m. tuberculosis isolates were obtained from the laboratory of mycobacteriology of the institut pasteur de tunis as part of the national tuberculosis surveillance program . all samples ( 884 specimens ) from patients with suspected tb residing in northern tunisia ( bizerte ) from august 2001 to october 2003 were forwarded to us by the referral regional hospital .
the incidence of tb in this area from 2001 to 2002 was 29/100,000 male patients and 11/100,000 female patients .
all patients received the standard chemotherapy regimen of the tunisian national tuberculosis program , i.e. , 2 months of rifampicin , isoniazid , pyrazinamide , and streptomycin , followed by 4 months of rifampicin and isoniazid ( 2rhzs/4rh ) .
the corresponding patients were interviewed , and detailed epidemiologic investigations were conducted according to described protocols ( 6 ) . in april 2004 , while the study was in progress , a new mdr case was diagnosed .
analyses by is6110 restriction fragment length polymorphism ( is6110 rflp ) , ligation - mediated polymerase chain reaction ( pcr ) , and spoligotyping were carried out by using standard protocols ( 79 ) .
typing of the polymorphic gc - rich repetitive sequence ( pgrs ) with probe mtb484 ( 1 ) was conducted according to a previously reported protocol ( 10 ) , with the exception that dna was digested with alui .
isolates were assigned to principal genetic groups according to the polymorphisms in the katg and gyra genes ( 11 ) .
the following primer pairs were used to sequence rpob , katg , and pnca gene mutations that confer resistance to rifampicin , isoniazid , and pyrazinamide , respectively : rpob ( 5-atcacaccgcagacgttg-3  , 5-tgcatcacagtgatgtagtcg-3  ) ; katg ( 5- cgtcgaaacagcggcgctga-3  , 5-caagcgccagcagggctctt-3  ) ; and pnca ( 5-ggcgcacacaatgatcggtg-3  , 5-gctttgcggcgagcgctcca-3  ) .
the recently described single nucleotide polymorphisms in putative m. tuberculosis mutator genes mutt1 , mutt2 , mutt3 , and ogt were investigated with the same protocol reported by rad et al . ( 12 ) .
dna sequencing was conducted directly on the purified pcr products by using the prism ready reaction dye deoxy terminator cycle sequencing kit on an abi prism 377 dna sequencer ( applied biosystems , foster city , ca , usa ) .
epidemiologic and clinical data indicated that all patients with mdr - tb were male with a mean age of 31 years at diagnosis ( table 1 ) .
all were tunisians and permanently resided in the northern part of the country ( bizerte ) .
all patients were seronegative for hiv with no documented history of travel abroad , and none had a history of immunosuppression , diabetes , or respiratory diseases other than tb .
mapping of the 21 patients with mdr - tb according to their residence sites showed that they were mostly scattered over the northeastern part of the region ( surface area 1,000 km ) with no concentration in a particular locality ( data not shown ) .
resistance to 5 first - line drugs was observed for most isolates ( table 2 ) . * pt , previously treated ; nc , new case ; nd , not determined .
* rflp , restriction fragment length polymorphism ; pgg , principal genetic grouping ; wt , wild type ( identical to strain h37rv ) ; nd , not determined . 
h , isoniazid ; s , streptomycin ; r , rifampicin ; e , ethambutol ; z , pyrazinamide . 
number of is6110 bands .   principal genetic grouping according to gyra and katg polymorphisms ( 11 ) . 
# is6110 typing was determined by ligation - mediated polymerase chain reaction and the profile was identical to the other outbreak - associated strains .  * * absence of spacers 15 , 2124 , and 3336 .
as indicated in table 1 , with the exception of patient p20 , the dna samples subjected to molecular typing were obtained from the initial isolate of all new patients .
rflp showed that 18 patients had nearly identical is6110 profiles ( figures 1 and 2 ) .
the predominant profile ( occurring in 13 patients ) showed 11 bands , while the remaining 5 patients had an additional is6110 band . the presence or absence of the additional is6110 band
the rflp pattern of patient p11 , a new patient , clearly showed a mixture of the 12-band profile and some additional is6110 bands ( figure 2a ) .
typing of his follow - up culture , which was obtained after 6 months of directly observed short - course therapy , as recommended by the world health organization , yielded only the 12-band profile ( figure 2a ) .
is6110 restriction fragment length polymorphism ( rflp ) analysis of mycobacterium tuberculosis isolates from 16 patients associated with the multidrug - resistant tuberculosis outbreak , bizerte , tunisia , 20012004 .
