Mycobacteremia in AIDS patients

Summary The importance of blood cultures in diagnosing disseminated mycobacteriosis in AIDS patients was evaluated. Blood samples were screened for mycobacteria by culture and microscopic techniques. Mycobacteremia was proven in 20/136 (14.7%) AIDS patients, the agent being M. avium-M. intracellulare (MAI) in 16 cases and M. tuberculosis in four cases. The rate of cases with positive blood samples in disseminated MAI infection was 59.3% (16/27 cases) and in disseminated tuberculosis 57.1% (4/7 cases). To detect mycobacteria buffy-coat was slightly superior to lysated cell pellets, obtained by a lysis-centrifugation technique. In 4/16 cases with MAI bacteremia, the agent was proven by positive blood smears for acid-fast bacilli only; in these four patients MAI was demonstrated at other body sites. These results illustrate the diagnostic role of blood culture and its use in early diagnosis of disseminated mycobacteriosis, with microscopic examination of blood smears being an important adjunct.Abbreviations AFB acid-fast bacilli - AIDS acquired immunodeficiency syndrome - CDC Centers for Disease Control - CSF cerebrospinal fluid - ELISA enzyme linked immunosorbent assay - GI-tract gastrointestinal tract - HIV human immunodeficiency virus - HIV human immunodeficiency virus - M Mycobacterium - MAI M. avium-M. intracellulare - MOTT mycobacteria other than tuberculosis     

Non tuberculous mycobacteria isolated from clinical specimens at a tertiary care hospital in South India

Purpose: This is a retrospective analysis of the isolation rates of nontuberculous mycobacteria (NTM) from various clinical specimens and their antimicrobial susceptibility patterns. Methods: All NTM isolated between 1999 and 2004 at Christian Medical College, Vellore, South India, were identified with various biochemical tests. Antimicrobial susceptibility test for all NTM was performed by standard methods. Results: A total of 32,084 specimens were received for culture, of which 4473 (13.9%) grew acid fast bacilli (AFB). Four thousand three hundred (96.1%) of the AFB were M. tuberculosis while 173 (3.9%) were NTM. Of the 173 NTM, 115 (66.5%) were identified to the species level. Pus, biopsy specimens and sputum specimens yielded most of the NTM of which M. chelonae (46%) and M. fortuitum (41%) accounted for majority of them. M. chelonae and M. fortuitum, showed highest susceptibility to amikacin (99.2%). NTM were repeatedly isolated from seven sputum specimens, 15 biopsy and pus specimens, two CSF and two blood cultures. Six were isolated from patients with AIDS and five from post transplant patients. Conclusions: The isolation of NTM from various clinical specimens is reported in this study to highlight the associated diseases and therapeutic options in these infections.     

A Radioreceptor Assay for Neuroleptic Drugs in Plasma

A simple and rapid method for measuring neuroleptic drugs in plasma by the estimation of dopamine receptor blocking activity is described. The assay has the advantages that it can be used without modification for all neuroleptic drugs and measures both parent compound as well as pharmacologically active metabolites.

Intra-assay and inter-assay coefficients of variation are 20% and 30% respectively and sensitivity is adequate to measure drug activity in plasma from patients treated with oral neuroleptics but not for those receiving depot injections.

Preliminary clinical investigations indicate that the ranges of dopamine receptor blocking activity in plasma vary for different drugs. Interference in the assay has been seen with some samples from patients receiving amitriptyline and nortriptyline, but not with other psychotropic drugs.

The receptor assay is considered suitable for routine monitoring of neuroleptic drug therapy.     

耻垢分枝杆菌作为免疫调节剂的研究

目的 探讨耻垢分枝杆菌制剂对不同免疫状态动物的免疫调节作用.方法 注射环磷酰胺建立免疫功能低下的动物模型,以结核分枝杆菌感染（早期）或猪血清致敏建立免疫功能亢进的动物模型,然后将耻垢分枝杆菌制剂分别注射于正常、免疫功能低下和免疫功能亢进的动物,研究耻垢分枝杆菌制剂对不同模型动物T淋巴细胞增殖反应、迟发型超敏反应或速发型超敏反应的影响.结果 耻垢分枝杆菌制剂可增强正常动物的T淋巴细胞增殖反应和迟发型超敏反应,促进免疫功能低下小鼠T淋巴细胞增殖功能的恢复,抑制免疫功能亢进豚鼠的迟发型超敏反应和猪血清致敏小鼠的速发型超敏反应.结论 耻垢分枝杆菌制剂具有双向免疫调节作用.     

