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Introduction: Zika virus (ZIKV) is a re-emerging flavivirus that poses a significant public health threat. ZIKV exhibits a wide array of non-vector borne human transmission routes, such as sexual transmission, transplacental transmission and blood transfusion. Detection and surveillance of ZIKV is considered paramount in prevention of major outbreaks. With the majority of cases reported in low-resource locations, simple, lowcost detection methods are considered highly desirable. Materials and Methods: Here we have developed a sensitive and specific ZIKV diagnostic using reverse transcription recombinase-aided amplification (RT-RAA) coupled with lateral flow detection (LFD) targeting a highly conserved region of the ZIKV NS1 gene. Results: We show our rapid, isothermal-ZIKV-diagnostic (Iso-ZIKV-Dx) can detect 500 copies of synthetic ZIKV RNA/µL in under 30min at a constant 39°C. Using simulated urine samples, we observed that Iso-ZIKV-Dx also detects as low as 34.28 RNA copies/reaction of ZIKV (MR766 strain). Specificity testing confirmed that our test does not detect any co-circulating flaviviruses (dengue, West Nile, Japanese encephalitis, Murray Valley encephalitis and yellow fever viruses) or chikungunya virus. Sample processing results show complete inactivation of ZIKV (MR766 strain) in 5min at room temperature using our novel viral RNA sample preparation reagent. Furthermore, lateral flow strips testing demonstrates positive diagnoses in as little as 5min in running buffer. Discussion: Contrary to conventional RT-qPCR, our Iso-ZIKV-Dx does not require expensive machinery, specialised laboratory settings or extensively trained personnel. Pre-clinical evaluation demonstrates that our test exhibits robust, in-field capabilities without compromising sensitivity or specificity. When compared to the gold-standard RT-qPCR, our Iso-ZIKV-Dx test offers an array of applications that extend beyond diagnostics alone, including potential for surveillance and monitoring of ZIKV vector competency. [ABSTRACT FROM AUTHOR]

Vector surveillance of Plasmodium falciparum is critical for monitoring and reducing one of the most severe forms of malaria, which causes high morbidity and mortality in children under five and pregnant women. Here we developed a rapid and highly sensitive test for the detection of P. falciparum (Pf)-infected mosquitoes (Rapid Pf test), with high suitability for low-resource vector surveillance implementation. The Rapid Pf test had similar analytical sensitivity to laboratory-based tests, detecting down to 4 copies/μL of a 18S rRNA DNA standard. In addition, the Rapid Pf test could be completed in less than 30 minutes, and only required a liquid sample preparation reagent, pestle, tube, and 39°C heating block for operation, indicating amenability for low-resource implementation. Diagnostic testing was performed using Anopheles stephensi mosquitoes, either uninfected, or fed with P. falciparum gametocyte cultures. These P. falciparum fed mosquitoes were determined to have 79% infection prevalence based on parallel microscopy and qPCR testing on a subset of 19 mosquitoes. However, our Rapid Pf test determined a 90% positive test rate when testing individual infected mosquitoes (n=30), and did not detect 40 uninfected mosquitoes regardless of blood-fed status (n=40), suggesting the true prevalence of infection in the mosquitoes may have been higher than calculated by qPCR and microscopy. The Rapid Pf test was demonstrated to detect infection in individual mosquitoes (both fresh and frozen/thawed), as well as pools of 1 infected mosquito mixed with 19 known uninfected mosquitoes, and individual mosquitoes left in traps for up to 8 days. After testing on infected and uninfected mosquitoes (n=148) the Rapid Pf test was conservatively estimated to achieve 100% diagnostic sensitivity (95% confidence interval, CI: 91%-100%) and 97% diagnostic specificity (CI: 92%-99%) compared to the estimated prevalence from combined microscopy and qPCR results. These results indicate the Rapid Pf test could provide a highly effective tool for weekly surveillance of infected mosquitoes, to assist with P. falciparum monitoring and intervention studies.

