A rapid influenza diagnostic test based on detection of viral neuraminidase activity.

Current methods used for diagnosis of acute infection of pathogens rely on detection of nucleic acids, antigens, or certain classes of antibodies such as IgM. Here we report a virus enzyme assay as an alternative to these methods for detection of acute viral infection. In this method, we used a luciferin derivative as the substrate for detection of the enzyme activity of influenza viral neuraminidase as a means for diagnosis of influenza. The resulting commercial test, the qFLU Dx Test, uses a different supply chain that does not compete with those for the current tests. The assay reagents were formulated as a master mix that accommodated both the neuraminidase and luciferase reactions, thereby enabling rapid and prolonged production of stable light signal in the presence of influenza virus in the sample. The assay was evaluated using depository throat swab specimens. As expected, the assay exhibited similar detection rates for all influenza types and subtypes except for A(H7N9), which exhibited lower detection rate due to lower viral titer in the specimens. When throat swab specimens were diluted with the sample buffer of the test kit and tested with the qFLU Dx Test. The sensitivity and specificity were 82.41% (95% confidence interval: 79.66-85.84%) and 95.39% (95% confidence interval: 94.32-96.46%), respectively, for these diluted specimens in comparison to a real-time polymerase chain reaction assay. The uniqueness of the qFLU Dx Test as an enzymatic assay makes it highly complementary with currently available methods.

Evaluation of the PrioCHECK™ Trichinella AAD kit to detect Trichinella spiralis, T. britovi, and T. pseudospiralis larvae in pork using the automated digestion method Trichomatic-35.

Trichinellosis is a potentially deadly parasitic zoonosis that is contracted by consuming undercooked infected meat. Reliable detection of infectious Trichinella spp. larvae in meat is therefore pivotal to ensure consumer's safety. The recently authorised PrioCHECK™ Trichinella Alternative Artificial Digestion (AAD) test kit appears promising when used with the standard magnetic stirrer method, but evaluation with other apparatus types is lacking. In this study, the performance of the AAD kit in an adapted Trichomatic-35 (TM35) instrument was evaluated, first, at the Swiss National Reference Laboratory for trichinellosis (NRL); second, in a ring trial involving four Swiss official laboratories. Proficiency pork samples spiked with larvae of Trichinella spiralis, T. britovi, or T. pseudospiralis were tested with the AAD kit and with the reference pepsin-HCl digestion method in TM35 instruments. At the NRL, both methods yielded identical qualitative and similar quantitative results independently of the Trichinella species. In the ring trial, satisfactory results were obtained for 47/50 (94.0%) (AAD) and 62/67 (92.5%) (reference method) of the analysed samples. Technical problems impairing analysis were more frequently observed with the AAD kit (n = 22) than with the reference method (n = 5) and were mainly (16/22) reported by one of the external labs. When no technical issues were recorded, the performance of both methods was comparable, in agreement with the observations at the NRL; however, these results suggest a need for further training with the kit and standardisation of the adapted TM35 instruments.

Lateral flow devices for samples collected by straw sampling method for postmortem canine rabies diagnosis.

The direct fluorescent antibody test (dFAT) using brain sample after opening the skull is the standard rabies diagnostic test in animal rabies. However, it is not feasible in many resource-limited settings. Lateral flow devices (LFD) combined with a simple sampling methodology is quicker, simpler, and less hazardous than the standard test and can be a useful tool. We conducted a prospective on-site study to evaluate the diagnostic accuracy of the LFD with the straw sampling method compared with that of the dFAT with the skull opening procedure for post-mortem canine rabies diagnosis. We collected 97 rabies-suspected animals between December 1, 2020 and March 31, 2021. Among the 97 samples, 53 and 50 cases were positive tests for dFAT and LFD, respectively. The sensitivity and specificity of LFD with straw sampling method were 94.3% (95% confidence interval [CI], 84.3-98.8%) and 100% (95% CI, 92.0-100%), respectively. The performance of LFD by the straw sampling method showed relatively high sensitivity and 100% specificity compared with that of dFAT performed on samples collected after opening the skull. This methodology can be beneficial and is a strong tool to overcome limited animal surveillance in remote areas. However, because of our limited sample size, more data using fresh samples on-site and the optimizations are urgently needed for the further implementation in endemic areas.

