Mycobacterium tuberculosis Lecture Dec. 9, 2023 PDF

Mycobacterium tuberculosis and Nontuberculous Mycobacteria VIRGINIA P. MESOLA, MD, MS, SM(AAM), FPSMID, ASSOC PSP LECTURED TO UCMT DEC. 9, 2023  OBJECTIVES After reading and studying this chapter, you should be able: 1. To compare the general characteristic...

Mycobacterium tuberculosis and Nontuberculous Mycobacteria VIRGINIA P. MESOLA, MD, MS, SM(AAM), FPSMID, ASSOC PSP LECTURED TO UCMT DEC. 9, 2023  OBJECTIVES After reading and studying this chapter, you should be able: 1. To compare the general characteristics of mycobacteria to those of other groups of bacteria. 2. Clinical significance of the nontuberculous mycobacteria. 3. Discuss the safety precautions to be followed while working in a mycobacteriology laboratory. 4. Describe the appropriate specimen collection and processing procedures to recover mycobacteria from clinical samples. 5. Justify the digestion and decontamination of certain clinical specimens for the isolation of mycobacteria.  OBJECTIVES 6. Describe the principles and procedures for the stains used to demonstrate mycobacteria in clinical samples and isolates. 7. Compare the different culture media used for the isolation of mycobacteria. 8. Discuss the different tests used to identify mycobacteria. 9. Compare continuous monitoring systems to those of conventional media for detecting mycobacterial species in clinical samples. 10. Develop a protocol for the isolation and identification of Mycobacterium tuberculosis from a sputum specimen. 11. Discuss the clinical disease caused by Mycobacterium tuberculosis. 12. Discuss the use of the tuberculin test.  Genus Mycobacteria 1. Species that cause most dreaded diseases: a. Mycobacterium tuberculosis (tuberculosis) b. Mycobacterium leprae (Hansen’s disease, leprosy) 2. Immunocompromised patients cause resurgence of TB and diseases caused by nontuberculous mycobacteria (NTM) or mycobacteria other than tuberculosis (MOTT). 3. Some species of environmental saprophytes called atypical mycobacteria or MOTT cause human infections that often resembles TB.  General characteristics: 1. Slender, slightly curved or straight rods. 2. Cell wall has high lipid content (mycolic acid) which enables organism to resist staining and called acid fast bacilli (AFB). 3. Strictly aerobic. 4. Pathogenic mycobacteria grow slowly. 5. Require 2-6 weeks of incubation on complex media. 6. M. leprae fails to grow in vitro. 7. MTB complex consists of: M. tuberculosis, M. bovis (including the vaccination strain bacillus Calmette-Guerin), M. africanum, M. canettii.  Mycobacterium tuberculosis 1. Robert Koch in 1882 first described Mycobacterium tuberculosis. 2. WHO in 2011 estimated 12 million people worldwide has TB. 3. TB is usually a disease of the respiratory tract.  Primary tuberculosis: 1. After exposure to MTB, the development of tuberculosis depends on the cellular immune response of the patient, amount of exposure and virulence of the strain. 2. Tubercle bacilli are acquired from active cases, through sneezing or talking. Airborne droplets containing 1-5 um in size will enter the respiratory tract of exposed individuals and reach the lung alveoli. 3. MTB are phagocytosed by alveolar macrophages. 4. Capable of intracellular multiplication.  Primary tuberculosis: 4. In persons with adequate CMI, T cells arrive in 4-6 weeks due to macrophage activation (lymphokines), macrophage in the area of infection will destroy the intracellular mycobacteria leading to regression and healing. 5. In some exposed individuals the immune system does not eliminate the bacteria causing pathologic features of TB. 6. Granuloma formation may occur leading to healing.. 7. Without granuloma, lesions may heal without obvious pathology.  Primary tuberculosis: 8. After healing of first degree infection the bacilli are not totally eradicated and can remain viable in granulomas for months or years. 9. In infected individuals, there is a potential for reactivation of tuberculosis. 10. Clinical diagnosis of primary tuberculosis is usually limited to signs and symptoms and a positive PPD skin test. 11. Some infected individuals will develop progressive (active) pulmonary disease due to failed CMI which failed to stop multiplication of the bacilli. 12.10% of young adults may progress to active disease from their primary infection.  Reactivation Tuberculosis: 1. Risk of reactivation tuberculosis is 3.3% during the first year of a positive PPD. 2. Development of disease is slow (insidious) and consist of fever, shortness of breath, night sweats, chills, fatigue, anorexia, weight loss. 3. 20% of individuals are asymptomatic. 4. Diagnosis is confirmed by: a. stained smear b. culture of sputum, gastric aspirates or bronchoscopy specimens (95%).  