IntroductionZika virus (ZIKV) is a re-emerging flavivirus that poses a significant public health threat. ZIKV exhibits a wide array of non-vector borne human transmission routes, such as sexual transmission, transplacental transmission and blood transfusion. Detection and surveillance of ZIKV is considered paramount in prevention of major outbreaks. With the majority of cases reported in low-resource locations, simple, low-cost detection methods are considered highly desirable.Materials and MethodsHere we have developed a sensitive and specific ZIKV diagnostic using reverse transcription recombinase-aided amplification (RT-RAA) coupled with lateral flow detection (LFD) targeting a highly conserved region of the ZIKV NS1 gene.ResultsWe show our rapid, isothermal-ZIKV-diagnostic (Iso-ZIKV-Dx) can detect 500 copies of synthetic ZIKV RNA/μL in under 30 min at a constant 39°C. Using simulated urine samples, we observed that Iso-ZIKV-Dx also detects as low as 34.28 RNA copies/reaction of ZIKV (MR766 strain). Specificity testing confirmed that our test does not detect any co-circulating flaviviruses (dengue, West Nile, Japanese encephalitis, Murray Valley encephalitis and yellow fever viruses) or chikungunya virus. Sample processing results show complete inactivation of ZIKV (MR766 strain) in 5 min at room temperature using our novel viral RNA sample preparation reagent. Furthermore, lateral flow strips testing demonstrates positive diagnoses in as little as 5 min in running buffer.DiscussionContrary to conventional RT-qPCR, our Iso-ZIKV-Dx does not require expensive machinery, specialised laboratory settings or extensively trained personnel. Pre-clinical evaluation demonstrates that our test exhibits robust, in-field capabilities without compromising sensitivity or specificity. When compared to the gold-standard RT-qPCR, our Iso-ZIKV-Dx test offers an array of applications that extend beyond diagnostics alone, including potential for surveillance and monitoring of ZIKV vector competency.

Accurate and timely diagnosis of Nipah virus (NiV) requires rapid, inexpensive, and robust diagnostic tests to control spread of disease. Current state of the art technologies are slow and require laboratory infrastructure that may not be available in all endemic settings. Here we report the development and comparison of three rapid NiV molecular diagnostic tests based on reverse transcription recombinase-based isothermal amplification coupled with lateral flow detection. These tests include a simple and fast one-step sample processing step that inactivates the BSL-4 pathogen, enabling safe testing without the need for multi-step RNA purification. The rapid NiV tests targeted the Nucleocapsid protein (N) gene with analytical sensitivity down to 1,000 copies/μL for synthetic NiV RNA and did not cross-react with RNA of other flaviviruses or Chikungunya virus, which can clinically present with similar febrile symptoms. Two tests detected 50,000–100,000 TCID50/mL (100–200 RNA copies/reaction) of the two distinct strains of NiV, Bangladesh (NiVB) and Malaysia (NiVM), and took 30 min from sample to result, suggesting these tests are well suited for rapid diagnosis under resource-limited conditions due to rapidity, simplicity, and low equipment requirements. These Nipah tests represent a first step toward development of near-patient NiV diagnostics that are appropriately sensitive for first-line screening, sufficiently robust for a range of peripheral settings, with potential to be safely performed outside of biohazard containment facilities. [ABSTRACT FROM AUTHOR]

Development and validation of rapid and easy-to-perform diagnostics continue to be a high priority during the current COVID-19 pandemic. Although vaccines are now widely available, early detection and consistent transmission control provide ideal means to mitigate the spread of SARS-CoV-2. Nucleic acid-based real‐time PCR tests are widely acknowledged as the gold standard for reliable diagnosis of COVID-19 infection. These tests are based on detecting viable or nonviable viral nucleic acids. SARS-CoV-2 spike protein is an alternative and ideal target for SARS-CoV-2 diagnosis in the early phase of infection, but point-of-care kits to detect the SARS-CoV-2 spike protein are limited. Here we describe a rapid and convenient method based on Lateral Flow Immunoassay (LFIA) to detect SARS-CoV-2 spike proteins, including SARS-CoV-2 variants (A.23.1, B.1.1.1, 1.617.2, B.1.1.7, B.1.351, P.1, N501Y, R.1, P681H, P3, UK, and South African) within 5 to 10 minutes. We generated highly specific monoclonal antibodies (mAbs) against rationally designed SARS-CoV-2 spike protein. Matched pair mAbs were selected by epitope mapping and employed as antigen capture reagents by spotting onto a nitrocellulose membrane and as detector reagents by conjugation with colloidal gold nanoparticles. We evaluated the performance of the LFIA using recombinant spike proteins of SARS-CoV-2 and several SARS-CoV-2 variants. The specificity of the LFIA was assessed using heat-inactivated SARS-CoV-2 and related human coronaviruses (HCoV-OC43, HCoV-229E, HCoV-HKU1, and HCoV-NL63) and an FDA-approved respiratory pathogens (RP) panel. The assay exhibited 98% specificity and acceptable performance with respect to the minimum limit of detection (25 ng/test) in validation tests. This new LFIA provides improved performance for the early diagnosis of SARS-CoV-2, particularly for home monitoring and in situations with limited access to molecular methods.

