Improved diagnosis of systemic lupus erythematosus with human-derived double-stranded DNA antigen.

Anti-double-stranded DNA antibodies (anti-dsDNA) serve as a crucial serological indicator for systemic lupus erythematosus (SLE). Chemiluminescent immunoassay (CIA) is mainly used in clinical diagnosis of SLE, but suffers from low specificity, partially because the use of dsDNA antigens of varied sources in current CIA kits that sometimes led to controversial results. On the basis that anti-dsDNA in healthy individuals tend to selectively bind with dsDNA originating from pathogens, whereas pathogenic anti-dsDNA in SLE patients bind all forms of dsDNA, here we proposed the use of dsDNA fragment derived from human genome as antigen (synthesized via PCR using the human genomic DNA as the template). A magnetic bead-based immunofluorescence assay (IFA) was thus developed for SLE diagnosis, which exhibited improved sensitivity and specificity over CIA using the WHO reference reagent (15/174) as standard. For clinical serum sample analysis (n = 590), IFA exhibited an accuracy of 71.9% that was higher than CIA (65.3%). Crucially, the IFA results exhibited stronger correlations with the activity of SLE, renal involvement, and its prognosis. Besides the improved clinical diagnosis, the proposed IFA also holds great promise in assay standardization due to the high homogeneity of the synthetic dsDNA.

Immunofluorescence detection of Ecytonucleospora hepatopenaei (EHP) in Penaeus vannamei.

Hepatopancreatic microsporidiosis (HPM), caused by the microsporidium Ecytonucleospora hepatopenaei (EHP) leads to retarded growth and enhanced susceptibility to other diseases in shrimp resulting in a major loss for the shrimp industry worldwide. It is little understood how EHP infects its host and hijacks its cellular machinery to replicate and exert clinical manifestations in infected shrimp. Since the initial record of HPM, histopathology and polymerase chain reaction (PCR)-based assays were developed for the detection of EHP to prevent spread of the disease. Availability of an antibody-based detection method would complement these existing diagnostic tools and be useful in studying EHP pathogenesis. We describe here an immunofluorescence assay (IFA) for detecting EHP using monoclonal antibodies (mAbs) that were originally developed against Cryptosporidium parvum, a coccidian parasite that infects calves (Bos taurus), other agriculturally important animals, and humans. Forty-one mAbs were screened and two mAbs, 3E2 and 3A12, were found to detect EHP successfully. The utility of these mAbs in detecting EHP was further assessed by testing 36 experimentally challenged EHP-infected shrimp (Penaeus vannamei). EHP-detection data from infected shrimp were compared by Hematoxylin and Eosin (H&E) histology, real-time PCR, and immunofluorescence. The data show IFA using mAbs 3E2 and 3A12 could successfully detect EHP and that the sensitivity of detection is comparable to H&E histology and quantitative PCR. Availability of mAbs that can detect EHP is expected to be immensely beneficial in HPM diagnosis. Since the pathobiology of C. parvum has been so widely studied, these cross-reactive mAbs may also aid in gaining some insight into EHP pathogenesis and disease.

Detection and differentiation of antinuclear antibodies in serum of dengue suspected patients with or without systemic autoimmune disease in Kolkata, India.

The pathophysiology of dengue may be influenced by antibodies released during infection. Several autoimmune diseases are accompanied by antinuclear antibodies (ANAs) but 8-10% of the general population have positive ANA tests. To test the hypothesis that an ANA-positive test indicates an immune dysregulated state that modifies the risk for certain clinical disorders in people with or without an autoimmune disease, we examined the various ANA profiles and their relationships to various autoimmune disorders, as well as the severity of these relationships, in patients infected with dengue fever. Enzyme-linked immunosorbent assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR) methods were used. Indirect immunofluorescence assay (IIFA) and line immunoassay (LIA) were performed to detect and differentiate the ANAs among dengue infected patients. Out of 135 dengue virus-positive patients, 94.07% were positive by ELISA and 5.93% positive by RT-PCR method. ANAs by IIFA and LIA were detected in 54.8% and 18.5% of the dengue positive patients, respectively, and 10.3% and 7.1% of the 126 dengue negative patients, respectively. This study showed that dengue was associated with an increased risk of autoimmune myositis and mixed connective tissue disease (MCTD), a rare complication of dengue. The risk of other autoimmune diseases did not seem to increase after DENV infection.