Extrapulmonary tuberculosis: 1. Extrapulmonary tuberculosis is less common. 2. Miliary tuberculosis is seeding of many organs outside the pulmonary tree with AFB through hematogenous spread. 3. Children account for most cases of miliary tuberculosis. 5. Resolution is common, AFB rarely seen in pleural fluid, culture + in 20-50% of cases, higher + yields with pleural biopsies.  Identification of Mycobacterium tuberculosis: 1. Colonies – slowly growing spp. a. Typically raised with a dry, rough appearance b. Non-pigmented, buff colored c. Cord formation due to elaboration of cord factor d. Optimal growth – 35-370C Cording Cording – electron microscope Electron microscope 2. Biochemical reactions: a. niacin test +, reduction of nitrate to nitrite b. production of catalase (isoniazid-resistant strains catalase -) 3. Inhibited by nitroimidazopyran or p-nitroacetylamine- B- propiophenone (NAP). Distinguished from M. bovis Treatment: 1. Involves the use of more than one anti-mycobacterial agent. Multidrug Resistant Mycobacterium tuberculosis (MDR-TB): 1. Drug resistance is usually acquired by spontaneous mutation. 2. MDR-TB (multidrug resistant TB) – resistance to at least INH and rifampin. 3. XDR-TB (extensively drug-resistant TB) – resistance to INH and rifampin plus resistance to any fluoroquinolone and at least one of three injectable second line of anti-TB drugs. 4. The laboratory must identify the MTB species rapidly and perform AST so that appropriate therapy can be administered as soon as possible.  Mycobacterium bovis: 1. Produces tuberculosis primarily in cattle and other ruminants also in dogs, cats, swine, parrots and humans. 2. The disease in humans closely resembles that caused by MTB. 3. M. bovis belongs to the MTB complex. 4. Grow very slowly on egg-based media. 5. On Middlebrook 7H10 medium, colonies are similar to Mycobacterium tuberculosis but slower to mature. 6. Niacin negative,  Clinical Significance of Nontuberculous Mycobacteria (NTM): 1. Most NTM are found in soil and water. 2. Opportunistic pathogens. 3. Chronic pulmonary disease resembling tuberculosis. 4. Some are associated with cutaneous infections. 5. Infections caused by NTM are not considered transmissible from person to person.  Slowly growing Mycobacteria 1. Photochromogen ◦M. kansasii ◦M. marinum 2. Scotochromogen ◦M. scrofulaceum 3. Non-photochromogen or nonchromogen ◦M. avium-intracellulare  Mycobacterium avium complex (MAC): Slowly Growing Species 1. Epidemiology: a. Mycobacterium avium and M. intracellulare complex (MAC). b. Common environmental saprophytes and recovered from soil, water, house dust, etc. c. Cause disease in poultry and swine. d. Animal to human transmission has not been shown. e. Environmental sources especially natural waters – reservoir for most human infections. f. Zoonotic g. Associated with AIDS infection. h. Most common NTM causing disease similar to TB in the United States. Mycobacterium avium complex (MAC): 2. Clinical Infections: a. Pulmonary disease similar to MTB. b. Most common systemic bacterial infection in patients with AIDS. 3. Laboratory Diagnosis: a. Reported as MAC and not M. intracellulare or M. avium. b. Colonies grow slowly, c. Microscopic – short, coccobacilli, uniformly stained no beading or banding. d. Heat stable catalase +. e. Nucleic acid probes – available. Thin, transparent Yellow with age  Mycobacterium kansasii: 1. Epidemiology: a. Second to MAC as the cause of NTM lung disease. b. Isolated from water. c. Contagious from person to person. 2. Clinical Infections: a. Chronic pulmonary disease involving the upper lobes with evidence of cavitation and scarring. 3. Laboratory diagnosis: a. Slow growing organisms b. Long rods with cross banding c. Some cording d. Strongly catalase +  Mycobacterium kansasii: Laboratory diagnosis: 1. Some colonies may be: a. photochromogenic – form pigment when exposed to light but nonpigmented in the dark b. scotochromogenic – produce pigment in light and dark c. nonchromogenic or nonphotochromogenic – no pigment in light and dark 2. Nucleic acid probe available  Mycobacterium marinum: 1. Implicated with diseases of the fish 2. Cutaneous infection in humans – tender red subcutaneous nodule, or swimming pool granuloma 3. Cutaneous infections in humans 4. Photochromogen 5. Microscopic – long rods with cross barring. 6. Niacin +, urease +  Mycobacterium ulcerans: 1. A rare cause of Mycobacteriosis, called Buruli ulcer. Difficulty to isolate. 2. Worldwide, the third most common Mycobacterium spp, behind M. tuberculosis and M. leprae. 3. Disease – painless nodule under the skin after previous trauma. No fever or systemic symptoms. 4. Colonies after 6-12 weeks, nonphotochromogenic.  