Dengue: A review of laboratory diagnostics in the vaccine age.

Background. Dengue is an important arboviral infection of considerable public health significance. It occurs in a wide global belt within a variety of tropical regions. The timely laboratory diagnosis of Dengue infection is critical to inform both clinical management and an appropriate public health response. Vaccination against Dengue virus is being introduced in some areas.Discussion. Appropriate diagnostic strategies will vary between laboratories depending on the available resources and skills. Diagnostic methods available include viral culture, the serological detection of Dengue-specific antibodies in using enzyme immunoassays (EIAs), microsphere immunoassays, haemagglutination inhibition or in lateral flow point of care tests. The results of antibody tests may be influenced by prior vaccination and exposure to other flaviviruses. The detection of non-structural protein 1 in serum (NS1) has improved the early diagnosis of Dengue and is available in point-of-care assays in addition to EIAs. Direct detection of viral RNA from blood by PCR is more sensitive than NS1 antigen detection but requires molecular skills and resources. An increasing variety of isothermal nucleic acid detection methods are in development. Timing of specimen collection and choice of test is critical to optimize diagnostic accuracy. Metagenomics and the direct detection by sequencing of viral RNA from blood offers the ability to rapidly type isolates for epidemiologic purposes.Conclusion. The impact of vaccination on immune response must be recognized as it will impact test interpretation and diagnostic algorithms.

Driving consistency: CEPI-Centralized Laboratory Network's conversion factor initiative for SARS-CoV-2 clinical assays used for efficacy assessment of COVID vaccines.

To date, thousands of SARS-CoV-2 samples from many vaccine developers have been tested within the CEPI-Centralized Laboratory Network. To convert data from each clinical assay to international standard units, the WHO international standard and the CEPI standard generated by the Medicines and Healthcare products Regulatory Agency were run in multiple facilities to determine the conversion factor for each assay. Reporting results in international units advances global understanding of SARS-CoV-2 immunity and vaccine efficacy, enhancing the quality, reliability, and utility of clinical assay data.

ADLM Guidance Document on Laboratory Diagnosis of Respiratory Viruses.

Respiratory viral infections are among the most frequent infections experienced worldwide. The COVID-19 pandemic has highlighted the need for testing and currently several tests are available for the detection of a wide range of viruses. These tests vary widely in terms of the number of viral pathogens included, viral markers targeted, regulatory status, and turnaround time to results, as well as their analytical and clinical performance. Given these many variables, selection and interpretation of testing requires thoughtful consideration. The current guidance document is the authors' expert opinion based on the preponderance of available evidence to address key questions related to best practices for laboratory diagnosis of respiratory viral infections including who to test, when to test, and what tests to use. An algorithm is proposed to help laboratories decide on the most appropriate tests to use for the diagnosis of respiratory viral infections.

Monkeypox Diagnosis in Clinical Settings: A Comprehensive Review of Best Laboratory Practices.

An outbreak of monkeypox (Mpox) was reported in more than 40 countries in early 2022. Accurate diagnosis of Mpox can be challenging, but history, clinical findings, and laboratory diagnosis can establish the diagnosis. The pre-analytic phase of testing includes collecting, storing, and transporting specimens. It is advised to swab the lesion site with virus transport medium (VTM) containing Dacron or polyester flock swabs from two different sites. Blood, urine, and semen samples may also be used. Timely sampling is necessary to obtain a sufficient amount of virus or antibodies. The analytical phase of infectious disease control involves diagnostic tools to determine the presence of the virus. While polymerase chain reaction (PCR) is the gold standard for detecting Mpox, genome sequencing is for identifying new or modified viruses. As a complement to these methods, isothermal amplification methods have been designed. ELISA assays are also available for the determination of antibodies. Electron microscopy is another effective diagnostic method for tissue identification of the virus. Wastewater fingerprinting provides some of the most effective diagnostic methods for virus identification at the community level. The advantages and disadvantages of these methods are further discussed. Post-analytic phase requires proper interpretation of test results and the preparation of accurate patient reports that include relevant medical history, clinical guidelines, and recommendations for follow-up testing or treatment.

