Rapid Laboratory Diagnosis Research Articles

Rapid laboratory diagnosis of urinary tract infection, with or without antibiotic decision support-a small pilot study investigating accuracy and clinical impact.

The study evaluated the accuracy and clinical impact of rapid diagnostics (RD) with or without antibiotic decision support (ADS) for hospitalized patients with urinary tract infections. A two-centre prospective intervention was conducted with 230 patients divided into three groups: RD-only (n = 59), RD plus ADS (n = 56) and a control group (n = 115). Mean laboratory turnaround time for RD was 10 h and 50 min. Of 115 microorganisms, 108 were correctly identified. The error rate for rapid susceptibility determination was 0.85%. Total antibiotic consumption, measured in defined daily doses (DDD), was lower in the intervention groups compared to the control group (ADS: 10.3 DDD, p = 0.01; RD: 10.9 DDD, p = 0.06; control: 13.0 DDD). No significant differences were observed in the use of broad-spectrum antibiotics (p = 0.816). Adherence to antibiotic guidelines was significantly better in the ADS group compared to the control group (p = 0.015) (RD vs control; p = 0.261). The ADS group also received fewer doses of ineffective antibiotics (ADS: 1.8 doses, p = 0.012; RD: 2.4 doses, p = 0.195; control: 3.4 doses). Length of hospital and ICU stays or 30-day readmission rates did not differ across groups. No in-hospital mortality was observed in any group.

Evaluation of Molecular Assays for Diagnosis of Amoebic Liver Abscess in India with Bayesian Latent Class Analysis.

Amoebic liver abscess (ALA) is the most common extra-intestinal complication of Entamoeba histolytica, accounting for 50,000 deaths annually, and is endemic in South Asia. Diagnosis based on microscopic examination is insensitive, and serological assays are not discerning of current infections in endemic settings with high exposure. For a rapid and confirmatory laboratory diagnosis of ALA, the performance of a polymerase chain reaction (PCR), quantitative real time PCR (qPCR), digital droplet PCR (ddPCR), and a loop-mediated isothermal amplification (LAMP) assay that detects E. histolytica DNA in liver abscess pus, and a lectin antigen detection ELISA were evaluated against clinical diagnosis (based on predefined criteria) as the gold standard. Owing to the lack of a laboratory gold standard, a Bayesian latent class analysis approach was also used to determine sensitivity and specificity of these assays. In the latent class analysis, qPCR and ddPCR showed the highest sensitivity (98% and 98.1%) and specificity (both 96.6%), and although clinical diagnosis had a comparable sensitivity to qPCR and ddPCR (95.2%), poorer specificity (64.3%) was seen. Kappa agreement analysis showed that qPCR and ddPCR had a perfect agreement of 1 followed by an agreement of 0.76 (95% CI: 0.64-0.88) with PCR. Considering the performance characteristics and relative ease of setting up qPCR as well as the wide availability of qPCR equipment needed, this would be the most optimal assay for rapid, confirmatory, molecular diagnosis of ALA in the tertiary care laboratory setting in India, whereas further optimization of LAMP or antibody-based detection is required for use at smaller or secondary hospitals.

HRP2 and PLDH Performance Comparison as Rapid Diagnostic Tools for Symptomatic Malaria Patients at Rugazi HC IV in Rubirizi District