Mycobacterium scrofulaceum: 1. The most common cause of cervical lymphadenitis in children before MAC. 2. Infection – one or more enlarged nodes often adjacent to mandible and high in the neck with little or no pain. 3. Colonies are scotochromogen. 4. Urease +, catalase +, do not reduce nitrate 5. Microscopic – uniformly stained, AFB, medium to long rods.  Laboratory diagnosis 1. Many species of mycobacteria both saprophytes and potential pathogens may be isolated from humans. 2. If the isolate is mycobacteria other than M. tuberculosis, the lab should be able to identify the species in by in vitro tests. 3. Diagnosis of specific organism – crucial in determining therapy. 4. ID by Runyon classification is inadequate. 5. Do bacteriologic ID of organism and repeated demonstration in patient secretions in the absence of other potential pathogens. 5. Tuberculin test is not helpful. 6. A + AF smear and a (–) tuberculin test may lead one to suspect the disease.  Identification 1. Grow well on Lowenstein-Jensen (LJ), Middlebrook 7H10 and other media used for culturing Mycobacterium tuberculosis. 2. Niacin test negative. 3. Only a few of these are known human pathogens  Epidemiology 1. Ubiquitous and found in all parts of the world. 2. Endemic in certain geographic areas. 3. No known 1o animal host but apparently exist in the soil. 4. No direct transmission from man to man. 5.Disease results from 2 coinciding events: a. Colonization of a large # of mycobacteria b. Impairment of body’s defense mechanism  Disease (MOTT) is group according to organ involvement 1. Pulmonary disease a. Most common manifestation in adults in USA. b. Most common cause: M. kansasii, M. fortuitum, M. avium-intracellulare complex (MAC) 2. Lymphadentis – usually seen in children. a. M. scrofulaceum 3. Cutaneous lesions (skin lesions). a. USA – M. marinum 4. Disseminated infections. a. M. kansasii, M. fortuitum, MAC b. AIDS - disseminated infection usually caused by M. avium-intracellulare complex (MAC).  Rapidly growing mycobacteria 1. Most species are purely environmental saprophytes. 2. M. fortuitum and M. chelonei – occasional pathogens of humans, birds and animals 3.M. fortuitum-M. chelonei complex – share similar characteristics and seen in same type of infection 4. M. smegmatis and M. phlei – do not cause disease  Mycobacterium leprae 1. Is the causative agent of Hansen’s disease (leprosy), an infection of the skin, mucous membranes and peripheral nerves. 2.12 million persons have leprosy worldwide. 3. Hansen’s disease is not highly contagious. 4. Man is only natural host. 5. Infection thru contact with patients with Lepromatous leprosy who shed organisms in nasal secretions. 6. Major portal of entry is the respiratory tract. 7. Genetic factors contributes to susceptibility and type of response to infection with M. leprae.  Ridley and Jopling classification 1.Tuberculoid leprosy (TT) 2. Borderline tuberculoid leprosy (BT) 3. Borderline leprosy (BB) 4. Borderline lepromatous leprosy (BL) 5. Lepromatous leprosy (LL) Only TT and LL are stable, other forms are unstable Spectrum of disease characterized by pronounced variations in clinical, histopathologic and immunologic findings  Clinical manifestations 1. Immunologic status of patient – determines the prognosis. 2. TT – benign, few skin lesions 3. BB – intermediate in position 4. LL – most severe, extensive form of disease – leonine facies, trauma and 2o infection 5. Long incubation period – 2-3 yrs (40 yrs) 6. Earliest symptoms – asymptomatic, slightly hypopigmented macule usually in trunk or distal portion of extremities 7. ¾ of patients – has solitary lesion and heal spontaneously 8. Disease progress from 1 form to another  Mycobacterium leprae: 1. 2 major forms: a. Tuberculoid leprosy b. Lepromatous leprosy 2. Tuberculoid leprosy: a. Symptoms – skin lesions and nerve involvement that can produce areas with less sensation. b. Develop and effective CMI response c. Optimal growth temperature - 300C d. Bacteria tend to remain in the extremities. e. Spontaneous recovery often occurs.  Lepromatous leprosy Lepromatous leprosy do not produce an effective CMI response. a. The disease is slowly progressive and life threatening if untreated. b. Findings – skin lesions and progressive symmetric nerve damage, lesions of the mucous membranes of the nose may lead to destruction of the cartilaginous septum, resulting in nasal and facial deformities (leonine facies). c. Skin smears - AFB are rod shaped bacterium abundant in samples.  M. leprae – nerve involvement 1. M. leprae is an obligate intracellular parasite that multiplies very slowly within the mononuclear phagocytes, especially the histiocytes of the skin and Schwann cells of the nerves. 2. It has strong predilection for the nerves. 3. TT – nerve damage is non specific due to CMI 4. LL – nerves are infected with numerous bacteria in Schwann cells. Lepromin test  Lepromin test 1. Not diagnostically useful – of value in determining the position of the patient on the immunologic spectrum. 