Serum Enzyme-linked Immunosorbent Assay Research Articles

Seropositivity for Trypanosoma cruzi in domestic dogs from Sonora, Mexico

BackgroundChagas disease is an important health problem in Latin America due to its incapacitating effects and associated mortality. Studies on seropositivity for Trypanosoma cruzi in Mexican dogs have demonstrated a direct correlation between seropositivity in humans and dogs, which can act as sentinels for the disease in this region. The objective of this study was to determine the seropositivity for T.cruzi infection in dogs from Sonora, a northern borderstate of Mexico.MethodsResponsible pet owners were selected at random from an urban area of Empalme municipality, Sonora, Mexico, and from there, 180 dog samples were collected. Anti-T. cruzi antibodies were determined using the enzyme-linked immunosorbent assay (ELISA) method. Reactive ELISA sera were processed by indirect immunofluorescence to confirm the presence of anti-T. cruzi antibodies. For the statistical analysis, chi-square tests were conducted.ResultsDogs’ sera showed a seropositivity rate of 4.44%. The rate of seropositivity was not associated with the dogs’ age, sex, or socioeconomics pertaining to the geographical area. One sample (1/180, 0.55%) showed the acute state of the disease.ConclusionsThe study found a presence of anti-T. cruzi antibodies in dogs in this area, which suggests vector transmission. There is a need for active surveillance programs throughout the state of Sonora and vector control strategies should also be implemented in endemic regions.

Comparison of real-time PCR, nested PCR, and galactomannan antigen detection by enzyme-linked immunosorbent assay in sera for diagnosis of invasive aspergillosis

Conventional methods for diagnosis of invasive aspergillosis (IA) lack sensitivity and specificity. Serological methods still have many cases of cross-reactivity. However, molecular techniques seem to arise as a rapid approach, specific and direct that could be used in the diagnosis of IA. In this study, we analyzed 88serum samples from patients of having IA using GM-ELISA test, nested PCR with primers for the rRNA 18S of Aspergillus genus and real time PCR specific for A.fumigatus. Among the 88samples, 64of them had positive GM titers and 23had positive nested PCRs; 18of the 23PCR-positive samples were also GM-positive. On the other hand, 18samples were detected positive by reel time PCR; 13positive samples were also detecting positive by nested PCR. QPCR revealed 26% of the patients with IA, while nested PCR and galactomannan ELISA revealed respectively 34% and 94% of the patients with IA. Probable IA was diagnosed in 18and possible IA was diagnosed in 6episodes. Forty-four episodes were defined as not having IA. The positive and negative predictive values were respectively 100%, and 88% for QPCR, 100% and 97% for nested PCR, and 28% and 60% for GM test. These results suggesting that combined use of methods might improve the diagnosis of IA.

Adiponectin exerts a potent anti-arthritic effect and insulin resistance in collagen-induced arthritic rats.

Previous research has shown that adiponectin (AD) induces severe insulin resistance (IR) and exhibits pro-inflammatory effect, so it could serve as a useful risk biomarker in rheumatoid arthritis (RA). The present study aims to evaluate the effect of AD on IR and anti-arthritis in collagen-induced arthritic (CIA) rats. After immunization with bovine type II collagen (CII), Wistar rats were administered with AD (60μg/kg/day) or saline into the ankle joint cavity of the left hind leg for 15days. The severity of arthritis was clinically and histologically assessed. Arthritis score was recorded every other day for each paw. Paw volume was measured on alternate days to monitor the progression of the disease in the arthritic control group. Tumor necrosis factor (TNF)-α, interleukin (IL)-1, AD, insulin and fasting glucose were measured in sera. Histopathology of joint synovial tissues was also examined. Treatment with AD resulted in significantly delayed onset of arthritis as well as decreased clinical arthritis and histopathological severity scores. AD reduced both serum fasting glucose, TNF-α, IL-1 and IR. Histological analysis confirmed treatment with AD suppressed joint synovial inflammation and immunohistochemical expression of TNF-α compared to the CIA group. Surprisingly, adiponectin levels measured by enzyme-linked immunosorbent assay in serum were significantly increased in CIA rats compared to the normal group. Adiponectin might display anti-inflammatory effects. These results suggest that AD may be a potential immunosuppressant for the treatment of RA linked to metabolic disease.

