Experimental tuberculosis infection of guinea pigs in the necropsy room and examination of infection risk using guinea pigs with tuberculosis

Three hundred thirty-six guinea pigs of the Hartley strain were infected with tubercle bacilli isolated from 7 patients in South India, all of whom had moderate to far advanced pulmonary tuberculosis. In addition, one control group of 24 animals was injected with H37Rv and another control group of 24 animals was injected with saline. All inocula used for infection contained more than 105 viable units, and all infected animals acquired a pattern of skin sensitivity to six mycobacterial antigens typical for tuberculous infection. Mastitis, orchitis, or both, and skin lesions developed in many infected animals. The possibility that these were due to a contaminant was evaluated and rejected. Based on the mortality curves, the tubercle bacilli isolated from 2 patients were virulent whereas the bacilli isolated from 5 patients were less virulent to the guinea pig. Despite homogeneity of the animals with respect to age, sex, and genetic background, individual susceptibility to tuberculosis varied appreciably. Th...

Resistance to infections with Mycobacterium tuberculosis has been studied in a number of experimental animals, but recently the suitability of mice for immunity tests has been emphasized. Stamatin and Stamatin (1) concluded that mice were less susceptible than guinea pigs. The use of mice for studies of resistance to tuberculosis was given great impetus by Dubos and associates (2a-2i) , who showed that, by controlling the size of the challenge dose of virulent bacilli injected intravenously, differences in reactions of nonvaccinated mice could be detected. Lethal infections were avoided because differences between the two groups of animals were difficult to detect. In tests to evaluate the immunogenicity of various strains of BCG, the number of organisms in selected tissues was determined because proliferation of the immunizing agent was necessary to produce protection. The degree of resistance was measured by: the comparative numbers of virulent organisms in lungs and spleens; the number of tuberculous lesions in the lungs; and the number of acid-fast organisms in each nodule of control and immunized mice. The application of these methods has been described by various writers (3-7). Crowle (8) has shown that the relative density of the lungs of vaccinated and control mice challenged with virulent tubercle bacilli may be used as an index of immunity. The survival time of control versus vaccinated mice after intravenous challenge with large numbers of virulent bacilli has also been used as a measure of resistance (9-11). Glover (12) showed that mice were susceptible to infection by aerosols of virulent bovine tubercle bacilli and that approximately 100 organisms were an infectious dose and 120,000 a lethal

Studies of the effects of serum from various mammals upon the tubercle bacillus have generally been limited in the time of contact between the bacillus and the investigated serum, and in the use of diluting media which can neutralize tuberculostasis. It was shown in previous reports that the latter limitation is mainly responsible for failures to demonstrate a strong antimycobacterial activity in immune guinea pig and normal human serum (1, 2)when these sera were diluted in various citrateor phosphate-containing media, tuberculostasis was vitiated. The use of different media for the dilution of investigated serum is the principal cause of the contradictory results reported in the literature. Thus, Kirchner (3) and Boissevain (4) diluted human, guinea pig, sheep, rabbit, and horse serum in synthetic media and concluded that these sera stimulated the growth of human tubercle bacilli, and that such serummedium mixtures could serve as an excellent medium for the growth of these organisms. Raffel (5), in discussing immunity in tuberculosis, indicated that there is no specific evidence in the literature to suggest that the serum of normal animals is tuberculostatic-virulent as well as avirulent bacilli grow very well in vitro in normal serum as a medium. Contrary to these findings, Hedvall (6) reported that serum specimens from man, horse, cow, dog, cat, rabbit, and guinea pig act as inhibitors of bacillary growth. In the present work, the effects of various mammalian serum samples upon the growth of tubercle bacilli were tested and an investigation made of ways of purification of the tuberculostatic factor which is present in most of the tested serum.

The recognition of tuberculosis or tuberculosis1ike disease in a wide variety of animals preceded the discovery of the tubercle bacillus by many years. Soon after the discovery of the tubercle bacillus it became evident that tuberculosis in various species was caused by organisms closely related to human tubercle bacilli but differing from them in specific and consistent ways. The existence of acid-fast bacilli producing pulmonary disease in humans, but differing from 111ycobacterium tuberculosis in cultural characteristics and in failure to produce progressive' disease in the guinea pig, has been reported sporadically for many years, but with increasing frequency recently (1-13). These bacilli differ from certain other acid-fast organisms such as 111. ulcerans and M, balnei (which have been reported to produce cutaneous lesions in humans occasionally), and also from 111. tuberculosis, in their ability to grow at room temperature (13). They differ from M, fortuitum, another lesionproducing Mycobacterium which also grows at room temperature, as they differ from nonpathogenic saprophytic acid-fact bacilli in that they require at least six to twenty-one days to grow out on culture while the latter two grow in three days or less (13, 14). Three different types of atypical mycobacteria have been described and have been classified according to pigment production when exposed to light (15).

