BORC complex specific components and Kinesin-1 mediate autophagy evasion by the autophagy-resistant Mycobacterium tuberculosis Beijing strain.

Autophagy induction by starvation has been shown to enhance lysosomal delivery to mycobacterial phagosomes, resulting in the restriction of the Mycobacterium tuberculosis reference strain H37Rv. In contrast to H37Rv, our previous study showed that strains belonging to the notorious M. tuberculosis Beijing genotype could evade autophagic elimination. Our recent RNA-Seq analysis also discovered that the autophagy-resistant M. tuberculosis Beijing strain (BJN) evaded autophagic control by upregulating the expression of Kxd1, a BORC complex component, and Plekhm2, both of which function in lysosome positioning towards the cell periphery in host macrophages, thereby suppressing enhanced lysosomal delivery to its phagosome and sparing the BJN from elimination as a result. In this work, we further characterised the other specific components of the BORC complex, BORC5-8, and Kinesin proteins in autophagy resistance by the BJN. Depletion of BORCS5-8 and Kinesin-1, but not Kinesin-3, reverted autophagy avoidance by the BJN, resulting in increased lysosomal delivery to the BJN phagosomes. In addition, the augmented lysosome relocation towards the perinuclear region could now be observed in the BJN-infected host cells depleted in BORCS5-8 and Kinesin-1 expressions. Taken together, the data uncovered new roles for BORCS5-8 and Kinesin-1 in autophagy evasion by the BJN.

Transcriptional response to the host cell environment of a multidrug-resistant Mycobacterium tuberculosis clonal outbreak Beijing strain reveals its pathogenic features.

Tuberculosis is a global public health problem with emergence of multidrug-resistant infections. Previous epidemiological studies of tuberculosis in Thailand have identified a clonal outbreak multidrug-resistant strain of Mycobacterium tuberculosis in the Kanchanaburi province, designated "MKR superspreader", and this particular strain later was found to also spread to other regions. In this study, we elucidated its biology through RNA-Seq analyses and identified a set of genes involved in cholesterol degradation to be up-regulated in the MKR during the macrophage cell infection, but not in the H37Rv reference strain. We also found that the bacterium up-regulated genes associated with the ESX-1 secretion system during its intracellular growth phase, while the H37Rv did not. All results were confirmed by qRT-PCR. Moreover, we showed that compounds previously shown to inhibit the mycobacterial ESX-1 secretion system and cholesterol utilisation, and FDA-approved drugs known to interfere with the host cholesterol transportation were able to decrease the intracellular survival of the MKR when compared to the untreated control, while not that of the H37Rv. Altogether, our findings suggested that such pathways are important for the MKR's intracellular growth, and potentially could be targets for the discovery of new drugs against this emerging multidrug-resistant strain of M. tuberculosis.

Genomic evidence supporting the clonal expansion of extensively drug-resistant tuberculosis bacteria belonging to a rare proto-Beijing genotype.

Tuberculosis disease (TB), caused by Mycobacterium tuberculosis, is a major public health issue in Thailand. The high prevalence of modern Beijing (Lineage 2.2.1) strains has been associated with multi- and extensively drug-resistant infections (MDR-, XDR-TB), complicating disease control. The impact of rarer proto-Beijing (L2.1) strains is less clear. In our study of thirty-seven L2.1 clinical isolates spanning thirteen years, we found a high prevalence of XDR-TB cases (32.4%). With ≤ 12 pairwise SNP distances, 43.2% of L2.1 patients belong to MDR-TB or XDR-TB transmission clusters suggesting a high level of clonal expansion across four Thai provinces. All XDR-TB (100%) were likely due to transmission rather than inadequate treatment. We found a 47 mutation signature and a partial deletion of the fadD14 gene in the circulating XDR-TB cluster, which can be used for surveillance of this rare and resilient M. tuberculosis strain-type that is causing increasing health burden. We also detected three novel deletion positions, a deletion of 1285 bp within desA3 (Rv3230c), large deletions in the plcB, plcA, and ppe38 gene which may play a role in the virulence, pathogenesis or evolution of the L2.1 strain-type.

Defective cytokine production from monocytes/macrophages of E-beta thalassemia patients in response to Pythium insidiosum infection.

