Applied Microbiology cases

Mycobacterium tuberculosis: Pulmonary tuberculosis
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A 55 year old man with persistent cough, fever, night sweats, loss of weight, anorexia, malaise and weakness since 3 months presents himself to the hospital. Chest X-ray suggested upper lobe consolidation. Hematological examination revealed mild leucocytosis.

What is your diagnosis?
It could be a case of pulmonary tuberculosis, however differential diagnosis includes aspergillosis, actinomycosis, bronchiectasis, histoplasmosis, lung abscess, blastomycosis etc.

What is the etiological agent of tuberculosis?
Typical pulmonary tuberculosis is caused by Mycobacterium tuberculosis, however other species of Mycobacteria such as M. bovis, M.avium-intercellulare complex too can cause tuberculosis.

What is the specimen collected?
Expectorated sputum specimen is the preferred specimen in the laboratory diagnosis of pulmonary tuberculosis. According to the revised guidelines of National Tuberculosis Control Programme (India), two sputa samples (spot-morning) must be collected from the patient. When the patient approaches the center to collect the container, spot specimen is collected. Patient expectorates the second sample on the next morning in the given container and submits it to the laboratory. After rinsing the mouth, patient should take deep breath, cough and expel the sputum into the container that is held close to the mouth.  In patients without spontaneous sputum production, sputum induction may be induced using hypertonic saline.

Which are the other specimen that can be obtained from patients?
In case of children (who tend to swallow sputum), a morning gastric lavage may be collected. Alternatively laryngeal swabs may also be collected.  Other invasive techniques that can be employed include fiberoptic bronchoscopy with transbronchial biopsy, bronchial brushings, and transtracheal aspirate.

How is the specimen processed?
A gram stained smear may be made from the thick part of the sputum to exclude other bacterial infection. Acid fast staining of the smear is considered the gold standard in the laboratory diagnosis. After assessing the suitability of the sample, the smear is stained with any of the acid fast staining techniques (Ziehl Neelsen, Kinyoun, Gabbett). Hundred fields must be observed in each smear before giving a negative report. If acid fast bacilli are seen, they should be counted and the smear is graded.

What are your observations?
Pink coloured, slightly curved bacilli in singles or clumps, with occasional branching and beaded appearance are seen against a blue background consisting of many pus cells and few epithelial cells. The given smear contains acid fast bacilli.

How is sputum graded?
According to the RNTCP, sputum sample should be graded in the following way:
>10 AFB/oil immersion field in at least 20 fields: 3+
1-10 AFB/oil immersion field in at least 50 fields: 2+
10-99 AFB/100 oil immersion field: 1+
1-9 AFB/100 oil immersion field: record exact number

What is the significance of grading the sputum smear?
The initial count gives a picture of the extent of disease. It has a very important role in monitoring the progress of treatment, as the counts decrease with successful treatment.

What is the sensitivity of sputum smears?
AFB smear is not a very sensitive technique, if numbers of bacilli are less than 1000/ml of sputum, they may be missed. The sensitivity of the smear can be increased by subjecting the sample to concentration techniques such as Petroff's method, Cetyl pyridinium chloride, Zepharin chloride method, or NALC method etc. The sensitivity of microscopic examination of sputum can be increased by using fluorescent dyes such as Auramine O.

How are Mycobacteria cultured?
Direct sputum sample (or sputum concentrate) is usually cultured on Lowenstein Jensen Medium and incubated at 37oC for 4-8 weeks. Cultures are not routinely performed, but may be done to identify the species or for drug susceptibility testing. Mycobacterium tuberculosis produces rough, buff and tough colonies on LJ medium. The acid fast smear of these colonies show acid fast bacilli. Conventional culture media used for Mycobacterial isolation are most often Lowenstein-Jensen (LJ), or Middlebrook 7H9, 7H10 or 7H11.Growth in these media is observed in a range of 3 – 56 days, depending on the species isolated and concentration of viable bacteria. The SEPTI-CHEK AFB system is intended for use as an integrated  in-vitro diagnostic system for the detection and isolation of Mycobacteria from various clinical specimens, which provides a presumptive identification as well as the ability to perform susceptibility testing. In miliary tuberculosis, Mycobacteria can be recovered from blood using BACTEC-460 TB system; a system employing radiometric technology providing rapid and accurate detection in as little as 4-8 days and susceptibility testing in as little as 4-12 days. Other system includes Mycobacterial growth indicator tube (MGIT) system. Culture using animal models are no longer employed for routine diagnosis.

How is Mycobacterium tuberculosis identified?
Conventional method of identification are aryl sulfatase test, niacin test, nitrate reduction, thermocatalase test etc. High pressure liquid chromatography for detection of mycolic acid, nucleic acid hybridization, PCR have replaced the conventional system in developed countries.

