Brain-eating amoebae (Acanthamoeba spp., Balamuthia mandrillaris, Naegleria fowleri) have gained increasing attention owing to their capacity to produce severe human and animal infections involving the brain. Early detection is a pre-requisite in successful prognosis. Here, we developed a nanoPCR assay for the rapid detection of brain-eating amoebae using various nanoparticles. Graphene oxide, copper and alumina nanoparticles used in this study were characterized using Raman spectroscopy measurements through excitation with a He-Ne laser, while powder X-ray diffraction patterns were taken on a PANanalytical, X'Pert HighScore diffractometer and the morphology of the materials was confirmed using high-resolution transmission electron microscopy (HRTEM). Using nanoparticle-assisted PCR, the results revealed that graphene oxide, copper oxide and alumina nanoparticles significantly enhanced PCR efficiency in the detection of pathogenic free-living amoebae using genus-specific probes. The optimal concentration of graphene oxide, copper oxide and alumina nanoparticles for Acanthamoeba spp. was determined at 0.4, 0.04 and 0.4 μg per mL respectively. For B. mandrillaris, the optimal concentration was determined at 0.4 μg per mL for graphene oxide, copper oxide and alumina nanoparticles, and for Naegleria, the optimal concentration was 0.04, 4.0 and 0.04 μg per mL respectively. Moreover, combinations of these nanoparticles proved to further enhance PCR efficiency. The addition of metal oxide nanoparticles leads to excellent surface effect, while thermal conductivity property of the nanoparticles enhances PCR productivity. These findings suggest that nanoPCR assay has tremendous potential in the clinical diagnosis of parasitic infections as well as for studying epidemiology and pathology and environmental monitoring of other microbes.

Acanthamoeba are widely distributed in the environment and are known to cause blinding keratitis and brain infections with greater than 90% mortality rate. Currently, polymerase chain reaction (PCR) is a highly sensitive and promising technique in Acanthamoeba detection. Remarkably, the rate of heating–cooling and convective heat transfer of the PCR tube is limited by low thermal conductivity of the reagents mixture. The addition of nanoparticles to the reaction has been an interesting approach that could augment the thermal conductivity of the mixture and subsequently enhance heat transfer through the PCR tube. Here, we have developed hexagonal boron nitride (hBN) nanoparticle-based PCR assay for the rapid detection of Acanthamoeba to amplify DNA from low amoeba cell density. As low as 1 × 10−4 wt % was determined as the optimum concentration of hBN nanoparticles, which increased Acanthamoeba DNA yield up to ~16%. Further, it was able to reduce PCR temperature that led to a ~2.0-fold increase in Acanthamoeba DNA yield at an improved PCR specificity at 46.2 °C low annealing temperature. hBN nanoparticles further reduced standard PCR step time by 10 min and cycles by eight; thus, enhancing Acanthamoeba detection rapidly. Enhancement of Acanthamoeba PCR DNA yield is possibly due to the high adsorption affinity of hBN nanoparticles to purine (Guanine—G) due to the higher thermal conductivity achieved in the PCR mixture due to the addition of hBN. Although further research is needed to demonstrate these findings in clinical application, we propose that the interfacial layers, Brownian motion, and percolation network contribute to the enhanced thermal conductivity effect.

The aim of this study was to determine the occurrence of free-living amoebae (FLA) in Peninsular Malaysia and to compare different methodologies to detect them from water samples. Water samples were collected from tap water, recreational places, water dispensers, filtered water, etc. and tested for FLA using both cultivation and polymerase chain reaction (PCR) via plating assays and centrifugation methods. Amoebae DNA was extracted using Instagene matrix and PCR was performed using genus-specific primers. Of 250 samples, 142 (56.8%) samples were positive for presence of amoebae, while 108 (43.2%) were negative. Recreational water showed higher prevalence of amoebae than tap water. PCR for the plating assays revealed the presence of Acanthamoeba in 91 (64%) samples and Naegleria in 99 (70%) of samples analysed. All samples tested were negative for B. mandrillaris. In contrast, the centrifugation method was less effective in detecting amoebae as only one sample revealed the presence of Acanthamoeba and 52 (29%) samples were positive for Naegleria. PCR assays were specific and sensitive, detecting as few as 10 cells. These findings show the vast distribution and presence of FLA in all 11 states of Peninsular Malaysia. Further studies could determine the possible presence of pathogenic species and strains of free-living amoebae in public water supplies in Malaysia.