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.     

Pulmonary Tuberculosis Apparently Caused by the Avian Tubercle Bacillus

Avian tubercle bacilli were repeatedly isolated from the sputum of a 65-year-old man who had cavitary disease in the upper lobe of the right lung. The clinical picture resembled that of pulmonary tuberculosis caused by the human type of tubercle bacilli, but the response to antituberculosis chemotherapy was unsatisfactory and the patient''s sputum remained positive. The bacilli were markedly pathogenic to chickens and rabbits but failed to produce progressive disease in guinea pigs. This is in accord with the properties of tubercle bacilli of the avian type.     

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.     

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.     

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.     

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.     

Detection and identification of mycobacteria by mycolic acid analysis of sputum specimens and young cultures

Ziehl–Neelsen acid-fast staining and mycolic acid analysis of concentrated samples and Middlebrook 7H9 cultures were carried out on 127 sputum specimens to evaluate a rapid method for detecting and identifying mycobacteria by analyzing fluorescent derivatives of mycolic acids in concentrated sputum specimens and in Middlebrook 7H9 cultures and compare with mycobacterial detection using Lowenstein–Jensen (LJ) cultures. All samples were classified into five groups according to the number of acid-fast bacilli observed in the smear. The group of samples with 3+ acid-fast bacilli in the smear had the highest number of positive detections of mycolic acids in the concentrated samples and the Middlebrook 7H9 cultures (81.8 and 100%, respectively). The overall percentages of mycolic acid detection for both sample types were 43.2 and 91.3%, respectively. The mycolic acid analysis of the Middlebrook 7H9 cultures had the fewest false negative detections with respect to the LJ cultures. The analysis of fluorescent derivatives of mycolic acids, using HPLC, is useful for concentrated sputum samples with large number of bacilli (3+) and is preferred for Middlebrook 7H9 cultures, even for clinical specimens with a low number of bacilli. Furthermore, with this analytical method, the simultaneous detection and identification of mycobacteria is usually possible.     

下呼吸道感染患者病原菌分布及耐药性

目的分析下呼吸道感染患者病原菌分布及耐药性,指导临床合理用药。方法收集我院2013年至2015年院内感染患者痰标本进行细菌培养和药物敏感性试验。结果 3年共收集下呼吸道痰液标本21 615份,分离病原菌5 621株,阳性率为26.0%;其中革兰阴性(G~-)菌4 249株,占75.6%,以铜绿假单胞菌居多(20.7%);真菌764株,占13.6%,以白假丝酵母居多(12.6%);革兰阳性(G~+)菌608株,占10.8%,以金黄色葡萄球菌居多(9.9%)。药物敏感试验结果显示:G~-杆菌对亚胺培南、美洛培南耐药率最低,对青霉素类、喹诺酮类和部分三代头孢类等抗菌药耐药率较高(50.0%)。G~+球菌对万古霉素、利奈唑胺耐药率为零,对青霉素类、喹诺酮类和红霉素等抗菌药耐药率较高(40.0%)。结论下呼吸道感染患者病原菌以G~-杆菌为主,耐药性呈增长趋势,临床应加大病原菌分布检测及其耐药性监测力度,及时调整抗菌药物用药。     

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.     

Infecc Ón Humana PorNocardia Asteroides,En México

Summary Although the existence ofNocardia asteroides as a human pathogen in Mexico has been known for several years, up to now there are only few reports in our country concerning the isolation of this microorganism both as a saprophyte as well as human or animal pathogen. This fact is more interesting when compared with the high incidence ofN. brasiliensis, causal agent of actinomycotic mycetoma, the most frequently observed deep mycosis in Mexico.In this paper, the isolation of eigth strains ofN. asteroides from clinical specimens is reported. Five strains were isolated from sputa, one from a fistulous lesion of the scrotum, one from a brain abscess and one more from a foot mycetoma.One of the strains isolated from sputum was in a case of pulmonary nocardiasis; another of these strains was isolated from a patient with coexistent pulmonary tuberculosis and nocardiasis. With the last three strains isolated from sputa, it was not possible to establish any correlation between the isolations and the clinical and radiological findings of the patients; two of the patients had pulmonary tuberculosis and the isolation ofN. asteroides was achieved only in one occasion in each case, with scant growth, and observed in routine cultures made for tubercle bacilli. The last strain was isolated from the sputum of a patient with chronic bronchitis. Some comments are made to the possibility that these three last isolations merely represent a saprophytic phase of the fungus.Some clinical and other features are discussed concerning the other three cases of scrotal fistula, brain abscess and mycetoma produced byN. asteroides.     

