Indirect Immunofluorescence Reaction Research Articles

Indirect Immunofluorescence Test for Detecting HSV-1, 2 in Patients with Esophageal Cancer: A Pilot Cross-Sectional Survey in Kazakhstan.

To evaluate the detectability of herpes simplex virus type 1 and 2 (HSV-1, 2) antigens in the esophageal mucosa in esophageal cancer. A cross-sectional pilot study with a control group was conducted from December 2022 to May 2023. The patients were divided into two groups: the main group with verified esophageal cancer and the control group without esophageal cancer based on the histological report. Diagnosis using fibroesophagogastroscopy (FEGS), histological analysis, and indirect immunofluorescence reaction (iIFR) was accomplished according to indications at the outpatient center of the endoscopic department of the Multidisciplinary Medical Center, Astana. Descriptive statistics and nonparametric methods were used to analyze the data. A total of 30 patients were recruited in the study, with 15 patients in the main group and 15 patients in the control group. HSV-1 and HSV-2 antigens were detected in the 13 cases (86.7%) during the study of biomaterial from patients with confirmed esophageal cancer in the main group and only in one case (6.8%) in the control group (95% CI 7.35 - 1126.9%, p=0.001). The presence of lymphocytic infiltration was detected in 13 cases (86.7%) in the main group and in 5 cases (33.3%) in the control group (95% CI 2.07 - 81.48%, p = 0.008). The process of keratinization was identified in HSV-1,2 antigens positive cases in the main group in 5 cases (35.7%) G1, in 5 cases (35.7%) G2, and in 3 cases (21.4 %) G3 (95% CI 1.66 - 656.23%, p=0.002). Overall, the local activity of herpesvirus infection to a certain extent influences the malignant potential of tumor cells and the resistance of surrounding healthy tissues. A large-scale study is still needed to confirm these results. The present pilot study provided an overview of a possible method for detecting HSV-1,2 activity in malignant tissue from esophageal cancer.

One Health Approach to Toxoplasmosis: Owner and Dog Seropositivity as Spatial Indicators of Risk Areas for Acquired, Gestational and Congenital Transmission.

Toxoplasmosis has been of public health concern due to direct associations with socioeconomic vulnerability and inadequate living conditions. Accordingly, the present study aimed to assess antibodies against T. gondii, historical reported toxoplasmosis cases and associated socio-environmental risk factors in Pinhais, a full urban area of Curitiba, currently the eighth biggest metropolitan area of Brazil. Anti-Toxoplasma gondii antibodies were assessed by an indirect immunofluorescence reaction (RIFI). Owner and dog samples were also tested by IFAT to anti-Leishmania spp. and anti-Trypanosoma cruzi antibodies. Overall, 20/135 (14.8%) persons and 13/133 (9.8%) dogs from 25 different households were considered seropositive to T. gondii. All samples were seronegative to Leishmania spp. and Trypanosoma cruzi. Although no significant covariates were found in the regression model, statistically associated risk factors in the bivariate analysis included no public water use (p = 0.016) and drinking raw milk (p = 0.041) for owners, and obesity (p = 0.028) and tick infestation (p = 0.03) for dogs. In addition, a spatial cluster of T. gondii seropositivity for both owners and their dogs overlapped the location of historic reported cases of human acquired, gestational and congenital toxoplasmosis. Finally, the results herein showed tick infestation as an indicator of socio-environmental risk for T. gondii exposure in the household environment, and dogs may be used as sentinels for human toxoplasmosis cases.

Comparison of immunological and molecular methods for laboratory diagnosis of ocular toxoplasmosis in blood, serum and tears in Brazil.

