Mycobacterium tuberculosis complex (MTC)

Epidemiology :

prevalence of infection : 1.7 billion people (dropped by > 20% since 1990 to 2005^ref), 12 million are coinfected with HIV-1 (66% live in Sub-Saharan Africa). Middle East accounted for 7% of all incident cases of tuberculosis globally as of 2002^ref

China : a 3-month-long survey covering nearly 400,000 school children has found that nearly 9% of students under the age of 14 in primary and middle schools in Harbin tested highly positive for tuberculosis (TB) at a tuberculin forearm skin test. The X-rays revealed > 100 children have already begun to show symptoms of TB. Chinese babies are usually given the BCG vaccine for the prevention of TB within 24 hours of birth. Those who tested negative have been advised to get vaccinated as soon as possible, albeit on a voluntary basis
Nepal : 60% of Nepal's adult human population is infected with Mycobacterium tuberculosis; > 80 000 have active TB infections; 44 000 people develop active disease every year, of whom 20 000 have active infectious TB; 6000-8000 people die from TB annually male-to-female ratio of registered TB patients is 2:1^ref
Italy : 12-13%
USA : foreign-born persons account for > 50% the cases of tuberculosis in the USA reported to the CDC, a rate that is 9 times that among those born in the USA^ref. In Massachusetts, 80% of the cases reported in 2003 occurred in foreign-born persons, most of whom had resided in the USA for 1-4 years^ref.
Botswana : 60% of TB patients are infected with HIV. Resistance to at least one drug in new patients rose from 3.7% in 1995 to 10.4% in 2002. Interventions for tuberculosis control are urgently needed in Botswana to prevent further emergence of drug resistance^ref.

incidence of infection : 8 million new cases / yr (12÷33%); reemerging in HIV-1 ⁺ individuals => chemoprophylaxis when [CD4⁺]_blood < 200 / mL ; people who are infected but have not developed disease are not infectious; risk of developing disease following infections : < 10%. Half of these, about 5%, will have active disease in the first 2 years and the other half, sometime during the rest of their lives. If, however, the contacts are profoundly immunocompromised such as HIV-infected individuals, the risk of active TB is closer to 10% per year as opposed to 10% per lifetime.

China 485,000 new cases every year
Italy 20 new cases per year every 100,000 inhabitants

prevalence of disease : 20÷30 millions (60÷80% acquire infection within age 20 : 20% in westernized countries (in Italy 5÷6 cases every 100,000 inhabitants, expecially reactivations in elderlies), 80% in developing countries (Latin America, subsaharian Africa and Southern Asia)) ; a person with active disease infects up to 10÷15 individuals / yr. 75% of male Indian smokers who become ill with TB would not have done so if they had not smoked : in some parts of the world the main way smoking kills is not via cancer and heart disease, but by damaging the lung's defenses against chronic TB infection. Smokers are about 4 times as likely to become ill with TB as are non-smokers, and consequently 4 times as likely to die from the disease. If infected, children aged < 2 years are at high risk for severe tuberculosis (TB) disease (e.g., TB meningitis)^ref.
mortality : 1.6÷3 million / yr (5% of infected; 50% of disease cases;

Italy : 0.5%
Asia : > 1 million

India has more TB deaths than any other country. 400,000 are co-infected with HIV-1

Africa : 400,000

outbreaks : the largest TB investigation in Dutch history begins in Zeist on Mon 31 Jan 2005. A 25-year-old employee of a C1000 supermarket in was diagnosed with a very infectious form of TB in November 2004. In Dec 2004, a "1st ring" contact investigation^ref, using tuberculin skin testing (Mantoux) and/or chest x-ray, revealed that all family members and close contacts of the index case (n=12) tested positive for LTBI, and 1 active case of TB was diagnosed. A "2nd ring" contact investigation was then performed, where all current and former colleagues of the index patient were tested and 44/77 current employees and 14/26 former employees tested positive for LTBI by Mantoux test. 1 active case of TB was also diagnosed in this group. Because of these results, an investigation of a "3rd ring" of contacts, which included all people who had visited this supermarket between 1 Jan and 18 Nov 2004, was launched. Zeist City Council and the health authority GGD Midden-Nederland decided to examine every customer who shopped at the supermarket between 18 Nov 2004 and 1 Jan 2005 based on a telephone survey. Between 31 Jan and 4 Feb 2005, over 21 000 people in the city of Zeist in the Netherlands were tested for latent tuberculosis infection (LTBI) or active tuberculosis (TB) : 5% were expected to have been infected with the bacteria, but it could also be 10% : the infection rate is dependent on many factors. This includes how closely the infected supermarket employee came into contact with customers. 75% of the C1000 customers urged to report for an examination will undergo Mantoux tests. Mantoux tests were performed on 15,515 people and 6000 people aged > 60 were screened by chest x-ray taken in a mobile health clinic. In total, 350/14 128 people (2.5%) whose Mantoux tests were evaluated had a positive reaction (> 14 mm). Those 350 people with suspected infections, and an additional 58 people (patients with suspected pulmonary problems, abnormal chest x-ray, or patients with risk factors for TB in conjunction with a Mantoux 5-14mm) were invited for further diagnostic tests at the tuberculosis clinic of the Municipal Health Department in Utrecht during Feb 2005. To date, about 2/3 of the 408 patients with either positive Mantoux test or abnormal chest x-ray have been examined for TB, and 5 new cases have been diagnosed. 1 of these 5 patients has open TB, and contact tracing linked to this case has also been initiated. So far, no conclusion can be drawn about the extent to which there has been an increased transmission of tuberculosis to customers in this supermarket. Data collection is continuing, and descriptive analysis of the 408 possible cases of LTBI has begun. It would be useful to assess a similar cohort of individuals in the community who did not shop at this supermarket for comparison with the group of 408 additional shoppers. It is not stated in the posting whether the index case had laryngeal tuberculosis or not. Having a positive Mantoux skin test for TB, assuming that the test was performed and read appropriately, reflects previous exposure to TB, not necessarily active infection at that time. Individuals with a positive skin test (Lutwick LI: Tuberculin skin testing. In, Tuberculosis, A Clinical Handbook. Lutwick LI (ed), Chapam & Hall, London, UK, 1995) have an approximately 10% chance of developing active tuberculosis in their lifetimes, 5% in the 1st 2 years and 5% afterwards. The risk of reactivation can be as high as 10% per year in AIDS patients. It is not clear how an estimate of 5-10% skin test reactivity after this exposure for those shopping in the store is suggested, as infectivity correlates with degree of exposure. Family and close friends having a high degree of skin test reactivity (it is likely the case that none of them have been found to have overt infection) does not necessarily suggest a high degree of transmission to much more casual contacts in less confined areas

