Clinical Diagnostic Laboratories Research Articles

Current state of chikungunya fever laboratory diagnosis (review of literature).

The article is about methods of chikungunya fever laboratory diagnosis. An algorithm for the study of biological material for the presence of antibodies against chikungunya virus and virus antigens is presented. The overview describes the information about commercial immunodiagnostic and genodiagnostic kits and their detailed specifications. The information presented in the review will be useful for doctors of clinical laboratory diagnostics to choose a method and an acceptable test system for laboratory confirmation of Chikungunya fever diagnosis, as well as differential diagnosis with other fevers, which have similar symptoms, common geographical distribution and carriers of infection.

EXTENDED SPECTRUM BETA LACTAMASE PRODUCING UROPATHOGENS IN A TERTIARY CARE HOSPITAL IN SOUTH INDIA: A CLINICO MICROBIOLOGICAL STUDY

Introduction: Urinary Tract Infection (UTI) is the most common bacterial infection encountered in both hospital and community setup and the presence of drug resistance carries remarkable clinical implication. Drug resistant phenotypes like ESBL (Extended Spectrum Beta lactamase producers) are difcult to treat causing increased morbidity. Aims: The present study aims to determine the prevalence of ESBL producing Enterobacteriacea among Uropathogens and to determine the Antibiotic susceptibility pattern. Materials and Methods :A total of 726 urine samples were obtained and processed as per standard microbiological techniques .Antibiotic susceptibility testing was done by Kirby Bauer disc diffusion method. All the isolates were screened for ESBL production and conrmed by Double Disc Synergy test (DDST).Differences between the antibiotic susceptibility pattern ,Various risk factors were assessed among the ESBL producing and non ESBLproducing bacteria and the signicance was analyzed . Statistical analysis used:Chi square test.(p < 0.05 signicant) Results: A total of 726 urine samples were processed of which 421 bacteria belonging to Enterobacteriaceae were included in the study. Most common organism isolated was Escherichia coli 279 (66.27%).Highest susceptibility was observed for Imipenem 361 (85.7%) and Amikacin 356 (84.5%).Out of 461 isolates, 235 (55.8%) isolates were positive for ESBL screening all the isolates were conrmed with DDST. There was a signicant difference for Diabetes mellitus, CAUTI and Inpatients among the antibiotic susceptibility pattern between ESBL and non ESBL producers.(p<0.0001) Conclusions: Early detection of Multidrug resistance isolates like ESBLproducers in routine testing could help avoid treatment failure. Screening for ESBL production needs to be carried out regularly in every clinical diagnostic laboratory to guide clinicians in proper selection of antibiotics. Continued monitoring of the susceptibility pattern of ESBLproducing bacteria will provide valuable information regarding the local antimicrobial susceptibility pattern which in turn will guide in choosing the initial empirical therapy

Validation of determination of human α-amylase activity for patients with pancreatic diseas

Blood α-amylase activity is a key laboratory marker of pancreatic diseases. At the same time, some of the common laboratory methods of its measurement have a number of limitations. Therefore new more effective laboratory methods are currently being developed over those methods that have been already available. The aim of this work was to compare two methods of determination of the activity of total and pancreatic α-amylase in human blood based on using 4,6-ethylidene-(G7)-p-nitrophenyl-(G1)-α,D-maltoheptaoside (EPS-G7) and 2-chloro-4-nitrophenyl-4-O-β-D-galactopyranosylmaltoside (GalG2CNP) as substrates. Blood serum samples from patients with different levels of α-amylase activity were analyzed. The main analytical characteristics of the method with the GalG2CNP substrate were determined using purified α-amylase specimens. A high-sized correlation and high accuracy of the α-amylase isoenzymes activity were observed for the both methods. Therefore, the method for determining the activity of α-amylase using GalG2CNP as a substrate has analytical characteristics similar to the common laboratory method with EPS-G7, but at the same time, the existing advantages allow it to be recommended for wider application in clinical laboratory diagnostics and scientific research.