BACKGROUND: Pythium insidiosum has been mainly reported to cause morbidity and mortality in thalassemia patients. P. insidiosum zoospores can germinate to be hyphae within a few hours; therefore, it is difficult to study the initial immune response that P. insidiosum zoospores induce. The present study aims to compare immune responses against P. insidiosum zoospore infection by comparing monocytes/macrophages from thalassemia patients with those from non-thalassemia controls. METHODS: In order to keepP. insidiosum in the zoospore stage in vitro for inoculation, the P. insidiosum zoospores were preserved without germination by treatment with inorganic hypochlorite solution. CD14+ cells were isolated from peripheral blood mononuclear cells of thalassemia and non-thalassemia donors and then left to transition to macrophages. Monocytes/macrophage culture was infected with P. insidiosum zoospores and culture supernatants were subjected to Th1/Th2 multiplex cytokine detection. RESULTS: Our study of cytokine production revealed that the basal level of GM-CSF produced by thalassemia monocytes/macrophages was lower than that observed in monocytes/macrophages of non-thalassemia individuals. Higher GM-CSF and IFN-γ response was also found when cells from non-thalassemia people were stimulated with P. insidiosum zoospores compared to thalassemia cells. It was also found that TNF-α, GM-CSF and IFN-γ productions from monocytes/macrophages of thalassemia patients who received iron chelator treatment were significantly higher than those produced from thalassemia patients without iron chelator treatment. CONCLUSION: For the first time, the present study demonstrates defective immune responses in monocytes/macrophages derived from thalassemia patients in response toP. insidiosum zoospore infection. The results also show an inverse correlation between iron overload and cytokine production in monocytes/macrophages of thalassemia patients. This finding could explain why thalassemia patients are susceptible to P. insidiosum infection.

Infection of multiple Mycobacterium tuberculosis strains among tuberculosis/human immunodeficiency virus co-infected patients: A molecular study in Myanmar.

Background: Appearance of Mycobacterium tuberculosis (MTB) in the sputum of a tuberculosis (TB)/human immunodeficiency virus (HIV) co-infected patient under treatment may indicate either failure or new infection. This study aims to evaluate whether TB treatment failure among TB/HIV co-infected patients is a real failure. Methods: A prospective cohort study was conducted among 566 TB/HIV co-infected patients who started TB treatment in 12 townships in the upper Myanmar. Among the 566 participants, 16 (2.8%) resulted in treatment failure. We performed a molecular study using mycobacterial interspersed repetitive-unit-variable number of tandem repeat (MIRU-VNTR) genotyping for them. The MIRU-VNTR profiles were analyzed using the web server, MIRU-VNTRplus. All data were entered into EpiData version 3.1 and analyzed using R version 3.4.3. Results: Among 16 failure patients, seven had incomplete laboratory results. Of the nine remaining patients, nobody had exactly the same MIRU-VNTR pattern between the initial and final isolates. Four patients had persistent East-African Indian (EAI) lineages and one each had persistent Beijing lineage, changing from EAI to Beijing, from Beijing to EAI, NEW-1 to Beijing, and NEW-1 to X strains. Female patients have significantly larger genetic difference between MTB of the paired isolates than male patients (t-test, P = 0.04). Conclusion: Thus, in our study patients, infection of multiple MTB strains is a possible cause of TB treatment failure. Explanation for the association between gender and distance of genotypes from the initial to subsequent MTB infection needs further studies.

Immune response in human chromoblastomycosis and eumycetoma - focusing on human interleukin-17A, interferon-gamma, tumour necrosis factor-alpha, interleukin-1 beta and human beta-defensin-2.

Knowledge regarding host immune response to chromoblastomycosis and eumycetoma is limited, particularly concerning cytokines and antimicrobial peptides production. This was a retrospective study of 12 paraffin-embedded tissue samples from patients diagnosed with chromoblastomycosis or eumycetoma from histological findings and tissue culture. DNA extraction and polymerase chain reaction (PCR) from tissues were done to evaluate human interleukin-17A (IL-17A), interferon-gamma (IFN-γ), tumour necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1ß) and human beta-defensin-2 (HBD-2) expressions. Human beta-actin primer was used for confirming DNA detection, and DNA extracted from psoriasis lesional skin samples was used as positive controls. The twelve paraffin-embedded sections used in this study consisted of five chromoblastomycosis and seven eumycetoma tissues. All PCR reactions showed beta-actin band at 51 bp in all clinical specimens, confirming adequate DNA levels in each reaction. As positive control, the psoriasis skin samples revealed bands for IL-17A at 174 bp, IFN-γ at 273 bp, TNF-α at 360 bp, IL-1ß at 276 bp and HBD-2 at 255 bp. For the chromoblastomycosis and eumycetoma tissues, PCR analyses showed IL-17A band at 174 bp in two eumycetoma tissues and HBD-2 band at 255 bp in a chromoblastomycosis tissue. This study demonstrated IL-17A expression in human eumycetoma and HBD-2 expression in human chromoblastomycosis for the first time. However, their role in immune response remains to be elucidated.

