Real-time single-base specific detection of the Haemonchus contortus S168T variant associated with levamisole resistance using loop-primer endonuclease cleavage loop-mediated isothermal amplification.

Haemonchus contortus is a parasitic haematophagous nematode that primarily affects small ruminants and causes significant economic loss to the global livestock industry. Treatment of haemonchosis typically relies on broad-spectrum anthelmintics, resistance to which is an important cause of treatment failure. Resistance to levamisole remains less widespread than to other major anthelmintic classes, prompting the need for more effective and accurate surveillance to maintain its efficacy. Loop-primer endonuclease cleavage loop-mediated isothermal amplification (LEC-LAMP) is a recently developed diagnostic method that facilitates multiplex target detection with single nucleotide polymorphism (SNP) specificity and portable onsite testing. In this study, we designed a new LEC-LAMP assay and applied it to detect the levamisole resistance marker S168T in H. contortus. We explored multiplexing probes for both the resistant S168T and the susceptible S168 alleles in a single-tube assay. We then included a generic probe to detect the acr-8 gene in the multiplex assay, which could facilitate the quantification of both resistance markers and overall genetic material from H. contortus in a single step. Our results showed promising application of these technologies, demonstrating a proof-of-concept assay which is amenable to detection of resistance alleles within the parasite population, with the potential for multiplex detection, and point-of-care application enabled by lateral flow end-point detection. However, further optimisation and validation is necessary.

Significance of cancer testis-associated antigens (SPAG9 and FBXO39) in colon cancer.

Background: Cancer testis antigens (CTA) are normally expressed in immune privileged tissues such as the testis. They are considered tumor-associated antigens because they are specifically expressed in different cancers. Their distinct nature rendered them appealing targets for cancer diagnosis, prognosis. and immunotherapy. We aimed to identify the association of two CTA genes with colon cancer (CC) in a cohort of Egyptian patients. Methods: We measured the relative gene expression levels of two CTAs: SPAG9 and FBXO39 in colonic tumor tissue and adjacent normal-appearing mucosa in 50 newly diagnosed colon cancer patients by real-time reverse transcription polymerase chain reaction. Gene expression was also studied in relation to demographic and pathological criteria. Results: SPAG9 and FBXO39 were overexpressed in 22% and 40% of cases, respectively. Overexpression of both genes was evident in 14% of cases. We report the significant expression of FBXO39 (P < 0.01) in tumor tissue compared to normal tissue. SPAG9 was significantly increased in large sized tumors compared to smaller sized tumors. Otherwise, there was no significant association between gene expression and the evaluated clinicopathological features (P > 0.05). Conclusions: SPAG9 and FBXO39 are possible CC diagnostic biomarkers. Further studies are warranted to validate our findings.

Thermal Inactivation of Hepatitis A Virus, Noroviruses, and Simian Rotavirus in Cows' Milk.

Consumption of raw or unpasteurized milk is a risk for the consumers because indirect contaminations such as fecal-cross-contamination could occur and determine the presence of enteric viruses. In this study, milk was treated with several temperature and time combinations chosen by performing a preliminary experiment to evaluate the intervals needed to inactivate Hepatitis A virus (HAV) HM175 strain, noroviruses genogroups I and II (GI and GII), and simian rotavirus SA11 at different temperatures. Results were obtained by measuring the genome copies and infectious units by real-time PCR and plaque assays respectively. At 85 °C, one minute and two minutes were needed to achieve 6.6 log10 ± 0.2 and 8 log10 ± 0 reductions of genome copies of HAV respectively. Similar genome copies reduction was also observed for noroviruses (GI and GII) and simian rotavirus. At higher temperatures, 90 s (s) at 90 °C and 60 s at 95 °C were needed to achieve 8 log10 ± 0 reductions of the genome copies of all studied viruses. Significant higher sensitivity of the infectious units of both HAV and simian rotavirus to heat treatment of milk than their genome copies was observed. At boiling point of milk (100.5 °C), 40 s were needed to achieve 8 log10 ± 0 reductions of genome copies of all the studied viruses, while 10 s were needed to achieve 6 log10 ± 0 reductions of the infectious units of HAV and simian rotavirus. Significant higher reduction of infectious units than genome copies was observed confirming that genome copies reduction does not correspond to infectious virus.