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Relevance. The problem of diagnosing hereditary neuromuscular diseases is one of the most difficult in the medical specialists’ practice. Molecular genetic diagnostics is one of the fundamental aspects in the classification and subsequent approaches to the treatment and prevention of hereditary diseases. Pathogenic variants identification leads to the formation of separate subtypes and phenotypically identical diseases syndromes. This review examines modern diagnostic methods and algorithmization of patients with neuromuscular diseases. Despite enormous research and clinical efforts, the molecular causes remain unknown for almost half of patients with neuromuscular diseases due to genetic heterogeneity and molecular diagnostics based on a gene-by-gene approach. Next-generation sequencing (NGS) is an effective and cost-effective strategy for accelerating patient diagnosis. However, the diagnostic value of conducting and prescribing whole- exome or whole- genome sequencing is largely dependent on the clinical picture of the disease and the professional competence of the doctor. Hereditary neuromuscular diseases have similar initial symptoms, and molecular genetic diagnostics can pinpoint the cause and pathogenesis of the observed disorders in the patient. Conclusion . The molecular diagnostics algorithm is based on sequential analysis, starting with the search for the most common pathogenic variants using inexpensive and rapid methods, and progressing to the search for rare, previously undescribed pathogenic variants using whole-g enome/whole-exome studies. The phasing allows science and medicine to uncover previously unknown causes of severe disease in patients with neuromuscular diseases, which often leading to disability or premature death. Earlier genetic diagnosis should provide more effective treatment of the disease and better genetic counseling for families and will also allow access to pathogenetic therapy for neuromuscular diseases.

Abstract Vancomycin resistant enterococci (VRE) are a leading cause of ICU-acquired bloodstream infections in Europe. The bacterial load in enteral colonization may be associated with a higher probability of transmission. Here, we aimed to establish a quantitative vanA/vanB DNA real-time PCR assay on a high-throughput system. Limits of detection (LOD), linear range and precision were determined using serial bacterial dilutions. LOD was 46.9 digital copies (dcp)/ml for vanA and 60.8 dcp/ml for vanB. The assay showed excellent linearity between 4.7 × 101 and 3.5 × 105 dcp/ml (vanA) and 6.7 × 102 and 6.7 × 105 dcp/ml (vanB). Sensitivity was 100% for vanA and vanB, with high positive predictive value (PPV) for vanA (100%), but lower PPV for vanB (34.6%) likely due to the presence of vanB DNA positive anerobic bacteria in rectal swabs. Using the assay on enriched VRE broth vanB PPV increased to 87.2%. Quantification revealed median 2.0 × 104 dcp/ml in PCR positive but VRE culture negative samples and median 9.1 × 104 dcp/ml in VRE culture positive patients (maximum: 107 dcp/ml). The automated vanA/B_UTC assay can be used for vanA/vanB detection and quantification in different diagnostic settings and may support future clinical studies assessing the impact of bacterial load on risk of infection and transmission.

Abstract Rapid and accurate identification of the bacteria responsible for sepsis is paramount for effective patient care. Molecular diagnostic methods, such as polymerase chain reaction (PCR), encounter challenges in sepsis due to inhibitory compounds in the blood, necessitating their removal for precise analysis. In this study we present an innovative approach that utilizes vancomycin (Van) and allantoin (Al)-conjugated polydopamine (PDA)-coated magnetic nanoparticles (MNPs) for the rapid and automated enrichment of bacteria and their DNA extraction from blood without inducing clumping and aggregation of blood. Al/Van-PDA-MNPs, facilitated by IMS, eliminate the need for preliminary sample treatments, providing a swift and efficient method for bacterial concentration and DNA extraction within an hour. Employing Al/Van-PDA-MNPs within an automated framework has markedly improved our ability to pre-concentrate various Gram-negative and Gram-positive bacteria directly from blood samples. This advancement has effectively reduced the detection threshold to 102 colony-forming unit/mL by both PCR and quantitative PCR. The method's expedited processing time, combined with its precision, positions it as a feasible diagnostic tool for diverse healthcare settings, ranging from small clinics to large hospitals. Furthermore, the innovative application of nanoparticles for DNA extraction holds promising potential for advancing sepsis diagnostics, enabling earlier interventions and improving patient outcomes.

GeneXpert MTB/RIF, a tool widely used for diagnosing tuberculosis, has limitations for detecting rifampin resistance in certain variants. We report transmission of a pre–extensively drug-resistant variant in Botswana that went undetected by GeneXpert. The public health impact of misdiagnosis emphasizes the need for comprehensive molecular testing to identify resistance and guide treatment.

Background: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a hereditary autosomal dominant vascular injury of the brain. Currently, the clinical diagnosis of CADASIL is based on the following criteria: the onset of the condition at a relatively young age (4050 years), recurrent lacunar infarcts, gradual progression of encephalopathy with the development of pseudobulbar syndrome and dementia, various emotional disorders, and autosomal dominant inheritance. Having a family history of relatives with similar symptoms. The diagnosis is verified on the basis of the results of the following studies: neuroimaging, instrumental, morphological, and laboratory. The presence of leukoaraiosis and multiple small bilateral infarctions in such anatomical structures as the basal ganglia and white matter of the cerebral hemispheres, visual tubercles and the pons, and brain stem during MRI diagnostics. Histologically, transmission electron microscopy reveals granular osmiophilic inclusions in the adventitial membrane of the blood vessels of the brain, and on the periphery in the adventitia of the vessels of skeletal muscles and somatic nerves and in vascular biopsies of skin flaps. A mutation in the Notch3 gene determines the clinic of the disease and morphological changes in blood vessels and is detected by a molecular genetic method. The article presents a clinical case, which was a manifestation of this rare hereditary microangiopathy. Clinical case description: A 43-year-old patient was admitted to the Department of Neurology of the Republican Clinical Hospital in Simferopol for ischemic stroke in the basin of the left middle cerebral artery. According to the anamnesis, the patient suffered from migraine without aura from a young age and lacunar strokes; examination revealed a decrease in cognitive function, and MRI showed signs of microangiopathy of cerebral vessels. For final diagnosis, the patient underwent an intravital pathomorphological examination of the biopsy of the musculoskeletal flap; the results obtained made it possible to make a reliable diagnosis of CADASIL. Conclusion: Understanding and knowledge of such a rare pathology as CADASIL is crucial for the rapid recognition of a characteristic clinical picture for further appointment and interpretation of the results of morphological and molecular genetic diagnostics, making it possible to make the correct diagnosis and treatment.