Performance of the Vetscan Imagyst in point-of-care detection of Giardia duodenalis in canine fecal samples.

Giardia duodenalis is a common parasite of the gastrointestinal tract of dogs, with an especially high prevalence in dogs <1-y-old. Methods for detecting G. duodenalis are point-of-care (POC) tests such as lateral-flow tests or fecal flotation. The Vetscan Imagyst (Zoetis) is a new POC device for the detection of G. duodenalis in fecal samples using zinc sulfate flotation, automated slide scanning, and image recognition with artificial intelligence. Vetscan results are the number of Giardia cysts per coverslip. We compared the performance of the Vetscan and another POC test (SNAP Giardia test; Idexx) with a direct immunofluorescence assay (IFA) performed in a specialized parasitology laboratory as the reference test. We included 164 dogs <19-mo-old. We used pooled fecal samples from 3 defecations gained within 2-3 d and tested the repeatability of the Vetscan by triplicate measurement. Compared to IFA, Vetscan had a diagnostic sensitivity of 88.4% and specificity of 98.1%; SNAP had a diagnostic sensitivity of 74.4% and specificity of 98.1%. A variation coefficient of 67.0% was determined for the Vetscan results. The performance of the Vetscan is acceptable for the qualitative evaluation of fecal samples (Giardia positive or negative), and the device can be used by untrained personnel. Given its high variation coefficient, we do not recommend the Vetscan for monitoring the number of cysts.

Serological and Molecular Survey of Rickettsial Agents in Wild Boars (Sus scrofa) from Midwestern Brazil.

Wild boars (Sus scrofa L.) are considered among the most harmful invasive species worldwide, causing irreversible ecosystem damage, acting as zoonotic spreaders and reservoirs, threatening human and animal health, and having an important economic impact. Accordingly, the present study has assessed the rickettsial exposure, tick infestation of wild boars, and rickettsial DNA presence in ticks from infested animals from the Cerrado biome in midwestern Brazil. Anti-Rickettsia spp. antibodies were detected in serum samples of wild boars by immunofluorescence assay. Overall, 106/285 (37.2%) wild boar serum samples from 13 to 18 (72.2%) municipalities showed seroreactivity to at least one of the four Rickettsia spp. antigens tested, the largest number of wild boars serologically tested to Rickettsia spp. in this type of study. Among the 106 seroreactive animals, 34 showed possible homologous reactions between R. parkeri, R. amblyommatis, and R. bellii, with endpoint titers between 128 and 512. A sample of 45 ticks collected from four culled wild boars was identified as Amblyomma sculptum, and all tested negative for rickettsial DNA presence. In conclusion, this study has provided a reliable sampling seroprevalence and indicated high exposure of wild boars to rickettsial agents, with a potential interaction with Rickettsia spp. from the spotted fever group within the Cerrado biome from midwestern Brazil.

Evaluation of the Accuracy of Estimated Endpoint Titer of NOVA View in Indirect Immunofluorescent Antinuclear Antibody Testing.

For antinuclear antibody (ANA) screening, the gold standard method is an indirect immunofluorescence assay (IIFA) using HEp-2 cells, and a serial dilution test is needed to determine the endpoint titer. We aimed to evaluate the accuracy of the estimated endpoint titer (eEPT) by the NOVA View system, by comparing it with the EPT by the serial dilution method (dEPT). The endpoint titers of a total of 1518 ANA positive cases with five major patterns including speckled, homogeneous, centromere, nucleolar, and nuclear dots patterns were determined using both the estimation function and the serial dilution method by the NOVA View system. A significant correlation between the light intensity unit (LIU) values and dEPTs was identified in all five patterns with high ρ values, ranging from 0.666 to 0.832. However, the overall exact match rate between dEPT and eEPT was 22.1% (336/1518), with the ±one-titer match rate being highest in the centromere pattern (62.8%, 81/129), and lowest in the homogeneous pattern (37.6%, 200/532). This suggests that while LIU values correlate well with dEPT, there are discrepancies in numerical agreement. Most cases that did not show an exact match, showed one-to-three-titer overestimations by eEPT. Therefore, adjusting eEPT downward significantly improved the concordance rates with dEPTs. Further investigation for an appropriate cutoff of LIU values for determining eEPT should be performed for clinical application and contribution to the standardization of the ANA titer.

