Diagnosis of Tuberculosis

Tuberculosis is an infectious, contagious, virulent and inoculable disease whose etiological agents are Mycobacterium tuberculosis. It is Robert Koch who described in 1882 the tubercle bacillus. This infection is common to humans, all species of domestic animals and some wildlife species. It is also a zoonosis.

Known since prehistoric times, tuberculosis has spread widely around the world. The first known precise description of the disease is that of the Hippocratic books. Tuberculosis was then called "Phthisis". However, tuberculosis is still not eradicated. On the contrary, it is nowadays ubiquitous (the industrialized countries are not spared) and is experiencing an upsurge engendered by demographic pressure, poverty and AIDS. Eight to ten million people in the world suffer from tuberculosis each year. The animal populations affected are numerous.

The clinical status of infected non-human primates might be altered, in spite of the fact that this is more likely to happen in the active form than in the latent form of tuberculosis. Weight loss may happen after the first month post-infection, as well as initial hyperthermia (three - eight weeks post-infection). Other clinical signs are scarcer but may include coughing or abdominal/pleural effusion. On a bloodwork panel, transient neutrophilia may occur within the first weeks. Erythrocyte sedimentation rate, fibrinogen, C-protein, and other inflammatory biomarkers may rise as well, while the albumin/alpha-2 ratio could diminish via plasmatic protein electrophoresis. Related to the clinical examination, imaging tools other than radiographs/computed tomographic-scan can help, such as endoscopy or ultrasound which could be used to envision granulomas or suspicious lymph nodes and to perform biopsies.

A dorsoventral chest radiograph can be used to detect thoracic irregularities. Lesions can be present anywhere in the lung and may differ in density, size, shape, and the manifestation of cavitations. Thoracic radiography may affirm pulmonary illness but cannot distinguish between tuberculosis and other pulmonary granulomatous or cavitary diseases such as echinococcosis, nocardiosis, aspergillosis. Therefore, chest radiographs might be utilized as an extra test method simply to screen for tuberculosis. Despite the fact that this extra test has a constrained sensitivity, it is valuable for the identification of non-human primates which have a negative tuberculin skin test (TST) response because of immunosuppression in an end-stage and fulminant sickness. Contrasted with other species, non-human primates rarely develop calcified lesions. Thus, chest radiographs can be hard to interpret and, in a perfect world are displayed to an accomplished radiologist for interpretation. There are no pathognomonic lesions, however enlargement of the bronchial lymph nodes might be an early albeit vague sign of pulmonary mycobacteriosis. Bigger tubercles or cavitations might be visualized radiographically. Radiographs of the abdomen may aid in identifying or confirming splenomegaly or mesenteric lymphadenopathy. Computed tomography scanning or magnetic resonance imaging can give comprehensive real-time imaging of disease progression and uncover small granulomas that might be overlooked despite regular radiographs.

The purpose of the intradermal reaction is to reveal a specific state of tuberculous hypersensitivity. Tuberculin reaction and pulmonary tuberculosis are different expressions of Type IV hypersensitivity (HS IV). The first corresponds to a localized expression and the second is a pathological reaction of the immune system.

Mechanism of the tuberculin reaction: The state of hypersensitivity results from the presence of specific antigens contained in the tubercle bacillus, in BCG or in other Mycobacterium tuberculosis (atypical especially) which have antigenic kinship. The tuberculin hypersensitivity reaction is delayed cell-mediated and peaks after 48-72 hours. Its presence means that there is an infection of the body by living Mycobacterium tuberculosis. If these Mycobacterium tuberculosis are pathogenic, there is an infection with tuberculosis.

Intradermal TST with bovine or weak tuberculins are carried out using an injection of 0.1mL (2.500 IU) of tuberculin. It is done with a syringe. The dosage is accurate and the penetration strictly intradermal. The injection is made proximal to the lateral canthus of the upper eyelid. In numerous laboratories, the in vivo diagnosis of tuberculosis depends on the intradermal TST using Mammalian Old Tuberculin (MOT) or purified protein derivates (PPD). In the United States, the only USDA approved tuberculin for non-human primates is MOT produced by Symbiotics, Inc. In large primates, intradermal injection of 0.1 ml of MOT using a 26 gauge needle in the palpebrae. In small primates, a reduced dose (0.05ml) can be used.

It is made in the hours following the 48-72nd hour (maximum reaction point). The allergic reaction is late, progressive and lasting. Inflammation results in (a) hot, red, circular or elliptical nodule(s). When the reaction is very strong, an oozing exudate surrounded by a hemorrhagic zone (it is a zone of necrosis) can be observed. Its drying leads to a pressure ulcer. Truncular lymphangitis and/or adenitis of pre-scapular ganglia may be associated.

