Perspective on the clone library method for infectious diseases

Recently, culture-independent molecular methods, such as DNA sequencing techniques targeting the 16S-ribosomal RNA (rRNA) gene and/or other housekeeping genes with Sanger method-based technologies, next generation sequencing (NGS), and metagenomic analysis, have been developed for detecting microorganisms in the human body; these can provide information on microbiomes of samples from individuals with or without infectious diseases. Determining the bacterial species is crucial in identifying causative bacteria of upper and lower respiratory tract infections, especially for Streptococcus species, but NGS analysis is often not precise enough to identify bacteria at the species level. This review briefly introduces previous observations of the microbiome of samples from various respiratory and other infections assessed using the clone library method with Sanger sequencing of the 16S-rRNA gene. On analysis of 16S-rRNA gene-sequence data of bronchoalveolar lavage fluid obtained from pneumonia lesions in patients with bacterial pneumonia and lung abscess, anaerobes are often detected in non-elderly patients with pneumonia, and the detection rate of Staphylococcus aureus in patients with hospital-acquired pneumonia is lower than that previously reported. Analysis of pleural effusion samples from patients with pleurisy indicated a more important role of anaerobes than previous believed. The other topics reviewed include microbiomes of nontuberculous mycobacteriosis and lower respiratory tract infections in children with permanent tracheostomy due to neuromuscular disorders, in nasal discharge, in bacterial vaginosis, in the intracystic fluid of postoperative maxillary cyst, and in bacterial conjunctivitis; urine microbiota in urethritis; fecal microbiota; and newly detected infectious organisms in the human respiratory tract.     

非结核分枝杆菌快速药敏试验研究

目的研究结核分枝杆菌BACTEC MGIT 960系统液体培养法和美国临床实验室标准化协会(Clinical and Laboratory Standards Institute,CLSIM)24-A微量肉汤稀释法的快速药敏试验与对非结核分枝杆菌的意义。方法分别采用BACTEC MGIT 960液体培养法联合微量肉汤稀释法的快速药敏试验与L-J固体培养法和绝对浓度药敏法分别对48份非结核分支杆菌(nontubeerculous mycobacteria,NTM)培养时间和药敏报告时间进行比较研究。结果 48份NTM在BACTEC MGIT 960培养仪的平均阳性报告时间(7.0±1.1)d,L-J培养基平均阳性报告时间(17.6±3.9)d,两者比较差异有统计学意义(t=-18,P0.01);微量肉汤稀释法结果报告时间为(7.2±1.2)d,L-J固体药敏试验报告时间为(27.3±2.4)d,两者比较差异有统计学意义(t=-52.2,P0.01),两种药敏试验方法对相同种类药物的耐药率均为90%以上,微量肉汤稀释法比L-J法可以检测的药物种类更多。结论 BACTEC MGIT 960液体培养法联合微量肉汤稀释法的快速药敏试验比L-J固体培养法和绝对浓度药敏法在培养时间和药敏结果种类及报告时间上有着明显优势。     

鱼刺伤肉芽肿四例分析

报道4例鱼刺伤所致肉芽肿,患者均为老年女性,均有鱼类接触史,皮疹特点均为暗红色丘疹、结节,其中一例伴有上肢串珠样皮下结节。组织病理示感染性肉芽肿。虽然微生物感染证据缺乏,但诊断考虑非结核分枝杆菌感染,予利福平、克拉霉素行经验性治疗有效。     

Endovascular Mycobacterium abscessus infection in a heart transplant recipient: a case report and review of the literature

Nontuberculous mycobacteria are ubiquitous in the environment. Although rarely a cause of infection in immunocompetent individuals, increased risk and severity of infection are seen in patients who are immunocompromised, such as those with solid organ transplants. In this report, we describe the first case of disseminated endovascular Mycobacterium abscessus in a heart transplant recipient. A review of the literature regarding this infection in heart transplant recipients and its therapeutic options and concerns are summarized.     

