GUIDED BY: DESAI P. BDIRECTOR & HEAD

DEPARTMENT OF MICROBIOLOGY,SHREE RAMKRISHNA INSTI. OF COMP. EDU. & APPL.

SCIENCES, SURAT.

“IN VITRO STUDY ON MULTI DRUG RESISTANT Mycobacterium

tuberculosis”

PRESENTED BY RAJANI S. D.

Lecturer LOKHAT SARVAJANIK TRUST, RAMPURA ,SURAT.

INTRODUCTIONTuberculosis is an ancient disease & it remains the leading cause of death of human being. It is mainly caused by Mycobacterium tuberculosis Mycobacteria causing human disease may be classified as follows:Mycobacteria

a). Typical tubercle bacilli.Human type M.tuberculosis.

Bovine type M.bovis.

Vole type M.microti.

Human type M.africanum.

b). Atypical mycobacteria (Runyon’s classification)

Group 1 PhotochromogenM. kansasiiM.marinumM. simiaeM. asiaticum

Group 2 Scotochromogen M.ScrofulsceumM. SzulgaiM. XenopiM.GordonaeM. Celatum

Group 3 Non photochromogens

M.avium-intracellular complex

M. Paratuberculosis

M.terrae-triviale

M.Shimoidae

Group 4 Rapid growers

M.fortuitum/ chelonae complex

M.thermoresistible

C). Saprophytic mycobacteria.

M.smegmatis

M.phlei

M.stercoris

M. thermo

National 75

North Zone 95

East Zone 65

West Zone 80**

South Zone 50

EPIDEMIOLOGY

The average prevalence of tuberculosis in India is estimated to be 5.05 per thousands. Prevalence of smear positive cause 2.7 per thousand and average. Annual incidence of smear positive cases at 84 per 1, 00,000 annually. [2]

The recent survey of WHO Says 98% of cases in developing countries with an increase of ~3% annually, 10% in African countries.

80% of cases seen in 22 countries; about half in 5 countries: India, China, Indonesia, Nigeria and Bangladesh.

In India every day more than 5,000 develop TB disease and more than 1,000 people die. [3]

It is estimated that yearly about 9 millions people in the world are attacked with TB, with 1.7 million deaths. [1]

AIM OF STUDY

To find out prevalence of Tuberculosis in South Gujarat region.

Identification and characterization of MDR in TB cases.

To find out MDR Pattern.

WHAT IS MDR-TB & XDR TB

Drug resistant TB is widespread and found in all countries surveyed. It emerges as a result of treatment mismanagement and is passed from person to person in the same way as drug sensitive TB.

Multidrug resistant TB [MDR-TB] is a form of TB that does not respond to the standard six month treatment using first line drugs [i.e. resistant to Isoniazid and Rifampicin]. It can take two years to treat with drugs that are more toxic, and 100 times more expensive. If the drugs to treat MDR-TB are mismanaged. Further resistance can occur.

Extensively drug resistant [XDR TB] is a form of TB caused by bacteria resistant to all the most effective drugs [i.e. MDR-TB plus resistance to any fluoroquinolone and any of the second line anti-TB injectable drugs: Amikacin, Kanamycin or Capreomycin].[18]

[19]

METHOD

The present study was conducted during Jan., 2009 to Nov., 2009.

We have analyzed all samples viz., sputum, pus, urine, fluid are collected and diagnosed by ZNCF stain and result noted as RNTCP gradation .

All samples were digested and decontaminated by N-Acetyl-l-cysteine sodium hydroxide [12] and cultured in the L.J medium.[13]

Isolated colonies were studied in detail for their morphological, cultural and biochemical characteristics.[14]

All positive culture samples, which were further tested for drug susceptibility towards first line drugs (rifampicin, isoniazid, streptomycin, ethambutol, pyrazinamide) and second line drugs (amikacin, kanamycin, ethionamide, sparfloxacin, levofloxacin, ciprofloxacin,) by MIC method. [15,16] .