Accurate and timely diagnosis of Nipah virus (NiV) requires rapid, inexpensive, and robust diagnostic tests to control spread of disease. Current state of the art technologies are slow and require laboratory infrastructure that may not be available in all endemic settings. Here we report the development and comparison of three rapid NiV molecular diagnostic tests based on reverse transcription recombinase-based isothermal amplification coupled with lateral flow detection. These tests include a simple and fast one-step sample processing step that inactivates the BSL-4 pathogen, enabling safe testing without the need for multi-step RNA purification. The rapid NiV tests targeted the Nucleocapsid protein (N) gene with analytical sensitivity down to 1,000 copies/μL for synthetic NiV RNA and did not cross-react with RNA of other flaviviruses or Chikungunya virus, which can clinically present with similar febrile symptoms. Two tests detected 50,000–100,000 TCID50/mL (100–200 RNA copies/reaction) of the two distinct strains of NiV, Bangladesh (NiVB) and Malaysia (NiVM), and took 30 min from sample to result, suggesting these tests are well suited for rapid diagnosis under resource-limited conditions due to rapidity, simplicity, and low equipment requirements. These Nipah tests represent a first step toward development of near-patient NiV diagnostics that are appropriately sensitive for first-line screening, sufficiently robust for a range of peripheral settings, with potential to be safely performed outside of biohazard containment facilities.

Phage therapy is one alternative to cure infections caused by antibiotic resistant bacteria. Due to the narrow host range of phages, hundreds to thousands of phages are required to cover the diversity of bacterial pathogens. In personalized phage therapy, fast selection of the phages for individual patients is essential for successful therapy. The aims of this study were to set up a rapid hydrogel-based liquid phage susceptibility assay (PST) for the selection of phages for therapeutic use and to establish a "ready-to-screen" plate concept, where phages are readily stored in hydrogel as small droplets in microtiter plate wells. We first tested four commercially available hydrogels (GrowDex, Askina, Purilon, and Intrasite) for their suitability as phage matrices in PSTs with four phages, two of which infecting Escherichia coli and two Staphylococcus aureus. Of these four hydrogels, GrowDex was the best matrix for PST, as it did not inhibit bacterial growth, released phages quickly when mixed with bacterial culture, and maintained phage viability well. We then optimized the assay for both optical density and microscopy readers using GrowDex as matrix with 23 bacterial strains representing 10 different species and 23 phages possessing different morphologies and genome sizes. When the bacterial growth was monitored by microscopy reader, the PST was executed in just 3 hours, and there was no need for overnight culturing bacterial cells prior to the assay, whereas using optical density reader, bacteria had to be pre-cultured overnight, and the assay time was five hours. Finally, we evaluated the effect of three different chemical stabilizers (trehalose, hyaluronic acid, and gelatin) in a six-month stability assay with six model phages. These phages assay behaved very differently in respect to the chemical stabilizers, and there was not a single stabilizer suitable for all phages. However, when gelatin (0.01%) or hyaluronic acid (0.2 mg/ml) was used as stabilizer, all tested phages were still considered as positives in PST after a sixmonth storage in 1 ml volume. In "ready-to-screen" plates, the differences in phage stabilities were even more profound, varying from two to six months for the most and least stable phages, respectively. [ABSTRACT FROM AUTHOR]

Phage therapy is one alternative to cure infections caused by antibiotic resistant bacteria. Due to the narrow host range of phages, hundreds to thousands of phages are required to cover the diversity of bacterial pathogens. In personalized phage therapy, fast selection of the phages for individual patients is essential for successful therapy. The aims of this study were to set up a rapid hydrogel-based liquid phage susceptibility assay (PST) for the selection of phages for therapeutic use and to establish a “ready-to-screen” plate concept, where phages are readily stored in hydrogel as small droplets in microtiter plate wells. We first tested four commercially available hydrogels (GrowDex, Askina, Purilon, and Intrasite) for their suitability as phage matrices in PSTs with four phages, two of which infecting Escherichia coli and two Staphylococcus aureus. Of these four hydrogels, GrowDex was the best matrix for PST, as it did not inhibit bacterial growth, released phages quickly when mixed with bacterial culture, and maintained phage viability well. We then optimized the assay for both optical density and microscopy readers using GrowDex as matrix with 23 bacterial strains representing 10 different species and 23 phages possessing different morphologies and genome sizes. When the bacterial growth was monitored by microscopy reader, the PST was executed in just 3 hours, and there was no need for overnight culturing bacterial cells prior to the assay, whereas using optical density reader, bacteria had to be pre-cultured overnight, and the assay time was five hours. Finally, we evaluated the effect of three different chemical stabilizers (trehalose, hyaluronic acid, and gelatin) in a six-month stability assay with six model phages. These phages assay behaved very differently in respect to the chemical stabilizers, and there was not a single stabilizer suitable for all phages. However, when gelatin (0.01%) or hyaluronic acid (0.2 mg/ml) was used as stabilizer, all tested phages were still considered as positives in PST after a six-month storage in 1 ml volume. In “ready-to-screen” plates, the differences in phage stabilities were even more profound, varying from two to six months for the most and least stable phages, respectively.