A person with recently diagnosed culture-confirmed, extensively drug-resistant pulmonary tuberculosis (XDR TB) traveled on the following 2 extended flights (more than 8 hours in duration) in May 2007:

12 May 2007 Atlanta, GA (ATL) Paris, France (CDG)/ Air France #385 / Delta #8517
24 May 2007 Prague, Czech Republic Montreal, Canada/ Czech Air #0104

A 2nd round of TB evaluation and testing should be done because a negative
TST or QFT-G result obtained less than 8 weeks after exposure may be
considered unreliable for excluding latent tuberculosis infection (LTBI).
Transmission :

inhalation of desiccated Pflüger droplets with Ø < 6 mm (1 droplet nucleus contains no more than 3 bacilli); Schrön-Much's granules (gram-positive, nonacid-fast granules and rods found in tuberculous sputum and thought to be modified tubercle bacilli)

human-to-elephant and elephant-to-human transmission of tuberculosis are known to occur^{ref1,
ref2}. These aerosols remain suspended in air for prolonged periods of time in closed, poorly ventilated settings, such as barns. It is in such a barn setting that zoonotic transmission of tuberculosis between handlers and infected elephants, confirmed by DNA fingerprinting, apparently occurred, as reported by Michalak et al 1998. Presumably, the risk of transmission would be small for persons with brief contacts with elephants in the outdoors, as would occur with tourists. Elephants in the wild are said to be free of tuberculosis. M. bovis has been isolated from an elephant in the USA^ref. Although the prevalence of M. bovis infection in people or other

M. bovis

M. tuberculos

M. bovis

M.bovis

^ref

ingestion of urine from patients affected by renal TB
through the conjunctiva or broken skin

Pathogenesis : the development of a strong T_h1-mediated adaptive immune response is considered of main importance for host defense against the intracellular pathogen Mycobacterium tuberculosis. The induction of a cellular immune response is not only dependent on the engagement of the TCR but also requires co-stimulation. In order to study the role of the co-stimulatory molecule of the tumor necrosis factor receptor family member CD27 during murine M. tuberculosis infection, we intranasally infected wild-type (WT) and CD27 knockout (KO) mice with 10⁵ colony-forming units M. tuberculosis. Whereas there were no differences in bacterial growth, inflammation and IFN-g production by CD4⁺ and CD8⁺ lymphocytes in the lungs early after infection, the number of splenic CD8⁺ T cells producing the key T_h1 cytokine IFN-g was lower in CD27 KO mice than in WT mice. After 6 weeks, CD27 KO mice had 3.6-fold higher mycobacterial counts in their lungs and displayed more pulmonary inflammation and increased numbers of infiltrated leukocytes. Despite these differences early in infection, an equal number of WT and CD27 KO mice died during a 43-week observation period and lung bacterial loads and inflammation were comparable in the surviving animals. CD27 does not contribute to the local IFN-g-mediated response and long-term protection against M. tuberculosis^ref. CD44^highCD62L^low effector CD4 T lymphocytes generated during the course of mycobacterial infection can be segregated into 2 subsets on the basis of CD27 receptor expression. Only the CD27^low subset exhibited a high capacity for IFN-g secretion, indicating that low CD27 expression is characteristic of fully differentiated effector CD4 T lymphocytes. CD27^low IFN-g-producing CD4 T lymphocytes accumulate in the lungs but are rare in LNs. Several factors contribute to their preferential accumulation. First, CD27^low CD4 T lymphocytes present in the LN are highly susceptible to apoptosis. Second, circulating CD27^low CD4 T cells do not enter the LN but efficiently migrate to the lungs. Third, CD27^high effector CD4 T cells that enter the lungs down-regulate CD27 expression in situ. In genetically heterogeneous mice that exhibit varying susceptibility to tuberculosis, the accumulation of mature CD27^low CD4 T cells in the lungs correlates with the degree of protection against infection. Thus, we propose that terminal maturation of effector CD4 T lymphocytes in the periphery provides the host with efficient local defense and avoids potentially harmful actions of inflammatory cytokines in lymphoid organs^ref.
=> tuberculosis (TB) / "White Plague" : the bacillus may colonize (primary tuberculosis) every organ (rare in exocrine glands and URT) except muscle tissues due to lactic acid sensitivity :

cutaneous tuberculosis / tuberculoderma
aerogenic or inhalation tuberculosis : tuberculosis caused by aspiration of the tubercle bacilli into the lungs => pulmonary tuberculosis (PTB) (68%)

diffusion by contiguity =>

tuberculosis of serous membranes : tuberculosis involving the pleura, peritoneum, pericardium, and cerebral meninges, producing inflammation of those structures.

tubercular pleurisy => pleural empyema
tubercular pericarditis

chronic laryngitis (tuberculous laryngitis / laryngeal tuberculosis / tuberculosis of larynx). It has been generally accepted that laryngeal TB is the most highly infectious form, but since most cases are associated with far advanced open cavitary pulmonary disease, the infectivity of laryngeal TB alone has not been clearly proven. In 2 patients with laryngeal TB without pulmonary disease, no evidence of intrafamilial spread was found, suggesting the laryngeal disease in itself may not be so infectious^ref. In a similar case isolated laryngeal TB was diagnosed, and despite a delay in making the diagnosis, no family and hospital spread was found
tubercular pharyngitis (pharyngeal tuberculosis)

disseminated tuberculosis : hematogenous (hematogenous tuberculosis) or lymphohematogenous spread of tubercle bacilli from a primary focus of infection; its incidence is increased among immunocompromised patients (< 20% in immunocompetent, 60-70% in immunocompromised) => extrapulmonary tuberculosis

disseminated or miliary tuberculosis : a type that varies from a chronic, slowly progressive debilitating infection to an acute fulminating disease; it is caused by hematogenous or lymphohematogenous dissemination of infected caseous material into the bloodstream and seeding of many organs (including tuberculosis miliaris disseminata ) with millet seed–like tubercles (Ø = 1÷2 mm). Complications : fibrothorax , bronchiectasia (=> increased bronchial secretions => Pseudomonas spp. and Aspergillus spp. infections), recurrent hemophtoe, coma vigil / "typhoid status".