Features of diagnosis of inflammatory lesions of central nervous system in HIV-infected patients at terminal stage of disease: clinical case

The authors of the article describe a case of the damage to the central nervous system by fungi of the genus Cryptococcus in a HIV-infected patient. The features of clinical manifestations and laboratory diagnostics are characterized. Special attention is paid to the discrepancy between the sharply changed appearance of the cerebrospinal fluid obtained during lumbar puncture and the clinical picture of the disease as well as the data of laboratory analysis of cerebrospinal fluid.

Diagnostic Utility of Gene Fusion Panel to Detect Gene Fusions in Fresh and Formalin-Fixed, Paraffin-Embedded Cancer Specimens

Somatic gene fusions are common in leukemias/lymphomas and solid tumors. The detection of gene fusions is crucial for diagnosis. NanoString fusion technology is a multiplexed hybridization method that interrogates hundreds of gene fusions in a single reaction. This study's objective was to determine the performance characteristics and diagnostic utility of NanoString fusion assays in a clinical diagnostics laboratory. Validation using 100 positive specimens and 15 negative specimens by a combined reference standard of fluorescence in situ hybridization (FISH)/RT-PCR/next-generation sequencing (NGS) assays achieved 100% sensitivity in leukemias/lymphomas and 95.0% sensitivity and 100% specificity in solid tumors. Subsequently, 214 consecutive clinical cases, including 73 leukemia/lymphoma specimens and 141 formalin-fixed, paraffin-embedded solid tumor specimens, were analyzed by gene fusion panels across 638 unique gene fusion transcripts. A variety of comparator tests, including FISH panels, conventional karyotyping, a DNA-based targeted NGS assay, and custom RT-PCR testing, were performed in parallel. The gene fusion assay detected 31 gene fusions, including 16 in leukemia/lymphoma specimens and 15 in solid tumor specimens. The overall sensitivity, specificity, and accuracy of gene fusions detected by the gene fusion panel in all 329 specimens (validation and consecutive clinical specimens) tested in this study were 94.8%, 100%, and 97.9%, respectively, compared with FISH/RT-PCR/NGS assays. The gene fusion panel is a reliable approach that maximizes molecular detection of fusions among both fresh and formalin-fixed, paraffin-embedded cancer specimens.

Benchmark of thirteen bioinformatic pipelines for metagenomic virus diagnostics using datasets from clinical samples.

Metagenomic sequencing is increasingly being used in clinical settings for difficult to diagnose cases. The performance of viral metagenomic protocols relies to a large extent on the bioinformatic analysis. In this study, the European Society for Clinical Virology (ESCV) Network on NGS (ENNGS) initiated a benchmark of metagenomic pipelines currently used in clinical virological laboratories. Metagenomic datasets from 13 clinical samples from patients with encephalitis or viral respiratory infections characterized by PCR were selected. The datasets were analyzed with 13 different pipelines currently used in virological diagnostic laboratories of participating ENNGS members. The pipelines and classification tools were: Centrifuge, DAMIAN, DIAMOND, DNASTAR, FEVIR, Genome Detective, Jovian, MetaMIC, MetaMix, One Codex, RIEMS, VirMet, and Taxonomer. Performance, characteristics, clinical use, and user-friendliness of these pipelines were analyzed. Overall, viral pathogens with high loads were detected by all the evaluated metagenomic pipelines. In contrast, lower abundance pathogens and mixed infections were only detected by 3/13 pipelines, namely DNASTAR, FEVIR, and MetaMix. Overall sensitivity ranged from 80% (10/13) to 100% (13/13 datasets). Overall positive predictive value ranged from 71-100%. The majority of the pipelines classified sequences based on nucleotide similarity (8/13), only a minority used amino acid similarity, and 6 of the 13 pipelines assembled sequences de novo. No clear differences in performance were detected that correlated with these classification approaches. Read counts of target viruses varied between the pipelines over a range of 2-3 log, indicating differences in limit of detection. A wide variety of viral metagenomic pipelines is currently used in the participating clinical diagnostic laboratories. Detection of low abundant viral pathogens and mixed infections remains a challenge, implicating the need for standardization and validation of metagenomic analysis for clinical diagnostic use. Future studies should address the selective effects due to the choice of different reference viral databases.