Validation of Selective Agars for Detection and Quantification of Escherichia coli Strains Resistant to Critically Important Antimicrobials.

Success in the global fight against antimicrobial resistance (AMR) is likely to improve if surveillance can be performed on an epidemiological scale. An approach based on agars with incorporated antimicrobials has enormous potential to achieve this. However, there is a need to identify the combinations of selective agars and key antimicrobials yielding the most accurate counts of susceptible and resistant organisms. A series of experiments involving 1,202 plates identified the best candidate combinations from six commercially available agars and five antimicrobials, using 18 Escherichia coli strains as either pure cultures or inocula-spiked feces. The effects of various design factors on colony counts were analyzed in generalized linear models. Without antimicrobials, Brilliance E. coli and CHROMagar ECC agars yielded 28.9% and 23.5% more colonies, respectively, than MacConkey agar. The order of superiority of agars remained unchanged when fecal samples with or without spiking of resistant E. coli strains were inoculated onto agars with or without specific antimicrobials. When antimicrobials were incorporated at various concentrations, it was revealed that ampicillin, tetracycline, and ciprofloxacin were suitable for incorporation into Brilliance and CHROMagar agars at all defined concentrations. Gentamicin was suitable for incorporation only at 8 and 16 µg/ml, while ceftiofur was suitable only at 1 µg/ml. CHROMagar extended-spectrum ß-lactamase (ESBL) agar supported growth of a wider diversity of extended-spectrum-cephalosporin-resistant E. coli strains. The findings demonstrate the potential for agars with incorporated antimicrobials to be combined with laboratory-based robotics to deliver AMR surveillance on a vast scale with greater sensitivity of detection and strategic relevance. IMPORTANCE Established models of surveillance for AMR in livestock typically have a low sampling intensity, which creates a tremendous barrier to understanding the variation of resistance among animal and food enterprises. However, developments in laboratory robotics now make it possible to rapidly and affordably process large volumes of samples. Combined with modern selective agars incorporating antimicrobials, this forms the basis of a novel surveillance process for identifying resistant bacteria by chromogenic reactions, including accurately detecting and quantifying the presence of bacteria even when they are present at low concentrations. Because Escherichia coli is a widely preferred indicator bacterium for AMR surveillance, this study identifies the optimal selective agar for quantifying resistant E. coli strains by assessing the growth performance on agars with antimicrobials. The findings are the first step toward exploiting laboratory robotics in an up-scaled approach to AMR surveillance in livestock, with wider adaptations in food, clinical microbiology, and public health.

Discordant results between genotypic and phenotypic assays (Xpert MTB/RIF vs. BACTEC MGIT 960 system) for detection of RIF-resistant Mycobacterium tuberculosis isolates in a high burden region.

Clinical isolates with discordant phenotypic and genotypic results were submitted to DNA sequencing to identify which were genuinely resistant to rifampin and determine the frequency of silent and disputed mutations in our region. We present the retrospective analysis of all the culture-proven TB cases tested with the Xpert®MTB/RIF assay at the Tuberculosis Clinic and Laboratory of the Tijuana General Hospital, Mexico. Clinical isolates showing a discrepancy between phenotypic and molecular tests were analyzed by DNA sequencing. Thirteen isolates tested as rifampin susceptible on the MGIT system were rifampin-resistant according to Xpert®MTB/RIF assay. DNA sequencing showed that seven (53.8%) isolates had a silent (P514P) mutation; three isolates showed different missense (L511P, D516Y, and S531L) mutations. Three isolates showed no mutations. The existence of heteroresistance and silent or disputed mutations warrants that all rifampin-resistance cases diagnosed with the Xpert®MTB/RIF should be referred to specialized centers for DNA sequencing.

On-farm colorimetric detection of Pasteurella multocida, Mannheimia haemolytica, and Histophilus somni in crude bovine nasal samples.