Objectives: This study aimed to report the protocol and results from the pilot phase of an opportunistic CP-based CD screening program in Barcelona, Spain.Methods: Three strategies according to recruitment approach were designed: passive, active and active-community. The study process consisted of signing the informed consent form, recording the patient’s data in a web-based database system, and performing the rapid test and blood collection on dry paper.Results: Nineteen pharmacies participated and 64 patients were included during the pilot phase of the study. The rapid diagnostic test (RDT) was positive in 2/64 (3.13%) cases. Of the 49 DBS samples that arrived at the laboratory, 22 (45%) were collected incorrectly. After quantitative and qualitative assessment of the program, the dry paper sample and passive strategy were ruled out.Conclusion: DBS sampling and the passive strategy are not suitable for CD screening in community pharmacies. There is a need to expand the number of participating pharmacies and individuals to determine whether conducting a RDT in community pharmacies is an effective screening method to increase access to CD diagnosis in a non-endemic area.

Highly virulent Klebsiella pneumoniae often causes invasive infections with high morbidity and mortality rates, posing an immense clinical challenge. Rapid and accurate detection of pathogenic bacteria is of great significance for treatment and preventive control. Conventional detection by polymerase chain reaction (PCR) is limited by a dependence on laboratory equipment and professional staff. Recombinase polymerase amplification (RPA) combined with a lateral flow strip (LFS) can rapidly amplify and visualize target genes in a short period of time. The aim of this study was to develop an RPA-LFS technique for detection of the K. pneumoniae virulence gene rmpA2. Primers were designed against conserved sequences specific to the virulence gene, and primer probe design was optimized by introducing base substitution to obtain a specific and sensitive primer-probe combination for clinical detection. We tested 65 actual samples collected from clinics to evaluate the performance of the newly established RPA-LFS system in comparison with conventional PCR methods and qPCR methods. The RPA-LFS assay was performed at for 25 min a constant temperature of 37°C, and results could be observed without instrumentation. The system could specifically identify highly virulent K. pneumoniae carrying the virulence gene rmpA2 with a minimum detection limit of 10−1 ng/μL and 10 copies/μL. For the 65 clinical samples tested, The RPA-LFS assay results were in complete agreement with the qPCR results and PCR results. The RPA-LFS assay provides a rapid, accurate, and simple method for identification of highly virulent K. pneumoniae carrying rmpA2. [ABSTRACT FROM AUTHOR]

Highly virulent Klebsiella pneumoniae often causes invasive infections with high morbidity and mortality rates, posing an immense clinical challenge. Rapid and accurate detection of pathogenic bacteria is of great significance for treatment and preventive control. Conventional detection by polymerase chain reaction (PCR) is limited by a dependence on laboratory equipment and professional staff. Recombinase polymerase amplification (RPA) combined with a lateral flow strip (LFS) can rapidly amplify and visualize target genes in a short period of time. The aim of this study was to develop an RPA-LFS technique for detection of the K. pneumoniae virulence gene rmpA2. Primers were designed against conserved sequences specific to the virulence gene, and primer probe design was optimized by introducing base substitution to obtain a specific and sensitive primer-probe combination for clinical detection. We tested 65 actual samples collected from clinics to evaluate the performance of the newly established RPA-LFS system in comparison with conventional PCR methods and qPCR methods. The RPA-LFS assay was performed at for 25 min a constant temperature of 37°C, and results could be observed without instrumentation. The system could specifically identify highly virulent K. pneumoniae carrying the virulence gene rmpA2 with a minimum detection limit of 10−1 ng/μL and 10 copies/μL. For the 65 clinical samples tested, The RPA-LFS assay results were in complete agreement with the qPCR results and PCR results. The RPA-LFS assay provides a rapid, accurate, and simple method for identification of highly virulent K. pneumoniae carrying rmpA2.

Dengue is among the most rapidly spreading arboviral disease in the world. A low-cost, easy to use point-of-care diagnostic tool for the detection and differentiation of dengue virus serotypes could improve clinical management, disease prevention, epidemiological surveillance, and outbreak monitoring, particularly in regions where multiple serotypes co-circulate. Despite widespread deployment, no commercial dengue antigen diagnostic test has proven effective in differentiating among dengue virus serotypes. In the current study, we first established mAb pairs and developed a multiplex lateral flow immunoassay for the simultaneous detection of the dengue viral NS1 antigen and identification of serotype. The proposed system, called Dengue serotype NS1 Multiplex LFIA, provides high sensitivity and specificity. In testing for JEV, ZIKV, YFV, WNV, and CHIKV, the multiplex LFIA gave no indication of cross- reactivity with cell culture supernatants of other flaviviruses or chikungunya virus. In analyzing 187 samples from patients suspected of dengue infection, the detection sensitivity for serotype D1 to D4 was 90.0%, 88.24%, 82.61%, and 83.33% and serotype specificity was 98.74%, 96.13%, 99.39%, and 97.04%, respectively. Our multiplex LFIA can also identify mono- and co-infection of different serotype of dengue viruses in mosquitoes. The proposed Multiplex LFIA provides a simple tool for the rapid detection of dengue serotypes and in the differential diagnosis of fever patients in regions where medical resources are limited and/or multiple DENVs co-circulate. [ABSTRACT FROM AUTHOR]