Ocular toxoplasmosis (OT) is caused by protozoan T. gondii. Ophthalmological examination is considered the gold standard for OT diagnosis, and laboratory tests are used for diagnostic confirmation. However, these tests can present different results, which change depending on their basis, on sample type and on patients' clinical alteration. Thus, the aim of the present study is to assess immunodiagnostic and molecular techniques applied in blood, serum and tear fluid to diagnose T. gondii infection in patients seen at an Ophthalmology Clinic. In total, 160 patients were included in the study, 40 of them had OT with active lesions (G1); 40 had OT with healed lesions (G2), 40 had non-toxoplasmic uveitis (G3) and 40 had no ocular alterations (G4). Serum samples were subjected to Immunoenzymatic Assay (ELISA) and to Indirect Immunofluorescence Reaction (IFAT) to search for anti-T. gondii IgM and IgG. Tear fluid samples were analyzed through ELISA for IgA research. All blood and tear fluid samples were subjected to conventional polymerase chain reaction (cPCR) and in a Nested PCR model for T. gondii DNA amplification with targets B1, GRA7 and REP 529. IgG and IgM anti-T. gondii was detected in serum samples from 106 and 15 patients, respectively, when combining ELISA and IFAT results. Anti-T.gondii IgA antibodies were detected in 9.2% of the tear material. Nested PCR with GRA7 target showed higher positivity in blood samples (24.4%); Nested PCR with B1 target showed a higher frequency of positivity in tears (15%). Biological samples of patients with active lesions showed the highest positivity frequencies in all immunodiagnostic assays, as well as in most PCR models. The present results highlighted the need of associating techniques with different fundamentals to confirm OT diagnosis. Furthermore, further tear fluid analyses should be performed to validate this biological material as lesser invasive alternative for the more accurate OT diagnosis.

Diagnóstico molecular de infecção por Ehrlichia canis e Babesia vogeli e diagnóstico sorológico de infecção por Neospora caninum e Toxoplasma gondii em cães provenientes do município de Cândido Sales-BA e microrregião

ABSTRACT The occurrence of Toxoplasma gondii, Neospora caninum, Ehrlichia canis and Babesia vogeli in dogs from the municipality of Cândido Sales, Bahia, was investigated. Total and peripheral blood samples were obtained from 131 dogs. Blood smears were performed to check for morulae. Toxoplasma gondii and N. caninum infections were determined by an Indirect Immunofluorescence Reaction. Nested-PCR and PCR were used for the diagnoses of E. canis and B. vogeli infections, respectively. Additionally, the risk factors associated with infection by these agents were analyzed. The frequency of infection was 70.2% for N. caninum, 67.9% for T. gondii and 37.4% for E. canis. None of the dogs tested positive for B. vogeli. Morulae of Ehrlichia spp. were identified in two animals and the piroplasm in one animal. Age (> 3 years) was considered a risk factor for infections by E. canis and N. caninum and the rural habitat for infections by N. caninum. Co-infections were frequent, mainly with N. caninum and T. gondii (45.03% of dogs). Ehrlichia canis infection was significantly associated with N. caninum and T. gondii infection. These findings indicated a high occurrence of T. gondii, N. caninum, and E. canis in the studied region.

The Role of Anti-DFS70 in the Diagnosis of Systemic Autoimmune Rheumatic Diseases

The diagnosis of systemic autoimmune rheumatic disease (SARD) or its exclusion is carried out taking into account the results of immunological studies, primarily antinuclear antibodies (ANA) and specific autoantibodies. Often, during ANA analysis via indirect immunofluorescence reaction on cellular and tissue substrates, a dense fine speckled 70 (DFS70) fluorescence pattern is observed. Studies on the diagnostic significance of antibodies to anti-DFS70 allow for optimizing the stepwise diagnosis of SARD. Currently, a two-step strategy for laboratory diagnostic investigation is recommended: in the first step, ANA screening is performed, and in the second step, patients with positive results undergo confirmatory tests to detect specific antibodies against individual nuclear antigens. The detection of anti-DFS70 in ANA-seropositive patients without clinical and/or other specific serological markers characteristic of a particular disease within the SARD group may be considered a negative prognostic marker. Also, in the process of decision making in clinical practice, we should remember that anti-DFS70 can be found in the blood of patients with a different, non-SARD pathology and that most people showing anti-DFS70 are healthy individuals.