superficial lymph nodes tuberculosis (painless lymphadenitis ; when laterocervical and/or submandibular lymph nodes are involved, they appear adherent to derma and create a sow-like look (scrofulosis / scrofula) evolving to draining fistulae)
cerebral tuberculosis / tubercular meningitis (expecially in babies and immunodepressed) : even in resource-rich countries, where there is easy access to paediatric subspecialty care, infants with tuberculous meningitis continue to have high mortality and morbidity, with deaths happening in about 15% of patients and up to 50% of infants left with serious neurological sequelae^{ref1,
ref2}
tuberculosis of bones and joints : tuberculosis involving the bones and joints, producing strumous arthritis, or white swelling, and cold abscess.

tubercular osteomyelitis in dorsolumbar spine
tuberculous dactylitis (also called spina ventosa), more common in children than in adults. Differential diagnoses include osteomyelitis, sarcoidosis, and bone neoplasms (e.g., enchondroma)^ref
tubercular septic arthritis (1% of all TB cases, 10% of extrapulmonary TB cases)

chronic granulomatous monoarthritis (including carpal tunnel syndrome (CTS))
Grocco-Poncet disease
tubercular spondylitis / Pott's disease infiltrating psoas muscle => hunchback

adrenal glands tuberculosis (primary Addison disease )
genitourinary tuberculosis : tuberculosis involving the genitourinary tract, often the result of hemic dissemination of pulmonary tuberculosis

genital tuberculosis

in females : tuberculous endometritis => female sterility
in males : prostatitis (hematogenous >> urinary route), epididymitis (retrograde spreading of urinary tuberculosis) => fistulizing orchitis (tuberculocele)

urinary tuberculosis => Mycobacteria in urines

renal tuberculosis (chronic suppurative nephritis )
cicatricial ureteral stenosis
ulcerative urethrocystitis (cystophthisis) => fibrosis

ocular tuberculosis (dacryocystitis , scrofulous ophthalmia , diffuse scleritis , nodular iritis , miliary or disseminated choroiditis )
oral tuberculosis : a rare condition usually occurring as a bloodborne complication of pulmonary tuberculosis, most often involving the gingivae (tuberculous gingivitis ) and tongue, and characterized by the presence of small, crateriform, painless ulcers that bleed readily and are surrounded by edema or reddish nodules

swallowing of phlegm =>

intestinal tuberculosis / tuberculosis of intestines : a form that involves the intestines, with diarrhea , formation of spreading ulcers (especially of the lymphoid tissue as Mycobacteria localize into isolated lymphoid follicles (ILFs) disposed along transversal axis), and sometimes eventual cicatricial stricture => tabes mesenterica or mesaraica (tuberculosis of the mesenteric glands in children, resulting in digestive derangement and wasting of the body) => Crohn -like disease in terminal ileum and caecum => tubercular peritonitis .

reinfection tuberculosis : a new infection with tuberculosis in a patient who was previously infected and cured
Ranke's stages : the hypothesis that tuberculosis of the lungs develops in 3 stages: (1) the primary focus, (2) generalized spread of the tubercle bacillus, and (3) isolated organ tuberculosis, chiefly of the lungs.

Resistance in HLA-DR4 haplotype, susceptibility in HLA-Bw15⁺. The supersusceptibility to tuberculosis 1 (sst1) genetic locus on mouse chromosome 1 contains the intracellular pathogen resistance 1 (Ipr1) gene, whose expression is upregulated in the sst1 resistant macrophages after activation and infection (but is not expressed in the sst1 susceptible macrophages) limiting the multiplication not only of M. tuberculosis but also of other intracellular bacteria as Listeria monocytogenes, switching a cell death pathway of the infected macrophages from necrosis to apoptosis^ref. RF

in 10-25%.
Susceptibility : genetic defects in the IFN-g

response pathway cause unique susceptibility to intracellular pathogens, particularly mycobacteria, but are rare and do not explain mycobacterial disease in the majority of affected patients. Acquired defects such as anti-IFN-g autoantibodies may cause a similar immunological phenotype and thus explain the occurrence of disseminated intracellular infections in some patients without identifiable immune deficiency^ref.

Laboratory examinations :

direct diagnosis :

culturing (sputum, BAL, CSF, gastric juices, stools) : women in Pakistan are less likely than men to test positive for tuberculosis because they don't know how to expectorate sputum properly. In this study, 771 men and 749 women were randomly assigned to get a 2-minute set of instructions on how to give a proper sputum sample and then were asked to provide a spot sample and an early-morning specimen taken upon awakening. Patients in the control group were asked for the 2 samples only. Instruction significantly increased the proportion of women who were smear-positive for TB by 63%. There were no significant differences among the men^ref.

