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Serological detection of antibodies to SARS-CoV-2 is essential for establishing rates of seroconversion in populations, and for seeking evidence for a level of antibody that may be protective against COVID-19 disease. Several high-performance commercial tests have been described, but these require centralised laboratory facilities that are comparatively expensive, and therefore not available universally. Red cell agglutination tests do not require special equipment, are read by eye, have short development times, low cost and can be applied at the Point of Care. Here we describe a quantitative Haemagglutination test (HAT) for the detection of antibodies to the receptor binding domain of the SARS-CoV-2 spike protein. The HAT has a sensitivity of 90% and specificity of 99% for detection of antibodies after a PCR diagnosed infection. We will supply aliquots of the test reagent sufficient for ten thousand test wells free of charge to qualified research groups anywhere in the world. Serological detection of antibodies against SARS-CoV-2 can help establish rates of seroconversion. Here the authors develop a red cell agglutination test to detect antibodies against the receptor binding domain for distribution free of charge to qualified research groups.

Background Leptospirosis is an underdiagnosed infectious disease with non-specific clinical presentation that requires laboratory confirmation for diagnosis. The serologic reference standard remains the microscopic agglutination test (MAT) on paired serum samples. However, reported estimates of MAT’s sensitivity vary. We evaluated the accuracy of four index tests, MAT on paired samples as well as alternative standards for leptospirosis diagnosis: MAT on single acute-phase samples, polymerase chain reaction (PCR) with the target gene Lfb1 , and ELISA IgM with Leptospira fainei serovar Hurstbridge as an antigen. Methods We performed a systematic review of studies reporting results of leptospirosis diagnostic tests. We searched eight electronic databases and selected studies that tested human blood samples and compared index tests with blood culture and/or PCR and/or MAT (comparator tests). For MAT selection criteria we defined a threshold for single acute-phase samples according to a national classification of leptospirosis endemicity. We used a Bayesian random-effect meta-analysis to estimate the sensitivity and specificity of MAT in single acute-phase and paired samples separately, and assessed risk of bias using the Quality Assessment of Studies of Diagnostic Accuracy Approach- 2 (QUADAS-2) tool. Results For the MAT accuracy evaluation, 15 studies were included, 11 with single acute-phase serum, and 12 with paired sera. Two included studies used PCR targeting the Lfb1 gene, and one included study used IgM ELISA with Leptospira fainei serovar Hurstbridge as antigen. For MAT in single acute-phase samples, the pooled sensitivity and specificity were 14% (95% credible interval [CrI] 3–38%) and 86% (95% CrI 59–96%), respectively, and the predicted sensitivity and specificity were 14% (95% CrI 0–90%) and 86% (95% CrI 9–100%). Among paired MAT samples, the pooled sensitivity and specificity were 68% (95% CrI 32–92%) and 75% (95% CrI 45–93%) respectively, and the predicted sensitivity and specificity were 69% (95% CrI 2–100%) and 75% (2–100%). Conclusions Based on our analysis, the accuracy of MAT in paired samples was not high, but it remains the reference standard until a more accurate diagnostic test is developed. Future studies that include larger numbers of participants with paired samples will improve the certainty of accuracy estimates.

The modified agglutination test (MAT) is one of the most commonly used tests for the detection of antibodies to Toxoplasma gondii in animal and human sera. The objective of the present study was to evaluate the diagnostic accuracy of the MAT and bioassay in free-range/backyard (FR) chickens (Gallus domesticus). Previously-published T. gondii test results from 2066 chickens from 19 countries were compiled for the present study. The frequency of isolation of T. gondii increased for MAT titres between 1:5 and 1:160, and ranged from 61 to 75% for antibody titres of 1:160, 1:320, and ⩾1:640. Twenty-three cats fed pooled hearts from a total of 802 FR seronegative (MAT, <1:5) chickens from several countries did not excrete oocysts, indicating a high negative predictive value of MAT because FR chickens would have been exposed to many microbes; cats are the most sensitive indicators of T. gondii infection in tissues and can excrete millions of oocysts after ingesting even a few bradyzoites. Of the 29 cats in this study, six cats, fed hearts pooled from 15–122 FR chickens, excreted oocysts; but these identifications were likely related to misidentification or prozone. Results of the present study support the validity of MAT for the detection of T. gondii infection in chickens.