Significane of determining antinuclear antibodies in systemic connective tissue disorders in children

Systemic connective tissue diseases (SCTD) are characterized by systemic autoimmune inflammation and are accompanied by development of antinuclear antibodies (ANA). Our aim was a comparative analysis of ANA in blood serum in children with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). The study included 50 patients with SLE, 50 patients with RA who were treated at the National Medical Research Center of Childrens Health. Median age was 12,2 y. o. (9-15.5). The titers of ANA and the cell fluorescence type were determined with the indirect immunofluorescence reaction (IFR) using the HEp-2 cell line (Immco Diagnostics, Inc, USA), as well as the concentration of antibodies to double-stranded DNA (adsDNA) in blood serum samples of the children detected by immunochemiluminescence (ICM) woth Elia dsDNA (Thermo Fisher Scientific, USA). A positive ANA titer and adsDNA were found, respectively, in 98% and in 48% in children with SLE. A positive ANA titer and adsDNA was detected in 100% and in 4% of children with RA, respectively. Highly positive ANA titers ( 1/1280) have been detected in 68% of children with SLE, and in 30% of children with RA. None of the RA patients with highly positive ANA titers had adsDNA. But, in patients with SLE, highly positive ANA titers and a positive dsDNA level were simultaneously detected in 16% of cases. There are both single types of cell fluorescence and their combinations in children with SLE and RA. Nuclear dot-like fluorescence was more common in children with SLE, cytoplasmic type, in children with RA, nucleolar type of glow was found only in children with RA. The revealed combinations of ANA and adsDNA titers in children with SLE and RA confirm the need for simultaneous use of RNIF and ICM.

Thymoquinone-loaded self-nano-emulsifying drug delivery system against ischemia/reperfusion injury.

In the present study, a self-nano-emulsifying drug delivery system (SNEDDS) was developed to evaluate the efficiency of thymoquinone (TQ) in hepatic ischemia/reperfusion. SNEDDS was pharmaceutically characterized to evaluate droplet size, morphology, zeta potential, thermodynamic stability, and dissolution/diffusion capacity. Animals were orally pre-treated during 10days with TQ-loaded SNEDDS. Biochemical analyses, hematoxylin-eosin staining, indirect immunofluorescence, and reverse transcription polymerase chain reaction (RT-PCR) were carried out to assess cell injury, oxidative stress, inflammation, and apoptosis. The TQ formulation showed good in vitro characteristics, including stable nanoparticle structure and size with high drug release rate. In vivo determinations revealed that TQ-loaded SNEDDS pre-treatment of rats maintained cellular integrity by decreasing transaminase (ALT and AST) release and preserving the histological characteristics of their liver. The antioxidant ability of the formulation was proven by increased SOD activity, reduced MDA concentration, and iNOS protein expression. In addition, this formulation exerted an anti-inflammatory effect evidenced by reduced plasma CRP concentration, MPO activity, and gene expressions of TLR-4, TNF-α, NF-κB, and IL-6. Finally, the TQ-loaded SNEDDS formulation promoted cell survival by enhancing the Bcl-2/Bax ratio. In conclusion, our results indicate that TQ encapsulated in SNEDDS significantly protects rat liver from I/R injury.

Prevalence of natural infection by Trypanosoma evansi in Crioula LAGEANA cattle

Cattle trypanosomiasis negatively impacts animal husbandry due to high morbidity, productivity losses, and mortality rates. Knowledge regarding Trypanosoma evansi infections in locally adapted breeds remains limited. Some cattle breeds exhibit trypanotolerance, requiring the determination of prevalence, as well as related tolerance and resistance characteristics, for disease control programs. This study aimed to determine T. evansi prevalence in Crioula Lageana cattle and associate clinical, hematological, and biochemical aspects with the infection to further research on tolerance in this population. Blood samples from 310 Crioula Lageana cattle were tested using Polymerase Chain Reaction (PCR) and Indirect Immunofluorescence Reaction (IIFR). T. evansi prevalence was 8% (24/310) using PCR and 4% (11/310) using IIFR. Positive animals showed increased ruminal movements, elevated eosinophil counts, and reduced monocyte numbers, but both latter within the reference range for the species. Albumin concentrations were low in positive cases and remained below the reference range limit for both groups. However, triglycerides exceeded the physiological range for the species in both positive and negative groups. Increased gamma-glutamyltransferase (GGT) activity was observed in positive animals. In conclusion, Crioula Lageana cattle exhibited enzootic instability with a low T. evansi infection prevalence when assessed using PCR and IIFR techniques. Furthermore, the animals did not display clinical, hematological, or biochemical alterations attributable to the presence of hemoparasites.