traditional culture on solid culture media :

albumin and oleic acid
Petragnani medium
Löwenstein-Jensen medium : After inoculation of 250 µl of decontaminant, Löwenstein–Jensen slants were incubated at 37°C and examined twice weekly from day 7 through day 60^ref. The median time was 26 days for the detection of growth on culture and 68 days for testing direct-drug susceptibility^ref
Middlebrook's medium

automated mycobacterial culture system

BACTEC system (a medium containing radio-labeled palmitate as the sole C source. As it multiplies, it breaks down the palmitate and liberates radio-labeled CO₂. Using the BACTEC system, growth can be detected in 9-16 days vs 4-8 weeks using conventional media). It is important to create an antibiogram
MB/BacT (bioMérieux) : bottles are inoculated with 500 µl of decontaminant, and cultures are monitored continuously for 42 days. The median time was 13 days for the detection of growth on culture and 13 days for testing direct-drug susceptibility^ref

microscopic-observation drug-susceptibility (MODS) assay, originally described in 2000^ref, with additional data reported in 2004^ref, for the detection of tuberculosis and multidrug-resistant tuberculosis, directly from sputum, relies on 3 principles: first, that Mycobacterium tuberculosis grows faster in liquid medium than in solid medium; second, that characteristic cord formation can be visualized microscopically in liquid medium at an early stage; and third, that the incorporation of drugs permits rapid and direct drug-susceptibility testing concomitantly with the detection of bacterial growth. In proof-of-principle studies, the MODS assay distinguished patients with tuberculosis from healthy controls. MODS is based on direct inoculation of the selective 7H9 liquid culture medium in 24-well plates with a sputum specimen subjected to the digestion–decontamination procedure with the use of a mixture of 2 reagents, N-acetyl-L-cysteine and sodium hydroxide, for 2 purposes (Kent PT, Kubica GP. Public health mycobacteriology: a guide for the level III laboratory. Atlanta: Centers for Disease Control, 1985; Laboratory services in TB control. Parts I, II, and III. Geneva: World Health Organization, 1998. (Publication no. WHO/tb/98.258.)). The first purpose is digestion, or liquefaction, accomplished with N-acetyl-L-cysteine and is required for the subsequent concentration of the mycobacteria by centrifugation. The second purpose is decontamination, which is performed with sodium hydroxide and is required to kill microbes that otherwise would interfere with the isolation of a pure mycobacterial culture. Concentration by centrifugation is presumed to be the final step of the procedure. Broth cultures are prepared in 24-well tissue-culture plates (Becton Dickinson), each containing 720 µl of decontaminant, Middlebrook 7H9 broth (Becton Dickinson), oxalic acid, albumin, dextrose, and catalase (OADC) (Becton Dickinson), and polymyxin, amphotericin B, nalidixic acid, trimethoprim, and azlocillin (PANTA) (Becton Dickinson). For each sample, 12 wells were used: in 4 control wells, no drug was added, and each of the remaining 8 wells contained 1 of 4 drugs at one of 2 concentrations tested. The cultures were examined under an inverted light microscope at a magnification of 40x every day (except Saturday and Sunday) from day 4 to day 15, on alternate days from day 16 to day 25, and twice weekly from day 26 to day 40. To minimize cross-contamination and occupational exposure, plates were permanently sealed inside plastic ziplock bags after inoculation and were subsequently examined within the bag. Positive cultures were identified by cord formation, characteristic of M. tuberculosis growth, in liquid medium in drug-free control wells, as described previously^ref. Nontuberculous mycobacteria were recognized by their lack of cording or, for M. chelonae (which is the only nontuberculous mycobacteria that does form cords), by rapid overgrowth by day 5. Fungal or bacterial contamination was recognized by rapid overgrowth and clouding in wells. Detection of the typical cord formation ("microcolonies") of M. tuberculosis in the wells on microscopical examination (under an inverted light microscope at a magnification of x40) constitutes the basis of diagnosis. Growth (or the lack thereof) in drug-containing wells, as compared with growth in drug-free wells, is the basis for reporting the results as "susceptible" or "resistant" to medication. The reported^ref sensitivity of this method in the recovery of mycobacteria from sputum specimens was higher than the sensitivity of either the MB/BacT automated mycobacterial system or traditional culture on Löwenstein–Jensen medium. The results of Moore et al. complement 2 previous publications from a group in Peru^{ref1,
ref2}, in one of which^ref the sensitivity of the MODS assay appears to be equal to that of the mycobacteria growth-indicator tube system and showed higher sensitivity than culture on agar plates. Results of testing for drug susceptibility with four drugs (isoniazid, rifampin, ethambutol, and streptomycin) agreed well with those obtained with the use of the MB/BacT system^ref. The most impressive data reported by Moore et al. are on the turnaround time of the assay: the median time was 7 days for the detection of growth on culture and testing direct-drug susceptibility, whereas the MB/BacT system required 13 and 22 days, respectively, and culture on Löwenstein–Jensen medium required 26 and 68 days, respectively^ref. Anyway in its current form, the MODS assay will require further improvement and standardization before it can be recommended for broad application in such countries. Reports of the identification of M. tuberculosis on the basis of the "string-and-tangle appearance"^ref of growth detected on microscopical examination of the wells should be considered provisional, rather than final. Some strains of M. tuberculosis may not produce this serpentine formation. Also, many isolates of M. kansasii also may produce cording in broth and have morphologic features and a timing of appearance indistinguishable from those of isolates of M. tuberculosis. An additional problem in countries where there is a significant prevalence of bovine tuberculosis is the differentiation between M. tuberculosis and M. bovis. These problems of identification can be addressed if isolation on a solid medium to obtain a pure culture is implemented along with the MODS assay, and such cultures can be used, if necessary, for subsequent confirmation of the diagnosis. Critical drug concentrations implemented for the MODS assay were adopted by the authors from those developed for the Bactec system (BACTEC 960, Becton Dickinson). Although the results obtained correlated well with results obtained with the use of two other methods, in general, each technology is known to require special calibration of critical concentrations halfway between the highest minimal inhibitory concentrations for susceptible strains and the lowest minimal inhibitory concentrations for resistant strains. The major difficulty in the implementation of the MODS assay or any other new cultivation method is that of biosafety, which is not fully addressed by Moore et al. Although it is not stated in their report, we assume that the sputum digestion–decontamination procedure was performed in conjunction with concentration of the specimens, as required by the original description of this technique (Kent PT, Kubica GP. Public health mycobacteriology: a guide for the level III laboratory. Atlanta: Centers for Disease Control, 1985). Concentration must be performed in aerosol-contained centrifuges. In addition, the manipulation of infected well plates is a potential hazard requiring special caution and should be performed only in biosafety cabinets. Use of these measures, along with other biosafety issues, can be properly addressed only in a well-organized tuberculosis laboratory using mandatory implementation of biosafety level 3 standards (Heifets LB, Richmond JY. Modern biological safety standards in tuberculosis diagnostic (TB) laboratories. In: Richmond JY, ed. Anthology in biosafety. IX. Exploring the performance envelope for BSL-3 and BSL-4 laboratories. Mundelein, IL: American Biological Safety Association, 2006:155-66). Regardless of how simple and inexpensive the newly introduced technique is, there is the issue of the cost of the materials needed for the test and the total cost of the procedure, including the costs of labor, laboratory equipment (centrifuges, biosafety cabinets, inverted light microscopes, among other types of equipment), and overhead. In other words, implementation of an "inexpensive" test can be successful only if it is incorporated into the overall algorithm of the laboratory protocol. Thus, the introduction of a new procedure (such as the MODS assay) requires the existence of a basic tuberculosis laboratory.