Streptococcus agalactiae (also known Group B Streptococcus or GBS) represents the main pathogen responsible for early- and late-onset infections in newborns. The present study aimed to determine the antimicrobial susceptibility pattern and the capsular serotypes of GBS isolated in Eastern Sicily over 5 years, from January 2015 to December 2019. A total of 3494 GBS were isolated from vaginal swabs of pregnant women (37–39 weeks), as recommended by the Centers for Disease Control and Prevention. Capsular polysaccharide’s typing of GBS was determined by a commercial latex agglutination test containing reagents to serotypes I–IX. The antimicrobial resistance pattern of GBS was determined through the disk diffusion method (Kirby-Bauer) and the double-disk diffusion test on Mueller-Hinton agar plates supplemented with 5% defibrinated sheep blood, according to the guidelines of the Clinical and Laboratory Standards Institute. Serotypes III (1218, 34.9%) and V (1069, 30.6%) were the prevalent colonizers, followed by not typable (570, 16.3%) and serotypes Ia (548, 15.7%), Ib (47, 1.3%), II (40, 1.1%), and IV (2, 0.1%). All 3494 clinical isolates were susceptible to cefditoren and vancomycin. Resistance to penicillin, ampicillin, levofloxacin, clindamycin, and erythromycin was observed in 6 (0.2%), 5 (0.1%), 161 (4.6%), 1090 (31.2%), and 1402 (40.1%) of the strains, respectively. Most of erythromycin-resistant GBS (1090/1402) showed the cMLSB phenotype, 276 the M phenotype, and 36 the iMLSB phenotype. Our findings revealed a higher prevalence of serotype III and a relevant resistance rate, among GBS strains, to the most frequently used antibiotics in antenatal screening.

Background Direct agglutination test (DAT) as a simple, accurate and reliable method, has been widely used for serodiagnosis of visceral leishmaniasis (VL) during the last three decades. The present study is a systematic review and meta-analysis to evaluate the diagnostic accuracy of DAT for serodiagnosis of human VL. Methods Electronic databases, including MEDLINE (via PubMed), SCOPUS, Web of Science, SID and Mag Iran (two Persian scientific search engines) were searched from December 2004 to April 2019. We determined the pooled sensitivity and specificity rates of DAT for the diagnosis of human VL, calculated positive and negative likelihood ratios (LR+ and LR-), and constructed summary receiver operating characteristic (ROC) curves parameters across the eligible studies. Results Of the 2928 records identified in the mentioned electronic databases and after examining reference lists of articles, 24 articles met inclusion criteria and were enrolled in the systematic review and out of them 20 records qualified for meta-analysis. The pooled sensitivity and specificity rates of DAT was 96% [95% CI, 92–98] and 95% [CI95% 86–99], respectively. The likelihood ratio of a positive test (LR+) was found to be 21 [CI95%, 6.6–66.5] and the likelihood ratio of a negative test (LR−) was found to be 0.04 [(CI95%, 0.02–0.08]. The combined estimate of the diagnostic odds ratio for DAT was high [467 (CI95%, 114–1912]). We found that the summary receiver operating characteristic curve (SROC) is positioned near the upper left corner of the curve and the area under curve (AUC) was 0.98 (95% CI, 0.97 to 0.99). Conclusion Referring to our analysis, we determined that DAT can be considered as a valuable tool for the serodiagnosis of human VL with high sensitivity and specificity. As DAT is a simple, accurate and efficient serological test, it can be recommended for serodiagnosis of human VL particularly in endemic areas.

Background Yeasts are ubiquitous microorganisms found both endogenously within the human body and in the environment. Humoral responses against yeasts lead to the production of yeast-binding antibodies, which can agglutinate yeast cell targets. These antibodies affect the accuracy of immunodiagnostic tools employing yeast cells as in recombinant surface antigen display. Aim To improve the applicability of such tools, the study aims to determine the abundance and characterize the agglutinating behavior of yeast-binding antibodies. Methodology The study employed the use of a yeast agglutination assay to determine the prevalence of agglutination across human fresh frozen plasma samples ( n  = 36). The mean area of the agglutinin complex served as the basis for differentiating positive and negative samples. Indirect ELISA set-ups using protein A, protein G, and anti-IgM horseradish peroxidase conjugates were used to quantify titers and characterize the isotypes driving agglutination. Results The results of the agglutination assays and indirect ELISA revealed that the formation of agglutinin complexes was promoted by a low pH and inhibited by a high ionic strength. Coagulation factors and complement proteins did not significantly contribute to agglutination. Finally, elution of agglutinating proteins was performed and the resulting eluate was tested further for re-binding and re-agglutination with yeast cells, suggesting the presence of antibodies. Conclusion Findings from the current study suggest that antibody-mediated yeast cell agglutination driven by immunoglobulins (i.e., IgM, IgG) present in human plasma can be affected by various physicochemical factors such as pH (i.e., acidity) and ionic strength (i.e., NaCl concentration) but is independent of the activity of coagulation factors. These conditions must be carefully optimized in the development of cell-based immunoassays and yeast surface display technologies, which utilize antibody-mediated yeast agglutination.

Host defense cationic Antimicrobial Peptides (AMPs) can kill microorganisms including bacteria, viruses and fungi using various modes of action. The negatively charged bacterial membranes serve as a key target for many AMPs. Bacterial cell death by membrane permeabilization has been well perceived. A number of cationic AMPs kill bacteria by cell agglutination which is a distinctly different mode of action compared to membrane pore formation. However, mechanism of cell agglutinating AMPs is poorly understood. The outer membrane lipopolysaccharide (LPS) or the cell-wall peptidoglycans are targeted by AMPs as a key step in agglutination process. Here, we report the first atomic-resolution structure of thanatin, a cell agglutinating AMP, in complex with LPS micelle by solution NMR. The structure of thanatin in complex with LPS, revealed four stranded antiparallel β-sheet in a ‘head-tail’ dimeric topology. By contrast, thanatin in free solution assumed an antiparallel β-hairpin conformation. Dimeric structure of thanatin displayed higher hydrophobicity and cationicity with sites of LPS interactions. MD simulations and biophysical interactions analyses provided mode of LPS recognition and perturbation of LPS micelle structures. Mechanistic insights of bacterial cell agglutination obtained in this study can be utilized to develop antibiotics of alternative mode of action.

