Molecular Diagnostic Techniques Research Articles

Overview of Mycotic Keratitis

Abstract: Keratomycosis is a serious corneal infection associated with high ocular morbidity that can lead to severe vision loss. It is estimated to affect more than 1 million patients annually, most commonly occurring in tropical climates, and represents a growing threat to patients worldwide. Despite aggressive medical management, fungal infections have a higher rate of perforation requiring surgical intervention compared with other infectious etiologies. Early diagnosis and appropriate treatment are keys to preserving vision and saving patients' eyes. Timely diagnosis of fungal keratitis helps minimize corneal damage and scarring and increases the likelihood of a favorable outcome. Studies have shown that correct identification of fungal infections is often delayed up to 2 to 3 weeks after initial presentation. This leads to incorrect or ineffective treatment for many patients. Diagnostic techniques explored in this study include corneal scrapings with staining and culture, visualization with in vivo confocal microscopy, molecular diagnostic techniques including polymerase chain reaction, and recently developed omics-based technologies. Treatment of fungal keratitis begins with topical antifungals. Medical management has been proven to be effective, but with limitations including poor drug penetration and low bioavailability. Cases that do not respond to topical therapy require more invasive and novel treatments to control the infection. We review the clinical trials that have shaped current practice patterns, with focus on the efficacy of topical natamycin as the primary therapy for filamentous fungal keratitis. We explore additional management strategies such as localized intrastromal and intracameral injections of antifungal medications, photodynamic therapy, and surgical intervention.

A Machine learning model for predicting sepsis based on an optimized assay for microbial cell-free DNA sequencing

ObjectiveTo integrate an enhanced molecular diagnostic technique to develop and validate a machine-learning model for diagnosing sepsis. MethodsWe prospectively enrolled patients suspected of sepsis from August 2021 to August 2023. Various feature selection algorithms and machine learning models were used to develop the model. The best classifier was selected using 5-fold cross validation set and then was applied to assess the performance of the model in the testing set. Additionally, we employed the Shapley Additive exPlanations (SHAP) method to illustrate the effects of the features. ResultsWe established an optimized mNGS assay and proposed using the copies of microbe-specific cell-free DNA per milliliter of plasma (CPM) as the detection signal to evaluate the real burden, with strong precision and high accuracy. In total, 237 patients were eligible for participation, which were randomly assigned to either the training set (70 %, n = 165) or the testing set (30 %, n = 72). The random forest classifier achieved accuracy, AUC and F1 scores of 0.830, 0.918 and 0.856, respectively, outperforming other machine learning models in the training set. Our model demonstrated clinical interpretability and achieved good prediction performance in differentiating between bacterial sepsis and non-sepsis, with an AUC value of 0.85 and an average precision of 0.91 in the testing set. Based on the SHAP value, the top nine features of the model were PCT, CPM, CRP, ALB, SBPmin, RRmax, CREA, PLT and HRmax. ConclusionWe demonstrated the potential of machine-learning approaches for predicting bacterial sepsis based on optimized mcfDNA sequencing assay accurately.

Application of Epithelial Growth Factor Receptor-Targeted Magnetic Resonance Imaging and Near-Infrared II Dual-Modal Probe in Lung Cancer Diagnosis and Surgical Resection.

There has been an increase in the use of molecular probe diagnostic techniques for lung cancer, and magnetic resonance imaging (MRI) offers specific advantages for diagnosing pulmonary carcinoma. Furthermore, advancements in near-infrared II (NIR-II) fluorescence have provided a new method for precise intraoperative tumor resection. However, few probes combine preoperative diagnosis with intraoperative imaging. This study aims to fill this research void by employing a dual-modal probe that targets the epidermal growth factor receptor for MR and NIR-II imaging, enabling the preoperative diagnosis of lung cancer using MRI and precise intraoperative tumor localization using NIR-II with a single probe. The imaging effects and targeting ability of the probe were confirmed in cell lines, mouse models, and clinical samples. The MR signal decreased within 24 h in the patient-derived xenograft mouse model. The average signal-to-background ratio of NIR-II reached 3.98 ± 0.27. The clinical sample also showed a decrease in the T2 signal using MRI, and the NIR-II optical signal-to-background ratio was 3.29. It is expected that this probe can improve the diagnostic rate of lung cancer using MRI and enable precise intraoperative tumor resection using NIR-II.

