RAPID POINT-OF-CARE TESTING FOR DETECTION OF ANTIBODIES TO TOXOPLASMA GONDII IN BLACK VULTURES AND RING-BILLED GULLS FROM PENNSYLVANIA.

Toxoplasma gondii is a zoonotic protozoan parasite that infects most warm-blooded animals, including birds. Scavenging birds are epidemiologically important hosts because they can serve as indicators of environmental T. gondii levels. A rapid point-of-care (POC) test that detects antibodies to T. gondii in humans is commercially available. In this research, we assessed the ability of the human POC test to detect anti-T. gondii antibodies in 106 black vultures (Coragyps atratus) and 23 ring-billed gulls (Larus delawarensis) from Pennsylvania, USA. Serum samples were tested with the POC test and compared to the modified agglutination test (MAT) in a blinded study. Overall, anti-T. gondii antibodies were detected in 2.8% (3/106) of black vultures and 60.9% (14/23) of ring-billed gulls by the POC test. One false-positive POC test occurred in a black vulture that was negative by MAT. False-negative results were obtained in 2 black vultures and 4 ring-billed gulls that had MAT titers of 1:25 or 1:50. The sensitivity and specificity of the POC for both black vultures and ring-billed gulls combined were 95.7% and 95.5%, respectively. This is the first study using human POC tests to detect antibodies to T. gondii in birds. Further study of the rapid test as a screening tool for serological surveillance of T. gondii in birds is warranted.

Seroprevalence of leptospirosis among stray dogs in Bosnia and Herzegovina.

Leptospirosis is a (re) emerging zoonosis that occurs worldwide. This study aimed to assess seroprevalence of leptospirosis and to identify the most common reactive serovars and risk factors for seropositivity in apparently healthy stray dogs of unknown vaccination status in the Sarajevo region of Bosnia and Herzegovina. Positive microscopic agglutination test titres (≥ 1:25) were detected in 3.87% (156/4028) of samples and most of the sera reacted against one serovar (85.9%). Dogs were most commonly reactive to Canicola (40.4%) and Hardjo (33.3%), followed by Pomona (15.4%) Tarassovi (14.7%), Icterohaemorrhagiae (8.3%), Grippotyphosa (5.8%), Bratislava (1.3%) and Saxkoebing (0.6%). Dogs older than one year had higher odds of seropositivity compared to younger dogs. The seropositivity was higher in spring and autumn than in summer. These results advocate for the need of a control strategy for this zoonosis in the country, which should include sero-surveillance, monitoring, and the inclusion of additional serovars in the testing.

Leptospira infection and carrier survey on rats from wet market areas in Kuala Lumpur, Malaysia.

BACKGROUND OBJECTIVES: Leptospirosis is an important zoonotic infection that has caused significant mortality and morbidity worldwide. This disease is endemic in Malaysia and as a developing tropical country, leptospirosis is concerning as it threatens Malaysian public health and the country's economic sectors. However, there is limited information on leptospirosis in Malaysia, especially regarding leptospiral seroepidemiology among carriers in Malaysia. Therefore, more epidemiological information on the source of the disease and reservoir are needed for better disease control and source intervention. The objectives of this study are to gather information on Leptospira infection and the carrier status of rats captured from selected wet markets of Kuala Lumpur metropolitan city in Malaysia. METHODS: Live rat trappings were performed in four major wet markets in Kuala Lumpur, namely, Pudu, Chow Kit, Datuk Keramat, and Petaling Street. Animal samplings were performed for 12 months in 2017, where blood and kidney samples were collected and tested for anti-leptospiral antibodies via Microscopic Agglutination Test (MAT) and pathogenic Leptospira screening via Polymerase Chain Reaction (PCR) amplification offlaB gene. RESULTS: MAT showed that 34.7% (n = 50/144) of the captured rats were positive for anti-leptospiral antibody of which the most prominent serovar was Malaya followed by a local strain, IMR LEP 175. In parallel, 50 rats were also positive for pathogenic Leptospira DNA. INTERPRETATION CONCLUSION: This study showed that there are persistent Leptospira infections among rats in Kuala Lumpur wet markets and these rats are important reservoir hosts for the bacteria.