Increasing reports of invasive Streptococcus pyogenes infections mandate surveillance for toxigenic lineage M1UK. An allele-specific PCR was developed to distinguish M1UK from other emm1 strains. The M1UK lineage represented 91% of invasive emm1 isolates in England in 2020. Allele-specific PCR will permit surveillance for M1UK without need for genome sequencing.

Fasciola hepatica has a complex lifecycle with multiple intermediate and definitive hosts and influenced by environmental factors. The disease causes significant morbidity in children and its prevalent worldwide. There is lack of data about distribution and burden of the disease in endemic regions, owing to poor efficacy of the different diagnostic methods used. A novel PCR-based test was developed by using a portable mini-PCR® platform to detect Fasciola sp. DNA and interpret the results via a fluorescence viewer and smartphone image analyzer application. Human stool, snail tissue, and water samples were used to extract DNA. Primers targeting the ITS-1 of the 18S rDNA gene of Fasciola sp. were used. The limit of detection of the mini-PCR test was 1 fg/μL for DNA samples diluted in water, 10 fg/μL for Fasciola/snail DNA scramble, and 100 fg/μL for Fasciola/stool DNA scramble. The product detection by agarose gel, direct visualization, and image analyses showed the same sensitivity. The Fh mini-PCR had a sensitivity and specificity equivalent to real-time PCR using the same specimens. Testing was also done on infected human stool and snail tissue successfully. These experiments demonstrated that Fh mini-PCR is as sensitive and specific as real time PCR but without the use of expensive equipment and laboratory facilities. Further testing of multiple specimens with natural infection will provide evidence for feasibility of deployment to resource constrained laboratories.

Abstract On October 23, 2020, a 69‐year‐old Chinese female patient was admitted to Yuncheng Hospital due to a history of postmenopausal bleeding and lower abdominal pain for 5 months. The HPV test and pathology results indicated the presence of independent HPV in primary serous carcinoma of the uterine cervix. The genetic testing identified variants of uncertain significance (PAX8 p.Tyr 410 Ter and TP53 p.Asn 247 Ile), microsatellite instability stable (MSI‐S), tumor mutational burden (TMB) 7.33Muts/Mb, and an elevated tumor neoantigen burden. Before undergoing radical hysterectomy treatment, the patient exhibited a positive response to three cycles of intravenous docetaxel (100 mg/3 h) and carboplatin (450 mg/1 h). Following the surgery, she received an additional three cycles of docetaxel (100 mg/3 h) and carboplatin (500 mg/1 h), accompanied by 25 cycles of radiation therapy (DT 46Gy/2Gy/23f). Concurrently, cisplatin (450 mg/1 h) was administered. As of now, the patient has achieved 20 months of disease‐free survival.

Abstract Background At present, the hereditary hearing loss homepage, ( https://hereditaryhearingloss.org/ ), includes 258 deafness genes and more than 500 genes that have been reported to cause deafness. With few exceptions, the region-specific distributions are unclear for many of the identified variants and genes. Methods Here, we used a custom capture panel to perform targeted sequencing of 518 genes in a cohort of 879 deaf Chinese probands who lived in Yunnan. Mutation sites of the parents were performed by high-throughput sequencing and validated by Sanger sequencing. Results The ratio of male to female patients was close to 1:1 (441:438) and the age of onset was mainly under six. Most patients (93.5%) were diagnosed with moderate to severe deafness. Four hundred and twenty-eight patients had variants in a deafness gene, with a detection rate of 48.7%. Pathogenic variants were detected in 98 genes and a number of these were recurrent within the cohort. However, many of the variants were rarely observed in the cohort. In accordance with the American College of Medical Genetics and Genomics, pathogenic, likely pathogenic and variants of uncertain significance accounted for 34.3%, 19.3% and 46.4% of all detected variants, respectively. The most common genes included GJB2, SLC26A4, MYO15A, MYO7A, TMC1, CDH23, USH2A and WFS1, which contained variants in more than ten cases. The two genes with the highest mutation frequency were GJB2 and SLC26A4, which accounted for 28.5% (122/428) of positive patients. We showed that more than 60.3% of coding variants were rare and novel. Of the variants that we detected, 80.0% were in coding regions, 17.9% were in introns and 2.1% were copy number variants. Conclusion The common mutation genes and loci detected in this study were different from those detected in other regions or ethnic groups, which suggested that genetic screening or testing programs for deafness should be formulated in accordance with the genetic characteristics of the region.

