Serological Survey of Paracoccidioidomycosis in Sheep

The objective of this study was to evaluate the prevalence of antibodies against Paracoccidioides brasiliensis in sheep from Guarapuava, Paraná State, Brazil. The seroepidemiological study was carried out in 262 sheep. The samples were analyzed by ELISA and immunodiffusion test using P. brasiliensis gp43 and exoantigen as antigens, respectively. Initially, two sheep were immunized with P. brasiliensis to evaluate whether contact with the fungal cells could induce a humoral immune response against gp43 and exoantigen from P. brasiliensis. Both animals produced antibodies against gp43 and exoantigen, the main antigens used for diagnosis and seroepidemiology of paracoccidioidomycosis. A reactivity of 37% was observed to the P. brasiliensis gp43 antigen by ELISA although no reactivity had been observed by the immunodiffusion test. Sheep under extensive grazing system showed higher frequency of positivity to P. brasiliensis (P ≤ 0.05) than those under intensive and semi-intensive systems. These data suggest that sheep may be a useful epidemiological marker of P. brasiliensis presence in the environment and reinforce that contact with soil is an important risk factor for infection.     

Serological Survey of Paracoccidioidomycosis in Cats

The objective of the present study was to evaluate infection of cats by Paracoccidioides brasiliensis. Serum samples of 136 cats from rural (n = 86) and urban areas (n = 50) were analyzed by indirect ELISA and immunodiffusion test using P. brasiliensis gp43 and exoantigen as antigens, respectively, and an overall reactivity of 31.6 % was observed by ELISA although no reactivity was detected by immunodiffusion. The positivity observed in animals living in rural areas (48.8 %) with free access to soil was significantly higher (P < 0.0001) than among urban animals (2 %) with limited access to soil, although no significant difference was observed in relation to age or sex. The high rates of positivity observed in cats from rural areas suggest that not diagnosed cases of this mycosis may be occurring in cats living in endemic areas for human paracoccidioidomycosis. This is the first report showing serological evidence of P. brasiliensis infection in cats.     

Indirect Fluorescent-Antibody and Quantitative Agar-Gel Immunodiffusion Tests for the Serological Diagnosis of Paracoccidioidomycosis

The value of various serological tests in the diagnosis of paracoccidioidomycosis was studied. Quantitative agar-gel immunodiffusion and indirect immunofluorescent tests were performed, and the results were compared with those of complement fixation and qualitative agar-gel procedures. The quantitative immunodiffusion procedure was found to serve as the simplest and safest quantitative test that could be performed for evaluation purposes, whereas the indirect fluorescent-antibody test gave nonspecific reactions and, as such, proved unsuitable.     

Serological Diagnosis of Paracoccidioidomycosis: High Rate of Inter-laboratorial Variability among Medical Mycology Reference Centers

Background

Serological tests have long been established as rapid, simple and inexpensive tools for the diagnosis and follow-up of PCM. However, different protocols and antigen preparations are used and the few attempts to standardize the routine serological methods have not succeeded.

Methodology/Principal findings

We compared the performance of six Brazilian reference centers for serological diagnosis of PCM. Each center provided 30 sera of PCM patients, with positive high, intermediate and low titers, which were defined as the “reference” titers. Each center then applied its own antigen preparation and serological routine test, either semiquantitative double immunodifusion or counterimmmunoelectrophoresis, in the 150 sera from the other five centers blindly as regard to the “reference” titers. Titers were transformed into scores: 0 (negative), 1 (healing titers), 2 (active disease, low titers) and 3 (active disease, high titers) according to each center''s criteria. Major discordances were considered between scores indicating active disease and scores indicating negative or healing titers; such discordance when associated with proper clinical and other laboratorial data, may correspond to different approaches to the patient''s treatment. Surprisingly, all centers exhibited a high rate of “major” discordances with a mean of 31 (20%) discordant scores. Alternatively, when the scores given by one center to their own sera were compared with the scores given to their sera by the remaining five other centers, a high rate of major discordances was also found, with a mean number of 14.8 sera in 30 presenting a discordance with at least one other center. The data also suggest that centers that used CIE and pool of isolates for antigen preparation performed better.

Conclusion

There are inconsistencies among the laboratories that are strong enough to result in conflicting information regarding the patients'' treatment. Renewed efforts should be promoted to improve standardization of the serological diagnosis of PCM.     

