DIAGNOSIS OF TUBERCULOSIS by Konrad T Juszkiewicz MD

DIAGNOSIS OF TUBERCULOSIS by Konrad T Juszkiewicz, MD, MPH Donald Burgess, Ph. D DRK Biomedical Research and Development LLC Almaty, November, 2013 1

TB or Not TB 2

Robert Koch Discovers Mycobacterium 3

Importance of Clinical services A clinicians knowledge Early diagnosis of the TB suspect Multiple diagnosis methods Proper treatment 4

TB suspect Productive cough longer than 2 -3 weeks. Fever for 1 month, or both Nigh sweats, Weight loss Fatigue Blood stained sputum. 5

Diagnosis Tuberculosis is a diversified, oportunistic disease Any organs can be involved. Any age group, gender no bar for Tuberculosis. Involvement of Lungs contribute to majority of tuberculosis spread. 6

Diagnosis of Pulmonary Tuberculosis Majority of Adults suffer with pulmonary tuberculosis. Microbiological examination of Sputum a quick method in proving the Diagnosis. Sputum examination in Children not as sensitive as in adult Diagnosis. Radiological examination of Lungs, most commonly prescribed investigation. Culture a reliable diagnostic tool 7

X - ray examination of chest a easily available Investigation tool 8

Microbiological Investigations Pulmonary Tuberculosis is the commonest presentation of Tuberculosis Sputum- the important specimen for identification and isolation of Acid fast bacilli. In the developing countries sputum the most important step in getting quick diagnosis 9

Sputum Specimens Well trained staff to obtain the appropriate specimen A few minutes of education to patients on importance of ideal sample make a great difference and improves the Diagnosis. Three sample collected 10

Sputum versus Saliva SPUTUM Specimens appear mucoid even, blood stained. Contains many polymorphonutrophils. SALIVA Appears clear, watery, and frothy. Contains many squamous epithelial cells Absence of polymorphoneutrophils 11

Microscopy in Tuberculosis Microscopy for Diagnosis of Tuberculosis initiated in 1880 The conceptions have not changed since then Best efforts should be put to obtain sputum Processing of saliva loses all valuable clues to diagnose 12

Microscopy and Tuberculosis Microscopy with Ziehl – Neelsen’s staining A century old procedure 13

Microscopy and Tuberculosis One need microscope, and few stains. Most rapid, economical, Can detect bacterial load. A diagnostic, and prognostic tool. A little of sputum 0. 2 µl is adequate. A prompt diagnosis after searching as few as 100 fields. 14

Limitation of Microscopy Repeated sample examinations (work load on technical staff) Training and dedication of microscopist The load of bacilli must be more than 10, 000 / 1 ml of sputum Low in sensitivity < 50 % Repeated requests for samples High drop out by patients for repeated samples Not dependable in pediatric age group 15

Smear showing Acid Fast Bacilli 16

Positive Smear by WHO All patients who have submitted three Specimens and at least two found to be positive for identification of AFB 17

Processing Direct Smear Negative Specimens Sputum Microscopy can be improved with its liquefaction, concentration and gravity sedimentation Popular solvents Sodium hypochlorite. Sodium hydroxide. Ammonium Sulphate N-acetyl-L-cysteine –sodium hydroxide. 18

Benefits of Liquefaction and Concentration Processing of sputum with chemicals and centrifugation improving sensitivity up to 18 %. Incremental yield ( positive with bleach minus positives with Ziehl – Neelsen stain) up to 9 %. Treating specimens with Sodium hypochlorite is Mycobactericidal and also kills HIV and improves the safety and acceptability by technical staff. 19

Microscopy weak side Smear negative tuberculosis patients HIV infected patients might prove negative in spite of presence of TB ( as few bacilli are expectorated) Needs concentration and liquefaction with chemicals Time consuming, needs more technical manpower 20

Acid Fast Bacilli Fluorescent Microscopy 21

Florescent Microscopy Advantages Useful when few bacilli are present Increases the sensitivity in HIV patients with tuberculosis Reduces the time needed for testing (good for screening) About 15 times as many fields of view can be scanned by fluorescent microscopy than by Ziehl – Neelsen’method in the same period Increases the sensitivity by 10 % Better conclusions with one or two specimens, unlike Ziehl Neelsen’s method needing 3 or > 3 specimens 22

Mycobacterium culture Culturing for isolation of Mycobacterium spp continues to be a Gold standard Need only 10 – 100 bacilli / 1 ml of sputum. 23

TB Culture Used In Surveillance Drug sensitivity testing patterns Identify treatment failures Useful in Patients presenting with respiratory symptoms, X- ray’s suggestive, but smear negative Cultures remain suggestive and helpful in early treatment periods or failed drug regimes. 24

Methods of Culturing on Lowenstein Jenson’s culture medium remain the affordable , economical method 25

Limitation in TB Culture Mycobacterium spp are slow growing Need 4 – 8 weeks for growing Specimens can be contaminated while growing, Need for repeated specimens, in turn patients loose confidence in laboratories. 26