Background Thalassaemia is one of the most common inherited monogenic diseases worldwide with a heavy global health burden. Considering its high prevalence in low and middle-income countries, a cheap, accurate and high-throughput screening test of thalassaemia prior to a more expensive confirmatory diagnostic test is urgently needed. Methods In this study, we constructed a machine learning model based on MALDI-TOF mass spectrometry quantification of haemoglobin chains in blood, and for the first time, evaluated its diagnostic efficacy in 674 thalassaemia (including both asymptomatic carriers and symptomatic patients) and control samples collected in three hospitals. Parameters related to haemoglobin imbalance (α-globin, β-globin, γ-globin, α/β and α-β) were used for feature selection before classification model construction with 8 machine learning methods in cohort 1 and further model efficiency validation in cohort 2. Results The logistic regression model with 5 haemoglobin peak features achieved good classification performance in validation cohort 2 (AUC 0.99, 95% CI 0.98–1, sensitivity 98.7%, specificity 95.5%). Furthermore, the logistic regression model with 6 haemoglobin peak features was also constructed to specifically identify β-thalassaemia (AUC 0.94, 95% CI 0.91–0.97, sensitivity 96.5%, specificity 87.8% in validation cohort 2). Conclusions For the first time, we constructed an inexpensive, accurate and high-throughput classification model based on MALDI-TOF mass spectrometry quantification of haemoglobin chains and demonstrated its great potential in rapid screening of thalassaemia in large populations.Key messages Thalassaemia is one of the most common inherited monogenic diseases worldwide with a heavy global health burden. We constructed a machine learning model based on MALDI-TOF mass spectrometry quantification of haemoglobin chains to screen for thalassaemia.

Beginning in May 2022, a rising number of monkeypox cases were reported in non–monkeypox-endemic countries in the Northern Hemisphere. We adapted 2 published quantitative PCRs for use as a dual-target monkeypox virus test on widely used automated high-throughput PCR systems. We determined analytic performance by serial dilutions of monkeypox virus reference material, which we quantified by digital PCR. We found the lower limit of detection for the combined assays was 4.795 (95% CI 3.6–8.6) copies/mL. We compared clinical performance against a commercial manual orthopoxvirus research use only PCR kit by using clinical remnant swab samples. Our assay showed 100% positive (n = 11) and 100% negative (n = 56) agreement. Timely and scalable PCR tests are crucial for limiting further spread of monkeypox. The assay we provide streamlines high-throughput molecular testing for monkeypox virus on existing broadly established platforms used for SARS-CoV-2 diagnostic testing.

Aporocotylid blood flukes Cardicola forsteri and C. orientalis are an ongoing health concern for the Southern Bluefin Tuna (Thunnus maccoyii, SBT) industry, where infections can lead to morbidity and mortality in ranched SBT populations. This study compared blood fluke infection in SBT from two companies during the 2021 ranching season. Both companies administered the same dosage of praziquantel approximately 5 weeks after transfer, feeding with frozen baitfish daily; the only difference in the company’s practices was that the pontoons were located 2.5 km apart. Infection severity was measured as prevalence and intensity by quantifying adult C. forsteri in SBT heart and copy numbers of C. forsteri and C. orientalis ITS-2 DNA in SBT heart and gills. Data from the 2018 and 2019 harvests of SBT were used to make comparisons with 2021 harvest data. Cardicola orientalis was detected at transfer and no longer detected after treatment with praziquantel. Cardicola spp. were present in 83% of sampled SBT in 2021. Both companies demonstrated similar patterns of infection, and Company A had higher prevalence and intensity of Cardicola spp. infection. Based on C. forsteri ITS-2 DNA, infection intensity at harvest was significantly greater for both companies in 2021 when compared to 2018 and 2019. Continued monitoring of Cardicola spp. in SBT and improvements in diagnostics contribute to our understanding of Cardicola spp. epizootiology and the detection of changes in treatment efficacy.

Summary: Background: Current methods for the detection and surveillance of urothelial carcinomas (UCs) are often invasive, costly, and not effective for low-grade, early-stage, and minimal residual disease (MRD) tumors. We aimed to develop and validate a model from urine sediments to predict different grade and stage UCs with low cost and high accuracy. Methods: We collected 167 samples, including 90 tumors and 77 individuals without tumors, as a discovery cohort. We assessed copy number variations and methylation values for them and constructed a diagnostic classifier to detect UC, UCseek, by using an individual read-based method and support vector machine. The performance of UCseek was validated in an independent cohort derived from three hospitals (n = 206) and a relapse cohort (n = 42) for monitoring recurrence. Findings: We constructed UCseek, which could predict UCs with high sensitivity (92.7%), high specificity (90.7%), and high accuracy (91.7%) in the independent validation set. The accuracy of UCseek in low-grade and early-stage patients reached 91.8% and 94.3%, respectively. Notably, UCseek retained great performance at ultralow sequencing depths (0.3X-0.5X). It also demonstrated a powerful ability to monitor recurrence in a surveillance cohort compared with cystoscopy (90.91% vs. 59.09%). Interpretation: We optimized an improved approach named UCseek for the noninvasive diagnosis and monitoring of UCs in both low- and high-grade tumors and in early- and advanced-stage tumors, even at ultralow sequencing depths, which may reduce the burden of cystoscopy and blind second surgery. Funding: A full list of funding bodies that contributed to this study can be found in the Acknowledgments section.

Routine testing for Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) in people with heightened risk is lacking in Thailand. This study aimed to assess the performance of the Cepheid Xpert CT/NG assay, conducted by key population (KP) lay providers, for CT and NG detection on single-site and pooled specimens from the pharynx, rectum, and urine. Between August and October 2019, 188 men who have sex with men and 11 transgender women were enrolled. Participants collected urine specimens while trained KP lay providers obtained pharyngeal and rectal swabs. Compared to single-site testing with the Abbott RealTime CT/NG assay by medical technologists, the Xpert assay missed one pharyngeal NG infection out of 199 single-site specimens, giving a 93.3% sensitivity for pharyngeal NG and one missed pharyngeal NG infection out of fifty pooled specimens, giving an 88.9% sensitivity for pharyngeal NG. There was no discrepancy between the two assays for CT detection. The Cohen’s Kappa coefficient of pooled specimen testing by the Xpert was 0.93 for NG and 1 for CT when compared to single-site testing by Abbott. Implementing pooled specimen testing by KP lay providers can be a cost-saving strategy to enhance the uptake of CT/NG services for populations facing increased risk.