Approximately one third of the global population is latently infected with tuberculosis (TB) and there are approximately 9.6 million new cases of TB disease per year resulting in 1.5 million deaths. Eleven percent of cases globally occur in children and 81% of the burden of TB disease is borne by the developing world and countries with emerging economies (BRICS). The African region accounts for 28% of new TB cases globally. TB remains a significant public health concern globally, particularly amongst children and immunocompromised individuals. Diagnosis of childhood TB is an on going challenge, as children usually do not present with the same signs and symptoms as adults, and are often misdiagnosed. Tuberculosis infection in children is seldom confirmed through sputum culture, as good sputum samples can rarely be collected. Only 15% of cases from children are sputum smear positive by acid-fast staining, and only 30%–40% are Mycobacterium tuberculosis (MTB) culture positive. Up to 40% of children present with extrapulmonary manifestations of TB disease. The most common manifestation is tuberculous lymphadenopathy. Good specimens for TB detection can be obtained from these cases through fine needle aspiration biopsy (FNAB), a cost-effective and practical out patient procedure for obtaining specimens from enlarged superficial lymph nodes. The conventional laboratory techniques that have been used globally for TB diagnosis are Ziehl-Neelsen (ZN) staining for acid fast bacilli (AFB) microscopy, culture and more commonly now LED microscopy, cytological determination with auto-fluorescence staining and molecular Xpert® MTB/RIF (Cepheid) detection. AFB-microscopy requires minimally 5000 AFB per millilitre of specimen to yield a consistently positive result and observation of between 100 and 300 microscopic fields in order to obtain accurate results. Culture for MTB is the gold standard diagnostic method but has a slow turnaround time and requires laboratory resources that are not available in most parts of the world. Recent systematic reviews of studies evaluating commercially available nucleic acid amplification test (NAAT) technologies confirm very high specificity, with sensitivity approaching, but not reaching, that of culture. The complexity and insufficient robustness of existing commercial NAAT protocols and their need for precision instruments, a high degree of technical support, and quality assurance make them unsuitable for most low resource TB endemic countries. In addition, none of these techniques have been fully validated for diagnosing TB in children and specifically not for extrapulmonary specimens. In light of these challenges, there is promise in two technologies that have been developed and under evaluation over the last few years; the Xpert® MTB/RIF kit (Cepheid) for rapid detection of MTB and rifampicin resistance endorsed for use by the WHO; and the Ustar EasyNATTM TB IAD kit (Ustar Biotechnologies, Hangzhou) for detection of MTB, selected by the WHO as a technology on assessment for use in TB endemic countries. In this study, their performance was assessed against the conventional laboratory diagnostic techniques of smear AFB microscopy, cytology and mycobacterial culture of fine needle aspirates from lymph nodes of children suspected of TB lymphadenopathy in Tanzania. Age defined clinical assessments were done for all 75 participants and TB treatment initiated based on these and/or laboratory diagnostic outputs. All laboratory diagnostic modalities were primarily assessed against TB culture as the conventional reference standard. As has been evidenced in earlier studies, the sensitivities for both smear microscopy and TB culture were very low in these extrapulmonary specimens. Lacking a true reference standard, composite reference standards (CRSs) were created to assess the performance of the test modalities under study. An alternative method for assessing diagnostic accuracy under these conditions is latent class analyses (LCA), which was utilized to further assess the performance of all diagnostic modalities in the study. The overall outcomes of the project demonstrated that cytomorphology was a feasible and effective technique for detection of TB in lymph node aspirates (sensitivity: LCA 100%, specificity: LCA 94.7%) that may complement TB culture (sensitivity: LCA 74.5%, specificity: LCA 90.3%). Further, it was shown that Xpert (sensitivity: CRS 58% LCA 70.7%, specificity: CRS 93% LCA 94.2%) was superior in performance to EasyNAT (sensitivity: CRS 19% LCA 29.2%, specificity: CRS 100% LCA 100%) and ZN (sensitivity: CRS 14% LCA 19.1%, specificity: CRS 100% LCA 100%) analyses, respectively. Combining two or more tests significantly improved the diagnostic efficacy, but including either EasyNAT testing or ZN microscopy to a diagnostic algorithm that already had Xpert testing added no value. These findings indicate that combining clinical assessment, cytology and Xpert MTB/RIF can provide for rapid and accurate diagnosis of childhood tuberculous lymphadenitis. Larger diagnostic evaluation studies on Xpert MTB/RIF would be required, to assess its use as a solitary initial test for tuberculous lymphadenitis in children.

