Automated identification of tubercle bacilli in sputum. A preliminary investigation

OBJECTIVE: To use an automated method to detect tubercle bacilli in sputum specimens. STUDY DESIGN: Using fluorescence microscopy, tubercle bacilli were identified on auramine-stained sputum specimens. Images were then captured with a digital camera and enhanced through imaging processing techniques. The bacilli were recognized using neural network classifiers. RESULTS: This preliminary investigation demonstrated a sensitivity of 94.1% for the identification of individual bacilli. As there are usually fairly numerous tubercle bacilli in the sputum of patients with active pulmonary tuberculosis, the overall diagnostic accuracy of sputum smear-positive patients can be expected to be very high. CONCLUSION: Potential benefits of automated screening for TB bacilli are: rapid, accurate, inexpensive diagnosis; the ability to screen larger numbers of people; increased resources to monitor patients; and reduction in health risks to staff.     

Survival of tubercle bacilli in heat-fixed and stained sputum smears

We used a slide culture technique to detect tubercle bacilli surviving in sputum smears (n=46) after conventional heat fixation and Ziehl-Neelsen staining. In all heat-fixed sputum smears, tubercle bacilli survived after time 0 (n=22), 24 h (n=7), 48 h (n=7), 72 h (n=4), and seven days (n=6). None of the stained sputum smears showed growth on slide cultures. Viable tubercle bacilli remaining in heat-fixed sputum smears for at least seven days may present an infection risk to laboratory staff. Thus, sputum smears should be stained immediately by the Ziehl-Neelsen method or stored in a safe container to avoid transmission of tuberculosis.     

Phage cocktail to control the exponential growth of normal flora in processed sputum specimens grown overnight in liquid medium for rapid TB diagnosis

The mechanical pressure exerted during centrifugation and the chemical pressure experienced when sputum specimens are processed, leave the tubercle bacilli in the sputum unsuitable for rapid detection especially in phage based assays. Thus, growing Mycobacterium tuberculosis in broth, at least overnight, is mandatory for allowing the tubercle bacilli to recoup. During this time the surviving colonizing flora grow faster and overgrow tubercle bacilli interfering with TB diagnosis. In the present study normal flora surviving the action of 4% NaOH was isolated and characterized. Phages capable of killing 14 different species representing this normal flora were isolated from soil and sewage samples and characterized. A novel and bio-friendly approach to treat sputum samples with a cocktail of three phages capable of killing most of the 14 representative organisms and not infecting mycobacteria is explored to control the overgrowth of colonizing bacteria in broth culture. While 26 of the 100 sputum samples processed by modified Petroff's procedure showed growth of colonizing flora on blood agar, all of them when grown in broth overnight showed mixed, confluent growth. The addition of phagebiotics controlled them all, showing a significant reduction in colony forming units but resulting in few discrete colonies in 54 samples. Isolation of phages capable of controlling these surviving organisms and including them in the phagebiotics mixture should lead to the control of colonizing bacteria effectively.     

THE TUBERCULOUS DISEASES Originating from Different Species of Acid-Fast Bacilli

Disease due to the typical human type tubercle bacillus is rapidly diminishing as a result of public health measures and specific chemotherapy. Lesions in man resulting from other kinds of acid-fast bacilli are now being recognized in increasing numbers. Some of these bacilli had been seen before but were confused with typical M. hominis, others were considered to be harmless saprophytes, while others could not be found with the methods used. Special culture media, different conditions for cultivation, new physical, chemical and biological tests, and inoculation into a variety of animal hosts are now available. With their use more than a dozen different strains of human type tubercle bacilli, and more than a score of other species of acid-fast bacilli may now be distinguished. A simple chemical test readily separates the human type tubercle bacilli from all other kinds of acid-fast bacilli. The differentiation of the different human and animal pathogenic acid-fast bacilli from the avirulent saprophytes and other harmless mycobacteria presents great difficulties, but methods are becoming available which usually make this possible. Since the distinction may be of great therapeutic and epidemiologic importance, the effort should be made.     

Rapid serodiagnosis of human mycobacteriosis by ELISA using cord factor (trehalose-6,6'-dimycolate) purified from Mycobacterium tuberculosis as antigen

IgG antibodies against purified cord factor (trehalose-6,6'-dimycolate, TDM) in sera of 99 patients infected with mycobacteria (42 patients with tuberculosis excreting tubercle bacilli in the sputum, 11 patients with non-tuberculous mycobacteriosis excreting acid-fast bacilli in the sputum, and 46 patients without bacilli in the sputum but diagnosed as having pulmonary tuberculosis by chest X-ray films and physical examination), five patients with lung cancer, and 100 healthy controls which included subjects positive and negative for the tuberculin test were tested by the ELISA with TDM purified from Mycobacterium tuberculosis H37Rv as the antigen. Of the 99 cases of mycobacteriosis, 83 patients (83.8%) had positive results (48 samples from 53 patients, or 90.5%, with bacilli in the sputum, and 35 samples from 46 patients (76%) with tuberculosis diagnosed clinically). The sera of the five patients with lung cancer and the 100 controls all gave negative results. Thus, the sensitivity and specificity were 83.8% and 100%, respectively. ELISA with TDM as the antigen is simple, reproducible, and useful for the rapid serodiagnosis of general mycobacterial infections including tuberculosis, because it does not involve the cultivation of bacteria.     