血清学指标检验在脂肪肝患者临床诊断中的应用价值研究

目的:探讨血清学指标检验在脂肪肝患者临床诊断中的应用价值。方法:收集2013年3月-2014年3月于我院接受治疗的脂肪肝患者42例,作为观察组;选取同一时间我院体检科健康查体者42例,作为对照组。比较两组血清学指标检验结果,并分析其在脂肪肝临床诊断中的价值。结果:观察组患者丙氨酸氨基转移酶(ALT)、天冬氨酸转氨酶(AST)水平明显高于对照组健康查体者,两组比较差异均具有统计学意义(t=11.42、13.17,P0.05);观察组患者血清甘油三酯(TG)、总胆固醇(TC)水平明显高于对照组健康查体者,两组比较差异均具有统计学意义(t=7.35、8.02,P0.05)。结论:血清ALT、AST、TG、TC指标在脂肪肝患者临床诊断中存在重要的临床应用价值,可作为临床监测脂肪肝的重要方法,值得推广。     

Paracoccidioidomycosis in women

A retrospective review of paracoccidioidomycosis cases seen between January 1966 to January 1997 revealled 27 of the 627 cases occurred in women (male to female ratio 27.3:1). Data for 23 of these 27 cases demonstrated the protective effect of female hormones.     

Application of PCR in Serum Samples for Diagnosis of Paracoccidioidomycosis in the Southern Bahia-Brazil

Paracoccidioidomycosis (PCM) cannot always be diagnosed by conventional means such as direct examination of histopathology or clinical samples, and serological methods, used as an alternative, still have many cases of cross-reactivity. In this scenario, molecular techniques seem to arise as a rapid approach, specific and direct that could be used in the diagnosis of this mycosis. In this study we analyzed 76 serum samples from patients in southern Bahia suspected of having paracoccidioidomycosis using a conventional PCR with primers for the ITS1 ribosomal DNA of P. brasiliensis. Of these 76 patients, 5 were positive for PCM by double immunodiffusion and/or direct examination and histopathology. To test specificity of PCR, we used human DNA and three isolates of P. lutzii (1578, 01 and ED01). Additionally, we analyzed by serial dilutions of DNA the limit of detection of the assay. The test of PCR proved specific, as only a 144 bp fragment of the three isolates of P. lutzii and no human DNA was amplified. Detection limit was 1.1 pg/µL of DNA. Despite the high detection limit and specificity of PCR none of the 76 serum samples were found positive by PCR, but a biopsy specimen obtained from one of the patients with PCM was positive. These results, albeit limited, show that PCR is not effective in detecting DNA of P. brasiliensis or P. lutzii in serum, but could perhaps be used with other types of clinical samples, especially in those instances in which conventional methods fail.     

Hepatic Involvement in Pediatric Patients with Paracoccidioidomycosis: A Clinical and Laboratory Study

The liver is one of the organs most affected by paracoccidioidomycosis, a systemic mycosis endemic in some Latin American countries. The majority of articles focused on adult populations and failed to describe any detailed experience of liver abnormalities in pediatric patients. Therefore, the aim of this study was to describe the frequency and characteristics of liver involvement in children with paracoccidioidomycosis. This study comprised 102 patients less than 16 years of age (median 104.3 months) diagnosed with paracoccidioidomycosis from 1980 to 2010. Diagnosis was established by the identification of fungus. Forty-one patients had liver involvement. The main clinical features were generalized lymph node enlargement (39/41), weight loss (34/41) and fever 32/41). Approximately, one-third of the patients had jaundice. Patients with hepatic involvement were younger. A predominant elevation of canalicular enzymes occurred. There was a statistically significant difference in albumin (p < 0.001) and hemoglobin (p = 0.002) values between patients with and without liver involvement, and the lowest values were found in the former group. Cutoff levels of albumin (<3.05 g/dL) and hemoglobin (<9.2 g/dL) can be used to infer hepatic involvement. Hypoalbuminemia (median 2.4 g/dl) is more severe in patients with hepatic involvement and may indicate a worse liver function or complication of the disease (intestinal lymphangiectasia). Deaths (6) occurred only among patients with liver involvement. Particular clinical and laboratory characteristics are present in pediatric patients with hepatic involvement. Younger patients and those with severe hypoalbuminemia are more likely to present liver involvement by Paracoccidioides brasiliensis.     

Synthetic Peptides Mimic gp75 from Paracoccidioides brasiliensis in the Diagnosis of Paracoccidioidomycosis

Paracoccidioidomycosis (PCM) is a systemic granulomatous disease, endemic in Latin America, caused by the thermal dimorphic fungus Paracoccidioides brasiliensis. Although some fungal antigens have already been characterized and used for serological diagnosis, cross-reactions have been frequently observed. Thus, the examination of fungal forms in clinical specimens or isolation of P. brasiliensis by culture is still the most frequent method for the diagnosis of this mycosis. In this study, a random peptide phage display library was used to select mimotopes of P. brasiliensis, which were employed as antigens in an indirect enzyme-linked immunosorbent assay. The protective monoclonal antibody against experimental PCM (anti-gp75) was used as molecular target to screen a phage display library. That approach led to a synthetic peptide named P2, which was synthesized and tested against PCM patients' sera to check whether it was recognized. There was significant recognition of P2 by sera of untreated PCM patients when compared with normal human sera. Sera from treated PCM group, patients with other mycosis or co-infected with HIV had much lower recognition of P2 than untreated patient group. The test showed a sensitivity of 100 and 94.59% of specificity in relation to human sera control. These data indicate a potential use of P2 as diagnostic tool in PCM. Its application for serological diagnosis of PCM may contribute to the development and standardization of simpler, faster and highly reproducible immunodiagnostic tests at low cost.     

