Distinct Pattern of Paracoccidioides lutzii, P. restrepiensis and P. americana Antigens Recognized by IgE in Human Paracoccidioidomycosis.

Paracoccidioidomycosis (PCM) is caused by the fungi Paracoccidioides spp. The main antigens recognized by IgE are known for P. brasiliensis species complex, but not for P. lutzii. Current research investigated the major P. lutzii (LDR2) antigens recognized by IgE, in comparison to P. restrepiensis and P. americana (former P. brasiliensis species complex), besides IgG recognition. Cell-free antigens (CFA) from P. lutzii (LDR2), P. restrepiensis (B339) and P. americana (LDR3) were analyzed by ELISA and immunoblotting (IB) by detecting specific IgG and IgE from sera from patients with PCM presumable by P. brasiliensis species complex (n = 24). Additionally, somatic antigen (SA) was analyzed by IB. P. lutzii (LDR2) antigens showed significantly lower reactivity than P. restrepiensis (B339) and P. americana (LDR3) in ELISA for both IgE and IgG (p < 0.05). The IgE-IB pattern was different between P. lutzii (LDR2) and the other species, regarding components with ~30 kDa and ~70 kDa in CFA and a ~200 kDa in SA. P. lutzii (LDR2) present at least three antigens recognized by IgE which mainly differ from P. restrepiensis (B339) and, to a lesser extent, from P. americana (LDR3). Current research evidenced for the first time the major P. lutzii (LDR2) antigens recognized by IgE.

IgG reactivity profile to Paracoccidioides spp. antigens in people with asymptomatic Paracoccidioidomycosis.

Introduction. Paracoccidioidomycosis (PCM) is a systemic mycosis caused by Paracoccidioides spp. As the disease is known to affect mostly men over 40 years old who previously worked handling soil, some cities of agricultural economy in endemic regions may have more cases of paracoccidioidal infection.Gap statement. The true frequency of PCM cannot be established in Brazil because it is not a disease of mandatory reporting. The detection of paracoccidioidal infection may assist in the planning of health services, in order to provide early detection of the disease and to prevent its worsening or even progression to death. In addition, little is described about sera reactivity with antigens from different species of Paracoccidiodes, especially P. lutzii.Aim. Current research was conducted in an inland municipality of southern Brazil, in order to assess infection rate within this endemic region of PCM disease.Methodology. ELISA was employed to evaluate 359 sera from random volunteers from Guarapuava, Paraná, Brazil, to detect IgG against cell-free antigens (CFA) from P. restrepiensis B339, P. americana LDR3 and P. lutzii LDR2. Confirmatory ELISA employed gp43 from B339. Reduction of cross-reactions was sought by treatment with sodium metaperiodate (SMP-CFA, SMP-gp43). Immunoblot was performed with 37 selected sera among those reactive in ELISA. Epidemiological profile was assessed by questionnaire.Results. ELISA reactivity was: CFA/SMP-CFA in general 37.3/17.8 %, B339 25.3/14.5 %, LDR3 24.5/1.4 %, LDR2 8.3/5.8 %; gp43/SMP-gp43 7.2/4.7 %. There were sera reactive with multiple CFAs. In immunoblot, five sera showed the same reaction profile with P. lutzii's antigens as PCM disease sera. Rural residence and soil-related professions were risk factors for paracoccidioidal infection.Conclusion. The low prevalence is in accordance with previous reports of lower PCM disease endemicity in Guarapuava than in other areas of Paraná. Although P. brasiliensis seems to be the prevalent strain of the region, 21 sera from people who only lived in Guarapuava reacted with P. lutzii LDR2. CFA-ELISA with whole antigens seems a good option for serological screening in epidemiological surveys.

Polyclonal antibodies to Paracoccidioides brasiliensis are able to recognise antigens from different strains from Paracoccidioides species complex, including Paracoccidioides lutzii LDR2.

Since the new species Paracoccidioides lutzii emerged in 2009, much has been researched on strains previously considered atypical. Still, there is no consensus about recognition of antigens from P. lutzii by antibodies directed to other Paracoccidioides species, which can have great impact on Paracoccidioidomycosis (PCM) diagnosis. Current research investigated soluble protein/carbohydrate epitopes from P. lutzii LDR2, Paracoccidioides restrepiensis B339 and Paracoccidioides americana LDR3 recognised by IgG directed to Paracoccidioides brasiliensis. Cell free antigens (CFA) were analysed by: (a)silver and periodic acid-Schiff staining of SDS-PAGE; (b)immunoblot (IB) with rabbit IgG anti-P. brasiliensis Pb18; (c)IB and ELISA with a pool of PCM patients' sera before and after treatment with sodium metaperiodate (SMP) to oxidise carbohydrate epitopes. Both rabbit and human immune sera recognised several antigens of P. lutzii LDR2, P. restrepiensis B339 and P. americana LDR3. P. lutzii's gp43 was not observed in IB or silver/PAS staining. SMP treatment affected reactions with all 3 CFAs, but more intensely with antigens from P. lutzii LDR2. In conclusion, antibodies directed to P. brasiliensis recognised antigens from P. lutzii LDR2. The use of any of the recognised antigens in a broad spectrum diagnostic model for Paracoccidioides species complex needs to be further investigated.