Detection of Colistin Resistance in Carbapenem Resistant Enterobacteriaceae by Reference Broth Microdilution and Comparative Evaluation of Three Other Methods.

Objective Challenges in susceptibility testing of colistin along with increase in the prevalence of colistin-resistant carbapenemase-producing Enterobacteriaceae (CRE) pathogens needs addressal. Evaluation of user-friendly methods is necessary as an alternative to broth microdilution (BMD), the reference susceptibility testing method, for routine implementation in diagnostic clinical microbiology laboratories. Genotypic detection of the plasmid-mediated colistin resistance is also needed for infection control purposes. Materials and Methods Colistin susceptibility of 200 nonduplicate clinical CRE isolates from December 2017 to June 2019 was determined by BMD, agar dilution (AD), E test, and rapid polymyxin NP test and interpreted as per the European Committee on Antimicrobial Susceptibility Testing. The results of AD, E test, and NP test were compared with that of BMD, considering minimal inhibitory concentration (MIC) ≤ 2 µg/mL as susceptible and > 2 µg/mL as resistant. Presence of any plasmid-mediated colistin resistance (mcr-1 and 2) was evaluated in 27 colistin-resistant CRE isolates by polymerase chain reaction. Statistical Analysis Performance of different phenotypic methods was analyzed by comparing MIC results of AD and E test with that of reference BMD method. Agreement between BMD and the other two methods was expressed in terms of categorical agreement and essential agreement. Errors were expressed as very major error (VME: false-susceptible) and major error (ME: false-resistance) by AD/E test. VME and ME of 3% disagreement were considered unacceptable. Results Colistin resistance was found in 27 (13.5%) isolates by BMD method. The VME rates of both AD (11%) and E test (37%) could not meet the Clinical and Laboratory Standards Institute recommendation (< 3% VME rate is acceptable) as alternative tests to the reference BMD. Colistin NP test showed sensitivity and specificity of 85% and 98%, respectively. The percentage discordant result in NP test was highest in Enterobacter spp. (17%). None of the 27 colistin resistant isolates showed presence of mcr-1 and mcr-2 genes. Conclusion High VME rate in AD and E tests precludes their use as alternatives to BMD for colistin susceptibility testing. NP test with moderate sensitivity but excellent specificity can be a good alternative for testing colistin susceptibility in CRE isolates, except in Enterobacter spp. Absence of mcr-1 and mcr-2 gene necessitates the exploration of other mechanisms of colistin resistance.

Prevalence of extended-spectrum beta-lactamase producing gram negative isolates in Enterobacteriaceae group of bacteria in clinical samples from a tertiary care hospital

Introduction: Extended spectrum β-lactamases (ESBLs) are enzymes that intervene resistance to extended-spectrum (third generation) cephalosporins (e.g., ceftazidime, cefotaxime, and ceftriaxone) and but do not affect carbapenems (e.g., meropenem or imipenem). Though the number of ESBLs producing organism has been increasing day by day, the detection methods and treatment option for them are extremely limited. Objective: Objective of the study was to investigate the rate of ESBLs production and their antibiotic susceptibility pattern. Materials and method: A total 200 Gram negative isolates from various clinical samples received in microbiology laboratory, Sir Takhtsinhji General Hospital, Bhavnagar were studied and Antibiotic susceptibility test was done for commonly used antibiotics. A hospital-based study was conducted in microbiology laboratory, Sir Takhtsinhji General Hospital, Bhavnagar from February 2012 to August 2012. A total of 200 Gram negative isolates from various clinical samples were collected and identified using the conventional biochemical tests following the Clinical and Laboratory Standard Institute (CLSI) guidelines. Antimicrobial susceptibility testing (AST) was performed using the standardized Kirby-Bauer disk diffusion method. Results: Among the total isolates 89(44.5%) were ESBLs producer, and the rate of ESBLs positivity was 39.8% for E. coli (33 out of 83), 10% for Proteus mirabilis (1 out of 10), 51.4% for Klebsiella spp (55 out of 107). ESBLs producing organisms were resistant to most of the antibiotics but 100% were sensitive to imipenem, meropenem, and cefoperazone + sulbactam. Conclusion: Screening for ESBLs production requires to be carried out regularly in all clinical diagnostic laboratories to direct clinicians in appropriate selection of antibiotics.