Canis lupus familiaris (domestic dog) represents a reliable sentinel for the occurrence of a well-established transmission cycle of Trypanosoma cruzi among wild mammals in the surroundings and, consequently, where the risk of human infection exists. Serological diagnosis is the chosen method to identify T. cruzi infection in dogs that, in Brazil, rarely present positive parasitological tests. The use of recombinant chimeric parasitic antigens results in a sensitive and specific serological diagnostic test in contrast to the use of crude T. cruzi antigens. Our objective was to evaluate the Chagas/Bio-Manguinhos Lateral Flow Immunochromatographic Rapid Test (Chagas-LFRT) for the diagnosis of T. cruzi infection in domestic dogs and the potential of application of this diagnostic platform to wild canid species. Two recombinant proteins (IBMP-8.1 and IBMP-8.4) that displayed the best performance in the enzyme immunoassay (ELISA) in previous studies were tested in a platform with two diagnostic bands. A panel of 281 dog serum samples was evaluated: 133 positive for T. cruzi by serological diagnosis, including 20 samples with positive blood cultures belonging to different discrete typing units (DTUs); 129 negative samples; and 19 samples from dogs infected by other trypanosomatids: Leishmania infantum , Trypanosoma rangeli , Trypanosoma caninum and Crithidia mellificae , in addition to samples infected by Anaplasma platys , Dirofilaria immitis and Erlichia sp. that were employed to evaluate eventual cross-reactions. We also evaluated the Chagas-LFRT to detect T. cruzi infection in 9 serum samples from six wild canid species. We observed that the intensity pattern of the bands was directly proportional to the serological titer observed in IFAT. The sensitivity was 94%, the specificity was 91% according to the ROC curve, and the defined cutoff was an optical density of 4.8. The agreement obtained was considered substantial by the kappa analysis (84%). From T. cruzi positive hemoculture samples, 88.9% were positive by Chagas-LFRT. The test was efficient in recognizing infections by five of the six T. cruzi DTUs. Cross-reactions were not observed in infections by L. infantum , T. rangeli , T. caninum and D. immitis ; however, they were observed in sera of dogs infected by Crithidia mellificae , Anaplasma sp. and Erlichia sp. A strong reaction was observed when serum samples from wild canids were submitted to the Protein A affinity test, confirming its applicability for these species. This test will allow rapid preventive actions in areas with high risk to the emergence of Chagas disease in a safer, reliable, low-cost and immediate manner, without the need for more complex laboratory tests. [ABSTRACT FROM AUTHOR]

Background: Although RT-qPCR remains the gold-standard for COVID-19 diagnosis, anti-SARS-CoV-2 serology-based assays have been widely used during 2020 as an alternative for individual and mass testing, and are currently used for seroprevalence studies. Objective: To study the clinical performance of seven commercial serological tests for COVID-19 diagnosis available in South America. Methods: We conducted a blind evaluation of five lateral-flow immunoassays (LFIA) and two enzyme-linked immunosorbent assays (ELISAs) for detecting anti-SARS-CoV-2 antibodies. Results: We found no statistically significant differences among ELISA kits and LFIAs for anti-SARS-CoV-2 IgG sensitivity (values ranging from 76.4% to 83.5%) and specificity (100% for the seven serological assays). For anti-SARS-CoV-2 IgM, the five LFIAs have a significantly higher sensitivity for samples collected 15 days after the first time RT-qPCR positive test, with values ranging from 47.1% to 88.2%; moreover, the specificity varied from 85% to 100%, but the only LFIA brand with a 100% specificity had the lowest sensitivity. Conclusion: The diagnostic performance of the seven serological tests was acceptable for the seven brands tested for anti-SARS-CoV-2 IgG detection for seroprevalence screening purposes. On the other hand, our results show the lack of accuracy of anti-SARS-CoV-2 IgM detection in LFIAs as a tool for SARS-CoV-2 acute-phase infection diagnosis. [ABSTRACT FROM AUTHOR]

