Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_2894_Библиотеки_им_академика_М_И_Перельмана

Mtb is a very slow-growing fastidious bacterium, which makes the culture of this important human pathogen difcult. Slow growth rate not only delays the diagnosis, but it also poses the problem of contamination by normal ora, making it even more difcult. Nonetheless, culture provides denitive diagnostics and is considered the “gold standard” of TB diagnosis [8]. With the availability of quick and rapid molec­ular diagnostics tests, culture may not be the preferred modality for the detection of Mtb, but it remains relevant for drug susceptibility testing (DST), genotyping, and the monitoring of patients’ response to treatments [7, 9]. DST is used to determine which antibiotics are effective at killing Mtb. This is important because TB is often treated with a combination of several different antibiotics to reduce the risk of the bacteria developing resistance. DST involves growing the bacteria on a solid media and adding different concentrations of antibiotics to see which ones can inhibit the growth of the bacteria [10, 11]. Genotyping is a process used to identify the specic strain of Mtb present in a sample. This is important because different strains of the bacteria may have different drug susceptibilities or be more or less virulent. Genotyping is typically done using techniques such as polymerase chain reaction (PCR) or DNA sequencing [12]. The continuous monitoring of patients’ response to treatment is very important for Mtb infections due to the emergence of various drug­resistant strains. After starting treatment, the patient’s sputum (a mixture of saliva and mucus that is produced by the respiratory tract) is collected and cultured to see if the bacteria are still present. If the bacteria are still present, it may indicate that the treatment is not effective and the patient’s treatment plan may need to be adjusted [13].

Egg-based Lowenstein-Jensen (LJ) and agar-based Middlebrook 7H10/7H11 (MB7H10/MB7H11) are the most commonly used solid media for growing Mtb. LJ culture media is widely used in the laboratory for the isolation and identication of mycobacteria. LJ medium is made up of fresh eggs from country hens, glycerol, mineral salts, and malachite green. Glycerol in the medium favors the growth of Mtb, and malachite green in the medium inhibits the growth of other types of bac­teria that may be present in a sample, making it selective for Mtb. The eggs provide nutrients in the form of proteins and lipids and work as solidifying agents. LJ medium is suitable for the growth of Mtb and allows for a visual inspection of the colony morphology of the isolate, making it easier to identify the culture [15]. It is also used for the antimicrobial susceptibility testing (AST) of rst-line and second­line drugs for TB [11]. Some of the limitations of this medium are slower growth of Mtb compared to the liquid medium, which delays the diagnosis, and shorter shelf life when compared to agar base media, such as MB7H10 and MB7H11. The aver­age time of growth of detectable macroscopic colonies of Mtb on the LJ medium is around 25.4days [16]. As LJ is always prepared as a slope or slant in culture tubes/ bottles, the effective surface area for the growth of mycobacteria is smaller than agar-based media, which may be prepared in Petri dishes as well.

BACTEC 460 TB (B460) system by Becton Dickinson Microbiology Systems, Sparks, Md., USA, was the rst automated culture system implemented for the diagnosis of TB.Despite having superior sensitivity, the use of radioisotopes in detection limited the use of this system [24]. The Mycobacteria Growth Indicator Tube (MGIT) system BACTEC MGIT 960 was then developed as an upgradation that utilizes a uorescent indicator, ruthenium pentahydrate. The uorescence is quenched by the oxygen in the medium, and as the bacteria grow, oxygen in the medium is depleted, resulting in the release of uorescence. This uorescence is detected by the instrument, and samples are labeled as positive for the presence of mycobacteria [25]. Prior to inoculation of the sample, each tube is supplemented with a growth supplement and an antibiotic mixture of polymyxin B, amphotericin B, nalidixic acid, trimethoprim, and azlocillin (PANTA) to avoid contamination. The tubes are incubated at 37°C and automatically monitored each hour for up to 42days or till the positive detection of uorescence [26]. The average detection time is around 9.7days in BACTEC MGIT 960 system [27]. Various studies have conrmed the increased sensitivity in the diagnosis of mycobacteria with this sys­tem as compared to the conventional culture detection method [28–32].

Before inoculation, MB/BacT antibiotic supplement (amphotericin B, azlocillin, nalidixic acid, polymyxin B, trimethoprim, vancomycin) in reconstitution uid (amaranth, glycerol, Tween-80, puried water) is added to the bottle. The tubes are incubated at 37°C and read every 10min. The results can be automatically read on the computer screen attached to the instrument (bioMérieux). The turnaround time is higher for BacT than the conventional culture methods [38, 39].The BacT alert 3D equipment can be implemented for detection of mycobacteria in high burden countries.

The current guidelines by the World Health Organization (WHO) have specically determined microscopy as the initial investigation for patients suspected of TB. WHO and the International Union Against Tuberculosis and Lung Disease (UNION) pan the technical guidelines for microscopy-based diagnosis [40]. Particularly in pulmonary TB, the guidelines suggest collecting sputum samples at three different time points. The quality of sputum samples is crucial for microscopy. Sputum, a sticky and thick sample taken with spontaneous expectoration, is consid­ered the ideal sample. Nasal, pharyngeal secretions or saliva with blood are not appropriate samples for microscopy. Bronchial lavage and gastric lavage are also taken as samples if medical professionals advise for it. On the other hand, in cases other than pulmonary TB, samples like cerebrospinal uid (CSF); pleural, ascitic, pericardial, joint, and other uids; biopsies; and resected material like pus, blood, and urine, are also tested initially with microscopy. The samples other than sputum have a low range of bacilli, therefore, are not relied on microscopy for the detection of TB [41, 42]. Microscopy is also considered for treatment monitoring as the pro­longed treatment of TB requires continuous monitoring. The low level of bacilli or dead bacilli detection at the last stage of intensive treatment is further conrmed by the solid culture technique to determine the viability of bacilli. Treatment failure is determined with repeated highly positive smear microscopy results even at the intensive phase of treatment [8]. Sputum smear microscopy, thus, provides a simple, fast, and cheap diagnosis method for the proper monitoring of patients. However, microscopic examination can identify the bacilli but are unreliable to differentiate between live and dead bacilli, similarly unreliable to differentiate between Mtb and

Depending on the use of heat during staining, ZN staining is further categorized into hot and cold staining [48]. Hot staining is widely used as the heating step helps in the solubilization of lipids present in the cell wall of mycobacteria and allows more penetration of the dye carbol fuchsin [49]. As per standard protocols, the smears are prepared directly from samples and dried. The slides are placed on a staining rack with smears facing up. The staining rack should have a gap of about 5cm. The whole smear is gently covered with 0.3% carbol fuchsin reagent. Then the slides are heated over the ame of a Bunsen burner, in swinging movements until the rst white steams are released. This is enough for carbol fuchsin to prop­erly penetrate the bacilli and bind to lipids. Care should be taken to not overheat, and the t slides/smear should not dry. Each slide is then rinsed individually in a gentle stream of running water until all free stains are washed away. Then comes the decolorizing step, in which the slides are ooded with 25% H2SO4 or 3% hydrochlo­ric acid in ethyl alcohol (decolorizing solution) for 2–3min. Slides are again thor­oughly rinsed with water, and excess water is drained from the slides. The slides are then counterstained by ooding the smear with 0.1% methylene blue for 30s and thoroughly rinsed with water. Smears are allowed to air-dry and observed under the microscope (100×) for AFB [50]. In 1915, Kinyoun published a method, which is