Multiple Soluble TGF-β Receptors in Addition to Soluble Endoglin Are Elevated in Preeclamptic Serum and They Synergistically Inhibit TGF-β Signaling.

Preeclampsia (PE) can be classified into early-onset (<34 weeks of gestation) and late-onset (>34 weeks of gestation) subtypes. Soluble endoglin, an auxiliary receptor for transforming growth factor (TGF)-β ligands, is increased in PE circulation and believed to inhibit TGF-β action by sequestering the ligands. However, soluble endoglin, with a low affinity to TGF-β ligands, has been demonstrated to have little effect by itself on TGF-β action. We examined whether multiple soluble TGF-β receptors are elevated in PE circulation and whether they synergistically block TGF-β signaling. TGF-β receptors were measured using enzyme-linked immunosorbent assay in sera collected from preeclamptic pregnancies and gestation-age-matched controls. TGF-β signaling was assessed using an in vitro bioassay and a tube formation assay. TGF-β type I, II, and III receptors were all identified in pregnant serum; all were substantially elevated in early-onset but not late-onset PE. Endoglin was increased in both subtypes. At the greatest concentrations detected in PE, none of these soluble TGF-β receptors alone, including endoglin, inhibited TGF-β signaling. However, when all four soluble receptors were present, signaling of both TGF-β1 and TGF-β2 was substantially reduced. Removal of any one of these soluble receptors alleviated TGF-β1 inhibition; however, removal of soluble TGFβRIII was necessary to relieve TGF-β2 inhibition. Multiple soluble TGF-β receptors are present in pregnant circulation and elevated in early-onset PE; they synergistically inhibit TGF-β signaling, which might be more likely to occur in early-onset than late-onset PE. Reducing soluble TGFβRIII, rather than endoglin, would be more effective in alleviating the inhibition of both TGF-β1 and TGF-β2 signaling in PE.

Calretinin as a blood-based biomarker for mesothelioma

BackgroundMalignant mesothelioma (MM) is a deadly cancer mainly caused by previous exposure to asbestos. With a latency period up to 50 years the incidence of MM is still increasing, even in countries that banned asbestos. Secondary prevention has been established to provide persons at risk regular health examinations. An earlier detection with tumor markers might improve therapeutic options. Previously, we have developed a new blood-based assay for the protein marker calretinin. Aim of this study was the verification of the assay in an independent study population and comparison with the established marker mesothelin.MethodsFor a case-control study in men, a total of 163 cases of pleural MM and 163 controls were available from Australia, another 36 cases and 72 controls were recruited in Germany. All controls had asbestosis and/or plaques. Calretinin and mesothelin were determined by ELISA (enzyme-linked immunosorbent assay) in serum or plasma collected prior to therapy. We estimated the performance of both markers and tested factors potentially influencing marker concentrations like age, sample storage time, and MM subtype.ResultsCalretinin was able to detect all major subtypes except for sarcomatoid MM. Calretinin showed a similar performance in Australian and German men. At a pre-defined specificity of 95% the sensitivity of calretinin reached 71% and that of mesothelin 69%, when excluding sarcomatoid MM. At 97% specificity, the combination with calretinin increased the sensitivity of mesothelin from 66% to 75%. Sample storage time did not influence the results. In controls the concentrations of calretinin increased 1.87-fold (95% CI 1.10–3.20) per 10 years of age and slightly more for mesothelin (2.28, 95% CI 1.30–4.00).ConclusionsCalretinin could be verified as a blood-based marker for MM. The assay is robust and shows a performance that is comparable to that of mesothelin. Retrospective analyses would not be limited by storage time. The high specificity supports a combination of calretinin with other markers. Calretinin is specific for epithelioid and biphasic MM but not the rarer sarcomatoid form. Molecular markers like calretinin and mesothelin are promising tools to improve and supplement the diagnosis of MM and warrant further validation in a prospective study.

Role of microRNA-7 and selenoprotein P in hepatocellular carcinoma.