Detection of tetanus toxoid with iron magnetic nanobioprobe.

Diagnosis of diseases with low facilities, speed, accuracy and sensitivity is an important matter in treatment. Bioprobes based on iron oxide nanoparticles are a good candidate for early detection of deadly and infectious diseases such as tetanus due to their high reactivity, biocompatibility, low production cost and sample separation under a magnetic field. In this study, silane groups were coated on surface of iron oxide nanoparticles using tetraethoxysilane (TEOS) hydrolysis. Also, NH2groups were generated on the surface of silanized nanoparticles using 3-aminopropyl triethoxy silane (APTES). Antibody was immobilized on the surface of silanized nanoparticles using TCT trichlorothriazine as activator. Silanization and stabilized antibody were investigated by using of FT-IR, EDX, VSM, SRB technique. UV/vis spectroscopy, fluorescence, agglutination test and ELISA were used for biosensor performance and specificity. The results of FT-IR spectroscopy showed that Si-O-Si and Si-O-Fe bonds and TCT chlorine and amine groups of tetanus anti-toxoid antibodies were formed on the surface of iron oxide nanoparticles. The presence of Si, N and C elements in EDX analysis confirms the silanization of iron oxide nanoparticles. VSM results showed that the amount of magnetic nanoparticles after conjugation is sufficient for biological applications. Antibody stabilization on nanoparticles increased the adsorption intensity in the uv/vis spectrometer. The fluorescence intensity of nano bioprobe increased in the presence of 10 ng ml-1. Nanobio probes were observed as agglomerates in the presence of tetanus toxoid antigen. The presence of tetanus antigen caused the formation of antigen-nanobioprobe antigen complex. Identification of this complex by HRP-bound antibody confirmed the specificity of nanobioprobe. Tetanus magnetic nanobioprobe with a diagnostic limit of 10 ng ml-1of tetanus antigen in a short time can be a good tool in LOC devices and microfluidic chips.

Levels of Cytokines in Leptospirosis Patients with Different Serovars and rfb Locus.

Leptospirosis has a wide spectrum of clinical manifestations ranging from mild to severe disease. The cytokine response is considered one of the key drivers for this varying manifestation. The different cytokine response observed in patients with leptospirosis could be due to the variation of infecting serovars. Since the rfb locus codes for the lipopolysaccharide synthesis of the bacterial cell wall, which also determines the serovar, this locus may play a role in driving a specific cytokine response in the host. We investigated 12 commonly used cytokine profiles in serum samples of culture, microscopic agglutination test (MAT), or polymerase chain reaction (PCR)-positive patients with leptospirosis. The sequences of the rfb locus in culture-positive samples were generated from whole genome sequencing and serovar status was drawn from original data published. Isolated cultures were subjected to whole genome sequencing using the PacBio RS II system, and the resulting data were used to determine the species. The recovered genomic data were annotated with the Rapid Annotation using Subsystem Technology (RAST) subsystem, and the rfb locus was extracted. The cytokine analysis was carried out using the Qiagen human ELISA kit. Eighteen samples were found to be positive by culture, while the other 7 samples were positive by PCR or MAT. Infections from Leptospira interrogans serovar Autumnalis (5), Pyrogens (3), Icterohaemorrhagiae (1) Leptospira borgpetersenii (all 7 samples clustered in same clonal group with serovar status not determined), Leptospira weilii (1 with serovar status not determined), and Leptospira kirschneri serovar Grippotyphosa (1) were included in the analysis. Three patients [infected with Leptospira interrogansserovar Autumnalis (2) and Pyrogens (1)] and 2 MAT-positive patients (highest titer against serovar Bratislava of L.interrognas) were reported to have severe clinical manifestations, while the rest had mild to moderate symptoms. Although the serum cytokine concentration of patients with severe clinical manifestation was comparatively higher, a statistically significant difference was observed only for interleukin (IL)-1ß (P < 0.05). IL-10/tumor necrosis factor-alpha (TNF-α) ratio was high in patients with severe complications. In general, patients infected with L. interrogans showed higher concentration of cytokines compared to L. borgpetersenii.