Metabolomics reveals early pregnancy biomarkers in sows: a non-invasive diagnostic approach.

In an effort to enhance reproductive management and reduce non-productive periods in swine breeding, this study presents a novel, non-invasive metabolomics approach for the identification of early pregnancy biomarkers in sows. Utilizing an untargeted metabolomics approach with mass spectrometry analysis, we examined saliva samples from pregnant (n = 6) and non-pregnant control sows (n = 6, artificially inseminated with non-viable sperm). Our analysis revealed 286 differentially expressed metabolites, with 152 being up-regulated and 134 down-regulated in the pregnant group. Among these, three metabolites, namely Hyodeoxycholic acid, 2'-deoxyguanosine, and Thymidine, emerged as potential early pregnancy biomarkers. These biomarkers were further evaluated using targeted LC-MS/MS quantification and qualification, accompanied by ROC curve analysis. The study confirmed Hyodeoxycholic acid and 2'-deoxyguanosine as promising biomarkers for early pregnancy detection, offering potential for future implementation in swine production environments. This research establishes a robust theoretical foundation for the development of innovative molecular diagnostic techniques and explores new avenues for molecular genetic breeding and non-invasive diagnostics, ultimately enhancing fertility and productivity in sow herds.

Immunological and molecular diagnostic techniques in fish health: present and future prospectus.

Fish health management is critical to aquaculture and fisheries as it directly affects sustainability and productivity. Fish disease diagnosis has taken a massive stride because of advances in immunological and molecular diagnostic tools which provide a sensitive, quick, and accurate means of identifying diseases. This review presents an overview of the main molecular and immunological diagnostic methods for determining the health of fish. The immunological techniques help to diagnose different fish diseases by detecting specific antigens and antibodies. The application of immunological techniques to vaccine development is also examined in this review. The genetic identification of pathogens is made possible by molecular diagnostic techniques that enable the precise identification of bacterial, viral, and parasitic organisms in addition to evaluating host reactions and genetic variation associated with resistance to disease. The combination of molecular and immunological methods has resulted in the creation of novel techniques for thorough evaluation of fish health. These developments improve treatment measures, pathogen identification and provide new information about the variables affecting fish health, such as genetic predispositions and environmental stresses. In the framework of sustainable fish farming and fisheries management, this paper focuses on the importance of these diagnostic techniques that play a crucial role in protecting fish populations and the aquatic habitats. This review also examines the present and potential future directions in immunological and molecular diagnostic techniques in fish health.

Sentinels in the shadows: Exploring Toxoplasma gondii and other Sarcocystidae parasites in synanthropic rodents and their public health implications

Synanthropic rodents play a crucial role in maintaining the life cycle of Toxoplasma gondii in anthropized regions and can serve as indicators of environmental oocyst contamination. This investigation aimed to explore the occurrence of T. gondii infection within synanthropic rodent populations using a molecular diagnostic technique targeting the 18S rDNA gene, which is generic for Coccidia, with subsequent specific PCR confirmation. We examined 97 brown rats (Rattus norvegicus), 67 black rats (R. rattus), 47 house mice (Mus musculus), and 1 common shrew (Sorex araneus). PCR tests were conducted on the brain, heart, and tongue tissues. PCR tested positive in at least one of the examined tissues in 26 R. norvegicus (26.8%), 13 R. rattus (19.4%), and 13 M. musculus (27.6%). Sequencing comparisons by BLAST allowed us to identify four different species of cyst-forming Apicomplexa. In particular, T. gondii DNA was detected in 13 (6.1%) rodents, Hammondia hammondi (including H. hammondi-like organisms) in 36 (17%) subjects, Besnoitia sp. (in two cases identified as B. besnoiti) in 8 (3.7%), and Sarcocystis gigantea in two (0.94%). Rodents from peri-urban and urban environments can act as indicators of environmental contamination by oocysts of apicomplexan parasites with cats as definitive hosts, such as T. gondii, H. hammondi, and S. gigantea, the latter of which has never been previously recorded in rodents. Moreover, the presence of B. besnoiti, a parasite with an unidentified definitive host in Europe, sheds light on the potential role of these hosts as infection sentinels.

