Molecular Diagnostic Techniques Research Articles

Advancements in LAMP-Based Diagnostics: Emerging Techniques and Applications in Viral Detection with a Focus on Herpesviruses in Transplant Patient Management

Loop-mediated isothermal amplification (LAMP) is a highly effective molecular diagnostic technique, particularly advantageous for point-of-care (POC) settings. In recent years, LAMP has expanded to include various adaptations such as DARQ-LAMP, QUASR, FLOS-LAMP, displacement probes and molecular beacons. These methods enable multiplex detection of multiple targets in a single reaction, enhancing cost-effectiveness and diagnostic efficiency. Consequently, LAMP has gained significant traction in diagnosing diverse viruses, notably during the COVID-19 pandemic. However, its application for detecting Herpesviridae remains relatively unexplored. This group of viruses is of particular interest due to their latency and potential reactivation, crucial for immunocompromised patients, including organ and hematopoietic stem cell transplant recipients. This review highlights recent advancements in LAMP for virus diagnosis and explores current research trends and future prospects, emphasizing the detection challenges posed by Herpesviridae.

Combined analysis of a serum mRNA/miRNA marker signature and CA 19-9 for timely and accurate diagnosis of recurrence after resection of pancreatic ductal adenocarcinoma: A prospective multicenter cohort study.

Timely and accurate detection of tumor recurrence in pancreatic ductal adenocarcinoma (PDAC) patients is an urgent and unmet medical need. This study aimed to develop a noninvasive molecular diagnostic procedure for the detection of recurrence after PDAC resection based on quantification of circulating mRNA and miRNA biomarkers in serum samples. In a multicentric study, serum samples from a total of 146 patients were prospectively collected after resection. Samples were classified into a "No Evidence of Disease" and a "Recurrence" group based on clinical follow-up data. A multianalyte biomarker panel was composed of mRNAs and miRNA markers and simultaneously analyzed in serum samples using custom microfluidic qPCR arrays (TaqMan array cards). A diagnostic algorithm was developed combining a 7-gene marker signature with CA19-9 data. The best-performing marker combination achieved 90% diagnostic accuracy in predicting the presence of tumor recurrence (98% sensitivity; 84% specificity), clearly outperforming the singular CA 19-9 analysis. Moreover, time series data obtained by analyzing successively collected samples from 5 patients during extended follow-up suggested that molecular diagnosis has the potential to detect recurrence earlier than routine clinical procedures. TaqMan array card measurements were found to be biologically valid and technically reproducible. The BioPac multianalyte marker panel is capable of sensitive and accurate detection of recurrence in patients resected for PDAC using a simple blood test. This could allow a closer follow-up using shorter time intervals than currently used for imaging, thus potentially prompting an earlier work-up with additional modalities to allow for earlier therapeutic intervention. This study provides a promising approach for improved postoperative monitoring of resected PDAC patients, which is an urgent and unmet clinical need.

Companion Diagnostics (CDx) Based on Molecular Biology Techniques

Molecular profiling based on genomic mutations provides clinically important diagnostic and prognostic information. Companion diagnostic (CDx) testing, which is based on targeted drug therapy, is being applied to a variety of molecular diagnostic techniques (e.g., fluorescent in situ hybridization—FISH; polymerase chain reaction—PCR; and next-generation sequencing—NGS) to diagnose complex etiologies using a minimal number of specimens, replacing immunohistochemical analysis, which may show bias at certain stages. The safety and effectiveness of CDx testing using molecular diagnostic technology in precision medicine is an important factor in determining the treatment outcome and prognosis of patients. Meeting minimum safety and effectiveness performance standards is essential for CDx testing, and a thorough understanding of regulatory considerations is necessary to plan and design the optimal product. In this review, we focus on the diagnostic field of precision medicine and discuss the safety and effectiveness that each molecular diagnostic technology must meet according to CDx testing diversity.

Confirmation of oat crown rust disease in Taiwan.