There is an obvious need to diagnose hepatocellular carcinoma using novel non-invasive and sensitive biomarkers. In this regard, the aim of this study was to evaluate and correlate both relative quantification of microRNA-7 using quantitative real time polymerase chain reaction and quantitative analysis of selenoprotein P using enzyme-linked immunosorbent assay in sera of hepatocellular carcinoma patients, chronic liver disease patients, as well as normal healthy subjects in order to establish a new diagnostic biomarker with a valid non-invasive technique. In addition, this study aimed to investigate whether changes in selenium supply affect microRNA-7 expression and selenoprotein P levels in human hepatocarcinoma cell line (HepG2). The results showed a highly significant decrease in serum microRNA-7 relative quantification values and selenoprotein P levels in malignant group in comparison with benign and control groups. The best cutoff for serum microRNA-7 and selenoprotein P to discriminate hepatocellular carcinoma group from benign and control groups was 0.06 and 4.30 mg/L, respectively. Furthermore, this study showed that changes in selenium supply to HepG2 cell line can alter the microRNA-7 profile and are paralleled by changes in the concentration of its target protein (selenoprotein P). Hence, serum microRNA-7 and selenoprotein P appear to be potential non-invasive diagnostic markers for hepatocellular carcinoma. Moreover, the results suggest that selenium could be used as an anticancer therapy for hepatocellular carcinoma by affecting both microRNA-7 and selenoprotein P.

Factors associated with Mycobacterium avium subsp. paratuberculosis in dairy cows from Northern Antioquia, Colombia

Background: Johne´s disease (JD) is a severe enteritis that affects ruminants and has been diagnosed in cattle and sheep in Colombia. However, epidemiological information on the disease in this country is scarce. Objective: to identify factors associated with the JD serum enzyme-linked immunosorbent assay (ELISA) status of dairy cows. Methods: a cross-sectional study was carried out in 307 asymptomatic adult Holstein dairy cows from 14 herds in nine districts of Belmira and San Pedro de los Milagros municipalities during November, 2007. From 19 to 25 cows, ≥ 2 years of age were randomly selected and blood sampled from every herd. A commercial ELISA kit was used to analyze sera. Information regarding cow related factors (age, farm-born, parity, and daily milk yield) and herd management practices (i.e. herd size, herd average milk production, current presence of symptomatic animals, cattle purchase, own animals grazing in foreign pastures, feeding of calves before weaning, manure spread on pastures, and sighting of birds in feed storing areas) was collected using questionnaires. Descriptive statistics were computed for all variables and a multivariable logistic regression model was constructed (p 1 parity. The OR was 0.74 times lower (OR = 0.26; 95% CI: 0.096-0.70; p<0.01) in herds feeding calves with pooled colostrum from several cows, compared to herds feeding calves with colostrum from their own dams. Conclusion: JD seroprevalence was 10 and 70% at animal and herd-level, respectively. Cow and herd factors “parity” and “feeding of calves before weaning” showed weak and strong association with positive results, respectively

Humoral Antibody Responses to HIV Viral Proteins and to CD4 Among HIV Controllers, Rapid and Typical Progressors in an HIV-Positive Patient Cohort.

The purpose of this study was to assess humoral antibody responses as a function of disease progression (DP) in a well-defined HIV+ cohort. We quantified antibodies to HIV-1 gp120, Gag, and CD4 receptor by enzyme-linked immunosorbent assay in sera from a cohort of 97 HIV+ subjects at defined stages of DP. We also measured antibody-dependent cellular cytotoxicity (ADCC) as a function of the clinical status of the patients. We purified antibodies to CD4 and gp120 and assessed them for specificity, ability to block gp120 binding to target cells, ability to block virus infection, and ability to facilitate ADCC. All of the HIV+ patient samples were positive for antibodies to HIV gp120 and p24 and 80% showed evidence of hypergammaglobulinemia. Approximately 10% of cohort members were positive for antibodies to CD4, but we noted no significant correlation relevant to DP. There were statistically significant differences between the groups concerning the level of humoral response to gp120 and Gag. However, we observed no distinction in ability of anti-gp120 antibodies purified from each group to neutralize infection. In addition, there was a statistically significant difference in ADCC, with elite controllers exhibiting significantly lower levels of ADCC than the other five groups. We detected IgA anti-gp120 antibodies, but did not correlate their presence with either DP or ADCC levels. The results are consistent with the interpretation that the humoral antibody response to the antigens assessed here represents a signature of the level of viremia but does not correlate with clinical status of HIV infection.