Protein A/G-based immunochromatographic test for serodiagnosis of pythiosis in human and animal subjects from Asia and Americas.

Pythiosis is a life-threatening infectious disease of both humans and animals living in Asia, Americas, Africa, and parts of Australia and New Zealand. The etiologic pathogen is the fungus-like organism Pythium insidiosum The disease has high mortality and morbidity rates. Use of antifungal drugs are ineffective against P. insidiosum, leaving radical surgery the main treatment option. Prompt treatment leads to better prognosis of affected individuals, and could be achieved by early and accurate diagnosis. Since pythiosis has been increasingly reported worldwide, there is a need for a rapid, user-friendly, and efficient test that facilitates the diagnosis of the disease. This study aims to develop an immunochromatographic test (ICT), using the bacterial protein A/G, to detect anti-P. insidiosum IgGs in humans and animals, and compare its diagnostic performance with the established ELISA. Eighty-five serum samples from 28 patients, 24 dogs, 12 horses, 12 rabbits, and 9 cattle with pythiosis, and 143 serum samples from 80 human and 63 animal subjects in a healthy condition, with thalassemia, or with other fungal infections, were recruited for assay evaluation. Detection specificities of ELISA and ICT were 100.0%. While the detection sensitivity of ELISA was 98.8%, that of ICT was 90.6%. Most pythiosis sera, that were falsely read negative by ICT, were weakly positive by ELISA. In conclusion, a protein A/G-based ICT is a rapid, user-friendly, and efficient assay for serodiagnosis of pythiosis in humans and animals. Compared to ELISA, ICT has an equivalent detection specificity and a slightly lower detection sensitivity.

Resistance to cellular autophagy by Mycobacterium tuberculosis Beijing strains.

Autophagy represents a key pathway in innate immune defense to restrict Mycobacterium tuberculosis growth inside host macrophages. Induction of autophagy has been shown to promote mycobacterial phagosome acidification and acquisition of lysosomal hydrolases, resulting in the elimination of intracellular M. tuberculosis reference strains such as H37Rv. The notorious Beijing genotype has been previously shown to be hyper-virulent and associated with increased survival in host cells and a high mortality rate in animal models, but the underlying mechanism that renders this family to have such advantages remains unclear. We hypothesize that autophagic control against M. tuberculosis Beijing strains may be altered. Here, we discovered that the Beijing strains can resist autophagic killing by host cells compared with that of the reference strain H37Rv and a strain belonging to the East African Indian genotype. Moreover, we have determined a possible underlying mechanism and found that the greater ability to evade autophagic elimination possessed by the Beijing strains stems from their higher capacity to inhibit autophagolysosome biogenesis upon autophagy induction. In summary, a previously unrecognized ability of the M. tuberculosis Beijing strains to evade host autophagy was identified, which may have important implications for tuberculosis treatment, especially in regions prevalent by the Beijing genotype.

Comparative Proteomics of Activated THP-1 Cells Infected with Mycobacterium tuberculosis Identifies Putative Clearance Biomarkers for Tuberculosis Treatment.

Biomarkers for determining clearance of Mycobacterium tuberculosis (Mtb) infection during anti-tuberculosis therapy or following exposure could facilitate enhanced monitoring and treatment. We screened for biomarkers indicating clearance of Mtb infection in vitro. A comparative proteomic analysis was performed using GeLC MSI/MS. Intracellular and secreted proteomes from activated THP-1 cells infected with the Mtb H37Rv strain (MOI = 1) and treated with isoniazid and rifampicin for 1 day (infection stage) and 5 days (clearance stage) were analyzed. Host proteins associated with early infection (n = 82), clearance (n = 121), sustained in both conditions (n = 34) and suppressed by infection (n = 46) were elucidated. Of the potential clearance markers, SSFA2 and CAECAM18 showed the highest and lowest protein intensities, respectively. A western blot of CAECAM18 validated the LC MS/MS result. For three clearance markers (SSFA2, PARP14 and PSME4), in vivo clinical validation was concordantly reported in previous patient cohorts. A network analysis revealed that clearance markers were enriched amongst four protein interaction networks centered on: (i) CD44/CCND1, (ii) IFN-ß1/NF-κB, (iii) TP53/TGF-ß and (iv) IFN-γ/CCL2. After infection, proteins associated with proliferation, and recruitment of immune cells appeared to be enriched possibly reflecting recruitment of defense mechanisms. Counteracting proteins (CASP3 vs. Akt and NF-κB vs. TP53) associated with apoptosis regulation and its networks were enriched among the early and sustained infection biomarkers, indicating host-pathogen competition. The BRCA1/2 network was suppressed during infection, suggesting that cell proliferation suppression is a feature of Mtb survival. Our study provides insights into the mechanisms of host-Mtb interaction by comparing the stages of infection clearance. The identified clearance biomarkers may be useful in monitoring tuberculosis treatment.

