Serology using rROP2 antigen in the diagnostic of toxoplasmosis in pregnant women

Toxoplasma gondii causes severe fetal disease during acute infection in pregnant women, thus demanding early diagnosis for effective treatment and fetus preservation. Fetal tests are inefficient and risky, and diagnosis is based on maternal IgM serology, which had weak screening ability due to increased sensitivity, with alternative IgG avidity tests. Here, we performed ELISA and avidity assays using a recombinant T. gondii antigen, rROP2, in samples from 160 pregnant women screened from a large public hospital who were referred due to positive IgM assays. IgG serology and avidity assays were compared using whole T. gondii extract or rROP2. ELISA IgG detection with rROP2 showed good agreement with assays performed with T. gondii extract, but rROP2 IgG avidity assays were unrelated to whole extract antigen IgG avidity, regardless of the chaotrope used. These data show that avidity maturation is specific to individual antigen prevalence and immune response during infection. ELISA rROP2 IgG assays may be an alternative serological test for the diagnosis of toxoplasmosis during pregnancy, although our data do not support their use in avidity assays.

Accidental infection by Trypanosoma cruzi follow-up by the polymerase chain reaction: case report

We report a case of accidental infection by Trypanosoma cruzi in a 42-year-old female patient who presented an inoculation chagoma. Laboratory confirmation was based on examination of fresh blood, Giemsa-stained blood smear, immunoenzyme test (EIA-IgG), indirect immunofluorescence (IIF-IgM, IgG) and polymerase chain reaction (PCR). Only the PCR gave a positive result, and the EIA test was inconclusive. Two treatments with benznidazole were necessary. PCR was the only technique that continued to give positive results for approximately two months (65 days, or 2.2 months) following the second treatment and negative results from 96 days (3.2 months) to 850 days (28.3 months). We concluded that the presence of an inoculation chagoma and use of PCR were important and decisive for diagnosis and follow-up of the case.

Potential immunological markers for diagnosis and therapeutic assessment of toxocariasis

In human toxocariasis, there are few approaches using immunological markers for diagnosis and therapeutic assessment. An immunoblot (IB) assay using excretory-secretory Toxocara canis antigen was standardized for monitoring IgG, IgE and IgA antibodies in 27 children with toxocariasis (23 visceral, three mixed visceral and ocular, and one ocular form) for 22-116 months after chemotherapy. IB sensitivity was 100% for IgG antibodies to bands of molecular weight 29-38, 48-54, 95-116, 121-162, >205 kDa, 80.8% for IgE to 29-38, 48-54, 95-121, > 205 kDa, and 65.4% for IgA to 29-38, 48-54, 81-93 kDa. Candidates for diagnostic markers should be IgG antibodies to bands of low molecular weight (29-38 and 48-54 kDa). One group of patients presented the same antibody reactivity to all bands throughout the follow-up study; in the other group, antibodies decayed partially or completely to some or all bands, but these changes were not correlated with time after chemotherapy. Candidates for monitoring patients after chemotherapy may be IgG antibodies to > 205 kDa fractions, IgA to 29-38, 48-54, 81-93 kDa and IgE to 95-121 kDa. Further identification of antigen epitopes related to these markers will allow the development of sensitive and specific immunoassays for the diagnosis and therapeutic assessment of toxocariasis.

Therapeutic Effects of Allium sativum and Allium cepa in Schistosoma mansoni experimental infection

The effects of both garlic (Allium sativum) and onion (Allium cepa) on some biochemical parameters in Schistosoma mansoni infected mice individually and mixed either with or without the currently used drug, praziquantel (PZQ) were investigated. These involved some immunological parameters, namely IgM, IgG, interleukins 2 and 6 (IL-2 and 6) and tumor necrosis factor (TNF-α), some antioxidant enzymes [catalase, superoxide dismutase (SOD) and glutathione peroxidase (GPX)]. In addition, parasitological and histopathological investigations were performed. No changes were observed in the normal control mice treated with dry extract of onion or garlic, individually or mixed, with or without PZQ, compared to the normal healthy control group. Infection with S. mansoni showed an increase in IgG, IgM, IL-2, IL-6, TNF-α and catalase enzyme, accompanied with a decrease in GPX and SOD antioxidant enzyme activities. Remarkable amelioration was noticed in the levels of all the measured parameters in S. mansoni infected mice after administration of the studied extracts. Moreover a significant reduction in worm burden, hepatic and intestinal eggs and oogram count was noticed which was reflected in normalization of liver architecture.