2. Lepromin – suspension of heat killed M. leprae. 3. Early reaction – Fernandez reaction (48 hours after). 4. Late reaction – Mitsuda reaction (21 days). 5. TT - + early and late reactions. 6. LL – negative. 7. Lacks specificity. 8. + reaction – persons with TB, healthy children with BCG.  Laboratory diagnosis 1. Laboratory diagnosis depends on microscopic demonstration of AFB that cannot be cultured from the skin biopsy specimens. 2. Suspect leprosy – from symptoms, type and distribution of lesion, history of living in endemic area. 3. Demonstration of AFB in smears of skin lesion, nasal scrapings, ear lobes, tissue secretions 4. LL – bacilli numerous 5. TT – very difficult to impossible to detect  M. leprae - immunity 1. Leprosy is a disease of low infectivity. 2. Lepers are immunologically defective with respect to M. leprae. 3. CMI and clinical forms: a. TT strong (CMI) DTH to lepromin b. LL loss of CMI 4. Antibodies to M. leprae has no protective role. 5. Antibodies to M. leprae cross react with other mycobacteria.  Mycobacterium leprae - Morphology 1. Acid fast in modified mononuclear or epitheloid cells called lepra cells arranged like packets of cigars. 2. Organisms are found singly or in large masses called globi. 3. Bacilli are usually straight or curved and may stain uniformly or show granular beads. 4. Uniformly stained bacilli are healthy bacilli; beaded bacilli are probably non viable.  Mycobacterium leprae 1. Acid fastness can be removed by extraction with pyridine – distinguishes M. leprae from other mycobacteria. 2. Structure resembles that of M. TB. 3. Not grown in tissue cultures of various types of human cells. 4. Grown experimentally only in animals – mice and armadillo. 5. Phenolase in M. leprae has been obtained from lepromatous nodules – separates M. leprae from other mycobacteria and nocardias.  M. leprae – experimental disease in animals 1. Animal models – extensively used for study of the organisms and experimental leprosy. 2. Footpads of normal mice – injected with materials from leprosy patients. 3. In mouse, infection can be initiated with as low as 1-10 bacilli. 4. Footpad temp. is 30oC – maintained by keeping room tempt at 200C-30oC, the secret in the footpad success. 5. Footpads of mice – generation time is 12 days. 6. The definitive laboratory diagnosis is the development of disease in laboratory mice following inoculation of patient biopsy material to the mouse foot pads.  M. leprae – experimental disease in animals footpads of mice 7. Multiplication continues for 150-180 days, until # of bacilli reaches 1 X 106 bacteria. Multiplication stops because of CMI. 8. Used for drug screening and vaccination experiments. 9. Nine-banded armadillos has been used. 10. Armadillos has disseminated leprosy. 11. Used to study immunologic factors that control development of disease. 12. Provide large numbers of M. leprae for vaccination study. 13. Man is highly resistant to experimental infection. Laboratory diagnosis Laboratory Diagnosis of Tuberculosis VIRGINIA P. MESOLA, M.D., M.S. FPSMID, SM(AAM/ASCP). FPASMAP PROFESSOR UNIVERSITY OF CEBU SCHOOL OF MEDICINE TB diagnosis and management depends upon a reliable and prompt laboratory service.  Specimen collection: 1. Acceptable specimens – respiratory specimens, urine, feces, blood, CSF, tissue biopsies, aspiration of any tissue or organs. 2. Successful isolation of mycobacteria from clinical specimens begins with properly collected and handled specimens. 3. When possible collect specimen before the initiation of therapy. 4. All specimens should be transported to the lab immediately after collection. 5. If immediate transport is not possible, refrigerate specimen overnight. 6. If possible process specimens daily because delay may lead to false negative cultures and increased bacterial contamination.  Specimen collection: 7. Each specimen should be collected in an individual container. 8. Most commonly recommended container is a sterile wide-mouth cup with a tightly fitted lid. Or a sterile 50 ml centrifuge container. 9. Use of swab for clinical specimen is discouraged. 10. Aseptic collection is important. 11. Specimen from any site is acceptable. 12. Each specimen type when properly collected, transported and processed may have an intrinsic maximal yield.  Sputum and other respiratory secretions: 1. Respiratory secretions as sputum and bronchial aspirates are the most common specimen received. 2. Early morning sputum is collected on 2 consecutive days. Recent studies show that 3 specimens does not significantly increase the sensitivity of detecting the bacteria. a. if 1 of 2 specimens are positive in direct smear, this is enough to confirm a diagnosis.  