Leishmania infantum heat shock protein 83 for the serodiagnosis of tegumentary leishmaniasis

The serologic assay is an important tool in the diagnosis of leishmaniasis. One of the most commonly used tests is enzyme-linked immunosorbent assay (ELISA). Since total Leishmania promastigotes are used as antigen in the routine assay, false-positive reactions are frequent due to cross-reaction with sera from other diseases, mainly Chagas' disease. Therefore, an antigen that determines less cross-reactivity has been pursued for the serodiagnosis of leishmaniasis. In the present study we analyzed the use of recombinant Leishmania infantum heat shock protein (Hsp) 83 in ELISA for the serodiagnosis of cutaneous (N = 12) and mucocutaneous leishmaniasis (N = 14) and we observed the presence of anti-L. infantum Hsp 83 antibodies in all samples as well as anti-Leishmania total antigen antibodies. When cross-reactivity was tested, chronic Chagas' disease patients (N = 10) did not show any reactivity. Therefore, we consider this L. infantum Hsp 83 to be a good antigen for routine use for serodiagnosis of tegumentary leishmaniasis.

Avaliação do teste do anel em leite na vigilância epidemiológica da brucelose bovina em rebanhos e em laticínios

Analisaram-se 464 amostras individuais de leite e 54 amostras de leite de latões, oriundos de leite dos mesmos animais, por meio do teste do anel do leite (TAL) visando à sua aplicação no diagnóstico individual e de rebanhos da brucelose bovina. Foram também avaliadas 464 amostras de soro sangüíneo por meio de provas do antígeno tamponado acidificado (ATA), soroaglutinação lenta em tubos (SAL) e 2-mercaptoetanol (2-ME), todas para brucelose. Cento e vinte e três amostras (26,5%) testadas pelo TAL apresentaram resultados positivos. Dessas, 30 resultaram positivas ao ATA, 28 ao ATA, à SAL e ao 2-ME e 18 à SAL. Das amostras positivas ao TAL, 95 pertenciam a animais sorologicamente negativos ao 2-ME, caracterizando 77,2% (95/123) das reações falso-positivas; dos resultados negativos ao TAL, 4 pertenciam a animais sorologicamente positivos, caracterizando 1,2% (4/341) de reações falso-negativas no TAL individual. Das 54 amostras de leite de latões analisadas pelo TAL, 17 foram consideradas positivas, das quais uma foi caracterizada como falso-positivo, pois todos os animais que a compunham foram negativos ao 2-ME. de 37 latões considerados negativos ao TAL, três continham leite de animais positivos ao 2-ME, caracterizando 8,1% de falso-negativos. O TAL individual demonstrou elevado percentual de resultados falso-positivos, enquanto o TAL em amostras de leite obtidas em latões detectou 84,2% de latões contaminados e 75% de rebanhos infectados.

Avaliação do teste do anel na vigilância epidemiológica da brucelose bovina em rebanhos e laticínios

Pós-graduação em Medicina Veterinária - FMVZ

Heteroduplex analysis of VDJ amplified segments from rearranged IgH genes for clonality assessments in B-cell non-Hodgkin's lymphoma. A comparison between different strategies.