Dengue is among the most rapidly spreading arboviral disease in the world. A low-cost, easy to use point-of-care diagnostic tool for the detection and differentiation of dengue virus serotypes could improve clinical management, disease prevention, epidemiological surveillance, and outbreak monitoring, particularly in regions where multiple serotypes co-circulate. Despite widespread deployment, no commercial dengue antigen diagnostic test has proven effective in differentiating among dengue virus serotypes. In the current study, we first established mAb pairs and developed a multiplex lateral flow immunoassay for the simultaneous detection of the dengue viral NS1 antigen and identification of serotype. The proposed system, called Dengue serotype NS1 Multiplex LFIA, provides high sensitivity and specificity. In testing for JEV, ZIKV, YFV, WNV, and CHIKV, the multiplex LFIA gave no indication of cross- reactivity with cell culture supernatants of other flaviviruses or chikungunya virus. In analyzing 187 samples from patients suspected of dengue infection, the detection sensitivity for serotype D1 to D4 was 90.0%, 88.24%, 82.61%, and 83.33% and serotype specificity was 98.74%, 96.13%, 99.39%, and 97.04%, respectively. Our multiplex LFIA can also identify mono- and co-infection of different serotype of dengue viruses in mosquitoes. The proposed Multiplex LFIA provides a simple tool for the rapid detection of dengue serotypes and in the differential diagnosis of fever patients in regions where medical resources are limited and/or multiple DENVs co-circulate.

BackgroundAlthough RT-qPCR remains the gold-standard for COVID-19 diagnosis, anti-SARS-CoV-2 serology-based assays have been widely used during 2020 as an alternative for individual and mass testing, and are currently used for seroprevalence studies.ObjectiveTo study the clinical performance of seven commercial serological tests for COVID-19 diagnosis available in South America.MethodsWe conducted a blind evaluation of five lateral-flow immunoassays (LFIA) and two enzyme-linked immunosorbent assays (ELISAs) for detecting anti-SARS-CoV-2 antibodies.ResultsWe found no statistically significant differences among ELISA kits and LFIAs for anti-SARS-CoV-2 IgG sensitivity (values ranging from 76.4% to 83.5%) and specificity (100% for the seven serological assays). For anti-SARS-CoV-2 IgM, the five LFIAs have a significantly higher sensitivity for samples collected 15 days after the first time RT-qPCR positive test, with values ranging from 47.1% to 88.2%; moreover, the specificity varied from 85% to 100%, but the only LFIA brand with a 100% specificity had the lowest sensitivity.ConclusionThe diagnostic performance of the seven serological tests was acceptable for the seven brands tested for anti-SARS-CoV-2 IgG detection for seroprevalence screening purposes. On the other hand, our results show the lack of accuracy of anti-SARS-CoV-2 IgM detection in LFIAs as a tool for SARS-CoV-2 acute-phase infection diagnosis.

Canis lupus familiaris (domestic dog) represents a reliable sentinel for the occurrence of a well-established transmission cycle of Trypanosoma cruzi among wild mammals in the surroundings and, consequently, where the risk of human infection exists. Serological diagnosis is the chosen method to identify T. cruzi infection in dogs that, in Brazil, rarely present positive parasitological tests. The use of recombinant chimeric parasitic antigens results in a sensitive and specific serological diagnostic test in contrast to the use of crude T. cruzi antigens. Our objective was to evaluate the Chagas/Bio-Manguinhos Lateral Flow Immunochromatographic Rapid Test (Chagas-LFRT) for the diagnosis of T. cruzi infection in domestic dogs and the potential of application of this diagnostic platform to wild canid species. Two recombinant proteins (IBMP-8.1 and IBMP-8.4) that displayed the best performance in the enzyme immunoassay (ELISA) in previous studies were tested in a platform with two diagnostic bands. A panel of 281 dog serum samples was evaluated: 133 positive for T. cruzi by serological diagnosis, including 20 samples with positive blood cultures belonging to different discrete typing units (DTUs); 129 negative samples; and 19 samples from dogs infected by other trypanosomatids: Leishmania infantum, Trypanosoma rangeli, Trypanosoma caninum and Crithidia mellificae, in addition to samples infected by Anaplasma platys, Dirofilaria immitis and Erlichia sp. that were employed to evaluate eventual cross-reactions. We also evaluated the Chagas-LFRT to detect T. cruzi infection in 9 serum samples from six wild canid species. We observed that the intensity pattern of the bands was directly proportional to the serological titer observed in IFAT. The sensitivity was 94%, the specificity was 91% according to the ROC curve, and the defined cutoff was an optical density of 4.8. The agreement obtained was considered substantial by the kappa analysis (84%). From T. cruzi positive hemoculture samples, 88.9% were positive by Chagas-LFRT. The test was efficient in recognizing infections by five of the six T. cruzi DTUs. Cross-reactions were not observed in infections by L. infantum, T. rangeli, T. caninum and D. immitis; however, they were observed in sera of dogs infected by Crithidia mellificae, Anaplasma sp. and Erlichia sp. A strong reaction was observed when serum samples from wild canids were submitted to the Protein A affinity test, confirming its applicability for these species. This test will allow rapid preventive actions in areas with high risk to the emergence of Chagas disease in a safer, reliable, low-cost and immediate manner, without the need for more complex laboratory tests.