Analytical and Clinical Performance of the NeuMoDx™ Platform for Cytomegalovirus and Epstein-Barr Virus Viral Load Testing.

DNA assays for viral load (VL) monitoring are key tools in the management of immunocompromised patients with cytomegalovirus (CMV) or Epstein-Barr virus (EBV) infection. In this study, the analytical and clinical performances of the NeuMoDx™ CMV and EBV Quant Assays were compared with artus CMV and EBV QS-RGQ Kits in a primary hospital testing laboratory. Patient plasma samples previously tested using artus kits were randomly selected for testing by NeuMoDx assays. The NeuMoDx CMV Quant Assay and artus CMV QS-RGQ Kit limits of detection (LoDs) are 20.0 IU/mL and 69.7 IU/mL, respectively; 33/75 (44.0%) samples had CMV DNA levels above the LoD of both assays. The Pearson correlation coefficient was 0.9503; 20 samples (60.6%) had lower NeuMoDx CMV quantification values versus the artus kit. The LoD of the NeuMoDx EBV Quant Assay and artus EBV QS-RGQ Kit are 200 IU/mL and 22.29 IU/mL, respectively; 16/75 (21.3%) samples had EBV DNA levels above the LoD of both assays. The Pearson correlation coefficient was 0.8990. EBV quantification values with the NeuMoDx assay were higher versus the artus kit in 15 samples (93.8%). In conclusion, NeuMoDx CMV and EBV Quant Assays are sensitive and accurate tools for CMV and EBV DNA VL quantification.

COVID-19 surveillance in a bone marrow transplantation unit: experience from a Brazilian tertiary-care teaching hospital.

PURPOSE: In this work, we aimed to describe the strategy of the weekly SARS-CoV-2 RT-PCR surveillance program that was implemented in our bone marrow transplantation (BMT) unit. METHODS: Our unit performed SARS-CoV-2 RT-PCR before admission and then weekly during hospitalization even if the patient was asymptomatic. From May 2021 to May 2022, we collected data from all patients that were admitted in the BMT unit to perform transplantation. The total of SARS-CoV-2 RT-PCR performed and the positive rate were described. RESULTS: During the study period, 65 patients were admitted for HSCT. A total of 414 SARS-CoV-2 RT-PCR were performed. Two cases were detected (positivity rate, 0.48%). After the positive test, both patients were isolated outside the BMT unit. CONCLUSION: We postulate that diagnosing these patients and isolating them outside the transplantation unit may have prevented secondary symptomatic cases.

Overview of Modern Commercial Kits for Laboratory Diagnosis of African Swine Fever and Swine Influenza A Viruses.

Rapid and early detection of infectious diseases in pigs is important, especially for the implementation of control measures in suspected cases of African swine fever (ASF), as an effective and safe vaccine is not yet available in most of the affected countries. Additionally, analysis for swine influenza is of significance due to its high morbidity rate (up to 100%) despite a lower mortality rate compared to ASF. The wide distribution of swine influenza A virus (SwIAV) across various countries, the emergence of constantly new recombinant strains, and the danger of human infection underscore the need for rapid and accurate diagnosis. Several diagnostic approaches and commercial methods should be applied depending on the scenario, type of sample and the objective of the studies being implemented. At the early diagnosis of an outbreak, virus genome detection using a variety of PCR assays proves to be the most sensitive and specific technique. As the disease evolves, serology gains diagnostic value, as specific antibodies appear later in the course of the disease (after 7-10 days post-infection (DPI) for ASF and between 10-21 DPI for SwIAV). The ongoing development of commercial kits with enhanced sensitivity and specificity is evident. This review aims to analyse recent advances and current commercial kits utilised for the diagnosis of ASF and SwIAV.

Inappropriate laboratory test utilization in outpatient tertiary care: Implications for value-based healthcare.

