Abstract:
Typhoid fever has become a major problem due to the emergence of
MDR strains of Salmonella enterica serovar Typhi, the causative agent, at
an alarming rate during recent years. The situation is worsened due to lack
of quick, sensitive and reliable diagnostic tools for determining the drug
resistance pattern. Conventional methods are time consuming and lack
sensitivity. It was envisaged that a multiplex PCR diagnosing typhoid and
detecting
resistance
against
standard
typhoid
drugs,
ampicillin,
chloramphenicol, trimethoprim-sulfamethoxazole and ciprofloxacin would
be very useful. After determining drug resistance patterns by standard disc
diffusion method on a pool of 23 MDR S. Typhi isolates, a PCR
amplification technique was used for various drug resistance related genes
found universally in S. Typhi. These were tem, catP, and sul2 genes. None
of these isolates was resistant to ciprofloxacin, so a fragment of gyrA gene
(related to ciprofloxacin resistance) was amplified from an MDR E.coli
isolate, cloned, and transformed into an MDR S. Typhi isolate that was
naturally resistant to other drugs. A regular multiplex PCR was
subsequently developed by using this cloned bacterium which was followed
by the development of a nested multiplex PCR for increasing specificity
and sensitivity. This diagnostic multiplex PCR has been successfully
optimized to be directly applicable to clinical samples.
