Apoptosis, production of MMP9, VEGF, TNF- and intracellular growth of M. tuberculosis for different genotypes and different pks15/1 genes.

Background: A previous study of IS6110 RFLP and spoligotyping of M. tuberculosis isolates from 152 Thai patients with tuberculous meningitis revealed a significantly higher percentage (57%) of the Beijing genotype as compared to isolates obtained from pulmonary tuberculosis. We postulated that the M. tuberculosis Beijing genotype is likely to be more virulent than others. Objectives: Ten M. tuberculosis cerebrospinal fluid (CSF) isolates from five RFLP groups, together with different characteristics of pks15/1, M. tuberculosis H37Rv and M. bovis BCG, were investigated for their virulence in vitro. Methods: In this study, THP-1 cells were used as host cells to determine the intracellular growth and the induction of MMP9, VEGF, TNF-α and apoptosis. Determinations of the cytokine production and apoptosis were based on available commercial kits using ELISA techniques. Results: No significant difference in intracellular multiplication was found between the M. tuberculosis CSF isolates. Three isolates, consisting of 2 Nonthaburi and 1 heterogeneous isolate, were found to stimulate high TNF-α and MMP-9 production during the early infection period.They were isolated from 3 different patients, 2 of whom died with initial stages II and III. This result suggested that there might be an association between TNF-α and MMP-9 production that could account for the specific virulent nature of Nonthaburi strains. VEGF production was determined and comparable levels were found in all isolates. No significant apoptosis was detected in M. tuberculosis CSF isolates. No significant differences suggesting that the 2 Beijing strains are more virulent than the others were observed. Conclusion: The predominance of the Beijing strains in cases of tuberculous meningitis (TBM) in Thai patients is not a result of their hypervirulence.

Random amplified polymorphic DNA typing and phylogeny of Pythium insidiosum clinical isolates in Thailand.

Forty-three Pythium insidiosum clinical isolates recovered from human pythiosis cases in Thailand were characterized by random amplified polymorphic DNA (RAPD) analysis. Three random oligonucleotide primers, OPW11, OPW12 and OPX13 generated 39, 34 and 35 DNA patterns with high value of typeability (100%), reproducibility (98.5, 88.8 and 93.3%) and discriminatory power (0.83, 0.82 and 0.77), respectively. Using GelCompar software based on band similarity, the 43 clinical isolates of P. insidiosum could be arranged into 9, 13 and 11 clades using OPW11, OPW12 and OPX13, respectively and the combination of all three primers revealed 36 RAPD patterns. Members in each RAPD pattern varied in both clinical forms and/or geographical locations. RAPD pattern 15 was found in 6 isolates, half of which were found in central region of Thailand. Isolates MCC15 and MCC16 isolated from different patients exhibited identical pattern with all three primers. Our results revealed high genetic heterogeneity among Pythium insidiosum isolates in Thailand. RAPD method should be appropriate for future epidemiological studies of P. insidiosum strains from patients and from natural habitats.

Mycobacterium avium and Burkholderia pseudomallei (Melioidosis) coinfection in an HIV-positive patient.

A 29 year old HIV positive Thai female with CD4 count of 10 cells/mm3 presented with chronic diffuse abdominal pain, fever, weight loss, anemia and leucopenia. Ultrasonography demonstrated diffuse upper abdominal lymphadenopathy with ascites. Microbiological and molecular work up of the specimen obtained by ultrasound-guided lymph node aspiration revealed co-infection with Burkholderia pseudomallei and Mycobacterium avium. Indirect hemagglutination, IgM-indirect fluorescent antibody, and IgG-indirect fluorescent antibody to Burkholderia pseudomallei were < 1:20, < 1:50 and < 1:50, respectively, at nine months, four months before the culture diagnosis and two months, eight months after the culture diagnosis of Burkholderia pseudomallei infection. The patient was treated initially with two weeks of intravenous ceftazidime, followed by oral cotrimoxazole, doxycycline and chloramphenicol. Clarithromycin and ofloxacin were added after the identification of Mycobacterium avium and its susceptibility test. The patients demonstrated clinical improvement with decreasing abdominal pain and resolution of fever.

Ganoderma lucidum: a cause of pseudoparasitosis.