Exposure to and infection by Leishmania infantum among domestic dogs in an area of the Cerrado biome, Maranhão, Brazil

Visceral Leishmaniasis is a serious public health problem and dogs are considered to be the main source of infection in urban areas. In Brazil, this disease is present in all regions, but with high concentration of cases in the Northeast, and the state of Maranhão is considered to be an endemic region. The aim of this study was to conduct an epidemiological, spatial, molecular and serological survey on Leishmania infantum among domestic dogs in the municipality of Belágua, Maranhão. Blood samples were collected from dogs and questionnaires were applied to their owners to obtain epidemiological data and risk factors relating to this zoonosis in the region. The coordinates of the dogs' homes were obtained to produce a disease risk map. Serological diagnoses were made using the indirect immunofluorescence reaction (IFAT) and the dual-path platform chromatographic immunoassay test (DPP®) (Bio-Manguinhos/FIOCRUZ, Brazil). A molecular investigation was undertaken using the polymerase chain reaction (PCR). Georeferencing was performed using the global positioning system (GPS) and cases of canine visceral leishmaniasis in the municipality were spatially represented and analyzed using QGIS version 3.16.6 (QGIS Development Team, 2021). A total of 205 blood samples were collected, of which 122 (59.51%) were seroreactive for L. infantum through IFAT, while the DPP test showed 84 reactive samples (40.97%). IFAT and DPP detected 16 positive animals simultaneously. One sample that was seroreactive through IFAT was also positive through PCR. In the clinical evaluation, it was observed that among the seropositive dogs, 112 (91.80%) were symptomatic and 10 (8.20%) were asymptomatic. In the spatial analysis, the Kernel density estimator enabled determination of the place at greatest risk of occurrence of the disease. The areas with the highest concentrations of cases were in districts with large quantities of precarious housing and lack of basic sanitation. This was the first report on the occurrence of L. infantum among dogs in the municipality of Belágua. The results show that canine visceral leishmaniasis is well dispersed in this municipality, thus putting at risk the human population.

Distribuição espacial e avaliação dos fatores de risco para neosporose bovina em Rondônia, Brasil

ABSTRACT Neospora caninum is an important worldwide parasite responsible for causing abortion in animals. Due to limited information on the occurrence of infection by this parasite in the state of Rondônia, Brazil, this study aimed to determine the seroprevalence and identify the risk factors associated with the infection in slaughtered cattle, from 19 municipalities distributed in seven microregions of the state. A total of 494 samples were obtained and subjected to anti-N. caninum antibodies, using the Indirect Immunofluorescence Reaction technique. Antibodies were detected in 5.06% (25/494) of the samples, in 30.30% (10/33) of farms, in nine municipalities located in four microregions of Rondônia. Of all the animals analyzed, 4.81% of the females (20/416) and 6.41% of the males (05/78) were seropositive for the parasite, with “abortion in the last 12 months” being considered an important risk factor for the occurrence of infection (OR = 9.54; p = 0.01). The present study points out the prevalence of anti-N. caninum antibodies in 5.06% of slaughtered animals and abortion as the main risk factor associated with infection by N. caninum, thus contributing to the elucidation of the epidemiology of this protozoan in Rondônia, Brazil.