Levels of Circulating Tumor Necrosis Factor-α in Children with Symptomatic Dengue Evaluated by ELISA and Bead-Based Assays.

Tumor necrosis factor (TNF)-α is a key cytokine in the pathogenesis of dengue virus infection, and its accurate detection in several types of human samples is critical. The enzyme-linked immunosorbent assay (ELISA) is the gold standard for the detection of TNF-α, but multiplexed bead-based assays such as cytometric bead array (CBA) are now frequently used. Here, using ELISA and two CBAs commercially available, we measured TNF-α concentrations in plasma and serum from children with acute dengue virus infection and healthy controls. To evaluate the detection efficiency and factors affecting it, spiked recovery and immune complex dissociation assays were also performed. The levels of TNF-α evaluated by ELISA in paired serum and plasma samples from children with dengue positively correlated (rho = 0.99, p < 0.0001). Children with dengue had higher levels of plasma TNF-α than those of healthy children (p = 0.004). The ELISA detected TNF-α in a higher number of plasma samples than the CBA (p < 0.0001), and both methods only correlated when TNF-α was evaluated in buffer-based solutions but not in plasma, indicating the presence of a factor interfering with the detection of TNF-α in plasma. The recovery of several types of human recombinant TNF-α was dramatically decreased in plasma but not in tissue culture media (p ≤ 0.01), and this effect was similar in the plasma obtained from the children with dengue or the healthy controls. The dissociation of immune complexes did not improve TNF-α recovery. Dilution of the plasma samples increased the recovery of TNF-α, but at high concentrations of the cytokine. In short, plasma affects the efficiency of TNF-α detection, and this effect should be considered in the measurement of this cytokine.

HMGB1 overexpression as a prognostic factor for survival in cancer: a meta-analysis and systematic review.

As there are millions of cancer deaths every year, it is of great value to identify applicable prognostic biomarkers. As an important alarm, the prognostic role of high mobility group box 1 (HMGB1) in cancer remains controversial. We aim to assess the association of HMGB1 expression with prognosis in cancer patients. Systematic literature searches of PubMed, Embase and Web of Science databases were performed for eligible studies of HMGB1 as prognostic factor in cancer. Pooled hazard ratios (HRs) and 95% confidence intervals (CIs) were calculated to evaluate the influence of HMGB1 expression on overall survival (OS) and progression-free survival (PFS) in cancer patients. 18 studies involving 11 different tumor types were included in meta-analysis. HMGB1 overexpression was significantly associated with poorer OS (HR: 1.99; 95% CI, 1.71-2.31) and PFS (HR: 2.26; 95% CI, 1.65-3.10) irrespective of cancer types including gastric cancer, colorectal cancer, hepatocellular carcinoma, pancreatic cancer, nasopharyngeal carcinoma, head and neck squamous-cell carcinoma, esophageal cancer, malignant pleural mesothelioma, bladder cancer, prostate cancer, and cervical carcinoma. Subgroup analyses indicated geographical area and size of studies did not affect the prognostic effects of HMGB1 for OS. Morever, HMGB1 overexpression had a consistent correlation with poorer OS when detected by immunohistochemistry in tissues and enzyme-linked immunosorbent assay in serum, whereas the correlation did not exist by quantitative real-time reverse-transcription polymerase chain reaction in tissues. HMGB1 overexpression is associated with poorer prognosis in patients with various types of cancer, suggesting that it is a prognostic factor and potential biomarker for survival in cancer.

The role of soluble tumor necrosis factor like weak inducer of apoptosis and interleukin-17A in the etiopathogenesis of celiac disease: A cross-sectional study.