Toxoplasma gondii Survey in Waterfowl and Gulls from Eight USA States.

Sera from 391 waterbirds from eight USA states were tested for Toxoplasma gondii antibodies using the modified agglutination test. Fifteen different waterbird species (26.6%; n=104) were seropositive. Of the adults, 25.4% (n=52) showed a significantly higher T. gondii seroprevalence compared with juveniles (13.4%; n=17); however, sex was not a significant factor.

Diagnosis of human leptospirosis: systematic review and meta-analysis of the diagnostic accuracy of the Leptospira microscopic agglutination test, PCR targeting Lfb1, and IgM ELISA to Leptospira fainei serovar Hurstbridge.

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.

Investigation of anti-Leptospira spp. antibodies and leptospiruria in cats attended to a veterinary teaching hospital in southern Brazil.

Leptospirosis is a bacterial zoonosis that affects both humans and animals worldwide. Currently, it is known that cats may be susceptible to infection. This study aims to investigate the presence of anti-Leptospira spp. antibodies and leptospiruria in cats, using Microscopic Agglutination Test (MAT) and Real-time Polymerase Chain Reaction (PCR) techniques, respectively. A total of 76 cats, undergoing comprehensive anamnesis, general physical examination, and complementary exams were included in the investigation. Among the 76 cats tested, 9.2% (7/76) exhibited the presence of anti-Leptospira spp. antibodies, while Leptospira spp. DNA was detected in at 1.3% (1/76) of the evaluated urine samples. No significant associations were observed between the serological and molecular diagnostic results and the assessed variables, including clinical data and laboratory results of cats testing positive. This study provides insight into the occurrence of Leptospira spp. infection and leptospiruria in cats treated at a veterinary teaching hospital in southern Brazil.

A novel Bayesian Latent Class Model (BLCM) evaluates multiple continuous and binary tests: A case study for Brucella abortus in dairy cattle.

Bovine brucellosis, primarily caused by Brucella abortus, severely affects both animal health and human well-being. Accurate diagnosis is crucial for designing informed control and prevention measures. Lacking a gold standard test makes it challenging to determine optimal cut-off values and evaluate the diagnostic performance of tests. In this study, we developed a novel Bayesian Latent Class Model that integrates both binary and continuous testing outcomes, incorporating additional fixed (parity) and random (farm) effects, to calibrate optimal cut-off values by maximizing Youden Index. We tested 651 serum samples collected from six dairy farms in two regions of Henan Province, China with four serological tests: Rose Bengal Test, Serum Agglutination Test, Fluorescence Polarization Assay, and Competitive Enzyme-Linked Immunosorbent Assay. Our analysis revealed that the optimal cut-off values for FPA and C-ELISA were 94.2 mP and 0.403 PI, respectively. Sensitivity estimates for the four tests ranged from 69.7% to 89.9%, while specificity estimates varied between 97.1% and 99.6%. The true prevalences in the two study regions in Henan province were 4.7% and 30.3%. Parity-specific odds ratios for positive serological status ranged from 1.2 to 2.2 for different parity groups compared to primiparous cows. This approach provides a robust framework for validating diagnostic tests for both continuous and discrete tests in the absence of a gold standard test. Our findings can enhance our ability to design targeted disease detection strategies and implement effective control measures for brucellosis in Chinese dairy farms.

Modelling variation in test sensitivity for monitoring leptospirosis in beef cattle.

When Bayesian latent class analysis is used for diagnostic test data in the absence of a gold standard test, it is common to assume that any unknown test sensitivities and specificities are constant across different populations. Indeed this assumption is often necessary for model identifiability. However there are a number of practical situations, depending on the type of test and the nature of the disease, where this assumption may not be true. We present a case study of using a microscopic agglutination test to diagnose leptospiroris infection in beef cattle, which strongly suggests that sensitivity in particular varies among herds. We develop and fit an alternative model in which sensitivity is related to within-herd prevalence, and discuss the statistical and epidemiological implications.

Accuracy of the direct agglutination test for diagnosis of visceral leishmaniasis: a systematic review and meta-analysis.