NON-TUBERCULOUS MYCOBACTERIAL LYMPHADENITIS

Background: Non-tuberculous mycobacterial lymphadenitis has been recognized since medieval times by an array of names. The condition is familiar to paediatricians and paediatric surgeons but it often is not recognized in its early stages. A paediatric surgeon's experience of the condition in Newcastle over 30 years is reviewed. Methods: A personal series of children with non-tuberculous mycobacterial lymphadenitis treated in Newcastle from 1966 to 1994 is reviewed. Clinical diagnosis was supported by multiple Mantoux skin testing in most patients using human purified protein derivative (PPD) and avian antigens. All were treated surgically with histological confirmation of the diagnosis. A total of 89 patients were encountered. Twenty-two were seen in hospital practice between 1966 and 1976 and have already been reported. The current paper presents the results of analysis of the clinical features, diagnosis and surgical treatment of the remaining 67 patients seen in paediatric surgical practice between 1976 and 1994. Results: There was equal sex distribution. Ages ranged from 1 to 10 years, with none under 1 year, and 82% of the patients were in the pre-school age group. Cervical lymph nodes were involved in all, the majority being jugulo-digastric or subman-dibular. Surgical excision by limited dissection of lymph nodes was performed in 55 patients with one recurrence, and by excision and curettage in eight patients with two recurrences. Simple bacterial wound infection occurred in four patients and two had prolonged postoperative suppuration as a result of mycobacterial wound infection. Paresis of the mandibular or cervical branch of the facial nerve occurred in 50% of patients where the nerve was at risk, but the majority of these recovered although it took over 6 months in some children. Culture for mycobacterial organisms was positive in only 29 patients. Conclusions: The diagnosis of non-tuberculous mycobacterial lymphadenitis is clinical and its early recognition requires an awareness of the condition. It can be confirmed by multiple Mantoux testing or fine needle aspiration biopsy. The treatment is local excision of the affected lymph nodes. Histological examination and mycobacterial culture should be performed.     

Morphometric analysis of Mycobacterium tuberculosis infection in mice suggests a genetic influence on the generation of the granulomatous inflammatory response.

There is evidence in natural human disease and experimental infection in mice that host genetic factors influence susceptibility to infection with Mycobacterium tuberculosis and the progress of the disease. In mouse models, both H-2 and non-H-2 genes have been implicated. In this study, four inbred strains of mice (Balb/b, Balb/k, B10, B10.BR), selected for combinations of two different H-2 haplotypes on two different non-H-2 backgrounds, were inoculated with M. tuberculosis, strain H37Rv, by intraperitoneal injection. The histological features of the granulomatous inflammatory response in the liver and lungs were investigated during the first 18 weeks of the infection. Granuloma fraction, mean granuloma area, bacillary load, and the density of acid-fast bacilli within granulomata were measured. Animals of all four strains showed the same general pattern of infection with an early, and later self-limiting, infection of the liver and delayed onset, but progressive, infection of the lung. The non-H-2 related genetic background appears to influence the morphology of the granulomatous inflammatory response. In comparison, H-2 differences appeared to be small and inconsistent.     

N1-Hetaryl Substituted Pyridine- and Pyrazinecarboxamidrazones with Antimycobacterial Activity

A series of amidrazones was prepared and characterized by ¹H NMR and mass spectroscopy. The substances were tested against M. tuberculosis, M. avium, M. intracellulare, and M. lufu. Compounds 11–13 exhibit a satisfactory inhibition of mycobacteria.     

Hypersensitivity pneumonitis due to metal working fluids: Sporadic or under reported?