Oat is a minor forage crop grown in Taiwan. Only a few historical records of oat rust disease have been reported in the country. Therefore, the pathogen population remains poorly characterized. A rust-like disease outbreak was detected at the Experimental Farm of National Taiwan University in 2019, which caused significant damage to field experiments. To determine the identity of the pathogen responsible for this disease outbreak, we collected infected foliar material. Disease signs suggested infection by the oat crown rust fungus. Hence, common procedures in rust pathology were applied to confirm the identity of the pathogen with phenotypic and molecular diagnostic techniques. A total of 50 field pathogen samples from infected oat cultivars were collected in 2019 and five single pustule rust isolates were obtained in 2020 and 2021. These isolates were initially identified as Puccinia coronata var. avenae f. sp. avenae (Pca) based on the phylogenetic analysis of nrITS sequence data. This identification was subsequently confirmed through whole-genome phylogeny, which showed that the representative Taiwanese isolate NTU1 clustered with other Pca representative strains in Basidiomycota. Phenotyping assays across 36 oat differential lines demonstrated that Taiwanese isolates are phenotypically similar with relatively low virulence. This study presents the first molecular confirmation of Pca in Taiwan and reports the virulence profiles of Taiwanese Pca population.

Detection of Filariid Infections in Mexican Primate Populations Through qPCR.

Filariae are parasitic nematodes of high veterinary and medical importance, responsible for some acute tropical diseases. They are transmitted through the bite of hematophagous vectors such as biting midges and blackflies. Filariae are among the most prevalent vector-borne parasitoses in Neotropical primates in which severe infections can cause inflammatory reactions and tissue damage. Given the location inside the host (peritoneal cavity, bloodstream, and lymphatics), the detection of filariid nematodes is challenging and is mostly postmortem; hence the scarcity of studies on the prevalence of filariae in wild primate populations. Here, we report the prevalence of filariid infections in free-ranging populations of Geoffroy's spider (Ateles geoffroyi) and black howler (Alouatta pigra) monkeys across southern Mexico, using a combination of noninvasive sampling and molecular diagnostic techniques. Fecal samples were screened for filariid DNA by qPCR protocols. A total of 88 samples were examined with an overall prevalence of 26%. Filariae were slightly more common in spider monkeys compared to howler monkeys. This study constitutes the first report of the prevalence of infection of filariid nematodes in populations of wild spider monkey across southern Mexico, and the first reporting of filariae in black howler monkeys, as part of a new era of primate parasitology and the diagnostics of parasite infections in light of the everyday more affordable molecular tools.

Monogenic Kidney Diseases in Adults With Chronic Kidney Disease (CKD).

According to current evidence, every 10th to 11th adult with chronic kidney disease (CKD) has a monogenic disease of the kidney. This review is based on reported studies in which molecular genetic diagnostic techniques were used to investigate monogenic kidney diseases in adults with CKD. The studies were identified by a selective literature search using predefined criteria. In 12 selected studies, diagnostic variants of 179 different genes were identified in 1467 out of 6607 study participants with CKD (22.2%). More than 60% of these variants affected 8 genes (PKD1, PKD2, COL4A3, COL4A4, COL4A5, UMOD, MUC1, HNF1B). Three diseases are associated with these genes: autosomal dominant polycystic kidney disease (ADPKD), Alport syndrome, and autosomal dominant tubulo-interstitial kidney disease (ADTKD). Physicians treating patients with CKD should be alert to the presence of any red flags, such as onset at a young age, a positive family history, or hematuria of unknown cause. When a genetic etiology is suspected, a specialized work-up is indicated, often including a molecular genetic investigation. A positive genetic finding usually leads to a modification of the patient's specific diagnosis and/or treatment. Awareness of the high prevalence of monogenic kidney diseases in adults with CKD and alertness to their suggestive clinical features are crucial for the timely initiation of targeted diagnostic testing. The molecular genetic identification of these diseases is a prerequisite for appropriate patient management.

Antibiotic Resistance in the Elderly: Mechanisms, Risk Factors, and Solutions.

Antibiotic resistance presents a critical challenge in healthcare, particularly among the elderly, where multidrug-resistant organisms (MDROs) contribute to increased morbidity, mortality, and healthcare costs. This review focuses on the mechanisms underlying resistance in key bacterial pathogens and highlights how aging-related factors like immunosenescence, frailty, and multimorbidity increase the burden of infections from MDROs in this population. Novel strategies to mitigate resistance include the development of next-generation antibiotics like teixobactin and cefiderocol, innovative therapies such as bacteriophage therapy and antivirulence treatments, and the implementation of antimicrobial stewardship programs to optimize antibiotic use. Furthermore, advanced molecular diagnostic techniques, including nucleic acid amplification tests and next-generation sequencing, allow for faster and more precise identification of resistant pathogens. Vaccine development, particularly through innovative approaches like multi-epitope vaccines and nanoparticle-based platforms, holds promise in preventing MDRO infections among the elderly. The role of machine learning (ML) in predicting resistance patterns and aiding in vaccine and antibiotic development is also explored, offering promising solutions for personalized treatment and prevention strategies in the elderly. By integrating cutting-edge diagnostics, therapeutic innovations, and ML-based approaches, this review underscores the importance of multidisciplinary efforts to address the global challenge of antibiotic resistance in aging populations.