OBJECTIVES: To assess the rate of inappropriate repetition of laboratory testing and estimate the cost of such testing for thyroid stimulating hormone (TSH), total cholesterol, vitamin D, and vitamin B12 tests. METHODS: A retrospective cohort study was carried out in the Family Medicine and Polyclinic Department at King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia. Clinical and laboratory data were collected between 2018-2021 for the 4 laboratory tests. The inappropriate repetition of tests was defined according to international guidelines and the costs were calculated using the hospital prices. RESULTS: A total of 109,929 laboratory tests carried out on 23,280 patients were included in this study. The percentage of inappropriate tests, as per the study criteria, was estimated to be 6.1% of all repeated tests. Additionally, the estimated total cost wasted amounted to 2,364,410 Saudi Riyals. Age exhibited a weak positive correlation with the total number of inappropriate tests (r=0.196, p=0.001). Furthermore, significant differences were observed in the medians of the total number of inappropriate tests among genders and nationalities (p<0.001). CONCLUSION: The study identified significantly high rates of inadequate repetitions of frequently requested laboratory tests. Urgent action is therefore crucial to overcoming such an issue.

[Laboratory diagnostics for vasculitis beyond antineutrophil cytoplasmatic autoantibodies]. / Labordiagnostik bei Vaskulitiden jenseits von antineutrophilen zytoplasmatischen Autoantikörpern.

The diagnosis of systemic vasculitis (SV) is a major clinical challenge due to the very different forms of presentation and requires an interdisciplinary approach. Targeted laboratory diagnostics support making the diagnosis, differential diagnosis and classification and are also a key component in the detection of active organ manifestations and treatment complications. The basic laboratory tests include the erythrocyte sedimentation rate (ESR), C­reactive protein (CRP), blood count, serum creatinine, urinalysis, specific autoantibodies, complement, immunoglobulins, cryoglobulins and hepatitis B and C serology. Antineutrophil cytoplasmic autoantibodies (ANCA), antiglomerular basement membrane antibodies (anti-GBM antibodies) and anti-C1q antibodies are valuable laboratory markers for the diagnosis of the various forms of small vessel vasculitis. There are no specific laboratory tests for the diagnosis of medium and large vessel vasculitis. Despite advances in our understanding of the pathogenesis of vasculitis, no biomarkers have yet been identified that can be reliably used to guide treatment or that are useful in distinguishing vasculitis from other inflammatory diseases such as infections or treatment complications.

Correlación entre los índices estandarizados SUVmáx de la PET/TC con [68Ga]Ga-PSMA-I&T pre-prostatectomía radical y los resultados histopatológicos finales: un análisis en profundidad / Correlation between pre-radical prostatectomy Standardized SUVmax ratios detected on 68Ga-PSMA-I&T PET/CT and final histopathology outcomes: An in-depth analysis

Objetivo: Evaluar el potencial predictivo del valor máximo de captación estandarizada (SUVmáx) de los tumores intraprostáticos obtenidos en la PET/TC preoperatoria con [68Ga]Ga-PSMA-I&T (SUVT), así como sus relaciones con el SUVmáx en el hígado (SUVTLR) y la glándula parótida (SUVTPR) con respecto a los hallazgos histopatológicos. Material y métodos: Se evaluaron los datos de pacientes sometidos a prostatectomía radical (PR) por cáncer de próstata (CaP) en nuestra clínica entre los años 2017-2020. Se excluyeron aquellos pacientes con una neoplasia maligna secundaria, antecedentes de resección transuretral de próstata, tratamiento previo para CaP o que fueron sometidos a una PR de rescate. Dos especialistas en medicina nuclear con más de una década de experiencia cada uno revisaron las imágenes del estudio de cuerpo completo obtenidas con el mismo equipo, según protocolo, para obtener el consenso en cada lesión. Se estudiaron las relaciones entre edad, antígeno específico de la próstata (PSA), volumen de la próstata, estadio clínico, el grado de la clasificación de la Sociedad Internacional de Anatomía Patología Urológica (ISUP, por sus siglas en inglés) en la biopsia, el grupo de riesgo de D’amico, el volumen tumoral intraprostático identificado en la revisión histopatológica final de la muestra (HPTV) y el grado HP-ISUP. Se analizó la invasión de vesículas seminales (SVI), la invasión extracapsular (ECI), el margen quirúrgico positivo (PSM), SUVT, SUVTLR y SUVTPR. Resultados: La edad media de los 64 pacientes incluidos fue de 64,1±5,3 años. Se observó una correlación estadísticamente significativa entre los valores de SUVT, SUVTLR, SUVTPR y los parámetros del estadio histopatológico, como el grado ISUP de la biopsia, la clasificación de riesgo D’amico, HP-ISUP, HPTV (p<0,05). PSMATV, SUVT y SUVTLR fueron predictores estadísticamente significativos de invasión extracapsular, mientras que PSA, PSMATV y SUVTLR fueron predictores significativos de SVI (p<0,05)... (AU)