Introduction: Coronavirus Disease-2019 (COVID-19), caused by the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is ravaging the globe due to its rapid spread. Since providing fast results is of critical importance in a time of shortage of medical personnel and beds in isolation wards and to ensure timely treatment for patients, developing high quality rapid Point of Care (POC) diagnostics is essential. Aim: To compare the diagnostic performance of chip based real time Reverse Trancriptase Polymerase Chain Reaction (RTPCR) (TrueNat) which has a shorter turnaround time compared to conventional real time RT-PCR in samples of suspected COVID-19 patients. Materials and Methods: The present cross-sectional observational study was carried out in a tertiary care hospital in New Delhi, India. Five hundred randomly selected Oropharyngeal (OP) swabs samples received from May-July 2020, were included in the study to compare the diagnostic performance of chip based real time RTPCR (TrueNat) with conventional real time RT-PCR for diagnosis of SARS-CoV-2 infection. All statistical analysis was performed using STATA version 16.1 software (College station, Texas, USA). Results: The sensitivity of TrueNat test was 100% while the specificity was found to be 99.12% at 95% confidence intervals. The positive predictive value was 91.84% and the negative predictive value was 100%. Conclusion: The short turnaround time, good sensitivity and specificity makes TrueNat a reliable and affordable option to provide rapid results in cases requiring urgent interventions and to augment SARS-CoV-2 testing capacity at peripheral settings where sample load is less.

Abstract Background Skeletal dysplasia is typically diagnosed using a combination of radiographic imaging, clinical examinations, and molecular testing. Identifying a molecular diagnosis for an individual with a skeletal dysplasia can lead to improved clinical care, guide future medical management and treatment, and inform assessment of risk for familial recurrence. The molecular diagnostic utility of multi-gene panel testing using next-generation sequencing (NGS) has not yet been characterized for an unselected population of individuals with suspected skeletal dysplasia. In this study, we retrospectively reviewed patient reports to assess the diagnostic yield, reported variant characteristics, impact of copy number variation, and performance in prenatal diagnostics of panel tests for variants in genes associated with skeletal dysplasia and growth disorders. Results Clinical reports of consecutive patients with a clinical indication of suspected skeletal dysplasia who underwent panel testing were examined. The 543 patients included in the study submitted samples for diagnostic genetic testing with an indication of suspected skeletal dysplasia or growth disorder and received one of three nested panel tests. A molecular diagnosis was established in 42.0% of patients (n = 228/543). Diagnostic variants were identified in 71 genes, nearly half of which (n = 35, 49.3%) contributed uniquely to a molecular diagnosis for a single patient in this cohort. Diagnostic yield was significantly higher among fetal samples (58.0%, n = 51/88) than postnatal samples (38.9%, n = 177/455; z = 3.32, p

Background: Different species of Campylobacter are the most common cause of bacterial gastroenteritis. There are many methods to detect the presence of Campylobacter, including PCR, but it takes no less than 5 -6 hours. Development of fast molecular diagnostic tests based on a loop-mediated amplification assay will allow simplifying the procedure and reducing the time of detection for a bedside application. Aims: To develop a loop-mediated isothermal amplification assay (LAMP) with a fluorescent probe for the diagnosis of campylobacteriosis. Methods: Stool suspensions were prepared and bacterial fractions were separated as described in the methodological recommendations of the Central Research Institute of Epidemiology. DNA was extracted using AmpliTest RIBO-prep (FSBI SPC FMBA, Russian Federation) according to the manufacturer's instruction and detected with AmpliSens OKI-screen-FL (FBIS CRIE, Russian Federation). Primers and probes were selected in a 16S rDNA gene region. Analytical specificity was confirmed on bacterial cultures, analytical sensitivity was assessed using a recombinant plasmid containing the target Campylobacter DNA sequence fragment. LAMP amplification was performed at 65C for 30 min. Results: An assay for the detection of Campylobacter spp. based on loop-mediated isothermal amplification has been developed, the reaction time does not exceed 30 minutes. The analytical sensitivity of the developed technique is comparable to the real-time PCR and is equal to 103 copies/ml, the analytical specificity is 100%. The evaluation of 127 clinical samples, previously characterized by a commercial kit, AmpliSens OKI-screen-FL (FBIS CRIE, Russian Federation), showed high diagnostic specificity and sensitivity of the developed LAMP-method. No false positive results were found, 108 samples were negative by LAMP and PCR. Campylobacter spp. DNA was detected by the LAMP method in 18 out of 19 PCR-positive samples. One discordant LAMP negative sample can be attributed to the low bacterial load of Campylobacter spp. for a given sample. Conclusions: A method for the rapid detection of Campylobacter spp. loop-mediated isothermal amplification has been developed, and its high analytical and diagnostic characteristics have been shown experimentally.