Commercial loop-mediated isothermal amplification (LAMP) assay for rapid diagnosis of Pneumocystis pneumonia: An alternative to immunofluorescence assays.

A commercial loop-mediated isothermal amplification (LAMP) assay is available for the detection of Pneumocytis jirovecii (Eazyplex®, Amplex diagnostics, Germany). Few centers currently use this LAMP assay in France. Recently, the commercialization of reagents used to perform the P. jirovecii immunofluorescence assay (IFA) was stopped. This study aimed to assess the position of the commercial LAMP P. jirovecii assay in the diagnostic strategy for Pneumocystis pneumonia. Over 24 months (August 1, 2021, to September 1, 2023), all bronchoalveolar lavage fluid (BALF) samples with a request for P. jirovecii detection were analyzed with the commercial Eazyplex® LAMP assay, using a Genie 2® device (Amplex, diagnostics), in parallel with the techniques used for direct examination. Specific P. jirovecii quantitative real-time PCR (qPCR) was subsequently performed. In total, 346 BALF samples were analyzed by Diff-Quik coloration, IFA, and the commercial Eazyplex® LAMP assay for initial screening. Twenty-six cases of PCP were retained based on radiological, biological and clinical criteria. Among the 26 cases of PCP, 11 BALF samples were positive using the initial screening techniques: four with the three techniques, six by IFA and Eazyplex®, and one only by IFA. The eleven BALF samples were positive with the specific P. jirovecii qPCR assay, with a mean quantification cycle (Cq) of 27 [19-32]. The commercial Eazyplex® LAMP assay is able to provide a result in 25 min and its sensitivity is similar to that of BALF direct examination techniques, such as IFA, which is a technique no longer available on the European market. The sensitivity of the commercial Eazyplex® LAMP assay is however clearly inferior to that of the specific P. jirovecii qPCR assay and, therefore, cannot replace the specific qPCR, but may have a place in the diagnostic strategy.

Characterising Eastern Grey Kangaroos (Macropus giganteus) as Hosts of Coxiella burnetii.

Macropods are often implicated as the main native Australian reservoir hosts of Coxiella burnetii (Q fever); however, the maintenance and transmission capacity of these species are poorly understood. The objective of this cross-sectional study was to describe the epidemiology of C. burnetii in a high-density population of eastern grey kangaroos (Macropus giganteus) in a peri-urban coastal nature reserve in New South Wales, Australia. Blood, faeces and swabs were collected from forty kangaroos as part of a population health assessment. Frozen and formalin-fixed tissues were also collected from 12 kangaroos euthanised on welfare grounds. Specimens were tested for C. burnetii using PCR, serology, histopathology and immunohistochemistry. A total of 33/40 kangaroos were seropositive by immunofluorescence assay (estimated true seroprevalence 84%, 95% confidence interval [CI] 69% to 93%), with evidence of rising titres in two animals that had been tested four years earlier. The PCR prevalence was 65% (95% CI 48% to 79%), with positive detection in most sample types. There was no evidence of pathology consistent with C. burnetii, and immunohistochemistry of PCR-positive tissues was negative. These findings indicate that kangaroos are competent maintenance hosts of C. burnetii, likely forming a significant part of its animal reservoir at the study site.

[Preparation and preliminary application of the polyclonal antibody against Toxoplasma gondii dense granule protein 24].