Objective: To evaluate the predictive potential of the maximum standardized uptake value (SUVmax) value of intraprostatic tumors derived from preoperative [68Ga]Ga-PSMA-I&T PET/CT (SUVT), and its ratios to SUVmax in the liver (SUVTLR) and parotid gland (SUVTPR) with respect to histopathological findings. Materials and methods: Data from patients who underwent radical prostatectomy (RP) for prostate cancer (PC) at our clinic between 2017-2020 were assessed. Patients with a secondary malignancy, a history of transurethral prostate resection, prior treatment for PC, or who received salvage RP were excluded. Whole-body images obtained using the same device, as per the guidelines, were reviewed by two nuclear medicine specialists with more than a decade of experience to reach a consensus for each lesion. The relationships between age, PSA, Prostate Volume, clinical T stage, biopsy International Society of Urological Pathology grade (ISUP), D’amico risk group, intraprostatic tumor volume (HPTV) identified in the final histopathological specimen review, HP-ISUP grade, seminal vesicle invasion (SVI), extracapsular invasion (ECI), positive surgical margine (PSM), SUVT, SUVTLR, and SUVTPR were analyzed. Results: The mean age of the 64 included patients was 64.1±5.3 y-o. A statistically significant correlation was found between SUVT, SUVTLR, SUVTPR values, and histopathologic stage parameters, such as biopsy ISUP, D’amico Risk Classification, HP-ISUP, HPTV (P<.05). PSMATV, SUVT, and SUVTLR were statistically significant predictors of extracapsular invasion, while PSA, PSMATV, and SUVTLR were significant predictors of SVI (P<.05). Conclusion: The standardized SUVT, SUVTLR, and SUVTPR values could be employed as noninvasive markers to assist in predicting postoperative histopathological findings, particularly ECI, SVI, and PSM. (AU)

Clinical validation of RCSMS: A rapid and sensitive CRISPR-Cas12a test for the molecular detection of SARS-CoV-2 from saliva.

Peru's holds the highest COVID death rate per capita worldwide. Key to this outcome is the lack of robust, rapid, and accurate molecular tests to circumvent the elevated costs and logistics of SARS-CoV-2 detection via RT-qPCR. To facilitate massive and timely COVID-19 testing in rural and socioeconomically deprived contexts, we implemented and validated RCSMS, a rapid and sensitive CRISPR-Cas12a test for the molecular detection of SARS-CoV-2 from saliva. RCSMS uses the power of CRISPR-Cas technology and lateral flow strips to easily visualize the presence of SARS-CoV-2 even in laboratories with limited equipment. We show that a low-cost thermochemical treatment with TCEP/EDTA is sufficient to inactivate viral particles and cellular nucleases in saliva, eliminating the need to extract viral RNA with commercial kits, as well as the cumbersome nasopharyngeal swab procedure and the requirement of biosafety level 2 laboratories for molecular analyses. Notably, RCSMS performed outstandingly in a clinical validation done with 352 patients from two hospitals in Lima, detecting as low as 50 viral copies per 10 µl reaction in 40 min, with sensitivity and specificity of 96.5% and 99.0%, respectively, relative to RT-qPCR. The negative and positive predicted values obtained from this field validation indicate that RCSMS can be confidently deployed in both high and low prevalence settings. Like other CRISPR-Cas-based biosensors, RCSMS can be easily reprogrammed for the detection of new SARS-CoV-2 variants. We conclude that RCSMS is a fast, efficient and inexpensive alternative to RT-qPCR for expanding COVID-19 testing capacity in Peru and other low- and middle-income countries with precarious healthcare systems.

