tuberculosis ( tb ) , one of the oldest and contagious infectious diseases is a major cause infection related morbidity and mortality .
worldwide , the disease tb ranks at 1 number as the infectious disease after human immunodeficiency virus ( hiv ) and india accounts for the second most populous country in the world with one - fourth of the global incident tb cases annually .
india accounted for 27% of global tb notifications in 2014 , followed by china ( 14% ) .
according to world health organization ( who ) reports ; worldwide , tb has engulfed about 9 million people out of which 1.1 million were hiv infected .
the most commonly observed age group among tb patients is between 15 and 59 years .
the causative agent of tb , mycobacterium tb ( mtb ) is acid fast bacilli , rod - shaped nonmotile ; obligate intracellular pathogen whose length and width are 24 and 0.20.5 m , respectively .
the bacterium may have killed more persons than any other microbial pathogen and is having the capacity to cause both symptomatic as well as asymptomatic infection .
the transmission takes place via inhalation of aerosol droplets by a healthy person expelled by the infected host . according to studies ,
around 70% of patients with sputum smear - positive cases of pulmonary tuberculosis ptb , died within 10 years .
 everyone infected with tb bacteria does not become sick and who are infected , but not sick , have latent tb infection . at times
the excreting bacilli become resistant to one or more anti - tubercular drugs , the case is then referred to as drug - resistant tb ( dr - tb ) .
dr - tb can take place in several forms : mono - resistance , poly resistance , multi - drug resistant tb ( mdr - tb ) , extensively drug resistant tb , and totally drug resistant tb ( tdr - tb ) .
when mtb become resistant to any one first line anti - tb drug ( fld ) the case is called confirmed mono - resistance .
the flds are isoniazid ( inh ) , rifampicin ( rif ) , pyrazinamide ( pza ) , ethambutol ( emb ) , and streptomycin ( sm ) .
direct repeat ( dr ) among previously treated patients is rising due to noncompliance to tb medications , lack of knowledge , poor management in health centers . according to who report , out of six other countries , india ( 2.0 million2.3 million )
was reported to be at the first position of high incidence of tb . according to who report statistics ( 2013 )
, the estimated prevalence and incidence for tb in india was recorded as 2.6 million and 2.1 million respectively out of the global incidence of tb recorded as 9 million .
the first - line drugs are considered as a boon in the treatment of tb patients and any mismanagement in consumption of these drugs results in a serious health hazard . to study the epidemiology of mtb , based on its dna polymorphism various molecular techniques
these include dr , variable number of tandem repeat typing , insertion sequence 6110 fingerprinting ( is6110 ) , spoligotyping , and much more .
the aim of this study was to check the mono , multi- and triple - drug resistance to flds among tb patients and to access their genetic profile using dr 3074 , dr 0270 , dr 0642 , dr 2068 , and dr 4110 using molecular techniques .
the aim of this study was to check the mono , multi- and triple - drug resistance to flds among tb patients and to access their genetic profile using dr 3074 , dr 0270 , dr 0642 , dr 2068 , and dr 4110 using molecular techniques .
after a verbal consent from one hundred fifty - nine ( 159 ) ptb patients showing zn stained smear positive who had visited the hospital for diagnosis and treatment were included in the study .
the study was carried out at king george 's medical university , lucknow , uttar pradesh , india , and sampling was done from april 2014 to october 2014 .
patients collected their sputum samples in a labeled sterile disposable container as directed and were also advised to collect it every morning after rinsing their mouths with plain water for 3 subsequent days .
subjects who satisfied the criteria were included in the study whereas ; those not fulfilling the criteria were excluded from the study .
new pulmonary tb patients of both the sexes , hiv , aged between 18 and 70 years who agreed to participate were included and patients who had eptb , hiv and did not agree to involve their selves were excluded from the study
isolation and identification was done by the conventional methods and thus subjected to drug susceptibility testing ( dst ) against the flds by the proportion method against inh ( 0.2 g / ml ) , emb ( 2 g / ml ) , sm ( 4 g / ml ) , and rif ( 40 g / ml ) .
incubation of samples was done at 37c for 6 weeks which therefore produced a visible growth on the lj slants and helped in the identification of the mycobacterium isolates .
dst using proportion method was performed to check the mono , multi , and triple dr among tb patients .
after a verbal consent from one hundred fifty - nine ( 159 ) ptb patients showing zn stained smear positive who had visited the hospital for diagnosis and treatment were included in the study .
the study was carried out at king george 's medical university , lucknow , uttar pradesh , india , and sampling was done from april 2014 to october 2014 .
patients collected their sputum samples in a labeled sterile disposable container as directed and were also advised to collect it every morning after rinsing their mouths with plain water for 3 subsequent days .