Bacterial acute pneumonia is responsible for an extremely large burden of death worldwide and diagnosis is paramount in the management of patients. While multidrug-resistant bacteria is one of the biggest health threats in the coming decades, clinicians urgently need access to novel diagnostic technologies. In this review, we will first present the already existing and largely used techniques that allow identifying pathogen-associated pneumonia. Then, we will discuss the latest and most promising technological advances that are based on connected technologies (artificial intelligence-based and Omics-based) or rapid tests, to improve the management of lung infections caused by pathogenic bacteria. We also aim to highlight the mutual benefits of fundamental and clinical studies for a better understanding of lung infections and their more efficient diagnostic management.

Bacterial acute pneumonia is responsible for an extremely large burden of death worldwide and diagnosis is paramount in the management of patients. While multidrug-resistant bacteria is one of the biggest health threats in the coming decades, clinicians urgently need access to novel diagnostic technologies. In this review, we will first present the already existing and largely used techniques that allow identifying pathogen-associated pneumonia. Then, we will discuss the latest and most promising technological advances that are based on connected technologies (artificial intelligence-based and Omics-based) or rapid tests, to improve the management of lung infections caused by pathogenic bacteria. We also aim to highlight the mutual benefits of fundamental and clinical studies for a better understanding of lung infections and their more efficient diagnostic management. [ABSTRACT FROM AUTHOR]

Trichinella spp., are amongst the most widespread parasitic nematodes, primarily live in the muscles of a wide range of vertebrate animals and humans. Human infection occurs by ingestion of raw or undercooked meat containing Trichinella larvae. Accurate diagnosis of Trichinella spp. infection in domestic animals is crucial for the effective prevention and control of human trichinellosis. In the present study, a simple, rapid and accurate diagnostic assay was developed combining recombinase polymerase amplification and a lateral flow strip (LF-RPA) to detect Trichinella spp. infection. The LF-RPA assay targets Trichinella spp. mitochondrial small-subunit ribosomal RNA (rrnS) gene and can detect as low as 100 fg DNA of Trichinella strains, which was approximately 10 times more sensitive than a conventional PCR assay. The LF-RPA assay can be performed within 10–25 min, at a wide range of temperatures (25–45°C) and showed no cross-reactivity with DNA of other parasites and related host species of Trichinella. The performance of the LF-RPA assay in the presence of high concentration of PCR inhibitor was better than that of a conventional PCR assay. Results obtained by LF-RPA assay for the detection of experimentally infected mice were comparable to the results obtained by using a conventional PCR, achieving 100% specificity and high sensitivity. These results present the developed LF-RPA assay as a new simple, specific, sensitive, rapid and convenient method for the detection of Trichinella infection in domestic animals.

Trichinella spp., are amongst the most widespread parasitic nematodes, primarily live in the muscles of a wide range of vertebrate animals and humans. Human infection occurs by ingestion of raw or undercooked meat containing Trichinella larvae. Accurate diagnosis of Trichinella spp. infection in domestic animals is crucial for the effective prevention and control of human trichinellosis. In the present study, a simple, rapid and accurate diagnostic assay was developed combining recombinase polymerase amplification and a lateral flow strip (LF-RPA) to detect Trichinella spp. infection. The LF-RPA assay targets Trichinella spp. mitochondrial small-subunit ribosomal RNA (rrnS) gene and can detect as low as 100 fg DNA of Trichinella strains, which was approximately 10 times more sensitive than a conventional PCR assay. The LF-RPA assay can be performed within 10–25 min, at a wide range of temperatures (25–45°C) and showed no cross-reactivity with DNA of other parasites and related host species of Trichinella. The performance of the LF-RPA assay in the presence of high concentration of PCR inhibitor was better than that of a conventional PCR assay. Results obtained by LF-RPA assay for the detection of experimentally infected mice were comparable to the results obtained by using a conventional PCR, achieving 100% specificity and high sensitivity. These results present the developed LF-RPA assay as a new simple, specific, sensitive, rapid and convenient method for the detection of Trichinella infection in domestic animals. [ABSTRACT FROM AUTHOR]