BACKGROUND: Occupational exposure to metal working fluids (MWF) is common with over 1.2 million workers in the United States involved in machine finishing, machine tooling, and other metalworking operations. MWF is a known cause of hypersensitivity pneumonitis (HP). Recent reports of outbreaks of hypersensitivity HP secondary to exposure to MWF are reported. DESIGN: Cases were identified through the Occupational Disease surveillance system in the State of Michigan and from referrals for evaluation to the Division of Occupational and Environmental Medicine at Michigan State University (MSU). Each patient underwent a clinical examination including an occupational history, lung function studies, radiographic imaging, and in some cases lung biopsies. Following the diagnosis of definite HP, an industrial hygiene investigation was carried out, which included a plant walk-through, and review of the "Injury and Illness" log. Air monitoring and microbial sampling results were reviewed. RESULTS: As part of Michigan's mandatory surveillance system for occupational illnesses, seven cases of suspected HP were identified in 2003-2004 from three facilities manufacturing automobile parts in Michigan. Each plant used semi-synthetic MWFs, and conducted a MWF management program including biocide additions. Two facilities had recently changed the MWF before the cases arose. Growth of mycobacteria was found in these two MWFs. Breathing zone samples for particulates of two employees in plant A (two cases) ranged from 0.48 to 0.56 mg/m3. In plant B (four cases), two employees' sampling results ranged from 0.10 to 0.14 mg/m3. No air sampling data were available from plant C. CONCLUSION: Hypersensitivity pneumonitis due to exposure to MWFs is under-recognized by health care providers, and current surveillance systems are inadequate to provide a true estimate of its occurrence. HP arose from environments with exposures well below the Occupational Safety and Health Administration (OSHA) permissible exposure limit (PEL) for MWF, and in one case from exposures well below the National Institute of Occupational Safety and Health (NIOSH) recommended exposure limit (REL). The sporadic nature of reports of HP in relationship to MWF probably represents a combination of workplace changes that cause the disease and inadequate recognition and reporting of the disease when it does occur. Physician awareness of HP secondary to MWF and an effective medical surveillance program are necessary to better understanding the epidemiology and prevention of this disease.     

Controlled trial of antituberculous chemotherapy in Crohn's disease: a five year follow up study

Background—It has been suggested thatMycobacterium paratuberculosis is the cause of Crohn'sdisease. In a previous report the immediate effect of two yearstreatment with antituberculous chemotherapy showed no clinical benefit.
Aims—To assess both the immediate and longer termeffect of treatment on the disease.
Methods—Patients were followed for five yearsfrom their date of entry to the study. One hundred and thirty patientsentered the initial study, and of these 111 (81%) were followed regularly.
Results—Overall, there was no evidence ofconsistent benefit or disadvantage from antituberculous chemotherapy inany of the assessments made, including the number of acute relapses,surgical episodes, hospital admissions, disease activity, blood tests, or medication required for Crohn's disease during the follow up period.
Conclusion—The absence of any benefit at the endof the initial two year trial period, and during the three yearsubsequent follow up, fails to support the hypothesis that mycobacteriaplay an important part in the pathogenesis of Crohn's disease, or that antituberculous chemotherapy may be of benefit.

Keywords:Crohn's disease; mycobacteria; antituberculuschemotherapy

     

MALDI-TOF MS Andromas strategy for the routine identification of bacteria, mycobacteria, yeasts, Aspergillus spp. and positive blood cultures

Clin Microbiol Infect 2012; 18: 1117-1125 ABSTRACT: All organisms usually isolated in our laboratory are now routinely identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) using the Andromas software. The aim of this study was to describe the use of this strategy in a routine clinical microbiology laboratory. The microorganisms identified included bacteria, mycobacteria, yeasts and Aspergillus spp. isolated on solid media or extracted directly from blood cultures. MALDI-TOF MS was performed on 2665 bacteria isolated on solid media, corresponding to all bacteria isolated during this period except Escherichia coli grown on chromogenic media. All acquisitions were performed without extraction. After a single acquisition, 93.1% of bacteria grown on solid media were correctly identified. When the first acquisition was not contributory, a second acquisition was performed either the same day or the next day. After two acquisitions, the rate of bacteria identified increased to 99.2%. The failures reported on 21 strains were due to an unknown profile attributed to new species (9) or an insufficient quality of the spectrum (12). MALDI-TOF MS has been applied to 162 positive blood cultures. The identification rate was 91.4%. All mycobacteria isolated during this period (22) were correctly identified by MALDI-TOF MS without any extraction. For 96.3% and 92.2% of yeasts and Aspergillus spp., respectively, the identification was obtained with a single acquisition. After a second acquisition, the overall identification rate was 98.8% for yeasts (160/162) and 98.4% (63/64) for Aspergillus spp. In conclusion, the MALDI-TOF MS strategy used in this work allows a rapid and efficient identification of all microorganisms isolated routinely.