A multicentre study to evaluate the diagnostic performance of a novel CAD software, DecXpert, for radiological diagnosis of tuberculosis in the northern Indian population

Tuberculosis (TB) is the leading cause of mortality among infectious diseases globally. Effectively managing TB requires early identification of individuals with TB disease. Resource-constrained settings often lack skilled professionals for interpreting chest X-rays (CXRs) used in TB diagnosis. To address this challenge, we developed “DecXpert” a novel Computer-Aided Detection (CAD) software solution based on deep neural networks for early TB diagnosis from CXRs, aiming to detect subtle abnormalities that may be overlooked by human interpretation alone. This study was conducted on the largest cohort size to date, where the performance of a CAD software (DecXpert version 1.4) was validated against the gold standard molecular diagnostic technique, GeneXpert MTB/RIF, analyzing data from 4363 individuals across 12 primary health care centers and one tertiary hospital in North India. DecXpert demonstrated 88% sensitivity (95% CI 0.85–0.93) and 85% specificity (95% CI 0.82–0.91) for active TB detection. Incorporating demographics, DecXpert achieved an area under the curve of 0.91 (95% CI 0.88–0.94), indicating robust diagnostic performance. Our findings establish DecXpert's potential as an accurate, efficient AI solution for early identification of active TB cases. Deployed as a screening tool in resource-limited settings, DecXpert could enable early identification of individuals with TB disease and facilitate effective TB management where skilled radiological interpretation is limited.

Clinical characteristics and mortality of mucormycosis in hematological malignancies: a retrospective study in Eastern China

BackgroundMucormycosis is a significant cause of morbidity and mortality in patients with hematological malignancies, but its characteristics are not fully understood. This study aimed to gain a better understanding of the clinical features of mucormycosis in patients with hematological malignancies in eastern China.MethodsA single-center retrospective analysis was conducted on the demographic profile, microbiology, management, and 90-day mortality of mucormycosis patients with hematological malignancies between 2018 and 2023.ResultsA total of 50 cases were included in the study, consisting of 11 proven and 39 probable cases of mucormycosis. The median age of the patients was 39.98 ± 18.52 years, with 52% being male. Among the cases, 46% had acute myeloid leukemia (AML), 16% had acute lymphoblastic leukemia (ALL), and 16% had myelodysplastic syndrome. The most common manifestations of mucormycosis were pulmonary (80%), disseminated (16%), and rhinocerebral (4%). The diagnosis was confirmed through histology, culture, microscopy, and molecular diagnostic techniques. The most commonly identified fungal species were Cunninghamella (40%), Rhizopus (26%), and Rhizomucor (22%). Treatment involved antifungals in 84% of cases and surgery in 10% of cases. The 90-day mortality rate was 76%. Logistic regression analysis revealed that treatment with amphotericin B and surgery was associated with improved survival, while neutropenia and administration of voriconazole prior to diagnosis was associated with higher mortality.ConclusionsMucormycosis continues to have a high mortality rate in patients with hematological malignancies. Early diagnosis using various techniques, including molecular biology, along with the appropriate use of amphotericin B and surgery when possible, is vital for the successful treatment of mucormycosis.

From Lab to Home: Ultrasensitive Rapid Detection of SARS-CoV-2 with a Cascade CRISPR/Cas13a-Cas12a System Based Lateral Flow Assay.