Our aim in this study was to determine soluble tumor necrosis factor (TNF)-like weak inducer of apoptosis (sTWEAK) and interleukin-17A (IL-17A) levels in celiac disease, and their association with the gluten diet and autoantibodies. Eighty patients with celiac diagnosis and 80 healthy control individuals with similar age, gender and body mass index to the patient group were included in the study. Serum sTWEAK and IL-17A levels were measured by the serum enzyme-linked immunosorbent assay kit. The median IL-17A (117.5 pg/mL vs. 56.7 pg/mL; P = 0.001) level in celiac patients was higher than in the control group, while the median sTWEAK (543 pg/mL vs. 643 pg/mL; P = 0.016) level in patients was determined to be lower. In the patient group, patients who complied with the gluten diet had a lower level of median IL-17A (98.1 pg/mL vs. 197.5 pg/mL; P = 0.034) and a higher level of sTWEAK (606 pg/mL vs. 522.8 pg/mL; P = 0.031) than those who did not adhere. Furthermore, the IL-17A level was higher and the sTWEAK level was lower in celiac patients with positive antibody than those with negative antibody. A positive correlation was determined among anti-gliadin antibody IgA, anti-gliadin antibody IgG, anti-tissue transglutaminase IgG levels and the IL-17A level, and a negative correlation was determined with the sTWEAK level. In celiac disease, the sTWEAK and IL-17A levels differ between patients who cannot adapt to the gluten diet and who are autoantibody positive, and patients who adapt to the diet and are autoantibody negative. We believe that sTWEAK and IL-17A are associated with the inflammation in celiac pathogenesis.

Seroprevalence of ovine brucellosis in Bangladesh

An investigation was carried out to study the seroprevalence and risk factors for Brucella seropositivity in sheep in Bangladesh. For this, highly sheep populated ten different districts including all seven divisions of Bangladesh were selected where sheep of Panchagarh (151), Naogaon (101), Tangail (83), Noakhali (56), Sunamganj (52), Patuakhali (119) and Dhaka (75) were tested but sheep of Chapai Nawabganj, Khulna, Noakhali and Feni has under tested for Brucellosis. In the present study, seroprevalence of ovine brucellosis was tested by Rose Bengal Plate Test (RBPT), Serum Agglutination Test (SAT) and Enzyme-Linked Immunosorbent Assay (ELISA). About 5% sheep might have got Brucella infection. The highest Brucella antibody was observed in sheep of Tangail district (8.4%) followed by the sheep of Savar (8.0%), where as sheep of Subarna Char and Kala Para were free from Brucella antibody. In relation to age and sex, adults (6.5%) were more positive than young (3.6%) and female (5.3%) were more susceptible than male (3.8%).Asian J. Med. Biol. Res. March 2016, 2(1): 13-18

Anti-Melanoma Differentiation-Associated Gene 5 Is Associated With Rapidly Progressive Lung Disease and Poor Survival in US Patients With Amyopathic and Myopathic Dermatomyositis.

Clinically amyopathic dermatomyositis (CADM) is a subset of dermatomyositis (DM) presenting with the characteristic rash of DM without objective muscle weakness. Asian studies report that anti-melanoma differentiation-associated gene 5 (anti-MDA-5) autoantibody in CADM is associated with interstitial lung disease (ILD), particularly rapidly progressive ILD (RPILD). These associations have not been established in US myositis patients. The goal of our study was to determine the association of anti-MDA-5 autoantibody with ILD, RPILD, and survival in US patients with CADM and classic DM. CADM patients were identified in the University of Pittsburgh Myositis Center Database and matched 1:1 (sex and age) to classic DM controls. Anti-MDA-5 was measured by serum enzyme-linked immunosorbent assay. Kaplan-Meier, log rank, and chi-square tests were used for analysis. We identified 61 CADM patients (62% women, mean age 48.2 years) and 61 classic DM controls (64% women, mean age 44.8 years). The frequencies of anti-MDA-5 positivity, ILD, and RPILD were similar in the 2 cohorts (MDA-5 positive: CADM 13.1% [8 of 61] and DM 13.1% [8 of 61], ILD positive: CADM 31.1% [19 of 61] and DM 26.2% [16 of 61], and RPILD positive: CADM 8.2% [5 of 61] and DM 5% [3 of 61]; P = 1, 0.55, and 0.46, respectively). Anti-MDA-5 positivity was significantly associated with ILD, since 50% of MDA-5-positive subjects (8 of 16) had ILD versus 25.5% of MDA-5-negative subjects (27 of 106; P = 0.04). Anti-MDA-5 was strongly associated with RPILD (P < 0.001). Anti-MDA-5-positive patients with ILD had worse baseline pulmonary function testing variables compared to anti-MDA-5-negative patients. Anti-MDA-5 positivity was significantly associated with poor survival (P = 0.007). Anti-MDA-5 antibody is significantly associated with ILD, RPILD, worse pulmonary outcome, and survival in US classic DM and CADM patients.

