Acanthamoeba spp. as a universal host for pathogenic microorganisms: One bridge from environment to host virulence.

Free-living amoebas (FLA) are ubiquitous environmental protists that have enormously contributed to the microbiological contamination of water sources. FLAs have displayed resistance to environmental adversities and germicides and have played important roles in the population control of microbial communities due to its predatory behavior and microbicidal activity. However, some organisms have developed resistance to the intracellular milieu of amoebas, as in the case of Acanthamoebas, which in turn, have been functioning as excellent reservoirs for amoeba-resistant microorganisms (ARMs), such as bacteria, viruses and fungi. Little is known about these relationships and interaction mechanisms, but it is speculated that the FLAs need a very broad repertoire or universal class of receptors to bind and recognize these diverse species of microorganisms. By harboring these organisms as a "Trojan Horse", the Achantamoeba has been working as an excellent vector for pathogens. Moreover, studies have demonstrated that the interaction of pathogens with Acanthamoeba results in environmental selective pressure responsible for induction and maintenance of virulence factors and increase in microbial pathogenicity. This phenomenon is correlated to the observation of higher gene number and DNA content of ARMs, when compared to their relatives which are adapted to other hosts, due to allopatric or sympatric gene transfer and acquisition, contradicting the overall genome reduction theory for intracellularly adapted pathogens. Thus, adaptation to FLAs indirectly provided a "learning" environment for pathogens to resist later to macrophages; besides the evolutionary distance, these phagocytes share similar predatory mechanisms, such as phagocytosis and phagolysossomal degradation. In this mini-review, we cover the most important aspects of Acanthamoeba biology and their interactions with endemically important human pathogens.

Schistosomiasis in areas of low endemicity: a new era in diagnosis.

Parasitological detection of Schistosoma is the cornerstone of schistosomiasis diagnosis in areas of transmission worldwide. However, a steep decrease of sensitivity in low-endemicity areas (LEAs) compromises estimation of schistosomiasis. Despite the restricted utilization of molecular and immunodiagnostic techniques, recent improvements and advances have been contributing to change this scenario, especially in LEAs. Nonetheless, the main issue in a new era of diagnosis overcomes technical advances per se and relates to the loss of 'gold standards' in schistosomiasis diagnosis in LEAs. Here, we review and discuss the current role of molecular and immunodiagnostic methods in schistosomiasis management.

Taenia crassiceps cysticerci: Characterization of the 14-kDa glycoprotein with homologies to antigens from Taenia solium cysticerci.

Glycoproteins from the total vesicular fluid of Taenia crassiceps (VF-Tc) were prepared using three different purification methods, consisting of ConA-lectin affinity chromatography (ConA-Tc), preparative electrophoresis (SDS-PAGE) (14 gp-Tc), and monoclonal antibody immunoaffinity chromatography (18/14-Tc). The complex composition represented by the VF-Tc and ConA-Tc antigens revealed peptides ranging from 101- to 14-kDa and from 92- to 12-kDa, respectively. Immunoblotting using lectins confirmed glucose/mannose (glc/man) residues in the 18- and 14-kDa peptides, which are considered specific and immunodominant for the diagnosis of cysticercosis, and indicated that these fractions are glycoproteins. Serum antibodies from a patient with neurocysticercosis that reacted to the 14 gp band from T. crassiceps (Tc) were eluted from immunoblotting membranes and showed reactivity to 14 gp from Taenia solium. In order to determine the similar peptide sequence, the N-terminal amino acid was determined and analyzed with sequences available in public databases. This sequence revealed partial homology between T. crassiceps and T. solium peptides. In addition, mass spectrometry along with theoretical M(r) and pI of the 14 gp-Tc point suggested a close relationship to some peptides of a 150-kDa protein complex of the T. solium previously described. The identification of these common immunogenic sites will contribute to future efforts to develop recombinant antigens and synthetic peptides for immunological assays.

Serological studies in Nectomys squamipes demonstrate the low sensitivity of coprological exams for the diagnosis of schistosomiasis.

The existence of wild rodents naturally infected by Schistosoma mansoni is a drawback for schistosomiasis control programs. As a consequence, it is necessary to have a precise diagnosis of S. mansoni infection in wild rodents (water rats; Nectomys squamipes), the species seemingly involved in the transmission of schistosomiasis at Sumindouro, Rio de Janeiro, Brazil. A total of 78 specimens of N. squamipes was captured in an endemic area at Vale do Pamparrão and Porteira Verde, Sumidouro, Brazil; 5 more were born in captivity and experimentally infected. The sensitivity and specificity of the coprological method of Kato-Katz and serological methods, i.e., enzyme-linked immunosorbent assay (ELISA) and western blot (WB), were compared. The rodents were subsequently killed and necropsied to confirm infection. The prevalences observed using ELISA (48%) and WB (41%) were equivalent to those found at necropsy (41%). The ELISA showed a sensitivity of 97% and a specificity of 87%, whereas the WB showed a sensitivity of 87% and a specificity of 89%. The Kato-Katz method exhibited 50% sensitivity and 100% specificity. The differences found among the ELISA, WB, and necropsy, when compared with Kato-Katz, may be related to the low sensitivity of the coprological method. Serological methods should be used for more reliable epidemiological information.