Performance Assessment of the Devyser High-Throughput Sequencing–Based Assay for Chimerism Monitoring in Patients after Allogeneic Hematopoietic Stem Cell Transplantation

Chimerism analysis is widely used to aid in the clinical management of patients after allogeneic hematopoietic stem cell transplantation. Many laboratories currently use assays based on PCR followed by capillary electrophoresis, with a limit of quantification of 1% to 5%. Assays with a lower limit of quantification could allow for earlier relapse detection, resulting in improved patient care. This study investigated the analytical, clinical, technical, and practical performance of the Devyser next-generation sequencing chimerism assay, a commercial high-throughput sequencing-based assay for chimerism analysis. Performance of this assay was compared with that of the Promega PowerPlex 16 HS assay, a commercial capillary electrophoresis-based assay. A limit of quantification of 0.1% was achievable with the Devyser assay. The repeatability, reproducibility, trueness, and linearity of the Devyser assay were acceptable. The Devyser assay showed potential for earlier relapse detection compared with the Promega assay. Conclusive analysis was not possible for 3% of donor-recipient pairs with the Devyser assay due to an insufficient number of informative markers; this factor was not an issue for the Promega assay. Further improvements in assay design or data analysis may allow the assay's applicability to be extended to all donor-recipient pairs studied. Technical performance criteria for chimerism analysis by high-throughput sequencing were suggested and evaluated. Both assays were found to be practical for use in a clinical diagnostics laboratory.

Iodine Deficiency Disorders: Current State of the Problem in the Republic of Crimea

Background: The article presents the results of a control and epidemiological study conducted in September 2020 by specialists of the Federal State Budgetary Institution «NMIC of Endocrinology» of the Ministry of Health of Russia, aimed at assessing the current state of iodine provision in the population of the Republic of Crimea. The study in Crimea is part of a number of activities and work carried out on behalf of the Ministry of Health of the Russian Federation within the framework of state assignments «Scientific assessment of the need for additional regulatory legal and other measures to eliminate iodine deficiency in pilot regions with severe iodine deficiency» and «Epidemiological and molecular-cellular characteristics of tumor, autoimmune and iodine deficiency thyropathies as a basis for prevention of complications and personalization of treatment.The data obtained reflect the state of the problem of iodine consumption on the territory of the Crimean Peninsula and indicate the relevance of the adoption of a regional preventive program aimed at eliminating iodine deficiency in the diet of the population and related diseases.Aim: Assessment of iodine supply of the population of the Republic of CrimeaMaterials and methods: The research was carried out in secondary schools of four districts of the Republic of Crimea — in the years. Simferopol, Belogorsk, Bakhchisarai and Saki.The volume of the study — 356 schoolchildren of 8-10 years old, all were completed: taking anamnesis and anthropometric parameters (height, weight), examination by an endocrinologist with palpation of the thyroid gland (thyroid gland), ultrasound examination of the thyroid gland (thyroid ultrasound), obtaining single portions of urine and samples of table salt (5-10 grams), which is used daily in the diet in their families. The measurements of the height and weight of the children by the standard method were carried out during the examination by a specialist. Thyroid ultrasound was performed in the supine position using a portable LOGIQe ultrasound machine (China) with a 10-15 MHz multifrequency linear transducer. All urine samples (n = 356) in disposable Eppendorf microtubes were immediately frozen at a temperature of minus 20-25°! for further determination of the concentration of iodine in urine using the cerium-arsenite method (based on the clinical diagnostic laboratory of the Federal State Budgetary Institution NMITs endocrinology «of the Ministry of Health of Russia). A qualitative study for the presence of potassium iodate in food table salt samples (n = 203) was carried out on site by the express method.Informed consent was obtained from all parents / guardians of children for the examination and processing of personal data. The permission of the local ethical committee of the Federal State Budgetary Institution «NMIC of Endocrinology» of the Ministry of Health of Russia was received on March 25, 2020, No. 5.Results: According to the results of a survey of 356 children of primary school age, the median concentration of iodine in urine (mCIM) is 97 pg / l and varies from 78 to 98 pg / l in the surveyed areas, the proportion of urine samples with a reduced iodine concentration was 51.2%. The share of iodized salt use in the families of schoolchildren was 12.3% (values range from 10% to 15%). The average incidence of goiter in children according to ultrasound data is 9.5% (range of values from 1.7% to 16.3%).Conclusion.The indicator of the median concentration of iodine in urine indicates an insufficient iodine supply of the population of the Republic of Crimea.The prevalence of goiter in children of primary school age according to the ultrasound examination of the thyroid gland corresponds to the mild severity of iodine deficiency in the mild degree of goiter endemic in the region.The share of households using iodized salt is extremely low and amounts to 12.3%, which does not meet the WHO recommendations for regions with natural iodine deficiency (from 90% or more).