Heterogeneity of phenotypic characteristics of the modern and ancestral Beijing strains of Mycobacterium tuberculosis.

BACKGROUND: The Beijing strain of Mycobacterium tuberculosis (MTB) is of great concern because this hypervirulent strain has caused numerous tuberculosis outbreaks. However, the mechanisms that allow the MTB Beijing strain to be highly pathogenic remain unclear and previous studies have revealed heterogeneity within this family. OBJECTIVE: To determine the association between some phenotypic characteristics and phylogroups of the Beijing strain of MTB. METHODS: Eight Beijing strains, 5 modern and 3 ancestral sublineages, were selected from the phylogroups of MTB. The selection was based on copy number of IS6110 at NTF, region of differences, and single nucleotide polymorphisms. The abilities of these strains to grow intracellularly in THP-1 macrophages, to induce apoptosis, necrosis, and cytokines production were examined using quantitative real-time PCR and commercially available ELISA kits, respectively. RESULTS: There were some significant differences between the two sublineages of the Beijing strain of MTB. The ancestral Beijing sublineages showed higher intracellular growth rates (p < 0.05) and necrosis induction rates (p < 0.01) than the modern Beijing sublineages. By contrast, the modern Beijing sublineages induced lower apoptosis and protective cytokine responses, i.e., TNF-α (p < 0.05) and IL-6 (p < 0.01) and higher non-protective IL-10 response. The modern Beijing sublineages may have evolved so that they have greater ability to diminish host defense mechanisms. The slower growth rate and reduced necrosis induction in host cells might allow the bacteria to cause a persistent infection. CONCLUSION: The results revealed a phylogroup-associated heterogeneity of phenotypes among MTB Beijing sublineages.

Detection of diketopiperazine and pyrrolnitrin, compounds with anti-Pythium insidiosum activity, in a Pseudomonas stutzeri environmental strain.

AIMS: Screening of bacterial flora for strains producing metabolites with inhibitory effects on the human pathogenic oomycete Pythium insidiosum. Separation and characterization of extracts from Pseudomonas stutzeri with anti-Pythium inhibitory activity. Search for genes with anti-Pythium effect within the genome of P. stutzeri. METHODS: A total of 88 bacterial strains were isolated from water resources in northeastern Thailand. Two screening methods were used to establish their inhibitory effects on P. insidiosum. One strain, P. stutzeri ST1302 was randomly chosen, and the extract with anti-P. insidiosum activity was fractionated and subfractionated using liquid column chromatography and purified by thin layer chromatography. The chemical structure of purified fractions was determined by Fourier transform infrared spectroscopy, nuclear magnetic resonance and mass spectrometry. Further, search for genes involved in the anti-Pythium activity (phenazine-1-carboxylic acid, 2,4-diacetylphloroglucinol, pyoluteorin and pyrrolnitrin) was undertaken in this P. stutzeri strain using primers described in the literature. RESULTS: Anti-P. insidiosum activity was detected in 16 isolates (18.2%). In P. stutzeri ST1302, a subfraction labeled PYK7 exhibited strong activity against this oomycete. It was assigned to the diketopiperazines as cyclo(D-Pro-L-Val). In the search for genes, one gene region was successfully amplified. This corresponded to pyrrolnitrin. The results suggest the possibility of using the related metabolites against P. insidiosum. This is the first report on the inhibitory effects of P. stutzeri against this oomycete. The results may contribute to the development of antimicrobial drugs/probiotics against pythiosis.

Apoptosis, production of MMP9, VEGF, TNF-alpha and intracellular growth of M. tuberculosis for different genotypes and different pks5/1 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-alpha 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-alpha 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-alpha 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.

Identification of a novel 74-kiloDalton immunodominant antigen of Pythium insidiosum recognized by sera from human patients with pythiosis.