subjects who satisfied the criteria were included in the study whereas ; those not fulfilling the criteria were excluded from the study .
new pulmonary tb patients of both the sexes , hiv , aged between 18 and 70 years who agreed to participate were included and patients who had eptb , hiv and did not agree to involve their selves were excluded from the study
isolation and identification was done by the conventional methods and thus subjected to drug susceptibility testing ( dst ) against the flds by the proportion method against inh ( 0.2 g / ml ) , emb ( 2 g / ml ) , sm ( 4 g / ml ) , and rif ( 40 g / ml ) .
incubation of samples was done at 37c for 6 weeks which therefore produced a visible growth on the lj slants and helped in the identification of the mycobacterium isolates .
dst using proportion method was performed to check the mono , multi , and triple dr among tb patients .
sputum sample of all the patients was collected aseptically and scientifically . of 200 patients ,
all the subjects were interviewed based upon for information on characteristics of education , occupation , and residence . among these patients ,
number of literate patients were 123 ( 77.3% ) and 36 ( 22.6% ) as illiterate .
25 ( 15.7% ) patients had farming as their occupation , and 80 ( 50.3% ) had nonagricultural occupation .
122 patients ( 76% ) were reported from urban areas and 37 ( 23.2% ) from rural areas .
were males [ table 1 ] . characteristics of patients on the basis of education and occupation in tb patients addiction to certain addictives was a common factor .
the addictives were bidi ( b ) , cigarette ( c ) , tobacco ( t ) , alcohol ( a ) , and ganja ( g ) .
some of the patients were addicted to mono addictives like b , t , and c and some patients were addicted to more than one addictives .
a number of patients who were addicted to b , t , and c were 67 ( 42.13% ) , 9 ( 5.66% ) , and 19 ( 11.94% ) , respectively .
on the other hand , patients addicted to c - b - g ; c - a ; and b - t were 3 ( 1.88% ) , 21 ( 13.20% ) , and 14 ( 8.80% ) respectively .
furthermore , number of patients who were addicted to b - a and b - c were 19 ( 11.94% ) and 7 ( 4.40% ) respectively [ table 2 ] .
distribution of patients according to their drug habits mono drug - resistant cases for inh , sm , emb , and rif were recorded as 62 , 45 , 47 , and 27 [ table 3 ] .
number of patients infected with mono direct repeat pattern whereas , mdr cases for inh + rif , inh + sm , inh + emb , and rif + sm was recorded as 49 , 53 , 52 , and 45 , respectively [ table 4 ] and tdr cases for inh + rif + sm , inh + rif + emb , inh + emb + sm were recorded as 40 , 36 , and 31 , respectively [ table 5 ] .
number of patients infected with multi - drug resistant tuberculosis pattern number of patients infected with triple drug resistance pattern number of patients who were resistant and sensitive to inh was 62 ( 38.9% ) and 97 ( 61% ) respectively .
furthermore , patients resistance and sensitivity toward sm was 45 ( 28.3% ) and 114 ( 71.6% ) respectively .
resistivity and sensitivity toward emb was found to be 47 ( 29.5% ) and 112 ( 70.4% ) respectively .
similarly , number of patients resistant and sensitive to rif was 27 ( 16.9% ) and 132 ( 83.01% ) respectively .
total number of patients calculated to be resistant and sensitive was 30 ( 18.8% ) and 74 ( 46.5% ) respectively [ table 6 ] .
number of patients resistant and sensitive to first - line antituberculosis drugs among all the mtb clinical isolates a noteworthy level of distinction was observed .
a total of 159 cases were studied out of which the polymorphism with various drs was seen in 47 patients [ table 7 ] .
patients with direct repeats polymorphism among mtb isolates was significantly observed by the amplification of primers .
the primers dr ( 3074 , 0272 , 2068 , and 0642 ) and is6110 were designed from mtb genome in such a way that it can help in studying the mtb epidemiology , detecting dna polymorphisms , and strain typing . the melting temperature and primer sequence of the same is given as below [ table 8 ] .
the polymorphism among the isolates was checked by running the pcr products on agarose gel .
ladder ( fermentas ) of 1 kb was run through the gel [ figures 1 - 3 ] .
the representative gel pictures depict the different band sizes which can be observed below : primers , their sequences , and melting temperatures representative gel pictures of clinical isolates : primer : dr0272 ; lane 1 : ladder ( 1 kb ) ; lane 11 : control ; lane 210 : clinical isolates ; primer : dr0642 ; lane 1 : ladder ( 1 kb ) ; lane 2 : control ; lane 315 : clinical isolates representative gel picture of clinical isolates ; primer : dr2068 ; lane 1 : ladder ( 1 kb ) ; lane 6 : control ; lane 25 ; 710 : clinical isolates ; primer : dr3074 ; lane 1 : ladder ( 1 kb ) ; lane 2 : control ; lane 313 : clinical isolates representative gel picture of clinical isolates .