Sputum and other respiratory secretions: b. if none or only one the first 3 sputum smear is positive, additional specimens are needed for culture confirmation 4. Pooled specimens are unacceptable because of increased contamination. 5. Specimens: a. 5-10 ml sputum by deep cough or expectorated sputum b. induced by inhalation of an aerosol of hypertonic solution c. Bronchoscopy if no sputum is produced. d. Bronchial washing or bronchoalveolar lavage (BAL) e. Bronchial brushing may be more diagnostic  Gastric aspirates and washings: 1. Gastric aspirates used to recover mycobacteria that may have been swallowed during the night. 2. Use for patients that do not produce sputum by aerosol induction, children below 3 years old and non-ambulatory patients. 3. Gastric lavage is better than bronchial alveolar lavage (BAL) to detect mycobacteria in children. 4. Gastric aspirates – obtained in the morning after an overnight fast.  Gastric aspirates and washings: 5. Instill 30-60 ml orally thru nasogastric tube aspiration. Collect 3 specimens in 3 days. 6. Process specimen immediately for gastric acid kills mycobacteria. 7. The specimen should be neutralized with sodium carbonate or another buffer at ph 7.0 as soon as possible after collection.  Urine: 1. Collect first morning midstream urine. Minimum of 15 ml. is collected in a sterile container. May be collected also through indwelling catheter with a sterile needle and syringe. 2. Refrigerate between collection and processing. Process immediately. 3. Pooled specimens collected over 12-24 hours are not recommended more subject to contamination and contain fewer viable tubercle bacilli.  Stool 1. Exam for AFB (Mycobacterium avium intracellulare) useful in cases of AIDS. 2. Culture of feces for mycobacteria from patients other than with AIDS is not warranted. 3. Collect in clean container without preservatives and process immediately, if not possible freeze at -200C.  Blood: 1. Mycobacteremia seen in patients with AIDS caused by MAC. 2. Cultured in: Isolator lysis-centrifugation system, MCO/F and BactT/Alert MB blood medium. 3. Tissue and Other Body Fluids: Volume: a. CSF - 2 ml b. 3-5 ml for exudates, pericardial and synovial fluids; c.10-15 ml for abdominal and chest fluids.  Tissue and Other Body Fluids: 1. Specimens from lung, pericardium, lymph nodes, bones, joints, bowel or liver may be used. If tissue is not processed immediately – add 10-15 ml sterile saline to1 prevent dehydration. 2. May collect fluid with fibrinogen (pleural, pericardial) – transfer to container with anticoagulant. 3. Histopathologic changes in tissues like caseating granuloma, are consistent but not specific for mycobacterial disease.  Transfer to Laboratory ▪Within 24 hours (or one working day, ▪max 48 hours) ▪Minimize overgrowth ▪Maintain AFB character ▪Potentially infected clinical samples ▪Routine procedures  Isolation and Identification of Mycobacteria 1. Traditional features used to differentiate species within the genus Mycobacteria. a. rate of growth b. colony morphology c. pigmentation d. nutritional requirements e. optimal incubation temperature f. biochemical test results Isolation and Identification of Mycobacteria 2. More rapid techniques include: a. broth-based culture system, 3. A limited number of species-specific nucleic acid probes offer rapid identification of the culture isolates. 4. Polymerase chain reaction (PCR) – increase the sensitivity of nucleic acid probes. 5. Other techniques – high pressure liquid chromatography (HPLC) used to distinguish mycobacterial species.  Laboratory Safety Considerations: The serious nature of tuberculosis and the usual airborne route of infection require that special safety precautions be used by anyone handling the mycobacterial specimens. Personal Safety: Mycobacteriology laboratory should ensure that every employee is: 1. Provided with adequate safety equipment. 2. Trained in safe laboratory procedures. 3. Informed of the hazard associated with the procedures. 4. Prepare for action following an unexpected accident. 5. Monitored regularly by medical personnel. 6. Lab personnel must use appropriate safety equipment and established procedures. Skin test (PPD) done on 1st day – if negative repeat regularly; If PPD + counsel regularly.  Laboratory Safety Considerations -Ventilation: 1. Lab design and ventilation play important roles in mycobacterial laboratory safety. Ideally, the mycobacteriology laboratory should be separate from the remainder of the laboratory and have a noncirculating ventilation system. 2. Area for processing of specimen and culture should have negative air pressure in relation to other areas – the airflow should be from the clean areas like corridor to less clean areas (mycobacteriology laboratory) 3. 