BACKGROUND: Parasitological investigation of bone marrow, splenic or lymph node aspirations is the gold standard for the diagnosis of visceral leishmaniasis (VL). However, this invasive test requires skilled clinical and laboratory staff and adequate facilities, and sensitivity varies depending on the tissue used. The direct agglutination test (DAT) is a serological test that does not need specialised staff, with just minimal training required. While previous meta-analysis has shown DAT to have high sensitivity and specificity when using parasitology as the reference test for diagnosis, meta-analysis of DAT compared to other diagnostic techniques, such as PCR and ELISA, that are increasingly used in clinical and research settings, has not been done. METHODS: We conducted a systematic review to determine the diagnostic performance of DAT compared to all available tests for the laboratory diagnosis of human VL. We searched electronic databases including Medline, Embase, Global Health, Scopus, WoS Science Citation Index, Wiley Cochrane Central Register of Controlled Trials, Africa-Wide Information, LILACS and WHO Global Index. Three independent reviewers screened reports and extracted data from eligible studies. A meta-analysis estimated the diagnostic sensitivity and specificity of DAT. RESULTS: Of 987 titles screened, 358 were selected for full data extraction and 78 were included in the analysis, reporting on 32,822 participants from 19 countries. Studies included were conducted between 1987-2020. Meta-analysis of studies using serum and DAT compared to any other test showed pooled sensitivity of 95% (95%CrI 90-98%) and pooled specificity of 95% (95%CrI 88-98%). Results were similar for freeze-dried DAT and liquid DAT when analysed separately. Sensitivity was lower for HIV-positive patients (90%, CrI 59-98%) and specificity was lower for symptomatic patients (70%, CrI 43-89%). When comparing different geographical regions, the lowest median sensitivity (89%, CrI 67-97%) was in Western Asia (five studies). CONCLUSIONS: This systematic review and meta-analysis demonstrates high estimated pooled sensitivity and specificity of DAT for diagnosis of VL, although sensitivity and specificity were lower for different patient groups and geographical locations. This review highlights the lack of standardisation of DAT methods and preparations, and the lack of data from some important geographical locations. Future well-reported studies could provide better evidence to inform test implementation for different patient populations and use cases. PROSPERO REGISTRATION: CRD42021240830.

Serological investigation of brucella infection using a dipstick assay among individuals with unexplained fever in farming-pastoral areas of Xinjiang, China.

OBJECTIVE: To evaluate the brucellosis detection of the dipstick assay coated with LPS antigen from Brucella melitensis vaccine strain M5 compared with Rose Bengal test (RB) and serum agglutination test (SAT), and investigate the brucella infection with the dipstick assay among people with unexplained fever in farming-pastoral areas of Xinjiang, China. METHODS: The dipstick assay was repeated to verify 130 positive and 200 negative serum samples, which had been confirmed by RB and SAT, for sensitivity and specificity analysis. Subsequently, 313 sera from people with unexplained fever in farming-pastoral areas including 6 counties in 3 regions where brucellosis is endemic and 200 sera from nonendemic city area (Urumqi City) in Xinjiang were detected with the dipstick assay for population infection rate survey. RESULTS: Sensitivity and specificity was 100% and 97% respectively with dipstick assay compared with RB and SAT. The average positive rate of sera from people with unexplained fever from farming-pastoral areas in Xinjiang was 18.5% (58/313) and the highest was 22.5% (9/40). CONCLUSIONS: The proportion of brucellosis infection among individuals with fever of unknown origin is relatively high in agricultural and pastoral areas of Xinjiang. The dipstick assay has a series of advantages such as low cost and fast speed, which make it suitable for the primary screening of high-risk populations.

Occurrence of serological reactions for Leptospira spp. in donkeys and mules from Minas Gerais, Brazil.