Background: Rapid and precise laboratory diagnosis is essential for effectively diagnosing and managing malaria patients. Malaria rapid diagnostic tests (mRDTs) play a critical role in malaria diagnosis in resource-constrained settings with limited access to labs and trained staff. The efficiency of two mRDTs (HRP2 and pLDH) was compared against microscopy, the gold standard for malaria diagnosis, in this study. Methods: In a cross-sectional study, 308 participants were tested for malaria parasites using two mRDTs and blood smear microscopy as the gold standard. STATA software version 17 was used for data analysis. Results: Out of the 308 participants, 82 tested positive for HRP2 mRDT (26.62% (95% CI: 21.97-31.86) positivity rate, 73.38% (95% CI: 68.14-78.83) negativity rate), and 99 tested positive for pLDH mRDT (32.14% (95% CI: 27.14-37.59) positivity rate, 67.86% (95% CI: 62.41-72.86) negativity rate). Using microscopy as the gold standard, the sensitivity, specificity, PPV, and NPV were determined. For HRP2 mRDT, sensitivity was 77.78% (95%CI: 73.13-82.42), specificity 97.61% (95%CI: 95.90 - 99.31), PPV 93.90% (95%CI: 91.23 – 96.56), and NPV 90.27% (95%CI: 86.96-93.58). For pLDH mRDT, the sensitivity was 94.95% (95% CI: 92.50-97.40), specificity was 97.61% (95% CI: 95.90-99.31), PPV was 94.95% (95% CI: 92.50 – 97.40), and NPV was 97.61% (95% CI: 95.90 - 99.31). The dominant malaria species was Plasmodium falciparum (74.7%), followed by Plasmodium vivax (12.1%) and Plasmodium ovale (1%). Mixed infections of plasmodium falciparum and plasmodium vivax (7.1%) and plasmodium falciparum and plasmodium ovale (5.1%) were also observed. Conclusion: There was no significant difference in the positivity rate, negativity rate, specificity or PPV in both mRDTs. However, a significant difference in the NPV and sensitivity of HRP2 and pLDH mRDTs was observed where HRP2’s sensitivity was below the WHO recommendation of ≥95% in this study population; thus, there is a need to improve the sensitivity of this mRDT kit. In our study, most malaria infections were caused by Plasmodium falciparum, followed by P. vivax and P. ovale. We therefore recommend the use of pLDH mRDTs over HRP2 mRDTs based on the results of our study

Viabilidade da substituição da técnica de isolamento viral em camundongos pela RT-qPCR para o diagnóstico da raiva

ABSTRACT: Rabies is a viral encephalitis that affects mammals, including humans. Rapid and effective laboratory diagnosis of the rabies virus is critical for public health. This study evaluated the operational, technical, and financial viability of the RT-qPCR in replacement of inoculation in mice for diagnosing rabies in the laboratory routine. A total of 316 samples of mammalian brains were analyzed, 121 positives and 195 negatives, previously diagnosed by direct immunofluorescence (DIF) and mouse inoculation test (MIT). The samples were submitted to the duplex TaqMan RT-qPCR technique. The accuracy, sensitivity, and specificity of RT-qPCR were analyzed. We analyzed the costs for performing the RT-qPCR technique and compared it with the cost of MIT. The results showed 99.37% accuracy, 99.17% sensitivity, and 99.49% specificity by RT-qPCR when related to DIF and MIT results, which proved to be a robust and repeatable technique. The minimum time for a positive diagnosis was reduced in RT-qPCR (1 day) if compared to MIT (9.64 days), with a 17.7% reduction in the cost of the molecular technique. The present study demonstrated that the molecular biology technique is an efficient tool to diagnose rabies in the laboratory routine, being able to replace MIT.

Rapid Laboratory Diagnosis of Epizootic Hemorrhagic Disease Virus Using a Loop-Mediated Isothermal Amplification Assay.

Reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) is a molecular diagnostic assay that is particularly useful for the detection of viral diseases of livestock. A major advantage of RT-LAMP is that it can be used either as a rapid field test or as a high-throughput screening tool in veterinary laboratories, with sensitivity comparable to the real-time RT-PCR assay. Unlike conventional or qPCR, RT-LAMP uses a strand displacement polymerase and a set of four to six primers that bind to several regions of the target nucleic acid. Amplification occurs without thermal cycling, and coupled with the numerous primers, RT-LAMP offers a rapid and highly specific molecular assay. In this chapter, we describe the RT-LAMP protocol for the detection of epizootic hemorrhagic disease virus (EHDV) as a low-cost, specific, and sensitive screening tool in veterinary diagnostic laboratories. We also provide guidance on how to adapt the RT-LAMP assay for rapid field testing.

Performance of a Novel Real-Time PCR-Based Assay for Rapid Monkeypox Virus Detection in Human Samples.

