Use of microscopic morphology in smears prepared from radiometric cultures for presumptive identification ofMycobacterium tuberculosis complex,Mycobacterium avium complex,Mycobacterium kansasii, andMycobacterium xenopi

The aim of this study was to assess the feasibility of a method for presumptive identification of mycobacteria, based on the morphology in smears prepared from radiometric Bactec-positive cultures (Becton Dickinson, USA) and to select the appropriate DNA probe (AccuProbe; Gen Probe, USA). The smear morphology of acid-fast bacilli was evaluated in 468 positive cultures from clinical samples: 313Mycobacterium tuberculosis complex, 67Mycobacterium avium complex, 32Mycobacterium kansasii, 49Mycobacterium xenopi, and sevenMycobacterium gordonae. The sensitivity and specificity for various morphological patterns were as follows: cord formation forMycobacterium tuberculosis complex 90% and 100%, respectively; striped bacilli forMycobacterium kansasii, 66% and 99%; sea urchin forMycobacterium xenopi, 96% and 99%; short bacilli forMycobacterium avium complex, 61% and 99%; fine-striped bacilli associated withMycobacterium avium complex from blood samples, 33% and 98%. This criterion was applied in the selection of a suitable DNA probe for the identification of 178 cultures. The correct probe was selected in 98%, 97%, and 72% of cultures, respectively, forMycobacterium tuberculosis complex,Mycobacterium avium complex, andMycobacterium kansasii. The observation of acid-fast bacilli morphology in radiometric cultures is a rapid and cost-efficient method for presumptive identification of common clinical isolates of mycobacteria.     

Real-time molecular epidemiology of tuberculosis by direct genotyping of smear-positive clinical specimens

We applied MIRU-VNTR (mycobacterial interspersed repetitive-unit-variable-number tandem-repeat typing) to directly analyze the bacilli present in 61 stain-positive specimens from tuberculosis patients. A complete MIRU type (24 loci) was obtained for all but one (no amplification in one locus) of the specimens (98.4%), and the allelic values fully correlated with those obtained from the corresponding cultures. Our study is the first to demonstrate that real-time genotyping of Mycobacterium tuberculosis can be achieved, fully transforming the way in which molecular epidemiology techniques can be integrated into control programs.     

Mycobacterium resembling Mycobacterium fortuitum that produces brown pigment.

Two cultures of acid-fast bacilli with characteristics most closely resembling those of Mycobacterium fortuitum were recovered as casual isolates from sputa of a patient with an apparent brochogenic tumor. One of the cultures was consistenly cream colored to rosy buff. The other, however, changed from buff to rust to dark brown and had the gross appearance of a fungus culture.     

Preliminary demonstration of human tuberculoimmunity in vitro.

In this paper a method for studying human tuberculoimmunity in vitro is described. Results from its use support an explanation for human tuberculoimmunity that is like that for mouse tuberculoimmunity: that immune lymphocytes are stimulated by being cultured with immunizing antigen to make a lymphokine which enables syngeneic macrophages to kill intracellular tubercle bacilli. This method uses antigen-responding lymphocytes and effector monocytes taken in the same original 20-ml sample of venous blood. These cells are cultured separately, the lymphocytes for 72 h with antigen to make immune lymphokine and the monocytes for 7 days to become macrophages. The macrophages are then infected with attenuated or virulent tubercle bacilli and cultured for 7 days more in medium with or without the immune lymphokine. Without it they are unable to control intracellular replication of the bacilli, whereas with it they do. This lymphokine was produced only by lymphocytes of immune subjects, of whom there were three kinds: tuberculin positive naturally immunized, Mycobacterium bovis BCG immunized, and trypsin-extracted bacillary antigen immunized. This method for detecting human tuberculoimmunity in vitro should be useful for comparing experimental vaccines and for studying cellular and molecular mechanisms of human tuberculoimmunity under better controlled conditions than hitherto have been possible.     