重组耻垢分枝杆菌对小鼠结核病免疫治疗效果的评价

目的 评估携带编码人天然颗粒溶素(GLS)和小鼠IL-12基因质粒(pZM03)的重组耻垢分枝杆菌对结核分枝杆菌感染的治疗效果,及其与临床抗痨药物疗效的比较.方法 结核分枝杆菌H37Rv感染Balb/c小鼠4周后分别用生理盐水、GLS/IL-12重组耻垢分枝杆菌(重组M.S)、INH PZA和重组M.S INH PZA治疗,于第1次治疗后3个月,处死小鼠,检测器官荷菌量、脾淋巴细胞IFN-γ和INF-α的分泌水平、血清IL-12和IFN-γ分泌水平、肺泡灌洗液SIgA的水平和肺、脾组织中GLS表达,同时观察小鼠肺、脾组织病理改变情况.结果 重组M.s、INH PZA和重组M.s INH PZA治疗组肺、脾组织荷菌量(log CFU/g)比生理盐水组显著降低.重组M.s组经PPD刺激后IFN-γ和TNF-α分泌水平明显高于其它组.重组M.s组血清IL-12和IFN-γ水平明显高于其它组.各组产生黏膜特异性SIgA明显高于未经感染组.用免疫组化检测到小鼠肺及脾组织中GKS的表达.生理盐水组肺组织病理改变以渗出为主,病变广泛,正常肺泡结构被破坏;其余组肺组织病变轻且局限.结论 重组M.s对小鼠结核病有一定免疫治疗作用,通过增强宿主Th1型免疫应答和GLS的抗菌活性有关;重组M.S对临床常用的抗结核药有协同作用,为结核病的综合治疗打下实验基础.     

Development and Prevention of Morphologic and Ultrastructural Changes in Uremia-induced Hyperplastic Parathyroid Gland

The detailed ultrastructural changes of uremia-induced hyperplastic parathyroid gland and the effects of current medical treatments for secondary hyperparathyroidism were investigated. Marked enlargement of parathyroid cell with accumulation of mitochondria and lipids and a significant increase in the thickness of the pericapillary area with increased fibrosis and appearance of fibroblast like cells were noted in the hyperplastic gland caused by uremia and phosphate retention. These ultrastructural changes and biochemical findings indicating hyperparathyroidism were significantly suppressed by all of the treatment using phosphate restriction, calcitriol, and cinacalcet. The characteristic ultrastructural changes, including the morphologic evidence of nodule formation, were indicated.     

不同月龄人胎心肌细胞超微结构变化

目的　探讨早、中及晚期胎儿心肌细胞的超微结构。方法　利用透射电镜和扫描电镜化学消化法。结果　随着心脏的胚胎发生发育 ,心肌细胞的形态及超微结构逐渐复杂。结论　其形态结构的变化与胎儿心脏供血功能密切相关     

The Macrophage Disappearance Reaction (MDR) as an in Vivo Test of Delayed Hypersensitivity in Mice

The macrophage disappearance reaction (MDR), as an in vivo analogue of in vitro tests for delayed hypersensitivity, was utilized in mice immunized with M. tuberculosis or mouse thyroid extract (MTE). The optimal schedule for the induction of macrophages in peritoneal exudates and the optimal concentration of antigen for the MDR were determined. Macrophage disappearance occurred 4 hr after immunized mice received an intraperitoneal injection of 200 μg of soluble antigen. The MDR was found to be antigen specific. Intraperitoneal injection of an unrelated antigen or tissue culture medium alone did not cause macrophage disappearance; however, the re-injection of the antigen used for immunization caused a 70-80% reduction of macrophages. Macrophage disappearance was greater in mice immunized with M. tuberculosis than in mice immunized with MTE. Comparison of the MDR with the footpad test in these 2 groups showed that mice immunized with M. tuberculosis developed a higher degree of delayed hypersensitivity than the group immunized with MTE. These results demonstrate that the MDR represents a specific and quantitative method for detecting the delayed type of cellular immune response in mice.     

A pulsed-field gel electrophoresis study of Mycobacterium fortuitum in a Caribbean setting underlines high genetic diversity of the strains and excludes nosocomial outbreaks

