Multiplexed electrochemical immunosensor for detection of celiac disease serological markers

Celiac disease (CD) is a gluten-induced autoimmune enteropathy characterized by the presence of antibodies against gliadin (AGA) and anti-tissue transglutaminase (anti-tTG) antibodies. A disposable electrochemical dual immunosensor for the simultaneous detection of IgA and IgG type AGA and antitTG antibodies in real patient’s samples is presented. The proposed immunosensor is based on a dual screen-printed carbon electrode, with two working electrodes, nanostructured with a carbon–metal hybrid system that worked as the transducer surface. The immunosensing strategy consisted of the immobilization of gliadin and tTG (i.e. CD specific antigens) on the nanostructured electrode surface. The electrochemical detection of the human antibodies present in the assayed serum samples was carried out through the antigen–antibody interaction and recorded using alkaline phosphatase labelled anti-human antibodies and a mixture of 3-indoxyl phosphate with silver ions was used as the substrate. The analytical signal was based on the anodic redissolution of enzymatically generated silver by cyclic voltammetry. The results obtained were corroborated with commercial ELISA kits indicating that the developed sensor can be a good alternative to the traditional methods allowing a decentralization of the analyses towards a point-of-care strategy.

The biological meaning of anti-HBC positive result in blood donors: relation to HBV-DNA and to other serological markers

In order to assess the potential risk of anti-HBc-positive blood donors for post-transfusional hepatitis and to investigate whether other HBV serological markers are capable of identifying the presence of the virus, 1000 first-time blood donors were enrolled between June and July 1997. These donors were screened using routine Brazilian blood center tests (HIV 1 and 2, HTLV 1 and 2, Chagas disease, Syphilis, HCV, HBsAg, anti-HBc and ALT ). The 120 (12%) found to be anti-HBc-positive underwent further tests: HBe, anti-HBe, anti-HBs and HBV-DNA by PCR. Ten cases were HBsAg positive and all were HBV-DNA positive by PCR. Three HBsAg-negative donors were HBV-DNA-positive. Two HBV-DNA-positive donors were also anti-HBs-positive. All the HBV-positive donors had at least one HBV marker other than anti-HBc. Anti-HBc is an important cause of blood rejection. Testing for HBsAg alone is not fully protective and anti-HBc remains necessary as a screening test. The presence of anti-HBs is not always indicative of absence of the virus. The addition of other HBV serological markers could represent an alternative in predicting the presence of the virus when compared with PCR. It is recommended that other studies should be carried out to confirm this finding.

Serological markers and risk factors for hepatitis B and C viruses in patients infected with human immunodeficiency virus

Both hepatitis B and hepatitis C viruses (HBV and HCV) infection are common in HIV-infected individuals as a result of shared risk factors for acquisition. A serological study for HBV and HCV was performed in 251 HIV-positive individuals from Medellín, Colombia. A qualitative RT-PCR for HCV was done in 90 patients with CD4+ T-cell count < 150 per mm³. Serological markers for HBV infection were present in 97 (38.6%) patients. Thirty six of them (37.1%) had isolated anti-HBc. A multivariate analysis indicated that the following risk factors were significantly associated with the presence of these markers: age (OR = 1.05, 95% CI: 1.01-1.08), pediculosis pubis (OR = 1.83, 95% CI: 1.01-3.33), men who have sex with men and women (OR = 3.23, 95% CI: 1.46-7.13) and men who have sex only with men (OR = 3.73, 95% CI: 1.58-8.78). The same analysis restricted to women showed syphilis as the only significant risk factor. Thus, HBV infection was considerably associated with high risk sexual behavior. HCV was present in only two (0.8%) of HIV patients. Both of them were positive by RT-PCR and anti-HCV. This low frequency of HIV/HCV coinfection was probably due to the uncommon intravenous drug abuse in this population. The frequent finding of isolated anti-HBc warrants molecular approaches to rule out the presence of cryptic HBV infection.