Evaluation of a New Enzyme-Linked Immunosorbent Assay (ELISA) for Johne's Disease in Goat Serum

Control of Johne’s Disease (JD) is a high priority, it causes significant economic losses in the livestock industry. An efficacious vaccine for JD is not currently available and no satisfactory treatment exists, so control programs depend on diagnostic tests based on detection of the immune response generated after infection with Mycobacterium avium subsp. paratuberculosis (MAP)-the causative agent of JD-to identify and remove infected animals and management practices to interrupt transmission. Objectives: The objective of this study was to evaluate a new, commercially-available, Enzyme-Linked, Immunosorbent Assay (ELISA) that was developed for cattle, for use with goat serum. For comparison, a United States Department of Agriculture (USDA)-approved ELISA kit for goats, an Agar Gel Immunodiffusion (AGID) test and a Polymerase Chain Reaction (PCR) test to detect MAP in fecal samples were also evaluated. Methods: Infection status at necropsy was taken as the reference standard, determined by gross, histological and microbiological examination of tissue specimens. The serum and fecal samples were taken from goat kids that had participated in a previous vaccine efficacy study. Eighty goat kids were vaccinated either once subcutaneously at eight weeks with a commercial vaccine, or orally at eight and 10 weeks with one of five experimental oral vaccines or a sham-control oral vaccine. Kids were challenged orally with a bovine isolate of MAP, three weeks after the last vaccination. Results: None of the vaccines prevented infection. Samples taken from 10 to 13 months post-challenge were used to compare the assays. The specificity of all tests was 100%. At 13 months, the new ELISA, approved ELISA, AGID test and fecal PCR test had sensitivities of 55.7%, 52.9%, 24.3% and 87.1% respectively. At 11 months the sensitivities were 48.6%, 38.6%, 10.0% and 78.6% respectively and the sensitivity of the new ELISA was significantly better than the approved ELISA (p = 0.0196). Conclusion: The new ELISA effectively detected circulating MAP antibody in the serum of infected goats and the preliminary results in this study indicate the kit has potential for use in goat JD control programs. The results for the new ELISA in goat serum are comparable with those reported when the ELISA test is used for cattle serum.

Analysis of IL-10 and IL-6 Gene Polymorphisms and Their Serum Levels in Patients with Brucellosis: A Case Control Study

ABSTRACTBackground: It seems that polymorphism in the regulatory areas of cytokine genes affects the cytokine production capacity and may play a role in the development of infectious diseases. Interleukin-10 (IL-10) and Interleukin-6 (IL-6), which are cytokines of Th2, cause the macrophage become inactive and patient conditions get worse.Methods: In this case‐control study, 60 patients with brucellosis and 60 healthy participants were recruited. IL-10 genotyping at positions -1082 (G/A), -819 (C/T), and -592 (C/A) and IL-6 genotyping at position -174 (G/C) were analyzed by amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) and restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR) methods. The levels of IL-10 and IL-6 were determined by a sandwich enzyme-linked immunosorbent assay in sera of study population.Results: The AA and CC genotypes of the IL-10 gene at positions -1082 G/A and -819 C/T were significantly more frequent in patients in comparison to controls, respectively. The AG genotype of the IL-10 gene at positions -1082 G/A was significantly more frequent in control groups than the patients. Serum levels of IL-10 and IL-6 were significantly more frequent in the patients than in the control groups.Conclusions: Our study showed that the AA and CC genotypes at positions -1082 and -819 are very important, respectively. These results suggest that IL-10 (-1082 G/A) GG genotype may be considered as a risk factor for brucellosis, while the AG genotype might be a protective factor against the disease.

Detection of Anti-Pentraxin-3 Autoantibodies in ANCA-Associated Vasculitis.