a ) is6110 restriction fragment length polymorphism ( rflp ) analysis ( left ) and polymorphic gc - rich repetitive sequence ( pgrs ) typing ( right ) of patient p11 . lane 1 , initial isolate ; lane 2 , follow - up isolate .
b ) is6110 rflp ( left ) and pgrs typing ( right ) of patient p20 ( lane 1 ) compared with patient p3 ( lane 2 ) , a typical outbreak - associated patient .
effective epidemiologic links were identified for 9 ( 47% ) patients ( table 1 ) .
it shows 10 is6110 bands ( figure 2b ) , 9 of which are common to the 12-band rflp pattern described for the other isolates .
the isolate from patient p19 displayed a 9-band is6110 profile that was clearly distinct from all the other patients with mdr - tb ( data not shown ) . with the exception of patient p19
, the mdr isolates were identical in their pgrs profile ( figure 2 ) and spoligotype patterns ( table 2 ) , which is characteristic of the haarlem3 type ( 4 ) .
sequence analysis of mutator and drug resistance genes conclusively confirmed that the 19 mdr isolates with nearly identical is6110 ( both 12- and 11-band profiles ) are genetically closely related .
they all harbor the l209l , t15s , s531l , and s315 t mutations in mutt3 , ogt , rpob , and katg genes , respectively ( table 2 ) , whereas mutt1 and mutt2 showed a wild type genotype ( data not shown ) .
the occurrence of an additional uncommon mutation in the rpob gene ( v610 m ) confirmed the clonality of this mdr haarlem strain since it was present only in 19 patients with mdr - tb .
the variability of resistance to pyrazinamide and the mutational profile within the pnca gene ( table 2 ) strongly suggest that primary transmission from person to person occurred mainly with a strain that was simultaneously resistant to isoniazid and rifampicin . to extend our analysis of the situation that prevailed in this region ,
samples from 143 ( 83% ) of 172 patients without mdr strains were spoligotyped . of these 143 patients , 41 ( 29% )
in contrast to the mdr - tb outbreak that involved only men , 6 women had a non - mdr haarlem3 strain . aside from the absence of clustering ,
ligation - mediated pcr typing showed that none of these non - mdr haarlem3 isolates displayed a profile similar to the 19 mdr isolates involved in the transmission chain .
sequencing of the rpob gene of 10 isolates randomly selected from the 31 non - mdr haarlem isolates showed the absence of the outbreak - associated mutation v610 m .
this finding is strongly indicative of a true clonal expansion and a typical mdr - tb outbreak .
the results indicate that an mdr strain of m. tuberculosis has been actively transmitted among 19 hiv - negative male patients in tunisia .
several observations indicate that this particular haarlem strain displays increased transmissibility , virulence , or both .
first , the outbreak peaked suddenly within a relatively short period of 21 months ; 17 new cases ( 89% ) were reported from september 2001 to june 2003 .
inspection of the hospital register for 2000 showed only 3 new patients with mdr isolates , including outbreak - associated patients p5 and p7 ( table 1 ) .
second , no epidemiologic links or contact points could be traced for several patients , which suggests that brief exposure would have been sufficient for effective transmission . because patients with mdr - tb do not respond to treatment
such a situation is likely to have occurred for the patients with established epidemiologic links .
third , the incidence of tb in the region in which the outbreak occurred is not particularly high .
fifth , no aids - associated tb outbreak that might have increased the adaptability of the strain within the indigenous population had occurred in the region .
sixth , although the haarlem strain was mdr , it was able to cause an outbreak in those vaccinated with bacille calmette - gurin and in persons who were not hospitalized . among the identified m. tuberculosis strain families ( 4,5 ) , the w /
the haarlem strain family appears to be widespread ( 4 ) , but its ability to cause outbreaks has been reported only twice , once in argentina ( 13 ) and once in the czech republic ( 14 ) .
the distinctive feature of the present haarlem mdr - tb outbreak is its accelerated transmission compared with the first 2 mdr - tb outbreaks .
alterations within dna repair genes ( mutator genes ) are thought to favor the emergence of mdr strains with an increased adaptability ( 12 ) . in this respect , both
widespread mdr strains might also benefit from their intrinsic adaptability ( 15 ) . from an epidemiologic point of view
, tb programs must conduct extensive surveillance of mdr strains of m. tuberculosis strain families because they might cause serious outbreaks .
 multidrug - resistant tuberculosis was diagnosed in 21 hiv - negative , nonhospitalized male patients residing in northern tunisia . 
 a detailed investigation showed accelerated transmission of a mycobacterium tuberculosis clone of the haarlem type in 90% of all patients . 
 this finding highlights the epidemic potential of this prevalent genotype . 
The Study
Conclusions