Evaluation of a rapid Loop Mediated Isothermal Amplification (LAMP) test for the laboratory diagnosis of sexually transmitted infections.

Sexually transmitted infections (STIs) have seen a considerable increase in the last years and given the health burden they may represent from both a personal and community perspective, they require surveillance and prevention programmes based on a timely and decentralized diagnosis. In this context, user-friendly rapid molecular tests may represent a good trade-off between diagnostic accuracy, accessibility and affordability. In this study we evaluated the diagnostic performance of a new real-time LAMP (Loop Mediated Isothermal Amplification) method for the rapid detection and differentiation of 7 major sexually transmissible pathogens by analysing real clinical samples (genital and extra-genital matrices) from individuals with suspected STIs. The assay showed good overall diagnostic performances in terms of sensitivity, specificity and concordance with a gold-standard PCR-based molecular method. This assay, not requiring specialised laboratory technicians or expensive instrumentation, but nonetheless capable of guaranteeing accurate results, is within the reach of outpatient settings, obstetrics, and gynaecology clinic, hence ensuring on-field access to early diagnosis.

Improved heat shock method for extracting total RNA from nasopharyngeal swab samples even with low viral load.

This study aimed to improve the heat shock method as a cost-effective and time-efficient for total RNA extraction. We compared the effectiveness of two total RNA extraction methods by using Real-Time PCR for nasopharynx swabs. Include: I; use of a commercial total RNA extraction kit as a standard. II; utilized a modified heat shock method (MHS). Time, centrifuge speed and duration, proteinase K, and RNA carrier were optimized. The optimized parameters included treating the sample with 5 µg/µL at 56°C for 5 minutes, heating at 95°C for 5 minutes followed by thermal shock in ice for 3 minutes, adding 4 µg/µL RNA carrier at room temperature for 3 minutes, and centrifuging at 7000 rpm for 10 minutes. This optimization demonstrated a sensitivity and specificity of 100% (CI: 95%) even in samples with low viral load. Our in-house method presents a rapid, and cost-effective alternative for total RNA extraction.

A Generalizable Decision-Making Framework for Selecting Onsite versus Send-out Clinical Laboratory Testing.

BACKGROUND: Laboratory networks provide services through onsite testing or through specimen transport to higher-tier laboratories. This decision is based on the interplay of testing characteristics, treatment characteristics, and epidemiological characteristics. OBJECTIVES: Our objective was to develop a generalizable model using the threshold approach to medical decision making to inform test placement decisions. METHODS: We developed a decision model to compare the incremental utility of onsite versus send-out testing for clinical purposes. We then performed Monte Carlo simulations to identify the settings under which each strategy would be preferred. Tuberculosis was modeled as an exemplar. RESULTS: The most important determinants of the decision to test onsite versus send-out were the clinical utility lost due to send-out testing delays and the accuracy decrement with onsite testing. When the sensitivity decrements of onsite testing were minimal, onsite testing tended to be preferred when send-out delays reduced clinical utility by >20%. By contrast, when onsite testing incurred large reductions in sensitivity, onsite testing tended to be preferred when utility lost due to delays was >50%. The relative cost of onsite versus send-out testing affected these thresholds, particularly when testing costs were >10% of treatment costs. CONCLUSIONS: Decision makers can select onsite versus send-out testing in an evidence-based fashion using estimates of the percentage of clinical utility lost due to send-out delays and the relative accuracy of onsite versus send-out testing. This model is designed to be generalizable to a wide variety of use cases. HIGHLIGHTS: The design of laboratory networks, including the decision to place diagnostic instruments at the point-of-care or at higher tiers as accessed through specimen transport, can be informed using the threshold approach to medical decision making.The most important determinants of the decision to test onsite versus send-out were the clinical utility lost due to send-out testing delays and the accuracy decrement with onsite testing.The threshold approach to medical decision making can be used to compare point-of-care testing accuracy decrements with the lost utility of treatment due to send-out testing delays.The relative cost of onsite versus send-out testing affected these thresholds, particularly when testing costs were >10% of treatment costs.