Determination of antibodies to pneumococcal C polysaccharide in patients with community-acquired pneumonia

Organized nerve cultures of dorsal root ganglia from neonatal mice were infected with Mycobacterium leprae, the causative agent of leprosy. A significant multiplication of the acid-fast bacilli was observed within the Schwann cell component of the culture. The growth of these bacilli was sensitive to antileprosy drugs and was not observed directly in bacteriological media. These organisms were brightly stained with the monoclonal antibody to phenolic glycolipid-I, a M. leprae-specific marker. The antigenic, pathogenic, and biochemical characteristics of this mycobacterium are under investigation.     

A novel enzyme-linked immunosorbent assay for diagnosis of Mycobacterium avium subsp. paratuberculosis infections (Johne's Disease) in cattle

Enzyme-linked immunosorbent assays (ELISAs) for the diagnosis of Johne's disease (JD), caused by Mycobacterium avium subsp. paratuberculosis, were developed using whole bacilli treated with formaldehyde (called WELISA) or surface antigens obtained by treatment of M. avium subsp. paratuberculosis bacilli with formaldehyde and then brief sonication (called SELISA). ELISA plates were coated with either whole bacilli or sonicated antigens and tested for reactivity against serum obtained from JD-positive and JD-negative cattle or from calves experimentally inoculated with M. avium subsp. paratuberculosis, Mycobacterium avium subsp. avium, or Mycobacterium bovis. Because the initial results obtained from the WELISA and SELISA were similar, most of the subsequent experiments reported herein were performed using the SELISA method. To optimize the SELISA test, various concentrations (3.7 to 37%) of formaldehyde and intervals of sonication (2 to 300 s) were tested. With an increase in formaldehyde concentration and a decreased interval of sonication, there was a concomitant decrease in nonspecific binding by the SELISA. SELISAs prepared by treating M. avium subsp. paratuberculosis with 37% formaldehyde and then a 2-s burst of sonication produced the greatest difference (7x) between M. avium subsp. paratuberculosis-negative and M. avium subsp. paratuberculosis-positive serum samples. The diagnostic sensitivity and specificity for JD by the SELISA were greater than 95%. The SELISA showed subspecies-specific detection of M. avium subsp. paratuberculosis infections in calves experimentally inoculated with M. avium subsp. paratuberculosis or other mycobacteria. Based on diagnostic sensitivity and specificity, the SELISA appears superior to the commercial ELISAs routinely used for the diagnosis of JD.     

Localization of Mycobacterium avium-intracellulare within a skin lesion of bacillary angiomatosis in a patient with AIDS.

We report a 39-year-old man who had AIDS and who presented with an unusual cutaneous vascular lesion, which was clinically thought to be Kaposi's sarcoma. Histologically, the lesion was characterized by capillary proliferation and a mixed inflammatory infiltrate that included numerous histiocytes. The lesion was found to contain slender intracellular acid-fast bacilli, as well as plump extracellular Warthin-Starry-positive bacilli. The acid-fast bacilli were confirmed to be Mycobacterium avium-intracellulare by subsequent positive blood cultures for this organism. To further investigate the lesion, polymerase chain reaction DNA amplification and sequencing was performed, and the lesion was found to contain DNA sequences identical to those previously established for the agent of bacillary angiomatosis. The lesion is thought to represent a lesion of bacillary angiomatosis with secondary involvement by M. avium-intracellulare.     

An in vitro model for sequential study of shiftdown of Mycobacterium tuberculosis through two stages of nonreplicating persistence.