Currently, CRISPR/Cas-based molecular diagnostic techniques usually rely on the introduction of nucleic acid amplification to improve their sensitivity, which is usually more time-consuming, susceptible to aerosol contamination, and therefore not suitable for at-home molecular testing. In this research, we developed an advanced CRISPR/Cas13a-Cas12a-based lateral flow assay that facilitated the ultrasensitive and rapid detection of SARS-CoV-2 RNA directly from samples, without the need for nucleic acid amplification. This method was called CRISPR LFA enabling at-home RNA testing (CLEAR). CLEAR used a novel cascade mechanism with specially designed probes that fold into hairpin structures, enabling visual detection of SARS-CoV-2 sequences down to 1 aM sensitivity levels. More importantly, CLEAR had a positive coincidence rate of 100% and a negative coincidence rate of 100% for clinical nasopharyngeal swabs from 16 patients. CLEAR was particularly suitable for at-home molecular testing, providing a low-cost, user-friendly solution that can efficiently distinguish between different SARS-CoV-2 variants. CLEAR overcame the common limitations of high sensitivity and potential contamination associated with traditional PCR-based systems, making it a promising tool for widespread public health application, especially in environments with limited access to laboratory resources.

Development of multiplex digital PCR based droplex NSCLC panel test for detecting major mutations in patients with non-small cell lung cancer (NSCLC).

46 Background: Recently, in the treatment of non-small cell lung cancer(NSCLC) patient, immunotherapies and targeted therapies aimed at various genes have been developed. Next-Generation Sequencing(NGS) technology is currently known as the only method for detecting genetic alterations in many different genes simultaneously. However, this technology requires a substantial amount of DNA or RNA input from patients to obtain high-quality results in a clinical setting. We have developed a Droplex NSCLC Panel Test Kit based on multiplex digital PCR, which is known as ideal molecular diagnostic technique due to its simple workflow, minimal sample input, high sensitivity and specificity. In addition, it has a one-day TAT (Turn around time) and low costs compared NGS, resulting in a lower financial burden for patients. Recently, the development of non-invasive liquid biopsy diagnostics as an alternative to tissue biopsy is emerging important issue. The kit, applying a digital PCR platform, enables multiple mutation detection using plasma samples of blood. The digital PCR-based Droplex NSCLC Panel Test Kit is designed to detect over 170 key mutations in 11 genes, namely EGFR, ALK, ROS1, BRAF, MET, RET, KRAS, HER2, NTRK1, NTRK2, and NTRK3, using DNA and RNA extracted from NSCLC FFPET and plasma samples. Methods: The Droplex NSCLC Panel Test Kit consists of four Oligo Mixtures (OMs) for detecting mutations of 4 genes on DNA and two OMs for detecting 7 gene rearrangements on RNA. After extracting DNA and RNA from a single sample, cDNA synthesis from RNA is prioritized, followed by simultaneous execution of droplet generation, PCR processes, and data analysis. The kit test was based on QX600 droplet digital PCR system of Bio-rad with 6 fluorescent channels. To validate the analytical performance of the Droplex NSCLC Panel Test Kit for each type of sample, DNA and RNA samples were extracted from FFPET sections and plasma isolated from NSCLC patients. Each type of samples, contrived samples were manufactured and tested for analytical performance. Results: The results showed that DNA/RNA multiple mutations can be detected simultaneously on a 6-fluorescent channel system. The kit was highly sensitive for low-input sample in dilutions studies ranging from 5 to 20ng DNA or RNA for FFPET samples and plasma sample. The Digital PCR NSCLC panel test detected an analytical specificity of 100% and detection limit of 0.25% or higher for all genes included in the kit. Conclusions: We demonstrated that the performance of the Droplex NSCLC Panel Test is reliable and effective for the detection of above 170 clinically relevant mutations of 11 genes in FFPET and plasma samples of patients with NSCLC. Additionally, it will be necessary to evaluate the utility of the kit through further clinical studies with NSCLC patients, as well as expanding its compatibility to various digital PCR platforms.

Evaluation of WHO catalog of mutations and five WGS analysis tools for drug resistance prediction of Mycobacterium tuberculosis isolates from China.