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Abstract. Recent pandemic caused by the SARS-CoV-2 virus has united researchers from all over the world. However, despite their efforts, many questions remain unsolved: how the immune system is effective against this viral infection, how long the immunity persists in convalescents, what are the consequences of the disease. The inflammatory process, which is based on multicomponent immunological disorders, is one of the leading pathogenetic links of the disease caused by the SARS-Cov-2 virus. In this regard, a precise study of the immune status peculiarities in patients � convalescents after COVID-19 and their possible role in the further development of chronic pathology are relevant. The aim of the work is to investigate peculiarities of immune disorders in convalescents after acute respiratory disease COVID-19 in relation to the severity of the disease and the timing of the examination after the acute period. Methods. We examined 91 convalescents after COVID-19, aged 21-67 years, treated as inpatients during the acute period of the disease. After the acute period these patients received a course of rehabilitation treatment in the Government Institution �The Scientific-Practical Medical Centre �Rehabilitation��. For comparison, a group of 45 patients with bacterial community-acquired pneumonia of medium severity were examined. As a control for laboratory indices, 24 practically healthy persons were examined. Cytokine status was studied by determining the levels of pro- and anti-inflammatory cytokines using immunoenzymatic method. Non-specific resistance was investigated on the base of phagocytic activity and phagocytic number of monocytes. Indicators of cellular immunity were evaluated using an indirect immunofluorescence reaction using monoclonal antibodies (CD3+, CD22+, CD4+, CD8+, CD16+) and on this basis index values were calculated that characterize the relationships of the studied subpopulations. When analyzing these data, the severity of the COVID-19 and the time of examination after the acute period of the disease were taken into account. Results. At the beginning of the course of recovery treatment, a pronounced imbalance in the ratio of pro- and anti-inflammatory cytokines with a significant predominance of the first one, was revealed. These changes can contribute to the persistence of the inflammatory process with further formation of the chronic pathology. This imbalance did not depend significantly on the severity of COVID-19, persisted for a long time and was 1.9 times more pronounced than in bacterial community-acquired pneumonia. In the period of convalescence, suppression of the monocytes� phagocytic activity and significant changes in the T-cells link of immunity were also revealed. It should be noted that disorders of cellular immunity depended on the severity of COVID-19 and were most pronounced in patients with its severe course. These changes persisted for a long time (examinations were carried out 2-3 weeks and 2-3 months after the acute period), which determines the need for recovery treatment during the convalescence period. Conclusions. 1. In convalescents after acute respiratory disease caused by the SARS-CoV-2 virus, certain disturbances of the immune status remain, which significantly affect the functional state of the organism as a whole and may be the basis for a persistent inflammatory process and the development of chronic pathology in the future. 2. Immune defense of convalescents after COVID-19 is characterized by a significant decrease in non-specific protection indices and the cellular link of immunity with manifestations of cytokine imbalance and these changes persist for a long time, which can contribute to the formation of post-COVID syndrome. 3. The obtained data serve as a pathogenetic basis for carrying out complex immunorehabilitation measures in convalescents after COVID-19 in order to recover the normal functioning of immune system, correcting protective reserves of the organism and preventing the development of chronic pathology. Key words: COVID-19, cytokine status, non-specific defense, cellular immunity

Seroprevalence and associated factors with Neospora caninum infection in sheep and goats slaughtered in the state of Paraíba, Brazil.

The objective was to describe the seroprevalence of anti-Neospora caninum antibodies in goats and sheep slaughtered in the state of Paraíba and to identify possible associated factors with the infections. Two hundred twenty-nine samples from goats and two hundred five from sheep were analyzed by Indirect Immunofluorescence Reaction (IFAT) using a cutoff point of 1:50. The presence of anti-N. caninum antibodies was identified in 28.4% (65/ 229; 95% Confidence Interval: 22.6-34.2) of the goat samples and in 12.7% (26/ 205; 95% CI: 8.2 - 17.2) of the sheep samples. Contact between goats and dogs (Odds ratio 4.81; CI 1.13 - 2.67; p = 0.041) and cattle (OR. 1.87; CI 1.13 - 2.67; p = 0.002) was identified as a risk factor for goats and contact between sheep and dogs (OR 2.32; CI 1.58 - 3.14; p = 0.026) and history of abortion (OR 1.94; CI 1.28 - 2.90; p = 0.001) was considered a risk factor for sheep. The study revealed a high seroprevalence of anti-N. caninum antibodies in slaughtered goats and sheep in Paraíba. Risk factors such as contact with dogs/cattle and abortion history underscore the need for preventive measures to control infection and enhance animal health management.

The role of herpesviruses and pneumocysts in infectious complications in children during liver transplantation

Infections occupy one of the central places among the complications of transplants. The frequency of fatal infectious complications during the first twelve months after transplantation ranges from 2.6 to 51.7%. Identifying markers of opportunistic infections before transplantation will help reduce the likelihood of developing these infections after induced immunosuppression. The aim of the study was to study the role of herpesviruses and pneumocysts in the occurrence of infectious complications in children before and after liver transplantation based on the detection of markers of a number of herpesvirus infections and pneumocystosis. The article presents the results of a comprehensive examination for markers of herpesvirus infections and pneumocystosis of 70 children who were treated at the Shumakov Transplantation Research Center. It should be noted that 55 patients (78.6%) were diagnosed with infectious complications, of which 46 people (65.7%) had pneumonia. To detect IgM and IgG antibodies to herpesviruses and pneumocysts, peripheral blood serum samples were examined by enzyme immunoassay (ELISA). Detection of common HBV antigens was carried out by indirect immunofluorescence reaction (NRIF). Early antigens and reproduction of herpesviruses were detected by rapid culture method (BCM) on Vero and M-19 cell cultures for CMVI. In the event of infectious complications (pneumonia) in children who underwent related liver transplantation, the number of patients with active EBI markers increased almost 7 times and active HCV-6 markers increased 3.5 times. The paper shows the need for a broader laboratory screening of opportunistic infections, which will serve to achieve better clinical results, contribute to the formation of more advanced diagnostic algorithms, as well as improve epidemiological surveillance of these infections.