OBJECTIVE: To prepare and characterize the mouse polyclonal antibody against the dense granule protein 24 (GRA24) of Toxoplasma gondii, and explore its preliminary applications. METHODS: The GRA24 coding sequences of different T. gondii strains were aligned using the MEGA-X software, and the dominant peptide of the GRA24 protein was analyzed with the Protean software. The base sequence encoding this peptide was amplified using PCR assay and ligated into the pET-28a vector, and the generated GRA24 truncated protein was transformed into Escherichia coli BL21. After induction by isopropyl-beta-D-thiogalactopyranoside (IPTG), the expression and purification of the recombinant GRA24 protein was analyzed using sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE). BALB/c mice were immunized by subcutaneous injection with the purified recombinant GRA24 truncated protein to generate the polyclonal antibody, and the titer of the polyclonal antibody was measured using enzyme linked immunosorbent assay (ELISA). The specificity of the polyclonal antibody was tested using Western blotting, and the intracellular localization of the polyclonal antibody was investigated using immunofluorescence assay (IFA). RESULTS: SDS-PAGE showed successful construction of the recombinant expression plasmid, and Coomassie brilliant blue staining showed the generation of the high-purity recombinant GRA24 truncated protein. ELISA measured that the titer of the polyclonal antibody against the GRA24 truncated protein was higher than 1:208 400, and Western blotting showed that the polyclonal antibody was effective to recognize the endogenous GRA24 proteins of different T. gondii strains and specifically recognize the recombinant GRA24 truncated protein. Indirect IFA showed that the GRA24 protein secreted 16 hour following T. gondii invasion in host cells. CONCLUSIONS: The polyclonal antibody against the T. gondii GRA24 protein has been successfully prepared, which has a widespread applicability, high titers and a high specificity. This polyclonal antibody is available for Western blotting and IFA, which provides the basis for investigating the function of the GRA24 protein.

Imaging Assays to Detect DNA Damage in Trypanosome Parasites Using γH2A.

Diseases caused by trypanosomatid parasites remain a significant unmet medical need for millions of people globally. Trypanosomatid parasites such as Trypanosoma cruzi and subspecies of Trypanosoma brucei cause Chagas disease and human African trypanosomiasis (HAT), respectively. Although efforts to find novel treatments have been successful for HAT, Chagas disease is still treated with decades-old therapies that suffer from long treatment durations and severe safety concerns. We recently described the identification and characterization of the cyanotriazole compound class that kills trypanosomes, in vitro and in vivo, by selective inhibition of the trypanosome nuclear topoisomerase II enzyme. To evaluate whether inhibition of the topoisomerase II enzyme led to parasite death due to lethal double-strand DNA breaks, we developed assays for detecting DNA damage in both intracellular amastigotes of T. cruzi and bloodstream-form T. brucei by using the canonical DNA damage marker γH2A. Herein, this article describes the protocols for detecting DNA damage using an immunofluorescence assessment of γH2A by microscopy in trypanosome parasites. Key features • Immunofluorescence-based assay to detect the γH2A response in T. brucei and T. cruzi parasites. • Robust DNA damage pathway-based cellular assays to evaluate topoisomerase II poisons' ability to cause DNA damage. • A 384-well plate-based T. cruzi protocol allows high-resolution and high-throughput evaluation of compounds that cause DNA damage by measuring γH2A in intracellular parasites. • This assay could be modifiable for evaluation of DNA damage responses in various intracellular and extracellular eukaryotic pathogens.

First laboratory-confirmed case of scrub typhus in Shijiazhuang City, Hebei Province.

Objective: Defining whether a suspected case was due to scrub typhus through laboratory testing, to understand the prevalence of scrub typhus in Shijiazhuang City, Hebei Province. Methods: An epidemiological investigation was conducted on the suspected case, utilizing Weil-Felix test and indirect immunofluorescence assay (IFA) to detect specific antibodies against O. tsutsugamushi in serum specimens. Additionally, PCR amplification of the 56-kDa and groEL genes was performed, followed by constructing a phylogenetic tree to identify the genotype. Results: The acute phase titer of the Weil-Felix test for the case was 1:160, which increased to 1:320 in the recovery phase. IFA assay revealed IgG titers against O. tsutsugamushi of 1:64 in the acute phase and 1:256 in the recovery phase. Sequence alignment of the PCR amplified fragment showed the highest similarity with the O. tsutsugamushi genotype. Kawasaki sequence, ranging from 99.71 to 100.00%. The strain exhibited the closest genetic relationship with the known O. tsutsugamushi Kawasaki genotype. Conclusion: This study confirms the presence of O. tsutsugamushi in Shijiazhuang City, Hebei Province, with the identified strain belonging to the Kawasaki genotype, marking the first diagnosis of this strain in the region.

Clinical Significance of Antibodies to DFS70 in Immunoinflammatory Rheumatic Diseases.