METHOD

RESULT & DISCUSSION TABLE: 1 SAMPLE WISE DISTRIBUTION:

SR.NO SAMPLE NO.OF SAMPLE

01 SPUTUM 4302 PUS 3003 URINE 0904 FLUID 08

Table 1 shows that among total 90 samples, majority were of sputum samples.

0.00%

10.00%

20.00%

30.00%

40.00%

50.00%

60.00%

SPUTUM PUS URINE FLUID

PERCENTAGE [%]

PERCENTAGE [%]

Chart-1

TABLE 2: DISTRIBUTION OF A.F.B. GRADATION BY RNTCP METHOD

SR.NO GRADATION NO.OF SAMPLES01 1+ 1502 2+ 1503 3+ 604 SCANTY 005 TOTAL 36

Table 2 shows among total 36 samples direct microscopic positive samples, majority sample were 1+ and 2+ . None of them was scanty.

Chart-2

TABLE 3: DISTRIBUTION BASED ON BIOCHEMICAL TEST

GROUP NO.OF CULTURE BIOCHEMICAL TEST

PNB CATALASE NIACIN

M.tuberculosis 32 - - +MOTT 01 + + -

Above table 3 suggested that total 33 strains, 32 strains were M.tuberculosis and 01 strain was MOTT.

PERCENTAGE (%)

M.tuberculosis

MOTT

Chart-3

SR.NO GROUP NO.OF STRAIN

01 MDR 1302 SENSITIVE TO ALL DRUGS 0203 MONO DRUG & OTHER THAN MDR 18

TABLE 4: DISTRIBUTION BASED ON SENSITIVITY PATTEREN:

Table 4 shows that total 33 strains, 13 strains were MDR, 02 strains were sensitive to all drugs and 18 strains were mono drug & other than MDR.

MDR ,Means in my study resistant to Rifampicin and Isoniazid with or without any other drug. Mono drug resistant means resistant to any one drug .

Chart-4

TABLE 5: MDR PATTERN [13 STRAINS]DRUGS NO.OF STRAINSHR 4HSR 3HER 2HESR 4TOTAL 13

MDR Pattern suggested equal no of HR & HESR resistance i.e. 4 while no of HSR resistance was 3 and HER resistance was 2.

0.00%5.00%

10.00%15.00%20.00%25.00%30.00%35.00%

HR HSR HER HESR

PERCENTAGE(%)

PERCENTAGE(%)

Chart-5

DISCUSSION

In present study,4 types of specimens were included. From 90 specimens, only 47.77% (n=43) were sputum samples whereas 52.23% (n=47) were non respiratory tract category. Among them 40% were smear positive and 36.66% were culture positive.

In our study, total 33 culture positive among them 96.96% were M.tuberculosis and 03.04 % were atypical Mycobacteria similar results were reported by Navarro V, Guix J, Roig P, Borras R. (10)

We found, out of the total 33 positive culture, 39.39 % (n=13) were MDR strain found that is higher than compare with Surveillance of drug-resistant tuberculosis in the state of Gujarat, India in 2009. (11)

Our results are similar to study conducted in Japan. At 2008 (19)

CONCLUSION

Our study shows that among isolated strains 96.96%were M.tuberculosis & 03.04% were atypical Mycobacteria.

The study revealed that direct smear positivity was 40. % and culture positivity was 36.36%, though tuberculosis has been clinically confirmed by physician but detail diagnosis was required to confirm the TB cases.Drug susceptibility testing was carried out with 33 culture positive isolates, 02 (6.06 %) strains were sensitive to all four drugs i.e., Rifampicin, Isoniazid, Ethambutol, Streptomycin. 13 (39.39%) strains were multi drug resistance, 18 (54.55%) strains were mono resistance and multi resist other than MDR.

So, Prevalence of MDR-TB infection was more in South Gujarat region and is necessary to find out the cause of spread of MDR in this region.

1. S.Singh: Scaling up Anti Mycobacterial drug susceptibility services in India. It is high time, Indian Journal of Medical Microbio. [2008] 26 [3] : 209-11

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