6-12 room air changes/hr effectively remove 99% o more of airborne particles within 35-45 minutes.  Laboratory Safety Considerations- Biological Safety Cabinet 1. To avoid the inhalation of airborne bacilli it is important that dispersal of bacilli into the air be minimized. This is the single most important piece of equipment in a mycobacteriology laboratory. a. Class I negative pressure cabinet b. Class II vertical laminar flow cabinet 2. Proper installation, maintenance and testing are essential to their performance. 3. Tested and recertify at least yearly. 4. Processing clinical specimens, transferring viable cultures outside a safety cabinet should not be permitted. 5. To prevent dispersal of infectious aerosols into the lab area, all potentially infectious materials should be tightly covered when outside the safety cabinet. 6. Specimens should be centrifuged in aerosol-free safety carriers, and remove tubes from carriers only inside the safety cabinet. 7. Specimens to be taken out from the cabinet for transport to decontaminated area must be covered. 8. After safety cabinet has been disinfected, the ultraviolet (UV) light should be used to eliminate any further contamination of surfaces and any airborne bacteria. 9. Due to harmful effect of UV light it should br turned on only when the safety cabinet is not in use. Laboratory Safety Considerations –Use of proper disinfectant 1. Covering the work surface with towel or absorbent pad soaked in disinfectant reduces the accidental creation of infectious aerosols. 2. Use a tuberculocidal disinfectant. Sodium hypochlorite of 0.1% -0.5% (1:50-1:10 dilution of most household bleaches). Freshly prepared daily, contact time 10-30 minutes. 3. 5% phenol with contact time of 10-30 minutes. Phenol-soap mixtures containing orthophenol with contact time of 10-30 minutes. 4. 3-8% formaldehyde or 2% alkaline glutaraldehyde with contact time of at least 30 minutes, Laboratory Safety Considerations – Other precautions 1. Sterilization of wire loop: a. Use electric incinerator in the safety cabinet. b. Use an alcohol-sand flask to avoid aerosols – c. Use splash-proof discard containers to prevent aerosol formation and possible cross contamination. 2. PPE provides extra safety to staff working in laboratory. a. Use gloves and lab coats for processing cultures or specimens. b. Use respiratory protection when performing procedures outside safety cabinet. c. Minimum respiratory protection – N95 mask Laboratory Diagnosis of Tuberculosis 1. Early diagnosis of tuberculosis and drug resistance improves survival and by identifying infectious cases. 2. The microbiological diagnosis of tuberculosis (TB) is an important tool for disease control.  Digestion and Decontamination of Specimens: To ensure optimal recovery of mycobacteria from clinical specimens, many specimens must be processed before inoculation into culture media. 1. Specimens from sterile body sites will be concentrated by centrifugation (if large volume) and inoculated. Specimens that may contain commensal bacteria should be decontaminated then concentrated. 2. Most clinical specimens like sputum has an abundance of nonmycobacterial organisms. When cultured the abundant non mycobacteria will overgrow the slowly growing mycobacteria. Digestion and Decontamination of Specimens: 3. Purpose of digestion-decontamination process a. liquify the sample b. allow the chemical decontaminating agent to contact and kill the nonmycobacterial organisms. 4. High lipid content of cell wall makes the killing 5. With liquefaction, surviving mycobacteria can be concentrated with centrifugation. Liquifying mucin enables the bacteria to come in contact with the nutrients in the medium and use for growth.  Digestion and Decontamination: 6. Specimens that contain mucus and require digestion and decontamination – sputum, gastric washing, BAL, bronchial washing, and transtracheal aspirate. 7. Specimens that require decontamination – voided urine, autopsy tissue, abdominal fluid and any contaminated fluid. 8. Specimens from sterile sites – blood, CSF, synovial fluid, and tissue biopsy from deep organs do not require decontamination. 9. Sterility should be strictly maintained in collection and transport. 10. Stool decontamination is especially difficult and may require repeated attempts.  Decontamination and digestion agents: 1. Each laboratory should maintain a balance between the rate of recovery of mycobacteria and suppression of contaminants. 2. A range considered acceptable is between 2% and 5% of bacterially contaminated mycobacterial cultures. 3. The optimal decontamination procedure requires an agent that is mild and yields growth of mycobacteria while controlling contaminants. 4. The use of selective media may diminish the need for harsh decontamination procedures.  