^ref

acid-fast (Ziehl-Neelsen stainable ) bacilli (AFB) in at least 1 out of 3 sputum, BAL, gastric juice, urine or pleural fluid samples : provide positive results in < 50% of patients with newly diagnosed pulmonary tuberculosis confirmed on culture
auramin O fluorescent staining
PCR : spacer oligonucleotide typing (spoligotyping), polymerase chain reaction with multiple primers^{ref1,
ref2}
rapid immunochromatographic assay based on the 38 kDa antigen of Mycobacterium tuberculosis^ref
urinary lipoarabinomannan^ref
neopterin levels in BAL, serum and particularly in urine may reflect PTB activity before exact diagnosis of the disease by culture results, and correlates with radiological extent

clinical diagnosis

risk factors : immunodepressions , COPDs .
symptoms & signs

fatigue, weight loss, afternoon fever , night sweats, chest pain, hemophtoe, dyspnea , anorexia => malnutrition => muscular hypotrophy => cachexia / phthisis , pallor.
chest auscultation : fine crackles => coarse crackles , amphoric breathing due to fistulas and pleural effusion

laboratory examinations

chest X-ray : atypical or typical (alternations of cavities and densities in post-primary TB, mediastinic lymphadenitis )
anemia, leukocytosis, increased ESR and CRP
⁶⁷Ga scintigraphy (to distinguish foci which are still active)

indirect diagnosis :

subcutaneous injection of Koch's Old tuberculin (OT) (a heat-concentrated filtrate of tubercle bacillus culture grown on a special medium. It was Robert Koch in 1891, following his identification of the human tubercle bacillus, who felt that he could develop a cure for TB using a filtrate of killed organisms (Koch R. Weitere mitteilungen uber ein Heilmittel gegen Tuberculose. Dtsch Med Wschr 1891; 17:101-102). This was not to be the case, but testing was found to be used as a diagnostic test, first observed by European veterinarians^ref; nowadays it has been replaced by the "purified" protein derivative (PPD^®) / Seibert's tuberculin, a soluble purified protein fraction precipitated by trichloracetic acid from filtrate of tubercle bacillus culture grown on a special medium) (tuberculin skin test (TST)) :

Koch's phenomenon or reaction : if a guinea pig that has been previously infected with tuberculosis organisms is reinjected intracutaneously, the skin over the injected area undergoes necrosis and a superficial ulcer develops; the ulcer heals quickly and infection of regional lymph nodes is retarded. The phenomenon demonstrates development of ability to localize tubercle bacilli, which is the principle underlying tuberculin tests
cockade reaction : the reaction of a sensitized guinea pig to intradermal injection of tuberculin; it consists of a large papule with a necrotic, hemorrhagic center.

von Pirquet reaction : deposition after scarification.

Moro-Vollmer strip-reaction : saturated strip (unknown absorbed [ ] !) over sternum or upper edge of trapezius muscle
Mantoux intradermoreaction (nowadays replaced by tine-test / multiple puncture TB test (MPT)): on the contrary of the 2 above mentioned methods, in this one the quantity of administered Ag doesn't depend on epidermis thickness and operative pressure. Within 48÷72 hrs after injection of 1 TU (first strength tuberculin, used for young children or persons in whom extreme hypersensitivity is expected : same potency as 0.01 mg OT prepared by Statens Seruminstitut in Copenhagen) in 0.1 mL, a type IV hypersensitivity (induration and erythema with Ø > 10 mm : 90% sensitivity; 100% sensitivity if Ø > 15 mm) occurs if within 3÷7 years after BCG vaccination or 4÷6 weeks after infection. If Ø < 5 mm, the result is negative, while if 10 < Ø < 5 mm, assay must be repeated using 5 TU (= 0.0001 mg of PPD, intermediate strength tuberculin = the standard test dose) and (if the second result is still doubtful) with 250 TU (second strength tuberculin) : sensitization during such a treatment shouldn't cause granuloma because immune responses against proteic antigens are usually humoral. Toning is late if infection is recent.
pe3yrbt^® (?, Russia)
Pirket^® (?, Russia)
RM^® (?, Russia)
Test-PPD^® (Chiron, Italy, Asia)
Tubergen Test/Tuberkulin^® (Chiron, Germany)

It generally requires about 6 weeks or more to convert a TST to positive after exposure.

serology :

IgMs and IgGs (TB-Test^®)
antibody detection kit (MycoDot test^®)^{ref1,
ref2}
veterinary tests : if the Elephant TB STAT-PAK is used as a screening test and the MAPIA is sequentially applied as a confirmatory assay, the accuracy of this testing algorithm is 100%