The continuous advancement of molecular diagnostic techniques, particularly whole-genome sequencing (WGS), has greatly facilitated the early diagnosis of drug-resistant tuberculosis patients. Nonetheless, the interpretation of results from various types of mutations in drug-resistant-associated genes has become the primary challenge in the field of molecular drug-resistance diagnostics. In this study, our primary objective is to evaluate the diagnosis accuracy of the World Health Organization (WHO) catalog of mutations and five WGS analysis tools (PhyResSE, Mykrobe, TB Profiler, Gen-TB, and SAM-TB) in drug resistance to 10 anti-Mycobacterium tuberculosis (MTB) drugs. We utilized the data of WGS collected between 2014 and 2017 in Zhejiang Province, consisting of 110 MTB isolates as detailed in our previous study. Based on phenotypic drug susceptibility testing (DST) results using the proportion method on Löwenstein-Jensen medium with antibiotics, we evaluated the predictive accuracy of genotypic DST obtained by these tools. The results revealed that the WHO catalog of mutations and five WGS analysis tools exhibit robust predictive capabilities concerning resistance to isoniazid, rifampicin, ethambutol, streptomycin, amikacin, kanamycin, and capreomycin. Notably, Mykrobe, SAM-TB, and TB Profiler demonstrate the most accurate predictions for resistance to pyrazinamide, prothionamide, and para-aminosalicylic acid, respectively. These findings are poised to significantly guide and influence future clinical treatment strategies and resistance monitoring protocols.IMPORTANCEWhole-genome sequencing (WGS) has the potential for the early diagnosis of drug-resistant tuberculosis. However, the interpretation of mutations of drug-resistant-associated genes represents a significant challenge as the amount and complexity of WGS data. We evaluated the accuracy of the World Health Organization catalog of mutations and five WGS analysis tools in predicting drug resistance to first-line and second-line anti-TB drugs. Our results offer clinicians guidance on selecting appropriate WGS analysis tools for predicting resistance to specific anti-TB drugs.

Prevalence of Toxoplasma gondii Infections in Pregnant Women and Small Ruminants: A Molecular Diagnostic Approach

Background: Toxoplasma gondii is a globally prevalent parasite with significant health implications, particularly for pregnant women and immunocompromised individuals. Infections during pregnancy can result in serious outcomes such as miscarriage, stillbirth, or congenital disabilities. This study aimed to assess the prevalence of T. gondii infection among pregnant women and small ruminants in Al-Anbar province, Iraq, using serological and molecular diagnostic techniques. Methods: Blood samples were collected from 165 pregnant women and 100 small ruminants (sheep and goats) between January and May 2021. Serological screening was conducted using the Humatex-Toxo rapid test to detect antibodies against T. gondii. DNA was extracted from the blood samples, and nested polymerase chain reaction (PCR) targeting the T. gondii B1 gene was used for molecular detection. The infection rates were analyzed statistically using ANOVA and Chi-square tests, with significance set at p<0.05. Results: The serological test identified T. gondii antibodies in 32.7% of pregnant women, while the PCR detected a higher infection rate of 40%. Among the small ruminants, PCR revealed an overall infection rate of 10%, with sheep showing a higher rate (16%) compared to goats (4%). The results indicate that PCR is more sensitive than serological testing, particularly for identifying active infections. Additionally, the higher infection rate in young women may be linked to lifestyle, environmental exposure, and dietary habits, while seasonal factors likely play a role in infection distribution. Conclusion: Nested PCR is a more effective method for detecting T. gondii in pregnant women, offering a precise tool for identifying infections and potential causes of abortion. The lower infection rates in small ruminants suggest a reduced zoonotic risk in the study area, although continued monitoring is essential. Negative PCR results may be due to inhibitors in the DNA extraction process or the parasite’s localization in tissues rather than blood. The findings highlight the importance of molecular diagnostics in public health strategies aimed at reducing T. gondii infections in high-risk populations.

Respiratory viral infections in children with cancer and febrile neutropenia and children undergoing hematopoietic stem cell transplantation.

The scope of this review is to understand the epidemiology and potential role of respiratory viral infections in children with cancer and febrile neutropenia, as well as in children, undergoing hematopoietic stem cell transplantation. Early detection of respiratory viral infections through molecular diagnostic techniques has allowed recent randomized clinical studies to advance the possibility of more rational use of antimicrobials in this susceptible population. Progress has been made in the early detection of respiratory viruses in episodes of fever and neutropenia in children with cancer. In selected patients who meet specific clinical safety criteria and have negative bacterial cultures, it has been possible to safely and effectively discontinue antimicrobials. This has been validated in recent randomized clinical studies. However, more evidence is still needed for a similar indication in children, undergoing hematopoietic stem cell transplantation with viral respiratory infection episodes. Understanding the role of respiratory viral infections in populations of immunocompromised children may contribute to a more rational use of antimicrobials and, in the near future, may help to decrease antimicrobial resistance in this susceptible population.