primer : dr4110 ; lane 1 : ladder ( 1 kb ) ; lane 2 : control ; lane 311 : clinical isolates
sputum sample of all the patients was collected aseptically and scientifically . of 200 patients ,
all the subjects were interviewed based upon for information on characteristics of education , occupation , and residence . among these patients ,
number of literate patients were 123 ( 77.3% ) and 36 ( 22.6% ) as illiterate .
25 ( 15.7% ) patients had farming as their occupation , and 80 ( 50.3% ) had nonagricultural occupation .
122 patients ( 76% ) were reported from urban areas and 37 ( 23.2% ) from rural areas .
were males [ table 1 ] . characteristics of patients on the basis of education and occupation in tb patients addiction to certain addictives was a common factor .
the addictives were bidi ( b ) , cigarette ( c ) , tobacco ( t ) , alcohol ( a ) , and ganja ( g ) .
some of the patients were addicted to mono addictives like b , t , and c and some patients were addicted to more than one addictives .
a number of patients who were addicted to b , t , and c were 67 ( 42.13% ) , 9 ( 5.66% ) , and 19 ( 11.94% ) , respectively .
on the other hand , patients addicted to c - b - g ; c - a ; and b - t were 3 ( 1.88% ) , 21 ( 13.20% ) , and 14 ( 8.80% ) respectively .
furthermore , number of patients who were addicted to b - a and b - c were 19 ( 11.94% ) and 7 ( 4.40% ) respectively [ table 2 ] .
distribution of patients according to their drug habits mono drug - resistant cases for inh , sm , emb , and rif were recorded as 62 , 45 , 47 , and 27 [ table 3 ] .
number of patients infected with mono direct repeat pattern whereas , mdr cases for inh + rif , inh + sm , inh + emb , and rif + sm was recorded as 49 , 53 , 52 , and 45 , respectively [ table 4 ] and tdr cases for inh + rif + sm , inh + rif + emb , inh + emb + sm were recorded as 40 , 36 , and 31 , respectively [ table 5 ] .
number of patients infected with multi - drug resistant tuberculosis pattern number of patients infected with triple drug resistance pattern number of patients who were resistant and sensitive to inh was 62 ( 38.9% ) and 97 ( 61% ) respectively .
furthermore , patients resistance and sensitivity toward sm was 45 ( 28.3% ) and 114 ( 71.6% ) respectively .
resistivity and sensitivity toward emb was found to be 47 ( 29.5% ) and 112 ( 70.4% ) respectively .
similarly , number of patients resistant and sensitive to rif was 27 ( 16.9% ) and 132 ( 83.01% ) respectively .
total number of patients calculated to be resistant and sensitive was 30 ( 18.8% ) and 74 ( 46.5% ) respectively [ table 6 ] .
number of patients resistant and sensitive to first - line antituberculosis drugs among all the mtb clinical isolates a noteworthy level of distinction was observed .
a total of 159 cases were studied out of which the polymorphism with various drs was seen in 47 patients [ table 7 ] .
patients with direct repeats polymorphism among mtb isolates was significantly observed by the amplification of primers .
the primers dr ( 3074 , 0272 , 2068 , and 0642 ) and is6110 were designed from mtb genome in such a way that it can help in studying the mtb epidemiology , detecting dna polymorphisms , and strain typing . the melting temperature and primer sequence of the same is given as below [ table 8 ] .
the polymorphism among the isolates was checked by running the pcr products on agarose gel .
ladder ( fermentas ) of 1 kb was run through the gel [ figures 1 - 3 ] .
the representative gel pictures depict the different band sizes which can be observed below : primers , their sequences , and melting temperatures representative gel pictures of clinical isolates : primer : dr0272 ; lane 1 : ladder ( 1 kb ) ; lane 11 : control ; lane 210 : clinical isolates ; primer : dr0642 ; lane 1 : ladder ( 1 kb ) ; lane 2 : control ; lane 315 : clinical isolates representative gel picture of clinical isolates ; primer : dr2068 ; lane 1 : ladder ( 1 kb ) ; lane 6 : control ; lane 25 ; 710 : clinical isolates ; primer : dr3074 ; lane 1 : ladder ( 1 kb ) ; lane 2 : control ; lane 313 : clinical isolates representative gel picture of clinical isolates .