Comparison of Methods for Tuberculosis Bacteriology

To improve efficiency of isolation of tubercle bacilli from clinical specimens, the following recommendations are presented. (i) Employ multiple specimens consisting of a combination of morning sputums for the early detection of positives, along with 24-hr sputum pools for the greatest total yield of positives. (ii) When timing is rigorously controlled, Zephiran-trisodium phosphate and sodium hydroxide-acetylcysteine are comparable, but if timing cannot rigidly be controlled, employ the Zephiran-trisodium phosphate digestion procedure to allow the greatest freedom in exposure time with the lowest kill rate to tubercle bacilli. (iii) Employ both an agar medium incubated in 5% CO₂, for the early detection of positives as well as positives in the presence of contaminants, and an egg medium, preferably with CO₂, to increase the yield of positives.     

Miliary Tuberculosis in Adults

Of 40 adults with miliary tuberculosis 24 had “overt” disease; in them miliary mottling was usually present on the chest radiograph, and tubercle bacilli were readily isolated from sputum, urine, or cerebrospinal fluid. In the remaining 16 patients the disease was termed “cryptic” because its usual clinical and radiographic features were absent. This cryptic type is as common as the overt type in patients over 60 years. In this series the peak age incidence was in the eighth decade, and possibly this increase in the incidence age is due to the breakdown of old tuberculous foci in patients with diminished immunological mechanisms.Cryptic miliary tuberculosis is a difficult diagnostic problem and should be suspected in any elderly patient, particularly a woman, who has an unexplained pyrexia, pancytopenia, or leukaemoid reaction. In 10 cases it was diagnosed by a therapeutic trial with para-aminosalicylic acid and isoniazid, a fall of temperature to normal (usually within a week), weight gain, a rise in haemoglobin, and increased well-being being the criteria of improvement The use of such a trial is strongly advocated as a specific method of diagnosing cryptic miliary tuberculosis.     

In Vitro Studies on the Mechanism of Acquired Resistance to Tuberculous Infection

Immune lymph node cells were obtained from mice immunized with bovine gamma globulin (BGG) in complete Freund's adjuvant or allogeneic MH134 tumor cells. They showed the capacity of conferring bactericidal activity on macrophages infected with Mycobacterium tuberculosis, H37Rv, when they were incubated on macrophage monolayers together with the corresponding antigen, i.e., BGG or solubilized cellular antigen of the tumor cells. However, such capacity was lower than that of tubercle bacilli-immune lymph node cells. Culture supernatants were harvested after incubation of tubercle bacilli-immune, BGG-immune or allogeneic tumor-immune lymph node cells with the corresponding antigen for 24 hr. Macrophages were altered so as to suppress intracellular bacillary growth when macrophage monolayers were exposed to the supernatants for more than 2 days. When normal lymph node cells were incubated on normal macrophage monolayers together with a mitogen such as PHA or concanavalin A, growth of tubercle bacilli within the macrophages was slightly but difinitely suppressed. The mechanism of elicitation of cellular immunity to the infection with tubercle bacilli is discussed on the basis of results presented in this and the preceding paper.     

On the Etiology of Sarcoidosis

Bacteriophages lytic for tubercle bacilli were isolated from tuberculous patients and patients with sarcoidosis. While tuberculous patients were found to raise phage-neutralizing antibodies, those with sarcoidosis appeared unable to do so. In vitro experiments showed that in the absence of neutralizing antibodies, phagolysis and lysogenic conversion of tubercle bacilli proceed. Lysogenic conversion results in the emergence of bacilli so modified in their morphological and antigenic properties that, on the basis of the classical bacteriological criteria, they can no longer be recognized as tubercle bacilli. From six lymph node biopsies collected from patients with sarcoidosis and cultured on a variety of media, including media containing anti-phage sera, five variant strains of tubercle bacilli were isolated. These observations support the view that certain cases of sarcoidosis are due to “modified” tubercle bacilli.     