This work modifies a loop-mediated isothermal amplification (LAMP) assay to detect the bovine respiratory disease (BRD) bacterial pathogens Pasteurella multocida, Mannheimia haemolytica, and Histophilus somni in a colorimetric format on a farm. BRD causes a significant health and economic burden worldwide that partially stems from the challenges involved in determining the pathogens causing the disease. Methods such as polymerase chain reaction (PCR) have the potential to identify the causative pathogens but require lab equipment and extensive sample processing making the process lengthy and expensive. To combat this limitation, LAMP allows accurate pathogen detection in unprocessed samples by the naked eye allowing for potentially faster and more precise diagnostics on the farm. The assay developed here offers 66.7-100% analytical sensitivity, and 100% analytical specificity (using contrived samples) while providing 60-100% concordance with PCR results when tested on five steers in a feedlot. The use of a consumer-grade water bath enabled on-farm execution by collecting a nasal swab from cattle and provided a colorimetric result within 60 min. Such an assay holds the potential to provide rapid pen-side diagnostics to cattle producers and veterinarians.

Evaluating the diagnostic test accuracy of molecular xenomonitoring methods for characterising the community burden of Onchocerciasis.

BACKGROUND: Molecular xenomonitoring (MX), the detection of parasite nucleic acid in the vector population, is recommended for onchocerciasis surveillance in elimination settings. However, the sensitivity of MX for detecting onchocerciasis-positive communities has not previously been evaluated. MX may have additional applications for control programmes but its utility is restricted by a limited understanding of the relationship between MX results and human prevalence. METHODS: We conducted a systematic review of studies reporting the prevalence of Onchocerca volvulus DNA in wild-caught Simulium spp. flies (MX rate) and corresponding prevalence of microfilaria (mf) in humans. We evaluated the sensitivity of MX for detecting onchocerciasis-positive communities and describe the characteristics of studies with reduced sensitivity. We conducted a linear regression to evaluate the relationship between mf prevalence and MX rate. RESULTS: We identified 15 relevant studies, with 13 studies comprising 34 study communities included in the quantitative analyses. Most communities were at advanced stages towards elimination and had no or extremely low human prevalence. MX detected positive flies in every study area with >1% mf prevalence, with the exception of one study conducted in the Venezuelan Amazonian focus. We identified a significant relationship between the two measurements, with mf prevalence accounting for half of the variation in MX rate (R2 0.50, p<0.001). CONCLUSION: MX is sensitive to communities with ongoing onchocerciasis transmission. It has potential to predict human mf prevalence, but further data is required to understand this relationship, particularly from MX surveys conducted earlier in control programmes before transmission has been interrupted.

Clinical utility of Abbott Alinity hq extended red blood cell parameters in differentiating ß-thalassemia trait and iron deficiency anemia.

INTRODUCTION: The objective of the study was to evaluate the performance of the Abbott Alinity hq advanced multi angle polarized scatter separation (MAPSSTM )-based optical RBC technology, for the differentiation between iron deficiency anemia (IDA) and ß-thalassemia carrier status. METHODS: Four hundred and sixty-four samples were analyzed. 228 were healthy controls, 30 were ß-thalassemia carriers, and 40 were IDA. Receiver operating characteristics analysis evaluated the performance of red cell parameters and mathematical formulas. RESULTS: RBC concentration was the most efficient discriminant (area under the curve; AUC of 0.963, Youden Index of 0.88) followed by red blood cell distribution width in size distribution (AUC of 0.960 and YI of 0.86), and red blood cell distribution width coefficient of variation (AUC of 0.924, and YI of 0.74). The absolute reticulocyte concentration showed good diagnostic efficiency, with AUC of 0.808. Hemoglobin distribution width, the %CV of directly measured cellular hemoglobin concentration, and CHCr, the average hemoglobin concentration of reticulocytes have emerged as novel discriminating parameters, with AUC of 0.749 and 0.785, respectively. The England and Fraser index was the best discriminating mathematical formula based on Youden Index of 0.91. The Ricerca, red blood cell distribution width index, Green and King, and Mentzner Index formulas also showed strong discriminative power. The Shine and Lal index, together with the recent mathematical formula M/H, (ratio of percent microcytic and hypochromic red blood cells) demonstrated moderate performance with AUC of 0.796 and 0.740, respectively. CONCLUSION: Extended red cell analysis delivered by the advanced optical technology on the Alinity hq hematology analyzer has efficient diagnostic utility in the initial discrimination of the two most common microcytic anemias: IDA and ß-thalassemia trait.

