MULTI DRUG RESISTANT MULTI DRUG RESISTANT TUBERCULOSISTUBERCULOSIS

(MDR – TB)(MDR – TB)Implications and Diagnosis

Dr.T.V.Rao MD

What is Tuberculosis ?What is Tuberculosis ?

• Tubrculosis is a disease caused by germs that are spread from person to person through the air. TB usually affects the lungs, but it can also affect other parts of the body, such as the brain, the kidneys, or the spine. In most cases, TB is treatable; however, persons with TB can die if they do not get proper treatment.

Tuberculosis continues to be important reemerging infection in era

of AIDS

Why Tuberculosis is Important

Disease• Tuberculosis is primarily a disease of men.• Occurs in locations where the transmission has

been stable or increasing for many years• The incidence of tuberculosis is highest in

among the adults• Most cases are due to recent infection or

reinfection• If controlled spread of transmission the caseload

shifts to older adults, and a higher proportion of cases are attributable to reactivation of latent infection.

Priorities to control Tuberculosis

• Today’s top priorities remain with Diagnosis and treatment of identified cases.

• The Global understanding leads to concrete plans to control the Disease.

• The Stop TB partnership’s Global plan for 2006 – 15 calls for a strengthening of Laboratory networks to facilitate detection of all forms of TB including smear negative Tuberculosis

Tuberculosis continues to be most Important Communicable

Disease

Problems in Diagnosis of Problems in Diagnosis of TuberculosisTuberculosis

• The persistent problem in difficulties in Tuberculosis management in Developing countries is based on

There is No Specific, Sensitive, Inexpensive and Rapid Method of Diagnosis of Disease

Why We Miss TuberculosisWhy We Miss Tuberculosis

• As many as 3 million patients who present every year with suspected Tuberculosis actively have sputum smear negative Pulmonary disease

or Extra pulmonary disease,which cannot be confirmed by sputum smear,

• We even miss Smear positive pulmonary tuberculosis, which cannot be addressed by

smear Microscopy.

ZN staining remianed the Gold standard for detection of AFB

Smear Microscopy continues to be backbone of DOTS programme

Mycobacterium as seen through Electron Microscopy

L J medium is a time tested medium for cultivation of Mycobacterium

tuberculosis

The Greater Challenges in Tuberculosis

• The Pediatric tuberculosis.• Multi drug resistant tuberculosis.Multi drug resistant tuberculosis.

( MDR – TB )( MDR – TB ) Tuberculosis in association Tuberculosis in association

with AIDSwith AIDS

Resistant Tuberculosis - A global Problem

Global control of Tuberculosis has been jeoparadised by two Major threats.

1 HIV/AIDS 2 Murltidrug resistant

tuberculosis

MDR TuberculosisMultidrug Resistant tuberculosis• Multi-drug resistant tuberculosis is defined

as resistance to Isoniazid and Rifampicin whether there is resistance to other drugs or not. It is therefore incorrect, by this definition, to classify a patient has having multi-drug resistant disease if they have an infection with a bacterium susceptible to Rifampicin but resistant to many other drugs.

Multi Drug Resistant Tuberculosis

• By definition Multi drug Resistant tuberculosis means

The Mycobacterium are resistant to Isoniazid Rifampicin There are varied statistical incidence It was found that only 3 % of all New

Tuberculosis cases that arise world wide every year are estimated to be Multi drug Resistant Tuberculosis

• Resistance to anti-TB drugs can occur when these drugs are misused or mismanaged. Examples include when patients do not complete their full course of treatment; when health-care providers prescribe the wrong treatment, the wrong dose, or length of time for taking the drugs; when the supply of drugs is not always available; or when the drugs are of poor quality.

• How does drug resistance happen?

• Drug resistance is more common in people who: • Do not take their TB medicine regularly • Do not take all of their TB medicines as told by

their doctor or nurse • Develop active TB disease again, after having

taken TB medicine in the past • Come from areas of the world where drug-

resistant TB is common • Have spent time with someone known to have

drug-resistant TB disease

• Who is at risk for getting MDR TB?

Detection of Drug resistance in Tuberculosis - is a priority

The development of new tools for improved diagnosis of TB including smear negative has been deemed to be a top priority by WHO

MDR TB strain have emerged in all regions of the world

Methods in detection of MDR - Tuberculosis

Traditionally drug susceptibility testing of mycobacterium isolates is performed on LJ medium,done using proportion method established as a gold standard

But cumbersome,lacks reproducabilityTime consuming

Drug susceptibility on L J Drug susceptibility on L J medium and Rapid Methodsmedium and Rapid Methods

In direct drug susceptibility testing was performed with use of proportion method for isolates from Lowenstein – Jensen culture.

Can also be reconfirmed with automated MBBacT system for isolates from automated mycobacterium cultures

Automation for MDR TB detection

Automation methods have reduced the time needed for performing drug susceptibility.

Testing possible in high resource laboratories MBBacT bottles were inoculated with 500 MBBacT bottles were inoculated with 500

microliters of decontaminated material and microliters of decontaminated material and cultures were monitored continuously for 42 cultures were monitored continuously for 42 days according to the recommendations of the days according to the recommendations of the manufacturer.manufacturer.