ObjectivesPentraxin 3 (PTX3), in common with myeloperoxidase and proteinase 3, is stored in human neutrophil granules and is expressed on apoptotic neutrophil surface. We therefore investigated the presence of anti-PTX3 autoantibodies (aAbs) in the sera of antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV) patients.MethodsPresence of anti-PTX3 autoantibodies was analysed by a specific enzyme-linked immunosorbent assay in sera from 150 patients with microscopic polyangiitis (MPA), granulomatosis with polyangiitis (GPA), and eosinophilic granulomatosis with polyangiitis (EGPA), and in sera of 227 healthy subjects (HS), 40 systemic sclerosis (SSc) patients, and 25 giant cell arteritis patients (GCA). Using indirect immunofluorescence on fixed human neutrophils, we also analyzed the staining pattern associated with the presence of anti-PTX3 aAbs.ResultsAnti-PTX3 aAbs were detected in 56 of 150 (37.3%) of the AAV patients (versus 12 of 227 (5.3%) of HS, p<0.001) and, interestingly, in 7 of 14 MPO and PR3 ANCA negative AAV patients. Moreover, by indirect immunofluorescence on fixed neutrophils, anti-PTX3 aAbs gave rise to a specific cytoplasmic fluorescence pattern distinct from the classical cytoplasmic (c-ANCA), perinuclear (p-ANCA), and atypical (a-ANCA) pattern. Anti-PTX3 aAbs levels were higher in patients with active AAV as compared to patients with inactive disease.ConclusionOur work suggests that PTX3 is as a novel ANCA antigen. Anti-PTX3 aAbs appear thus as a promising novel biomarker in the diagnosis of AAV, including in patients without detectable MPO and PR3 ANCA.

Development of Anti-bodies against Infliximab in Iraqi Patients with Rheumatoid Arthritis.

Background: Rheumatoid arthritis is a common chronic and destructive autoimmune arthropathy .Treatment with infliximab gives great improvement to a large numbers of patients with RA ,however, in some patients after prolonged treatment infliximab can induce anti-infliximab antibodies formation and result to loss of infliximab efficacy and active persistent disease.Objective: to investigate the frequency of anti-infliximab antibodies in Iraqi patients with rheumatoid arthritis.Patients and methods: fifty Iraqi RA patients(36 females and 14 males) compared with 50 control( 25 healthy control and 25 case control (patients with RA on other treatment) ) were included in this study from begging of March 2014 till end of September 2014.All patients were diagnosed by full history, complete clinical examination and laboratory test. Anti-infliximab antibodies were meatured using enzymelinked immunosorbent assay in serum of Iraqi patients with RA treated with infliximab more than 3 monthsduration.Results: Antibodies to infliximab were detected in 35(70%) Iraqi RA patients , while the patients without detectable antibodies against infliximab were 15(30%),also there were no anti-infliximab antibodies in the control groups.Conclusion: In this study, nearly three quarter of the Iraqi RA patients treated with infliximab developed anti-infliximab antibodies.

P09.37 Syphilis rapid test validation for treponemal diagnosis in guatemala, 2013–2014

Background International guidelines for syphilis testing recommend non-treponemal tests, and confirmation by more complex treponemal test such as Treponema pallidum haemagglutination (TPHA), enzyme-linked immunosorbent assay (ELISA) or others. Similar to HIV rapid tests, rapid treponemal tests can improve point-of-care testing in laboratories with limited capacities. We present main results of a validation for treponemal diagnosis performed in Guatemala. Methods A syphilis rapid test laboratory validation was performed in two phases during 2013–2014. First stage was performed using frozen serums from National Health Laboratory and second stage was performed using whole blood from pregnant women from peripheral health services from Guatemala City. Four treponemal rapid tests were evaluated in the first stage: Determine TP (DetTP), SD Syphilis (SDSyp), Omega Visitect (OVis), and Rapid Syphilis (RSyp). Only two tests were evaluated in the second stage: DetTP and SDSyp. For confirmation, TPHA was used for diagnosis of treponemal antibodies in serum and treponemal ELISA for plasma. Results A total of 460 serums were evaluated in first stage and 432 whole blood samples in second stage. Compared to TPHA, sensitivity ranged from 87.8% for RSyp to 99.2% in DetTP in serum and 100% for SDSyp and DetTP in whole blood. Specificity was lower, ranging from 74.8% for DetTP to 87.7% for SDSyp in serum and 100% for SDSyp and DetTP in whole blood. Conclusion Rapid treponemal tests are a useful diagnostic tool in syphilis. Sensibility was high enough to be used as a screening test, with good results in whole blood samples. Low prevalence of syphilis in the validation contributed to poor positive predictive values. The use of a reverse algorithm might be useful for implementation of this test in Guatemala. Disclosure of interest statement We declare that we have no conflicts of interest.