Obtaining hybridomas producing monoclonal antibodies with different specificities against Mycobacterium tuberculosis of the human and bovine types

A procedure for isolation of hybridomes producing monoclonal antibodies (McAB) to tubercle bacilli is described. Specificity of the McABs was studied with the solid phase radioimmune and immunoenzyme tests. Supernatant of tubercle bacilli destroyed with ultrasound was used as antigens. The McABs did not practically react with antigens of the tubercle bacilli atypical forms. Five ascitic monoclonal hybridomes were isolated. Four of them produced antibodies with selective specificity to antigens of bovine tubercle bacilli (M. bovis-8 and BCG) and one produced antibodies to antigens of human tubercle bacilli (H37Rv).     

Experimental study on the relapse of tuberculosis after the termination of antituberculous chemotherapy using immunodeficient nude mice.

The effect of cellular immunity induced by tuberculous infection on bacteriological relapse after the termination of antituberculous chemotherapy was studied experimentally using immunodeficient nu/nu mice and immunocompetent dd mice. The efficacy of intensive chemotherapy was excellent even in nu/nu mice; tubercle bacilli in the organs decreased below the detectable limit, but formidable regrowth of bacilli was seen after the termination of chemotherapy. On the other hand, in the case of dd mice that established antituberculous cellular immunity through tuberculous infection, bacteriological relapse was generally very slight. It was concluded that bacteriological relapse was related closely with the established cellular immunity induced by the infected tubercle bacilli.     

Inhibition of growth of tubercle bacilli by ali-esterase inhibitors in various culture media

Summary The effect of the potent ali-esterase inhibitor E 600 (paraoxon) on the growth of tubercle bacilli is potentiated by the addition of Tween 80 to the medium. This may be due to a change in the permeability of the bacteria for the inhibitor. Growth of tubercle bacilli from sputa is also inhibited by E 600 in the haemolysed human blood medium of Price. Cultures of tubercle bacilli in media with E 600 begin to grow after some time, when the inhibitor has broken down. The results obtained by the method of Price indicate that growth of the cultures does not originate from a few resistant cells but from the whole population which may have regained the ali-esterase activity essential to their growth.     

恶性淋巴瘤组织切片中结核菌L型的检出及其意义

本文应用Ｉｎｔｅｎｓｉｆｉｅｄｋｉｎｙｏｕｎ（ＩＫ）改良抗酸染色和结核菌抗休免疫组织化学染色检测了５１例恶性淋巴瘤组织功片内结核菌及其Ｌ型感染率。结果发现５１例恶性淋巴瘤组织功片中,ＩＫ染色阳性２５例（４９％）,免疫组化染色阳性２６例（５０．９％）,并发现结核菌Ｌ型感染与恶性淋巴瘤的疗效有关（Ｐ＜０．０１）。故认为恶性淋巴瘤中,结核菌Ｌ型感染是一个值得进一步研究的课题。     

A promoter mutation causes differential nitrate reductase activity of Mycobacterium tuberculosis and Mycobacterium bovis

The recent publication of the genome sequence of Mycobacterium bovis showed >99.95% identity to M. tuberculosis. No genes unique to M. bovis were found. Instead numerous single-nucleotide polymorphisms (SNPs) were identified. This has led to the hypothesis that differential gene expression due to SNPs might explain the differences between the human and bovine tubercle bacilli. One phenotypic distinction between M. tuberculosis and M. bovis is nitrate reduction, which not only is an essential diagnostic tool but also contributes to mycobacterial pathogenesis. We previously showed that narGHJI encodes a nitrate reductase in both M. tuberculosis and M. bovis and that NarGHJI-mediated nitrate reductase activity was substantially higher in the human tubercle bacillus. In the present study we used a genetic approach to demonstrate that an SNP within the promoter of the nitrate reductase gene cluster narGHJI is responsible for the different nitrate reductase activity of M. tuberculosis and M. bovis. This is the first example of an SNP that leads to differential gene expression between the human and bovine tubercle bacilli.     

Time to Culture Positivity and Sputum Smear Microscopy during Tuberculosis Therapy

Sputum smear microscopy is widely used for tuberculosis diagnosis and treatment monitoring. We evaluated the correlation between smear microscopy and time to liquid culture positivity during early tuberculosis treatment. The study included patients with smear-positive pulmonary tuberculosis hospitalized at a tuberculosis reference centre in Germany between 01/2012 and 05/2013. Patient records were reviewed and clinical, radiological and microbiological data were analysed. Sputum samples were collected before treatment initiation and weekly thereafter. A number of 310 sputum samples from 30 patients were analysed. Time to liquid culture positivity inversely correlated with smear grade (Spearman''s rho −0.439, p<0.001). There was a better correlation within the first two months vs. after two months of therapy (−0.519 vs. −0.416) with a trend to a more rapid increase in time to positivity between baseline and week 2 in patients who culture-converted within the first two months (5.9 days vs. 9.4 days, p = 0.3). In conclusion, the numbers of acid-fast bacilli in sputum smears of patients with pulmonary tuberculosis and time to culture positivity for M. tuberculosis cultures from sputum are correlated before and during tuberculosis treatment. A considerable proportion of patients with culture conversion after two months of therapy continued to have detectable acid-fast bacilli on sputum smears.     