Continuing Education of Animal Health Professionals in Uganda: A Training Needs Assessment.

In Uganda, delivery of veterinary services is vital to animal health and productivity, and is heavily dependent on well-trained and skilled animal health professionals. The purpose of this study was to identify and prioritize areas for refresher training and continuous professional development of animal health professionals (veterinarians and veterinary paraprofessionals), with the overarching aim of improving veterinary service delivery in Uganda. A survey was administered electronically to 311 animal health professionals during the period November 14-30, 2019. Data were collected on relevant parameters including demographics, knowledge on preventive medicine, diagnostics, disease control and treatment, epidemiology, and One Health, as well as participants' opinions on training priorities, challenges faced, and constraints to veterinary service delivery. Most respondents were veterinarians 26-35 years old, were male, and worked in clinical practice. Lowest perceived knowledge was reported on subjects relating to laboratory diagnostics, antimicrobial resistance (AMR), and nutrition. Training topics considered to be of most benefit to respondents included laboratory diagnostics, treatment of common livestock diseases, AMR, and practical clinical skills in reproductive and preventive medicine. Participants preferred to receive training in the form of practical workshops, in-practice training, and external training. This study highlights the need to prioritize training in practical clinical skills, laboratory diagnostics, and AMR. Wet labs and hands-on practical clinical and laboratory skills should be incorporated to enhance training. Provision of targeted and successful trainings will be dependent on the allocation of adequate resources and support by relevant public and private stakeholders across the veterinary sector.

The influence of biologically active additives on the morpho-biochemical parameters of the blood of broiler chickens

Abstract. The introduction of synbiotics and phytobiotics into the diet allows us to optimize metabolism, increase the level of use of feed components, and activate immune processes in the body. The purpose of the work is to determine the effect of “GerbaStor” and “ProStor” feed additives, including probiotic, prebiotic and phytobiotic components, on the morphological and biochemical parameters of the blood of broiler chickens of the “Ross-308” cross. Methods. In the course of a scientific and economic experiment at the age of 29 days, blood was taken from 5 medium-sized broilers by decapitation to determine morphological and biochemical parameters. The blood test was carried out at the Clinical and Diagnostic Center in Ekaterinburg. The morphological examination of blood was carried out in the hemostasis laboratory, on an automatic analyzer ADVIA 120, manufactured by BAYER, as well as by manual counting of formed elements in the Goryaev chamber. A biochemical blood test was performed in a clinical diagnostic laboratory using a Vitros 350 analyzer. Results. The inclusion of “GerbaStor” and “ProStor” in the diet did not adversely affect the metabolic processes of the bird. Morpho-biochemical blood parameters were within physiological values. In the experimental groups was observed an improvement in the assimilation of protein nitrogen, it is evidenced by a decrease in uric acid in the blood serum. In addition, the use of the studied feed was accompanied by the activation of lipid metabolism in the body of birds, which was characterized by a tendency to reduce the amount of triglycerides in the blood serum in individuals of the 1st and 2nd experimental groups. The results of the study showed that when “ProStor” is introduced into the feed concentrate, there is a tendency of decreasing in the activity of transaminases in the body of broilers, which demonstrates a higher resistance of their internal organs, in particular the liver and heart, to unfavorable exogenous and endogenous influences. The academic novelty lies in the fact that new data were obtained on the influence of the studied feed factors, including probiotic, prebiotic and phytobiotic components, on the morpho-biochemical parameters of the blood of broiler chickens.