Human Babesia odocoilei and Bartonella spp. co-infections in the Americas

BackgroundIn recent years, Babesia and Bartonella species co-infections in patients with chronic, nonspecific illnesses have continued to challenge and change the collective medical understanding of “individual pathogen” vector-borne infectious disease dynamics, pathogenesis and epidemiology. The objective of this case series is to provide additional molecular documentation of Babesia odocoilei infection in humans in the Americas and to emphasize the potential for co-infection with a Bartonella species.MethodsThe development of improved and more sensitive molecular diagnostic techniques, as confirmatory methods to assess active infection, has provided increasing clarity to the healthcare community.ResultsUsing a combination of different molecular diagnostic approaches, infection with Babesia odocoilei was confirmed in seven people suffering chronic non-specific symptoms, of whom six were co-infected with one or more Bartonella species.ConclusionsWe conclude that infection with Babesia odocoilei is more frequent than previously documented and can occur in association with co-infection with Bartonella spp.Graphical

P-557 The contribution of PGT-A to PGT-M and PGT-SR clinical outcomes – A single centre retrospective analysis

Abstract Study question To determine if the introduction of concurrent testing with PGT-A has improved clinical outcomes for PGT-M/SR patients. Summary answer This study demonstrates that the introduction of concurrent testing may have improved clinical outcomes for this patient demographic irrespective of age. What is known already Preimplantation Genetic Testing (PGT) is utilised to investigate the genetic constitution of embryos. PGT-M (monogenic) and PGT-SR (structural rearrangements) are screening techniques considered in treatment for couples at risk of conceiving offspring with known genetic or structural chromosomal abnormalities respectively. Preimplantation Genetic testing for Aneuploidy (PGT-A) was developed in order to screen embryos for numerical chromosomal abnormalities, a leading cause of failed implantation. Nevertheless, routine use of PGT-A is discouraged due to lack of evidence regarding its efficacy. However, PGT-A is routinely used alongside PGT-M and PGT-SR despite the lack of evidence of its effectiveness in improving patient outcomes. Study design, size, duration The retrospective analysis of 496 PGT-M/SR single embryo transfer patient cycles between December 2013 and December 2021. Participants/materials, setting, methods The retrospective analysis of 496 PGT-M/SR single embryo transfer cycles at CRGH Portland was undertaken (single testing n = 77; concurrent testing n = 419). The clinical outcomes (positive urine pregnancy test, clinical pregnancy, live birth and miscarriage rates) were compared between groups using IBM SPSS Statistics (premium 27). Binary logistic regression was performed for statistical analysis, results with p < 0.05 were determined statistically significant. Main results and the role of chance Overall, concurrent testing was associated with statistically significant improved clinical outcomes compared to the reference group. Statistical significance was seen in positive urine pregnancy test (49% vs 68%, p = 0.012), clinical pregnancy (32.5% vs 63%; p = 0.000013), live birth rate (32.5% vs 59%; p = <0.00016) and miscarriage rates (34.2% vs 13%; p = 0.001). The results imply that the introduction of concurrent testing has improved the probability of obtaining a positive clinical outcome and has reduced the chance of miscarriage in this patient demographic. Further analysis showed concurrent testing improved the chances of obtaining a positive clinical outcome irrespective of the patients age or quality of the embryo. Limitations, reasons for caution This study has produced promising results. However, there are several limitations such as the small sample size, embryo biopsy techniques used, and the different molecular diagnostic techniques and providers utilised. Wider implications of the findings The results imply a justification for the use of PGT-A alongside PGT-M/SR screening techniques to improve embryo selection and clinical outcomes for this cohort of patients. Additionally, they demonstrate the efficacy of PGT-A in a previously underrepresented demographic. However, further research is required to confirm the clinical value of PGT-A. Trial registration number not applicable

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

A simplified molecular tool for detecting the Chagas etiological agent using a vector feces sample in field conditions