The Prevalence in Canada of Drug-Resistant Tubercle Bacilli in Newly Discovered Untreated Patients with Tuberculosis

During the period February 1963 to September 1964, the incidence of strains of tubercle bacilli excreted by newly diagnosed, previously untreated patients with tuberculosis in Canada that were resistant to the major antituberculosis drugs, viz. streptomycin, para-aminosalicylic acid and isoniazid, was 4.8%. Resistance to a single drug (3.8%) was observed more commonly than was resistance to two drugs (0.9%) or to all three drugs (0.3%), and the drug most frequently involved in this regard was streptomycin (1.8%). Data for the province of Ontario were almost the same as those for Canada. These data are compared with results of surveys in other countries and it is concluded that while drug-resistant tubercle bacilli do not constitute a major problem in Canada at present, awareness of this potential hazard should be maintained.     

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).     

178例小儿肺炎分离菌分布和耐药性分析

目的 了解小儿肺炎病原菌谱及耐药性。方法 对2003～2004年178例小儿肺炎的痰标本进行细菌学培养,测定药物敏感性。结果 分离菌中,以革兰阴性菌居首位．占58．51％。肺炎克雷伯菌和大肠埃希菌为主琴芦种,对亚胺培南、美罗培南的耐药性最低;其次为革兰阳性菌,占40．43％,以金黄色葡萄球菌为主,耐甲氧西林的葡萄球菌为57．1％,对万古霉素、替考拉宁最敏感。结论 小儿肺炎病原菌以革兰阴性菌为主,并且出现多重耐药。临床上应避免盲目经验性用药,减少或减缓细菌耐药的发生。     

骨科患者伤口分泌物病原菌分布及耐药性分析

目的:了解我院骨科患者伤口分泌物病原菌分布及其耐药性情况,为临床上对骨科患者合理使用抗生素提供相关理论根据。方法:将2013年2月至2014年8月我院282例术后骨科患者的伤口分泌物标本接种培养,按要求分离纯菌,采用VITEK 2Compact全自动微生物分析仪进行鉴定及药敏试验。结果:282份标本中分离出致病菌186株(65.96%),其中革兰氏阴性球菌94株(50.54%),革兰氏阳性球菌83株(44.62%),真菌9株占4.83%。分离率排在前三位的致病菌分别为阴沟肠杆菌(19.35%),金黄色葡萄球菌(17.20%),表皮葡萄球菌(15.59%)。阴沟肠杆菌、大肠埃希菌、肺炎克雷伯菌对头孢唑林和氨苄西林的耐药率最高,其中阴沟肠杆菌对头孢唑林耐药率高达100%。但未发现主要革兰氏阴性球菌对亚胺培南耐药。革兰氏阳性球菌对青霉素的耐药率较高,但未发现革兰氏阳性球菌对万古霉素耐药。结论:术后骨科患者伤口优势菌种是阴沟肠杆菌,而且耐药性高;临床医生应根据病菌鉴定和药敏分析结果,对不同种类的病原菌使用不同的抗生素进行针对性治疗。     

Correlation between culture of Mycobacterium tuberculosis and antimycobacterial antibody in lumbar, ventricular and cisternal cerebrospinal fluids of patients with tuberculous meningitis.

In this study positive culture for M. tuberculosis were obtained, 20% in lumbar cerebrospinal fluid (CSF), 75% in ventricular CSF and 87.5% in cisternal CSFs of patients with tuberculous meningitis. Low culture positivity in lumbar CSF is due to the low density of circulating tubercle bacilli in lumbar CSF than in cisternal or ventricular CSFs. However antimycobacterial antibody in lumbar, cisternal and ventricular CSFs circulate in significant titres and are not statistically different from one another. Since specimens of CSF can not be obtained from cisternal or ventricular routes for the routine bacteriological investigations in patients with tuberculous meningitis, detection of antimycobacterial antibody of M. tuberculosis antigen 5 in lumbar CSF by an indirect ELISA may be considered as an aid for the diagnosis of tuberculous meningitis, particularly when repeated CSF cultures are negative for M. tuberculosis.     

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.     

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.     

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.