Among rapidly-growing opportunistic mycobacteria, organisms of the Mycobacterium fortuitum-Mycobacterium chelonae complex (M. fortuitum, M. chelonae, M. abscessus and M. peregrinum) were isolated in significantly higher numbers during the period 1993-99 from clinical samples in Guadeloupe, Martinique and French Guiana. Based on biochemical and cultural tests and PCR-restriction fragment length polymorphism (RFLP) of the hsp65 gene, 51 isolates from 47 patients were unambiguously identified as M. fortuitum. A molecular epidemiological study by pulsed-field gel electrophoresis (PFGE) using DraI and Xbal digestions of bacterial DNA revealed two clusters designated A and B; cluster A was composed of strains showing 10 bands that were isolated from 3 patients in Martinique within a 2-months period in 1999, and the cluster B was composed of 2 strains showing 9 bands from 2 patients in Martinique, also isolated within a 2-months period in 1999. The available epidemiological and clinical information neither incriminated M. fortuitum as a cause of disease in these patients, nor showed any potential epidemiolgical links between them, except for the fact that the samples were processed in the same microbiology laboratory within a short span of time. In conclusion, isolation of M. fortuitum from non-sterile sites in patients without predisposing conditions, and in absence of repeated isolation, may be caused by contaminants or colonizers that are picked up more easily due to improvement of techniques used for mycobacterial isolation and identification.     

分析5种结核杆菌耐药基因突变与耐药水平的关系

目的：分析5种结核杆菌（M.tb)耐药基因突变的情况，了解基因突变和耐药水平的关系。方法：134例临床分离株均做传统梯度药敏试验和聚合酶链反应－单链构象多态性I（PCR－SSCP）试验。结果：耐PZA(pncA),SM(rpsL),REP(rpoB),INH(katG),EMB(embB)基因突变率分别为42．7％、72％、78％、69％和43．9％，其中，上述高耐株基因突变率分别为70％、87．2％、93．4％、80％、43．9％。低耐株分别为12．5％、28．5％、45．4％、18．7％，EMB在低耐区无基因突变，结论：M.tb耐药基因变与耐药水平联系密切，多数M.tb耐药基因突变易发生在高耐药区，也有少数菌基因突变易发生在低耐药区。     

Morphologic and morphometric analyses of muscle in the neonatal myotonic dystrophy syndrome

Autopsy studies of three premature siblings who died soon after birth with the neonatal myotonic dystrophy syndrome revealed pulmonary hypoplasia and congenital pleural effusions. Neither of these findings has been described previously in this condition. New ultrastructural findings include focal diaphragmatic myofiber degeneration and necrosis, which were attributed to over-stretching of the fetal diaphragm. In addition, abnormally small stores of free and intravesicular glycogen were observed in skeletal muscle fibers. The morphometric features of control fetal and neonatal skeletal muscle were recorded for comparison with muscle fiber measurements in the three infants. Fiber diameters in the latter were much smaller than expected for body weights. The morphologic and morphometric findings support the concept that fetal muscle maturation is severely retarded in this syndrome.     

Detection of mutation in isoniazid-resistant Mycobacterium tuberculosis isolates from tuberculosis patients in Belarus

The aim of this study was to investigate the frequency, location and type of katG mutations in Mycobacterium tuberculosis strains isolated from patients in Belarus. Forty two isoniazid-resistant isolates were identified from sputum of 163 patients with active pulmonary tuberculosis. Drug susceptibility testing was determined by using CDC standard conventional proportional method and BACTEC system. Standard PCR method for detection of isoniazid resistance associated mutations was performed by katG gene amplification and DNA sequencing. Most mutations were found in katG gene codons 315, 316 and 309. Four types of mutations were identified in codon 315: AGC-->ACC (n=36) 85%, AGC-->AGG (n=1) 2.3%, AGC-->AAC (n=2) 4.7%, AGC-->GGC (n=1) 2.3%. One type of mutation was found in codon 316: GGC-->AGC (n=18) 41.4%, four types of mutations were detected in codon 309: GGT-->GGT (n=7) 16.1%, GGT-->GCT (n=4) 9.2%, GGT-->GTC (n=3)6.9%, GGT-->GGG (n=1) 2.7%. The highest frequency of mutations sharing between primary and secondary infections was found in codon 315.     

Ultrastructural studies of acute hypersensitivity pneumonitis in rabbits

Acute experimental hypersensitivity pneumonitis in rabbits was studied using scanning and transmission electron microscopy. Scanning electron microscopy reveals the three-dimensional architecture of lesion localization, alveolar cellular filling, and scptal thickening, which typify this animal model. Transmission electron microscopy has identified the macrophage as the dominant cell filling alveolar spaces with variable numbers of lymphocytes and granulocytes. Septal thickening is also accompanied by increased cellularity involving septal cells and fewer numbers of other mononuclear and polymorphonuclear cells. No evidence of proliferating alveolar lining cells was found.     

