Diagnostic evaluation of IgM ELISA and IgM Immunofluorescence assay for the diagnosis of Acute Scrub Typhus in central Nepal.

BACKGROUND: Scrub typhus is an acute febrile illness caused by the obligate intracellular bacterium, Orientia tsutsugamushi. Immunochromatography (ICT) and IgM ELISA are two of the routinely employed antibody based assays for diagnosis of Scrub typhus fever in Nepal, although the recommended gold standard diagnostic test is IgM Immunofluorescence assay (IFA). This study evaluated InBios Scrub Typhus Detect™ Immunoglobulin M (IgM) ELISA and IgM Immunofluorescence assays in single serum sample at the time of admission. METHOD: Study participants (1585 suspected cases), were enrolled based on acute febrile illness with suspected scrub typhus cases in central Nepal. Blood sample was collected from the suspected patients of scrub typhus, presenting with acute febrile illness. IgM antibody to Orientia tsusugamushi was detected by using Scrub Typhus Detect™ Kit and an in-house IgM IFA. The IFA assay was performed with the Gilliam, Karp, Kato strains and O. chuto antigens following the ARRL protocol. RESULT: Statistical analysis of IgM ELISA results when compared to reference test, IgM IFA results demonstrated the following characteristics, sensitivity 84.0% (95%CI: 79.73-87.68%), specificity 94.82% (95% CI: 93.43-95.99%), positive likelihood ratio 16.21% (95% CI: 12.71-20.67%), negative likelihood ratio 0.17% (95% CI: 0.13-0.21%), disease prevalence 22.08% (95% CI: 20.06 -24.21%), positive predictive value 82.12% (95% CI: 78.28-85.42%) and negative predictive value 95.44% (95% CI: 94.27-96.38%) respectively. CONCLUSION: Although IgM IFA is considered the gold standard test for the diagnosis of scrub typhus cases, it is relatively expensive, requires trained personal and a microscope with fluorescence filters. Scrub typhus IgM ELISA may be the best alternative test and possible viable option for resource limited endemic countries like Nepal.

Lateral flow fluorescent immunoassay based on isothermal amplification for rapid quantitative detection of Salmonella spp.

Salmonella spp. are zoonotic pathogens of substantial public health concern. To enable detection in the field or under instrument-free conditions, we developed a rapid and robust lateral flow fluorescent immunoassay based on strand exchange amplification (SEA-LFIA) for the quantitative detection of Salmonella spp. As far as we know, this work is the first report regarding the use of Bst DNA polymerase-assisted SEA for fluorescence sensing to detect Salmonella spp. The SEA method was further confirmed by enzymatic digestion and Sanger dideoxy sequencing. The specificity of SEA-LFIA assay was verified by 89 Salmonella strains (18 Salmonella reference strains and 71 clinical isolates) and 15 non-Salmonella reference strains (different genera). The sensitivity of SEA-LFIA assay was 6 × 100 CFU mL-1 of Salmonella pure culture or 3 × 104 CFU 25 g-1 of artificially spiked raw chicken meat. Using this assay, it was found that 37 (16%) of the 236 samples collected were positive, which was consistent with the results of conventional PCR. The cutoff value is 15 and SEA-LFIA assay only takes ∼30 min without high equipment and reagent cost. In addition, the proposed strategy can be easily extended by redesigning the corresponding amplification primers to detect target analytes. In conclusion, the optimized SEA-LFIA assay is an efficient and specific method for the detection of Salmonella spp., and can potentially serve as a new on-site diagnostic tool in life sciences.

Fluorescent Reporter Mice for Nerve Guidance Conduit Assessment: A High-Throughput in vivo Model.