It was demonstrated previously that abrupt transfer of vigorously aerated cultures of Mycobacterium tuberculosis to anaerobic conditions resulted in their rapid death, but gradual depletion of available O2 permitted expression of increased tolerance to anaerobiosis. Those studies used a model based on adaptation of unagitated bacilli as they settled through a self-generated O2 gradient, but the model did not permit examination of homogeneous populations of bacilli during discrete stages in that adaptation. The present report describes a model based on culture of tubercle bacilli in deep liquid medium with very gentle stirring that keeps them in uniform dispersion while controlling the rate at which O2 is depleted. In this model, at least two stages of nonreplicating persistence were seen. The shift into first stage, designated NRP stage 1, occurred abruptly at a point when the declining dissolved O2 level approached 1% saturation. This microaerophilic stage was characterized by a slow rate of increase in turbidity without a corresponding increase in numbers of CFU or synthesis of DNA. However, a high rate of production of glycine dehydrogenase was initiated and sustained while the bacilli were in this state, and a steady ATP concentration was maintained. When the dissolved O2 content of the culture dropped below about 0.06% saturation, the bacilli shifted down abruptly to an anaerobic stage, designated NRP stage 2, in which no further increase in turbidity was seen and the concentration of glycine dehydrogenase declined markedly. The ability of bacilli in NRP stage 2 to survive anaerobically was dependent in part on having spent sufficient transit time in NRP stage 1. The effects of four antimicrobial agents on the bacilli depended on which of the different physiologic stages the bacilli occupied at a given time and reflected the recognized modes of action of these agents. It is suggested that the ability to shift down into one or both of the two nonreplicating stages, corresponding to microaerophilic and anaerobic persistence, is responsible for the ability of tubercle bacilli to lie dormant in the host for long periods of time, with the capacity to revive and activate disease at a later time. The model described here holds promise as a tool to help clarify events at the molecular level that permit the bacilli to persist under adverse conditions and to resume growth when conditions become favorable. The culture model presented here is also useful for screening drugs for the ability to kill tubercle bacilli in their different stages of nonreplicating persistence.     

Glyoxylate metabolism and adaptation of Mycobacterium tuberculosis to survival under anaerobic conditions.

Tuberculosis is characterized by periods in which the disease may be quiescent or even clinically inapparent, but in which tubercle bacilli persist and retain the potential to reactivate the disease. The present study was carried out in pursuit of an in vitro model which might contribute to the understanding of the physiology of nonreplicating persisters, with oxygen limitation used as the means of inducing this state. When actively growing aerated cultures of Mycobacterium tuberculosis were suddenly placed under anaerobic conditions the bacilli died rapidly, with a half-life of 10 h. When the bacilli were grown in liquid medium without agitation, they adapted to the microaerophilic conditions encountered in the sediment; the adapted bacilli in the sediment did not replicate there but were tolerant of anaerobiosis, exhibiting a half-life of 116 h. Among the early events associated with the adaptation were the synthesis of an antigen designated URB, the function of which is not known, and a fourfold increase in isocitrate lyase activity. The bacilli later exhibited a 10-fold increase in synthesis of a glycine dehydrogenase that catalyzes the reductive amination of glyoxylate, concomitantly oxidizing NADH to NAD. Specific activities of other enzymes studied were either not affected or moderately diminished in the sedimented bacilli. It is proposed that the glyoxylate synthesis in this model serves mainly to provide a substrate for the regeneration of NAD that may be required for the orderly completion of the final cycle of bacillary replication before oxygen limitation stops growth completely. This orderly shutdown is essential to continued survival of M. tuberculosis in a quiescent form.     

Negative images of bacilli and mycobacterial infection: a study of fine-needle aspiration smears from lymph nodes in patients with AIDS

In patients with the acquired immunodeficiency syndrome (AIDS), four cases of mycobacterial infection in which bacilli appeared as cylindrical, nonstaining "negative images" have been previously described. These may have been extracellular or within histiocyte cytoplasm, and they have been described in aspirations from liver, lymph node, and bone marrow and in bronchoalveolar lavage fluid. We report three additional examples of this finding in fine-needle aspirations from lymph nodes in AIDS patients infected with Mycobacterium avium-intracellulare. Our findings support the concept that these negative images of bacilli are diagnostic of mycobacterial infection. Air-dried Romanovsky-stained material is required for their identification.     

Relationship Between the Staining Quality of Mycobacterium leprae and Infectivity for Mice

The proposal has been made that only solidly staining forms of Mycobacterium leprae are viable. On the basis of a previous study, solidly staining bacilli were defined as those that stained completely and darkly throughout their length. A study was carried out to correlate the proportion of solidly staining bacilli in inocula with the infectivity and rate of appearance of bacillary growth in inoculated mice. The inocula originated in skin biopsy specimens of patients and in mouse passage material; there were 347 inocula suitable for study. The rate of appearance of bacillary growth in inoculated mice confirmed the hypothesis that nonsolid bacilli are inert and nonviable and that all growth originates from the solidly staining organisms. The minimal infectious dose was determined in four suspensions and was found to range from 40 to 3 solidly staining bacilli.     