Lassa fever. Part 2. Laboratory diagnostics, treatment, development of medications

Globalization and high-speed means of transportation contribute to the spread of infections dangerous to humans. Airborne pathogens have pandemic potential as currently shown in case of the novel coronavirus SARS-CoV-2. Natural focal Lassa fever (LF) common in West African countries, in 35 cases was registered in non-endemic geographical areas because any person infected with Lassa virus (LASV) is a long-term source of infection (up to two months). Cases of person-to-person infection in endemic territories are described. In Germany, the facts of secondary virus transmission from patients to doctors have been recorded during the examination and blood collection from an apparently healthy person as well as during the autopsy of a deceased subjects due to severe LF course. Nonspecific malaise symptoms in LF are also characteristic of numerous other diseases common on the African continent, e.g., malaria and typhoid fever or viral infections such as yellow fever, Chikungunya, dengue and Zika, monkey pox and Ebola virus disease. In this regard, there may be similar dermatological manifestations. Timely detection of cases and differential diagnosis are crucial to ensure safe patient care and use of affordable antiviral therapy for LL provided by the drug Ribavirin. Research methods for studying LASV use polymerase chain reaction (PCR) for detecting viral RNA, electron microscopy, isolation of infectious virus cultured sensitive cells, indirect immunofluorescence reaction, enzyme immunoassay (ELISA) and immunochromatographic assays for the detection of antibodies and/or antigen as well as immunoblotting. Currently, test kits based on molecular and genetic methods are mainly used for LF laboratory diagnostics. Since the 1980s, ribavirin has been used to treat patients with LF. The serum accumulation of the drug in large quantities causes hemolysis, development of anemia and impaired renal function. In this regard, treatment options are being considered with decline in its concentration due to combined use with other antiviral drugs. A search for new therapeutic agents capable of inhibiting viral replication at disease early stage has been in progress due to lack of any approved vaccines.

Diagnostic value of screening methods for the determination of antinuclear antibodies using indirect immunofluorescence on HEp-2 cells and enzyme immunoassay in autoimmune liver diseases.

Antinuclear antibodies (ANA) are a heterogeneous group of autoantibodies that react with various components of the cell nucleus and cytoplasm. ANA is the main serological marker for autoimmune liver disease (AILD). The aim of the study was to compare the diagnostic value of two methods of screening for the determination of ANA (indirect immunofluorescence reaction on HEp-2 cells (IIF -HEp-2) and enzyme-linked immunosorbent assay (ELISA) in the sera of AILD patients. The sera of 118 patients with AILD (51 with autoimmune hepatitis - AIH, 19 with primary biliary cholangitis - PBC, 48 with overlapping syndrome - OVERLAP), 30 patients with non-alcoholic fatty liver disease (NAFLD) and 30 healthy donors (HD) were studied. Determination of ANA by the IIF-HEp-2 method was carried out by visual assessment of samples under an AXIOSKOP 40 microscope, by ELISA - on an Alegria automatic analyzer. A weak degree of agreement between the positive and negative results of the ANA screening study using IIF-HEp-2 and ELISA (Cohen's kappa coefficient æ=0.4) was noted. Screening determination of ANA in patients with AILD by the IIF-HEp-2 method was distinguished by greater diagnostic sensitivity (DS) (68.6%) and a lower frequency of false negative results (31.4%) compared with ELISA (35.6% and 64.4 % respectively, p<0.05). The overall diagnostic specificity (DS) of the ANA study in IIF-HEp-2 was lower than with ELISA (66.7% and 86.7%, respectively, p<0.05). Both screening methods for determining ANA (IIF-HEp-2 and ELISA) were useful for diagnosing AILD (positive likelihood ratio - LR+: 2.1 and 2.6, respectively). In terms of the negative likelihood ratio (LR-), screening for ANA by the IIF-HEp-2 method, in contrast to ELISA, served as a "useful" test to exclude the diagnosis of AILD (0.5 and 0.8, respectively). The determination of ANA using IIF-HEp-2 is the most sensitive and "useful" screening test for the diagnosis of AILD, and ELISA is classified as a less "useful" screening method due to low diagnostic sensitivity and a high false-negative rate.