OBJECTIVE: Use of cell culture and conventional in vivo mammalian models to assess nerve regeneration across guidance conduits is resource-intensive. Herein we describe a high-throughput platform utilizing transgenic mice for stain-free axon visualization paired with rapid cryosection techniques for low-cost screening of novel bioengineered nerve guidance conduit performance. METHODS: Interposition repair of sciatic nerve transection in mice expressing yellow fluorescent protein in peripheral neurons (Thy1.2 YFP-16) was performed with various bioengineered neural conduit compositions using a rapid sutureless entubulation technique under isoflurane anesthesia. Axonal ingrowth was assessed at 3 and 6 weeks using epifluorescent microscopy following cryosectioning. RESULTS: Mean procedure time (incision-to-closure) was less than 2½ minutes. Direct operational costs of a 3-week experiment was calculated at $21.47 per animal. Tissue processing steps were minimized to aldehyde fixation, cryoprotection and sectioning, and rapid fluorescent dye staining for conduit visualization. Fluorescent microscopy readily resolved robust axonal sprouting at 3 weeks, with clear elucidation of ingrowth-permissive, semipermissive, or restrictive nerve guidance conduit environments. CONCLUSION: A rapid and cost-efficient in vivo platform for screening of nerve guidance conduit performance has been described. LEVEL OF EVIDENCE: NA. Laryngoscope, E392-E392, 2018.

Comparative analysis of Mouse Inoculation Test and Virus Isolation in Cell Culture for rabies diagnosis in animals of Parana, Brazil.

INTRODUCTION: Rabies is an acute zoonotic disease, caused by a rhabdovirus that can affect all mammals, and is commonly transmitted by the bite of a rabid animal. The definitive diagnosis is laboratorial, by the Fluorescent Antibody Test (FAT) as a quick test and Mouse Inoculation Test (MIT) as a confirmatory test (gold standard). Studies conducted over the past three decades indicate that MIT and Virus Isolation in Cell Culture (VICC) can provide the same effectiveness, the latter being considered superior in bioethics and animal welfare. The aim of this study was to compare VICC with MIT, in terms of accuracy, biosafety and occupational health, supply and equipment costs, bioethics and animal welfare, in a Brazilian public health lab. METHODS: We utilized 400 samples of animal neurological tissue to compare the performance of VICC against MIT. The variables analyzed were accuracy, biosafety and occupational health, time spent in performing the tests, supply and equipment costs, bioethics and animal welfare evaluation. RESULTS: Both VICC and MIT had almost the same accuracy (99.8%), although VICC presented fewer risks regarding biosafety and mental health of the technicians, and reduced time between inoculation and obtaining the results (approximately 22 days less). In addition, VICC presented lower supply costs (86.5% less), equipment costs (32.6% less), and the advantage of not using animals. CONCLUSIONS: These results confirm that VICC can replace MIT, offering the same accuracy and better features regarding cost, results, biosafety and occupational health, and bioethics and animal welfare.

Comparative analysis of Mouse Inoculation Test and Virus Isolation in Cell Culture for rabies diagnosis in animals of Parana, Brazil

Abstract INTRODUCTION: Rabies is an acute zoonotic disease, caused by a rhabdovirus that can affect all mammals, and is commonly transmitted by the bite of a rabid animal. The definitive diagnosis is laboratorial, by the Fluorescent Antibody Test (FAT) as a quick test and Mouse Inoculation Test (MIT) as a confirmatory test (gold standard). Studies conducted over the past three decades indicate that MIT and Virus Isolation in Cell Culture (VICC) can provide the same effectiveness, the latter being considered superior in bioethics and animal welfare. The aim of this study was to compare VICC with MIT, in terms of accuracy, biosafety and occupational health, supply and equipment costs, bioethics and animal welfare, in a Brazilian public health lab. METHODS: We utilized 400 samples of animal neurological tissue to compare the performance of VICC against MIT. The variables analyzed were accuracy, biosafety and occupational health, time spent in performing the tests, supply and equipment costs, bioethics and animal welfare evaluation. RESULTS: Both VICC and MIT had almost the same accuracy (99.8%), although VICC presented fewer risks regarding biosafety and mental health of the technicians, and reduced time between inoculation and obtaining the results (approximately 22 days less). In addition, VICC presented lower supply costs (86.5% less), equipment costs (32.6% less), and the advantage of not using animals. CONCLUSIONS: These results confirm that VICC can replace MIT, offering the same accuracy and better features regarding cost, results, biosafety and occupational health, and bioethics and animal welfare.

GloSensor assay for discovery of GPCR-selective ligands.