BACKGROUND AND OBJECTIVE: The main difficulty of PCR-based clonality studies for B-cell lymphoproliferative disorders (B-LPD) is discrimination between monoclonal and polyclonal PCR products, especially when there is a high background of polyclonal B cells in the tumor sample. Actually, PCR-based methods for clonality assessment require additional analysis of the PCR products in order to discern between monoclonal and polyclonal samples. Heteroduplex analysis represents an attractive approach since it is easy to perform and avoids the use of radioactive substrates or expensive equipment. DESIGN AND METHODS: We studied the sensitivity and specificity of heteroduplex PCR analysis for monoclonal detection in samples from 90 B-cell non Hodgkin's lymphoma (B-NHL) patients and in 28 individuals without neoplastic B-cell disorders (negative controls). Furthermore, in 42 B-NHL and in the same 28 negative controls, we compared heteroduplex analysis vs the classical PCR technique. We also compared ethidium bromide (EtBr) vs. silver nitrate (AgNO(3)) staining as well as agarose vs. polyacrylamide gel electrophoresis (PAGE). RESULTS: Using two pair consensus primers sited at VH (FR3 and FR2) and at JH, 91% of B-NHL samples displayed monoclonal products after heteroduplex PCR analysis using PAGE and AgNO(3) staining. Moreover, no polyclonal sample showed a monoclonal PCR product. By contrast, false positive results were obtained when using agarose (5/28) and PAGE without heteroduplex analysis: 2/28 and 8/28 with EtBr and AgNO(3) staining, respectively. In addition, false negative results only appeared with EtBr staining: 13/42 in agarose, 4/42 in PAGE without heteroduplex analysis and 7/42 in PAGE after heteroduplex analysis. INTERPRETATION AND CONCLUSIONS: We conclude that AgNO(3) stained PAGE after heteroduplex analysis is the most suitable strategy for detecting monoclonal rearrangements in B-NHL samples because it does not produce false-positive results and the risk of false-negative results is very low.

A more reliable PCR for detection of Mycobacterium tuberculosis in clinical samples

Diagnostic techniques based on PCR have two major problems: false-positive reactions due to contamination with DNA fragments from previous PCRs (amplicons) and false-negative reactions caused by inhibitors that interfere with the PCR. We have improved our previously reported PCR based on the amplification of a fragment of the Mycobacterium tuberculosis complex-specific insertion element IS6110 with respect to both problems. False-positive reactions caused by amplicon contamination were prevented by the use of uracil-N-glycosylase and dUTP instead of dTTP. We selected a new set of primers outside the region spanned by the formerly used primers to avoid false-positive reactions caused by dTTP-containing amplicons still present in the laboratory. With this new primer set, 16 copies of the IS6110 insertion element, the equivalent of two bacteria, could be amplified 10(10) times in 40 cycles, resulting in a mean efficiency of 77% per cycle. To detect the presence of inhibitors of the Taq polymerase, which may cause false-negative reactions, part of each sample was spiked with M. tuberculosis DNA. The DNA purification method using guanidinium thiocyanate and diatoms effectively removed most or all inhibitors of the PCR. However, this was not suitable for blood samples, for which we developed a proteinase K treatment followed by phenol-chloroform extraction. This method permitted detection of 20 M. tuberculosis bacteria per ml of whole blood. Various laboratory procedures were introduced to reduce failure or inhibition of PCR and avoid DNA cross contamination. We have tested 218 different clinical specimens obtained from patients suspected of having tuberculosis. The samples included sputum (n=145), tissue biopsy samples (n=25), cerebrospinal fluid (n=15), blood (n=14), pleural fluid (n=9), feces, (n=7), fluid from fistulae (n=2), and pus from a wound (n=1). The results obtained by PCR were consistent with those obtained with culture, which is the "gold standard." We demonstrate that PCR is a useful technique for the rapid diagnosis of tuberculosis at various sites.

African swine fever virus transmission cycles in Central Europe: evaluation of wild boar-soft tick contacts through detection of antibodies against Ornithodoros erraticus saliva antigen.