Dormancy ofMycobacterium tuberculosis and latency of disease

There is ample circumstantial evidence from observation of the natural history of tuberculosis in humans and experimental animals thatMycobacterium tuberculosis is capable of adapting to prolonged periods of dormancy in tissues, and that these dormant bacilli are responsible for latency of the disease itself. Furthermore, the dormant bacilli are resistant to killing by antimycobacterial agents. A systematic evaluation of the mechanism of dormancy, and of attempts to abrogate latency will require a better understanding of the physiologic events that attend the shiftdown into dormancy. There are probably two or more stages in the shiftdown ofMycobacterium tuberculosis from active replication to dormancy as bacilli in unagitated cultures settle through a self-generated O₂ gradient into a sediment where O₂ is severely limited. One step involves a shift from rapid to slow replication. The other involves complete shutdown of replication, but not death. Presumably this last step includes completion of a round of DNA synthesis. The shiftup on resumption of aeration includes at least three discrete sequential steps, the production of RNA, the ensuing synchronized cell division and, finally, the initiation of a new round of synthesis of DNA. Three markers of the process of shiftdown ofMycobacterium tuberculosis to dormancy have been described, namely the changes in tolerance to anaerobiosis, the production of a unique antigen and the ten-fold increase in glycine dehydrogenase production. Additional markers represented in the shiftup and shiftdown process may yet be discovered, and determination of their specific functions should provide insights into the mechanisms of dormancy and latency in tuberculosis, and into strategies for preventing reactivation of the bacilli and development of disease.     

Macrophage Accumulation, Division, Maturation and Digestive and Microbicidal Capacities in Tuberculous Lesions: I. Studies Involving their Incorporation of Tritiated Thymidine and their Content of Lysosomal Enzymes and Bacilli

Dermal and pulmonary tuberculous lesions were produced in rabbits with BCG, biopsied, incubated in vitro with tritiated thymidine (³HT) under hyperbaric oxygen, quickly frozen, sectioned in a cryostat, stained for the lysosomal enzyme β-galactosidase, autoradiographed, stained for acid-fast bacilli and counterstained with hematoxylin. As macrophages developed into epithelioid cells, they could still divide—ie, incorporate ³HT. However, once they became fully mature epithelioid cells that were 4-plus in β-galactosidase, they could not do so. Tuberclebacilli did not stimulate macrophage division. On the contrary, macrophages containing bacilli did not divide, except when the lesions began. During the development of tuberculous lesions, macrophages (including those rich in enzymes and those containing bacilli) died, forming caseous centers. Therefore, local cell division did not seem to be the main mechanism by which macrophages reduced their bacillary load. Such division seemed mainly to occur in young macrophages that had recently immigrated into the lesions from the bloodstream and had not yet ingested bacilli.     

Effect of egg yolk on growth of Mycobacterium tuberculosis in 7H12 liquid medium.

Of 92 drug-resistant Mycobacterium tuberculosis strains isolated from sputum specimens, 86 showed growth in two types of 7H12 broth, one with egg yolk and the other without egg yolk. In addition, two strains grew only in plain 7H12 broth without yolk, and four others were recovered only in the medium supplemented with egg yolk. The radiometrically detected growth was higher in the presence of egg yolk, corresponding to a higher number of CFU per milliliter in these cultures. The improvement of growth in 7H12 broth supplemented with egg yolk was most noticeable in cultures isolated from sputum specimens having a low number of acid-fast bacilli in the smear and producing only a few colonies on solid media.     

Tuberculous lymphadenitis associated with human immunodeficiency virus (HIV) in Uganda.

Sixteen adults presented with lymphadenopathy which was tuberculous on biopsy; they were all seropositive for human immunodeficiency virus (HIV-1), but none had the clinical criteria of the acquired immunodeficiency syndrome (AIDS). The biopsy specimen showed caseating tuberculosis, with scanty or no visible acid fast bacilli in seven cases; the remaining nine had a poor cellular reactivity with numerous bacilli. Antituberculous chemotherapy for two months reduced the lymphadenopathy. Two patients subsequently developed AIDS. Mycobacterial cultures were not performed, but the infection was almost certainly Mycobacterium tuberculosis. The space-time clustering of tuberculous lymphadenitis now seen in Kampala, and the unusual non-reactive histopathology, are typical of the impairment of cellular immunity induced by HIV infection.     