G protein-coupled receptors (GPCRs) are modulators of almost every physiological process, and therefore, are most favorite therapeutic target for wide spectrum of diseases. Ideally, high-throughput functional assays should be implemented that allow the screening of large compound libraries in cost-effective manner to identify agonist, antagonist, and allosteric modulators in the same assay. Taking advantage of the increased understanding of the GPCR structure and signaling, several commercially available functional assays based on fluorescence or chemiluminescence detection are being used in both academia and industry. In this chapter, we provide step-by-step method and guidelines to perform cAMP measurement using GloSensor assay. Finally, we have also discussed the analysis and interpretation of results obtained using this assay by providing several examples of Gs- and Gi-coupled GPCRs.

Strategies to improve the efficiency of celiac disease diagnosis in the laboratory.

The demand for testing to detect celiac disease (CD) autoantibodies has increased, together with the cost per case diagnosed, resulting in the adoption of measures to restrict laboratory testing. We designed this study to determine whether opportunistic screening to detect CD-associated autoantibodies had advantages compared to efforts to restrict testing, and to identify the most cost-effective diagnostic strategy. We compared a group of 1678 patients in which autoantibody testing was restricted to cases in which the test referral was considered appropriate (G1) to a group of 2140 patients in which test referrals were not reviewed or restricted (G2). Two algorithms A (quantifying IgA and Tissue transglutaminase IgA [TG-IgA] in all patients), and B (quantifying only TG-IgA in all patients) were used in each group, and the cost-effectiveness of each strategy was calculated. TG-IgA autoantibodies were positive in 62 G1 patients and 69 G2 patients. Among those positive for tissue transglutaminase IgA and endomysial IgA autoantibodies, the proportion of patients with de novo autoantibodies was lower (p=0.028) in G1 (11/62) than in G2 (24/69). Algorithm B required fewer determinations than algorithm A in both G1 (2310 vs 3493; p<0.001) and G2 (2196 vs 4435; p<0.001). With algorithm B the proportion of patients in whom IgA was tested was lower (p<0.001) in G2 (29/2140) than in G1 (617/1678). The lowest cost per case diagnosed (4.63 euros/patient) was found with algorithm B in G2. We conclude that opportunistic screening has advantages compared to efforts in the laboratory to restrict CD diagnostic testing. The most cost-effective strategy was based on the use of an appropriate algorithm.

Immunohisto- and Cytochemistry Analysis of Connexins.

Immunohistochemistry (IHC) is a ubiquitous used technique to identify and analyze protein expression in the context of tissue and cell morphology. In the connexin research field, IHC is applied to identify the subcellular location of connexin proteins, as this can be directly linked to their functionality. The present chapter describes a protocol for fluorescent IHC to detect connexin proteins in tissues slices and cells, with slight modifications depending on the nature of biological sample, histological processing, and/or protein expression level. Basically, fluorescent IHC is a short, simple, and cost-effective technique, which allows the visualization of proteins based on fluorescent-labeled antibody-antigen recognition.

A novel approach for integrative studies on neurodegenerative diseases in human brains.

Despite a massive research effort to elucidate Alzheimer's disease (AD) in recent decades, effective treatment remains elusive. This failure may relate to an oversimplification of the pathogenic processes underlying AD and also lack of understanding of AD progression during its long latent stages. Although evidence shows that the two specific neuropathological hallmarks in AD (neuronal loss and protein accumulation), which are opposite in nature, do not progress in parallel, the great majority of studies have focused on only one of these aspects. Furthermore, research focusing on single structures is likely to render an incomplete picture of AD pathogenesis because as AD involves complete brain networks, potential compensatory mechanisms within the network may ameliorate impairment of the system to a certain extent. Here, we describe an approach for enabling integrative analysis of the dual-nature lesions, simultaneously, in all components of one of the brain networks most vulnerable to AD. This approach is based on significant development of methods previously described mainly by our group that were optimized and complemented for this study. It combines unbiased stereology with immunohistochemistry and immunofluorescence, making use of advanced graphics computing for three-dimensional (3D) volume reconstructions. Although this study was performed in human brainstem and focused in AD, it may be applied to the study of any neurological disease characterized by dual-nature lesions, in humans and animal models. This approach does not require a high level of investment in new equipment and a significant number of specimens can be processed and analyzed within a funding cycle.