The relevance of the problem of immunoinflammatory rheumatic diseases (IIRD) for modern medicine is determined by their high prevalence in the population, the difficulty of early diagnosis, the rapid development of disability and poor life prognosis. Recent data on the significance of anti-DFS70 have opened up new possibilities for optimizing the step-by-step diagnosis of IIRD. The detection of these antibodies can help in the interpretation of a positive result for antinuclear antibodies (ANA) by indirect immunofluorescence assay on HEp-2 cells (IIFA-HEp-2) in the absence of autoantibodies specific for IIRD. Detection of anti-DFS70 in antinuclear factor (ANF) seropositive patients without clinical and/or serological markers characteristic of a certain disease from the IIRD group can be considered as a potential marker that excludes this group of diseases.

Immunoantitumor Activity and Oxygenation Effect Based on Iron-Copper-Doped Folic Acid Carbon Dots.

Cancer metastasis and recurrence are closely associated with immunosuppression and a hypoxic tumor microenvironment. Chemodynamic therapy (CDT) and photothermodynamic therapy (PTT) have been shown to induce immunogenic cell death (ICD), effectively inhibiting cancer metastasis and recurrence when combined with immune adjuvants. However, the limited efficacy of Fenton's reaction and suboptimal photothermal effect present significant challenges for successfully inducing ICD through CDT and PTT. This paper described the synthesis and immunoantitumor activity of the novel iron-copper-doped folic acid carbon dots (CFCFB). Copper-doped folic acid carbon dots (Cu-FACDs) were initially synthesized via a hydrothermal method, using folic acid and copper gluconate as precursors. Subsequently, the nanoparticles CFCFB were obtained through cross-linking and self-assembly of Cu-FACDs with ferrocene dicarboxylic acid (FeDA) and 3-bromopyruvic acid (3BP). The catalytic effect of carbon dots in CFCFB enhanced the activity of the Fenton reaction, thereby promoting CDT-induced ICD and increasing the intracellular oxygen concentration. Additionally, 3BP inhibited cellular respiration, further amplifying the oxygen concentration. The photothermal conversion efficiency of CFCFB reached 55.8%, which significantly enhanced its antitumor efficacy through photothermal therapy. Immunofluorescence assay revealed that treatment with CFCFB led to an increased expression of ICD markers, including calreticulin (CRT) and ATP, as well as extracellular release of HMGB-1, indicating the induction of ICD by CFCFB. Moreover, the observed downregulation of ARG1 expression indicates a transition in the tumor microenvironment from an immunosuppressive state to an antitumor state following treatment with CFCFB. The upregulation of IL-2 and CD8 expression facilitated the differentiation of effector T cells, resulting in an augmented population of CD8+ T cells, thereby indicating the activation of systemic immune response.

Rapid diagnosis of acute myocardial infarction through integrated microfluidic chips for detection of characteristic targets.

Myoglobin (Myo), creatine kinase-MB (CKMB), and cardiac troponin I (cTnI) are crucial biomarkers for diagnosing acute myocardial infarction (AMI) The accurate and rapid detection of these three targets can greatly improve the prognosis of AMI patients. Herein, this study developed a microfluidic immunofluorescence method that can detect all three targets in 10-15 min. Ultrasonic atomization and spray technology are used to modify the surface of the injection-molded microfluidic chip (MFC), which effectively solves the problem of biological cross-linking and antibody immobilization on the MFC surface. In addition, it improves the hydrophilicity of the chip surface, thus enhancing fluid self-driving effect. The linear response towards Myo, CKMB and cTnI range from 5 ng/mL to 500 ng/mL, 1 ng/mL to 70 ng/mL, and 0.05 ng/mL to 30 ng/mL, respectively. The intra-batch precision is ≤ 10%, and the inter-batch precision is ≤ 15%. Furthermore, this method shows good consistency compared with the BECKMAN ACCESS2 chemiluminescent immunoanalyzer. The present work provides an AMI diagnostic method with high sensitivity, good repeatability, high accuracy and simple operation, which can satisfy the needs of clinical diagnosis, and shows promising application prospects.

Development of monoclonal antibodies targeting the conserved fragment of hexon protein to detect different serotypes of human adenovirus.