Summary: Background: The diagnosis of cancer in Bethesda III/IV thyroid nodules is challenging as fine-needle aspiration (FNA) has limitations, and these cases usually require diagnostic surgery. As approximately 77% of these nodules are not malignant, a diagnostic test accurately identifying benign thyroid nodules can reduce “potentially unnecessary” surgery rates. We have previously reported the development and validation of a microRNA-based thyroid classifier (mir-THYpe) with high sensitivity and specificity, which could be performed directly from FNA smear slides. We sought to evaluate the performance of this test in real-world clinical routine to support clinical decisions and to reduce surgery rates. Methods: We designed a real-world, prospective, multicentre study. Molecular tests were performed with FNA samples prepared at 128 cytopathology laboratories. Patients were followed-up from March 2018 until surgery or until March 2020 (patients with no indication for surgery). The final diagnosis of thyroid tissue samples was retrieved from postsurgical anatomopathological reports. Findings: A total of 435 patients (440 nodules) classified as Bethesda III/IV were followed-up. The rate of avoided surgeries was 52·5% for all surgeries and 74·6% for “potentially unnecessary” surgeries. The test achieved 89·3% sensitivity, 81·65% specificity, 66·2% positive predictive value, and 95% negative predictive value. The test supported 92·3% of clinical decisions. Interpretation: The reported data demonstrate that the use of the microRNA-based classifier in the real-world can reduce the rate of thyroid surgeries with robust performance and support clinical decision-making. Funding: The São Paulo Research-Foundation (FAPESP) and Onkos.

Abstract Objective In this study, the performance of a commercially available malaria LAMP assay (Alethia® Malaria Plus LAMP) was evaluated using retrospective clinical samples obtained from travelers returning to the United States of America (USA). Recently, several laboratories in non-malaria endemic countries evaluated the use of the loop mediated isothermal amplification (LAMP) assays for the diagnosis of imported malaria cases. These tests are simpler than polymerase-chain reaction (PCR)-based assays and were shown to have high sensitivity. Much of malaria diagnoses in the USA, is undertaken at the state level using mainly microscopy and rapid diagnostic tests (RDTs). However, molecular tools offer greater sensitivity over microscopy and RDTs. A reliable, easy to perform molecular assay can provide a test of choice for the accurate detection of malaria parasites in places where expert microscopy is lacking and/or for the detection of low-parasite density infections. Results The Alethia® Malaria Plus LAMP assay was easy to use, had similar test performances as the real-time PCR reference test and results were obtained faster (within 1 h) than the reference test. The sensitivity of the assay was 100% with a kappa score of 1 when compared to the reference PET-PCR assay.

Early diagnosis and personalised disease management remain serious challenges in the field of haematological malignancies, especially for acute conditions. Additionally, reliable and timely detection of minimal residual disease is the key to improving patients’ clinical management, including guiding therapy choice, monitoring treatment response, and detecting relapse. Genomic biomarkers provide valuable information in this regard. For example, chromosomal translocations and mutations in genes involved in haematopoietic proliferation and differentiation are considered the strongest predictors of treatment response and overall survival in acute myeloid leukaemia. Furthermore, NPM1 gene mutations and internal tandem duplications in FMS-like tyrosine kinase-3 (FLT3) are used to monitor minimal residual disease in acute myeloid leukaemia. Despite the growing availability of relevant molecular biomarkers, long turnaround times for genomic testing greatly impact the management of aggressive haematological disorders. Delayed access to laboratory test results both negatively influences patients’ psychological state and postpones therapy administration and adjustments. Depending on the technology used, next-generation sequencing allows high-throughput genome analyses within hours to days, at a relatively low cost. Simultaneously, it enables testing of large numbers and various types of biomarkers by targeted gene panels. At the present time, with the latest technological improvements, next-generation sequencing provides the means for advancing genomic-based diagnostics in haematological malignancies, by simplifying complex laboratory workflow and should be introduced more widely in routine clinical settings.

The COVID-19 pandemic took place during the years 2020–2022 and the virus, named SARS-CoV-2, seems likely to have resulted in an endemic disease. Nevertheless, widespread COVID-19 has given rise to several major molecular diagnostics’ facts and concerns that have emerged during the overall management of this disease and the subsequent pandemic. These concerns and lessons are undeniably critical for the prevention and control of future infectious agents. Furthermore, most populaces were introduced to several new public health maintenance strategies, and again, some critical events arose. The purpose of this perspective is to thoroughly analyze all these issues and the concerns, such as the molecular diagnostics’ terminologies, their role, as well as the quantity and quality issues with a molecular diagnostics’ test result. Furthermore, it is speculated that society will be more vulnerable in the future and prone to emerging infectious diseases; thus, a novel preventive medicine’s plan for the prevention and control of future (re)emerging infectious diseases is presented, so as to aid the early prevention of future epidemics and pandemics.

Viral infections, or their reactivations, are one of the most important groups of transplantation complications that can occur among recipients of both hematopoietic cells and solid organ transplants. They are the most commonly caused by cytomegalovirus (CMV). Currently, the use of whole blood or plasma samples is recommended for CMV viral load monitoring. The aim of the study was to assess and compare the level of CMV DNA, depending on the type of clinical material—whole blood or plasma fraction derived from the same patient. The studies were carried out on 156 whole blood samples in which the presence of CMV genetic material was confirmed and the corresponding plasma samples from the same rounds of sampling. CMV DNA was not present in 59 (37.8%) of plasma samples compared to whole blood-positive counterparts. Of the samples positive in both types of clinical specimen, 77 (79.4%) had higher viral DNA levels in the whole blood samples. There were statistically significant differences in the detected CMV DNA load in the whole blood compared to plasma fraction counterparts (p < 0.001). The detected CMV DNA value is usually higher in whole blood compared to plasma samples of the same patient. Due to the variability in CMV viral load depending on the clinical material used for a particular patient, one type of specimen should be always used consequently for CMV viremia monitoring.