Comparative cost-effectiveness of two-tiered testing strategies for serodiagnosis of lyme disease with noncutaneous manifestations.

The mainstay of laboratory diagnosis for Lyme disease is two-tiered serological testing, in which a reactive first-tier enzyme-linked immunosorbent assay (ELISA) or an immunofluorescence assay is supplemented by separate IgM and IgG immunoblots. Recent data suggest that the C6 ELISA can be substituted for immunoblots without a reduction in either sensitivity or specificity. In this study, the costs of 4 different two-tiered testing strategies for Lyme disease were compared using the median charges for these tests at 6 commercial diagnostic laboratories in 2012. The study found that a whole-cell sonicate ELISA followed by the C6 ELISA was the most cost-effective two-tiered testing strategy for Lyme disease with acute-phase serum samples. We conclude that the C6 ELISA can substitute for immunoblots in the two-tiered testing protocol for Lyme disease without a loss of sensitivity or specificity and is less expensive.

A faster immunofluorescence assay for tracking infection progress of human cytomegalovirus.

Immunofluorescence assay (IFA) is one of the most frequently used methods in the biological sciences and clinic diagnosis, but it is expensive and time-consuming. To overcome these limitations, we developed a faster and more cost-effective IFA (f-IFA) by modifying the standard IFA, and applied this method to track the progression of human cytomegalovirus (HCMV) infection in different cells. The f-IFA that we developed not only saves time, but also dramatically reduces the quantity of antibody (Ab), which will facilitate the application of IFA in clinic diagnosis. f-IFA requires only 15 min for blocking, 10 min incubation for each primary and secondary Abs, followed by 1 min extensive wash after each incubation. Only 25 µl of diluted Ab solution was needed for each coverslip at the primary and secondary Ab incubation steps. In addition, all steps were performed at room temperature. This f-IFA has been applied successfully to follow virion entry (pp65) and expression of viral genes (IE1, UL44, and pp65) in order to track the details of HCMV infection process. We found that ∼0.5% HCMV-infected T98G cells formed multiple-micronuclei (IE1 and nucleus staining) and had virus shedding (pp65 staining) by f-IFA, which could not be detected by the traditional IFA. Our results indicated that f-IFA is a sensitive, convenient, fast, and cost-effective method for investigating the details of virus infection progress, especially HCMV infection. The faster and cost-effective feature with higher sensitivity and specificity implies that f-IFA has potential applications in clinical diagnosis.

Immunosensor towards low-cost, rapid diagnosis of tuberculosis.

A rapid, accurate tuberculosis diagnostic tool that is compatible with the needs of tuberculosis-endemic settings is a long-sought goal. An immunofluorescence microtip sensor is described that detects Mycobacterium tuberculosis complex cells in sputum in 25 minutes. Concentration mechanisms based on flow circulation and electric field are combined at different scales to concentrate target bacteria in 1 mL samples onto the surfaces of microscale tips. Specificity is conferred by genus-specific antibodies on the microtip surface. Immunofluorescence is then used to detect the captured cells on the microtip. The detection limit in sputum is 200 CFU mL(-1) with a success rate of 96%, which is comparable to PCR.

Evaluation of PG-M3 antibody in the diagnosis of acute promyelocytic leukaemia.

