RESULTS: A set of SREHP gene specific LAMP primers were designed for the specific detection of Entamoeba histolytica. This set of primers recorded 100% specificity when it was evaluated against 3 medically important Entamoeba species and 75 other pathogenic microorganisms. These primers were later modified for conventional PCR, nPCR and qPCR applications. Besides, 3 different post-LAMP analyses including agarose gel electrophoresis, nucleic acid lateral flow immunoassay and calcein-manganese dye techniques were used to compare their limit of detection (LoD). One E. histolytica trophozoite was recorded as the LoD for all the 3 post-LAMP analysis methods when tested with E. histolytica DNA extracted from spiked stool samples. In contrast, none of the PCR method outperformed LAMP as both qPCR and nPCR recorded LoD of 100 trophozoites while the LoD of conventional PCR was 1000 trophozoites.
CONCLUSIONS: The analytical sensitivity comparison among the conventional PCR, nPCR, qPCR and LAMP reveals that the LAMP outperformed the others in terms of LoD and amplification time. Hence, LAMP is a relevant alternative DNA-based amplification platform for sensitive and specific detection of pathogens.
RESULTS: A 300 fecal samples were collected from village chickens (n = 100), layer chickens (n = 100) and captive birds (n = 100). Fecal samples were split into two aliquots for microbiological and molecular detection of MAA. Microbiology detection consisted of microscopy (Ziehl-Neelsen staining) and culture of samples decontaminated with 1% Cetylperidinium chloride and vancomycin, nalidixic acid and amphotericin B (VNA) antibiotic cocktail [vancomycin (VAN) 100 μg/ml, nalidixic acid (NAL) 100 μg/ml and amphotericin B (AMB) 50 μg/ml] onto Löwenstein-Jensen (L-J). Molecular detection (PCR-IS901) was performed to detect MAA DNA from the feces and PCR-16S rRNA and IS901 for identification of genus Mycobacterium and Mycobacterium avium sub species avium isolated onto L-J. All samples (296) were AFB negative smear. M. avium was isolated in 0.3% (1/296) samples by culture and detected in 2.5% (6/242) samples by PCR (IS901). Other mycobacteria were found in 1.7% (5/296) chickens. Of five isolates, two were identified as Mycobacterium terrae and M. engbaekii and remaining isolates were not sequenced. Birds positive for M. avium included White Pelican (n = 1) Black Hornbill (n = 1), Macaw (n = 2), Cockatoo (n = 2) and village chicken (n = 1).
CONCLUSION: It is concluded that chickens and birds were infected with M. avium in selected areas of Peninsular Malaysia. Although, PCR is rapid, reliable and cost effective method for detection of M. avium in a subclinical stage, the culture of the avian feces should still be used as a reference test for the diagnosis of avian tuberculosis.