Research concerning leptospirosis in donkeys and mules has been neglected around the world. Therefore, the aim of this study was to investigate the epidemiological situation of the prevalence of anti-Leptospira spp. antibodies in donkeys and mules from the state of Minas Gerais, Brazil. Blood serum samples were collected from 180 animals (109 donkeys and 71 mules) in two rural properties from the state of Minas Gerais, Brazil, and then submitted to a microscopic agglutination test (MAT). Urea and creatinine values were also quantified. Epidemiological variables such as age, breeding system, contact with other animal species, source of water and food, vaccination against leptospirosis, presence of reproductive alterations, and rodent control were also investigated. From 180 samples collected, 39 (21.67%) showed positive results in the MAT, at a dilution ≥ 1:100. Some animals were reactive for more than one serovar. The serovar Tarassovi was the most frequent (14.07%), followed by Hardjo (11.85%) and Wolffi (11.11%). There was a statistically significant difference between animals from 0 to 3 years of age reactive in the MAT in comparison to the other age groups. Most of the animals had urea and creatinine concentrations within the acceptable reference limit; however, there was a significant increase in creatinine levels in some of the test animals. The studied properties showed differences in some epidemiological aspects such as vaccination of the animals, presence of reproductive problems in the herd, and rodent control. Such aspects pointed as risk factors that may influence the frequency of positive serological results in property 1. The present study demonstrated that the prevalence of leptospirosis in donkeys and mules is high and several serovars are being maintained by these animals, representing a potential public health risk.

USE OF A POINT OF CARE TEST TO DETERMINE THE PREVALENCE OF ANTIBODIES TO TOXOPLASMA GONDII IN BLACK BEARS FROM NORTH CAROLINA AND PENNSYLVANIA.

Toxoplasma gondii is an important protozoan parasite of humans and animals throughout the world. Black bears are among the animals with the highest seroprevalence of T. gondii in the United States. A rapid point of care (POC) test is commercially available to detect antibodies to T. gondii in humans. We evaluated the utility of the POC test to detect anti-T. gondii antibodies in 100 wild black bears from North Carolina (n = 50) and Pennsylvania (n = 50). In a blind study, sera were tested by the POC test, and results were compared to the modified agglutination test (MAT). Overall, anti-T. gondii antibodies were detected in 76% (76/100) black bears by both MAT and POC tests. One false positive and one false negative result in the POC test were obtained in bears from Pennsylvania. The sensitivity and specificity of the POC test were both 99% when compared to the MAT. Results from our study indicate the POC test could be a useful screening tool for serological surveillance of T. gondii in black bears.

Accuracy of serological tests in diagnosing mucosal leishmaniasis.

In this serum panel-based study, we evaluated the accuracy of serological tests originally developed for visceral leishmaniasis (VL), for diagnosis of mucosal leishmaniasis (ML). A total of five tests were evaluated, four of which are registered at the National Agency of Sanitary Surveillance (Agência Nacional de Vigilância Sanitária-ANVISA) (RIDASCREEN® Leishmania Ab from R-Biopharm AG., Leishmania ELISA IgG + IgM from Vircell S.L., IFI Leishmaniose Humana-BioManguinhos, and IT-LEISH® from Bio-Rad Laboratories, Inc.), and the other a direct agglutination test (DAT-LPC) prototype kit developed at Fiocruz. The panel was composed of 40 serum samples from patients with confirmed ML and 20 from patients with mucosal involvement and negative parasitological/molecular tests for leishmaniasis and confirmation of another etiology. All cases were treated from 2009 to 2016 in a referral center for leishmaniasis in Belo Horizonte, Minas Gerais, Brazil (Instituto René Rachou, Fiocruz). Diagnostic accuracy, based on the cut-off point for VL diagnosis, was 86.2% with RIDASCREEN® Leishmania Ab, 73.3% with Leishmania ELISA IgG + IgM, and 66.7% with IFI Leishmaniose Humana, while IT-LEISH® and DAT-LPC had the lowest accuracy (38.3%), despite high specificity (100% and 95%, respectively). New cut-off points defined with sera from ML patients improved accuracy from 86.2 to 89% (p = 0.64) and 73.3 to 88% (p = 0.04) for RIDASCREEN® Leishmania Ab and Leishmania ELISA IgG + IgM, respectively. Moreover, these tests presented greater sensitivity and immunoreactivity in patients with moderate/severe clinical ML forms. The data of this study suggest that ELISA assays can contribute to laboratory diagnosis, especially for patients with moderate or severe mucosal involvement.