Macrophages derived from cortisone-treated or untreated guinea pigs were infected with virulent tubercle bacilli and maintained in tissue culture. The fate of intracellular bacilli was determined. It was shown that the rates of bacillary multiplication within these two populations of macrophages were practically identical. Cytologic studies, however, revealed that the intracellular infection was toxic to the host cells derived from cortisone-treated animals. Serum from EGG-vaccinated animals was shown to protect the infected cells from the cortisone-treated animals against toxic injury, but its presence in culture medium did not alter the rate of intracellular bacillary multiplica­ tion. Hence, the increased susceptibility of cortisone-treated animals to tuberculous in­ fection is not likely to be caused by an altered rate of intracellular multiplication of tubercle bacilli but by the enhanced susceptibility of the host cells to toxic injury imposed by the intracellular multiplication of the pathogen.

Although the clinical effects of successful tuberculosis chemotherapy are obviously beneficial and dramatic, the anatomic and histologic changes are inconspicuous and subtle. They consist, for the most part, in the absence of those lesions and reactions found in patients dying of pulmonary tuberculosis. It is the purpose of this report to compare the findings in tissue surgically resected for pulmonary tuberculosis in a group of patients who did receive chemotherapy with a group of patients who did not receive chemotherapy. Feldman and Hinshaw (1) first reported the lack of lesions or the presence of a few abortive tubercles in guinea pigs treated with streptomycin after being injected with virulent tubercle bacilli. Mahon (2) stated that with streptomycin therapy, miliary tubercles healed more rapidly, cavities contained unusually vascular granulation tissue, and bronchi were surprisingly free of tubercles; however he also noted that fundamental tissue reactions were not altered by the treatment. Koyama (3) reported that chemotherapy prevented spread of the disease and prevented cavitation and empyema. Auerbach (4) believed that chemotherapy produced specific qualitative and quantitative differences in tissue reaction to infection, which included a rapid and extensive decrease in perifocal reaction, a decrease in the width of the capsule and thickness of cavity wall, a decrease in pulmonary fibrosis, emphysema, aneurysms and hemorrhage, and, frequently, an unusual mode of cavity healing (open healing). Laennec (5) had described open healing and clean cavities and, though not unknown since then, it was considered a rare event

Institutional Control Measures for Tuberculosis in the Era of Multiple Drug Resistance: ACCP/ATS Consensus Conference

We examined the incidence of tuberculosis among necropsy staff and the environment in the necropsy rooms at five medical institutions carrying out a large number of necropsy annually in the metropolis of Tokyo. The following results were obtained: 1) Incidence of tuberculosis was high among necropsy workers. 2) The method of wearing face masks was inadequate. 3) Tubercle bacilli were detected from the necropsy workers' aprons and the air-conditioning equipment of the necropsy room. 4) Formalin treatment of resected organs, especially the lungs, was inadequate. 5) The air conditioners in necropsy rooms were not effective. Considering these findings, improvement of the working conditions and environment of the necropsy rooms is needed.

A study on orange-pigmented non-photochromogenic mycobacteria.

Results of experiments on a gamma -irradiated vaccine (GIV) are reported. Cells irradiated with 500,000 or 1,000,000 r were injected subcutaneously in mice. The tests for reproductive viability by culture and guinea pig inoculations indicated that 500,000 r was sufficient to inactivate the tubercle bacilli. No visible growth was observed in culture media inoculated with cells exposed to 500,000 or cells showed no gross signs of infection. A long-term vaccination study showed that the vaccinated mice became segregated into two nonoverlapping groups. One group consisted of animals which developed little or no immunity and died after challenge within the period Qf time during which all of the nonvaccinated controls died (15 to 28 days); in the other group, the mice developed sufficient immunity to live 2 to 6 times longer (55 to 191 days). In terms of survival rates on day 35 after challenge, vaccination with BCG or GIV, with or without adjuvant, protected a significant portion of the vaccinated population, as compared with the nonvaccinated controls, with the exception of a group of male mice vaccinated with 0.1 mg. of GIV with adjuvant and challenged with 0.75 mg of virulent tubercle bacilli. GIV with adjuvant protected significantly greater proportionsmore » of the mice than did the vaccine without adjuvant. BCG, protected a significantly greater proportion of the anlmals than 0.1 or 0.4 mg of GIV, with or without adjuvant, but the protection afforded by a single 1.6-mg dose or four doses of 0.1 mg of GIV with adjuvant was statistically equivalent to that of BCG, Cobalt-60-irradiated M. tuberculosis H37Rv, although unable to grow on culture media and to produce disease in lab animals, respires on suitable substrates. In acid-fast stained films the irradiated cells cannot be distinguished, morphologically or tinctorially, from the nonirradiated cells. Such gamma -irradiated cells, produced from a virulent pathogen, have the advantage of a living attenuated vaccine, i.e,, ability to metabolize, without its disadvantages, i.e., genetic differences from the virulent organism (such as with BCG) and the possibility of producing disease in susceptible animals (not a major problem with BCG vaccine). The major disadvantage of the BCG vaccine is its instability, since exposure to temperatures above 4 deg C for a few days or to light for a few hours or even a few minutes may affect its potency. Whether the same factors will adversely affect the gamma -irradiated vaccine has yet to be explored. (BBB)« less