BACKGROUND African swine fever (ASF) is one of the most complex viral diseases affecting both domestic and wild pigs. It is caused by ASF virus (ASFV), the only DNA virus which can be efficiently transmitted by an arthropod vector, soft ticks of the genus Ornithodoros. These ticks can be part of ASFV-transmission cycles, and in Europe, O. erraticus was shown to be responsible for long-term maintenance of ASFV in Spain and Portugal. In 2014, the disease has been reintroduced into the European Union, affecting domestic pigs and, importantly, also the Eurasian wild boar population. In a first attempt to assess the risk of a tick-wild boar transmission cycle in Central Europe that would further complicate eradication of the disease, over 700 pre-existing serum samples from wild boar hunted in four representative German Federal States were investigated for the presence of antibodies directed against salivary antigen of Ornithodoros erraticus ticks using an indirect ELISA format. RESULTS Out of these samples, 16 reacted with moderate to high optical densities that could be indicative of tick bites in sampled wild boar. However, these samples did not show a spatial clustering (they were collected from distant geographical regions) and were of bad quality (hemolysis/impurities). Furthermore, all positive samples came from areas with suboptimal climate for soft ticks. For this reason, false positive reactions are likely. CONCLUSION In conclusion, the study did not provide stringent evidence for soft tick-wild boar contact in the investigated German Federal States and thus, a relevant involvement in the epidemiology of ASF in German wild boar is unlikely. This fact would facilitate the eradication of ASF in the area, although other complex relations (wild boar biology and interactions with domestic pigs) need to be considered.

Skin reactions to thimerosal and Leishmania in dogs from a leishmaniasis endemic area: it is better to keep them apart

Positive Montenegro's skin test is a delayed type hypersensitivity reaction widely used as indicative of previous infection with Leishmania in both humans and dogs. Montenegro's antigen consists of a crude Leishmania antigen solution, usually containing thimerosal as preserving agent. In this work it is shown that a large proportion of dogs (11 out of 56) examined in an endemic area of leishmaniasis presented induration at the site of injection of a diluent containing thimerosal alone. This clearly demonstrates that thimerosal leads to a high number of false positive skin reactions in dogs and that its use in Montenegro's skin test antigenic preparations should be avoided.

False negative apraclonidine test in two patients with Horner syndrome

BACKGROUND: Because of denervation supersensitivity, a miotic pupil in a sympathetically-denervated eye dilates in response to a dilute or weak alpha-1-agonist drug. A reversal of anisocoria after topical apraclonidine is considered as a positive test result that diagnoses a unilateral Horner syndrome. HISTORY AND SIGNS: Two women aged 34 and 46 years with a cocaine-confirmed oculosympathetic defect (Horner syndrome) were tested with 1 % topical apraclonidine on separate days. THERAPY AND OUTCOME: In one patient, her miotic Horner pupil dilated marginally but not enough to reverse the baseline anisocoria. Additionally, the upper lid on the same side retracted. There was no discernable effect of apraclonidine on the normal, contralateral eye. In the second patient, there was no pupillary response to apraclonidine but there was resolution of her ptosis. CONCLUSIONS: Neither patient demonstrated a reversal of anisocoria, the current criterion for diagnosing a Horner syndrome using apraclonidine. Thus, these two patients with an established oculosympathetic defect were said to have a "negative test" for Horner syndrome. Yet both women showed subtle pupil and/or lid changes in response to apraclonidine that were consistent with sympathetic denervation supersensitivity. Reversal of anisocoria following topical apraclonidine does not occur in all patients with a unilateral oculosympathetic defect and more specific parameters for defining a positive test result might optimize apraclonidine's utility as a diagnostic test for Horner syndrome

Acute Hepatitis B in a patient previously positive for antibody to surface antigen (anti-HBs) determined by radioimmunoassay: case report and review of the literature

The determination of anti-HBs as a screening test before vaccination has been advisable in order to encounter immune individuals that don't need to receive vaccine protection. A case-report is presented and three other cases are reviewed from the literature. Anti-HBs was positive in these health-care personnels that developped typical acute B hepatitis. Different subtyping involving the d/y determinants were found in the first case, but false-positive anti-HBs even with high titres, determined by RIA, were found in the other cases. Concomitant determination of anti-HBc or absence of screening tests seem to be more reasonable policies until a low-cost and risk-free vaccine is produced.

