The second patient, a 63-year-old diabetic Kuwaiti (Middle Eastern) male was the father of the first patient and lived with his daughter. He was diagnosed with pulmonary TB nearly two years earlier (June 2000). The M. tuberculosis isolate recovered from this patient in the year 2000 was susceptible to INH, SM, EMB and PZA but resistant to RMP. He was treated with a four-drug regimen of INH, SM, PZA and EMB for six months. This patient was admitted to the Chest Diseases Hospital in February 2003 with relapsing pulmonary tuberculosis with cough, fever, loss of appetite and loss of weight. The chest radiogram showed pulmonary infiltrates. The sputum was positive for acid-fast bacilli. The drug susceptibility performed with BACTEC 460 TB system on the M. tuberculosis strain (isolate Kw 180) grown from sputum exhibited sensitivity to INH, SM, EMB and PZA but resistance to RMP. A repeat isolate recovered 15 days later (isolate Kw 181) yielded the same profile. The patient was started on daily therapy with INH, SM, PZA, EMB and ciprofloxacin (CIP). However, he could not tolerate PZA, so the treatment was continued with the regimen of SM, INH, EMB and CIP for 75 days after which the sputum was negative for acid-fast bacilli. The treatment was continued with the regimen of INH, EMB and CIP for four months. The patient was successfully treated and the last culture result at the end of therapy was negative.
Most (>95%) rifampin-resistant strains of M. tuberculosis contain mutations within an 81-base pair rifampin-resistance-determining region (RRDR) of the rpoB gene encoding the beta-subunit of RNA polymerase. The presence of rifampin resistance conferring mutations in the rpoB gene in clinical M. tuberculosis isolates (Kw 176, Kw 180 and Kw 181) was determined by direct DNA sequencing of the PCR amplified DNA fragments containing RRDR as described previously. The fingerprinting of the isolates was carried out by the modified double repetitive element (DRE)-PCR as described in detail elsewhere. The isolates were assigned to one of four genetic groups on the basis of the polymorphisms at katG codon 463 and gyrA codon 95. The presence of R463/L463 in the katG gene and S95/T95 in the gyrA gene were determined by amplification of the corresponding katG and gyrA gene DNA regions by PCR followed by restriction endonuclease digestion of the PCR amplified fragments with Nci I and Ale I, respectively, to generate restriction fragment length polymorphism as described previously.