Development of a new loop-mediated isothermal amplification test for the sensitive, rapid, and economic detection of different genotypes of Classical swine fever virus.

Classical swine fever virus (CSFV) remains one of the most important pathogens in animal health. Pathogen detection relies on viral RNA extraction followed by RT-qPCR. Novel technologies are required to improve diagnosis at the point of care. A loop-mediated isothermal amplification (LAMP) PCR technique was developed, with primers designed considering all reported CSFV genotypes. The reaction was tested using both fluorometric and colorimetric detection, in comparison to the gold standard technique. Viral strains from three circulating CSFV genotypes were tested, as well as samples from infected animals. Other pathogens were also tested, to determine the LAMP specificity. Besides laboratory RNA extraction methods, a heating method for RNA release, readily available for adaptation to field conditions was evaluated. Three primer sets were generated, with one of them showing better performance. This primer set proved capable of maintaining optimal performance at a wide range of amplification temperatures (60°C - 68°C). It was also able to detect CSFV RNA from the three genotypes tested. The assay was highly efficient in detection of samples from animals infected with field strains from two different genotypes, with multiple matrices being detected using both colorimetric and fluorometric methods. The LAMP assay was negative for all the unrelated pathogens tested, including Pestiviruses. The only doubtful result in both fluorometric and colorimetric LAMP was against the novel Pestivirus italiaense, ovine Italy Pestivirus (OVPV), which has proven to have cross-reaction with multiple CSFV diagnostic techniques. However, it is only possible to detect the OVPV in a doubtful result if the viral load is higher than 10000 viral particles. The results from the present study show that LAMP could be an important addition to the currently used molecular diagnostic techniques for CSFV. This technique could be used in remote locations, given that it can be adapted for successful use with minimal equipment and minimally invasive samples. The joined use of novel and traditional diagnostic techniques could prove to be a useful alternative to support the CSF control.

Molecular Identification and Prevalence of the Mite Carpoglyphus lactis (Acarina: Carpoglyphidae) in Apis mellifera in the Republic of Korea.

Apis mellifera, especially weak ones, are highly vulnerable to Carpoglyphus lactis mites, which can rapidly infest and consume stored pollen, leading to weakened colonies and potential colony collapse. This study aimed to ascertain and investigate the prevalence of this mite in honeybee colonies across nine provinces in the Republic of Korea (ROK). A total of 615 honeybee colony samples were collected from 66 apiaries during the spring and 58 apiaries during the summer of 2023. A 1242 bp segment of the Cytochrome c oxidase subunit 1 (COI) gene was amplified using the polymerase chain reaction method. The detection levels of C. lactis in the honeybees were compared between winter and summer. Based on the COI sequence analysis, the nucleotide sequence similarity of C. lactis mites isolated in the ROK with those from China (NC048990.1) was found to be 99.5%, and with those from the United Kingdom (KY922482.1) was 99.3%. This study is the first report of C. lactis in Korean apiaries. The findings of this study demonstrate a significantly higher detection rate in winter, which is 4.1 times greater than that in summer (p < 0.001). Furthermore, the results underscore the usefulness of molecular diagnostic techniques for detecting C. lactis mites.

Molecular Pathology of Hematologic Malignancies

Abstract Hematological malignancies represent a heterogeneous group of diseases that may have overlapping clinical manifestations. Successful and optimal management depends on early and accurate diagnosis of the disease. Differential diagnosis therefore requires methods of morphology, immunohistochemistry, flow cytometry, and also molecular and cytogenetic examinations. Molecular diagnostic techniques are becoming more accurate and sophisticated, which is why nowadays hematopathological diagnosis relies heavily on molecular and cytogenetic analyses. They are beneficial not only for diagnosis, but also for evaluating prognostic and risk markers, as well as treatment monitoring. This article presents an overview of selected hematological malignancies – mature lymphoid neoplasms, multiple myelomas, myeloproliferative neoplasms, myelodysplastic syndromes, and acute myeloid leukemias; their molecular pathology, risk and prognostic markers. Understanding of the biologic basis leds to a targeted therapy development for the treatment of these diseases.