Porcine epidemic diarrhea virus (PEDV), a member of the Coronaviridae family, is the etiological agent of an acute and devastating enteric disease that causes moderate-to-high mortality in suckling piglets. The accurate and early detection of PEDV infection is essential for the prevention and control of the spread of the disease. Many molecular assays have been developed for the detection of PEDV, including reverse-transcription polymerase chain reaction (RT-PCR), real-time RT-PCR (qRT-PCR) and loop-mediated isothermal amplification assays. Additionally, several serological methods have been developed and are widely used for the detection of antibodies against PEDV. Some of them, such as the immunochromatography assay, can generate results very quickly and in field conditions. Molecular assays detect viral RNA in clinical samples rapidly, and with high sensitivity and specificity. Serological assays can determine prior immune exposure to PEDV, can be used to monitor the efficacy of vaccination strategies and may help to predict the duration of immunity in piglets. However, they are less sensitive than nucleic acid-based detection methods. Sanger and next-generation sequencing (NGS) allow the analysis of PEDV cDNA or RNA sequences, and thus, provide highly specific results. Furthermore, NGS based on nonspecific DNA cleavage in clustered regularly interspaced short palindromic repeats (CRISPR)–Cas systems promise major advances in the diagnosis of PEDV infection. The objective of this paper was to summarize the current serological and molecular PEDV assays, highlight their diagnostic performance and emphasize the advantages and drawbacks of the application of individual tests.

Coronavirus disease (COVID-19) is an infectious disease caused by SARS-CoV-2. In Colombia, many commercial methods are now available to perform the RT-qPCR assays, and laboratories must evaluate their diagnostic accuracy to ensure reliable results for patients suspected of being positive for COVID-19. The purpose of this study was to compare four commercial RT-qPCR assays with respect to their ability to detect the SARS-CoV2 virus from nasopharyngeal swab samples referred to Laboratorio Carvajal IPS, SAS in Tunja, Boyacá, Colombia. We utilized 152 respiratory tract samples (Nasopharyngeal Swabs) from patients suspected of having SARS-CoV-2. The diagnostic accuracy of GeneFinderTM COVID-19 Plus RealAmp (In Vitro Diagnostics) (GF-TM), One-Step Real-Time RT-PCR (Vitro Master Diagnostica) (O-S RT-qPCR), and the Berlin modified protocol (BM) were assessed using the gold-standard Berlin protocol (Berlin Charité Probe One-Step RT-qPCR Kit, New England Biolabs) (BR) as a reference. Operational characteristics were estimated in terms of sensitivity, specificity, agreement, and predictive values. Using the gold-standard BR as a reference, the sensitivity/specificity of the diagnostic tests was found to be 100%/92.7% for GF-TM, 92.75%/67.47% for O-S RT-qPCR, and 100%/96.39% for the BM protocol. Using BR as a reference, the sensitivity/specificity for the diagnostic tests were found to be 100%/92.7% for the GF-TM assay, 92.72%/67.47% for the O-S RT-qPCR, and 100%/96.39% for BM. Relative to the BR reference protocol, the GF-TM and BM RT-PCR assays obtained similar results (k = 0.92 and k = 0.96, respectively), whereas the results obtained by O-S-RT-qPCR were only moderately similar. We conclude that the GF-TM and BM protocols offer the best sensitivity and specificity, with similar results in comparison to the gold-standard BR protocol. We recommend evaluating the diagnostic accuracy of the OS-RT-qPCR protocol in future studies with a larger number of samples.

Recent seroprevalence studies in animals detected Rocio virus in regions of Brazil, indicating risk for re-emergence of this pathogen. We identified Rocio virus RNA in samples from 2 human patients for whom dengue fever was clinically suspected but ruled out by laboratory findings. Testing for infrequent flavivirus infections should expedite diagnoses.

Abstract Background Monogenic diabetes (MgD) accounts for 1–2% of all diabetes cases. In adults, MgD is difficult to distinguish from common diabetes causes. We assessed the diagnosis rate and genetic spectrum of MgD using next-generation sequencing in patients with late adolescence/adult-onset diabetes referred for a clinical suspicion of MgD. Methods This cross-sectional study was performed in 1564 probands recruited in 116 Endocrinology departments. Inclusion criteria were the absence of diabetes autoantibodies, and at least two of the three following criteria: an age ≤ 40 years and a body mass index (BMI)

Mycoplasma genitalium infections of the urogenital tract are usually treated with azithromycin; however, for the past several years, rates of azithromycin treatment failure have increased. To document the occurrence and frequency of macrolide resistance–mediating mutations (MRMMs) in M. genitalium infections, we collected 894 M. genitalium–positive samples during April 2014–December 2017 and retrospectively tested them for MRMMs. We designated 67 samples collected within 6 weeks after a positive result as test-of-cure samples; of these, 60 were MRMM positive. Among the remaining 827 samples, the rate of MRMM positivity rose from 22.7% in 2014 and 22.3% in 2015 to 44.4% in 2016 but decreased to 39.7% in 2017. Because of these high rates of MRMMs in M. genitalium infections, we recommend that clinicians perform tests of cure after treatment and that researchers further explore the clinical consequences of this infection.

The resulted clinical example of genetic diagnostics of the causes of spinal muscular atrophy in a patient planning fertility. The revealed mutation made it possible to clarify the etiology and pathogenesis of the development of neuromuscular disorders, determine the prognosis of inheritance and prenatal diagnosis, and evaluate the possibilities of adequate treatment.