ISSUES OF EXTERNAL QUALITY CONTROL OF LABORATORY STUDIES OF SYPHILIS IN MEDICAL INSTITUTIONS OF UKRAINE

The objective of the work is to identify systemic and random errors in the formulation of controlled methods and to achieve comparative results obtained by laboratories participating in external quality control of laboratory tests for syphilis. Materials and methods. Laboratories of three levels took part in the activities of external quality control of serological diagnostics of syphilis: clinical diagnostic laboratories (CDL) of medical institutions of scientific research institutes of the NAMS of Ukraine, serological departments as part of the CDL of the dermatovenerological profile of the RDVD, CDVD. Results. The quality of serological tests for syphilis in 21 CDL (serological departments) was assessed using the following methods: RMP, RPR, Rapid-test, TPHA, FTA-abs, ELISA in three control serum samples according to formal and essential characteristics. The most significant differences in the results were revealed in the study of control serum samples with a low content of antibodies and those without antibodies to the causative agent of syphilis in the non-treponemal RMP test and the treponemal FTA-abs test. Conclusions. The conducted studies have shown the high efficiency of the practical application of the developed system of external quality control of laboratory methods for diagnosing syphilis.

Blood Culture Positive for Gram-Positive Rods: Contamination or a True Infection-A literature Review

Today, human bloodstream infections (BSIs) are recognized as a major cause of morbidity and mortality worldwide. The bacteria, responsible for bacteremia, are usually identified in diagnostic clinical laboratories, using blood cultures. True bacteremia is defined as a positive blood culture (>15 CFU/mL), with signs and symptoms of infection (e.g., fever and chills). A wide range of bacteria can cause true bacteremia and some bacterial isolates from BSIs may be responsible for contamination. Gram-positive bacilli, such as Bacillus species, and coryneform bacteria are suspected sources of contamination in blood cultures. However, in certain patients, such as immunocompromised patients and intravenous drug users, gram-positive bacilli can act as a true pathogen. Therefore, it is important to know when gram positive bacilli act as a true pathogen and when they act as contamination. So, the rapidly diagnosis of true pathogens and appropriate treatment play a very important role in controlling infection with these bacteria. Effective measures are especially important in patients with an underlying disease or an immunocompromised status. In this article, we reviewed the literature on common Gram-positive rod-shaped bacteria, which were isolated from blood cultures and were suspected to be true pathogens or contaminants.

Clinical epigenomics: genome-wide DNA methylation analysis for the diagnosis of Mendelian disorders