The Elephant TB STAT-PAK is a lateral-flow assay that incorporates a unique cocktail of mycobacterial antigens impregnated on a nitrocellulose membrane and placed in a plastic cassette similar to a pregnancy test kit. Serum, plasma, or whole blood may be used and results are available in 20 minutes (see Lyashchenko, K.P. et al, 2006. Tuberculosis in elephants: antibody responses to defined antigens of Mycobacterium tuberculosis, potential for early diagnosis, and monitoring of treatment. Clin Vaccine Immunol 13(7):722-732, and Lyashchenko, K. et al, 2005. Application of MAPIA (Multiple antigen print immunoassay) and rapid lateral flow technology for tuberculosis testing of elephants. 2005 Proceedings AAZV, AAWV, AZA Nutrition Advisory Group, pp. 64-65)
The MAPIA is a laboratory procedure for antibody detection that uses a panel of multiple purified and recombinant antigens of _M. tuberculosis_ and _M. bovis_ that are separately applied to a nitrocellulose membrane using an automated printing device (see Lyashchenko, K. et al., 2000. Multi-antigen print immunoassay for the development of serological diagnosis of infectious disease. Journal of Immunological Methods 242:91-100)

interferon-γ release assays (IGRA) by ELISpot to look for blood T lymphocytes specific for Mycobacterium tuberculosis antigens that are absent from Mycobacterium bovis BCG and most environmental mycobacteria. The test is less likely to give false positives than the skin-prick test, which includes proteins that are used in the BCG vaccine and is therefore confounded by TB vaccination. The test also works well in individuals with weak immune systems. As the test can monitor how the number of specific T cells changes with time, it might also be used to monitor treatment efficacy. Although the test is more expensive than the skin prick, it should save money in the long term by eliminating the unnecessary treatment of false positives^ref. Overall agreement with the skin test was similar (T-SPOT.TB k=0.508, QuantiFERON-TB Gold k=0.460), but fewer BCG-vaccinated individuals were identified as positive by the two blood assays than by the tuberculin skin test (p=0.003 for T-SPOT.TB and p<0.0001 for QuantiFERON-TB Gold). Indeterminate results were significantly more frequent with QuantiFERON-TB Gold (11%, 43 of 383) than with T-SPOT.TB (3%, 12 of 383; p<0.0001) and were associated with immunosuppressive treatments for both tests. Age younger than 5 years was significantly associated with indeterminate results with QuantiFERON-TB Gold (p=0.003), but not with T-SPOT.TB. Overall, T-SPOT.TB produced significantly more positive results (38%, n=144, vs 26%, n=100, with QuantiFERON-TB Gold; p<0.0001), and close contacts of patients with active tuberculosis were more likely to be positive with T-SPOT.TB than with QuantiFERON-TB Gold (p=0.0010)^ref.
diagnosis of active tuberculosis in children poses a major challenge. Clinical symptoms of tuberculosis in children are often non-specific and resemble those of common paediatric illnesses, including pneumonia and malnutrition. Adequate respiratory specimens are difficult to obtain for bacterial confirmation, particularly in very young children who are unable to expectorate and in whom diagnostic yields are poor because of the paucibacillary nature of the disease.2 and 5 Hence, the diagnosis is routinely made on the basis of a combination of clinical features, contact history, chest radiography, and tuberculin skin test, and often with scoring charts that have poor diagnostic accuracy.6 and 7
- Xpert MTB/RIF assay enables timely, sensitive, and specific molecular detection of pulmonary tuberculosis and rifampicin resistance in adults,8 but its value in young children is greatly reduced. In a recent meta-analysis, the calculated pooled sensitivity was 66% for Xpert MTB/RIF against culture in expectorated or induced sputa, or gastric lavage specimens from children with suspected tuberculosis. WHO strongly recommends Xpert MTB/RIF as the initial diagnostic test in children suspected of having multidrug-resistant or HIV-associated tuberculosis, and only conditionally in all children suspected of having tuberculosis
- immunodiagnostic tests do not depend on the presence of Mycobacterium tuberculosis in collected samples. Although these tests have use in screening special risk populations for latent tuberculosis infection, they cannot enable the crucial distinction between active tuberculosis disease and latent tuberculosis infection. 10 Failure to accurately identify cases with active tuberculosis restricts the clinical application of these tests in endemic regions where latent tuberculosis infection is ubiquitous.
- T-cell activation marker–tuberculosis (TAM-TB) assay consists of flow-cytometric analysis of the CD27 on circulating M tuberculosis-specific T cells can discriminate active tuberculosis from latent tuberculosis infection in adults and children. Loss of CD27 expression on M tuberculosis-specific CD4 T cells is a marker of active tuberculosis due to persistent antigenic stimulation and probably relates to increased cellular homing to the site of disease^ref
in pericardial, pleural or ascitis exudate, ADA > 72 U/l has a sensitivity of 100% and a specificity of 94% in the diagnosis of tuberculous pericarditis. Similarly for pericardial lysozyme, a value of 6.5 mg/dl had a sensitivity and specificity of 100 and 91.17%, respectively^ref

ex adiuvantibus criterion : improvement within 90 days of treatment with a polychemotherapy. Drug development against Mycobacterium tuberculosis has been an overlooked area of tuberculosis research for some time. The first breakthrough—to obtain an active antituberculosis agent—occurred in the mid-1940s with the seemingly reluctant discovery of the activity of streptomycin (SM) (JH Comroe Jr., Pay dirt: the story of streptomycin. Part I: from Waksman to Waksman, Am Rev Respir Dis 117 (1978), pp. 773–781) and the more focused path towards the synthesis of para-aminosalicylic aci (J Lehmann, Twenty years afterward: historical notes on the discovery of the antituberculosis effect of para-aminosalicylic acid (PAS) and the first clinical trials, Am Rev Respir Dis 90 (1964), pp. 953–966). The second breakthrough came when three drug companies simultaneously and independently applied to patent isonicotinic acid hydrazide (now known as isoniazid (INH)), only to learn that this drug had already been synthesised 50 years earlier (H Meyer and J Mally, On hydrazine derivatives and pyridine carbonic acids, Monatshefte Chemie verwandte Teile anderer Wissenschaften 23 (1912), pp. 393–414). The third and perhaps last specific attempt at finding an antituberculosis agent came with the isolation of an antibiotic from the rifamycin class, published in 1959 (P Sensi, P Margalith and MT Timbal, Rifomycin, a new antibiotic—preliminary report, Farmaco Ed Sci 14 (1959), pp. 146–147). Almost two decades passed before a regimen based on isoniazid plus rifampicin underwent rigorous assessment (G Brouet and G Roussel, Trial 6.9.12. Overall methodology and summary of results, Rev Fr Mal Respir 5 (suppl 1) (1977), pp. 5–13). Although so-called short-course treatment can effectively cure drug-susceptible tuberculosis in 6 months, a large proportion of patients in whom tuberculosis is diagnosed do not complete a course of treatment.2 New drugs that shorten the duration of tuberculosis treatment would substantially reduce the likelihood of disease recurrence and death caused by inadequate therapy. Additionally, since every year there are 500 000 reported cases of tuberculosis caused by strains of Mycobacterium tuberculosis that are resistant to the key first-line drugs isoniazid and rifampicin, agents that are active in multidrug-resistant tuberculosis are also needed. Moxifloxacin is a fluoroquinolone that has potent in-vitro activity against M tuberculosis. Early studies of moxifloxacin in murine models of tuberculosis showed that the drug had good bactericidal activity that was additive to isoniazid. On the basis of these studies, we designed a phase II clinical trial to test the hypothesis that the substitution of D-ethambutol (EMB), an agent used primarily to prevent the emergence of drug resistance, with moxifloxacin would significantly increase the proportion of patients with negative sputum cultures after 8 weeks of treatment. 8-week sputum conversion has proved a useful surrogate marker of the sterilising activity of tuberculosis regimens^ref.