We report a pseudoparasitosis case due to Ganoderma lucidum, (lingzhi or reishi mushroom); we believe this to be a first reported case in Thailand. A 49-year-old male patient with non-Hodgkins lymphoma presented with chronic watery diarrhea. He had a history of consumption of powdered lingzhi extract as a dietary supplement and herbal medicine. Stool examination demonstrated many spores of G. lucidum, which must be differentiated from intestinal helminth ova and coccidia. After discontinuation of mushroom spores ingestion, the diarrheal symptoms improved and fecal examination subsequently showed no Ganoderma spores. Many artifacts in the stool may be confused with parasites. Differentiation of parasites from artifacts depends on characterization of the size, shape, structure, and reactivity with common stains.

One-tube multiplex PCR method for rapid identification of mycobacterium tuberculosis.

A rapid, inexpensive, simple, and accurate multiplex polymerase chain reaction (PCR) was developed in a single tube for identification of Mycobacterium tuberculosis. Assessment of sensitivity and specificity of simple PCR was performed with 116 strains of M. tuberculosis complex (MTC) and 144 strains of nontuberculous mycobacteria (NTM) compared with the biochemical method. Specific amplification of KS4, MTC-specific DNA fragment, was found in 98% (114/116) of MTC and not detected in 99% (143/144) of NTM. Amplification of the mtp40 gene revealed 95% sensitivity (100/105 strains of M. tuberculosis) and 77% specificity (not found in 119/155 mycobacterial strains). A multiplex PCR method based on the combination of KS4- and mtp40-derived primers was used for identification of M. tuberculosis. Crude DNA from slow growing mycobacteria with cream rough colonies that showed both 768-bp amplified product for KS4 and 396-bp for mtp40 was identified as M. tuberculosis whereas that from MTC gave only the 768-bp product.

Susceptibility of mycobacterium tuberculosis to non-toxic level ozone.

Decontamination of Mycobacterium tuberculosis (MTB) in the environment by specific methods, such as ultraviolet light and the toxic level ozone, has been effectively used. However, the efficacy of the non-toxic level ozone in the decontamination of MTB is not well documented. This study focused on the impact of non-toxic level (0.03-0.05 part per million) ozone in normal room air on MTB on a solid medium, M7H10. The non-toxic level ozone can reduce the number of MTB colonies as compared to the control. The number of MTB colonies was rapidly reduced, more than 95 percent, during the first two hours and gradually reduced during the remaining time of the experiment. The results suggest that the non-toxic level ozone, produced by specific devices, is promising for the decontamination of solid surfaces from MTB.

Treatment of tinea capitis caused by Microsporum ferrugineum with itraconazole.

A prospective, non-randomized, open clinical trial was conducted to determine the efficacy of itraconazole for treatment of Microsporum ferrugineum tinea capitis. Itraconazole capsules were given every day in continuous group and every day for 1 week on and 3-week off in pulse therapy group. Concomitant topical therapy with 2% ketoconazole shampoo was used daily. Clinical evaluation consisted of assessing the degree of hair loss, scaling, erythema, pustule, and crust. In both groups, the treatment was stopped when the clinical signs of inflammation had resolved and the mycological examination had become negative or at week 12. There were 81 patients consisted of 49 boys and 32 girls enrolled and average dose of itraconazole was 4.5 mg/kg/day. During the 16-week study period (with 4-week follow-up visit) the overall clinical severity score decreased every visit (p < 0.001). The improvement of the scores showed no statistical difference between both groups. The cumulative cure rate using combined clinical and mycological cure at week 16 in patients treated with continuous and pulse regimen was 54.3% (19/35) and 37.0% (17/46), respectively. The cumulative percentage of all cure rates including clinical cure, mycological cure and combined clinical and mycological cure of the continuous group was significantly higher than in the pulse therapy group (p < 0.001). The superior efficacy of the continuous therapy group was observed after week 8. The cumulative cure rate increased with the longer treatment duration but decreased with the larger infected area involvement (p = 0.001). All patients who were not cured showed improvement. There was no significant adverse effect. The higher dosage or the longer treatment duration of itraconazole may be required for treatment of tinea capitis from M. ferrugineum to achieve more cure rate.

Evaluation of polymerase chain reaction and restriction enzyme analysis for routine identification of mycobacteria: accuracy, rapidity, and cost analysis.