SEROLOGICAL MARKERS OF VIRAL, SYPHILITIC AND TOXOPLASMIC INFECTION IN CHILDREN AND TEENAGERS WITH NEPHROTIC SYNDROME: CASE SERIES FROM MATO GROSSO STATE, BRAZIL

Some infections can be the cause of secondary nephrotic syndrome. The aim of this study was to describe the experience of a Renal Disease Reference Clinic from Central Brazil, in which serological markers of some infectious agents are systematically screened in children with nephrotic syndrome. Data were obtained from the assessment of medical files of all children under fifteen years of age, who matched nephrotic syndrome criteria. Subjects were tested for IgG and IgM antibodies against T. gondii and cytomegalovirus; antibodies against Herpes simplex, hepatitis C virus and HIV; and surface antigen (HBsAg) of hepatitis B virus. The VDRL test was also performed. 169 cases were studied. The median age on the first visit was 44 months and 103 (60.9%) patients were male. Anti-CMV IgG and IgM were found in 70.4% and 4.1%, respectively. IgG and IgM against Toxoplasma gondii were present in 32.5% and 5.3%, respectively. Two patients were positive for HBsAg, but none showed markers for HIV, hepatitis C, or Treponema pallidum. IgG and IgM against herpes simplex virus were performed on 54 patients, of which 48.1% and 22.2% were positive. IgM antibodies in some children with clinical signs of recent infection suggest that these diseases may play a role in the genesis of nephrotic syndrome.

Prevalence of serological markers of hepatitis B virus in pregnant women from Paraná State, Brazil

The prevalence of hepatitis B virus (HBV) in Brazil increases from South to North but moderate to elevated prevalence has been detected in the Southwest of Paraná State. The prevalence of serological markers of HBV was evaluated in 3188 pregnant women from different counties in Paraná State and relevant epidemiological features were described. The prevalence of HBV markers in pregnant women for the state as a whole was 18.5% (95% CI = 17.2-19.9), ranging from 7.2% in Curitiba to 38.5% in Francisco Beltrão. The endemicity of HBV marker prevalence in pregnant women was intermediate in Cascavel, Foz do Iguaçu, and Francisco Beltrão, and low in Curitiba, Londrina, Maringá, and Paranaguá. Multiple logistic regression showed that HBV marker prevalence increased with age, was higher among black women, among women of Italian and German descent, and among women who had family members in neighboring Rio Grande do Sul State. Univariate analysis showed that HBV marker prevalence was also higher among women with no education or only primary education, with a lower family income and whose families originated from the South Region of Brazil. Pregnant women not having positive HBV markers (anti-HBc, HBsAg or anti-HBs detected by ELISA) corresponded to 73.7% of the population studied, implying that HBV vaccination needs to be reinforced in Paraná State. The highest prevalence was found in three counties that received the largest number of families from Santa Catarina and Rio Grande do Sul, where most immigrants were of German or Italian ascendance. This finding probably indicates that immigrants that came to this area brought HBV infection to Southwestern Paraná State.

Prevalence of hepatitis B and C serological markers among first-time blood donors in Brazil: A multi-center serosurvey

Little data are available on the seroprevalence of, and risk factors for hepatitis B and C viruses (HBV and HCV) infection in Latin American countries. A multi-center serosurvey was conducted among 3,598 first-time blood donors (65% men) from Sao Paulo, Salvador and Manaus in Brazil. The gender-specific seroprevalences of antibodies against hepatitis B core antigen (anti-HBc) and of the hepatitis B surface antigen (HBsAg) in anti-HBc-positive sera were measured, and risk factors analyzed by gender. The gender-specific seroprevalences of antibodies against HCV (anti-HCV) were measured, but risk factors for HCV were not determined. Anti-HBc and HBsAg seroprevalences were not significantly different in men [101/2,341 (4.31%) and 4/2,229 (0.18%), respectively] and women [65/1,237 (5.25%) and 8/ 1,169 (0.68%), respectively], whereas the seroprevalence of anti-HCV was higher in women (12/1,238 [0.97%] vs. 9/2,353 [0.38%]; odds ratio [OR] = 2.49; 95% confidence interval [Cl]: 1.0-6.0). No significant difference for HBV infection was found across the three study sites or by ethnic group. The seroprevalence of anti-HBc increased with age, but decreased with education level in both genders. Lifetime number of sexual partners was associated with anti-HBc prevalence among men (OR = 1.95; 95% Cl: 1.2-3.1), but not women. The seroprevalence of HBV and HCV was low among Brazilian blood donors, and exposure increased with age in both genders.