PurposeWe describe the clinical implementation of genome-wide DNA methylation analysis in rare disorders across the EpiSign diagnostic laboratory network and the assessment of results and clinical impact in the first subjects tested. MethodsWe outline the logistics and data flow between an integrated network of clinical diagnostics laboratories in Europe, the United States, and Canada. We describe the clinical validation of EpiSign using 211 specimens and assess the test performance and diagnostic yield in the first 207 subjects tested involving two patient subgroups: the targeted cohort (subjects with previous ambiguous/inconclusive genetic findings including genetic variants of unknown clinical significance) and the screening cohort (subjects with clinical findings consistent with hereditary neurodevelopmental syndromes and no previous conclusive genetic findings). ResultsAmong the 207 subjects tested, 57 (27.6%) were positive for a diagnostic episignature including 48/136 (35.3%) in the targeted cohort and 8/71 (11.3%) in the screening cohort, with 4/207 (1.9%) remaining inconclusive after EpiSign analysis. ConclusionThis study describes the implementation of diagnostic clinical genomic DNA methylation testing in patients with rare disorders. It provides strong evidence of clinical utility of EpiSign analysis, including the ability to provide conclusive findings in the majority of subjects tested.

Substantiation of organ-preserving surgical treatment of children with nonparasitic spleen cysts

BACKGROUND: The urgency of surgical treatment of children with nonparasitic spleen cysts is determined by the lack of consensus in the professional community, lack of regulatory documents governing the treatment of these patients, frequency of postoperative complications, and unfavorable outcomes.&#x0D; AIM: This study aimed to improve the efficiency and safety of organ-preserving minimally invasive interventions in children with nonparasitic spleen cysts based on the development of a multifactorial preoperative planning system and substantiation of an algorithm for choosing the optimal surgical strategies.&#x0D; MATERIALS AND METHODS: Results of surgical treatment of 60 children aged 218 yrs with nonparasitic spleen cysts are presented. The spleen cyst volume varied from 3 ml to 1000 ml (Me 50 ml). Preoperative examination included clinical examination, laboratory diagnostics, ultrasonography, computed tomography or magnetic resonance imaging, and angiography of the spleen vessels. The range of surgical technologies included percutaneous puncture (n = 2) and percutaneous puncture drainage (n = 28), followed by sclerosing of the cyst with 96% ethyl alcohol, combined interventions, supplemented by superselective embolization of the spleen arteries feeding the pathological formation (n = 15), laparoscopic fenestrations of cysts with physical de-epithelialization of the inner lining (n = 14), and laparoscopic resection of the spleen pole (n = 1).&#x0D; RESULTS: The analysis of postoperative complications was carried out depending on the chosen technology of surgical treatment. The follow-up period of 44 patients varied from 6 mon to 3 yrs, which made it possible to reveal the regularities of the reduction of residual cyst cavities and the course of the regeneration processes with an objective assessment of the volumetric characteristics of the spleen. Obliteration of the residual cyst cavities was observed in 79.1% of the patients during the first month after surgery. Subsequent total obliteration of the residual cyst cavities was observed within 1 yr after surgery in 91.7% of children and residual pathological formations persisted in five patients, which accounted for 8.3% of clinical observations. The volume of residual cysts ranged from 1.2% to 10.0% of the initial value, which was regarded as a satisfactory treatment result.&#x0D; CONCLUSION: Results of a retrospective multivariate analysis made it possible to develop an algorithm for substantiating surgical techniques, providing a radical cure for 95.5% of children with nonparasitic spleen cysts.

SARS-CoV-2 detection by a clinical diagnostic RT-LAMP assay.

The ongoing pandemic of SARS-CoV-2 calls for rapid and cost-effective methods to accurately identify infected individuals. The vast majority of patient samples is assessed for viral RNA presence by RT-qPCR. Our biomedical research institute, in collaboration between partner hospitals and an accredited clinical diagnostic laboratory, established a diagnostic testing pipeline that has reported on more than 252,000 RT-qPCR results since its commencement at the beginning of April 2020. However, due to ongoing demand and competition for critical resources, alternative testing strategies were sought. In this work, we present a clinically-validated procedure for high-throughput SARS-CoV-2 detection by RT-LAMP that is robust, reliable, repeatable, specific, and inexpensive.

