Search Results (10)

Swine infectious diseases, often caused by multiple co-infecting agents, pose severe global threats to pig health and industry economics. Conventional single-plex testing assays, whether relying on pathogen antigens or nucleic acids, exhibit limited efficacy in the face of co-infection events. The modern nucleic acid-based multiplex testing (NAMT) methods demonstrate substantial strengths in the simultaneous detection of multiple pathogens involving co-infections owing to their remarkable sensitivity, exceptional specificity, high-throughput, and short turnaround time. The development, commercialization, and application of NAMT assays in swine infectious disease surveillance would be advantageous for early detection and control of pathogens at the onset of an epidemic, prior to community transmission. Such approaches not only contribute to saving the lives of pigs but also aid pig farmers in mitigating or preventing substantial economic losses resulting from infectious disease outbreaks, thereby alleviating unwanted pressure on animal and human health systems. The current literature review provides an overview of some modern NAMT methods, such as multiplex quantitative real-time PCR, multiplex digital PCR, microarrays, microfluidics, next-generation sequencing, and their applications in the diagnosis of swine infectious diseases. Furthermore, the strengths and weaknesses of these methods were discussed, as well as their future development and application trends in swine disease diagnosis. Full article

Despite numerous studies on haemosporidians in wild birds from Brazil, the presence of other vector-borne agents (VBA) such as Anaplasma spp., Bartonella spp., and Onchocercidae filariids in avian hosts remains largely unknown. The low occurrence of these VBAs might be due to the low sensitivity of traditional molecular techniques. The microfluidic real-time PCR assay, known for its high sensitivity, has emerged as a promising method to detect and study the occurrence and diversity of VBAs in both arthropod vectors and vertebrate hosts. To validate previously and standardize newly designed microfluidic real-time PCR protocols, selected positive avian blood DNA samples for Anaplasma spp., Bartonella spp., haemosporidians, and filariids were used. The molecular occurrence rates for the selected VBAs were 18.2% for Anaplasma spp., 0.36% for Bartonella spp., 6.2% for Plasmodium spp., 4.7% for Haemoproteus spp., and 6.5% for Onchocercidae filariids. The Plasmodium spp. cytB sequence detected in a Volatinia jacarina clustered with Plasmodium tejerai, whereas the Haemoproteus spp. cytB sequence detected in a Columbina squamata clustered with Haemoproteus columbae. While Onchocercidae filariid cox-1 sequences were detected in specimens of Ramphocelus carbo, Turdus amaurocalinus and Synallaxis albilora grouped with Aproctella spp., one sequence detected in R. carbo was ancestral to the clade comprising Splendidofilaria spp. and Eufilaria spp. High-throughput microfluidic real-time PCR assay can be used for screening VBAs in avian hosts from South America, but new primers/probe sets should be designed for VBA genotypes present in Brazil. Full article

Polymerase chain reaction (PCR) chips are advanced, microfluidic platforms that have revolutionized biomarker discovery and validation because of their high sensitivity, specificity, and throughput levels. These chips miniaturize traditional PCR processes for the speed and precision of nucleic acid biomarker detection relevant to advancing drug development. Biomarkers, which are useful in helping to explain disease mechanisms, patient stratification, and therapeutic monitoring, are hard to identify and validate due to the complexity of biological systems and the limitations of traditional techniques. The challenges to which PCR chips respond include high-throughput capabilities coupled with real-time quantitative analysis, enabling researchers to identify novel biomarkers with greater accuracy and reproducibility. More recent design improvements of PCR chips have further expanded their functionality to also include digital and multiplex PCR technologies. Digital PCR chips are ideal for quantifying rare biomarkers, which is essential in oncology and infectious disease research. In contrast, multiplex PCR chips enable simultaneous analysis of multiple targets, therefore simplifying biomarker validation. Furthermore, single-cell PCR chips have made it possible to detect biomarkers at unprecedented resolution, hence revealing heterogeneity within cell populations. PCR chips are transforming drug development, enabling target identification, patient stratification, and therapeutic efficacy assessment. They play a major role in the development of companion diagnostics and, therefore, pave the way for personalized medicine, ensuring that the right patient receives the right treatment. While this tremendously promising technology has exhibited many challenges regarding its scalability, integration with other omics technologies, and conformity with regulatory requirements, many still prevail. Future breakthroughs in chip manufacturing, the integration of artificial intelligence, and multi-omics applications will further expand PCR chip capabilities. PCR chips will not only be important for the acceleration of drug discovery and development but also in raising the bar in improving patient outcomes and, hence, global health care as these technologies continue to mature. Full article