PREVALENCE OF HEPATITIS B AND HEPATITIS C MARKERS IN ALCOHOLICS WITH AND WITHOUT CLINICALLY EVIDENT HEPATIC CIRRHOSIS

We assessed the frequency of serological markers of hepatitis B virus (HBV) and hepatitis C virus (HCV) infections in 365 alcoholics by determining, by ELISA, the presence of HBsAg, anti-HBc, anti-HBs and anti-HCV. Fifty patients were cirrhotics and 315 had no evidence of hepatic cirrhosis; of the latter HBsAg was assessed in all, anti-HBc and anti-HBs in 130, and anti-HCV in 210. Among the alcoholics the frequencies of HBsAg (1.9%), anti-HBc (28.3%) and anti-HCV (3.8%) were higher (p<0.001) than among the controls (N=17,059), 0.4%, 4.0% and 0.4% respectively. The frequency of positive HBsAg was higher (p<0.001) in the cirrhotic patients (8.0%) than in alcoholics without cirrhosis (0.95%) and in controls (0.4%), and similar between the latter; of anti-HBc in alcoholics without cirrhosis (28.5%) was similar in cirrhotics patients (28.0%) and higher (p<0.001) than in the controls (4.0%); of anti-HBs in alcoholics without cirrhosis (20.8%) was similar to that of the cirrhotic patients (10.0%), and the anti-HCV was similar between alcoholics with (6.0%) and without cirrhosis (3.3%) and higher (p<0.001) than in controls (0.4%). We concluded that: a) alcoholics with or without cirrhosis have similar frequencies of infection with HBV and HCV between them, and higher than in nonalcoholics; b) alcoholics without cirrhosis had a frequency of HBV active infection (HBsAg+) which was similar to the controls, whereas among those who progressed to cirrhosis this frequency was significantly higher, what suggests that HBV may be implicated in the pathogenesis of cirrhosis in a few alcoholic individuals.

Seroprevalence of hepatitis B and C infection markers among children and adolescents in the southern Brazilian region

Hepatitis B virus (HBV) and hepatitis C virus (HCV) infections account for a substantial proportion of liver diseases worldwide. The aim of this study was to determine the prevalence of HBV and HCV serological markers among children and adolescents and verify the epidemiology of the HBV infection over than a decade of the introduction of vaccination program. Serologic markers to HBsAg, total anti-HBc and anti-HCV had been tested in 393 samples. The seropositivity for HBsAg was 0.76% and for total anti-HBc was 1.02%. Copositivity between HBsAg and total anti-HBc was verified in 0.76% of the analyzed samples. There was no seropositivity for anti-HCV marker. The seroprevalence of HBV infection markers among children and adolescents in the southern Brazilian region is high compared to that reported in other countries. Preventive measures, such as educational activities in addition to the universal childhood HBV vaccination, should be initiated in order to reduce the morbimortality and the economic burden associated with the disease.

