biopsy was performed on lesions that were more than 1 day old.

Nontuberculous mycobacterial skin infections: clinical and bacteriological studies.

OBJECTIVE: Nontuberculous mycobacterial (NTM) skin infections were analysed in terms of clinical manifestation in different species to provide clues for the clinical diagnosis and sensitivity patterns of these species were studied for planning appropriate therapy. DESIGN: A retrospective study was performed in 123 suspected cases of NTM infections from January 1994 to December 2000. NTM infection was documented by culture result of the infected tissue obtained by skin biopsy. Drug susceptibility test was done as requested. RESULT: Rapid growers (M. fortuitum-chelonae) were found in 26 cases (65%) and M. marinum was responsible for 12 cases (30%) and caused only localized skin lesions on arms or legs as indurated plaque, Disseminated skin infections manifested as multiple abscesses were found in 2 cases caused by M. avium in an HIV-infected male patient and mixed infection of M. szulgai and M. terrae in an immunocompetent female patient after a dental procedure. Both sexes were affected equally in overall number but male predominated in M. marinum infection and females predominated in rapid growers. All ages can be affected but most cases were middle aged. Scrofuloderma-like cervical lymphadenitis and cutaneous abscesses were the common manifestation of rapid grower infections. Hyperkeratotic verrucous plaques (tuberculosis verrucosa cutis-like) and sporotrichoid lesions were the common manifestations of M. marinum infection. M. marinum is sensitive to minocyclin, clarithromycin, amikacin, rifampicin and ethambutol and a good clinical response was obtained with doxycyclin 100 mg orally twice a day for 3 months. Clarithromycin and amikacin showed in vitro activity against the same strain of M. fortuitum but most strains of rapid growers resisted antituberculous drugs and also various antibiotics. CONCLUSION: Clinical manifestations can be used as clues for diagnosis. Medical therapy is recommended for M. marinum infection and surgical treatment is recommended for rapid growers.

Clinical efficacy of lemon grass oil cream in the treatment of ring worm.

A randomized controlled trial was conduct to determine the efficacy of lemon grass (Cymbopogon citratus,DC) oil cream in the treatment of dermatophytosis at any site of the body except nail and scalp infections in adult Thai patients. Eight-one patients were enrolled in the study in which 38 cases were allocated to receive lemon grass oil cream and 43 cases to receive clotrimazole cream in an identical package for local application at the skin lesions twice a day for 4 weeks. The outcome was measured clinically and microbiologically by KOH preparation and culture before treatment and 2 and 4 weeks during treatment. Twenty-five cases (30.9%) were lost to follow up. Fifty-six cases were treatment completely, 30 cases in the clotrimazole group with a 53.3% cure rate and 26 cases in lemon grass oil group with a 26.9% cure rate. Although the clinical signs and symptom had improved in the later group, they stilled a positive KOH-preparation and / or positive culture. Contact dermatitis from the lemon grass oil cream was found in 1 case but no serious adverse effect was found. The high temperature in Thailand may reduce the concentration of the volatile oil "citral" in lemon grass oil cream which could influence the clinical outcome and lead it to less effective than in a previous in vitro study.

Pathological study of pigmented dermatofibrosarcoma protuberans.

In this paper we performed morphological studies of Bednar tumor, which is a rare pigmented variant of dermatofibrosarcoma protuberans (DFSP) for evidence of histogenetic origin. Presence of melanosome-containing cells is the only histologic criterion of Bednar tumor, which is different from the ordinary DFSP. Immunohistochemical and ultrastructural examinations revealed that the melanosome-containing cells gave positive reactivity to antibodies to vimentin and S-100 protein, but negative to HMB-45, CD38, Factor VIII, NSE, and KP-1. The melanosomes are in Stage II, III, and IV, suggesting that the pigment was produced within the cell rather than phagocytosed from other cells. The rest of the tumor had similar pattern of immunoreactivity to DFSP such as positivity for CD34 and vimentin, indicating that Bednar tumor and DFSP originated from the same cell line. The ultrastructural feature of non-pigmented cells was similar to perineural cells in the first group and similar to fibroblasts in the second group. DNA flow cytometry in our cases revealed diploid cells with low to intermediate S-phase fraction and no aneuploid cells which were reported in a previous study.