Human adenovirus (HAdV) infects the respiratory system, thus posing a threat to health. However, immunodiagnostic reagents for human adenovirus are limited. This study aimed to develop efficient diagnostic reagents based on monoclonal antibodies for diagnosing various human adenovirus infections. Evolutionary and homology analyses of various human adenoviral antigen genes revealed highly conserved antigenic fragments. The prokaryotic expression system was applied to recombinant penton, hexon, and IVa2 conserved fragments of adenovirus, which were injected into BALB/c mice to prepare human adenovirus-specific monoclonal antibodies. Enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence assay (IFA), and Western blotting were used to determine the immune specificity of the monoclonal antibodies. Indirect ELISA showed that monoclonal antibodies 1F10, 8D3, 4A1, and 9B2 were specifically bound to HAdV-3 and HAdV-55 and revealed high sensitivity and low detection limits for various human adenoviruses. Western blotting showed that 1F10 and 8D3 specifically recognized various human adenovirus types, including HAdV-1, HAdV-2, HAdV-3, HAdV-4, HAdV-5, HAdV-7, HAdV-21, and HAdV-55, and 4A1 specifically recognized HAdV-1, HAdV-2, HAdV-3, HAdV-5, HAdV-7, HAdV-21, and HAdV-55. IFAs showed that 1F10, 8D3, and 4A1 exhibited highly selective localization to A549 cells infected with HAdV-3 and HAdV-55. Finally, two antibody pairs that could detect hexon antigens HAdV-3 and HAdV-55 at low concentrations were developed. The monoclonal antibodies developed in this study show potential for detecting human adenoviruses. IMPORTANCE: In this study, we selected the three most conserved antigenic fragments of human adenovirus to prepare a murine monoclonal antibody for the first time, and human adenovirus antigenic fragments with heretofore unheard of degrees of conservatism were isolated. The three monoclonal antibodies with the ability to recognize human respiratory adenovirus over a broad spectrum were screened by hybridoma and monoclonal antibody preparation. Human adenovirus infections are serious; however, therapeutic drugs and diagnostic reagents are scarce. Thus, to reduce the serious consequences of human viral infections and adenovirus pneumonitis, early diagnosis of infection is required. The present study provides three monoclonal antibodies capable of recognizing a wide range of human adenoviruses, thereby offering guidance for subsequent research and development.

Seasonal Testing, Results, and Effect of the Pandemic on Coxsackievirus Serum Studies.

Coxsackieviruses (CVs) are common causes of infections and can be life-threatening. Unfortunately, rigorous studies guiding the clinician in interpreting CV serum antibody titer testing is lacking. To explore the epidemiology of circulating CVs and the serological test utility in aiding diagnosis of CV infections in our community, we obtained results of CV immunologic diagnostic tests between 2018 and 2022 from a regional healthcare database. For CV type A, rare individuals had positive CF (complement fixation) tests whereas all 16 individuals with IFA testing showed at least one positive serotype. For CV type B CF testing, 52.2% of 222 patients had at least one serotype positive, with B5 being most common and also the most common with higher titers (14.8% with ≥1:32). We found a significant reduction in seropositivity rate during the pandemic in 2020 compared to 2018, which continued through 2022 (OR: 0.2, 95% CI: 0.08-0.49, p-value < 0.001). During the pandemic, the seasonal pattern of positive tests varied from the pre-pandemic pattern. Testing for CVs was increased after the first year of the pandemic. Overall, the variability by month and seasonal change in our data support that CF testing can be used to identify recent CVB infection.

Serosurvey of Coxiella burnetii in Descendants of Former Black Slaves (Quilombola Communities) of Southern Brazil.