Polymerase chain reaction and restriction enzyme analysis (PCR-REA) of the hsp65 gene was evaluated for use as a routine identification method for identifying mycobacteria. The accuracy, rapidity, and cost were assessed compared with the conventional biochemical method. Five hundred and forty-one mycobacterial clinical isolates obtained from the Department of Microbiology, Faculty of Medicine at Siriraj Hospital, Mahidol University, were submitted for PCR-REA and biochemical identification. PCR-REA showed high concordant result with 100, 96.2, and 94.1% for identification of Mycobacterium tuberculosis, rapid- and slow-growing mycobacteria, respectively. Discordant results were obtained from 24 (4.4%) out of 541 isolates, consisting of 9 rapid growers (6 M. chelonae, 2 M. abscessus, and 1 M. fortuitum) and 15 slow growers (9 M. scrofulaceum, 2 M. gordonae, 1 M. avium, 1 M. kansasii, 1 M. malmoense, and 1 M. terrae complex). PCR-REA demonstrated not only accurate results but was also less expensive (2.1 US dollars/sample). This method was rapid with a turn-around time of 30 hours compared with 2-4 weeks for the conventional method.

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.

Six-year report of mycobacteria recovered from automated culture system at Siriraj Hospital.

An increased incidence of tuberculosis and other mycobacterial infections among immunocompromised patients has created a serious health crisis, especially in resource-poor countries. In addition, disseminated disease occurs more frequently in these patients. Rapid isolation and accurate identification of causative agents are necessary for selecting an appropriate treatment regimen. Since an isolation of Mycobacterium tuberculosis and slowly growing mycobacteria require 3-4 weeks for conventional culture, the automated system can reduce the detection time to 7-10 days. The present study demonstrated the mycobacteria recovered from hemocultures and other sterile body fluids, using the BACTEC 9000 system. Overall, 5,490 samples during the period 1998-2003 were submitted for hemocuture and the isolated mycobacteria were identified by using molecular techniques, like multiplex PCR and PCR-REA. The results demonstrated that~18-28% of hemocultures were positive for mycobacteria. Of these, M. tuberculosis appeared to be the most common species among mycobacteria isolates whereas the M. avium complex was found to be the second most common. The combined use of an automated culture system and molecular techniques as shown in this study is a useful procedure for rapid isolation and identification of mycobacteria that can reduce the time from 6-8 weeks to 2-3 weeks.

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.

Thrombocytopenic purpura associated with miliary tuberculosis.

The authors report a case of thrombocytopenia associated with miliary tuberculosis. The patient was a 28-year-old woman who was admitted because of massive upper gastrointestinal hemorrhage and acute respiratory failure. Chest radiographs revealed diffuse bilateral reticulonodular infiltration and complete blood count was significant for severe thrombocytopenia. Bone marrow biopsy was performed to investigate the cause of thrombocytopenia and demonstrated multiple tiny caseating granulomas suggesting miliary tuberculosis (TB). She received anti-TB therapy and a short course of steroid with good response. Platelet count returned to normal limit within 10 days. Although isolated thrombocytopenia is uncommon in TB, it is still important to consider TB in the differential diagnosis of thrombocytopenia, particularly in patients with abnormal chest radiographs. Bone marrow examination is very helpful in this situation.

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.

Evaluation of in-house optimized semi-nested PCR and EIA for direct detection of mycobacterial DNA in CSF.

A rapid and correct diagnosis of mycobacterial infections is important for effective patient treatment. Semi-nested-PCR with Fl-16 SOL, 16SOR and 16SNSR primers based on the 16S rRNA gene, under optimized conditions, can detect 499 bp amplified products from all tested mycobacteria. The assay could detect as little as 100 fg of mycobacterial DNA except for rapid growing mycobacteria, whose detection limits ranged from 1 ng to 10 pg. The specificities of the capture probes were assessed with 96 mycobacterial strains (22 species) and 33 nonmycobacterial strains (30 species). The specificities of pAll1, pTbc1 and pMar1 were 94%, 93% and 82%, respectively, and that of pAvi1, pInt1, pChe1 and pFor1 were 100%. The pTbc1 and pAvi1 were tested with DNA from 108 CSF samples, and the sensitivity and specificity of the detection method were 56% and 84% compared to culture and patient histories. The assay should be used for rapid detection and concurrent identification of slow growing mycobacteria without parallel conventional culture verification.