Genotyping of Leptospira spp. in wild rats leads to first time detection of L. kirshneri serovar Mozdok in Serbia.

This study aimed to investigate the prevalence and molecular characterization of Leptospira species in Belgrade, Serbia, an area where this disease is underexplored. Specifically, the study sought to employ molecular and multilocus sequence typing analyses to fill the gap in understanding the diversity and distribution of Leptospira species within the region. A comprehensive molecular analysis was conducted on kidney samples obtained from Norway rats (Rattus norvegicus) in the urban environment. The study utilized molecular diagnostic techniques including real-time PCR targeting the lipL32 gene and performing sequence-based typing schemes utilizing adk, icdA, lipL32, lipL41, rrs2, and secY genes. These methodologies were applied to ascertain the presence and characterize different Leptospira species and serovars, respectively. The findings revealed the presence of two Leptospira species and three separate serovars in the Belgrade area. This study identified the presence of L. kirschneri serovar Mozdok in Serbia for the first time, a significant discovery previously undocumented in the region. This pioneering investigation sheds light on the molecular diversity and prevalence of Leptospira species in Serbia. The study underscores the importance of employing molecular typing methods to gain insights into the epidemiology and characterization of Leptospira species. These findings significantly contribute to both local and global perspectives on leptospirosis epidemiology, providing vital insights for the development of effective control strategies and interventions. In our recent study, we explored the presence and performed molecular typing of the Leptospira species, the bacteria responsible for leptospirosis, in wild rats in Serbia. This was the first time such a study was conducted in the region. Leptospirosis is a serious disease that affects both animals and humans, often transmitted through contact with water contaminated by infected animals. Our focus was on understanding which types of Leptospira were present in these animals. Excitingly, we discovered a particular strain of Leptospira, known as L. kirshneri serovar Mozdok, for the first time in Serbia. This finding is significant because it sheds light on the presence and spread of different Leptospira serovars in Serbia. It also raises awareness about the potential health risks associated with this serovar, which was previously unknown in the area. Our work fits into a broader context of disease surveillance and public health. By identifying the types of Leptospira present in a specific region, we can better understand the risks to public health and take steps to prevent and control the spread of leptospirosis. This discovery is not just important for scientists studying infectious diseases; it has real implications for public health officials, veterinarians, and anyone concerned with preventing and treating leptospirosis. Our findings highlight the need for ongoing monitoring of Leptospira in wildlife and synanthropic fauna, to protect both animal and human health.

Amebic liver abscess: An update.

Amebic liver abscess (ALA) is still a common problem in the tropical world, where it affects over three-quarters of patients with liver abscess. It is caused by an anaerobic protozoan Entamoeba hystolytica, which primarily colonises the cecum. It is a non-suppurative infection of the liver consisting primarily of dead hepatocytes and cellular debris. People of the male gender, during their reproductive years, are most prone to ALA, and this appears to be due to a poorly mounted immune response linked to serum testosterone levels. ALA is more common in the right lobe of the liver, is strongly associated with alcohol consumption, and can heal without the need for drainage. While majority of ALA patients have an uncomplicated course, a number of complications have been described, including rupture into abdomino-thoracic structures, biliary fistula, vascular thrombosis, bilio-vascular compression, and secondary bacterial infection. Based on clinico-radiological findings, a classification system for ALA has emerged recently, which can assist clinicians in making treatment decisions. Recent research has revealed the role of venous thrombosis-related ischemia in the severity of ALA. Recent years have seen the development and refinement of newer molecular diagnostic techniques that can greatly aid in overcoming the diagnostic challenge in endemic area where serology-based tests have limited accuracy. Metronidazole has been the drug of choice for ALA patients for many years. However, concerns over the resistance and adverse effects necessitate the creation of new, safe, and potent antiamebic medications. Although the indication of the drainage of uncomplicated ALA has become more clear, high-quality randomised trials are still necessary for robust conclusions. Percutaneous drainage appears to be a viable option for patients with ruptured ALA and diffuse peritonitis, for whom surgery represents a significant risk of mortality. With regard to all of the aforementioned issues, this article intends to present an updated review of ALA.