BACKGROUND & OBJECTIVES: Acute promyelocytic leukaemia (APL) is a distinct subtype of acute myeloid leukaemia (AML) characterized by a reciprocal translocation, t(15;17) and a high incidence of life-threatening coagulopathy. APL diagnosis is considered a medical emergency. As reverse transcription-polymerase chain reaction (RT-PCR) for PML-RAR fusion oncoprotein is time consuming, there is a need for a rapid and accurate diagnostic test for APL. This study evaluates the role of PG-M3 monoclonal antibody using immunofluorescence (IF) in the early diagnosis of APL. MATERIALS AND METHODS: Thirty-six new untreated APL cases diagnosed with RT-PCR for PML-RAR as the gold standard and 38 non-APL controls (28 non-APL AMLs and 10 non-leukaemic samples) were evaluated by routine morphology and cytochemistry, RT-PCR and IF using PG-M3 monoclonal antibody. RESULTS: Using IF, 34 of 36 (94·4%) APL cases showed a microgranular pattern suggestive of APL and two cases (5·6%) showed a speckled pattern typical of wild-type PML protein (False negative). By comparison, two of 28 (7·1%) non-APL AMLs showed microgranular pattern (false positive). Hence, IF as a diagnostic test for APL resulted in a sensitivity of 94·4%, specificity of 92·9% and positive and negative predictive values of 94·4% and 92·9% respectively. All 10 non-leukaemic samples showed a speckled pattern. CONCLUSIONS: IF using PG-M3 antibodies can be used as a rapid (takes 2 h), cheap, sensitive and specific method to identify APL. It can be a useful adjunct for diagnosis of APL especially if facilities for RT-PCR are not available, particularly in resource-limited settings.

Development of a fluorescent-microsphere immunoassay for detection of antibodies specific to equine arteritis virus and comparison with the virus neutralization test.

The development and validation of a microsphere immunoassay (MIA) to detect equine antibodies to the major structural proteins of equine arteritis virus (EAV) are described. The assay development process was based on the cloning and expression of genes for full-length individual major structural proteins (GP5 amino acids 1 to 255 [GP5(1-255)], M(1-162), and N(1-110)), as well as partial sequences of these structural proteins (GP5(1-116), GP5(75-112), GP5(55-98), M(88-162), and N(1-69)) that constituted putative antigenic regions. Purified recombinant viral proteins expressed in Escherichia coli were covalently bound to fluorescent polystyrene microspheres and analyzed with the Luminex xMap 100 instrument. Of the eight recombinant proteins, the highest concordance with the virus neutralization test (VNT) results was obtained with the partial GP5(55-98) protein. The MIA was validated by testing a total of 2,500 equine serum samples previously characterized by the VNT. With the use of an optimal median fluorescence intensity cutoff value of 992, the sensitivity and specificity of the assay were 92.6% and 92.9%, respectively. The GP5(55-98) MIA and VNT outcomes correlated significantly (r = 0.84; P < 0.0001). Although the GP5(55-98) MIA is less sensitive than the standard VNT, it has the potential to provide a rapid, convenient, and more economical test for screening equine sera for the presence of antibodies to EAV, with the VNT then being used as a confirmatory assay.

Quantitative immunohistochemistry of fluorescence labelled probes using low-cost software.

This paper describes simple procedures to process digital images in quantitative immunofluorescence microscopy. Monoclonal antibodies directed against the sarcoplasmic myosin heavy chain isoforms and against laminin, located on the basement membranes, were applied to sections of human skeletal muscle. The localisation and staining intensity of a fluorescent secondary antibody were recorded using an indirect histochemical method. The digitised images were pre-processed and the luminosities of appropriate structures were determined using existing tools in the widely used image processing software Photoshop from Adobe. Procedures to obtain a quantitative measure for the specific fluorescence signal (the background corrected fluorescence in the object) were developed. In addition, antibody binding to individual cells could be quantified whether these cells are well separated or not. The relation between the specific fluorescence signal and the dilution factor of the primary antibody could be measured to determine a suitable concentration of the antibody for incubation of the sections. The potential fading of the fluorescence signal with time and prolonged exposure to light from the microscope was explored and analysed. With the tools described in the present report it is thus possible also to optimize the topical immunohistochemical protocol in order to quantify the fluorescence signal.

Impact of the new Beckman Coulter Cytomics FC 500 5-color flow cytometer on a regional flow cytometry clinical laboratory service.