In mammals, the serum proteins and constituent fractions, separated by electrophoresis, are significantly altered by a wide variety of physiologic and pathologic factors. Among these, the effects of experimental infection with 111 ycobacterium tuberculosis have been studied in the rabbit (1), the guinea pig (2), the mouse (3), and the rat (4). Although the serum proteins of birds have been the subject of several investigations by free (5-7) and zone (8) electrophoresis, these studies have dealt primarily with factors other than pathologic states. Reports are available concerning the effects of vitamin E deficiency (9) and of erythroblastosis (10) on the electrophoretic distribution of serum proteins in the chick. Since dietary fat level has been reported (11) to influence resistance to experimental infection with M. tuberculosis in mice, the present investigation was designed to study the serum protein response in the chick to infection with avian tubercle bacilli; and to assess the possible effects of dietary fat level on this response. Data regarding mortality and tissue reactions to infection are the subjects of other reports (12); those presented here concern the serum protein and lipoprotein responses to infection studied by paper electrophoresis.

Index case is a 17-year-old boy who was admitted to our hospital with pleurisy and a minimal pulmonary lesion, and tubercle bacilli were recovered from pleural fluid. A diagnosis of primary tuberculosis was made based on the onset by pleurisy and the existence of hilar and mediastinal lymph node swelling. On the same day, a 76-year-old man, grandfather of the index case was admitted for precise examination of suspected extensive pneumonia. Tubercle bacilli were also isolated from the pus of infected bulla obtained by puncture. Neither of these two cases, however, seemed to be the source of the familial tuberculous infection because of such sudden onset of the disease as pleurisy and pneumonia. Two months later, a 46-year-old man, father of the index case was examined at our hospital. He was considered to be the source of the familial infection because he was diagnosed as tuberculosis with positive smear and a thick wall cavity (3.2 cm in diameter) on the left apex, and abnormal shadow was detected on his chest X-ray already two years ago. The fourth case was a mother of the index case, and wife of the third case, whose chest radiography revealed an infiltrative shadow on the right apex by a family contacts examination. Though tubercle bacilli were not isolated from her sputum, pulmonary lesions considered to be tuberculosis due to their typical location and nature, a positive PPD skin test, and the response to antituberculous drugs.

The relationship of atypical acid-fast bacteria to human disease; a preliminary report.

Human infection with M. tuberculosis val'. bovis in the United States is no longer an important problem. In other countries, however, this is not so. Urech (1) in 1957 reported 4.5 per cent bovine infections among 441 tuberculous patients in Germany. The major differences between the human and bovine strains are based on cultural characteristics and animal pathogenicity studies. Konno and associates (2-4) reported a biochemical method which would differentiate human tubercle bacilli from all other types of mycobacteria. This method is based on the fact that the human strains have a significantly greater niacin content than all of the other mycobacteria tested. Halpern and Grossowicz (5) studied the amidase activities of cell-free extracts from M. phlei and BeG, using various substrates, and reported a higher nicotinamidase activity in M. phlei than in BeG. Bonicke and Lisboa (6) compared the nicotinamidase activity of bacterial emulsions of human and bovine tubercle bacilli and reported no nicotinamidase activity in the bovine variety. The purpose of this report is to distinguish bovine tubercle bacilli from other mycobacteria (human and avian tubercle bacilli, unclassified mycobacteria, and nonpathogenic acid-fast bacilli) by determining the nicotinamidase activity of the cell-free extracts.