From degraded to deciphered: ATAC-seq's application potential in forensic diagnosis.

In recent years, molecular biology-based diagnostic techniques have made remarkable strides and are now extensively utilized in clinical practice, providing invaluable insights for disease diagnosis and treatment. However, forensic medicine, especially forensic pathology, has witnessed relatively limited progress in the application of molecular biology technologies. A significant challenge in employing molecular techniques for forensic diagnoses lies in the quantitative and qualitative changes observed in diagnostic markers due to sample degradation-a recognized and formidable obstacle. Inspired by the success of DNA sequencing in forensic practices, which enables accurate individual identification even in cases involving degraded and deteriorated tissues and organs, we propose the application of the assay for transposase-accessible chromatin with sequencing (ATAC-seq) to identify targets at the transcriptional onset, exploring chromatin and DNA-level alterations for injury and disease inference in forensic samples. This study employs ATAC-seq to explore alterations in chromatin accessibility post-injury and their subsequent changes over a 2-h degradation period, employing traumatic brain injury (TBI) as a representative model. Our findings reveal high sensitivity of chromatin accessibility sites to injury, evidenced by shifts in thousands of peak positions post-TBI. Remarkably, these alterations remain largely unaffected by early degradation. Our results robustly endorse the notion that integrating and incorporating these specific loci for injury and disease diagnosis in forensic samples holds tremendous promise for practical application. We further validated the above results using human cortical tissue, which supported that early degradation did not significantly affect chromatin accessibility. This pioneering advancement in molecular diagnostic techniques may revolutionize the field of forensic science, especially forensic pathology.

Lymphocytic Choriomeningitis Virus (LCMV): Current Status and Future Directions for Clinical and Molecular Diagnostic Techniques

Lymphocytic Choriomeningitis Virus (LCMV): Current Status and Future Directions for Clinical and Molecular Diagnostic Techniques

Advancements in the Detection and Treatment of Rare ALK Fusion Mutations in Hepatocellular Carcinoma: A Case Report and Literature Review

This case report and comprehensive literature review highlights the significance of advanced molecular diagnostic techniques, particularly next-generation sequencing (NGS), in the identification and treatment of rare ALK fusion mutations in hepatocellular carcinoma (HCC). Through a detailed analysis of a single patient case, accompanied by a review of existing literature, we underscore the diagnostic challenges and therapeutic potential associated with rare ALK fusions in HCC. Our findings demonstrate the superior capability of NGS, both DNA-based and RNA-based, in detecting these mutations, leading to targeted treatment approaches with ALK inhibitors. The case report illustrates the practical application of precision oncology in HCC, showcasing significant clinical benefit and improved treatment outcomes with the use of ALK inhibitors for rare fusion types. This study not only contributes to the existing body of knowledge by documenting a rare instance of ALK fusion in HCC but also advocates for the integration of comprehensive molecular profiling into standard clinical practice to enhance personalized treatment strategies and patient care.

Identification of TP53 aberrations in chronic lymphocytic leukaemia using different molecular diagnostic techniques

Identification of TP53 aberrations in chronic lymphocytic leukaemia using different molecular diagnostic techniques

Molecular Genetic Profile of Myelofibrosis: Implications in the Diagnosis, Prognosis, and Treatment Advancements