Lysosomal enzymes and microbicidal capacity of activated macrophage.

A relation was sought between acid phosphatase contents and the presence of tubercle bacilli inside the peritoneal exudate cells (PEC) of normal guinea pigs and those immunized with BCG. This was done to investigate the role lysosomal enzymes play in the microbicidal capacity of the cell. In both normal and immune animals tubercle bacilli were present only in those PEC that contained acid phosphatase. Cells without acid phosphatase did not contain bacilli. Thus, only activated cells ingested bacilli. Under the conditions of these experiments, macrophage activation, as indicated by the presence of acid phosphatase, was not related to the immune status of the animal. Similarly, stimulation by ingestion of tubercle bacilli was not significant. Also, the number of acid phosphatase grains/cell did not influence the number of bacilli/cell. Thus, the acid phosphatase content of the cell did not correlate with the number of bacilli inside the cell. It was concluded that acid phosphatase may not be one of the factors that contribute to the microbicidal capacity of the cell.     

In vitro studies on the mechanism of acquired resistance to tuberculous infection. II. The effects of the culture supernatants of specifically stimulated-sensitized lymphocytes on the growth of tubercle bacilli within macrophages.

Immune lymph node cells were obtained from mice immunized with bovine gamma globulin (BGG) in complete Freund's adjuvant or allogeneic MH134 tumor cells. They showed the capacity of conferring bactericidal activity on macrophages infected with Mycobacterium tuberculosis, H37Rv, when they were incubated on macrophage monolayers together with the corresponding antigen, i.e., BGG or solubilized cellular antigen of the tumor cells. However, such capacity was lower than that of tubercle bacilli-immune lymph node cells. Culture supernatants were harvested after incubation of tubercle bacilli-immune, BGG-immune or allogeneic tumor-immune lymph node cells with the corresponding antigen for 24 hr. Macrophages were altered so as to suppress intracellular bacillary growth when macrophage monolayers were exposed to the supernatants for more than 2 days. When normal lymph node cells were incubated on normal macrophage monolayers together with a mitogen such as PHA or concanavalin A, growth of tubercle bacilli within the macrophages was slightly but difinitely suppressed. The mechanism of elicitation of cellular immunity to the infection with tubercle bacilli is discussed on the basis of results presented in this and the preceding paper.     

Disinfecting endoscopes: how not to transmit Mycobacterium tuberculosis by bronchoscopy.

Mycobacterium tuberculosis was cultured from the bronchial washings of two patients who underwent bronchoscopy consecutively with the same bronchoscope. Active pulmonary tuberculosis was later confirmed in the first patient, whereas the second patient had clinical and serologic evidence of infection with respiratory syncytial virus. The bronchoscope had been cleaned with an iodophor disinfectant, which had not destroyed the tubercle bacilli. The agent recommended for chemical disinfection of fibreoptic bronchoscopes is 2% glutaraldehyde solution; the instrument should be immersed in it for 10 to 30 minutes. Five hours'' exposure to ethylene oxide is recommended for sterilization of instruments. These procedures must be preceded by adequate mechanical cleaning. Then transmission of pathogenic organisms during endoscopy, which can result in nosocomial disease, misdiagnosis or inappropriate treatment, will be avoided.     

肾综合征出血热少尿期的肺部感染菌群分布及其耐药分析

目的了解肾综合征出血热少尿期的肺部细菌感染对临床治疗的意义。方法送检的所有痰液标本按常规方法进行细菌分离鉴定,药敏试验为K-B法,ESBLs以双纸片协同试验进行,MRS检测为纸片筛选法。结果痰液标本338份,细菌培养检出率68.9%,革兰阴性菌占优势,以铜绿假单胞菌、大肠埃希菌、肺炎克雷伯菌为主。对碳青霉烯类、头孢哌酮/他唑巴坦均分别低于9.09%、12.5%,头孢吡肟为27.4%,其余抗生素的耐药率均高于31.3%。3种主要产ESBLs菌对碳青霉烯类、头孢哌酮/他唑巴坦均分别低于15.8%、17.2%,其余抗生素的耐药率均高于55.2%。革兰阳性菌主要以金黄色萄葡球菌为主。结论肺部感染以革兰阴性菌占优势,碳青霉烯类、头孢哌酮/他唑巴坦是抗感染治疗的首选药物。