Current Programs:

1. Preparation of conjugate vaccine for Salmonella typhi and Salmonella paratyphi A: 
Traditional typhoid vaccines have failed to provide long term protection. Conjugate vaccines hold good promise because protein carrier provides adequate immunity. Two years ago, we started work on development of  a multivalent conjugate vaccine that will cover  all local variants of typhoidal pathogens. As an initial step, we  developed  a multiplex PCR for rapid and definitive identification of various Salmonellae relevant to this project.  We screened  NIBGE stock cultures, one strain of each species was selected for further studies and after growing  the bacteria at large scales and extracting antigens  by complicated procedures we ascertained that the  purity level was well above the WHO standards.   Then we proceeded to separate O-SP from CP, characterized them and proceeded for conjugation with proteins. The results were checked by FTIR spectroscopy and immunodiffusion assays.

2. Detection, molecular analysis and expressional studies of local Vi negative strains of Salmonella typhi:
Vi antigen is the main focus of vaccine development but Vi negative strains have been reported in many places globally in recent years. We have succeeded in proving natural occurrence of these strains is nearly 15% of local population in a collaborative study with Imperial College, London.   We also found that local strains were different in nature from those analyzed in West as in most of them the pathogenicity island SPI7 is present but Vi operon which is responsible for Vi antigen production is mutated or absent. On the other hand , in strains analyzed elsewhere, in most cases whole island is missing.

Our studies thus showed that the local Vi negative strains of Salmonella typhi are different and fully capable as pathogens. As the present vaccines will suppress Vi positive strains, these Vi negative pathogens will take their opportunity and emerge as a big threat. Therefore, it is essential to study their drug resistance abilities and  invasive abilities. We have carried out using HeLa cell line. Comparison of Vi positive and Vi negative isolates showed that they had equal invasive power.

3. Identification of integrons and drug resistance genes in clinical isolates of extraintestinal E.coli:
Extraintesitnal E.coli are the most important urinary pathogens in humans. We have studied their phylogenicity, nature of integrons, and the genes associated with them. Our results show that phylogenetic group B2 is most common followed by A, B1, and D.   We have also found that majority of isolates have Class 1 integrons. According to our findings, these pathogens are highly resistant to cephradine, ampicillin, streptomycin, tetracycline and sulfonamide, where as cefoperazone, cefixime, aztreonam, nalidixic acid, ciprofloxacin were moderately effective. Chloramphenicol, gentamicin, ceftriaxone and amikacin were most effective drugs. The virulence factors of these isolates were also investigated. These included papC, sfa, afa, hly and cnf1. Cytotoxicity  studies were carried out on Vero cells. Generally, it was found that isolates positive for  hly and  cnf1 were extremely cytotoxic.  

4. Detection of Shiga toxin producing Escherichia coli by using  multiplex PCR:
Shiga toxin producing E coli (STEC) are a major pathogen associated with outbreak of diarrhea, hemorrhagic colitis and hemolytic uremic syndrome (HUS) in humans. A couple of years ago, we initiated a study to determine the prevalence of these pathogens in Faisalabad region and characterize the isolates. This was the first study of its kind in Pakistan. A multiplex PCR for detection of various virulence genes simultaneously was used. A total of 200 samples of diarrhea patients were investigated. It was found that 21% carried Shiga toxin genes. 

It has been reported that use of antibiotics as a treatment may actually have adverse effect on STEC infections. We have studied the release of Shiga toxin (Stx) from STEC in response to different antibiotics by using ELISA and PCR.  The drug sensitivity for 11 different antibiotics were determined by disc diffusion methods. The MiC of different drugs were determined by tube dilution method. Vero cells were used for cytotoxicity studies.

We found that there was marked increase in the release of stx with inhibitory and sub-inhibitory concentration of ampicillin and vancomycin. Exposure to gentamicin and cefatoxime markedly decreased the release of stx in tested strains.

5. Molecular typing of rotavirus infection in local population and development of    relevant  multiplex PCR diagnostic procedure:
Rotaviruses are the cause of 60% of all cases of gastroenteritis in children. There are at  least six different serotypes. We plan to develop a multipex PCR that will be able to detect the relevant serotype causing infection in a patient. The data thus generated will provide a base on which strategies for control of this disease can be developed.

Out of 183 patients with diarrhea, 42. 07% (77) were positive for rotavirus by G type specific PCR. Prevalence was greatest in age group less than 1 year (43 cases, 51.19% cases) followed by 1-5 years (28 cases, 38.35%) and 5-12 years (6 cases, 23.07%).  G1 was most prevalent (38 cases, 49.35%), followed by G4 (22 cases, 28.57%).  Respective figures for G2, G3, G8 and G9 were 6 (7.79%), 3 (3.89%), 3 (3.89%) and 5 (6.49%) cases (Table 2).  No significant age related difference was seen in G type distribution. 
Currently we are working on P typing.

Programs just Iinitiated:

1. Molecular characterization of local isolates of Shigella species.

2. Molecular studies of E.coli from wound infections.

Completed Projects:

1. PCR-based diagnosis of typhoid

2. Characterization of biofilm produced by clinical isolates of S. Typhi

3. Development of Multiplex PCR for diagnosis of S. Typhi and its drug resistance

4. Discrimination of S. Typhi and related bacteria by PCR-ribotyping

5. Incidence of typhoid in cases of Hepatitis C

6. Determination of typhoid carriers in normal human population by PCR

7. Relative efficacy of fliC gene and Vi-gene based methods for the diagnosis of suspected cases of typhoid

8. Discrimination of Salmonella species by multiplex PCR

9. Isolation and characterization of hemolysin produced by Escherichia coli  causing urinary tract and wound infections

10. Detection of Escherichia coli virulence genes in clinical samples using multiplex PCR

11. Identification of integrons and drug resistance genes in the clinical isolates of extraintestinal E.coli

12. Molecular ribotyping of clinical isolates of enteric bacteria by PCR

13. Molecular analysis of genetic elements causing drug resistance in clinical isolates of MDR S. Typhi

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