The ongoing epidemic of mpox, namely human monkeypox virus (MPXV) infection, requires rapid and reliable laboratory diagnosis. We report on the QIAstat-Dx viral vesicular panel PCR assay that allows the detection of (within 75 min) six vesicular disease-causing viruses, including MPXV. We analyzed 168 clinical samples, known to be positive (51 samples) or negative (117 samples) for MPXV clade II, obtained from patients at their mpox diagnosis or follow-up. QIAstat assay results were compared to those of a MPXV-specific reference PCR assay. The QIAstat assay detected MPXV (clade II) in 51 (100%) of 51 samples and did not detect MPXV in 117 (100%) of 117 samples, resulting in a positive or negative agreement of 100% (95% CI, 93.0-100) and 100% (95% CI, 96.8-100), respectively. Of the 20 patients diagnosed with mpox, 18 (90.0%) had at least a vesicular swab and 1 (5.0%) had only an oropharyngeal swab positive for MPXV. At mpox follow-ups, 2 (10.0%) of 20 patients had first-time positive whole blood samples. Thirteen MPXV-negative samples were positive for mpox-mimicking viruses. Our findings show the excellent performance of the QIAstat-Dx assay for MPXV detection in clinical samples. Further studies are needed before considering a large-scale application of the QIAstat-Dx assay.

Zika virus disease: an alarming situation resurfacing on the radar - a short communication.

On the 13th of December 2022, a 5-year-old girl from Karnataka, India, tested positive for Zika virus. The first Zika virus was isolated from the serum of a rhesus monkey in the Zika Forest of Uganda in 1947. Zika virus was largely dormant for about 70 years before suddenly resurfacing across all of America, from Brazil to the Pacific Islands and is connected to a grouping of microcephaly phenotypes based on a complete virus genome analysis. All of the aforementioned research provides an overview of the migration of this virus from the Americas to continental Africa via mosquitoes. The current study, therefore, aims to evaluate the virologic characteristics, prophylaxis, transmitting mechanisms, diagnosis, clinical manifestations, and treatment of ZIKV infection in light of the virus's widespread dissemination and deadly nature. The investigation's findings aim to demonstrate that in order to prevent further outbreaks, there is a national requirement for active epidemiological and entomological observation of Zika. Data were extracted from academic journals of medicine published in MEDLINE, PubMed, ScienceDirect, Ovid, and Embase inventory databases with a predetermined search strategy. Articles considering the Zika virus and its clinical manifestations, especially neurological, were included. The Zika virus has been declared a public health emergency of global significance by the World Health Organization (WHO). It is of alarming concern that it is now one of the most prevalent infectious diseases associated with birth abnormalities discovered in the past five decades. The onset and accelerated spread of disease to other parts of the world is attributed to the migration of infected hosts and climate change. Rapid laboratory diagnosis, evaluation of serological techniques, and virus isolation are urgently needed, along with newer modalities such as mathematical modeling as prediction devices to curb this issue. Due to its grave neurological manifestations, it is mandated to engineer peptide therapies and a virus-specific vaccination to treat this neurotropic virus. There is currently no vaccination against Zika virus infection. If societies are not adequately prepared, the epidemiological wave will have an impact on the workforce and could pose a serious threat. To alleviate the significant cost on health systems and manage its promotion globally, improved investigation and response activities are needed.

The Changing Global Epidemiology of Re-emerging Human Monkeypox Virus Infection: A Systematic Review.