Factors associated to infection by Toxoplasma gondii in pregnant women attended in Basic Health Units in the city of Rolândia, Paraná, Brazil

The aim of the present work was to determine the prevalence of IgG and IgM anti-Toxoplasma gondii antibodies and the factors associated to the infection in pregnant women attended in Basic Health Units in Rolândia, Paraná, Brazil. The sample was divided in two groups: group I (320 pregnant women who were analyzed from July 2007 to February 2008) and group II (287 pregnant women who were analyzed from March to October 2008). In group I, it was found 53.1% of pregnant women with IgG reactive and IgM non-reactive, 1.9% with IgG and IgM reactive, 0.3% with IgG non-reactive and IgM reactive and 44.7% with IgG and IgM non-reactive. In group II, it was found 55.1% with IgG reactive and IgM non-reactive and 44.9% with IgG and IgM non-reactive. The variables associated to the presence of IgG antibodies were: residence in rural areas, pregnant women between 35-40 years old, low educational level, low family income, more than one pregnancy, drinking water which does not originate from the public water supply system and the habit of handling soil or sand. Guidance on primary prevention measures and the quarterly serological monitoring of the pregnant women in the risk group are important measures to prevent congenital toxoplasmosis.

Acute inflammatory demyelinating polyradiculoneuropathy (Guillain-Barré syndrome) following dengue fever

This paper reports a case of dengue in a six-year-old female child who suddenly developed excruciating headaches, fever, myalgia and paresis. Laboratory examinations included blood count, platelet count, biochemical tests (BUN, creatinine, aminotransferases, and total bilirubin and bilirubin fractions) and specific IgM titers (enzyme-immunoassay with recombinant tetravalent dengue). After ten days of hospitalization and having already been in a home environment, a new clinical image emerged, characterized by dysphagia, dysphonia, weakness, peripheral facial palsy and paresthesia. The diagnosis of Guillain-Barré Syndrome was based on clinical findings, cerebrospinal fluid examination, electrophysiological findings and the exclusion of other pathologies. Our case, as some shown in previous reports, calls attention to the possibility that Guillain-Barré Syndrome may occur in association with dengue.

Evaluation of dengue NS1 antigen detection for diagnosis in public health laboratories, São Paulo State, 2009

The present work evaluated the diagnostic accuracy of detection of Dengue NS1 antigen employing two NS1 assays, an immunochromatographic assay and ELISA, in the diagnostic routine of Public Health laboratories. The results obtained with NS1 assay were compared with virus isolation and, in a subpopulation of cases, they were compared with the IgM-ELISA results obtained with convalescent samples. A total of 2,321 sera samples were analyzed by one of two NS1 techniques from March to October 2009. The samples were divided into five groups: groups I, II and III included samples tested by NS1 and virus isolation, and groups IV and V included patients with a first sample tested by NS1 and a second sample tested by IgM-ELISA. Sensitivity, specificity, positive and negative predictive values, Kappa Index and Kappa Concordance were calculated. The results showed that NS1 testing in groups I, II and III had high sensitivity (98.0%, 99.5% and 99.3%), and predictive values and Kappa index between 0.9 - 1.0. Groups IV and V only had Kappa Concordance calculated, since the samples were analyzed according to the presence of NS1 antigen or IgM antibody. Concordance of 92.1% was observed when comparing the results of NS1-negative samples with IgM-ELISA. Based on the findings, it is possible to suggest that the tests for NS1 detection may be important tools for monitoring the introduction and spread of Dengue serotypes.