Evaluation of nitrate reductase assay for direct detection of drug resistance in Mycobacterium tuberculosis: rapid and inexpensive method for low-resource settings

The aim of this study was to evaluate a nitrate reductase assay (NRA) for the direct detection of multidrug resistance (MDR) in Mycobacterium tuberculosis from 100 smear-positive sputum samples. The NRA results were compared with the reference proportion method for 100 sputum specimens for which comparable results were available. NRA results were obtained at day 7 for 61 specimens, results for 26 specimens were obtained at day 10, and the results for 13 specimens were obtained at day 14. Thus, 87% of NRA results were obtained in 10 days. NRA is a rapid, accurate, and cost-effective method for the detection of MDR in M. tuberculosis isolates as compared to the proportion method, which is time consuming. Therefore, NRA constitutes a useful tool for detection of tuberculosis drug resistance in low-resource countries with limited laboratory facilities due to its low-cost, ease of performance and lack of requirement of sophisticated equipment.     

Different types of pulmonary granuloma necrosis in immunocompetent vs. TNFRp55-gene-deficient mice aerogenically infected with highly virulent Mycobacterium avium

The immunopathogenesis of mycobacterial infections frequently involves the formation of caseating granulomas which cause tissue destruction and, in the case of tuberculosis (TB), may lead to cavity formation. Both intravenous and aerosol models of Mycobacterium tuberculosis infection in mice do not reflect the pulmonary lesions characteristic of TB patients. Using both low-dose (10² colony-forming units, cfu) and high-dose (10⁵ cfu) aerosol infection with a highly virulent strain of Mycobacterium avium (TMC724) in C57BL/6 mice, it is now shown that these mice are capable of developing centrally caseating necrosis in lung granulomas after approximately 4 months of infection. In contrast, mice infected intravenously with the high dose never developed this type of lesion, although bacterial counts in their lungs reached levels comparable to those attained by aerosol-infected mice (10¹⁰ cfu). To study the relevance of events signalled by tumour necrosis factor (TNF) in this model, TNFRp55 gene-deficient and syngeneic C57BL/6 immunocompetent mice were infected with 10⁵ cfu M. avium via aerosol. In gene-deficient mice, newly formed pulmonary granulomas acutely disintegrated, showing signs of apoptotic cell death and neutrophil influx, and TNFRp55 knock-out mice all succumbed to infection just beyond the stage of granuloma initiation. Aerogenic infection with M. avium in mice is a suitable model to study the immunopathogenesis of granuloma necrosis because it closely mimicks the histopathology of mycobacterial infections in humans, including TB. Furthermore, the use of TNFRp55 gene-deficient mice in this model establishes a role for TNF in maintaining the integrity of a developing pulmonary granuloma. Copyright © 1999 John Wiley & Sons, Ltd.     

四种卡介苗重组疫苗对小鼠免疫原性的研究

目的研究esat6-卡介苗重组疫苗、mpt64-卡介苗重组疫苗、ag85a-卡介苗重组疫苗、ag85b-卡介苗重组疫苗的免疫原性。方法通过ELISA法检测小鼠血清中特异性抗体,用MTS法分析其脾淋巴细胞增殖指数。结果以生理盐水、卡介苗为对照,用各卡介苗重组疫苗免疫小鼠后,各组小鼠体重变化与对照组相比差异无统计学意义。应用不同抗原检测各组小鼠不同时间采集的血清,各组有其特异性抗体产生;ag85a-卡介苗重组疫苗能提高卡介苗刺激B细胞产生抗体的水平;各卡介苗重组疫苗免疫后15d,抗体水平已有升高,45d时达到最高水平。用不同抗原刺激各组小鼠脾淋巴细胞,平均刺激指数均达2.0以上,esat6-卡介苗重组疫苗组小鼠脾淋巴细胞的刺激指数稍高,其它各组之间差异无统计学意义。结论结核分枝杆菌esat6、mpt64、ag85a、ag85b基因在卡介苗中都能够表达特异的蛋白,刺激小鼠产生特异性抗体,均能刺激T淋巴细胞产生细胞免疫。     

恶性疟原虫FCC-1/HN株裂殖子表面抗原2基因在M.smegmatis mc2155中的表达

目的研究裂殖子表面抗原2(merozoitesurfaceantigen2,MSA2)基因重组BCG疫苗的保护作用.方法采用电穿孔转化法将重组质粒pBCG/MSA2导入耻垢分枝杆菌(M.smegmatis)mc2155中,通过卡那霉素抗性筛选并经PCR鉴定的重组M.smegmatismc2155培养于middlebrook7H9broth(M7H9)培养基,并添加10%M7H9enrichmentADC和0.05%Tween80,48～72?h后于45℃进行30?min的诱导表达,表达产物进行十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)及Westernblot分析.结果SDS-PAGE及Westernblot分析结果均显示在相对分子质量(Mr)约31×103的位置上可见明显的蛋白条带,并与MSA2基因编码序列推断的Mr相符.结论恶性疟原虫裂殖子表面抗原2可在M.smegmatis中表达,这些结果将为恶性疟原虫多价BCG疫苗的研制提供有用的资料.     