Human monkeypox virus (MPVX) infection represents an emerging zoonotic disease caused by an orthopoxvirus, resulting in a condition reminiscent of smallpox. More recent developments have witnessed a notable surge in global MPVX outbreaks, eliciting significant concerns. We aimed to investigate the epidemiological factors of the emerging human monkeypox virus infection, including the number of suspected, confirmed, and fatal cases, as well as the risk factors for contracting monkeypox infection.We performed a systematic review of peer-reviewed literature by following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. An electronic database search (PubMed, Wiley Online Library, and Science Direct) was undertaken. For monkeypox-related studies, we included 25 peer-reviewed articles from 2018 and 2022, and data were extracted on the current evidence on the cases and the risk factors for MPVX infection, to develop public health advisories.Our reports show a rapid rise of MPVX cases in the highly endemic African regions after the 1970s, spread to other countries, and an increase in the median age from young children to young adults. The cessation of smallpox vaccination might have been one of the factors responsible for these findings.As of 2022, the genomic sequences of ten MPVX strains associated with the recent countrywide outbreak have been determined. While the West African Clade has been primarily implicated in the recent viral surge, data were insufficient to determine which mutation contributed to increased transmissibility. In the Democratic Republic of the Congo (DRC), sleeping on the floor was significantlyassociated with contracting MPVX,while eating or processing of animal foods was not a significant risk factor. In the United States, cleaning the cages and bedding of sick animals, touching infected animals, and daily exposure to sick animals were associated with an increased probability of contracting the MPVX infection. Recent global outbreaks and the rising incidence of MPVX infections among young adults in the endemic zones might be a result of the cessation of the smallpox vaccine. The increased risk associated with exposure to sick animals or sleeping on the floor suggests high infectivity from animal excretions. Increasing awareness, strict surveillance, and contact tracing can help contain global outbreaks. The ring vaccination approach for exposed individuals is another potential disease containment strategy. Future studies should investigate measures for rapid laboratory diagnosis, maintaining lab safety, and transmissibility.

Increased serum interleukin-6 and lactate dehydrogenase levels among nonsurvival severe COVID-19 patients when compared to survival ones

BackgroundAccurate and rapid laboratory diagnosis of COVID-19 infection and its deterioration is one of the milestones of pandemic control. Therefore, this study aimed to compare the diagnostic and prognostic accuracy of the mainly used laboratory biomarkers (WBCS, neutrophil and lymphocyte percentages, CRP, ferritin, IL-6, D-dimer, procalcitonin, and LDH) in the sera of severe COVID-19 Egyptian patients to assess the most appropriate biomarker used in severe COVID-19 patients. MethodsA total of 180 unvaccinated severe COVID-19 patients were enrolled in our study. Demographic data, hospitalization time, medical history, oxygen saturation, respiratory rate, oxygen supply, laboratory findings, and thorax tomography of the patients were obtained retrospectively from the hospital's electronic information system. ResultsOur results revealed that the levels of neutrophil percentage, CRP, IL-6, PCT, and LDH were significantly increased while lymphocyte percentage was significantly decreased among nonsurvival severe COVID-19 patients when compared with survival ones. By using ROC curve analysis, IL-6, and LDH are the most sensitive and specific markers for the prediction of bad prognosis and mortality among severe COVID-19 patients with 100% and 93% sensitivity and 93.7% specificity; respectively. IL-6 and LDH showed significant correlations with the other parameters, which suggested their association with the severity of COVID-19. ConclusionBy using survival severe COVID-19 patients as a control group, our results showed that blood neutrophil percentage, serum CRP, IL-6, PCT, and LDH were significantly increased in non-survivors as compared to survivors. As biomarkers, our results revealed that IL-6 and LDH are good predictors of mortality among severe COVID-19 patients.

Coronin-1A serves as a serum biomarker for supportive diagnosis of Mycobacterium tuberculosis infection.

The severity and spread of tuberculosis, a major burden, can be prevented by more rapid and accurate laboratory diagnosis. The purpose of this study is to systematically explore candidate serum proteins in patients with Mycobacterium tuberculosis infection for further application as novel biomarkers. Our study was performed in two major steps: screening of the literature for potential biomarkers, and then validation of their levels in patients and controls. Many serum/plasma proteins previously reported to be abnormally expressed in patients with tuberculosis between 2012 and 2021 were comprehensively assembled. The biological role in tuberculosis was also predicted for each using the bioinformatics tool STRING. Candidate proteins found to have the same expression in other related diseases were excluded. Subsequently, the serum level of the candidate serum/plasma protein that met the aforementioned criteria was validated by sandwich ELISA; diagnostic performance was analysed by the area under the curve (AUC) of the receiver operating characteristic (ROC). From 103 collected serum/plasma proteins, coronin-1A was found to have abnormal expression only in patients with tuberculosis and was associated with tuberculosis. In addition, the validation of coronin-1A in the serum of patients with pulmonary tuberculosis revealed a higher level than in that of healthy individuals. Furthermore, the area under the ROC curve for diagnostic power of coronin-1A was 0.866, with high sensitivity and specificity at a cut-point of approximately 52.7 ng/mL. We concluded that the level of serum coronin-1A might serve as a novel biomarker for alternative laboratory examination to effectively distinguish patients with tuberculosis from those with other related diseases and healthy individuals.