Many acute viral infections cause similar clinical symptoms, therefore, establishing the etiology of a viral disease requires the use of whole complexes of serological or PCR tests designed to detect a particular type of pathogen. Modern methods of molecular biology allow early diagnosis of viral diseases at a time when serological diagnostic methods are not yet effective. The aim of the work was to analyze molecular diagnostic methods that allow the determination of viral nucleic acids in human blood. The article presents the classification of molecular methods for the diagnosis of viral particles in clinical specimens. Methods such as in situ hybridization, reverse transcription reaction (RT-PCR), nested PCR, multiplex PCR, as well as DNA microarray technology, and the method of massive parallel sequencing are considered in detail. Particular attention is paid to NGS-technologies that were used in virology almost immediately after their appearance and allowed for detection of a number of new types of human viruses (including representatives of anelloviruses, picornaviruses, polyomaviruses, etc.). The advantages and problems associated with the application of these methods in clinical practice, as well as the prospects for their improvement are discussed.

Bacterial meningitis is a major global cause of morbidity and mortality. Rapid identification of the aetiological agent of meningitis is essential for clinical and public health management and disease prevention given the wide range of pathogens that cause the clinical syndrome and the availability of vaccines that protect against some, but not all, of these. Since microbiological culture is complex, slow, and often impacted by prior antimicrobial treatment of the patient, molecular diagnostic assays have been developed for bacterial detection. Distinguishing between meningitis caused by Neisseria meningitidis (meningococcus), Streptococcus pneumoniae (pneumococcus), Haemophilus influenzae, and Streptococcus agalactiae and identifying their polysaccharide capsules is especially important. Here, we review methods used in the identification of these bacteria, providing an up-to-date account of available assays, allowing clinicians and diagnostic laboratories to make informed decisions about which assays to use.

Abstract Objectives Acute febrile illness is a common cause of hospital admission, and its associated infectious causes, of which a key bacterial causative agent is Streptococcus pneumoniae, contribute to substantial morbidity and mortality. We sought to evaluate the utility of real-time (rt)-PCR on dried blood spots (DBS) for diagnosis of S. pneumoniae in acute febrile illness among children presenting to hospitals in Nigeria. We previously described preliminary results in a sample of 537 patients. Here we present data from a larger collection of 1038 patients. Results Using rt-PCR for Streptococcus pneumoniae on 1038 dried blood spots from children prospectively enrolled with acute febrile illness, including 79 healthy controls, we detected pneumococcal DNA in nine of 15 blood culture-positive specimens, one culture-negative specimen from a high-risk group, a culture-confirmed non-pneumococcal specimen and a healthy control. Six culture-positive isolates (40%) were negative. Sensitivity was 60%, specificity 99.7%, positive predictive value 75% and negative predictive value 99.4%. Rt-PCR of DBS has limited sensitivity in blood specimens from acute febrile illness in children.

Abstract Objective Diagnostic testing for tuberculosis depends on microbiological detection of Mycobacterium tuberculosis (Mtb) in sputum. For patients unable to expectorate sputum, such as children and individuals living with HIV, this poses barriers to rapid diagnosis and treatment initiation. Therefore, this study aimed to use oral swabs as an alternative sample type for Mtb detection via molecular testing. Results In a pilot study, we aimed to evaluate sensitivity of Mtb detection via oral swabs using Xpert MTB/RIF ULTRA. We enrolled 33 TB cases and 30 controls from Lima, Peru, and detected Mtb from oral swabs with a sensitivity of 45% (95% confidence interval (CI) 29–62%) and specificity of 100% (95% CI 89–100%) using liquid culture of sputum as reference test. Our current protocol will need optimization, but these results support future exploration of the use of oral swabs for Mtb detection.

Introduction: Spine deformity formed as a result of anomalies in its development and this is one of the common orthopaedic pathologies of childhood. The results of some genetic studies did not show significant relationship between the different polymorphisms studied and the rate of progression of Idiopathic Scoliosis (IS). The study on new genetic markers for IS is an important and urgent task. Aim: To study the molecular genetic analysis of folate cycle genes and Interleukin 6 (IL-6) gene polymorphism in children with progressive type of IS. Materials and Methods: The case-control study was conducted on 110 children with IS between the age of 14 years to 18 years and 110 healthy volunteers without pathology of the spine. The study was carried out by the methods of molecular genetics analysis. Molecular diagnostics were carried out by analysing multiple polymorphic regions in the folate group and IL-6 gene, which are of clinical importance due to their predisposing factors in spine malformations. Genotyping of the gene polymorphism was performed using Polymerase Chain Reaction (PCR) in conventional and real-time method and the PCR products were analysed directly on the CFX96 Touch RealTime PCR Detection System or by gel electrophoresis. Statistical analysis was performed with software package Statistica 6.0. Results: The polymorphisms of the genes MTHFR, MTR, MTRR, IL-6 and their frequency distribution among patients with IS and control group were investigated. Some differences in the distribution of genotypes compared with the control group were found in the MTHFRA1298C, MTHFRC677T, MTRA2756G, MTRRA66G, and IL-6G (-174)C polymorphisms. The most significant differences were revealed for MTRA2756G polymorphism, and especially when dividing the group of patients with IS by gender. Conclusion: The results suggested that some polymorphisms of the folate group and IL-6 genes are involved in the pathogenesis of IS. This is also indicated by the well-known role that these genes do play in the ontogeny of the spine.

Rickettsial infections are an emerging public health concern in many parts of India with several undiagnosed or misdiagnosed cases. Despite its widespread prevalence in India, lack of suspicion, nonspecific signs and symptoms, and absence of widely available sensitive and specific diagnostic test contributes to a high fatality rate. This is a case study of a 44-year-old male patient with a severe Rickettsial infection that turned fatal. Aim of this case study is to highlight the importance of timely diagnosis of Rickettsial infections which in this case could have saved the patient’s life.