Myelofibrosis (MF) is an essential element of primary myelofibrosis, whereas secondary MF may develop in the advanced stages of other myeloid neoplasms, especially polycythemia vera and essential thrombocythemia. Over the last two decades, advances in molecular diagnostic techniques, particularly the integration of next-generation sequencing in clinical laboratories, have revolutionized the diagnosis, classification, and clinical decision making of myelofibrosis. Driver mutations involving JAK2, CALR, and MPL induce hyperactivity in the JAK-STAT signaling pathway, which plays a central role in cell survival and proliferation. Approximately 80% of myelofibrosis cases harbor additional mutations, frequently in the genes responsible for epigenetic regulation and RNA splicing. Detecting these mutations is crucial for diagnosing myeloproliferative neoplasms (MPNs), especially in cases where no mutations are present in the three driver genes (triple-negative MPNs). While fibrosis in the bone marrow results from the disturbance of inflammatory cytokines, it is fundamentally associated with mutation-driven hematopoiesis. The mutation profile and order of acquiring diverse mutations influence the MPN phenotype. Mutation profiling reveals clonal diversity in MF, offering insights into the clonal evolution of neoplastic progression. Prognostic prediction plays a pivotal role in guiding the treatment of myelofibrosis. Mutation profiles and cytogenetic abnormalities have been integrated into advanced prognostic scoring systems and personalized risk stratification for MF. Presently, JAK inhibitors are part of the standard of care for MF, with newer generations developed for enhanced efficacy and reduced adverse effects. However, only a minority of patients have achieved a significant molecular-level response. Clinical trials exploring innovative approaches, such as combining hypomethylation agents that target epigenetic regulators, drugs proven effective in myelodysplastic syndrome, or immune and inflammatory modulators with JAK inhibitors, have demonstrated promising results. These combinations may be more effective in patients with high-risk mutations and complex mutation profiles. Expanding mutation profiling studies with more sensitive and specific molecular methods, as well as sequencing a broader spectrum of genes in clinical patients, may reveal molecular mechanisms in cases currently lacking detectable driver mutations, provide a better understanding of the association between genetic alterations and clinical phenotypes, and offer valuable information to advance personalized treatment protocols to improve long-term survival and eradicate mutant clones with the hope of curing MF.

The Sensitization Profile for Selected Food Allergens in Polish Children Assessed with the Use of a Precision Allergy Molecular Diagnostic Technique.

Individual populations show a variety of sensitization patterns, which may be associated with the geographic region, climate, dietary habits, or ways of preparing food. The purpose of this study was to comprehensively assess the food allergy sensitization profile in Polish children, particularly to eight food allergens (so-called "the Big 8"): cow milk, eggs, wheat, soybeans, fish, crustacean shellfish, tree nuts, and peanuts. To assess the prevalence and serum levels of specific immunoglobulins E (sIgE), we analyzed the results obtained from selected laboratories located in all regions of Poland that used the multiplex ALEX® test in the period from 2019 to 2022. Results from 3715 children were obtained. The mean age of the study population was 7.0 years. The results were stratified by age: <12 months (3.63%), 1-5 years (39.54%), 6-13 years (46.32%), and 14-18 years (10.0%). The final analysis included the sIgE results obtained with 95 food extracts and 77 food allergen molecules. The highest rates of sIgE to food allergen extracts were found for peanut (29.20%), hazel (28.20%), and apple (23.60%), and those to allergenic molecules were found for the PR-10 family of molecules (Cor a 1.0401 (23.77%), Mal d 1 (22.37%), Ara h 8 (16.93%), and globulin 7/8S (Ara h 1; 15.59%)). The lowest rates of sIgE reactivity to extracts were found for strawberry (0.40%), oregano (0.30%), and thornback ray (0.16%), and those to allergenic molecules were found for Mal d 2 (0.27%) (thaumatin-like protein, TLP), Ani s 1 (0.30%) (Kunitz-type serine protease inhibitor), and Che a 1 (0.43%) (Ole e 1 family). The rates of sensitization to storage proteins of the analyzed "the Big 8" molecules decreased significantly (p < 0.05) with age. Conversely, the rates of sensitization to PR-10 family proteins increased significantly with age. The three most common allergens in Poland, regardless of whether IgE was assayed against extracts or molecules of food allergens, were peanut, hazel, and apple (in different order depending on the ranking). A detailed analysis of sensitization to the extracts and molecules of main food allergens based on the results of a multiplex ALEX® test demonstrated the sensitization profile in Polish children (including molecular sensitization, particularly the "the Big 8" food allergen molecules), which shows considerable differences in comparison with those in other countries. Serum sIgE analysis of children from all regions of Poland revealed a food allergen molecular sensitization profile that changes with age.

Microbiology of Severe Community-Acquired Pneumonia and the Role of Rapid Molecular Techniques.