Assessment of diagnostic and prognostic laboratory biomarkers in severe COVID-19 patients admitted to intensive care unit

Coronavirus disease (COVID-19) is a global pandemic. In the first two years of the pandemic, nearly 15 million people died worldwide. Accurate and rapid laboratory diagnosis of COVID-19 infection is one of the milestones of pandemic control. Therefore, this study aimed to compare the diagnostic and prognostic accuracy of mainly used laboratory biomarkers (CRP, ferritin, IL-6, D-dimer, procalcitonin, and LDH) in the sera of severe COVID-19 Egyptian patients, to assess the most appropriate biomarker used in severe COVID-19 patients. A total of 120 COVID-19 patients and 50 normal controls were enrolled into our study. Demographic data, hospitalization time, medical history, oxygen saturation, respiratory rate, oxygen supply, laboratory findings and thorax tomography of the patients were obtained from the hospital electronic information system retrospectively. Our results revealed that the serum levels of CRP, ferritin, IL-6, D-dimer, PCT and LDH were highly significantly increased in severe COVID-19 patients as compared to normal controls (p < 0.001), and in non-survivors as compared to survivors (p < 0.001). By using ROC curve analysis, IL-6 appeared to be the most sensitive and specific marker with 80.9% sensitivity and 84.9% specificity; followed by LDH with 85.1% sensitivity and 82.8% specificity in the prediction of death. In conclusion CRP and IL-6 could be the most appropriate biomarkers in the diagnosis of severe COVID-19 disease, while IL-6 and LDH may be good predictors of mortality between severe COVID-19 patients.

Comparison of Genexpert MTB/RIF Assay and AFB Smear Microscopy in Diagnosis of Pulmonary Tuberculosis among HIV Patients at a Tertiary Care Hospital

Introduction: Tuberculosis (TB) has been the most common acute opportunistic infection in HIV positive patients and accounts for more than half of all AIDS cases in underdeveloped nations. Hence, rapid laboratory diagnosis of M. tuberculosis is needed for vulnerable patients. Methodology: In this study, 72 sputum samples were collected from ART-naive and patients using ART for 6 months. The samples were tested with the GeneXpert MTB / RIF assay to diagnose TB and drug resistance and AFB smear microscopy as per NTP guidelines. Results: Among 72 sputum samples studied, males were more diagnosed with TB than females. People aged 25-56 years were more infected with HIV-TB co-infection. Four patients (5.56%) were found to be TB positive with both techniques, 7 (9.72%) were GeneXpert MTB/RIF positive but smear-negative and 61 patients (84.72%) were negative with both methods. The positivity for MTB detected for GeneXpert MTB/RIF method in females was 3 (15%) and the males were 8 (15.38%), while for the ZN staining method the positivity in females was 2 (10%) and the males were 2 (3.85%). The GeneXpert MTB/RIF assay was also capable of detecting TB in smear-negative cases. Conclusion: This study showed that the GeneXpert MTB/RIF assay is an effective tool for the early diagnosis of TB among HIV patients as compared to AFB smear staining method.

VIRAL PNEUMOENTERITIS OF CATTLE IN FARMS OF THE REPUBLIC OF BELARUS

The article describes the etiological structure of pneumoenteritis in cattle (cattle) in livestock farms of the Re-public of Belarus for the period 2020–2021. based on the analysis of the results of our own research and describes the role of associations of viruses involved in their occurrence.&#x0D; Among the etiological causes of pneumoenteritis in cattle, there are such infections as BVDV, IBR, PIV-3, BRSV, bovine rotavirus and bovine coronavirus infections that cause significant economic damage to livestock. Preven-tion of diseases is the most important task of modern animal husbandry. In many cases, preventive measures can pre-vent the development of diseases or make their prognosis and consequences more favorable. Prevention and treatment requires rapid and accurate laboratory diagnosis. In addition to establishing the etiology of the disease, laboratory diagnostics is important in establishing the epizootic situation and in organizing anti-epizootic measures.