Whole blood IGRAs ought to be believed to be as an alternate or as a complementary tool to TST. These tests evaluate the cellular-mediated immunologic response to mycobacteria. IGRAs perceive cellular reactivity to antigens by gauging interferon-gamma (IFN?) production by live lymphocytes found in a whole blood sample. IFN? is a crucial cytokine involved in the cell-mediated immunologic response towards mycobacteria. The assays can distinguish between M. avium and M. bovis infection by baring blood samples to bovine and avian PPD. Instead of PPDs, single antigens could also be utilized in the test to supply additional specificity. Recently, a whole blood stimulation assay has become commercially accessible as a PRIMAGAM test which is approved for usage in rhesus and cynomolgus macaques. The assays can distinguish between M. bovis and M. tuberculosis complex infection by incorporating bovine and avian PPD separately in the test. The amount of interferon created via stimulation of different antigens are often wont to be used to differentiate a reaction due to M. tuberculosis/bovis or atypical mycobacteria. It is a quantitative assay with respectable sensitivity and specificity compared with illness status determined by pathologic examination.

Recent results advise that the IFN? response to tuberculin antigen might not be dependable in cynomolgus macaques and that another cut-off ought to be used to read the analysis. PRIMAGRAM, when compared to TST, has a low sensitivity and high specificity. A combination of these two tests could increase the overall sensitivity and specificity. Whole blood with anticoagulant, ideally lithium-heparin, is required for testing. As the test depends on stimulation of primed T-lymphocytes, the first obligatory step is to ensure that blood cells remain viable for in vitro culture. Thus, samples should be available for testing not later than eight-ten hours post-sampling and ought to be kept at ambient temperature in the meantime. Particular care should be paid to ensure homogenous and full mixing of blood and anticoagulant at collection. IGRA is an in-vitro test, and so discards some effects of in vivo tuberculin injections. Over the past few years, comparable assays for humans (T-SPOT-tuberculosis) and oxen (Bovigam) gave dependable and enlightening results on the status of tuberculosis infection. There is scant data about the usage of Quantiferon Gold test in non-human primates. A comparison of this test with the TST showed that the Quantiferon Gold test would possibly be a highly sensitive tool for the identification of mycobacterial infection in rhesus macaques, but it was unimaginable to discover specific infection with M. tuberculosis with this test. However, three vital caveats ought to be known regarding IGRAs used in non-human primates.

First, there are many different interpretation guidelines, based on diverse calculation methods with varied cut-off thresholds. In humans, IGRAs are better-known to have a high variability between individuals. Thus, caps might be influenced by the species and as the individual knowledge. Experienced technicians are required for the completion of IGRAs, and raw data will be communicated and documented properly. It is essential to possess a balanced interpretation scheme. The test implies the use of a mitogen as a positive control. An aliquot of the sample is bared to this mitogen, which in turn provokes an undefined application and IFN? creation in lymphocytes. However, this mitogen is typically not provided by the test and ought to be selected by the laboratory. The level of undefined stimulation is species-dependent and must be gauged when an IGRA gets established. For instance, a human IGRA could be used for some species of non-human primates but includes PHA as a mitogen, which is regrettably known to be a frail stimulant in non-human primates. Lab technicians who perform IGRA must be mindful of mitogen/species distinctions. Unfortunately, however, the majority of non-human primate species lacks this information. It is thus suggested to utilize two or more diverse mitogens to test non-human primate species with scarcity of information about the most appropriate mitogen. The third caveat is the recognition of IFN? itself. IGRAs rely on ELISPOT or ELISA which are designed to uncover IFN? molecules for specific species.

Throughout the non-human primate taxonomy, IFN? shifts in configuration and structure, and there’s no broad ELISA that’s capable of distinguishing interferon of all species. For example, ELISA within PRIMAGAM is a “simian INF?” ELISA but is incapable of detecting IFN? in most New World monkeys. Hence, the parallel utilization of TST and PRIMAGAM is fitting for an ideal coverage in a diagnostic procedure. TST might impact IGRA results when performed a short time before blood is sampled. As observed in other species, a “booster effect” may happen with the previous tuberculin injection reinforcing the immune response acquired on consequent IGRA. Said to extend sensitivity of the test, questions may emerge on the impact on its specificity, as the “boost effect” may be made only by tuberculin sensitization. Therefore, the utilization of a booster method cannot be suggested at that stage. Further studies are required to reply to those questions.

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