Triatomine bugs are vectors of Trypanosoma cruzi, the etiologic agent of Chagas disease in the American continent. Here, we have tested a loop-mediated isothermal amplification (LAMP) test for a direct detection of T. cruzi in feces of Triatoma infestans, the main vector of this parasite in the Southern Cone of America. The analytical evaluation showed positive results with samples of triatomine feces artificially inoculated with DNA from strains of T. cruzi corresponding to each Discrete Typing Units (I-VI), with a sensitivity of up to one parasite per reaction. Conversely, the reaction yielded negative results when tested with DNA from Trypanosoma rangeli and other phylogenetically related and unrelated organisms. In triatomines captured under real field conditions (from urban households), and defined as positive or negative for T. cruzi using the reference microscopy technique, the LAMP test achieved a concordance of 100 %. Our results demonstrate that this LAMP reaction exhibits excellent analytical specificity and sensitivity without interference from the fecal matrix, since all the reactions were conducted without purification steps. This simple molecular diagnostic technique can be easily used by vector control agencies under field conditions.

Detection of Duffy blood group genotypes and submicroscopic Plasmodium infections using molecular diagnostic assays in febrile malaria patients

BackgroundMalaria remains a severe parasitic disease, posing a significant threat to public health and hindering economic development in sub-Saharan Africa. Ethiopia, a malaria endemic country, is facing a resurgence of the disease with a steadily rising incidence. Conventional diagnostic methods, such as microscopy, have become less effective due to low parasite density, particularly among Duffy-negative human populations in Africa. To develop comprehensive control strategies, it is crucial to generate data on the distribution and clinical occurrence of Plasmodium vivax and Plasmodium falciparum infections in regions where the disease is prevalent. This study assessed Plasmodium infections and Duffy antigen genotypes in febrile patients in Ethiopia.MethodsThree hundred febrile patients visiting four health facilities in Jimma town of southwestern Ethiopia were randomly selected during the malaria transmission season (Apr–Oct). Sociodemographic information was collected, and microscopic examination was performed for all study participants. Plasmodium species and parasitaemia as well as the Duffy genotype were assessed by quantitative polymerase chain reaction (qPCR) for all samples. Data were analysed using Fisher’s exact test and kappa statistics.ResultsThe Plasmodium infection rate by qPCR was 16% (48/300) among febrile patients, of which 19 (39.6%) were P. vivax, 25 (52.1%) were P. falciparum, and 4 (8.3%) were mixed (P. vivax and P. falciparum) infections. Among the 48 qPCR-positive samples, 39 (13%) were negative by microscopy. The results of bivariate logistic regression analysis showed that agriculture-related occupation, relapse and recurrence were significantly associated with Plasmodium infection (P < 0.001). Of the 300 febrile patients, 85 (28.3%) were Duffy negative, of whom two had P. vivax, six had P. falciparum, and one had mixed infections. Except for one patient with P. falciparum infection, Plasmodium infections in Duffy-negative individuals were all submicroscopic with low parasitaemia.ConclusionsThe present study revealed a high prevalence of submicroscopic malaria infections. Plasmodium vivax infections in Duffy-negative individuals were not detected due to low parasitaemia. In this study, an improved molecular diagnostic tool was used to detect and characterize Plasmodium infections, with the goal of quantifying P. vivax infection in Duffy-negative individuals. Advanced molecular diagnostic techniques, such as multiplex real-time PCR, loop-mediated isothermal amplification (LAMP), and CRISPR-based diagnostic methods. These techniques offer increased sensitivity, specificity, and the ability to detect low-parasite-density infections compared to the employed methodologies.

The Application of Clinical and Molecular Diagnostic Techniques to Identify a Rare Haemoglobin Variant.

Haemoglobin disorders represent a heterogeneous group of inherited conditions that involve at least one genetic abnormality in one or more of the globin chains, resulting in changes in the structure, function, and/or amount of haemoglobin molecules, which are very important for their related clinical aspects. Detecting and characterizing these disorders depends primarily on laboratory methods that employ traditional approaches and, when necessary, newer methodologies essential for solving a number of diagnostic challenges. This review provides an overview of key laboratory techniques in the diagnosis of haemoglobinopathies, focusing on the challenges, advancements, and future directions in this field. Moreover, many haemoglobinopathies are benign and clinically silent, but it is not uncommon to find unexpected variants during routine laboratory tests. The present work reported a rare and clinically interesting case of identification of haemoglobin fractions in an adult man by the determination of glycated haemoglobin (HbA1c) during a routine laboratory assessment, highlighting how the correct use of laboratory data can modify and improve the patient's clinical management.