Evaluating the interchangeability of infrared and digital devices with the traditional mercury thermometer in hospitalized pediatric patients: an observational study.

Gradual replacement of the mercury thermometers with alternative devices is ongoing around the world in a bid to protect human health and the environment from the adverse effects of mercury. However, to reduce the risks of misdiagnosis, unnecessary treatments, and omission of care in pediatric populations, more evidence on the reliability of alternative thermometers is needed. The aim of this comparative observational study was to detect any differences in temperature measurements between the use of the axillary mercury thermometer and the alternative techniques. Temperature values in degree Celsius (°C) were measured in a group of Albanian children aged up to 14 years using mercury and digital axillary thermometers, as well as forehead and tympanic infrared thermometers. The digital axillary device, compared with the mercury one, showed no clinically significant difference in the mean values (- 0.04 ± 0.29 °C) and the narrowest 95% level of agreement (+ 0.53 °C to - 0.62 °C) in the paired comparisons. For cut-off point of 37.5 °C, the digital axillary thermometer showed the highest levels of sensitivity (72.5%) and specificity (99.1%) in detecting fever. This study indicates that the digital axillary thermometer may be the better option since it adequately balances accuracy, safety, and children's comfort.

Saliva Testing Is Accurate for Early-Stage and Presymptomatic COVID-19.

Although nasopharyngeal samples have been considered the gold standard for COVID-19 testing, variability in viral load across different anatomical sites could cause nasopharyngeal samples to be less sensitive than saliva or nasal samples in certain cases. Self-collected samples have logistical advantages over nasopharyngeal samples, making them amenable to population-scale screening. To evaluate sampling alternatives for population screening, we collected nasopharyngeal, saliva, and nasal samples from two cohorts with varied levels and types of symptoms. In a mixed cohort of 60 symptomatic and asymptomatic participants, we found that saliva had 88% concordance with nasopharyngeal samples when tested in the same testing lab (n = 41) and 68% concordance when tested in different testing labs (n = 19). In a second cohort of 20 participants hospitalized for COVID-19, saliva had 74% concordance with nasopharyngeal samples tested in the same testing lab but detected virus in two participants that tested negative with nasopharyngeal samples on the same day. Medical record review showed that the saliva-based testing sensitivity was related to the timing of symptom onset and disease stage. We find that no sample site will be perfectly sensitive for COVID-19 testing in all situations, and the significance of negative results will always need to be determined in the context of clinical signs and symptoms. Saliva retained high clinical sensitivity for early-stage and presymptomatic COVID-19 while allowing easier collection, minimizing the exposure of health care workers, and need for personal protective equipment and making it a viable option for population-scale testing. IMPORTANCE Methods for COVID-19 detection are necessary for public health efforts to monitor the spread of disease. Nasopharyngeal samples have been considered the best approach for COVID-19 testing. However, alternative samples like self-collected saliva offer advantages for population-scale screening. Meta-analyses of recent studies suggest that saliva is useful for detecting SARS-CoV-2; however, differences in disease prevalence, sample collection, and analysis methods still confound strong conclusions on the utility of saliva compared to nasopharyngeal samples. Here, we find that the sensitivity of saliva testing is related to both the timing of the sample collection relative to symptom onset and the disease stage. Importantly, several clinical vignettes in our cohorts highlight the challenges of medical decision making with limited knowledge of the associations between laboratory test data and the natural biology of infection.

International Multicenter Validation Study of the SAGIT® Instrument in Acromegaly.