Efficiency of the glutaraldehyde test strip for monitoring the concentration of glutaraldehyde in reused solutions for disinfecting endoscopes.

BACKGROUND: Glutaraldehyde has been widely used for low-temperature disinfection of endoscopes. The current practice at Siriraj Hospital is to change the glutaraldehyde solution every 21 days or when the solution appears turbid. The disadvantages of this practice include inadequate disinfection of endoscopes if the concentration of glutaraldehyde in a reused solution is insufficient or wasted if the discarded solution is still active. OBJECTIVE: To determine the efficiency of a glutaraldehyde test strip (GTS) in monitoring the amount of glutaraldehyde in a reused solution for disinfecting endoscopes. METHOD: Reused glutaraldehyde solutions for disinfecting bronchoscopes, gastroscopes and colonoscopes were tested for the concentration of glutaraldehyde with a GTS thrice weekly for the first week and then every working day up to 56 days. If the GTS indicated a concentration of glutaraldehyde > or = 1.8 per cent after 21 days, 5 ml of the solution was taken to the laboratory to determine its mycobactericidal activity. RESULTS: All samples of the reused glutaraldehyde solution up to 56 days with a concentration of > or = 1.8 per cent glutaraldehyde on GTS from testings showed mycobactericidal activity. If the glutaraldehyde solution was reused for up to 28, 42 or 56 days, it could save 9,603; 22,813 and 29,415 baht per year respectively for the gastroscopy and colonoscopy units. The corresponding figures were -949; 2,726 and 4,564 baht per year for the bronchoscopy unit. It is estimated that up to 400,000 baht per year could be saved by adopting the strategy of GTS monitoring in all endoscopy units at Siriraj Hospital. CONCLUSION: The current strategy of discarding reused glutaraldehyde solution in the gastroscopy, colonoscopy and bronchoscopy units at Siriraj Hospital may be inappropriate since the reused solution is still mycobactericidal for up to 56 days.

Mycobacterium avium complex in HIV-infected Thai children.

Of the 169 human immunodeficiency virus (HIV)-infected children being cared for at Siriraj Hospital from January 1998 to September 2000, 10 had Mycobacterium avium complex (MAC) infection; seven had disseminated disease and three had MAC pneumonia. Nine children were in the advanced stage of HIV disease at the time of diagnosis with the median CD4 count of 7 cells/mm3 and 127 cells/mm3 and the median age of 65 months and 63 months in disseminated MAC and MAC pneumonia respectively. None of these children had received prior chemoprophylaxis. Common clinical findings included prolonged fever, weight loss, lymphadenopathy, hepatosplenomegaly, diarrhea, anemia and leukopenia. The outcome of MAC infection was poor, with a mortality rate of 60 per cent. In in vitro susceptibility testing, clarithromycin was the least resistant drug. With the incidence rate of 2.15 per 100 person-years, the high rate of antimicrobial resistance, and the poor outcome, primary chemoprophylaxis for MAC infection in conjunction with effective antiretroviral therapy should be considered for Thai children in the advanced stage of HIV infection.

Infection due to nontuberculous Mycobacterium other than MAC in AIDS patients at Siriraj hospital during 1998-2000: saprophyte vs pathogen.

HIV is a major health problem in Thailand. These patients are vulnerable to opportunistic infections, especially Mycobacterium tuberculosis and MAC infection. However, NTM was considered a rare disease in Thailand before the AIDS era. In this study, there were 38 HIV seropositive patients with NTM (other than MAC) identified from clinical specimens during the 3 year period 1998-2000 at Siriraj Hospital, which has a higher prevalence than the previous report. Among these patients, 29 cases were likely to have had definite infection from NTM, 5 cases possibly had NTM as a pathogen, and 4 cases had NTM as colonization. The most common site of infection was the lung (87%) and most common symptoms were cough (62.2%), fever (34.2%), weight loss (42.1%), and lymphadenopathy (5.3%). The outcome was poor because many NTM are not susceptible to standard medication for tuberculosis which is the empirical treatment for the majority of HIV seropositive patients with a clinical finding suspected of mycobacterial infection. The fatality rate was as high as 58.6 per cent. Awareness of NTM as a potential pathogen in HIV seropositive patientsand adjustment of medications even before the availability of culture results may improve the outcome of treatment of NTM infection in HIV seropositive patients.

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.

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.