Seroepidemiological markers of enterically transmitted viral hepatitis A and E in individuals living in a community located in the North Area of Rio de Janeiro, RJ, Brazil

We investigated the seroprevalence of hepatitis A virus (HAV) and hepatitis E virus (HEV) infection in subjects living in the community of Manguinhos, Rio de Janeiro, Brazil, and assisted at the Health Unit of Escola Nacional de Saúde Pública, Fundação Oswaldo Cruz. After formal consent, individuals were submitted to an interview using a standardized questionnaire. Anti-HAV and anti-HEV antibodies were detected by ELISA. Statistical analysis was carried out using the Epi-Info 6.04b software, to investigate possible associations between serological markers and risk factors. Results were regarded as significant when p value < 0.05. Although a high prevalence of anti-HAV was observed (87%), almost 50% of subjects under the age of 10 were susceptible to HAV infection, an unexpected rate in endemic areas. This fact could be attributed to improvements in environmental sanitation, occurring in this area in the last years. The increasing proportion of susceptible people may result in outbreaks of HAV infection, since the virus still circulates in this area, as verified by the detection of anti-HAV IgM in some individuals. No statistical association was met between HAV infection and the risk factors here assessed. The anti-HEV IgG prevalence found in this population was 2.4%, consistent with the one found in non-endemic areas.

Hepatitis B and C in the hemodialysis unit of Tocantins, Brazil: serological and molecular profiles

A survey was conducted in the hemodialysis population of the state of Tocantins, Brazil, aiming to assess the prevalence of hepatitis B virus (HBV) and hepatitis C virus (HCV) infections, to analyze associated risk factors, and also to investigate these viruses genotypes distribution. During January and March 2001, all patients (n = 100) were interviewed at the unique dialysis unit in Tocantins. Blood samples were collected and serum samples were screened for HBV serological markers. Hepatitis B surface antigen positive samples were tested for HBV DNA. All samples were also tested for anti-HCV antibodies and HCV RNA. An overall prevalence of 45% was found for HBV infection (4% were HBsAg/anti-HBc positive, 2% were anti-HBc only and 39% had anti-HBc/anti-HBs markers). Concerning HCV infection, anti-HCV and HCV RNA were detected in 13% and 14% of the subjects, respectively. Three patients were HCV RNA positive and anti-HCV negative, resulting in an overall HCV prevalence of 16%. Univariate analysis of risk factors showed that only shift and length of time on hemodialysis were associated with HBV and HCV positivity, respectively. Among the four HBsAg-positive samples, HBV DNA was detected in three of them, which were identified as genotype A by restriction fragment length polymorphism (RFLP) analysis. All 14 HCV RNA-positive samples were genotyped by INNO-LiPA. Genotypes 1a and 3a were found in 85% and 15%, respectively. The present data show low HBsAg and HCV prevalence rates. The risk factors associated with HBV and HCV positivity suggest that nosocomial transmission may influence in spreading these viruses in the dialysis unit studied.

Congenital toxoplasmosis: evaluation of serological methods for the detection of anti-Toxoplasma gondii IgM and IgA antibodies

A study was carried out to evaluate the presence of serological markers for the immunodiagnosis of the vertical transmission of toxoplasmosis. We tested the sensitivity, specificity and predictive values (positive and negative) of different serological methods for the early diagnosis of congenital toxoplasmosis. In a prospective longitudinal study, 50 infants with suspected congenital toxoplasmosis were followed up in the ambulatory care centre of Congenital Infections at University Hospital in Goiânia, Goiás, Brazil, from 1 January 2004-30 September 2005. Microparticle Enzyme Immunoassay (MEIA), Enzyme-Linked Fluorescent Assay (ELFA) and Immune-Fluorescent Antibody Technique (IFAT) were used to detect specific IgM anti-Toxoplasma gondii antibodies and a capture ELISA was used to detect specific IgA antibodies. The results showed that 28/50 infants were infected. During the neonatal period, IgM was detected in 39.3% (11/28) of those infected infants and IgA was detected in 21.4% (6/28). The sensitivity, specificity and predictive values (positive and negative) of each assay were, respectively: MEIA and ELFA: 60.9%, 100%, 100%, 55.0%; IFAT: 59.6%, 91.7%, 93.3%, 53.7%; IgA capture ELISA: 57.1%, 100%, 100%, 51.2%. The presence of specific IgM and IgA antibodies during the neonatal period was not frequent, although it was correlated with the most severe cases of congenital transmission. The results indicate that the absence of congenital disease markers (IgM and IgA) in newborns, even after confirming the absence with several techniques, does not constitute an exclusion criterion for toxoplasmosis.