Pathergy test in Thai patients with behcet's disease: A clinical, histopathological and direct immunofluorescence study.

To evaluate the clinical, histopathological and immunopathological findings of the pathergy test in Thai patients with Behcet's disease. Methods : A pathergy test using the needle prick method with a 20G disposable needle after cleaning with 70% alcohol was performed in 5 patients (4 females and 1 male), with Behcet's disease according to International Study Group (ISG) criteria. The skin was pricked making a 45oangle with the skin surface to a depth of 3 mm at an avascular and hairless point of both forearms symmetrically. After 4 and 48 hours the test sites were evaluated clinically and skin biopsies were performed at a prick site on the right forearm after 4 hours for direct immunofluorescence study and after 48 hours at the prick site on the left forearm for histopathological study. Results : Clinical pathergy tests were positive (a single papular lesion at the needle prick site) in 3 out of 5 patients at 48 hours. No response was observed at 4 hours. Direct immunoflurorescence study showed no deposition of immnoglobulins (IgG, IgM, IgA) and C3 in any case. The histopathological findings 48 hours after the test revealed definite neutrophilic infiltration around a superficial vascular plexus in 4 cases, in addition, one of these had endothelial cell swelling with red blood cell extravasation. The rest (1 case) showed nonspecific finding. Conclusion : We conclude that the pathergy test is a useful tool for the diagnosis of Behcet's disease. The intradermal needle prick method using 20G disposable needle after cleaning the skin with 70% alcohol, yields results of moderately high sensitivity.

Disseminated penicilliosis marneffei in immunocompetent hosts.

Two cases of disseminated Penicilliosis marneffei are reported; both were middle-aged female patients from the central part of Thailand who presented with multiple cystic skin lesion. Their systemic symptoms included chronic fever, weight loss, malaise, anemia, cervical lymphadenopathy and osteolytic bone lesions. They had no underlying disease causing immunosupression and both were HIV-negative. Skin manifestations occurred frequently in disseminated penicilliosis and abscesses were the most common manifestation in HIV-negative patients whereas umbillicated papules were common in HIV-positive ones. A biopsy from the skin lesions was good specimens for histopathological study and frequently yielded positive culture results. The characteristic histopathological feature is granulomatous inflammation with macrophages containing yeast-like organisms with septa which show a lack of budding. The characteristic mycologic feature of P.marneffei is a thermally dimorphic fungus which produces a mycelial phase colony appearing within 2 days at room temperature (25-30oC) and which produces a bright, purple-red, water-soluble pigment into the surrounding agar. The yeast form grows at 37oC as a whitish colony produced in 4 days and this produces less red pigment compared with the mycelial form. The first case was treated with oral itraconazole intermittently as a result of multiple recurrent episodes until she died of the disease after one year. The second case was treated with amphotercin B intravenously followed by oral itraconazole with a satisfactory result.

Etiologic agents of cutaneous infectious granuloma: 2 year-experience from granuloma clinic, Siriraj Hospital.

Etiological diagnosis of cutaneous infectious granuloma is difficult to achieve and leads to difficult in patient management. We proposed to search for etiologic agents by special stain of histologic sections, cultural method and PCR technique for tuberculous and non-tuberculous mycobacteria. The skin biopsy specimens were obtained from dermatologic patients who attended Granuloma Clinic, Siriraj Hospital from January 1994 to December 1996. Sixty-nine cases of mixed cell granuloma were found during the 2-year-period. The causative agents had been found in 45 cases (65.22%). Mycobacterial infections were documented in 25 cases. Five cases of tuberculous infection were diagnosed by PCR method. Non-tuberculous mycobacterial infections were diagnosed by cultural method in 14 cases and the other 6 cases were diagnosed by acid-fast stain in histologic section alone. Actinomycotic mycetoma were diagnosed in 4cases. Fungal infections were documented in 16 cases, which caused by hyaline fungi 7 cases and dematiaceous fungi 9 cases. About one-third of all cases (24 cases) the etiologic agent could not be identified.