CONTEXT: The SAGIT® instrument (SAGIT) has been developed to enable accurate characterization of acromegaly disease activity. OBJECTIVE: We evaluated the ability of SAGIT to discriminate acromegaly disease control status. METHODS: This multicenter, noninterventional, prospective and retrospective, longitudinal study, conducted at academic and private clinical practice sites, included patients aged ≥ 18 years with a diagnosis of controlled (n = 109) or non-controlled (n = 105) acromegaly, assessed by clinical global evaluation of disease control (CGE-DC) questionnaire, investigator therapeutic decision, and international guidelines. Control status was not determined at baseline for 13 patients. Since 9 patients were enrolled retrospectively, all presented analyses are based on the prospective population (N = 227). Patients were assessed over a 2-year follow-up period. Classification and regression tree (CART) analyses were performed to investigate how SAGIT components at baseline (signs/symptoms [S], associated comorbidities [A], growth hormone levels [G], insulin-like growth factor 1 levels [I], tumor features [T]) discriminate between controlled and non-controlled acromegaly. RESULTS: Baseline mean subscores S, G, I, and T were significantly lower in patients with CGE-DC controlled vs CGE-DC non-controlled acromegaly. SAGIT components I and G for CGE-DC and S, A, G, I, and T for the clinician's therapeutic decision were retained by CART analyses. For international guidelines, only SAGIT component I was retained. The risk for undergoing ≥ 1 treatment change during the study was 3.44 times greater for CGE-DC non-controlled acromegaly relative to CGE-DC controlled acromegaly. CONCLUSION: The SAGIT instrument is a valid and sensitive tool to comprehensively and accurately assess acromegaly severity.

Effect of pH on the Quantification of Common Chemistry Analytes in Body Fluid Specimens Using the Roche cobas Analyzer for Clinical Diagnostic Testing.

OBJECTIVES: To determine the influence of pH on recovery of analytes in body fluids (BFs), investigate the mechanism of pH interference, measure the frequency of abnormal-pH BFs received, and compare pH measured by meter and paper. METHODS: We performed pH titration in residual BFs. A low-pH BF was spiked and neutralized to investigate pH interference. We measured analytes on a Roche cobas c501 analyzer (Roche Diagnostics) and calculated the percent recovery. Measurement of pH using a meter and paper was conducted on 122 BF samples received in the laboratory. RESULTS: Enzyme activity in BFs was unaffected when pH = 7.4-8.5 lactate dehydrogenase, pH = 7.3-10.2 amylase, pH = 6.0-9.9 lipase, and pH = 1.3-11.7 all other analytes. BFs had mean (range) pH of 8.0 (5.1-8.9), with a mean (range) difference (paper ‒ meter) of ‒0.4 (‒0.6 to 1.1). CONCLUSIONS: Irreversible loss of enzyme activity occurs in BFs at low pH. Few clinical BFs have pH < 7.0, but laboratories should incorporate pH measurement in BF workflows.

An updated RT-qPCR assay for the simultaneous detection and quantification of chikungunya, dengue and zika viruses.

The real-time reverse transcription-polymerase chain reaction (real-time RT-qPCR) has become a leading technique for the detection and quantification of arboviruses, including Chikungunya, Dengue, and Zika viruses. In this study, an updated real-time RT-qPCR assay was designed and evaluated together with a synthetic positive-control chimeric RNA for the simultaneous detection and quantification of Chikungunya, Dengue, and Zika viruses. Amplification assays were performed to verify the construct integrity and optimal reaction/thermal cycling conditions. The analytical sensitivity of the assay was determined for each virus in single and multiplex reactions, as well as the performance in the detection and viral load quantification of experimental samples. The real-time RT-qPCR assay presented here allowed for the simultaneous detection and quantification of Chikungunya, Dengue, and Zika viruses and could be applied in several studies where the accurate quantification of viral genomes is required.

RT-PCR/MALDI-TOF mass spectrometry-based detection of SARS-CoV-2 in saliva specimens.

As severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infections continue, there is a substantial need for cost-effective and large-scale testing that utilizes specimens that can be readily collected from both symptomatic and asymptomatic individuals in various community settings. Although multiple diagnostic methods utilize nasopharyngeal specimens, saliva specimens represent an attractive alternative as they can rapidly and safely be collected from different populations. While saliva has been described as an acceptable clinical matrix for the detection of SARS-CoV-2, evaluations of analytic performance across platforms for this specimen type are limited. Here, we used a novel sensitive RT-PCR/MALDI-TOF mass spectrometry-based assay (Agena MassARRAY®) to detect SARS-CoV-2 in saliva specimens. The platform demonstrated high diagnostic sensitivity and specificity when compared to matched patient upper respiratory specimens. We also evaluated the analytical sensitivity of the platform and determined the limit of detection of the assay to be 1562.5 copies/ml. Furthermore, across the five individual target components of this assay, there was a range in analytic sensitivities for each target with the N2 target being the most sensitive. Overall, this system also demonstrated comparable performance when compared to the detection of SARS-CoV-2 RNA in saliva by the cobas® 6800/8800 SARS-CoV-2 real-time RT-PCR Test (Roche). Together, we demonstrate that saliva represents an appropriate matrix for SARS-CoV-2 detection on the novel Agena system as well as on a conventional real-time RT-PCR assay. We conclude that the MassARRAY® system is a sensitive and reliable platform for SARS-CoV-2 detection in saliva, offering scalable throughput in a large variety of clinical laboratory settings.

Performance Evaluation of Biozentech Malaria Scanner in Plasmodium knowlesi and P. falciparum as a New Diagnostic Tool.

The computer vision diagnostic approach currently generates several malaria diagnostic tools. It enhances the accessible and straightforward diagnostics that necessary for clinics and health centers in malaria-endemic areas. A new computer malaria diagnostics tool called the malaria scanner was used to investigate living malaria parasites with easy sample preparation, fast and user-friendly. The cultured Plasmodium parasites were used to confirm the sensitivity of this technique then compared to fluorescence-activated cell sorting (FACS) analysis and light microscopic examination. The measured percentage of parasitemia by the malaria scanner revealed higher precision than microscopy and was similar to FACS. The coefficients of variation of this technique were 1.2-6.7% for Plasmodium knowlesi and 0.3-4.8% for P. falciparum. It allowed determining parasitemia levels of 0.1% or higher, with coefficient of variation smaller than 10%. In terms of the precision range of parasitemia, both high and low ranges showed similar precision results. Pearson's correlation test was used to evaluate the correlation data coming from all methods. A strong correlation of measured parasitemia (r2=0.99, P<0.05) was observed between each method. The parasitemia analysis using this new diagnostic tool needs technical improvement, particularly in the differentiation of malaria species.

Machine Learning and Intelligent Diagnostics in Dental and Orofacial Pain Management: A Systematic Review.

Purpose: The study explored the clinical influence, effectiveness, limitations, and human comparison outcomes of machine learning in diagnosing (1) dental diseases, (2) periodontal diseases, (3) trauma and neuralgias, (4) cysts and tumors, (5) glandular disorders, and (6) bone and temporomandibular joint as possible causes of dental and orofacial pain. Method: Scopus, PubMed, and Web of Science (all databases) were searched by 2 reviewers until 29th October 2020. Articles were screened and narratively synthesized according to PRISMA-DTA guidelines based on predefined eligibility criteria. Articles that made direct reference test comparisons to human clinicians were evaluated using the MI-CLAIM checklist. The risk of bias was assessed by JBI-DTA critical appraisal, and certainty of the evidence was evaluated using the GRADE approach. Information regarding the quantification method of dental pain and disease, the conditional characteristics of both training and test data cohort in the machine learning, diagnostic outcomes, and diagnostic test comparisons with clinicians, where applicable, were extracted. Results: 34 eligible articles were found for data synthesis, of which 8 articles made direct reference comparisons to human clinicians. 7 papers scored over 13 (out of the evaluated 15 points) in the MI-CLAIM approach with all papers scoring 5+ (out of 7) in JBI-DTA appraisals. GRADE approach revealed serious risks of bias and inconsistencies with most studies containing more positive cases than their true prevalence in order to facilitate machine learning. Patient-perceived symptoms and clinical history were generally found to be less reliable than radiographs or histology for training accurate machine learning models. A low agreement level between clinicians training the models was suggested to have a negative impact on the prediction accuracy. Reference comparisons found nonspecialized clinicians with less than 3 years of experience to be disadvantaged against trained models. Conclusion: Machine learning in dental and orofacial healthcare has shown respectable results in diagnosing diseases with symptomatic pain and with improved future iterations and can be used as a diagnostic aid in the clinics. The current review did not internally analyze the machine learning models and their respective algorithms, nor consider the confounding variables and factors responsible for shaping the orofacial disorders responsible for eliciting pain.