Serological Diagnosis of Classical Swine Fever

Antibody levels in post-infection sera from a pig inoculated with a low virulent strain of classical swine fever virus (Hannover 62) and in sera from two pigs inoculated with another low virulent strain (Spielbach 66) and from an in-contact pig were assayed by complement fixation and immunofluorescence using classical swine fever virus (ALD strain) and bovine virus diarrhoea virus (UG 59 strain) as antigens. The complement fixation test used was modified by addition of a preparation of porcine Glq to the complement and by mercaptoethanol treatment of the immune serum before use. The mercaptoethanol treatment of the immune serum resulted in complete elimination of a haemolytic prozone often seen with porcine immune sera. In the sera from the inoculated animals complement-fixing antibodies appeared earlier than neutralizing antibodies. A few weeks after inoculation there was a correlation between the presence of complement-fixing and neutralizing antibodies. During the entire observation period of 13 weeks it was not possible to demonstrate complement-fixing or neutralizing antibodies in serum from a pig exposed to infection by contact with the two pigs inoculated with the Spièlbach 66 strain of classical swine fever virus.     

Paracoccidioidal Infection in HIV Patients at an Endemic Area of Paracoccidioidomycosis in Brazil

The association between paracoccidioidomycosis (PCM) and AIDS is relatively rare in contrast to the higher incidence of other systemic mycosis. The explanation may be that AIDS is still predominantly an urban disease, and the PCM is endemic in Latin American rural areas. The aim of this study was to detect the prevalence of Paracoccidioides brasiliensis infection in HIV-positive patients at an endemic area of paracoccidioidomycosis in Brazil. Skin test with purified 43 kD glycoprotein (gp43) was performed in 90 HIV/AIDS patients. The prevalence found was 12.2% and it may be even greater, considering that HIV/AIDS patients may not respond to the intradermal test, which depends on cellular immunity for its positivity.     

脓毒症诊断与预后的血清学标志物研究进展

脓毒症可引起患者的多器官功能衰竭,从而导致严重烧伤、手术后、孕产妇、重症监护病房和新生儿监护病房的患者死亡。缺乏脓毒症急性相反应的患者病死率更高。寻找快速、简洁、敏感性和特异性高的脓毒症实验室诊断指标,已成为临床实验室长期以来的研究课题。     

Study on Post-Treatment Relapse in HBeAg Positive CHB Patients

Background

Many factors are associated with post-treatment relapse in CHB patients, and there are no effective factors to predict relapse. In this study, we investigate the influence factors associated with post-treatment relapse and their predictive value in HBeAg positive CHB (eP-CHB).

Methods

The factors associated with post-treatment relapse were analyzed firstly by a retrospective study in eP-CHB. Variables included age, sex, regimen, baseline HBeAg and HBV DNA level, total course of treatment as well as duration of consolidation therapy after HBeAg seroconversion. The predictive effects of the influence factors were evaluated in an eP-CHB prospective cohort.

Results

89 patients were enrolled in the retrospective study, 42(47.2%) relapsed after discontinuation of treatment. Factors related to post-treatment relapse were total course of treatment, duration of consolidation therapy and baseline HBV DNA level. Relapse rates in patients with total course >36 months, consolidation duration >12 months and baseline HBV DNA level < 1.0E+5IU/ml were lower than those of total course <24 months (P = 0.002), consolidation duration≤12 months (P = 0.011) and baseline HBV DNA level > 1.0E+7IU/ml (P = 0.01) respectively. Patients with HBV DNA≥1.0E+7IU/ml plus HBeAg<200COI at baseline had the highest relapse rate and cumulative relapse rate than the other three arms (P = 0.048 and 0.008 respectively). Logistic regression analysis demonstrated that baseline HBV DNA level, duration of consolidation therapy and combination of baseline HBV DNA and HBeAg (Ig^DNA/Ig^HBeAg) were independent factors to predict post-treatment relapse. The model based on baseline Ig^DNA/Ig^HBeAg and consolidation duration worked well in predicting post-treatment relapse in the prospective study and the accuracy, specificity, sensitivity, PPV and NPV for prediction were 80.3%, 81.1%, 79.2%, 73.1% and 85.7% respectively.

Conclusions

Virological factors including baseline HBV DNA, HBeAg and treatment course were major influence factors associated with post-treatment relapse in eP-CHB. Patients with higher HBV DNA and lower HBeAg levels at baseline, shorter total course as well as consolidation therapy were more likely to develop relapse after discontinuation of therapy. The antiviral therapy in eP-CHB patients should be individually managed at different levels. It is better to treat those with higher viral load and lower HBeAg levels at baseline for a longer course, especially longer consolidation duration so as to decrease the relapse rate.