The Accelerate Pheno™ System-A New Tool in Microbiological Diagnostics of Bloodstream Infections: A Pilot Study from Poland.

The aim of this study was to evaluate the usefulness of the Accelerate Pheno™ system (APS) (Accelerate Diagnostics, Denver, CO, USA) for rapid laboratory diagnosis of bloodstream infections. The study included 45 positive blood samples obtained from patients hospitalized in University Hospital No. 1 in Bydgoszcz, Poland. In 40 (88.9%) blood samples, the APS was capable of identification of at least one microorganism at the genus or species level and in 38 (84.4%) of them additionally assessed antimicrobial susceptibility. The time of identification and the time to result of antimicrobial susceptibility ranged from 1:32 to 1:42 and 5:02 to 5:36 h, respectively. Six positive blood samples revealed a poly-microbial culture. In these cases, only one out of two or three microorganisms was detected by the APS, and the system assessed antimicrobial susceptibility only for them. For 78.6% positive blood samples, agreement on identification compared to mass spectrometry was found. For all but one sample, a 96-100% compliance of the resistance category was achieved when comparing the antimicrobial susceptibility testing results to conventional methods. Using the APS, the total time to report was reduced from 13:34 to even 63:47 h compared to the standard microbiological laboratory workflow. The APS is a very useful system, especially for the rapid assessment of antimicrobial susceptibility of bacteria directly from positive blood samples, offering the greatest potential for microbiology laboratories operating around the clock.

Rapid Multilateral and Integrated Public Health Response to a Cross-City Outbreak of Salmonella Enteritidis Infections Combining Analytical, Molecular, and Genomic Epidemiological Analysis.

On September 21, 2019, the Shenzhen and Dongguan Centers for Disease Control and Prevention received notification of a large cluster of suspected gastroenteritis involving primarily children who sought medical care at hospitals throughout two adjacent cities in China, Shenzhen, and Dongguan. A joint outbreak response was promptly initiated across jurisdictions in a concerted effort between clinical microbiologists, epidemiologists, and public health scientists. Concurrently, multiplex PCRs were used for rapid laboratory diagnosis of suspected cases; epidemiological investigations were conducted to identify the outbreak source, complemented by near real-time multicenter whole-genome analyses completed within 34 h. Epidemiological evidence indicated that all patients had consumed egg sandwiches served on September 20 as snacks to children and staff at a nursery in Dongguan, located near Shenzhen. Salmonella Enteritidis was isolated from case-patients, food handlers, kitchenware, and sandwiches with kitchen-made mayonnaise. Whole-genome single-nucleotide polymorphism (SNP)-based phylogenetic analysis demonstrated a well-supported cluster with pairwise distances of ≤1 SNP between genomes for outbreak-associated isolates, providing the definitive link between all samples. In comparison with historical isolates from the same geographical region, the minimum pairwise distance was >14 SNPs, suggesting a non-local outbreak source. Genomic source tracing revealed the possible transmission dynamics of a S. Enteritidis clone throughout a multi-provincial egg distribution network. The efficiency and scale with which multidisciplinary and integrated approaches were coordinated in this foodborne disease outbreak response was unprecedented in China, leading to the timely intervention of a large cross-jurisdiction Salmonella outbreak.

Performance evaluation of routine blood and C-reactive protein analysis using Mindray BC-7500 CRP auto hematology analyzer.