Ribonucleic acid (RNA) viruses are one of the major classes of pathogens that cause human diseases. The conventional method to detect RNA viruses is real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), but it has some limitations. It is expensive and time-consuming, with infrastructure and trained personnel requirements. Its high throughput requires sophisticated automation and large-scale infrastructure. Isothermal amplification methods have been explored as an alternative to address these challenges. These methods are rapid, user-friendly, low-cost, can be performed in less specialized settings, and are highly accurate for detecting RNA viruses. Microfluidic technology provides an ideal platform for performing virus diagnostic tests, including sample preparation, immunoassays, and nucleic acid-based assays. Among these techniques, nucleic acid isothermal amplification methods have been widely integrated with microfluidic platforms for RNA virus detection owing to their simplicity, sensitivity, selectivity, and short analysis time. This review summarizes some common isothermal amplification methods for RNA viruses. It also describes commercialized devices and kits that use isothermal amplification techniques for SARS-CoV-2 detection. Furthermore, the most recent applications of isothermal amplification-based microfluidic platforms for RNA virus detection are discussed in this article. Full article

In mountainous regions, diverse ecosystems provide a habitat for numerous species of organisms. In this study, we focused on ixodid ticks and their presence in the Western Carpathians, Poland. Our objectives were to investigate the impact of environmental factors on tick occurrence and activity, the prevalence of vectored pathogens, and tick hosts, and their role as reservoir organisms for tick-borne pathogens (TBPs). To this end, we collected ticks from the vegetation and from animals (Apodemus agrarius, A. flavicollis, Capreolus capreolus, Microtus spp., Myodes glareolus, Ovis aries). In addition, we collected blood samples from rodents. The collected material underwent molecular analysis, utilizing the high-throughput microfluidic real-time PCR technique, to detect the presence of TBPs. Our findings confirmed the occurrence of only two species of ixodid ticks in the study area: the dominant Ixodes ricinus, and Dermacentor reticulatus with very limited abundance. Temperature significantly influenced tick activity, and the number of I. ricinus nymphs varied with altitude. We also observed a circadian pattern of questing activity in I. ricinus ticks. The main hosts for juvenile tick stages were M. glareolus and A. agrarius, while adult stages were frequently found on C. capreolus. I. ricinus ticks collected from the vegetation were often infected with Rickettsia helvetica (up to 35.71%), Borrelia afzelii (up to 28.57%), and Ehrlichia spp. (up to 9.52%). In contrast, juvenile stages frequently carried Bartonella spp. (up to 10.00%), Mycoplasma spp. (up to 16.67%) and R. helvetica (up to 16.67%). Moreover, we detected genetic material of Mycoplasma spp. (up to 100.00%), Ehrlichia spp. (up to 35.71%), Bartonella spp. (up to 25.00%), and Borrelia spp. (up to 6.25%) in rodent blood samples. The obtained results indicate A. agrarius and M. glareolus as reservoir animals for TBPs in the studied region. Full article

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused an ongoing coronavirus disease (COVID-19) outbreak and a rising demand for the development of accurate, timely, and cost-effective diagnostic tests for SARS-CoV-2 as well as other viral infections in general. Currently, traditional virus screening methods such as plate culturing and real-time PCR are considered the gold standard with accurate and sensitive results. However, these methods still require sophisticated equipment, trained personnel, and a long analysis time. Alternatively, with the integration of microfluidic and biosensor technologies, microfluidic-based biosensors offer the ability to perform sample preparation and simultaneous detection of many analyses in one platform. High sensitivity, accuracy, portability, low cost, high throughput, and real-time detection can be achieved using a single platform. This review presents recent advances in microfluidic-based biosensors from many works to demonstrate the advantages of merging the two technologies for sensing viruses. Different platforms for virus detection are classified into two main sections: immunoassays and molecular assays. Moreover, available commercial sensing tests are analyzed. Full article

The impact of mosquito-borne diseases on human and veterinary health is being exacerbated by rapid environmental changes caused mainly by changing climatic patterns and globalization. To gain insight into mosquito-borne virus circulation from two counties in eastern and southeastern Romania, we have used a combination of sampling methods in natural, urban and peri-urban sites. The presence of 37 mosquito-borne viruses in 16,827 pooled mosquitoes was analyzed using a high-throughput microfluidic real-time PCR assay. West Nile virus (WNV) was detected in 10/365 pools of Culex pipiens (n = 8), Culex modestus (n = 1) and Aedes vexans (n = 1) from both studied counties. We also report the first molecular detection of Sindbis virus (SINV) RNA in the country in one pool of Culex modestus. WNV infection was confirmed by real-time RT-PCR (10/10) and virus isolation on Vero or C6/36 cells (four samples). For the SINV-positive pool, no cytopathic effectwas observed after infection of Vero or C6/36 cells, but no amplification was obtained in conventional SINV RT-PCR. Phylogenetic analysis of WNV partial NS5 sequences revealed that WNV lineage 2 of theCentral-Southeast European clade, has a wider circulation in Romania than previously known. Full article