Case-control study of an outbreak of acute toxoplasmosis in an industrial plant in the state of São Paulo, Brazil

Foodborne diseases represent operational risks in industrial restaurants. We described an outbreak of nine clustered cases of acute illness resembling acute toxoplasmosis in an industrial plant with 2300 employees. These patients and another 36 similar asymptomatic employees were diagnosed with anti-T. gondii IgG titer and avidity by ELISA. We excluded 14 patients based on high IgG avidity and chronic toxoplasmosis: 13 from controls and one from acute disease other than T. gondii infection. We also identified another three asymptomatic employees with T.gondii acute infection and also anti-T. gondii IgM positive as remaining acute cases. Case control study was conducted by interview in 11 acute infections and 20 negative controls. The ingestion of green vegetables, but not meat or water, was observed to be associated with the incidence of acute disease. These data reinforce the importance of sanitation control in industrial restaurants and also demonstrate the need for improvement in quality control regarding vegetables at risk for T. gondii oocyst contamination. We emphasized the accurate diagnosis of indexed cases and the detection of asymptomatic infections to determine the extent of the toxoplasmosis outbreak.

IgG AVIDITY WESTERN BLOT USING Toxoplasma gondii rGRA-7 CLONED FROM NUCLEOTIDES 39-711 FOR SERODIAGNOSIS OF ACUTE TOXOPLASMOSIS

Toxoplasmosis is an important cause of congenital infection. The present study was performed to evaluate the usefulness of recombinant (r) GRA-7 cloned from nucleotides (n) 39-711 in discriminating between acute and chronic toxoplasmosis. First, commercial IgM, IgG and IgG avidity ELISAs were used to determine the serological profile of the sera. Serum samples were from 20 symptomatic patients with acute infection (low IgG avidity, IgM positive), 10 with chronic infection (high IgG avidity, IgM negative) and 10 with indeterminate IgG avidity (IgM positive) which were tested for IgG avidity status with an in-house developed IgG avidity Western blot using the rGRA-7 recombinant antigen. All 20 sera from cases of probable acute infection showed bands which either faded out completely or reduced significantly in intensity after treatment with 8 M urea, whereas the band intensities of the 10 serum samples from chronic cases remained the same. Of the 10 sera with indeterminate IgG avidity status, after treatment with 8 M urea the band intensities with six sera remained the same, two sera had completely faded bands and another two sera had significantly reduced band intensities. Discrimination between acute and chronic toxoplasmosis was successfully performed by the in-house IgG avidity Western blot.

Active search for leprosy cases in Midwestern Brazil: a serological evaluation of asymptomatic household contacts before and after prophylaxis with bacillus Calmette-Guérin

Leprosy is a disease caused by Mycobacterium leprae that carries a high risk of disability, making early diagnosis mandatory. This study aimed to determine the applicability of anti-PGL-1 IgM antibody detection, using the ML FLOW technique, as an assistant tool for the detection of leprosy infection in asymptomatic household contacts (AHHC) of multibacillary leprosy index cases from Midwest Brazil. Serological changes induced by the prophylaxis of these household contacts with Bacillus Calmette-Guérin (BCG) were also verified. A total of 91 AHHC were assessed, among which, 18.68% (n = 17) presented both positive bacilloscopy and positive anti-PGL-1 IgM serology. Positivity concordance between these two laboratorial exams (Kappa Index = 1; p < 0.001) was indicated, however, one case did not demonstrate concordance between the semiquantitative assessment of anti-PGL-1 IgM and the bacilloscopy index (Kappa Index = 0.96; p < 0.001). Among the 17 AHHC with positive bacilloscopy, eight were reassessed after prophylaxis with BCG and two of them presented negative anti-PGL-1 IgM serology, being these patients who had presented a bacilloscopy index of < 2[+] in the initial assessment. This study shows that anti-PGL-1 IgM detection may be used as a tool to determine the bacillary load in AHHC and to detect immune changes related to prophylaxis by nonspecific vaccination.