Other Methods in Detection of MDR TB (Colorometric

method) Colorimetric method based on oxidation

reduction with indicator Tetrazolium bromide.

Easier to implement with advent of rapid tetrazolium microplated assasy (TEMA )

Emerging methods in Emerging methods in detection of MDR - TBdetection of MDR - TB

MODS ( Microscopic observation of drug susceptibility test) developed in Laboratory at Universidad Peruna Cayetano Heredia in Lima depends on two long features of mycobacterial microbiology

1 M tuberculosis grwos rapidly in liquid (broth) medium than on solid phase and

2 Characterstic “ tangles “ of M.tuberculosis can be visualised under microscope long before colonies to the naked eye.

MODS for detection of MDR - TB

The scientific observations have proved that a single MODS culture of sputum sample offers more rapid and sensitive detection of tuberculosis and Murltidrug-resistant tuberculosis than the existing gold standard methods

used.

Advantages of MODS methodology in MDR detection

• All the chemical ingredients are available locally, except few which can be acquired easily.

• Existing infrastructure in District and Teaching hospital can be adopted for implementation of MODS

• Risk to technician handling the specimens is minimal, there is no absolute need to obtain grade III safety cabinets,

• Technology transfer is easier all the new technical manpower can be trained easily.

• The greatest advantage of MODS is earliest growth results can be read > 5 days of incubation in the Medium.

• A suggestive positive results can be given within 15 days, unlike routine cultures done on LJ medium

• Samples from patients receiving effective chemotherapy are rarely positive after after 21 days of incubation, however the MODS plates are retained for 40 days.

Advantages of MODS Assay

Performing MODS Assay

The MODS assay was performed as described in standard protocols,

Broth cultures were prepared in 24 well tissue culture plates ( Becton Dickinson) each containing 720 micoliters of decontaminant, 7H9 broth (Becton Dickinson), oxalic acid, albumin, dextrose, and catlase (OADC) (Becton Dickinson) and polymyxin,amphotericin B, nalidixic acid,trimethoprim and azlocillin (PANTA)

MODS Assay ( Contd)

For each sample, 12 wells were used; Four in control wells, no drug was used

and each of the remaining 8 wells, contained one of the four drugs at one of the two concentrations tested.

The cultures were examined under an inverted light microscope at magnification of 40x every day ( except weekends ) from 4 to day 15, on alternative days from 16 today 25 and twice weekly from 26 to 40day.

In MODS growth is identified by cording on Microscopy

MODS assay ( Contd)

To minimize cross contamination and occupational exposure, plates were permanently sealed inside plastic zip lock bags after inoculation and were subsequently examined with in the bag

Observation of growth in Observation of growth in MODSMODS

Positive cultures Positive cultures were identified by were identified by cord formation, cord formation, characteristic of characteristic of M.tuberculosis M.tuberculosis grwoth,in liquid grwoth,in liquid medium in drug medium in drug free control wells.free control wells.

MODS in Atypical Mycobacterium

Non tuberculous mycobacterium were recognised by their lack of cording or, for M,chelonae ( which forms cords) by rapid overgrwoth by day 5.

Contamination in MODS Assay

Fungal or bacterial Fungal or bacterial contamination was contamination was recognised by rapid recognised by rapid overgrowth and overgrowth and clouding in wells.clouding in wells.

If contamination was If contamination was detected, the detected, the original samples original samples was cultured again was cultured again after being after being decontaminated decontaminated once moreonce more

Confirming MODS results Spacer

oligonucleotide typing Spligotyping, polymerase chain reaction with multiple primers, or both were applied to all isolates from each of the three types of cultures in order to confirm the presence of M.tuberculosis.

Drug susceptibility TestingIn MODS

Drug susceptibility testing was performed with the use of MODS assay,

Growth in the drug free control wells but not in drug containing wells, indicates susceptibility

The drug concentration were as follows Isoniazid , 0.1 and 0.4 µg/milliliter Rifampicin 1 and 2 µg per millilitre

MODS and MDR detection

• The drug sensitivity for Rifampicin and Isoniazid can be tested and established the presence of MDR.

• In view of being chronic disease it is highly essential to establish MDR Tuberculosis at centers serving DOTS under WHO guidelines

• Starting and establishing centers to identify MDR at every district and Teaching Medical centers leads to better control of Tuberculosis

Why MODS is a better method for MDR TB

detection Increased sensitivity carries the risk of

increased bacterial overgrwoth ( for MODS culture and automated mycobacterial cuture ) though even after repeated decontamination,the sensitivity and specificity of MODS culture were unaffted.

If a MODS culture was negative on day 15,there is 99.7% chance that the sample is truly culture negative.

The negative MODS cultures can be discarded after 3 weeks

In spite of several advances In spite of several advances Tuberculosis continues to be a Tuberculosis continues to be a

difficult diseasedifficult disease

Created for awareness on Created for awareness on MDR TB in Developing worldMDR TB in Developing world

Dr.T.V.Rao MDemail

doctortvrao@gmail.com