Small wild mammals are an important element in the emergence and transmission of vector-borne pathogens (VBPs). Among these species, hedgehogs have been found to be a reservoir of VBPs and host of arthropod vectors. Surveillance of VBPs in wildlife and their arthropods are crucial in a one health context. We conducted an exploratory study to screen Atelerix algirus hedgehogs and their infesting ticks and fleas for VBPs using a high throughput microfluidic real-time PCR system. Tested biopsies from hedgehogs were found to be naturally infected by Theileria youngi, Hepatozoon sp., Ehrlichia ewingii, Coxiella burnetii, and Candidatus Ehrlichia shimanensis. Similarly, Haemaphysalis erinacei and Rhipicephalus sanguineus tick species were infected by Ehrlichia ewingii, Rickettsia spp., Rickettsia massiliae, Borrelia sp., Coxiella burnetii, Rickettsia lusitaniae and Anaplasma sp. Archaeopsylla erinacei fleas were infected by Rickettsia asembonensis, Coxiella burnetii, and Rickettsia massiliae. Co-infections by two and three pathogens were detected in hedgehogs and infesting ticks and fleas. The microfluidic real-time PCR system enabled us not only to detect new and unexpected pathogens, but also to identify co-infections in hedgehogs, ticks, and fleas. We suggest that hedgehogs may play a reservoir role for VBPs in Tunisia and contribute to maintaining enzootic pathogen cycles via arthropod vectors. Full article

Ixodid ticks are hematophagous arthropods considered to be prominent ectoparasite vectors that have a negative impact on cattle, either through direct injury or via the transmission of several pathogens. In this study, we investigated the molecular infection rates of numerous tick-borne pathogens in ticks sampled on cattle from the Kabylia region, northeastern Algeria, using a high-throughput microfluidic real-time PCR system. A total of 235 ticks belonging to seven species of the genera Rhipicephalus, Hyalomma, and Ixodes were sampled on cattle and then screened for the presence of 36 different species of bacteria and protozoans. The most prevalent tick-borne microorganisms were Rickettsia spp. at 79.1%, followed by Francisella-like endosymbionts (62.9%), Theileria spp. (17.8%), Anaplasma spp. (14.4%), Bartonella spp. (6.8%), Borrelia spp. (6.8%), and Babesia spp. (2.5%). Among the 80.4% of ticks bearing microorganisms, 20%, 36.6%, 21.7%, and 2.1% were positive for one, two, three, and four different microorganisms, respectively. Rickettsia aeschlimannii was detected in Hyalomma marginatum, Hyalomma detritum, and Rhipicephalus bursa ticks. Rickettsia massiliae was found in Rhipicephalus sanguineus, and Rickettsiamonacensis and Rickettsia helvetica were detected in Ixodesricinus. Anaplasma marginale was found in all identified tick genera, but Anaplasma centrale was detected exclusively in Rhipicephalus spp. ticks. The DNA of Borrelia spp. and Bartonella spp. was identified in several tick species. Theileria orientalis was found in R. bursa, R. sanguineus, H. detritum, H. marginatum, and I. ricinus and Babesia bigemina was found in Rhipicephalus annulatus and R. sanguineus. Our study highlights the importance of tick-borne pathogens in cattle in Algeria. Full article

Despite the high burden of vector-borne disease in (sub)tropical areas, few information are available regarding the diversity of tick and tick-borne pathogens circulating in the Caribbean. Management and control of vector-borne disease require actual epidemiological data to better assess and anticipate the risk of (re)emergence of tick-borne diseases in the region. To simplify and reduce the costs of such large-scale surveys, we implemented a high-throughput microfluidic real-time PCR system suitable for the screening of the main bacterial and parasitic genera involved in tick-borne disease and potentially circulating in the area. We used the new screening tool to perform an exploratory epidemiological study on 132 adult specimens of Amblyomma variegatum and 446 of Rhipicephalus microplus collected in Guadeloupe and Martinique. Not only the system was able to detect the main pathogens of the area—Ehrlichia ruminantium, Rickettsia africae, Anaplasma marginale, Babesia bigemina and Babesia bovis—but the system also provided evidence of unsuspected microorganisms in Caribbean ticks, belonging to the Anaplasma, Ehrlichia, Borrelia and Leishmania genera. Our study demonstrated how high-throughput microfluidic real-time PCR technology can assist large-scale epidemiological studies, providing a rapid overview of tick-borne pathogen and microorganism diversity, and opening up new research perspectives for the epidemiology of tick-borne pathogens. Full article