Detection of malaria in Saudi Arabia by real-time PCR.

Malaria transmission occurs in Saudi Arabia and mainly endemic in the lowlands of Asir region, the Southwester Province. Imported cases have been reported. Sensitive routine laboratory techniques for rapid and accurate malaria diagnosis are therefore desirable to facilitate the identification of individuals infected with the malarial parasites and to follow up the progress of treatment of such cases with appropriate drugs. Traditional diagnosis, based on the microscopic examination of Giemsa-stained thick and thin films remains the main standard method of diagnosis used for malaria diagnosis in Saudi Arabia. Molecular diagnostic techniques based on the detection of nucleic acids (as PCR; Real-time PCR) are now highly considered. Real time-PCR a new methodology has been recently applied to detect human malaria. In this study a total of forty four samples, using whole-blood, dried blood and thick smears were examined by PCR and Real-time PCR. Both techniques showed a higher sensitivity than the microscopy. Parasites were detected in twenty nine samples out of forty four, compared to twenty six of thirty nine were positive with thin blood film. The real-time PCR assay offers a practical and positive alternative for rapid and accurate diagnosis for malaria infection. The application of such technique will be significantly valuable especially for screening for malaria infection in endemic areas.

Evaluation of 2 real-time PCR assays for the investigation of mecA gene in clinical isolates of MRSA in Western Saudi Arabia.

OBJECTIVE: In diagnostic microbiology laboratories, Methicillin resistant Staphylococcus aureus (MRSA) is identified by positive coagulase test and positive deoxyribonuclease (DNase) activity followed by demonstration of oxacillin resistance on susceptibility testing on agar plate. This usually takes an approximately 48-72 hours. The purpose of this study is to evaluate 2 real-time polymerase chain reaction (PCR) assays for the presence of mecA gene in a population of MRSA strains circulating in Jeddah, Western Saudi Arabia, in order to determine their usefulness in the speedy diagnosis of MRSA in our clinical setting and their contribution to optimal patient management. METHODS: Ninety MRSA isolates obtained from clinical samples were identified by using conventional methods. They were collected between February 2004 and August 2004, from 2 major hospitals in Jeddah; King Abdul-Aziz University Hospital, Jeddah (50 strains) and King Khalid National Guard Hospital, Jeddah (40 strains). All isolates were confirmed as MRSA using Gram stain, catalase and coagulase activity, confirmatory DNAse activity and Kirby Bauer disc diffusion method with resistance to oxacillin by the agar disc method. The DNA extract was tested by 2 assays. The first was the commercial IVD Roche kit, which detects the mecA gene using the Light Cycler system. The other method employs multiplex PCR which detects As442 fragment and mecA optimized for the Smart Cycler system (Cephied). The length of time taken to perform the assays was recorded. RESULTS: All isolates were positive for Sa442 fragment and the coa gene specific for Staphylococcus aureus (S. aureus). However, 88/90 isolates (97.7%) tested were positive for mecA gene with both systems. The amplification, detection and melting curve analysis took 59.2 minutes for 32 samples on the Light Cycler and 46.7 minutes for 16 samples on the Smart Cycler. CONCLUSION: The 2 methods studied were equally specific and sensitive for the detection of mecA gene in confirmed S. aureus isolates and capable of identifying MRSA much earlier than conventional methods. The detection of 2 targets in the multiplex PCR assay reduces the 2-hour time required for DNase testing and may be used as a primary screening test for the detection of MRSA in clinical samples, such as blood cultures and sterile body fluids.