Introduction: Spine congenital curvatures formed as a result of anomalies in the development of vertebral bodies leads to severe and rigid deformities of the spinal column in the adolescent age and are often accompanied by irreversible neurologic disorders. Therefore genetic markers study of the congenital malformations is an important and urgent task. Aim: The aim of the study is to determine the frequency of detoxification and reparation genes polymorphism in congenital spine deformities patients. Materials and Methods: The study of 200 children with Congenital Spine Deformities (CSD) between the age of 1 year 2 months to 16 years and 96 healthy children aged from 2 to 16 years without pathology of the spine was carried out by the methods of clinical and radiographic diagnostics. Molecular diagnostics was carried out by analysing multiple polymorphic regions in the genes of detoxification and DNA repair, which are of clinical importance due to their predisposing factors in various congenital malformations. Genotyping of the gene polymorphism was performed using Polymerase Chain Reaction (PCR) and the PCR products were visualised by gel electrophoresis. Results: The polymorphisms of the genes CYP1A2, GSTM1, GSTT1, GSTP1, NAT2, XRCC1, XRCC3 and their frequency distribution among patients with CSD and control group were investigated. Significant differences in the distribution of genotypes compared with the control group were found in the polymorphic regions of the genes CYP1A2, GSTM1, GSTT1, NAT2, XRCC3. However, in the GSTP1 and XRCC1 genes, no significant differences between the CSD patients and the control group was observed. Conclusion: All patients with congenital spine deformities had significant changes in some candidate genes alleles. It was found that the number of minor alleles in the genes under investigation correlate with the severity of congenital deformity and the variability of vertebral anomalies. It was determined that in children with multiple and combined defects in the spine development, there is a greater number of mutations in the detoxification and DNA-repair genes compared to children with isolated vertebral anomalies.

Few studies have examined whether the Xpert MTB/RIF test improves time to treatment initiation for persons with multidrug-resistant tuberculosis (MDR TB). We determined the impact of this test in Latvia, where it was introduced in 2010. After descriptive analyses of pulmonary MDR TB patients in Latvia during 2009–2012, time to treatment initiation was calculated, and univariate and multivariable accelerated failure time models were constructed. Univariate results showed strong evidence of an association between having rifampin-resistant TB detected by Xpert MTB/RIF and reduced time to treatment initiation versus the test not being used. A multivariable model stratifying by previous TB showed similar results. Our finding that in Latvia, time to treatment initiation was decreased for MDR TB cases that were rifampin-resistant TB by XpertMTB/RIF has implications for the use of this test in other settings with a high burden of MDR TB in which rifampin resistance is highly predictive of MDR TB.

Nucleic acid-based tests (NABTs) were developed to decrease the time required to identify microorganism in the pathological specimens. The commercially available NABTs are of two types – one those can be applied to positive cultures grown in blood culture bottles and other those can be applied directly to blood samples. The latter tests are polymerase chain reaction (PCR)-based assays which can amplify the existing load of microorganism nucleic acids many a times like a culture in a blood culture bottle. Both tests then identify the pathogenic organisms with the use of specific nucleic acid probes. These tests have proven useful in management of heart transplant sepsis management.

Streptococci and enterococci are significant opportunistic pathogens in epidemiology and infectious medicine. High genetic and taxonomic similarities and several reclassifications within genera are the most challenging in species identification. The aim of this study was to identify Streptococcus and Enterococcus species using genetic and phenotypic methods and to determine the most discriminatory identification method. Thirty strains recovered from clinical samples representing 15 streptococcal species, five enterococcal species, and four nonstreptococcal species were subjected to bacterial identification by the Vitek® 2 system and Sanger-based sequencing methods targeting the 16S rRNA, sodA, tuf, rpoB, and recA genes. Phenotypic methods allowed the identification of 10 streptococcal strains, five enterococcal strains, and four nonstreptococcal strains (Leuconostoc, Granulicatella, and Globicatella genera). The combination of sequencing methods allowed the identification of 21 streptococcal strains, five enterococcal strains, and four nonstreptococcal strains. The 16S rRNA and rpoB genes had the highest identification potential. Only a combination of several molecular methods was sufficient for unambiguous confirmation of species identity. This study will be useful for comparison of several identification methods, both those used as a first choice in routine microbiology and those used for final confirmation.

Yaws is a skin debilitating disease caused by Treponema pallidum subspecies pertenue with most cases reported in children. World Health Organization (WHO) aims at total eradication of this disease through mass treatment of suspected cases followed by an intensive follow-up program. However, effective diagnosis is pivotal in the successful implementation of this control program. Recombinase polymerase amplification (RPA), an isothermal nucleic acid amplification technique offers a wider range of differentiation of pathogens including those isolated from chronic skin ulcers with similar characteristics such as Haemophilus ducreyi (H. ducreyi). We have developed a RPA assay for the simultaneous detection of Treponema pallidum (T. pallidum) and H. ducreyi (TPHD-RPA). The assay demonstrated no cross-reaction with other pathogens and enable detection of T. pallidum and H. ducreyi within 15 min at 42 °C. The RPA assay was validated with 49 clinical samples from individuals confirmed to have yaws by serological tests. Comparing the developed assay with commercial multiplex real-time PCR, the assay demonstrated 94% and 95% sensitivity for T. pallidum and H. ducreyi, respectively and 100% specificity. This simple novel TPHD-RPA assay enables the rapid detection of both T. pallidum and H. ducreyi in yaws-like lesions. This test could support the yaws eradication efforts by ensuring reliable diagnosis, to enable monitoring of program success and planning of follow-up interventions at the community level.