Decontamination and digestion agents: 1. Sodium hydroxide (NaOH) – usual concentration is 2%, 3%,or 4% - serves as a digestant and decontaminating agent. 2. N-Acetyl-L-cysteine – A liquifying agent + NaOH is commonly used. Liquifaction of the samples allow the decontaminating chemical to come in contact with the bacteria. Contamination can be controlled by lower concentration of NaOH, this improves the recovery of the mycobacteria. Laboratory Diagnosis tuberculosis A. Conventional methods B. Molecular methods for laboratory diagnosis (Genotypic methods)  Conventional Methods 1. Microscopy A. The most rapid diagnostic method. ◦Standard light microscopy (LM) ◦Flourescent microscopy (FM) ◦LED FM microscopes  Staining for Acid Fast Bacilli (AFB): 1. When gram stained, Mycobacterium spp – stain faintly or not at all. 2. Acid fast smears are prepared directly from clinical specimens and digested, decontaminated and concentrated. 3. Acid fast staining methods used are Ziehl-Neelsen and Kinyoun stains.. 4. Ziehl-Neelsen procedure involves the application of heat and Kinyoun acid fast stain is a cold stain. 5. Ziehl-Neelsen method tends to provide more consistent results. 6. Examine slides under OIO for 15 minutes, viewing a minimum of 300 fields before a slide is called negative.  Staining for Acid Fast Bacilli (AFB): 6. Auramine or auramine-rhodamine fluorochrome stains are more sensitive than the carbolfuchsin stains. 18% of all culture positive specimens are positive on auramine-rhodamine stain but negative on Kinyoun or Ziehl-Neelsen stain. 7. If rapidly growing mycobacteria are suspected smears should be stained with carbolfuchsin and a weaker decolorizing process used. The sensitivity of acid fast smear varies from 20% to 80% depending on the extent of the infection.  Microscopy An AFB smear report is not only of great value in diagnosis and prognosis, but also helps to grade the infection. The scale recommended by RNTCP (Revised National Tuberculosis Control Programme) is as follows: 3+ : > 10 AFB/ oil immersion field 2+ : 1-10 AFB/ oil immersion field 1+ : 10-99 AFB/ 100 oil immersion fields Positive scanty: 1-9 AFB/ 100 oil immersion fields Negative: no AFB/ 100 oil immersion fields Acid Fast Stain  Conventional Methods 2. Culture 1) Culture and drug resistance testing (DST) a. Phenotypic methods b. microscopic observation drug c. susceptibility assay (MODS)*  Limitation in culturing 1. Mycobacterium species are slow growing 2. Need 6-8 weeks for growing 3. Specimens can be contaminated while growing, need repeated specimens, in turn patients lose confidence in the laboratory Standards for Laboratory Diagnosis Current best practices Simple and straightforward Not replicate or replace the National SOP Culture Isolation and Identification Conventional solid culture Automated liquid culture on all samples ◦Set up within 24 hours of receipt ◦Plus conventional solid culture Complete identification of most mycobacterial isolates within 21 days.  Culture media and isolation methods: 1. Mycobacteria are strictly aerobic. Generation time is longer than 12 hours. It has the longest replication time, at 20-22 hours. 2. Most pathogenic mycobacteria require 2-6 weeks of incubation. 3. The growth of MTB is enhanced by 5% -10% CO2. pH is between 6.5 and 6.8 for growth medium. 4. A pathogenic mycobacteria for humans, M. genavense does not grow on media routinely used for mycobacteria. And requires extended incubation (6-8 weeks). 5. M. leprae fails to grow on artificial media.  Culture media and isolation methods: 5. Three general types of culture media a. egg based media – Lowenstein Jensen (LJ) with a shelf life of 1 year b. serum albumin agar media -Middlebrook 7H10 and 7H11. Addition of antimicrobial agents makes the media suppress the growth of contaminating bacteria. May perform drug susceptibility on this media. c. liquid media 6. Solid based medium – LJ media with liquid based medium recommended for routine culturing of specimens for recovery of AFB.  Culture media: 7. Liquid media: a. Mycobacteria grow more rapidly in liquid medium 8. BACTEC 460TB an automated radiometric method using Middlebrook 7H12. a. Improve the isolation of Mycobacteria and reduces recovery time compared with conventional methods. 9. In smear + MTB may be detected in 7-8 days. 10. MGIT 960 system a nonradiometric microbial detection using Middlebrook 7H9.  BACTEC MGIT 960 CULTURE SYSTEM FOR MYCOBACTERIA MGIT Lowenstein Jensen Medium Niacin Test TBC ID  Laboratory Identification: 1. Preliminary ID of mycobacteria: 1st confirm that isolate is AFB. 2. Presumptive ID in speciating mycobacteria. a. Colony morphology – smooth and soft or dry and friable appearance. Cording (curved strands of bacilli) seen in rough colonies. b. Growth rate – growth rate and recovery time depend (3-60 days) on species of mycobacteria c. Temperature – Optimal temp may be narrow – 300C to 320C. M. xanopi grows best at 420C. d. Photoreactivity – photochromogens, scotochromogens, nonchromogenic or nonphotochromogenic.  