Human descending aorta injury caused by brucellosis: A case report.

BACKGROUND: Brucellosis is one of the most common zoonotic diseases in the world. Although cardiovascular complications of human brucellosis account for only 3% of morbidity, they are the leading cause of death. Peripheral vascular disease due to brucellosis is rare and under-reported in the literature. CASE PRESENTATION: Two patients with previous brucellosis, both of whom had been treated with anti-brucellosis, were admitted to vascular surgery for thoracic aortic ulcer and abdominal aortic pseudoaneurysm, respectively, with positive IgG antibody to brucellosis and negative IgM antibody to brucellosis, tube agglutination test, and blood culture. These 2 patients were successfully treated with aortic stent-graft implantation and followed up for 8 and 10 weeks without complications. CONCLUSIONS: Chronic damage to human blood vessels by brucellosis may not disappear with brucellosis treatment, and peripheral blood vessels should be examined annually in people previously diagnosed with brucellosis. Clinicians in related departments should pay attention to peripheral vascular complications of brucellosis.

Utility of rapid chromatographic Immunoassay to rule out typhoid Fever among malaria negative patients.

Enteric fever, an endemic disease, is a significant health problem in developing low- and middle-income countries (LMICs). We studied the utility of Typhoid IgM/IgG assay in Widal titre positive samples among malaria negative patients. A total of 30 febrile patients were included. A blood sample was collected for performing the Widal test and a rapid lateral flow immune assay (Typhoid IgG/IgM tests). A total of 13/30 were positive on blood culture; however, Salmonella typhi grew on only two (6.6%). Of the 30 samples, 24 (80%) were positive for the rapid immunochromatographic (ICT) test None of the samples negative by the rapid ICT test grew Salmonella typhi. The rapid ICT test has better sensitivity and is easy to perform with minimal infrastructure; hence, it is a practical alternative to the age old Widal test.

Application value of antibody titres and RNA detection in the early prediction of Mycoplasma pneumoniae pneumonia in children: a retrospective study.

BACKGROUND: Children with Mycoplasma pneumoniae pneumonia (MPP) are prone to a missed diagnosis at the early stages of the disease, which greatly affects the prognosis of children. In this study, the application value of Mycoplasma pneumoniae (MP) antibody titres and RNA detection for diagnosing MP infection in children with community-acquired pneumonia (CAP) was evaluated. The present study aimed to seek appropriate detection methods and strategies for early rapid diagnosis in children with MPP. METHODS: A retrospective study was conducted on 563 paediatric patients aged 1 month to 15 years with CAP who were admitted to Wuhan Children's Hospital, Tongji Medical College, Huazhong University of Science and Technology between July 2021 and February 2022. In all patients, throat swabs were collected for MP-RNA detection (simultaneous amplification and testing, SAT), and paired serum samples were collected for MP total antibody detection (particle agglutination, PA). RESULTS: The classification as MPP or non-MPP was based on clinical diagnosis, serum MP antibody titre, and clinical or laboratory evidence of infection by other pathogen(s). Among the 563 patients with pneumonia, 187 patients were in the MPP group, and 376 patients were in the non-MPP group. The Kappa values between the particle agglutination test at different titres (1:80, 1:160) and MP-RNA detection were 0.612 and 0.660 (P<0.01), and the consistency of the three methods was acceptable. When the single screening method was used, MP-RNA had the highest sensitivity (93.05%), while PA (1:160) had the highest specificity (100%). PA (1:80), with an area under the curve (AUC) of 0.822, was better than PA (1:160), with an AUC of 0.783, and there was a significant difference. When the combined screening methods were used, the AUC of MP-RNA parallel PA (1:160) was significantly higher than that of titres (1:80) (z=-4.906, P < 0.01). Except for MP-80, the efficacy of the other three test methods in females was slightly better than that in males. Among the differences in age distribution, PA (1:80) was slightly less effective in the 13-72 months age group than at other ages, and MP-RNA parallel PA (1:160) was slightly better than the younger age group (≤ 36 m). In the older age group (> 36 m), PA (1:160) was just the opposite, while MP-RNA was slightly better than other age groups in the 13-72 months age group. CONCLUSIONS: For the diagnosis of MPP in children at the early of the disease, the antibody titre (1:160) parallel MP-RNA should be given preference, and then the disease should be further classified according to the antibody titre level and the age of the child. The combined application of the two detection methods could complement each other and strengthen the advantages, providing reliable laboratory evidence for the clinical diagnosis and timely treatment of MPP. When using the PA method alone to provide a reference standard to clarify MP infection, the differential diagnosis ability of 1:80 for MPP is better than 1:160, especially for children younger than 36 months.