Pulmonary Bovine-Type Tuberculosis in Rabbits: Bacillary Virulence, Inhaled Dose Effects, Tuberculin Sensitivity, and Mycobacterium vaccae Immunotherapy

This report elucidates four aspects of the immunology of pulmonary tuberculosis produced in rabbits: (i) the virulence of bovine-type tubercle bacilli, strain Ravenel S, (ii) systemic factors influencing the generation of visible primary pulmonary tubercles, (iii) differences in tuberculin sensitivity of rabbits and humans, and (iv) the effect of Mycobacterium vaccae immunotherapy on cavitary tuberculosis. Laboratory strain Ravenel S (ATCC 35720) was not fully virulent. Fully virulent strains produce one visible primary pulmonary tubercle for each three bacillary units inhaled. Strain ATCC 35720 produced one such tubercle for each 18 to 107 bacillary units inhaled, indicating that its virulence was reduced by 6- to 36-fold. When a low dose of this Ravenel S strain was inhaled, the host resistance (measured by the number of inhaled bacilli needed to generate one visible primary pulmonary tubercle) was increased at least 3.5-fold compared to the host resistance when a high dose was inhaled. Rabbits and humans differ in the degree and in the maintenance of their dermal sensitivities to tuberculin. Compared to rabbits, humans are 100 times more sensitive to tuberculin. Also, at 33 weeks rabbits with well-controlled cavitary tuberculosis usually showed a decrease in their tuberculin reactions of about 50% from peak values, whereas humans with such well-controlled tuberculosis are thought to maintain strong reactions for many years. These species differences may be due to desensitization to group II mycobacterial antigens in the rabbits because they have a different diet and a different type of digestive tract. M. vaccae immunotherapy of rabbits with cavitary tuberculosis produced no statistically significant effects. Experiments with many more rabbits would be required to prove whether or not such immunotherapy is beneficial.     

Histochemical studies relating the activation of macrophages to the intracellular destruction of tubercle bacilli.

Dermal tuberculous lesions, both primary and those of reinfection, were produced in rabbits with 14C-labeled BCG and biopsied once at various times. Macrophage activation was evaluated by the indolyl histochemical test for beta-galatosidase, the number of bacilli in macrophages by acid-fast staining, and the breakdown of bacilli by autoradiography. After the rabbits became tuberculin positive, the stongly activated macrophage population contained a) fewer parasitized cell, b) fewer bacilli in each parasitized cell, and c) more "free" 14C-label (not associated with intact bacilli) than the weakly activated macrophage population. These results suggest that the more highly activated macrophages had destroyed many of the bacilli that they once contained and that their power to do so was enhanced by immunologic mechanisms.     

Simple and Rapid Identification of the Mycobacterium tuberculosis Complex by Immunochromatographic Assay Using Anti-MPB64 Monoclonal Antibodies

A newly developed immunochromatographic assay (MPB64-ICA) for identification of the Mycobacterium tuberculosis complex was evaluated with 20 reference strains of mycobacterial species and 111 clinical isolates. MPB64-ICA displayed a very strong reaction band with organisms belonging to the M. tuberculosis complex but not with mycobacteria other than M. tuberculosis (MOTT bacilli), except for one of four M. marinum strains tested and one M. flavescens strain, both of which gave very weak signals. The effectiveness of MPB64-ICA in combination with two liquid culture systems (MB-REDOX and MGIT) was tested. A total of 108 of 362 sputum specimens processed were positive for acid-fast bacilli. Samples taken from the cultures on the same days when either of the two culture systems became positive for mycobacteria were assayed with MPB64-ICA. Of 108 cultures with mycobacteria, 51 showed a positive signal with the test, in which the presence of the M. tuberculosis complex was demonstrated later by the Accuprobe for M. tuberculosis complex. In addition, MPB64-ICA could correctly detect the M. tuberculosis complex in mixed cultures of the M. tuberculosis complex and MOTT bacilli. These results indicate that MPB64-ICA can be easily used for rapid identification of the M. tuberculosis complex in combination with culture systems based on liquid media without any technical complexity in clinical laboratories.