Exploration of an Efficient Simultaneous Molecular Detection Method of HIV, HCV, and Syphilis from a Single Dried Blood Spot.

OBJECTIVE: The aim of the present study was to evaluate the performance of the simultaneous detection of HIV-1 RNA, HIV-1 DNA, and HCV RNA using one dried blood spot (DBS) as an alternative sample to plasma. METHOD: A total of 571 paired DBS/plasma samples were collected from men who have sex with men (MSM) and injection drug users (IDUs), and serological and molecular assays were performed. Using plasma results as the reference standard, the performance of DBS tests for HIV-1 RNA, HIV-1 DNA, and HCV RNA was evaluated. Pearson's correlation coefficients and Bland-Altman analysis were performed to assess the correlation and concordance between DBS and plasma. RESULTS: Among paired plasma/DBS samples with detectable HIV-1 RNA and HCV RNA, five samples (5/32) were not detectable in DBS, while measurable HIV-1 RNA levels were present in plasma (1.44 to 3.99 log 10 copies/mL). There were two samples (2/94) with undetectable HCV RNA in DBS, while measurable HCV RNA levels were present in plasma (-5 to 5.99 log 10 copies/mL). The correlation between HIV-1 RNA light chain variable region (VL) values obtained from plasma and DBS showed that r = 0.683 ( P < 0.01), n = 27 and r = 0.612 ( P < 0.01), n = 89 in HCV RNA. Bland-Altman analysis revealed that in HIV-1 RNA, the mean (± SD) difference between HIV-1 RNA in plasma and DBS was 1.00 ± 1.01 log 10 copies/mL, and all samples were within ± 1.96 SD (-0.97 to 2.97 log 10 copies/mL) for DBS. The mean difference (± SD) in HCV RNA was 0.15 ± 1.08 log 10 copies/mL, and 94.38% (84/89) were within ± 1.96 SD (-1.96 to 2.67 log 10 copies/mL). Overall, HIV-1 RNA and HCV RNA levels obtained from a DBS were lower than those obtained from plasma. HIV-1 DNA in a DBS showed concordant results with HIV-1 RNA in plasma. HIV-1 DNA RT-PCR using a DBS showed acceptable performance. CONCLUSION: The performance of the simultaneous detection of HIV-1 RNA, HIV-1 DNA, and HCV RNA using one DBS was acceptable. DBS, as an alternative sample to plasma, may be a viable option for the simultaneous detection of HIV-1 RNA, HIV-1 DNA, and HCV RNA in resource-limited settings or for individuals living in areas that are difficult to access.

HRP2 and HRP3 cross-reactivity and implications for HRP2-based RDT use in regions with Plasmodium falciparum hrp2 gene deletions.

