Protein A/G-based immunochromatographic test for serodiagnosis of pythiosis in human and animal subjects from Asia and Americas.

Pythiosis is a life-threatening infectious disease of both humans and animals living in Asia, Americas, Africa, and parts of Australia and New Zealand. The etiologic pathogen is the fungus-like organism Pythium insidiosum The disease has high mortality and morbidity rates. Use of antifungal drugs are ineffective against P. insidiosum, leaving radical surgery the main treatment option. Prompt treatment leads to better prognosis of affected individuals, and could be achieved by early and accurate diagnosis. Since pythiosis has been increasingly reported worldwide, there is a need for a rapid, user-friendly, and efficient test that facilitates the diagnosis of the disease. This study aims to develop an immunochromatographic test (ICT), using the bacterial protein A/G, to detect anti-P. insidiosum IgGs in humans and animals, and compare its diagnostic performance with the established ELISA. Eighty-five serum samples from 28 patients, 24 dogs, 12 horses, 12 rabbits, and 9 cattle with pythiosis, and 143 serum samples from 80 human and 63 animal subjects in a healthy condition, with thalassemia, or with other fungal infections, were recruited for assay evaluation. Detection specificities of ELISA and ICT were 100.0%. While the detection sensitivity of ELISA was 98.8%, that of ICT was 90.6%. Most pythiosis sera, that were falsely read negative by ICT, were weakly positive by ELISA. In conclusion, a protein A/G-based ICT is a rapid, user-friendly, and efficient assay for serodiagnosis of pythiosis in humans and animals. Compared to ELISA, ICT has an equivalent detection specificity and a slightly lower detection sensitivity.

Detection assay for HPV16 and HPV18 by loop­mediated isothermal amplification with lateral flow dipstick tests.

Cervical cancer is the third highest cause of death in developing countries and most commonly results from high­risk human papillomavirus (HR­HPV) infection. Among HR­HPV genotypes, HPV16 and HPV18 are the most prevalent in cervical cancers. Therefore, the present study aimed to develop a detection assay for HPV16 and HPV18 infection using loop­mediated isothermal amplification (LAMP) with lateral flow dipstick (LFD) tests. This assay is a simplified, user­friendly method for the visual detection of HPV genotypes. DNA was extracted from clinical tissue samples, and HPV genotyping was performed using nested polymerase chain reaction (PCR). The clinical samples were demonstrated to include 44 HPV16­positive, 18 HPV18­positive and 80 HPV­negative samples. All DNA samples were also used as templates for a LAMP reaction (30 min at 65˚C), and subsequently, a fluorescein isothiocyanate­labelled probe was hybridized with the reaction product. Finally, the LFD test was performed. The sensitivity of the LAMP­LFD test was higher than LAMP­turbidity, exhibiting up to 100­fold higher sensitivity for HPV16 and 10­fold higher sensitivity for HPV18. All HPV16 and HPV18­positive samples generated positive results in both assays; however, 22 samples detected as HPV­negative by LAMP­turbidity exhibited positive results by LAMP­LFD test (22 of 80 samples). Therefore, these samples were further examined using quantitative (q)PCR. The results demonstrated that 20 out of the 22 samples designated positive by LAMP­LFD, but negative by LAMP turbidity, gave a positive result with qPCR, while the remaining 2 samples were negative by qPCR. The present results suggested that LAMP­LFD provided higher sensitivity than LAMP­turbidity and nested PCR. Thus, the LAMP­LFD test developed in the present study might be useful for the detection of HPV16 and HPV18 in local hospitals.

High sensitivity, loop-mediated isothermal amplification combined with colorimetric gold-nanoparticle probes for visual detection of high risk human papillomavirus genotypes 16 and 18.

High-risk human papillomavirus (HR-HPV) causes cervical cancer. HPV16 and HPV18 are the most prevalent strains of the virus reported in women worldwide. Loop-mediated isothermal amplification (LAMP) is an alternative method for DNA detection under isothermal conditions. However, it results in a turbid amplified product which is not easily detected by the naked eye. This study aimed to develop an improved technique by using gold nanoparticles (AuNPs) attached to a single-stranded DNA probe for the detection of HPV16 and HPV18. Detection of the LAMP product by AuNP color change was compared with detection by visual turbidity. The optimal conditions for this new LAMP-AuNP assay were an incubation time of 20min and a temperature of 65°C. After LAMP amplification was complete, its products were hybridized with the AuNP probe for 5min and then detected by the addition of magnesium salt. The color changed from red to blue as a result of aggregation of the AuNP probe under high ionic strength conditions produced by the addition of the salt. The sensitivity of the LAMP-AuNP assay was greater than the LAMP turbidity assay by up to 10-fold for both HPV genotypes. The LAMP-AuNP assay showed higher sensitivity and ease of visualization than did the LAMP turbidity for the detection of HPV16 and HPV18. Additionally, AuNP-HPV16 and AuNP-HPV18 probes were stable for over 1year. The combination of LAMP and the AuNP-probe colorimetric assay offers a simple, rapid and highly sensitive alternative diagnostic tool for the detection of HPV16 and HPV18 in district hospitals or field studies.

Gold Nanoparticles Enhance the Anticancer Activity of Gallic Acid against Cholangiocarcinoma Cell Lines.

Gold nanoparticles (GNPs) were conjugated with gallic acid (GA) at various concentrations between 30 and 150 µM and characterized using transmission electron microscopy (TEM) and UV-Vis spectroscopy (UV-VIS). The anticancer activities of the gallic acid-stabilized gold nanoparticles against well-differentiated (M213) and moderately differentiated (M214) adenocarcinomas were then determined using a neutral red assay. The GA mechanism of action was evaluated using Fourier transform infrared (FTIR) microspectroscopy. Distinctive features of the FTIR spectra between the control and GA-treated cells were confirmed by principal component analysis (PCA). The surface plasmon resonance spectra of the GNPs had a maximum absorption at 520 nm, whereas GNPs-GA shifted the maximum absorption values. In an in vitro study, the complexed GNPs-GA had an increased ability to inhibit the proliferation of cancer cells that was statistically significant (P<0.0001) in both M213 and M214 cells compared to GA alone, indicating that the anticancer activity of GA can be improved by conjugation with GNPs. Moreover, PCA revealed that exposure of the tested cells to GA resulted in significant changes in their cell membrane lipids and fatty acids, which may enhance the efficacy of this anticancer activity regarding apoptosis pathways.