Calgary Laboratory Services (CLS) in Alberta, Canada, is the regional reference laboratory providing flow cytometry services for southern Alberta and southeastern British Columbia. As a busy reference flow laboratory we provide flow cytometry immunophenotyping for investigation and diagnosis of acute and chronic leukemias, lymphomas, immunodeficiencies, neuroblastoma, platelet disorders, and interstitial lung disease (ILD). Because of increasing workload and the continual effort to improve the service to our health care providers, CLS invested in the new Beckman Coulter Cytomics FC 500 5-color flow cytometer. In addition to time and labor savings due to reduced maintenance and operating system design, this new flow cytometer automates many of the previous manual steps involved in quality control and flow cytometric analysis. It also incorporates 2 lasers and is capable of measuring 5-color antibody combinations in a single tube, enabling us to reduce the number of tubes and overall costs, giving us better gating options for minimal residual disease analysis. We present the first published evaluation, an assessment of the overall productivity and cost impact of the new state-of-the-art Cytomics FC 500 flow cytometer. Implementation of the Cytomics FC 500 has resulted in a 20% reduction in reagent costs and shorter turnaround time for analysis and diagnosis. This instrument has allowed us to reduce our acute leukemia panel from 17 to 13 tubes, our lymphoma panel from 13 to 7 tubes, and our ILD panel from 4 to 2 tubes. The availability of 2 lasers provides more flexibility in choosing antibodies and conjugates to customize immunophenotyping panels. It also allows us to use the DRAQ5 dye and simultaneously analyze the immunophenotype and DNA content of cells with very little compensation. Many of the arduous, time-consuming flow operator tasks often associated with previous generation flow cytometry instruments, such as color compensation, list mode analysis, sample repeats, and interpretations, have been substantially reduced with the Cytomics FC 500 5-color flow cytometer. In conclusion the Cytomics FC 500 5-color flow cytometer is a major advance in flow cytometry instrumentation and has reduced our overall reagent costs by 20%, provided better information and speedier turnaround time to our health care professionals. It is an ideal flow cytometer for any busy clinical or research flow cytometry service.

Rapid enhanced tissue culture immunofluorescence: a new, rapid method for detection of viruses.

The detection of a large variety of viruses requires a number of different susceptible cell lines in a shell vial method--currently considered to be the most enhanced method of viral detection. However, this method is unsuitable in its standard form for handling a large volume of specimens. We have developed a time and cost saving new method, named rapid enhanced tissue culture immunofluorescence (RETCIF) and incorporates the attributes of the current methods. It is specific, uses the most sensitive cell lines and monoclonal antibodies and does not require the use of cover slips. The RETCIF method, in our hands, is a time saving procedure, with higher isolation ratio than the shell vial method. We recommend the RETCIF method for busy diagnostic virology laboratories.

'RETCIF': a rapid, sensitive method for detection of viruses, applicable for large numbers of clinical samples.

Rapid detection of viruses in clinical samples is important for continuing appropriate antiviral treatment and discontinuing unnecessary antibacterial treatment, as well as for excluding viral pathogens. Yet detection of viral agents may require numerous susceptible cell lines. Even with the shell vial culture method, it is cumbersome for handling large volumes of specimens. A procedure has been developed, which is time and cost-saving and uses specific cell lines in a 96-well microtitre plate and monoclonal antibodies (RETCIF-rapid enhanced tissue culture immunofluorescence). Each clinical sample was inoculated into 12 different wells with five different cell lines. Enhancement was achieved by sonication, centrifugation and hormonal supplementation to the medium used. Cytomegalovirus (CMV), herpes simplex virus (HSV) and respiratory viruses were detected by monoclonal antibodies on day 2, whilst varicella zoster virus (VZV) and enteroviruses were detected on days 5 and 7, respectively. During July-December 1998, 3298 patient specimens were compared by RETCIF and a modified shell vial method. Either or both methods isolated 779 viruses (24% positivity rate), whilst both methods detected 621. Of the 779 viruses, 87% (679) were isolated by the shell vial method in an average time of 4.9 days. For RETCIF the respective rate was 92.5% (721), in an average time of 3.0 days. The RETCIF method is a time-saving procedure, with higher isolation rates than the shell vial method.

[Avidity of IgG anti-Toxoplasma gondii. Study to establish a new decision tree for screening]. / Avidité des IgG anti Toxoplasma gondii. Etude en vue d'établir un nouvel arbre décisionnel dans le dépistage de la maladie.

Toxoplasmosis infection is commonly asymptomatic, but it may have severe teratogenic consequences. The authors review the literature on serologic screening in the first trimester of pregnancy.