primer : dr4110 ; lane 1 : ladder ( 1 kb ) ; lane 2 : control ; lane 311 : clinical isolates
consumption of anti - tb drugs in a prescribed and regular manner helps combating tb .
discontinuation of drugs as advised increases the risk of dr - tb , treatment failure and relapse .
hence , this study was undertaken to study three types of drug resistance patterns , i.e. , mono drtb , mdr - tb , and tdr - tb to flds in newly diagnosed cases of ptb .
it reduces the time of patient 's ailment and prevents the transmission of infection to others .
insertion sequence - is6110 initially described by thierry et al . is distributed throughout the mtb complex and has been in great use in the epidemiological applications of restriction fragment length polymorphism analysis . a study carried out by gupta et al . ,
2013 , showed the dr pattern to inh , rifampin , sm and emb as 18.3% , 4.7% , 10.1% , and 10.7% , respectively which did not coincide with our study .
our study was totally different from the study done by , the percentage of patients resistant to inh , rif , emb , and sm was 1.4% , 0.2% , 0% , and 7.3% , respectively .
mdr cases for inh + rif , inh + sm , inh + emb , and rif + sm was recorded as 49 , 53 , 52 , and 45 , respectively [ table 4 ] .
a study carried out at portugal showed resistance to inh + rif and inh + sm as 1.1% and 3.3% respectively which did not match with our study .
a similar type of study carried out at belgaum showed the highest resistance to rif ( 80.4% ) , while resistance to inh , pza , emb , and sm were 60% , 58.7% , 52.1% , and 63% , respectively .
resistance to one drug , three drugs , four drugs were ( 17.9% ) , ( 17.9% ) , and ( 8.7% ) , respectively .
according to a study carried out in karnataka , 24 ( 52.2% ) isolates showed mdr strains while 8 ( 17.9% ) and 4 ( 8.7% ) isolates confirmed mono and poly resistance , respectively .
similarly , in another study being carried out at hyderabad 28% of the cases were confirmed with mdr - tb whereas polydrug resistance was reported in 42% of the cases .
a study was carried out at lucknow , india , in which a total of 69 patients were studied , and five types of dr 's were amplified out of the total number of patients . in a study carried out at thailand , polymorphism with various drs was observed in 39 out of 91 patients .
literate patients were in majority and non - agricultural occupation was seen in most of the patients .
dr - tb is a major public health problem because treatment is complicated , cure rates are well below those for drug susceptible tb , and patients may remain infectious for months or years despite receiving the best available therapy .
the data showed significant level of dissimilarities among all the dr isolates of mtb and number of repeats of is6110 were present in different clinical isolates . over the years
, the identification method based on is6110 has been established as the standard for typing strains of mtb .
is6110 genotyping is very convincing when it is applied to classify mtb isolates harboring a large number of is6110 in their chromosomes .
 background : tuberculosis ( tb ) , a highly contagious disease that sees no gender , age , or race is mainly a disease of lungs . according to world health organization , a tb patient can be completely cured with 69 months of anti - tb treatment under directly observed treatment short course.objectives:the aim of this study was to check the mono , multi- and triple - drug resistance to first line drugs ( flds ) among tb patients and to access their genetic profile using dr 3074 , dr 0270 , dr 0642 , dr 2068 , and dr 4110 using molecular techniques.material and methods : to gain a better understanding of drug resistant tb , we characterized 121 clinical isolates recovered from 159 drug resistant pulmonary tuberculosis patients by is6110 genotyping . 
 mtb isolates recovered from hiv- negative , and smear positive cases of both genders , age varied from 18 to 70 years with drug resistant - tb that was refractory to chemotherapy given for > 12 months . of a total of 159 sputum smear 
 positive patients sum number of male and female patients was 121 ( 76.10% ) and 38 ( 23.89% ) , respectively . among these patients , 
 number of literate and illiterate patients were 123 ( 77.3% ) and 36 ( 22.6% ) . 
 25 ( 15.7% ) patients had farming as their occupation , 80 ( 50.3% ) had nonagricultural occupation and 54 ( 33.9% ) women were housewives.results:mono drug resistant , multi - drug resistant , and totally drug resistant ( tdr ) cases of tb were calculated as 113.83% , 125.1% , and 67.9% . 
 isoniazid showed the highest percentage of resistance among the patients.conclusion:any noncompliance to tb medications , lack of knowledge , and poor management in health centers , etc . 
 , results in the emergence of deadly direct repeat forms of tb , which are further complicated and complex to treat . 
INTRODUCTION
Study design
MATERIALS AND METHODS
Study population
RESULTS
Patients
DISCUSSION
CONCLUSION
Financial support and sponsorship
Conflicts of interest