Interleukin-28B polymorphisms and interferon gamma inducible protein-10 serum levels in seronegative occult hepatitis C virus infection.

Polymorphisms upstream interleukin (IL)-28B gene and serum levels of interferon gamma inducible protein-10 (IP-10) are associated with spontaneous and treatment-induced hepatitis C virus (HCV) clearance. Patients with seronegative occult HCV infection are anti-HCV and serum HCV-RNA negative but have viral RNA in liver and abnormal values of liver enzymes. We examined if the rs12979860 polymorphism of IL-28B and serum IP-10 levels differ between chronic and seronegative occult CV infection. IL-28B polymorphism was determined with allele specific TaqMan probes in total DNA isolated from peripheral blood mononuclear cells and IP-10 by an enzyme-linked immunosorbent assay in serum from 99 patients with seronegative occult HCV infection and 130 untreated patients with chronic hepatitis C. IL-28B genotypes were also determined in 54 healthy volunteers. Prevalence of the IL-28B CC genotype was significantly higher in seronegative occult HCV infection (52/99; 52.5%) than in chronic hepatitis C (32/130; 24.6%, P < 0.0001) or healthy controls (19/54: 32.5%, P = 0.039). Among patients with seronegative occult HCV infection, HCV-RNA load in liver was significantly lower in those with the IL-28B CC genotype than in those with CT + TT genotypes (2.8 × 10(5) ± 5.8 × 10(4) vs. 4.1 × 10(5) ± 5.9 × 10(4) copies/μg of total RNA respectively; P = 0.023). Mean serum IP-10 levels were significantly lower in patients with seronegative occult HCV infection than in patients with chronic hepatitis C (160.8 ± 17.9 vs. 288.7 ± 13.3 pg/ml respectively; P < 0.0001). These findings suggest that the host immune response plays an important role in seronegative occult HCV infection in comparison with chronic hepatitis C.

Changes in Serum Strongylus Vulgaris-Specific Antibody Concentrations in Response to Anthelmintic Treatment of Experimentally Infected Foals.

Strongylus vulgaris is the most pathogenic nematode parasite of horses. Its extensive migration in the mesenteric blood vessels can lead to life-threatening intestinal infarctions. Recent work has shown that this parasite is still identified among managed horse populations. A serum enzyme-linked immunosorbent assay (ELISA) has been developed for the detection of migrating larvae of S. vulgaris. Previous work has documented an increase in ELISA values following larvicidal treatment with ivermectin and suggested that the target parasite antigen is primarily produced by the later larval stages. The aim of this study was to experimentally inoculate cohorts of foals with S. vulgaris, and then compare ELISA responses to early or later ivermectin treatments. Fifteen foals were held in confinement and infected orally with ~25 S. vulgaris third-stage larvae on Days 0, 7, 14, and 21. Foals were weaned on Day 43 and turned out to a pasture not previously grazed by horses. Foals remained at pasture continuously until the study was terminated on Day 196. On Day 55, foals were randomly allocated to three treatment groups of five each. Group 1 received ivermectin on Day 56, Group 2 received ivermectin on Day 112, and Group 3 foals served as untreated controls. Serum and fecal samples were collected at 28-day intervals throughout the study. Serum samples were analyzed with the S. vulgaris-specific ELISA and fecal samples were processed for fecal egg counting. The ELISA values of Group 1 foals were significantly lower than Groups 2 or 3 on Days 140–196. Both treated groups exhibited increased ELISA values following ivermectin treatment. Results indicate that the target diagnostic antigen is produced throughout the course of arterial infection with S. vulgaris, but that an early ivermectin treatment can reduce the cumulative antigen produced over the course of an infection.