BackgroundMindray’s newly developed BC-7500 CRP auto hematology analyzers can simultaneously measure the routine blood and C-reactive protein (CRP) results, thereby significantly improving the efficiency and quality of clinical examination. This study was designed to evaluate the basic performance of the BC-7500 CRP as well as the performance characteristics of the new measurement mode combines routine blood and CRP.MethodsVenous whole blood samples with the anticoagulant ethylene diamine tetraacetic acid K2 (EDTA-K2) were collected at the Fujian Medical University Union Hospital according to the guidelines of the American Clinical Laboratory Standards Institute (CLSI) and International Standards Committee for Hematology (ISCH). We analyzed and evaluated the performance of blank counts, carryover contamination rate, precision, comparability, flagging capability, sample stability, and automatic micro-whole blood (MWB) mode analysis in the BC-7500 CRP hematology analyzer.ResultsAll parameters measured by the BC-7500 CRP showed good blank counts (CRP, 0.008–0.02 mg/L). The carryover contamination rates for each mode combination did not exceed 1%, it indicates a low carryover effect. The CRP showed high precision while the within-laboratory precision is 6.91% and 4.38% in two different levels. Relatively good correlation was found for the routine blood results between the BC-7500 CRP and Sysmex-XN (r=0.99), and CRP result between the BC-7500 CRP and IMMAGE 800 (r=0.99). The sensitivities of abnormal cell flagging for Blast, Immature Gran, and nucleated red blood cell (NRBC) were 93.9%, 91.6%, and 86.7%, while their specificities were 83.9%, 62.7%, and 97.5%. The test results of CRP at room temperature for 24 hours and refrigerated for 48 hours were −0.04% and −2.94%, which showed very stable changes. Also, the automatic micro-whole blood mode performed well (P=0.2014), which was very close to the test results of the automatic mixing test of peripheral blood and the manual mixing of open sampling.ConclusionsThe BC-7500 CRP analyzer provided reliable results on routine blood and CRP, which can meet the testing requirements of routine blood and CRP items in clinical whole blood samples and provide a guarantee for rapid and accurate laboratory diagnosis.

Fungal corneal ulcer complicated by endophthalmitis: clinical and laboratory diagnostics, treatment tactics

Purpose: to study the clinical signs and effectiveness of laboratory diagnostics of fungal corneal ulcer complicated by endophthalmitis aimed at treatment tactics optimization.Materials and methods. 15 patients aged 20–55 with severe monocular fungal ulcer complicated by endophthalmitis were examined by microscopy and culture of corneal scraping on Sabouraud Agar; microscopy and culture of intraocular fluid and corneal fragments, obtained during the surgical treatment. Corneal scrapings from the corneal ulcer and corneal fragments removed during keratoplasty were tested in polymerase chain reaction (PCR) for the presence of deoxyribonucleic acid (DNA) of Candida albicans and total Fungi DNA.Results. Before treatment start, 2.5 hours after the biomaterial was taken, PCR revealed Fungi DNA in all 15 cases. Symptoms of the severe fungal corneal ulcer complicated by endophthalmitis and caused by Aspergillus spp have been identified and described. Conservative therapy and total penetrating keratoplasty (PKP) helped retain the eye in 13 out of 15 patients (86.7 %) and partially restore the visual functions.Conclusion. Rapid laboratory diagnosis of ophthalmomycosis using PCR offers an advantage over the culturing technique: the results are ready within 2.5 hours instead of 10-14 days (culture method); it is highly sensitive and allows identifying the fungi not only in the tissue of the removed cornea, but also in the сorneal scrapings. The effective treatment tactics of fungal corneal ulcer complicated by endophthalmitis combining PKP with pre-operative and long-term postoperative antifungal therapy was proposed.

RT-qPCR Detection of SARS-CoV-2: No Need for a Dedicated Reverse Transcription Step.