Directly observed therapy, short course (DOTS)

^ref

first choice (per os, all days in early phases or every 2 days in late phases, for 2 months) :

isoniazid (INH) 300 mg/day
rifampin (RIF) 450 mg/day (weight <50 kg) or 600 mg (weight >50 kg)
pyrazinamide (PZA / PIZ) 20–25 mg/kg
moxifloxacin 400 mg/day > D-ethambutol (EMB) 15–20 mg/kg

second choice (for multi-drug resistant TB (MDR-TB / MDRTB), which does not respond to treatment with either of the 2 main TB drugs, isoniazid (INH) and rifampin (RIF). About 450 000 get MDR-TB each year. Although other drugs are available, the treatment takes up to 2 years and can cost anything between $500 and $6,000. 14% of new cases in eastern Europe and central Asia involve MDR strains. The top 10 hotspots for MDR strains are in 6 Republics of the old Soviet Union (Kazakhstan, Uzbekistan, Estonia, Lithuania, Latvia and Russia) and 2 China provinces (Liaoning and Henan). In May 2004 the EU opens its borders to 3 of the top 10 geographical hotspots (Estonia, Latvia, and Lithuania). 'Hot zones' are areas that have >5% prevalence (or incidence) of MDRTB. Paradoxically areas with programs that successfully reduced wild-type pansensitive strains often evolved into hot zones. Second, some hot zones emerged even when MDR strains were substantially less fit (and thus less transmissible) than wild-type pansensitive strains. Third, levels of MDR are driven by case-finding rates, cure rates and amplification probabilities^ref. Even when the average relative fitness of MDR strains is low and a well-functioning control program is in place, a small subpopulation of a relatively fit MDR strain may eventually outcompete both the drug-sensitive strains and the less fit MDR strains. These results imply that current epidemiological measures and short-term trends in the burden of MDRTB do not provide evidence that MDRTB strains can be contained in the absence of specific efforts to limit transmission from those with MDR disease^ref.

p-aminosalicylic acid (PAS) for 6÷12 months
kanamycin
quinolones (ofloxacin, moxifloxacin )
pleuromutilins
isocitrate lyase inhibitors

extensively drug-resistant TB (XDR-TB) : the definition of XDR TB until the fall of 2006 was an isolate of M.tuberculosis resistant to isoniazid and rifampin and at least 3 of the 6 main classes of 2nd-line drugs (aminoglycosides, polypeptides, fluoroquinolones, thioamides, cycloserine, and para-aminosalicyclic acid). After the WHO Report of the meeting of the WHO Global Task Force on XDR-TB, Geneva, Switzerland, 9-10 Oct 2006, the definition was modified to: an isolate resistant to INH and rifampin (also called rifampicin) which was also resistant to a fluoroquinolone and to 1 or more of the following 3 injectable antituberculosis drugs: capreomycin, kanamycin and amikacin. According to the report, a number of considerations were used in revising the definition, including^ref :

technical feasibility and reproducibility of testing for second-line drugs
efficacy and availability of second line drugs
the need for a definition with significant worse treatment outcome than MDR-TB alone.

South Africa^ref : High Prevalence and Mortality from Extensively-Drug Resistant (XDR) TB in TB/HIV Coinfected Patients in Rural South Africa N.R. Gandhi et al. In a rural district in Kwazulu Natal, South Africa, where the TB/HIV coinfection rate is > 80%, antiretroviral therapy (ARV) has significantly reduced mortality. Of the remaining deaths, 67% have been documented to be due to MDR TB. Surveillance with sputum culture and drug susceptibility testing was performed for patients with known or suspected TB in a rural district hospital (at the Church of Scotland Hospital in a KwaZulu-Natal hamlet called Tugela Ferry). Spoligotyping was performed on isolates resistant to all tested TB drugs (isoniazid, rifampin, ethambutol, streptomycin, ciprofloxacin, kanamycin). Between January 2005 and March 2006, sputum collected from 1540 patients revealed that 536 (35%) patients were culture positive for M.tb. Of these, 221 (41%) had MDR TB, and 53 (24% of MDR isolates, 10% of all positive cultures) had resistance to all 1st and 2nd line drugs tested (XDR TB). Spoligotyping revealed 90% of XDR TB patients were infected with a genetically similar strain. 52 of 53 (98%) XDR TB patients have died; the median survival after sputum collection was 25 days (range: 11-136). All 47 XDR TB patients with known HIV status were HIV⁺. Only 34% of patients were previously treated for TB, and 56% had been previously hospitalized (presented in mid-August 2006 at the XVI International AIDS Conference in Toronto as a late-breaker abstract^ref. Since then a total of 78 patients have been confirmed with XDR-TB in the province, 74 of whom have died. Another 9 cases have been identified in Gauteng (South Africa's premier economic region, which includes Johannesburg and Pretoria^ref), 10 in North West, 6 in Eastern Cape and 3 in Limpopo^ref. As of Nov 23 2006, a total of 303 cases of XDR-TB were confirmed across the country : 263 cases in KwaZulu-Natal, 10 cases each in the Eastern Cape and the North West, 9 in Gauteng, 6 in the Free State, 3 in Limpopo and 2 in the Northern Cape. None were reported in Mpumalanga and the Western Cape^ref. found at 39