Identification of the most effective serovars to be included in the MAT antigen panel to optimize the serodiagnosis of Leptospira infection in Northern Italy.

The microscopic agglutination test (MAT) assay is adopted as a world-wide reference test for the serodiagnosis of leptospirosis in humans and animals. One of the main limitations of MAT is the lack of sensitivity and serodiagnostic antigens should be periodically updated with locally circulating serovars in order to optimise its performance. The aim of this study was to determine the need to implement the antigen panel currently adopted in Northern Italy for the diagnosis of Leptospira infection in dogs. For this purpose, a group of 288 dogs with and without clinical signs potentially consistent with Leptospira infection or found to have an increased C-reactive protein (CRP) serum concentration, sampled in 2013-2016 in Northern Italy, were tested by MAT comparing the results obtained with a nine antigens panel (Australis-Bratislava, Ballum-Ballum, Canicola-Canicola, Grippotyphosa-Grippotyphosa, Icterohaemorrhagiae-Copenhageni, Icterohaemorrhagiae-Icterohaemorrhagiae, Sejroe-Hardjo, Pomona-Pomona and Tarassovi-Tarassovi serovars) routinely adopted and a panel expanded to 27 antigens. In general, the antigen panel currently adopted in Northern Italy for the routine MAT assay resulted adequate for the diagnosis of Leptospira infection in dogs. The main exception concerns the Sejroe serogroup, with the Saxkoebing and Sejroe serovars that were more effective than Hardjo for diagnosis in dogs and whose inclusion in the antigen panel is recommended. Among other antigens evaluated in this study, Cynopteri serovar was detected with high frequency but its pathogenic role in dogs and as public health threat deserve further investigation.

Evaluating the efficacy of serological testing of clinical specimens collected from patients with suspected brucellosis.

BACKGROUND: This study aims to evaluate the efficacy of the standard agglutination test (SAT), the Brucellacapt test and enzyme-linked immunosorbent assay (ELISA) in clinical specimens collected from patients with suspected brucellosis. METHODS: A prospective study was conducted from December 2020 to December 2021. Brucellosis was diagnosed on the basis of clinical evidence, and confirmed by isolation of Brucella or a four-fold rise in SAT titer. All samples were tested by the SAT, ELISA and the Brucellacapt test. Titers ≥1:100 were considered as SAT positive; ELISA was considered positive when an index greater than 11 was detected, while titers ≥1/160 indicated positivity on the Brucellacapt test. The specificity, sensitivity, and positive (PPVs) and negative predictive values (NPVs) of the three different methods were calculated. RESULTS: A total of 149 samples were collected from patients with suspected brucellosis. The sensitivities for the SAT, IgG, and IgM detection were 74.42%, 88.37% and 74.42%, respectively. The specificities were 95.24%, 93.65%, and 88.89%, respectively. The simultaneous measurement of IgG and IgM improved the sensitivity (98.84%) but reduced the specificity (84.13%) compared to each antibody test separately. The Brucellacapt test had excellent specificity (100%) and a high PPV (100%); however, the sensitivity and NPV were 88.37% and 86.30%, respectively. The combination of IgG detection by ELISA and the Brucellacapt test had excellent diagnostic performance, with 98.84% sensitivity and 93.65% specificity. CONCLUSION: This study showed that the simultaneous performance of IgG detection by ELISA and the Brucellacapt test has the potential to overcome the current limitations of detection.