Objective Commercial kits of column tests for pre-transfusion testing have progressively replaced conventional tube tests in most laboratories. Aim of this study was to compare three commercial test cell panels for the identification of irregular red blood cell (RBC) alloantibodies. Overall, 44 samples with a positive indirect antiglobulin test (IAT) by routine testing were used for comparison of following panels: Ortho RESOLVE ® panelC (Ortho Clinical Diagnostics (OCD), Milan, Italy), ID-DiaPanel(-P) (Bio-Rad Laboratories, CA, USA) and Identisera Diana(P) (Grifols, Barcelona, Spain). Column agglutination techniques were used, with microtubes containing either microgel (Bio-Rad/Grifols) or glass bead microparticles (Ortho). Results Alloantibody identification was possible in 38 samples, of which identical identification was shown in 33 samples by all methods. The remaining samples showed differences between certain methods, with the gel card system being superior to the glass card system for analyzing stored samples Considering that not all samples were evaluated in all three methods, the concordance rate reached 100% between Bio-Rad and Grifols, 90.5% between Bio-Rad and OCD, 86.5% between OCD and Grifols and 90.5% between all methods. Although differences in sensitivities were seen for specific antibodies, the three methods showed comparable performance for the identification of RBC alloantibodies.

Visceral leishmaniasis (VL) is the most severe form of leishmaniasis and is potentially fatal if not diagnosed and treated. Accurate and timely diagnosis is considered one of the pillars needed for the reduction in disease-related lethality. Brazil is currently one of the three eco-epidemiological hotspots for this disease. Several serological tests are commercially available in this country for VL diagnosis, although information on the performance of these tests is fragmented and insufficient. The aim of this study was to directly compare the performance of six commercial kits: three enzyme-linked immunosorbent assays (ELISAs), two immunofluorescence antibody tests (IFATs), one immunochromatographic test (ICT), besides one ICT, currently not commercially available in Brazil and one in-house direct agglutination test (DAT-LPC), not yet marketed. A panel of 236 stored samples from patients with clinically suspected VL, including 77 HIV-infected patients, was tested. IT-LEISH and DAT-LPC showed the highest accuracy rate among the non-HIV-infected patients, 96.2% [CI95%: 92.8-99.7%] and 95.6% [CI95%: 91.9-99.3%], respectively. For the ELISA tests evaluated, the maximum accuracy was 91.2%, and in the inter HIV-status group analysis, no significant differences were observed. For both IFATs evaluated, the maximum accuracy was 84.3%, and a lower accuracy rate was observed among the HIV-infected patients (p = 0.039) than among the non-HIV-infected patients. The DAT-LPC was the most accurate test in the HIV-infected patients (p≤0.115). In general, no significant difference in accuracy was observed among the VL-suspected patients stratified by age. In summary, the differences in the performance of the tests available for VL in Brazil confirm the need for local studies before defining the diagnostic strategy.

Although tissue aspiration procedures (TAPs) are considered the gold standard for visceral leishmaniasis (VL) diagnosis, they often fail to detect disease progression before the amastigote-demonstrable phase. To overcome this limitation, a primary direct agglutination test (DAT) was developed using intact Leishmania donovani promastigotes initially treated with trypsin as the antigen. To enhance the exposure of specific epitopes on the promastigote surface and antibody-binding sites, alternative proteolytic agents were evaluated and incorporated into antigen processing and test execution. This approach led to significant or complete inhibition of agglutination in most known cross-reacting disorders. The LQ-DAT consistently demonstrated highly reproducible results across diverse geographical regions, regardless of the L. donovani sub-species or strain. To facilitate routine implementation and local production, the LQ-DAT processing know-how was disseminated to all major VL-endemic areas. Sensitivities comparable to TAP outcomes were demonstrated in 2,224 of 2,697 VL cases successfully diagnosed and treated over 35 years of routine, epidemic, and outbreak evaluations. Notably, 473 (17.5%) of these cases were symptomatic, with TAP-negative but LQ-DAT-positive results, and responded favorably (98.0%−100%) to specific treatment. Given the lower sensitivity also demonstrated by LQ-DAT, TAP does not meet the criteria for classification as the gold-standard VL diagnostic. Consequently, a positive response to specific anti-leishmanial treatment is recommended as a benchmark for diagnostic reliability. Beyond its advantage in detecting VL at earlier stages compared to TAP, the improved LQ-DAT described here also exhibited feasibility and stability required for local production in low-resource settings.