Behavioral Intention of Receiving Monkeypox Vaccination and Undergoing Monkeypox Testing and the Associated Factors Among Young Men Who Have Sex With Men in China: Large Cross-Sectional Study.

BACKGROUND: The worldwide human monkeypox (mpox) outbreak in 2022 mainly affected men who have sex with men (MSM). In China, young men who have sex with men (YMSM) were at a potential high risk of mpox infection due to their sexual activeness and the eased COVID-19 restrictions at the end of 2022. OBJECTIVE: This study aimed to investigate the behavioral intention of receiving mpox vaccination and undergoing mpox testing in 4 different scenarios and explore their associations with background and behavioral theory-related factors among Chinese YMSM. METHODS: An online cross-sectional survey was conducted among YMSM aged 18-29 years from 6 representative provinces of China in September 2022. Participants recruited (recruitment rate=2918/4342, 67.2%) were asked to self-administer an anonymous questionnaire designed based on prior knowledge about mpox and classic health behavior theories. Data on the participants' background, mpox knowledge and cognition, mpox vaccination and testing cognition, and the behavioral intention of receiving mpox vaccination and undergoing mpox testing were collected. Descriptive analysis and univariate and multivariate linear regressions were performed. Geodetector was used to measure the stratified heterogeneity of behavioral intention. RESULTS: A total of 2493 YMSM with a mean age of 24.6 (SD 2.9) years were included. The prevalence of having a behavioral intention of receiving mpox vaccination ranged from 66.2% to 88.4% by scenario, varying in epidemic status and cost. The prevalence of having an mpox testing intention was above 90% in all scenarios regardless of the presence of symptoms and the cost. The positive factors related to vaccination intention included mpox knowledge (ba=0.060, 95% CI 0.016-0.103), perceived susceptibility of mpox (ba=0.091, 95% CI 0.035-0.146), perceived severity of mpox (ba=0.230, 95% CI 0.164-0.296), emotional distress caused by mpox (ba=0.270, 95% CI 0.160-0.380), perceived benefits of mpox vaccination (ba=0.455, 95% CI 0.411-0.498), self-efficacy of mpox vaccination (ba=0.586, 95% CI 0.504-0.668), and having 1 male sex partner (ba=0.452, 95% CI 0.098-0.806), while the negative factor was perceived barriers to vaccination (ba=-0.056, 95% CI -0.090 to -0.022). The positive factors related to testing intention were perceived severity of mpox (ba=0.283, 95% CI 0.241-0.325), perceived benefits of mpox testing (ba=0.679, 95% CI 0.636-0.721), self-efficacy of mpox testing (ba=0.195, 95% CI 0.146-0.245), having 1 male sex partner (ba=0.290, 95% CI 0.070-0.510), and having in-person gatherings with MSM (ba=0.219, 95% CI 0.072-0.366), while the negative factor was emotional distress caused by mpox (ba=-0.069, 95% CI -0.137 to -0.001). CONCLUSIONS: Among Chinese YMSM, the intention of undergoing mpox testing is optimal, while the mpox vaccination intention has room for improvement. A future national response should raise YMSM's mpox knowledge, disseminate updated information about mpox and preventive measures, improve preventive service accessibility and privacy, and provide advice on positively coping with the associated emotional distress.

Clinical and laboratory diagnosis of Mayaro virus (MAYV): Current status and opportunities for further development.

The recent outbreaks related to Mayaro virus (MAYV) infection in the Americas have brought this neglected virus as a potential threat to global public health. Given the range of symptoms that can be associated with MAYV infection, it can be challenging to diagnose individuals based on clinical signs, especially in countries with simultaneous circulation of other mosquito-borne viruses, such as dengue virus (DENV) and chikungunya virus (CHIKV). With this challenge in mind, laboratory-based diagnosis assumes a critical role in the introduction of measures to help prevent virus dissemination and to adequately treat patients. In this review, we provide an overview of the clinical features reported in infected patients and currently available laboratory tools that are used for MAYV diagnosis, discussing their advances, advantages, and limitations to apply in the field. Moreover, we explore novel point-of-care (PoC) diagnostic platforms that can provide de-centralised diagnostics for use in areas with limited laboratory infrastructure.