Detection of Microsatellite Instability by High-Resolution Melting Analysis in Colorectal Cancer

Background: Colorectal cancer (CRC) is the third most common cancer worldwide. microsatellite instability (MSI) is a molecular marker of a deficient mismatch repair system and happens in almost 15% of CRCs. Because of a wide frequency of MSI+ CRC in Iran compared to other parts of the world, the importance of screening for this type of cancer is highlighted. Methods: : The most common MSI detection technique is a fluorescent PCR-based method in which fragments are analyzed by capillary electrophoresis (CE). This technique is very time-consuming, difficult, and expensive. We sought to develop and evaluate a proper method with high accuracy, specificity, and sensitivity to screen the MSI+ CRC. A high-resolution melting (HRM) analysis procedure is relying on the analysis of the melting curve attributes. Low cost, feasibility, high specificity, and sensitivity are outstanding attributes of HRM analysis. Results: Five mononucleotide microsatellite markers, including BAT-25, BAT-26, NR-21, NR-24, and NR-27, in 25 archival CRC tumor tissue samples were compared with normal tissue adjacent using HRM method. The specificity and sensitivity of BAT-25 with HRM method were 100% compared to CE, while other markers had lower sensitivity. However, when all the markers were considered together, the sensitivity and specificity became 100%. The number of MSI+ samples was 56%, which shows a higher ratio than previous Iranian studies. The highest MSI was related to BAT-26 (52%). Conclusion: The HRM method is much simpler and more cost-effective than current MSI techniques, and its sensitivity and accuracy are comparable. Therefore, it can serve as an alternative method in cases where CE is unavailable.

Direct detection of Pseudomonas aeruginosa from patients with healthcare associated pneumonia by real time PCR.

Using oprL sequences, a TaqMan real time PCR was developed and used for quantitative detection of Pseudomonas aeruginosa from 99 broncoalveolar lavage and 11 sputum specimens collected from patients with health care associated pneumonia. All specimens were cultured on appropriate media to isolate bacteria. Twenty five specimens were positive by both methods. Polymicrobial infections were found in 13 specimens. Amplification of oprL in serial dilutions ranged from 10(9)CFU/ml to 10(2)CFU/ml. Standard curve of duplicated every dilution had slope 3.25±0.1 and R(2)>0.99 with SD 0.1. Our real time PCR assay showed high sensitivity (100%) and specificity (98.85%). This technique could detect and enumerate 100 bacteria directly from clinical specimens and showed that the threshold is 10(3)CFU/ml in cases with clinical symptoms. Our method can be used for quantitative detection of P. aeruginosa from BAL and sputum specimens in 1h and 10min.

Distribution of bla(TEM), bla(SHV), bla(CTX-M) genes among clinical isolates of Klebsiella pneumoniae at Labbafinejad Hospital, Tehran, Iran.

Extended-spectrum beta-lactamase (ESBL)-producing isolates of Klebsiella pneumoniae have been increasingly recognized in the hospital settings in Iran as well as throughout the world. The aim of this study was to detect and determine the genes encoding the ESBLs including bla(TEM), bla(SHV), and bla(CTX-M) groups among the K. pneumoniae isolates at Labbafinejad Hospital by polymerase chain reaction (PCR) and characterize them by direct sequencing of PCR products. Eighty-nine isolates were isolated from patients at different wards during March 2008-March 2009. They were identified as K. pneumoniae using biochemical tests. Susceptibility of isolates to 17 different antimicrobial agents was determined using agar disk diffusion method. The phenotypic confirmatory test was used to screen the isolates for production of ESBLs. To amplify the bla(SHV) the template DNA was extracted by boiling method. Plasmid DNA was extracted using minipreparation kit and used as template in PCR for detection of bla(TEM) and bla(CTX-M). The selected PCR products were sequenced and analyzed. All 89 strains were susceptible to imipenem. The rates of resistance to different antibiotics were in the following order: aztronam (79.7%), cefexime (67.4%), cefpodoxime (66.2%), cefotaxime (65.1%), ceftazidime (61.7%). The phenotypic confirmatory test detected 62 isolates (69.7%) as ESBL-producing K. pneumoniae. The prevalence of genes encoding ESBLs were as follows: bla(TEM) 54% (n = 48), bla(SHV) 67.4% (n = 60), bla(CTX-M-I) 46.51% (n = 40), and bla(CTX-M-III) 29% (n = 25). The bla(CTX-M-II) and bla(CTX-M-IV) were not detected. All bla(TEM) types were characterized as bla(TEM-1) and all bla(CTX-M-I) were identified as bla(CTX-M-15). The SHV types were characterized as SHV-5, SHV-11, and SHV-12. The rate of ESBL at Labbafinejad Hospital was 25% increase in a 4-year study that ended in March 2009. It appears that bla(TEM-1), bla(SHV-5), bla(SHV-11), bla(SHV-12), and bla(CTX-M-15) are the dominant ESBLs among the resistant strains of K. pneumoniae in Iran.