HBV markers in haemodialysis Brazilian patients: a prospective 12-month follow-up

The aim of this study was to determine the prevalence and the incidence of hepatitis B virus (HBV) among haemodialysis (HD) subjects and to evaluate whether testing for serological markers at the time of admission is suitable for HBV screening in this population. One hundred twenty-three patients belonging to two HD centres from São Paulo, Brazil, were tested prospectively. HBV DNA was detected by polymerase chain reaction (PCR) in each of the prospective subjects (n = 123) during one year. Additionally, all samples (n = 1,476) were analysed for HBV serological markers. The prevalence of hepatitis B core antibody (anti-HBc), hepatitis B surface antigen (HBsAg) and HBV DNA were 34.1%, 15.4% and 8.1%, respectively, while the incidence was null. Fluctuation in HBV serology was observed in one patient. Only 37.8% (17/45) of cases responded to the HBV vaccine. Our results suggest that employing more than one HBV marker and repeated follow-up evaluations may improve HBV screening in HD units.

Allergic bronchopulmonary aspergillosis: the hunt for a diagnostic serological marker in cystic fibrosis patients.

The diagnosis of allergic bronchopulmonary aspergillosis (ABPA) in cystic fibrosis patients remains challenging, mainly owing to overlapping symptoms of the underlying lung disease with clinical symptoms of ABPA. In addition, a varying mixture of diagnostic criteria, including clinical status, radiological findings and immunological measurements, has led to confusion and differing recommendations. In order to help simplify as well as standardize the diagnostic criteria for ABPA, different serological markers have been evaluated in the last 20 years and their usefulness has been assessed in many clinical studies. This review presents current diagnostic criteria of ABPA, with a special focus on serum markers supporting the diagnosis and explains why the hunt for a serological marker for ABPA is still ongoing.

Evaluation of cardiovascular disease markers in patients submitted to carotid artery stenting or endarterectomy

Introduction. Microembolization during the carotid artery revascularization procedure may cause cerebral lesions. Elevated C-Reactive Protein (hsCRP), Vascular endothelial growth factor (VEGF) and serum amyloid A protein (SAA) exert inflammatory activities thus promoting carotid plaque instability. Neuron specific enolase (NSE) is considered a marker of cerebral injury. Neoangiogenesis represents a crucial step in atherosclerosis, since neovessels density correlates with plaque destabilization. However their clinical significance on the outcome of revascularization is unknown. This study aims to establish the correlation between palque vulnerabilty, embolization and histological or serological markers of inflammation and neoangiogenesis. Methods. Serum hsCRP, SAA, VEGF, NSE mRNA, PAPP-A mRNA levels were evaluated in patients with symptomatic carotid stenosis who underwent filter-protected CAS or CEA procedure. Cerebral embolization, presence of neurologicals symptoms, plaque neovascularization were evaluated testing imaging, serological and histological methods. Results were compared by Fisher’s, Student T test and Mann-Whitney U test. Results. Patients with hsCRP<5 mg/l, SAA<10mg/L and VEGF<500pg/ml had a mean PO of 21.5% versus 35.3% (p<0.05). In either group, embolic material captured by the filter was identified as atherosclerotic plaque fragments. Cerebral lesions increased significantly in all patients with hsCRP>5mg/l and SAA>10mg/l (16.5 vs 2.8 mean number, 3564.6 vs 417.6 mm3 mean volume). Discussion. High hsCRP, SAA and VEGF levels are associated with significantly greater embolization during CAS and to the vulnerabiliy of the plaque. This data suggest CAS might not be indicated as a method of revascularization in this specific group of patients.