The microbiology of severe community acquired pneumonia (SCAP) has implications on management, clinical outcomes and public health policy. Therefore, knowledge of the etiologies of SCAP and methods to identify these microorganisms is key. Bacteria including Streptococcus pneumoniae, Staphylococcus aureus and Enterobacteriaceae continue to be important causes of SCAP. Viruses remain the most commonly identified etiology of SCAP. Atypical organisms are also important etiologies of SCAP and are critical to identify for public health. With the increased number of immunocompromised individuals, less common pathogens may also be found as the causative agent of SCAP. Traditional diagnostic tests, including semi-quantitative respiratory cultures, blood cultures and urinary antigens continue to hold an important role in the evaluation of patients with SCAP. Many of the limitations of the aforementioned tests are addressed by rapid, molecular diagnostic tests. Molecular diagnostics utilize culture-independent technology to identify species-specific genetic sequences. These tests are often semi-automated and provide results within hours, which provides an opportunity for expedient antibiotic stewardship. The existing literature suggests molecular diagnostic techniques may improve antibiotic stewardship in CAP, and future research should investigate optimal methods for implementation of these assays into clinical practice.

Multiple cross displacement amplification combined with nanoparticle-based lateral flow biosensor for rapid and sensitive detection of Epstein-Barr virus.

Epstein-Barr virus (EBV) is a highly dangerous virus that is globally prevalent and closely linked to the development of nasopharyngeal cancer (NPC). Plasma EBV DNA analysis is an effective strategy for early detection, prognostication and monitoring of treatment response of NPC. Here, we present a novel molecular diagnostic technique termed EBV-MCDA-LFB, which integrates multiple cross displacement amplification (MCDA) with nanoparticle-based lateral flow (LFB) to enable simple, rapid and specific detection of EBV. In the EBV-MCDA-LFB system, a set of 10 primers was designed for rapidly amplifying the highly conserved tandem repeat BamHI-W region of the EBV genome. Subsequently, the LFB facilitate direct assay reading, eliminating the use of extra instruments and reagents. The outcomes showed that the 65°C within 40 minutes was the optimal reaction setting for the EBV-MCDA system. The sensitivity of EBV-MCDA-LFB assay reached 7 copies per reaction when using EBV recombinant plasmid, and it showed 100% specificity without any cross-reactivity with other pathogens. The feasibility of the EBV-MCDA-LFB method for EBV detection was successfully validated by 49 clinical plasma samples. The complete detection process, consisting of rapid template extraction (15 minutes), MCDA reaction (65°C for 40 minutes), and LFB result reading (2 minutes), can be finalized within a 60-minutes duration. EBV-MCDA-LFB assay designed here is a fast, extremely sensitive and specific technique for detecting EBV in field and at the point-of-care (PoC), which is especially beneficial for countries and regions with a high prevalence of the disease and limited economic resources.

Human rickettsial infections in India - A review

Rickettsial infections are emerging and/or re-emerging disease that poses a serious global threat to humans and animals. Transmission to humans and animals is through the bite of the ectoparasites including ticks, fleas and chigger mites. Most of the rickettsial diseases are endemic in India, but underdiagnosed. This review is aimed at analyzing the prevalence of rickettsiosis in India and the advancement of rickettsial diagnosis. We have conducted a systematic review on the prevalence of rickettsial disease in India ranging from 1.3% to 46.6% for spotted fever, 2.4% to 77.8% for scrub typhus and 1% to 46.4% for Q fever, based on the literature published with the evidence of isolation, serological, and molecular diagnostics. Search engines Medline/PubMed, Science Direct, ProQuest, and EBSCO were used to retrieve the articles from electronic databases by using appropriate keywords to track the emergence of these rickettsial diseases in India for the period of 1865 to till date. We retrieved 153 published rickettsial articles on hospital-based studies from India that were purely made on the basis of prevalence and the laboratory parameters viz., Weil-Felix test (WF) and Rapid Immunochromatographic tests (RICT) with reference to the gold standard IFA and ELISA. More epidemiological studies are required for epidemic typhus to know the exact prevalence status of this louse-borne rickettsiosis in India. Currently, there is no confirmed specific inflammatory marker for rickettsial diseases. Moreover, serological cross-reactivity is an important aspect, and it should be investigated in endemic areas, there is also a need to include molecular diagnostic techniques for further confirmation in healthcare settings.