Brazilian descendants of former Black-slave (quilombola) communities have been predisposed to several zoonotic diseases due to social vulnerability, characterized by subsistence and close contact with livestock and companion animals. Accordingly, the present study has assessed anti-Coxiella burnetii antibodies in 200 individuals and 20 dogs from four quilombola communities located in Paraná State, southern Brazil. Serum samples were tested by indirect immunofluorescence assay (IFA) using in-house and commercial diagnostic protocols, with analysis of seropositive titers and antibody type. Fisher's exact test was used to compare seropositivity to C. burnetti with binary variables, with variables with three or more possible responses submitted to logistic regression. In total, 44/200 (22%; 95% CI 16.82-28.24) people tested positive, and 4.5% had titers higher than 128, indicating a recent onset of C. burnetii infection. Seropositive individuals were statistically associated with the Limitão community (p = 0.0013), urban workers as occupations (p = 0.0475), consumption of undercooked meat (p = 0.0159), and contact with animal abortion (p = 0.0276). No seropositivity association was found for age, sex, education, habit of entering forest areas, consumption of game meat, consumption of raw milk, flea and tick bites, dog contact, or history of female miscarriage. Only one of 20 dogs was seropositive with a titer of 128, probably related to an acute animal infection. Despite the prevalence here being higher than previous Brazilian reports, including with symptomatic populations, the results were within range for worldwide outbreaks and occupational risk populations. To the reader's knowledge, this is the first human survey of Q fever in southern Brazil and should be considered a warning for C. burnetii in vulnerable populations, particularly Quilombola communities.

Direct Detection of Borrelia Species in Tissues.

Among the controversies in Lyme disease is the potential for Borrelia spirochetes to persist after guideline-directed antimicrobial therapy. Direct detection of the spirochetes has been essential to explore this phenomenon, given that the infection is often occult and infrequently observed in blood and other body fluids. In addition, the role of spirochetal infection has been examined in the etiology of neurodegenerative diseases through detection in affected tissues. In this chapter, we describe methodology to specifically identify Borrelia DNA, RNA, and intact organism (via protein) in tissue for studies of Lyme Borreliosis.

Adopting the International Consensus on ANA Patterns (ICAP) classification for reporting: the experience of Italian clinical laboratories.

The indirect immunofluorescence assay on HEp-2 cells (HEp-2 IFA) is still considered the reference method to detect anti-nuclear antibodies (ANA) because of its high sensitivity and represents a relevant tool for the diagnosis of autoimmune rheumatic diseases. During the last decade, the International Consensus on ANA Patterns (ICAP) initiative promoted harmonization and understanding of HEp-2 IFA staining pattern nomenclature, as well as promoting their use in patient care by providing interpretation for HEp-2 IFA test results. In conjunction with a nationwide survey on the evolution of autoantibody diagnostics in autoimmune rheumatic diseases, we focused on the adherence of the Italian laboratories to the ICAP nomenclature analyzing its lights and shadows. The recent ICAP-oriented report, largely used today among Italian laboratories, also represents a further step in harmonizing and improving communication with the clinicians, adding value to laboratory findings and helping with critical clinical decisions.

Extended autoantibody panel in Turkish patients with early-stage systemic sclerosis: Coexpressions and their influences on clinical phenotypes.

BACKGROUND/AIM: To investigate the frequency and clinical relevance of an extended autoantibody profile in patients with systemic sclerosis (SSc). MATERIALS AND METHODS: In this cross-sectional study, serum from 100 consecutive patients was subjected to indirect immunofluorescence (IIF) (HEp-20-10/primate liver mosaic) and Systemic Sclerosis Profile by EUROIMMUN to evaluate anti-nuclear antibodies (ANA) and autoantibodies against 13 different autoantibodies in patients with SSc less than 3 years. RESULTS: Ninety-three of 100 patients were positive for ANA by IIF. Fifty-three patients showed single positivity, 26 anti-topoisomerase antibodies (anti-Scl70 ab), 16 anticentromere antibodies (ACAs), six anti-RNA polymerase III antibodies (anti-RNAPIII ab), one anti-Ku antibody, one anti-PM/Scl100 antibody, two anti-PM/Scl75 antibodies, one anti-Ro52 antibody, whereas 32 patients had multiple autoantibody positivities. Among classic SSc-specific autoantibodies, anti-Scl70 and anti-RNAPIII abs showed the highest cooccurrence (n = 4). One patient was simultaneously positive for anti-RNAPIII ab and ACA, and one was positive for ACA and anti-Scl70 ab. The clinical features were not statistically different between single and multiple autoantibody-positivity for classic SSc-specific autoantibodies (ACA, anti-Scl70 ab, and anti-RNAPIII ab), except for digital ulcer in the multiantibody positive ACA group (p = .019). CONCLUSION: Based on our results, coexpression of autoantibodies is not uncommon in SSc patients. Although autoantibodies specific to SSc in early disease show generally known clinical features, it remains to be investigated how the coexpression of autoantibodies will affect clinical presentation.