Lung-brain axis: Metabolomics and pathological changes in lungs and brain of respiratory syncytial virus-infected mice.

The lung-brain axis is an emerging area of study that got its basis from the gut-brain axis biological pathway. Using Respiratory Synctial Virus (RSV) as the model of respiratory viral pathogen, this study aims to establish some biological pathways. After establishing the mice model, the inflammation in lung and brain were assayed using Hematoxylin-eosin staining, indirect immunofluorescence (IFA), and quantitative reverse-transcription polymerase chain reaction. The biological pathways between lung and brain were detected through metabolomics analysis. In lung, RSV infection promoted epithelial shedding and infiltration of inflammatory cells. Also, RSV immunofluorescence and titerss were significantly increased. Moreover, interleukin (IL)-1, IL-6 and tumor necrosis factor-α (TNF-α) were also significantly increased after RSV infection. In brain, the cell structure of hippocampal CA1 area was loose and disordered. Inflammatory cytokines IL-6 and IL-1β expression in the brain also increased, however, TNF-α expression showed no differences among the control and RSV group. We observed an increased expression of microglia biomarker IBA-1 and decreased neuronal biomarker NeuN. In addition, RSV mRNA expression levels were also increased in the brains. 15 metabolites were found upregulated in the RSV group including nerve-injuring metabolite glutaric acid, hydroxyglutaric acid and Spermine. ɑ-Estradiol increased significantly while normorphine decreased significantly at Day 7 of infection among the RSV group. This study established a mouse model for exploring the pathological changes in lungs and brains. There are many biological pathways between lung and brain, including direct translocation of RSV and metabolite pathway.

Chagas disease in northern Chile: Detection of Trypanosoma cruzi in children, dogs and triatomine bugs

Chagas disease is an anthropozoonotic disease caused by the protozoan Trypanosoma cruzi, transmitted by triatomine vectors. In Chile, there are four species of triatomine bugs that are potential vectors of T. cruzi, being Triatoma infestans the main vector in endemic areas of the country. The “Programa Nacional de Control Vectorial de la Enfermedad de Chagas de Chile” has significantly reduced the rates of home infestation to less than 1% and has interrupted vectorial transmission since 1999. The objective of this study was to evaluate the impact of vectorial control and the continuity of the interruption of vectorial transmission in northern Chile (provincia de El Loa, región de Antofagasta). The study comprised fingerstick blood samples of 2104 children, attending local school, venous blood samples of 65 dogs, associated to houses with T. infestans unique findings and vector infestation, and intestine samples of 284 T. infestans specimens, from the provincia de El Loa, during 2014–2016 period. The samples were analyzed by enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence and/or polymerase chain reaction (PCR) techniques. A total of 5 children (0.24%), 7 dogs (10.8%), and 6 specimens of T. infestans (2.1%) resulted positive to T. cruzi infection. This study showed that the risk of transmission of Chagas disease is low in the north of Chile (provincia de El Loa), detected a low positive rate of chagasic children and of infected triatomine bugs, and showed the existence of T. cruzi transmission in dogs, which are used as natural sentinels for the detection of T. cruzi infection, being especially useful during surveillance program in human population characterized by low seroprevalence.