The oomycetous, fungus-like, aquatic organism Pythium insidiosum is the etiologic agent of pythiosis, a life-threatening infectious disease of humans and animals that has been increasingly reported from tropical, subtropical, and temperate countries. Human pythiosis is endemic in Thailand, and most patients present with arteritis, leading to limb amputation and/or death, or cornea ulcer, leading to enucleation. Diagnosis of pythiosis is time-consuming and difficult. Radical surgery is the main treatment for pythiosis because conventional antifungal drugs are ineffective. The aims of this study were to evaluate the use of Western blotting for diagnosis of human pythiosis, to identify specific immunodominant antigens of P. insidiosum, and to increase understanding of humoral immune responses against the pathogen. We performed Western blot analysis on 16 P. insidiosum isolates using 12 pythiosis serum samples. These specimens were derived from human patients with pythiosis who had different forms of infection and lived in different geographic areas throughout Thailand. We have identified a 74-kDa immunodominant antigen in all P. insidiosum isolates tested. The 74-kDa antigen was also recognized by sera from all patients with pythiosis but not by control sera from healthy individuals, patients with thalassemia, and patients with various infectious diseases, indicating that Western blot analysis could facilitate diagnosis of pythiosis. Therefore, the 74-kDa antigen is a potential target for developing rapid serodiagnostic tests as well as a therapeutic vaccine for pythiosis. These advances could lead to early diagnosis and effective treatment, crucial factors for better prognosis for patients with pythiosis.

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.

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.

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.

A simple C-reactive protein measurement for the differentiation between tuberculous and malignant pleural effusion.

OBJECTIVE: The aim of this study was to determine the validity of pleural fluid C-reactive protein (CRP) concentrations and/or pleural fluid to serum CRP ratio for differentiating tuberculous pleuritis (TBP) from malignant pleural effusion (MPE) in patients presenting with lymphocytic exudative pleural effusions. METHODOLOGY: A cross-sectional study was conducted on 161 patients with pleural effusion who underwent diagnostic evaluation at Siriraj Hospital, Bangkok, Thailand, between April 2001 and March 2002. The complete biochemical analysis of pleural fluid, cultures of pleural fluid, and pathological examinations of pleural fluid and pleural tissue were performed. The CRP concentrations were then measured in stored sera and pleural fluid samples from patients with a lymphocytic exudative pleural effusion and with a definite diagnosis. RESULTS: Among the 148 patients with lymphocytic exudative pleural effusions, 55 were diagnosed with TBP, 60 with MPE, and 33 with non-specific pleuritis. Pleural fluid and serum CRP levels were significantly higher in the TBP group than in the MPE group (54.58 +/- 4.50 mg/L and 106.93 +/- 9.54 mg/L vs 12.66 +/- 3.52 mg/L and 49.66 +/- 8.84 mg/L, respectively, P < 0.001). The ratio of pleural fluid to serum CRP was significantly higher in the TBP group than in the MPE group (0.52 +/- 0.18 vs 0.30 +/- 0.16, P < 0.001). The optimum cut-off value for pleural fluid CRP level of > or =30 mg/dL had a sensitivity of 72% with 93% specificity, and the pleural fluid to serum CRP ratio cut-off value of 0.45 had a sensitivity of 60% with 89% specificity. A correlation between serum and pleural fluid CRP levels was observed in TBP patients but not in MPE patients. CONCLUSION: In patients presenting with lymphocytic exudative pleural effusion, a simple marker of raised pleural fluid CRP level may be helpful in discriminating between TBP and MPE.

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.

Scrofuloderma and Sweet's syndrome.

BACKGROUND: In recent years, the rare association of Sweet's syndrome with nontuberculous mycobacterial lymphadenitis has been reported. OBJECTIVE: To report the clinical, demographic, and bacteriologic data and association with Sweet's syndrome of 18 patients with scrofuloderma and scrofuloderma-like condition caused by nontuberculous mycobacterial infections seen during the past 7 years (1994-2000). METHODS: In all patients, a biopsy specimen was obtained for histopathologic and microbiologic studies. Patients from whom Mycobacterium tuberculosis and nontuberculous mycobacteria were isolated from the culture of skin biopsy specimens were included. Deep fungal infection was excluded by the lack of a fungal element in histologic section and cultural methods. The patients were treated with antimicrobials or antituberculous drugs according to the causative species. RESULTS: Eighteen cases of scrofuloderma (nine male, nine female; mean age, 36.9 years) were found among 104 patients with cutaneous tuberculosis and nontuberculous mycobacterial cutaneous infections. Sixteen of the 18 cases had lymphadenitis as the underlying focus of scrofuloderma: 15 cases occurred in the cervical group and one case in the inguinal area. One case drained from the soft tissue and one from the paranasal air sinus. Five cases had multiple episodes of Sweet' s syndrome during the course of treatment. Most cases in this group (four of the five) were middle-aged women with cervical lymphadenitis, and the most common species were rapid growers. CONCLUSIONS: Age, sex, and the site of infection may have some influence on the association with Sweet's syndrome in nontuberculous mycobacterial infections.