^ref

Canada : since 2004, the tuberculosis service at West Park Healthcare Centre in Toronto has treated 5 or 6 patients with XDR TB. the

^ref

China
many republics of the former Soviet Union
emergence of Mycobacterium tuberculosis with Extensive Resistance to 2nd-Line Drugs --- Worldwide, 2000--2004 : 17 690 isolates from the period 2000-2004 were tested for susceptibility to > 3 of the 6 2nd line drugs (SLD) classes. Of these, 11 939 were from South Korea, of which 1298 (11%) were multidrug-resistant (MDR, defined as resistance to at least isoniazid and rifampin). From the other Global Supranational TB Reference Laboratory (SRLs), 2222 (39%) of 5751 isolates were MDR. Of the 3520 MDR isolates, 347 (10%) were XDR (defined as cases in persons with TB whose isolates were resistant to isoniazid and rifampin and at least 3 of the 6 main classes of SLDs (aminoglycosides, polypeptides, fluoroquinolones, thioamides, cycloserine, and para-aminosalicyclic acid), including 200 (15%) of 1298 from South Korea and 147 (7%) of 2222 from other SRLs. The drug-susceptibility testing results were tabulated by year and geographic region (on the basis of the country of origin of the isolates). XDR TB was identified in all regions but was most common in South Korea (n = 200; 15% of all MDR TB isolates) and countries of eastern Europe/western Asia (Armenia, Azerbaijan, Czech Republic, Republic of Georgia, and Russia, n = 55; 14% of all MDR TB isolates). The total number and proportion of XDR TB isolates observed worldwide (excluding South Korea) increased from 14 (5% of MDR TB isolates) in 2000 to 34 (7% of MDR TB isolates) in 2004. Year-specific proportions were stratified by geographic region. Increasing proportions of XDR TB were found among isolates from countries of eastern Europe/western Asia (n = 5 [9%] in 2000; n = 11 [17%] in 2003) and the group of industrialized nations (Australia, Belgium, Canada, France, Germany, Ireland, Japan, Portugal, Spain, UK, and USA, n = 3 [3%] in 2000; n = 25 [11%] in 2004). USA national TB surveillance data included 169 654 patients with drug-susceptibility testing results. During 1993-2004, a total of 689 (1.6%) MDR TB cases were identified, of which 1814 (67%) had results reported for 3 or more SLD classes. Of these, 74 (4.1%) had resistance to 3 or more SLD classes and thus met the criteria for XDR TB. Despite an overall decline in MDR TB incidence in the USA, the proportion of XDR TB increased slightly, from 37 (3.9%) of 944 cases during 1993-1996 to 20 (4.1%) of 489 during 1997-2000, to 17 (4.5%) of 381 in 2001-2004 (chi-square test for trend = 0.20; p = 0.66). During 1993-2002, patients with XDR TB were 64% more likely to die during treatment (relative risk [RR] = 1.6; 95% confidence interval [CI] = 1.2-2.2) than patients with MDR TB. Among 605 MDR TB patients in Latvia who initiated therapy during 2000-2002, 115 (19%) had XDR TB. The proportion with XDR TB increased from 30 (15%) of 204 in 2000, to 46 (21%) of 215 in 2001, to 39 (21%) of 186 in 2002 (chi-square test for trend = 2.57; p = 0.11). Patients with XDR were 54% more likely to die or have treatment failure (RR = 1.5; CI = 1.1-2.2)."^ref

^ref

^{ref1,
ref2,
ref3,
ref4}

^ref

^ref

the restriction is provided for and carried out in accordance with the law;
the restriction is in the interest of a legitimate objective of general interest;
the restriction is strictly necessary in a democratic society to achieve the objective;
there are no less intrusive and restrictive means available to reach the same objective;
the restriction is based on scientific evidence and not drafted or imposed arbitrarily, i.e. in an unreasonable or otherwise discriminatory manner.

^ref

surgical tuberculosis : tuberculosis that can be treated by surgical means

therapeutic pneumothorax is no longer used

^ref

Relapse

resistance

Glucocorticoids

meningitis

pericarditis

Prevention :

attenuated live vaccine . Good overall health and adequate living conditions without crowding are the 2 most important historical parameters in decreasing the amount of tuberculosis in a community. This is best shown by the decrease in Mycobacterium tuberculosis infections in the USA starting at the beginning of the 20th century, 4 decades before the 1st anti-tuberculosis drugs were utilized
the role of secretory proteins of Mycobacterium tuberculosis in pathogenesis and stimulation of specific host responses is well documented. They are also shown to activate different cell types, which subsequently present mycobacterial antigens to T cells. Therefore identification of T cell epitopes from this set of proteins may serve to define candidate antigens with vaccine potential. 52 secretory proteins of M. tuberculosis H37Rv were analyzed computationally for the presence of HLA class I binding nonameric peptides. All possible overlapping nonameric peptide sequences from 52 secretory proteins were generated in silico and analyzed for their ability to bind to 33 alleles belonging to A, B and C loci of HLA class I. 15% of generated peptides are predicted to bind to HLA with halftime of dissociation t_1/2 >/= 100 min and 73% of the peptides predicted to bind are mono-allelic in their binding. The structural basis for recognition of nonamers by different HLA molecules was studied employing structural modeling of HLA class I-peptide complexes and there exists a good correlation between structural analysis and binding prediction. Pathogen peptides that could behave as self- or partially self-peptides in the host were eliminated using a comparative study with the human proteome, thus reducing the number of peptides for analysis. The implications of the finding for vaccine development are discussed vis-a-vis the limitations of the use of subunit vaccine and DNA vaccine^ref.
post-exposure chemoprevention

Web resources:

National Center for HIV, STD and TB prevention (NCHSTP) at CDC