Reverse transcription of RNA coupled to amplification of the resulting cDNA by the polymerase chain reaction (RT-PCR) is one of the principal molecular technologies in use today, with applications across all areas of science and medicine. In its real-time, fluorescence-based usage (RT-qPCR), it has long been a core technology driving the accurate, rapid and sensitive laboratory diagnosis of infectious diseases. However, RT-qPCR protocols have changed little over the past 30 years, with the RT step constituting a significant percentage of the time taken to complete a typical RT-qPCR assay. When applied to research investigations, reverse transcription has been evaluated by criteria such as maximum yield, length of transcription, fidelity, and faithful representation of an RNA pool. Crucially, however, these are of less relevance in a diagnostic RT-PCR test, where speed and sensitivity are the prime RT imperatives, with specificity contributed by the PCR component. We propose a paradigm shift that omits the requirement for a separate high-temperature RT step at the beginning of an RT-qPCR assay. This is achieved by means of an innovative protocol that incorporates suitable reagents with a revised primer and amplicon design and we demonstrate a proof of principle that incorporates the RT step as part of the PCR assay setup at room temperature. Use of this modification as part of a diagnostic assay will of course require additional characterisation, validation and optimisation of the PCR step. Combining this revision with our previous development of fast qPCR protocols allows completion of a 40 cycle RT-qPCR run on a suitable commercial instrument in approximately 15 min. Even faster times, in combination with extreme PCR procedures, can be achieved.

Quantitation of Neuroblastoma Markers Homovanillic Acid (HVA) and Vanillylmandelic Acid (VMA) in Urine by Gas Chromatography-Mass Spectrometry (GC/MS).

Neuroblastoma and other neural crest tumors can be characterized by the increased production and excretion of catecholamines and their metabolites. Homovanillic acid (HVA) and vanillylmandelic acid (VMA) are important catecholamine metabolites that can be measured to provide relatively rapid laboratory diagnosis and clinical follow-up of neuroblastoma. We present a procedure to quantify HVA and VMA in urine samples which have been diluted to a creatinine concentration of 2mg/dL. Diluted samples are spiked with deuterated internal standards, acidified, and extracted with an organic solvent. A bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane (TMCS) and pyridine mixture is added to the dried extract to create trimethylsilyl derivatives of HVA and VMA. The derivatized compounds are measured using gas chromatography-mass spectrometry (GC/MS).

Development and Evaluation of a Nested PCR for Improved Diagnosis and Genetic Analysis of Peste des Petits Ruminants Virus (PPRV) for Future Use in Nascent PPR Eradication Programme.

Simple SummaryPeste des petits ruminants (PPR) is a highly contagious and economically important viral disease of small ruminants and a large number of wildlife species. The causative agent, PPR virus (PPRV) is mainly circulating across large areas in Africa, the Middle East and Asia and is, fortunately, currently subject to a global eradication program. A robust vaccination program along with a rapid and accurate laboratory diagnosis play a major role in the effective control and eradication of the disease. Furthermore, a genetic analysis of the circulating PPRV can help to identify the epidemiological linkage of the virus spread between neighboring countries. Here, we have developed an improved polymerase chain reaction (PCR) that can detect low viral loads and help in genetic identification, particularly in resource-limited settings.Peste des petits ruminants (PPR) is a highly contagious viral disease of small ruminants caused by PPR virus (PPRV). PPR is endemic in Asia, the Middle East and across large areas of Africa and is currently targeted for global eradication by 2030. The virus exists as four different lineages that are usually limited to specific geographical areas. However, recent reports of spread of PPRV, in particular of lineage IV viruses to infection-free countries and previously PPR endemic areas are noteworthy. A rapid and accurate laboratory diagnosis and reports on its epidemiological linkage for virus spread play a major role in the effective control and eradication of the disease. Currently, molecular assays, including conventional reverse transcription-polymerase chain reaction (RT-PCR) and real-time RT-PCR (RT-qPCR) are usually used for diagnosis of PPR while the sequencing of part of the nucleocapsid gene is usually carried out for the viral lineage identification. However, it is difficult to diagnose and sequence the genetic material if the animal excreted a low level of virus at the initial stage of infection or if the PPRV is degraded during the long-distance transportation of samples to the reference laboratories. This study describes the development of a novel nested RT-PCR assay for the detection of the PPRV nucleic acid by targeting the N-protein gene, compares the performance of the assay with the existing conventional RT-PCR and also provides good-quality DNA suitable for sequencing in order to identify circulating lineages. The assay was evaluated using cell culture propagated PPRVs, field samples from clinically infected animals and samples from experimentally infected animals encompassing all four lineages (I–IV) of PPRV. This assay provides a solution with an easy, accurate, rapid and cost-effective PPR diagnostic and partial genome sequencing for use in resource-limited settings.