Which are the other investigation techniques employed in the laboratory diagnosis of tuberculosis?
Serological methods detecting mycobacterial antigens or IgA, IgG or IgM antibodies against Mycobacterium using ELISA have been employed but have not met with great success. Skin testing (tuberculin test such as PPD, Mantoux) have been employed to test for sensitivity to Mycobacteria. Reading the test has been prone to error and are subjected to many false positive and negative results.

What is tuberculin test?
Please read this notes.

What is the pathogenesis of tuberculosis?
Tuberculosis (TB) is spread from person to person through the air by droplet nuclei that contain M. tuberculosis. Droplet nuclei are produced when persons with pulmonary tuberculosis cough, sneeze, or speak.Infectious dose is less and few bacilli can cause infection. Droplet nuclei are small enough to reach the alveoli within the lungs. Alveolar macrophages ingest the bacilli and enclose them in phagosomes. If these macrophages are activated, the mycobacteria containing phagosomes fuse with lysosomes, and the bacteria are killed. If, on the other hand, the alveolar macrophages are not activated, the bacilli survive and multiply within the phagosomes. The macrophages lyse and the mycobacteria are released into the surrounding lung tissue, where they are phagocytized by tissue macrophages. Again, if the macrophages are activated, the bacteria are killed. However, if these tissue macrophages are not activated, the mycobacteria continue to multiply within the phagosomes and, upon release, are phagocytized by additional tissue macrophages and the infection spreads. As this process continues, a primary lesion forms. As the primary lesion enlarges, some mycobacteria are transported to the regional draining lymph nodes and the lymph nodes enlarge as the bacilli multiply intracellularly. Extension from the lung parenchyma or the lymph nodes lead to progressive primary tuberculosis. They may also become dormant and remain asymptomatic, or may proliferate after a latency period (reactivation disease). The main determinant of the pathogenicity of tuberculosis is its ability to escape host defense mechanisms, including macrophages. Among the several virulence factors in the mycobacterial cell wall are the cord factor, lipoarabinomannan, and a 65-kd heat shock protein. Progression of the primary complex may lead to enlargement of hilar and mediastinal nodes. Lymphohematogenous dissemination of the mycobacteria to other body parts and their multiplication results in miliary or disseminated tuberculosis. Tubercular meningitis may also result from hematogenous dissemination. Bacilli may remain dormant in the apical posterior areas of the lung for several months or years, which may later progress resulting in the development of reactivation-type tuberculosis.

How is the diagnosis made using sputum smears?
The following is the recommendation of RNTCP: If one or two samples of sputa are smear positive it may be considered as sputum smear positive tuberculosis and the patient is put on anti-tuberculosis treatment. If only one smear is positive, but X-ray findings is suggestive of TB, the patient is considered smear positive and put on treatment. If the x-ray is not suggestive, then TB is ruled out. If all the smears turn out to be smear negative but X-ray finding suggest tuberculosis, the patient is considered to be sputum smear negative tuberculosis and is put on treatment. Tuberculosis is ruled out if all the smears are negative and x-ray finding too is not suggestive.

How is tuberculosis treated?
Directly observed short-course chemotherapy for newly diagnosed cases and sputum smear negative but seriously ill with tuberculosis are subjected to intensive phase treatment regimen comprising of isoniazid, rifampicin, pyrazinamide and ethambutol that is administered three times a week for two months. When the patient has completed the initial intensive phage of two months and the sputum smear becomes negative, the continuation phase is begun. If the sputum remains positive despite the intensive phase, then the four drugs of intensive phase is continued for another month. After this period, continuation phase is begun irrespective of smear status. The continuation phase consists of isoniazid and rifampicin given three times a week for four months.

Why are anti-tubercular drugs given in combination?
Spontaneous mutation can result in development of resistant strains even during the course of treatment. Inclusion of more than one drug ensures that strains resistant to one drug are killed by the other drug.

What is MDR-TB?
Please read this notes.

Are there any rapid commercial methods of diagnosis?
Interferon-Gamma Release Assays (IGRAs) measure a person’s immune reactivity to M. tuberculosis. Freshly drawn patient's blood samples are mixed with purified M. tuberculosis antigen (synthetic peptides). White blood cells of the patient that are reactive to M.tuberculosis will release interferon-gamma, which is quantitatively measured. Results of this test can be available within 24 hours. However, this test does not help in differentiating latent tuberculosis infection from tuberculosis disease. Prior vaccination with BCG does not cause a false-positive result.

How can antibiotic resistance be detected in short time?
Line probe assay is a PCR and DNA hybridization based molecular tool that can detect resistance to rifampin and isoniazid. This test can be performed directly on smear-positive sputum samples and results would be available in five hours.

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