Recent facts on TB Culture Useful in HIV infected patients with Tuberculosis Even few bacilli can be grown in spite of smear negativity The specimens to be incubated for longer time if only few bacilli present 27

Pitfalls in TB Culture Specificity might be lost due to contamination False positive results in 1 – 4 % of the cases Negative culture may in spite of x ray or clinical symtoms suggestive of tuberculosis 28

Growth of Acid Fast Bacilli on L J Medium 29

ADVANCES IN CULTURING TECHNIQUES Emerging Modern Media with accurate detection Replacing the Egg and Agar based medium 30

Emerging methods in Culturing MGIT – Mycobacterium growth incubator tube method Growth occurs in shorter time than egg medium Useful in HIV patients Less contamination cases Expensive method for developing countries 31

Blood TB culture Useful in HIV patients, and children Effective in isolation of Atypical mycobacterium Cost not effective Potential tool in the future for diagnosing Tb in HIV infected 32

Molecular Methods in Diagnosis of Tuberculosis Several methods available Mainly used as research tools Used to define TB origin (finger printing) Dr. T. V. Rao MD 33

Real Time PCR 34

PCR Method PCR ( Polymerase chain reaction ) used by investigators Most TB cases can be diagnosed with simple methods Not cost effective High specificity 35

Emerging Rapid Methods Fast Plaque TB uses phage amplification technology. ELISA ( Quanti. FERON – TB ) Enzyme-Linked immuno-spot ( ELISPOT ) ELISPOT proved highly useful to detect active tuberculosis in adults and children. 36

Emerging Technologies (MODS) Microscopic Observation Drug Susceptibility assay ( MODS ) A new method, gained importance in several reviews Use a tissue culture plate based assay with use of Middle Brook 7 HG Needs a inverted light microscope Even the drug resistance can be tested with Rifampicin, and Isoniazid Safe to work with cultures 37

Non Specific tests, Tuberculin Test ( Mantoux Test or PPD) Test to be interpreted in relation to clinical evaluation Results after 3 days Often false positive or false negative Even the induration of 5 mm to be considered positive when tested on HIV patients Lacks specificity 38

Serology in Tuberculosis Several serological methods evaluated Never gained the acceptance of the majority of the clinicians Serological tests has low sensitivity Many physicians depend on serology in extra pulmonary tuberculosis. 39

TB Diagnosis for HIV/AIDS Patients Consider the HIV status Identify the severity of Tuberculosis Early use of chest radiography Maximal number of sputum smear examinations Sputum concentration methods to be encouraged even by smaller laboratories Explore the use of Florescent Microscopy All smear negative specimens should be cultured 40

Limitations of Rapid Tests The testing needs advanced and sophisticated infrastructure. Unable to diagnose between active disease and latent infection Exclusively used in developing countries 41

Extra Pulmonary Tuberculosis Poses several challenges No optimal, specific diagnostic methods 42

Extra pulmonary Tuberculosis A real challenge to Clinicians and Laboratories Optimal specimen collection a priority Molecular Methods are growing need Clinicians start drug regimes on empirical basis Several serological tests for antibody determinations are evaluated 43

Identification of Atypical Mycobacterium A growing concern on infections with less known, uncommon Mycobacterium in immunosuppressed, an emerging infectious disease. 44

Atypical Mycobacteriae Need the help of reference laboratories Need different drug regimes, unlike typical Mycobacterium isolates Growing concern in the era of HIV/AIDS Dr. T. V. Rao MD 45

Gene. Xpert MTB/RIF A cartridge-based, automated diagnostic test allowing to identify MTB and it’s resistance to rifampicin (RIF). Co-developed by Cepheid, Inc. and Foundation for Innovative New Diagnostics with additional financial support from the US National Institutes of Health (NIH) and technical support from the University of Medicine and Dentistry of New Jersey 46

How the test works The Xpert MTB/RIF detects DNA sequences specific for Mycobacterium tuberculosis and rifampicin resistance by polymerase chain reaction It is based on the Cepheid Gene. Xpert system, a platform for rapid and simple-to-use nucleic acid amplification tests (NAAT) 47

How the test works The Xpert® MTB/RIF purifies, concentrates, amplifies (by real-time PCR) and identifies targeted nucleic acid sequences in the Mycobacterium tuberculosis genome It provides results from unprocessed sputum samples in 90 minutes, with minimal biohazard and very little technical training required to operate 48

TB Diagnosis Concerns and it’s Future Essential to explore and discover rapid, simple, accurate and unexpensive tuberculosis diagnostic tools An investment, greater scientific interest, political commitment a top priority Man power development and human resource utilization a greater concern Microscopy and Florescent Microscopy utilization as an immediate concern, and strengthening of treatment initiation protocols Effective methods in diagnosing smear negative patients as a growing priority 49

Thanks Spasiba Rakhmet Deburgess@drkbiomed. org Kjuszkiewicz@drkbiomed. org Cell. : +7 701 218 2377