BACKGROUND: The Plasmodium falciparum antigen histidine rich protein 2 (HRP2) is a preferred target for malaria rapid diagnostic tests (RDTs) because of its abundant production by the parasite and thermal stability. As a result, a majority of RDTs procured globally target this antigen. However, previous reports from South America and recent reports from sub-Saharan Africa and Asia indicate that certain P. falciparum parasites have deletions of the gene coding for HRP2. The HRP2 antigen is paralogous to another P. falciparum antigen HRP3 and some antibodies to HRP2 cross-react with HRP3. Multiple parasites have been described with deletions of one or both hrp2 and hrp3 genes. It is unclear how the various combinations of hrp2 and hrp3 deletion genotypes affect clinical sensitivity of HRP2-based RDTs. METHODS: Cross-reactivity between HRP2 and HRP3 was tested on malaria RDTs using culture-adapted P. falciparum parasites with both hrp2 and hrp3 intact or with one or both genes deleted. Ten-fold serial dilutions of four culture-adapted P. falciparum parasites [3D7 (hrp2+/hrp3+), Dd2 (hrp2-/hrp3+), HB3 (hrp2+/hrp3-) and 3BD5 (hrp2-/hrp3-)] ranging from 100,000 to 0.01 parasites/µL were prepared. HRP2, Plasmodium lactate dehydrogenase (pLDH) and aldolase concentrations were determined for the diluted samples using a multiplex bead assay. The samples were subsequently tested on three RDT products designed to detect P. falciparum by HRP2 alone or in combination with pLDH. RESULTS: At parasite densities of approximately 1000 parasites/µL, parasites that expressed either hrp2 or hrp3 were detected by all three RDTs. Multiplex based antigen measurement using HRP2- conjugated beads demonstrated higher antigen concentration when both hrp2 and hrp3 genes were intact (3D7 parasites, 47.9 ng/ml) compared to HB3 (3.02 ng/mL) and Dd2 (0.20 ng/mL) strains that had one gene deleted. 3D7 at 10 parasites/µL (0.45 ng/mL) was reactive on all three RDT products whereas none of the other parasites were reactive at that density. CONCLUSIONS: Above a certain antigen threshold, HRP3 cross-reactivity on HRP2-based RDTs is sufficient to mask the effects of deletions of hrp2 only. Studies of hrp2 deletion and its effects on HRP2-based RDTs must be studied alongside hrp3 deletions and include clinical sample reactivity on HRP2-based tests.

Comparison of saliva with oral and nasopharyngeal swabs for SARS-CoV-2 detection on various commercial and laboratory-developed assays.

The accurate laboratory detection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a crucial element in the fight against coronavirus disease 2019 (COVID-19). Reverse transcription-polymerase chain reaction testing on combined oral and nasopharyngeal swab (ONPS) suffers from several limitations, including the need for qualified personnel, the discomfort caused by invasive nasopharyngeal sample collection, and the possibility of swab and transport media shortage. Testing on saliva would represent an advancement. The aim of this study was to compare the concordance between saliva samples and ONPS for the detection of SARS-CoV-2 on various commercial and laboratory-developed tests (LDT). Individuals were recruited from eight institutions in Quebec, Canada, if they had SARS-CoV-2 RNA detected on a recently collected ONPS, and accepted to provide another ONPS, paired with saliva. Assays available in the different laboratories (Abbott RealTime SARS-CoV-2, Cobas® SARS-CoV-2, Simplexa™ COVID-19 Direct, Allplex™ 2019-nCoV, RIDA®GENE SARS-CoV-2, and an LDT preceded by three different extraction methods) were used to determine the concordance between saliva and ONPS results. Overall, 320 tests were run from a total of 125 saliva and ONPS sample pairs. All assays yielded similar sensitivity when saliva was compared to ONPS, with the exception of one LDT (67% vs. 93%). The mean difference in cycle threshold (∆C t ) was generally (but not significantly) in favor of the ONPS for all nucleic acid amplification tests. The maximum mean ∆​​​​​C t was 2.0, while individual ∆C t varied importantly from -17.5 to 12.4. Saliva seems to be associated with sensitivity similar to ONPS for the detection of SARS-CoV-2 by various assays.

Real-Time Diagnosis of Helicobacter pylori During Endoscopy by Gastric Juice Analysis.

EndoFaster is a high precision device for gastric juice analysis in real time during gastroscopy that enables detection of Helicobacter pylori infection and hypochlorhydric/achlorhydric conditions through the measurement of ammonium concentration and gastric pH. The high accuracy and feasibility of this technology enables a more accurate diagnosis and a reduced number of histologies, focusing the attention of the endoscopist on patients with high risk for cancer progression and limiting or avoiding biopsies on the low-risk ones while also saving costs and time.