Contamination Assessment for Cancer Next-Generation Sequencing

The presence of allogeneic contamination impacts clinical reporting in cancer next-generation sequencing specimens. Although consensus guidelines recommend the identification of contaminating DNA as a part of quality control, implementation of contamination assessment methods in clinical molecular diagnostic laboratories has not been reported in the literature. To develop and implement a method to assess allogeneic contamination in clinical cancer next-generation sequencing specimens. We describe a method to detect contamination based on the evaluation of single-nucleotide polymorphic sites from tumor-only specimens. We validate this method and apply it to a large cohort of cancer sequencing specimens. Identification of specimen contamination was validated via in silico and in vitro mixtures, and reference range and reproducibility were established in a panel of normal specimens. The algorithm accurately detects an episode of systemic contamination due to reagent impurity. We prospectively applied this algorithm across 7571 clinical cancer specimens from a targeted next-generation sequencing panel, in which 262 specimens (3.5%) were predicted to be affected by greater than 5% contamination. Allogeneic contamination can be inferred from intrinsic cancer next-generation sequencing data without paired normal sequencing. The adoption of this approach can be useful as a quality control measure for laboratories performing clinical next-generation sequencing.

Guideline-Adherent Clinical Validation of a Comprehensive 170-Gene DNA/RNA Panel for Determination of Small Variants, Copy Number Variations, Splice Variants, and Fusions on a Next-Generation Sequencing Platform in the CLIA Setting.

We describe the clinical validation of a targeted DNA and RNA-based next-generation sequencing (NGS) assay at two clinical molecular diagnostic laboratories. This assay employs simultaneous DNA and RNA analysis of all coding exons to detect small variants (single-nucleotide variants, insertions, and deletions) in 148 genes, amplifications in 59 genes, and fusions and splice variants in 55 genes. During independent validations at two sites, 234 individual specimens were tested, including clinical formalin-fixed, paraffin-embedded (FFPE) tumor specimens, reference material, and cell lines. Samples were prepared using the Illumina TruSight Tumor 170 (TST170) kit, sequenced with Illumina sequencers, and the data were analyzed using the TST170 App. At both sites, TST170 had ≥98% success for ≥250× depth for ≥95% of covered positions. Variant calling was accurate and reproducible at allele frequencies ≥5%. Limit of detection studies determined that inputs of ≥50 ng of DNA (with ≥3.3 ng/μl) and ≥50 ng RNA (minimum of 7 copies/ng) were optimal for high analytical sensitivity. The TST170 assay results were highly concordant with prior results using different methods across all variant categories. Optimization of nucleic acid extraction and DNA shearing, and quality control following library preparation is recommended to maximize assay success rates. In summary, we describe the validation of comprehensive and simultaneous DNA and RNA-based NGS testing using TST170 at two clinical sites.

Centralised or Localised Pathogen Whole Genome Sequencing: Lessons Learnt From Implementation in a Clinical Diagnostic Laboratory.

Whole genome sequencing (WGS) has had widespread use in the management of microbial outbreaks in a public health setting. Current models encompass sending isolates to a central laboratory for WGS who then produce a report for various levels of government. This model, although beneficial, has multiple shortcomings especially for localised infection control interventions and patient care. One reason for the slow rollout of WGS in clinical diagnostic laboratories has been the requirement for professionally trained personal in both wet lab techniques and in the analysis and interpretation of data, otherwise known as bioinformatics. A further bottleneck has been establishment of regulations in order to certify clinical and technical validity and demonstrate WGS as a verified diagnostic test. Nevertheless, this technology is far superior providing information that would normally require several diagnostic tests to achieve. An obvious barrier to informed outbreak tracking is turnaround time and requires isolates to be sequenced in real-time to rapidly identify chains of transmission. One way this can be achieved is through onsite hospital sequencing with a cumulative analysis approach employed. Onsite, as opposed to centralised sequencing, has added benefits including the increased agility to combine with local infection control staff to iterate through the data, finding links that aide in understanding transmission chains and inform infection control strategies. Our laboratory has recently instituted a pathogen WGS service within a diagnostic laboratory, separate to a public health laboratory. We describe our experience, address the challenges faced and demonstrate the advantages of de-centralised sequencing through real-life scenarios.