Progression of chronic pulmonary tuberculosis in mice intravenously infected with ethambutol resistant Mycobacterium tuberculosis

Purpose: Ethambutol (EMB) is an important first line drug, however little information on its molecular mechanism of resistance and pathogenicity of resistant isolates is available. Present work was designed to study virulence of the EMB resistant M. tuberculosis strains and the host responses in-vivo on infection of EMB resistant M. tuberculosis using Balb/c mouse model of infection. Methods: Three groups of Balb/c mice (female, age 4-6 wk; 21 mice in each group) were infected intravenously with 106 CFU of M. tuberculosis H37Rv and two EMB resistant clinical isolates. Age and sex matched control animals were mock inoculated with Middlebrook 7H9 broth alone. At 10, 20, 30, 40, 50, 60, and 70 days post-infection three animals from each group were sacrificed by cervical dislocation and lung tissue was collected for further analysis. Results: Infection with EMB resistant M. tuberculosis led to progressive and chronic disease with significantly high bacillary load (p=0.02). Massive infiltration and exacerbated lung pathology with increased expression of IFN-γand TNF-αwas observed in lungs of mice infected with EMB resistant strains. The present study suggests that infection with EMB resistant M. tuberculosis leads to chronic infection with subsequent loss of lung function, bacterial persistence with elevated expression of TNF-αresulting in increased lung pathology. Conclusion: These findings highlight that EMB resistant M. tuberculosis regulates host immune response differentially and its pathogenicity is different from drug sensitive strains of M. tuberculosis.     

Phosphorylated STAT3 and PD‐1 regulate IL‐17 production and IL‐23 receptor expression in Mycobacterium tuberculosis infection

We studied the factors that regulate IL‐23 receptor expression and IL‐17 production in human tuberculosis infection. Mycobacterium tuberculosis (M. tb)‐stimulated CD4⁺ T cells from tuberculosis patients secreted less IL‐17 than did CD4⁺ T cells from healthy tuberculin reactors (PPD⁺). M. tb‐cultured monocytes from tuberculosis patients and PPD⁺ donors expressed equal amounts of IL‐23p19 mRNA and protein, suggesting that reduced IL‐23 production is not responsible for decreased IL‐17 production by tuberculosis patients. Freshly isolated and M. tb‐stimulated CD4⁺ T cells from tuberculosis patients had reduced IL‐23 receptor and phosphorylated STAT3 (pSTAT3) expression, compared with cells from PPD⁺ donors. STAT3 siRNA reduced IL‐23 receptor expression and IL‐17 production by CD4⁺ T cells from PPD⁺ donors. Tuberculosis patients had increased numbers of PD‐1⁺ T cells compared with healthy PPD⁺ individuals. Anti‐PD‐1 antibody enhanced pSTAT3 and IL‐23R expression and IL‐17 production by M. tb‐cultured CD4⁺ T cells of tuberculosis patients. Anti‐tuberculosis therapy decreased PD‐1 expression, increased IL‐17 and IFN‐γ production and pSTAT3 and IL‐23R expression. These findings demonstrate that increased PD‐1 expression and decreased pSTAT3 expression reduce IL‐23 receptor expression and IL‐17 production by CD4⁺ T cells of tuberculosis patients.     

Translating the role of vitamin D3 in infectious diseases

Vitamin D₃ affects both the innate as well as adaptive immune responses. Epidemiological studies have established that vitamin D₃ deficiency plays an important role in tuberculosis (TB) and viral influenza prevalence as well as susceptibility to active disease in TB. Vitamin D₃ status has been associated with the clinical course of HIV infection and drug interaction with anti-retroviral therapy. This article reviews the immunomodulatory capacity of vitamin D₃ and examines the impact of vitamin D₃ supplementation as a preventive or therapeutic intervention with the intent to uncover its potential therapeutic application in infectious diseases and to identify novel areas for future research. We present a review of randomized, controlled clinical studies conducted in humans which included assessment of the immune function or clinical outcome as study end points. Current data support vitamin D₃ supplementation as risk-modifying intervention in tuberculosis and viral respiratory tract infection, but the optimal dosage regimen remains to be determined. However, to date the knowledge on its role in fungal infection and sepsis is limited although a potential benefit could be harnessed from its ability to curtail the unrestrained pro-inflammatory response and therefore prevent excessive collateral tissue damage.