Serological studies on shistosomiasis mansoni in the Northeast Brasil

Sera from the patients (N = 10) with schistosomiasis mansoni of the hospital of Federal University of Pernambuco, the Schistosoma mansoni egg-positive (N = 51) and -negative (N = 452) inhabitants in Cabo City area, out-patients (N = 37) of the IMIP hospital and Japanese immigrants (N = 127) in Petrolina City area of northeast Brazil as well as Japanese healthy subjects (N = 30) were examined by serological tests including an enzyme-linked immunosorbent assay with antigens prepared from eggs (ELISA-egg) and adult worms (ELISA-adult). The ELISA with egg or adult antigen correctly identified 100% of the uninfected individuals lived in non-endemic area of schistosomiasis. Moreover, when examined cross-reactivity of our ELISA with sera isolated from 78 subjects infected with various intestinal parasitic infections, only one of these sera reacted with the egg and adult antigens. On the examination of 51 sera from the egg-positive subjects, the ELISA-egg revealed the highest sensitivity (98.0%), whereas a large number of false negative reactions of ELISA-adult, Ouchterlony method using adult antigen, circumoval precipitation and immediate intradermal skin test were observed. A low sensitivity of these serologic tests except for ELISA-egg appears to be primarily due to their inability to detect antibody in the sera from egg-positive infantiles. There was no positive correlation between the absorbance values of these two types of ELISA among the sera isolated from ELISA-positive subjects. Rather, by the reactivity of these sera to egg or adult antigen, they could be divided into two subgroups; one reacted more positively with egg antigen and the other with adult antigen. Moreover, it was confirmed that the sera from young subjects (under 20 years old) appear to be highly reactive to the egg antigen than did aged ones. These data suggest that the ELISA with egg antigen, but not with the adult antigen, appears to be useful for the serological survey of schistosomiasis mansoni in the endemic area of northeast Brazil.

Incidência de "Falsos-Positivos" em Idosos com Fibrilhação Auricular Paroxística Submetidos a Teste de Inclinação

The autonomic nervous system (ANS) plays a role as a modulator in the pathogenesis of paroxysmal atrial fibrillation (PAF). The clinical pattern of vagally mediated PAF has been observed mainly in young patients. Neurocardiogenic responses during orthostatic stress are related to autonomic reflexes in which the vagal influence predominates. AIM: To evaluate the susceptibility of elderly patients with PAF to activation of vasovagal syncope mechanisms. METHODS: We performed passive head-up tilt testing (HUT) in 34 patients (62% women, aged 72 +/- 7 years), with > or = 1 year of clinical history of PAF--19 without structural heart disease, 11 with hypertensive heart disease and 4 with coronary artery disease (who had no previous myocardial infarction, had undergone myocardial revascularization, and had no documented ischemia) (PAF group), and compared the results with those obtained in a group of 34 age-matched patients (53% women, aged 74 +/- 6 years), who underwent HUT due to recurrent syncope (Sc group). In this group, 21 had no documented heart disease and none had a clinical history of AF. There was no diabetes, congestive heart failure or syncope in the PAF group. After a supine resting period, the subjects were tilted at 70 degrees for 20 minutes while in sinus rhythm. No provocative agents were used to complement the HUT. ECG and blood pressure were continuously monitored (Task Force Monitor, CNSystems). The test was considered positive when syncope or presyncope occurred with bradycardia and/or arterial hypotension. Abnormal responses were classified as cardioinhibitory, vasodepressor or mixed. RESULTS: HUT was positive in seven patients of the PAF group--vasodepressor response in five and mixed in two (20.5% of the total; 26.3% of those without heart disease)--and in eight patients (vasodepressor in six and mixed in two) of the Sc group (p=NS). During HUT, three patients of the PAF group had short periods of self-limited PAF (in one, after vasodepressor syncope). There were no differences in gender distribution, age or heart disease. No cardioinhibitory responses or orthostatic hypotension were observed. CONCLUSION: In elderly patients with PAF, a significant number of false positive results during passive HUT may be expected, suggesting increased vasovagal reactions despite aging. This suggests that ANS imbalances may be observed in this population.