Luteolin restricts ASFV replication by regulating the NF-κB/STAT3/ATF6 signaling pathway

African swine fever (ASF) is a devastating infectious disease that causes significant economic losses to the pig industry worldwide. Luteolin is abundant in onion leaves, carrots, broccoli, and apple skin and exerts various biological activities, including anti-cancer and anti-virus effects. Our aim was to demonstrate the mechanism of action and potent antiviral activity of luteolin against ASF virus (ASFV) in porcine alveolar macrophages. We performed cell viability, hemadsorption, indirect immunofluorescence, western blotting, and quantitative real-time polymerase chain reaction assays to investigate the effect of luteolin on ASFV. Notably, luteolin restricted ASFV replication in a dose-dependent manner. The anti-ASFV activity of luteolin was maintained for 24–72 h. Subsequent experiments revealed that luteolin could block multiple stages of the ASFV replication cycle, including those at 6–9 h and 12–15 h after infection, instead of directly interacting with ASFV. Moreover, ASFV infection stimulated the expression of phosphorylated nuclear factor (NF)-κB, interleukin (IL)− 6, and phosphorylated signal transducer and activator of transcription 3 (STAT3). However, luteolin downregulated ASFV-induced NF-κB, IL-6, and STAT3 expression. Importantly, NF-κB agonist CU-T12–9 weakened the inhibitory effects of luteolin on NF-κB and STAT3. Moreover, CU-T12–9 partially restored the inhibitory effect of luteolin on ASFV. Similarly, luteolin reduced ASFV-induced activating transcription factor 6 (ATF6) expression, and CU-T12–9 weakened the inhibitory effect of luteolin on ATF6. Our findings suggested that luteolin inhibited ASFV replication by regulating the NF-κB/STAT3/ATF6 signaling pathway and might provide a rationale for anti-ASFV drug development.

FATORES DE RISCO E SOROPREVALÊNCIA DE ASSINTOMÁTICOS DA LEISHMANIOSE VISCERAL HUMANA EM ÁREA DE TRANSMISSÃO INTENSA EM PETROLINA/PE, BRASIL

OBJECTIVES: To identify the seroprevalence of asymptomatic individuals in areas of disease transmission and analyze the environmental, socioeconomic and demographic risk factors in the municipality of Petrolina/PE, Brazil. METHODS: We approached 92 individuals in 19 localities with VL cases recorded in the Information System for Notifiable Diseases (SINAN) between 2016 and 2017. In each visit, two questionnaires were applied seeking to obtain clinical, social, economic and environmental variables. Viable blood samples collected during the visits (n=90) were submitted to the Indirect Immunofluorescence Reaction to identify anti-Leishmania antibodies. For data analysis, descriptive statistical tests were performed using Stata® 14.0 software. RESULTS: The presence of antibodies was detected in 2.2% (2/90) of the samples, in which one individual presented symptoms and the other was classified as asymptomatic. No risk factors related to the presence of anti-Leishmania antibodies were identified. CONCLUSION: The identification of one asymptomatic individual reinforces the importance of expanding human prevalence studies in endemic areas. Despite the low prevalence, it was possible to identify a possible case with manifestation of the clinical form of chronic VL that needs proper care.

Necessity for modern methods of diagnosing Hepatitis C, especially when overlapping with secondary infection

In this study, we focused on the antimicrobial peptides and T-lymphocyte clusters indicating the immune system ability to react violently leading to reduce the organism sensitivity to bacteria tested in 87 individuals with chronic hepatitis C. All patients were divided into two groups: with chronic hepatitis C, and the group in which hepatitis C overlaps with secondary infection leading to pneumonia. Endotoxin and lipopolysaccharide-binding protein (LBP) were determined using the ELISA technique. Determination of the clusters of differentiation (CD) carried out by indirect immunofluorescence reaction, while the circulating immune clusters (CIC) identified by method of sedimentation with a 3.5% solution of polyethylene glycol. Statistical processing of the results carried out using the Wilkinson U-test (Mann - Whitney). In the group of patients with hepatitis C, CD25+ was nearly halved, while in group with hepatitis aggravated by pneumonia this value lowered approximately three times. CD25+ indicator in II group was even 1.4 times less than in the group without pneumonia. Defensin levels were significantly higher in the I group, where endotoxin raises up to 24.4 vs normal levels. In the II group, aggravated by pneumonia, endotoxin elevated even up to 57.7 IU/ml, the same direction changed defensin concentration. The results of this study show, that instead of standard tests for liver damage: bilirubin, AST and γ-glutamyl transpeptidase, it is much more expedient to use the antimicrobial peptides defensin and LBP, which are more informative in the diagnosis of hepatitis C, especially when it overlaps with secondary infection.