Paracoccidioides restrepiensis B339 (PS3) and P. lutzii LDR2 yeast cells and soluble components display in vitro hemolytic and hemagglutinating activities on human erythrocytes.

Paracoccidioidomycosis (PCM) is a systemic mycosis caused by thermodimorfic fungi of Paracoccidioides species complex. Several pathogenic fungi produce hemagglutinins and hemolysins, which are virulence factors involved in adhesion of pathogens to host tissues or cells and in destruction of erythrocytes. The present research investigated hemolytic and hemagglutinating activities of yeast cells and soluble components from P. restrepiensis (PS3; former P. brasiliensis B339) and P. lutzii (LDR2). Different concentrations of live and heat-killed yeast cells and soluble components from cell free antigen preparation (CFA) (native or heated - 56 and 100 °C, 30 min) were mixed with 1% human erythrocyte suspension. Yeast cells from both species caused hemolysis, but P. lutzii LDR2 was more hemolytic than P. restrepiensis B339, while the opposite phenomena occurred with soluble components in most conditions. Live or heat-killed yeast cells of both fungi agglutinated erythrocytes, but only heated soluble components from P. restrepiensis B339 showed hemagglutinating activity. In conclusion, yeast cells of P. restrepiensis B339 and P. lutzii LDR2 produce hemolysins and hemagglutinins, which most likely are more restricted to yeast cells in P. lutzii LDR2 and are more released in soluble form byP. restrepiensis B339, requiring further study.

High prevalence rate of extended-spectrum beta-lactamases (ESBL) among Enterobacteriaceae in a small Brazilian public hospital

The production of extended-spectrum beta-lactamases (ESBL) is considered one of the most important resistance mechanisms that impair antimicrobial treatment of infections caused by Enterobacteriaceae. Data on culture and susceptibility tests were collected from the Clinical Analyses and Research Laboratory charts reporting on patients admitted to the University Hospital of Maringá (HUM) from January 2004 to December 2009. The following Enterobacteriaceae were selected: Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter spp. and Proteus mirabilis. All tests were performed according to the recommendations of the Clinical and Laboratory Standards Institute (biochemical identification; susceptibility tests; initial screening and phenotypic confirmatory tests for ESBL). For Enterobacter spp. isolates, a disk approximation test was carried out, adding a cefepime disk. Seven hundred samples were analyzed, and E. coli was the most prevalent bacteria (n= 356). ESBLs were detected phenotypically in 7.3 percent of E. coli, 61.7 percent of K. pneumoniae, 33.3 percent of K. oxytoca, 7.1 percent of P. mirabilis, and 13.4 percent of Enterobacter spp samples. Overall ESBL prevalence reached 22 percent when all producers were taken together. Although HUM is considered a small-sized hospital, it showed high levels of resistance to antimicrobial agents, similar to those observed in bigger hospitals, which demonstrated the need for careful epidemiological surveillance.

A produção de beta-lactamases de espectro ampliado (ESBL) é considerada um dos mais importantes mecanismos de resistência aos antimicrobianos, o que dificulta o tratamento de infecções causadas por enterobactérias. Dados sobre cultura e testes de sensibilidade foram coletados das fichas do Laboratório de Ensino e Pesquisa de Análises Clínicas de pacientes atendidos no Hospital Universitário de Maringá (HUM), de janeiro de 2004 a dezembro de 2009. As enterobactérias escolhidas foram: Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter spp. e Proteus mirabilis. Os testes foram realizados de acordo com as recomendações do CLSI (identificação bioquímica; testes de suscetibilidade; triagem e confirmação fenotípica de produção de ESBL). Para isolados de Enterobacter spp., utilizou-se a técnica de disco aproximação, acrescentando um disco de cefepima. Setecentas amostras foram analisadas e E. coli foi a bactéria mais prevalente (n=356). ESBLs foram detectadas fenotipicamente em 7,3 por cento das amostras de E. coli, 61,7 por cento das de K. pneumoniae, 3,3 por cento das de K. oxytoca, 7,1 por cento das de P. mirabilis e em 13,4 por cento das de Enterobacter spp. A prevalência geral de ESBL chegou a 22 por cento, somando-se todos os isolados produtores. O HUM, mesmo sendo considerado um hospital de pequeno porte, apresenta níveis altos de resistência a antimicrobianos, semelhantes àqueles observados em hospitais maiores, demonstrando a necessidade de cuidadosa vigilância epidemiológica.