BackgroundWestern blot (WB) assay is considered the gold standard test for HIV infection confirmation. However, it requires technical expertise and is quite time-consuming. WHO recommends blood-based rapid diagnosis to achieve same-day test and treatment. However, this rapid testing strategy has not been promoted worldwide due to inadequate research evaluating the effectiveness of rapid tests (RTs) as an alternative confirmatory HIV test for WB. This study aims to compare the diagnostic performance of rapid HIV tests compared with WB.MethodsPubMed and Web of Science were searched for publications on rapid HIV tests using blood specimen. A meta-analysis was performed to quantitatively evaluate the diagnostic performance of rapid HIV tests compared with the WB assay in terms of pooled sensitivity, specificity, area under summary receiver operating characteristic (SROC) curve, and diagnostic odds ratio (DOR).ResultsTwenty articles involving 27,343 fresh specimens for rapid HIV tests were included in the meta-analysis. Regarding Capillus HIV-1/HIV-2, the pooled sensitivity, specificity, area under SROC curve, and DOR derived from six studies were 0.999 (95% CI, 0.956–1.000), 0.999 (95% CI, 0.991–1.00), 1.00 (95% CI, 0.99–1.00), and 1.0 × 106 (95% CI, 2.6 × 104–3.9 × 107) compared with the WB assay, respectively. With respect to Determine HIV-1/2, the pooled sensitivity, specificity area under SROC, and DOR derived from eight studies were 1.00 (95% CI, 0.789–1.000), 0.992 (95% CI, 0.985–0.996), 1.00 (95% CI, 0.99–1.00), and 1.8 × 106 (95% CI 406.049–7.8 × 109) compared with the WB assay, respectively. Regarding two-step serial RTs, the pooled sensitivity, specificity area under SROC, and DOR derived from eight studies were 0.998 (95% CI, 0.991–1.000), 0.998 (95% CI, 0.994–0.999), and 1.00 (95% CI 0.99–1.00) compared with the WB assay, respectively.ConclusionOur meta-analysis results may provide evidenced-based support for substituting RT for WB. Blood-based rapid HIV tests have comparable sensitivity and specificity to WB for HIV early therapy.

Background: Western blot (WB) assay is considered the gold standard test for HIV infection confirmation. However, it requires technical expertise and is quite time-consuming. WHO recommends blood-based rapid diagnosis to achieve same-day test and treatment. However, this rapid testing strategy has not been promoted worldwide due to inadequate research evaluating the effectiveness of rapid tests (RTs) as an alternative confirmatory HIV test for WB. This study aims to compare the diagnostic performance of rapid HIV tests compared with WB. Methods: PubMed and Web of Science were searched for publications on rapid HIV tests using blood specimen. A meta-analysis was performed to quantitatively evaluate the diagnostic performance of rapid HIV tests compared with the WB assay in terms of pooled sensitivity, specificity, area under summary receiver operating characteristic (SROC) curve, and diagnostic odds ratio (DOR). Results: Twenty articles involving 27,343 fresh specimens for rapid HIV tests were included in the meta-analysis. Regarding Capillus HIV-1/HIV-2, the pooled sensitivity, specificity, area under SROC curve, and DOR derived from six studies were 0.999 (95% CI, 0.956–1.000), 0.999 (95% CI, 0.991–1.00), 1.00 (95% CI, 0.99–1.00), and 1.0 × 106 (95% CI, 2.6 × 104–3.9 × 107) compared with the WB assay, respectively. With respect to Determine HIV-1/2, the pooled sensitivity, specificity area under SROC, and DOR derived from eight studies were 1.00 (95% CI, 0.789–1.000), 0.992 (95% CI, 0.985–0.996), 1.00 (95% CI, 0.99–1.00), and 1.8 × 106 (95% CI 406.049–7.8 × 109) compared with the WB assay, respectively. Regarding two-step serial RTs, the pooled sensitivity, specificity area under SROC, and DOR derived from eight studies were 0.998 (95% CI, 0.991–1.000), 0.998 (95% CI, 0.994–0.999), and 1.00 (95% CI 0.99–1.00) compared with the WB assay, respectively. Conclusion: Our meta-analysis results may provide evidenced-based support for substituting RT for WB. Blood-based rapid HIV tests have comparable sensitivity and specificity to WB for HIV early therapy. [ABSTRACT FROM AUTHOR]

The presence of Vibrio cholerae in the environment is key to understanding the epidemiology of cholera. The gold standard for laboratory confirmation of V. cholerae from water is a culture method, but this requires laboratory infrastructure. A rapid diagnostic test that is simple, inexpensive, and can be deployed widely would be useful for confirming V. cholerae in samples of environmental water. Here, we evaluated a dipstick test to detect V. cholerae O1 and O139 from environmental water samples in spiked samples and under field conditions. When environmental water samples were incubated in alkaline peptone water for 24 hours at room temperature, samples spiked with as few as 10 CFU could be detected using the dipstick test. When compared to culture, the test was 89% sensitive and 100% specific with environmental samples.