Biochemical identification: A panel of biochemical tests can identify most mycobacteria isolate but slow growth of bacteria is so slow, molecular technology is being used. No single biochemical test should be relied on for the identification as a species. 1. Niacin test – 95% of MTB produce free nacin (nicotinic acid) because this species lacks the niacin connecting enzyme. 2. Nitrate reduction – uncommon among the Mycobacterium spp. 3. Catalase – Mycobacteria are catalase positive. Hybridization and Nucleic Acid amplification test for Mycobacterium tuberculosis 1. The use of the nucleic acid hybridization techniques allow for the rapid identification of several common mycobacterial spp. 2. The sensitivity of the assay varies from 95% to 100% depending on the species and species complex. 3. Many laboratories are using PCR test. 4. Automated DNA sequencing is the most accurate method for identification of Mycobacteria. The most commonly target is gene coding for 16S rRNA.  How could GeneXpert help? Low sputum smear positivity of PTB in HIV patients is common due to: ◦Poor immunity to localize the lesion ◦High possibility for TB dissemination to other organs GeneXpert can detect more TB among PLHIV GeneXpert  GeneXpert Mycobacterium tuberculosis and Rifampicin resistance Sputum, 2-3 ml or 3 consecutive days in sterile container (no food or solid particles) OR Body fluids, aspirate samples, 5-10 ml in sterile container OR Urine, 30 ml first morning specimen in sterile container OR Cerebrospinal fluid, 2-3 ml in sterile container OR Tissue specimen, 25-50 mg in sterile NSS Transport on ice Tissue specimen - room temperature Store at 4oC for 5 days  GeneXpert result Test processed in 2 hours Perform with microscopy Positive test with Xpert, smear may be + or - = MTBC Negative Xpert, positive smear = NTM Determine RIF resistance  Molecular Methods Nucleic acid amplification test (NAAT) ◦May provide results in a couple of days or even hours upon receipt of the specimen by the laboratory ◦PCR is the common format of NAAT ◦GeneXpert MTB/RIF ◦Speed up the diagnosis of both TB and its resistance to the major anti-TB drugs  Immunodiagnosis of MTB infection Tuberculin Skin test- used to determine exposure to M tuberculosis. 1. Use – protein extract and purified from cell wall of culture grown MTB. Purified protein derivative (PPD). 2 Inject intradermally 0.1 ml into patient’s forearm and read after 48 hours. a. raised firm area (induration) 10 mm or larger (reactive). In immunocompetent individual indicates past exposure to MTB. b. induration of 10 mm or less. In immunocompromised patient with previous MTB infection, or has Mycobacterium spp infection. 4. Detects a patient’s CMI response to the bacterial antigen in type IV hypersensitive reaction.  Immunodiagnosis of MTB infection Serology (IGRAS) 1. Clinical, radiologic and microbiological tests are currently used for the diagnosis of M. tuberculosis infection. 2. Quantiferon-TB Gold Assay and the T.SPOT.TB measure the CMI response in whole blood samples to mycobacterial antigens. 3. These assays are not affected by BCG vaccination and do not cross react with antigens from most other mycobacterial spp.  Serology (IGRAS) 4. Results are available in 2-3 days. They are more expensive that tuberculin testing. 5. These tests are called the interferon-gamma released assays measure interferon gamma production by cells that have been stimulated by 2 or 3 secretory, low molecular weight mycobacterial peptides. 6. IGRAS have sensitivity and specificity greater than 96%. However less reliable in children, making tuberculin testing recommended for children younger than 5 years.  Susceptibility testing Complete within 30 days of initial receipt of clinical sample for primary agents ◦Isoniazid, rifampicin, pyrazinamide, ethambutol ◦Takes 10-20 days by liquid proportion (automated) or resistance ration Molecular detection ◦Rifampicin within 24h if MDRTB suspected ◦Isoniazid under development Done at RCM with accreditation, IQC, EQA Susceptibility Testing of Mycobacterium tuberculosis: 1.With increased incidence of mycobacterial diseases and development of MDR strains, patient must be on appropriate therapy. 2.CDC recommends isolates to be tested for susceptibility to INH, rifampin, ethambutol and streptomycin.Pyrazinamide is considered. 3. Absolute concentration method determines the MIC. 4.Currently there are 4 commercial methods approved by FDA for susceptibility testing of MTB. 5. Susceptibility testing of most NTM is not performed routinely. Thank You!!!