Standardization and evaluation of a duplex real-time quantitative PCR for the detection of Leishmania infantum DNA: a sample quality control approach

Abstract INTRODUCTION: Molecular techniques have been shown to be alternative methods for the accurate detection of infectious and parasitic diseases, such as the leishmaniases. The present study describes the optimization and evaluation of a duplex real-time quantitative PCR (qPCR) protocol developed for the simultaneous detection of Leishmania infantum DNA and sample quality control. METHODS: After preliminary tests with the newly designed TaqMan® probes for the two targets ( L. infantum and glyceraldehyde 3-phosphate dehydrogenase (G3PD) gene), the duplex qPCR protocol was optimized. For the evaluation of the standardized protocol, human blood samples were tested (n=68) and the results were compared to those obtained by reference diagnostic techniques. Statistical analyses included percentage agreement and the Kappa ( k ) coefficient. RESULTS: The detection limit of L. infantum DNA reached 2x10 2 fg (corresponding to ~1 parasite) per µL of blood (ε: 93.9%). The percentage agreement obtained between the duplex VL qPCR and the reference techniques was individually obtained as follows: molecular: 88.3% ( k =0.666; 95% CI 0.437-0.894, good), and serological: 81.7% ( k =0.411; 95% CI 0.125-0.697, moderate). Between the reference techniques, the percentage agreement was 86.7% ( k =0.586; 95% CI 0.332-0.840, moderate). CONCLUSIONS: The new duplex VL qPCR protocol indicated good potential for the accurate, fast, and reliable detection of L. infantum DNA, when applied as a complement to the classical diagnostic tools already available, especially in health or research reference centers.

Influence of sample collection techniques on test results / Эрүүл мэндийн лаборатори

Background:Laboratory test are done on clinical specimens in order to get information about the health of a patient as pertaining to the diagnosis, treatment and prevention of disease. Laboratory test gives 70% of information to get a right diagnosis. By some study, physiological factors such as diet, stress, exercise and sample collection techniques are influencing 32%-75% of test accuracy and reliability. That is why we want to studyhow nurses follow standard of sample collection techniques in UB.Our study conducted in 5 different hospitals. We observed nursesway of collecting sample from 150 patientsResult:Nurses did not identify patients ID in 80% of patients and did not ask test preparation and diet of 100%. They prepare necessary items to blood draw 95%. Also nurses did not fully follow blood draw standard in such way: hand sterilization, asepticize place of puncture and using bandage. Conclusion:Nurses don’t follow standard of draw blood from vein and did not clarifies patients test preparation. Pre-analysis process is the most influencing factor in the test result. So we have to train nurses to follow their standard of sample collection procedure.