Clinical and epidemiological profile of female blood donors with positive serology for viral hepatitis B

ABSTRACTINTRODUCTION:Since women are frequently the minority among blood donors worldwide, studies evaluating this population usually reflect male features. We assessed the features of female blood donors with positive serology for HBV and compared them with those of men.METHODS The study comprised consecutive blood donors referred to a specialized liver disease center to be evaluated due to HBsAg- and/or anti-HBc-positive tests.RESULTS: The study encompassed 1,273 individuals, 219 (17.2%) of whom were referred due to positive HBsAg test and 1,054 (82.8%) due to reactive anti-HBc test. Subjects' mean age was 36.8±10.9 years, and 28.7% were women. Female blood donors referred for positive HBsAg screening tests demonstrated higher prevalence of healthcare workers (9.3% vs 2.5%) and lower prevalence of sexual risk behaviors (15.1% vs 41.1%) and alcohol abuse (1.9% vs 19.8%) compared to men. Women had lower ALT (0.6 vs 0.8×ULN), AST (0.6 vs 0.8×ULN), direct bilirubin (0.2 vs 0.3mg/dL), and alkaline phosphatase (0.5 vs 0.6×ULN) levels and higher platelet count (223,380±50,293 vs 195,020±53,060/mm3). Women also had a higher prevalence of false-positive results (29.6% vs 17.0%). No differences were observed with respect to liver biopsies. Female blood donors referenced for reactive anti-HBc screening tests presented similar clinical, epidemiological, and biochemical characteristics to those reported for positive HBsAg screening tests and similarly had a higher prevalence of false-reactive results.CONCLUSIONS: Compared to men, female blood donors with positive HBsAg and/or anti-HBc screening tests demonstrated higher prevalence of professional risk and false-positive results and reduced alteration of liver chemistry.

Serological Evidence of HTLV-I and HTLV-II Coinfections in HIV-1 Positive Patients in Belém, State of Pará, Brazil

The occurrence of HTLV-I/II and HIV-1 coinfections have been shown to be frequent, probably in consequence of their similar modes of transmission. This paper presents the prevalence of coinfection of HTLV among HIV-1 infected and AIDS patients in Belém, State of Pará, Brazil. A group of 149 patients attending the AIDS Reference Unit of the State Department of Health was tested for the presence of antibodies to HTLV-I/II using an enzyme immunoassay and the positive reactions were confirmed with a Western blot that discriminates between HTLV-I and HTLV-II infections. Four patients (2.7%) were positive to HTLV-I, seven (4.7%) to HTLV-II and one (0.7%) showed an indeterminate pattern of reaction. The present results show for the first time in Belém not only the occurrence of HTLV-II/HIV-1 coinfections but also a higher prevalence of HTLV-II in relation to HTLV-I. Furthermore, it also enlarges the geographical limits of the endemic area for HTLV-II in the Amazon region of Brazil.

Development and Preliminary Evaluation of a Rapid Oligochromatographic Assay for Specific Detection of New Human Influenza A H1N1 Virus

A new oligochromatographic assay, Speed-Oligo Novel Influenza A H1N1, was designed and optimized for the specific detection of the 2009 influenza A H1N1 virus. The assay is based on a PCR method coupled to detection of PCR products by means of a dipstick device. The target sequence is a 103-bp fragment within the hemagglutinin gene. The analytical sensitivity of the new assay was measured with serial dilutions of a plasmid that contained the target sequence, and we determined that down to one copy per reaction of the plasmid was reliably detected. Diagnostic performance was assessed with 103 RNAs from suspected cases (40 positive and 63 negative results) previously analyzed with a reference real-time PCR technique. All positive cases were confirmed, and no false-positive results were detected with the new assay. No cross-reactions were observed when other viral strains or clinical samples with other respiratory viruses were tested. According to these results, this new assay has 100% sensitivity and specificity. The turnaround time for the whole procedure was 140 min. The assay may be especially useful for the specific detection of 2009 H1N1 virus in laboratories not equipped with real-time PCR instruments