FATAL OUTCOME OF INFECTION BY DENGUE 4 IN A PATIENT WITH THROMBOCYTOPENIC PURPURA AS A COMORBID CONDITION IN BRAZIL

Dengue is currently a major public-health problem. Dengue virus (DENV) is classified into four distinct serotypes, DENV 1-4. After 28 years of absence, DENV-4 was again detected in Brazil in 2010 in Roraima State, and one year later, the virus was identified in the northern Brazilian states of Amazonas and Pará, followed by Rio de Janeiro and São Paulo. In Minas Gerais, the first confirmed case of DENV-4 occurred in the municipality of Frutal in 2011 and has now been isolated from a growing number of patients. Although DENV-2 is associated with the highest risk of severe forms of the disease and death due to the infection, DENV-4 has also been associated with severe forms of the disease and an increasing risk of hemorrhagic manifestations. Herein, the first fatal case of confirmed DENV-4 in Brazil is reported. The patient was an 11-year-old girl from the municipality of Montes Claros in northern Minas Gerais State, Brazil. She had idiopathic thrombocytopenic purpura as a comorbid condition and presented with a fulminant course of infection, leading to death due to hemorrhagic complications. Diagnosis was confirmed by detection of Dengue-specific antibodies using IgM capture enzyme-linked immunosorbent assay and semi-nested RT-PCR. Primary care physicians and other health-care providers should bear in mind that DENV-4 can also result in severe forms of the disease and lead to hemorrhagic complications and death, mainly when dengue infection is associated with coexisting conditions.

FALSE-NEGATIVE DENGUE CASES IN RORAIMA, BRAZIL: AN APPROACH REGARDING THE HIGH NUMBER OF NEGATIVE RESULTS BY NS1 AG KITS

Serum samples from 150 NS1-negative (Platelia ELISA) patients presumptively diagnosed with dengue were analyzed by the TaqMan probed real-time reverse transcription PCR (TaqMan qRT-PCR) method. The qRT-PCR positive samples were tested for serotype by semi-nested RT-PCR and a qualitative immunochromatographic assay for IgG and IgM. Molecular detection methods showed 33 (22%) positive samples out of 150 NS1-antigen negative samples. Of these, 72% were collected up to day 2 after the onset of symptoms, when diagnostic sensitivity of NS1-antigen test assays is significantly enhanced. Most of the cases were not characterized as secondary infection. Twenty-eight samples were successfully serotyped, 75% of which for DENV-4, 14% for DENV-2, 7% for DENV-3 and 4% for DENV-1. These findings reaffirm the hyperendemic situation of the state of Roraima and suggest a lower sensitivity of the NS1 test, mainly when DENV-4 is the predominant serotype. Health care providers should therefore be aware of samples tested negative by NS1 antigen assays, especially when clinical symptoms and other laboratory data results show evidence of dengue 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.

A NEW POSSIBILITY FOR SURVEILLANCE: DO WE IDENTIFY ALL CASES OF LEPTOSPIROSIS?

SUMMARY Leptospirosis is a febrile disease with a typically underestimated global incidence, especially in regions where dengue is endemic. Therefore, it is difficult to accurately determine the number of leptospirosis cases in these areas, which contributes to significant under-reporting this disease. In this study, we estimated the number of possible leptospirosis cases among dengue-like cases that were reported during 2008, 2010, and 2012 in the city of Fortaleza, northeast Brazil. Patients were evaluated for dengue and leptospirosis using immunoenzymatic tests for IgM antibodies that were specific to each pathogen. Among the suspected cases of dengue that resulted as negative in laboratory tests, 10.8% (2008), 19.2% (2010), and 30.8% (2012) were confirmed to be leptospirosis. Considering the cases reported by the surveillance authority as dengue that were subsequently discarded based on the laboratory test results, we estimate that the number of actual leptospirosis cases may be 26 to 49 times higher than those diagnosed and reported by the Health Services. Furthermore, we believe that approximately 20% of dengue-like cases may be leptospirosis cases in areas where the two diseases are endemic.

SEROLOGICAL DETECTION OF HEPATITIS A VIRUS IN FREE-RANGING NEOTROPICAL PRIMATES (Sapajus spp., Alouatta caraya) FROM THE PARANÁ RIVER BASIN, BRAZIL

Nonhuman primates are considered as the natural hosts of Hepatitis A virus (HAV), as well as other pathogens, and can serve as natural sentinels to investigate epizootics and endemic diseases that are of public health importance. During this study, blood samples were collected from 112 Neotropical primates (NTPs) (Sapajus nigritus and S. cay, n = 75; Alouatta caraya, n = 37) trap-captured at the Paraná River basin, Brazil, located between the States of Paraná and Mato Grosso do Sul. Anti-HAV IgG antibodies were detected in 4.5% (5/112) of NTPs, specifically in 6.7% (5/75) of Sapajus spp. and 0% (0/37) of A. caraya. In addition, all samples were negative for the presence of IgM anti-HAV antibodies. These results suggest that free-ranging NTPs were exposed to HAV within the geographical regions evaluated.