Identification of a 26-kDa protein fraction as an important antigen for application in the immunodiagnosis of strongyloidiasis.

Strongyloidiasis caused by the intestinal nematode Strongyloides stercoralis typically occurs in the asymptomatic form. The definitive diagnosis is usually done by detection of larvae on fecal samples. However, as the parasite load is often low in most cases, microscopy is not usually sensitive and specific, and diagnosis becomes extremely difficult. Thus, development of reliable serological methods is imperative. In the present study, a diversity of epitopes from S. stercoralis larva were characterized by analysis of reactivity with serum samples obtained from individuals with and without the infection by using Western blot technique. A total of 91 serum samples belonging to 5 groups were analyzed. Different reactivity profiles were observed, representing recognition of proteins with molecular mass varied from 6 to 129 kDa. A protein band of approximately 26 kDa presented a high frequency of reactivity with serum samples from the strongyloidiasis patients group (18/23). Reactivity with this protein band was also observed in only 7 of 64 non-infected individuals or individuals infected with other helminthes. Reactivity with 2 other bands, 1 of approximately 33 kDa and a duplet of approximately 21 kDa, were also found in high frequency (17/23 and 9/23, respectively). However, reactivity with these bands was also observed in all the other serum groups studied. The results indicate that the 26-kDa band maybe be an important tool for the development of diagnostic techniques for strongyloidiasis.

Immunoassays as an auxiliary tool for the serodiagnosis of Schistosoma mansoni infection in individuals with low intensity of egg elimination.

In an attempt to improve the screening of Schistosoma mansoni-infected individuals from a low-transmission area, we established a protocol that includes three independent coproscopy examinations and two serological assays. Three stool samples were examined using the Kato-Katz and free sedimentation methods and serum samples were tested by IgG-ELISA and IgM-immunofluorescence. Two hundred and sixty-nine individuals participated in the survey: 132 individuals (49%) showed positive serological test results. Of these, 16 (6%) had positive results in stool examination in the first sample batch. However, there were also cases with positive serological test results in spite of negative Kato-Katz stool examinations. Additional stool samples were obtained from these subjects and in this way an additional 11 egg-excretors were found. Our findings suggest that a screening method that combines antibody isotype detection and repeated parasitological stool examinations could increase the chances of detecting S. mansoni-infected patients.

Accidental infection of laboratory worker with vaccinia virus.

We report the accidental needlestick inoculation of a laboratory worker with vaccinia virus. Although the patient had previously been vaccinated against smallpox, severe lesions appeared on the fingers. Western blot and polymerase chain reaction-restriction fragment length polymorphism were used to analyze the virus recovered from the lesions. The vaccinia virus-specific immunoglobulin G levels were measured by enzyme-linked immunosorbent assay. Our study supports the need for vaccination for laboratory workers that routinely handle orthopoxvirus.

Evaluation of an antigen from Taenia crassiceps cysticercus for the serodiagnosis of neurocysticercosis.

We report here the evaluation of an antigen from Taenia crassiceps cysticercus as a potential reagent in an enzyme-immunoelectrotransfer blotting assay (EITB) and an enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of neurocysticercosis (NC) using clinical specimens obtained from patients in different phases of the disease. Serum and cerebrospinal fluid (CSF) samples from 64 patients suspected of having NC according to clinical manifestation and brain computed tomography were tested by ELISA with Taenia solium total saline antigen (ELISA-Tso) and by immunoblotting with T. crassiceps glycoproteins antigen (EITB-gpTcra). Forty-five serum samples were also tested immunoblotting with T. solium glycoproteins antigen (EITB-gpTso) and 30 were tested by ELISA with T. crassiceps 14 kDa glycoprotein (ELISA-gp14Tcra). Serum samples from apparently healthy individuals without any parasitic disease and from patients with other parasitic diseases were included as controls. The results of ELISA-Tso analysis with CSF obtained from 64 patients with NC showed that 53 (83%) were reactive. EITB-gpTcra analysis with serum from the same group of patients showed a sensitivity of 91%. Results of EITB-gpTso and EITB-gpTcra analysis with serum samples demonstrated an agreement of 100% between both tests. ELISA-gp14Tcra was positive in 23 (77%) sera, 22 with paired CSF positive. When ELISA-gp14Tcra results were compared to EITB-Tso results, a relative sensitivity of 95% was observed. All serum samples from the control group were negative in ELISA-gp14Tcra and only one serum from an individual with Taenia saginata was reactive in this assay, showing a specificity of 99% for ELISA-gp14Tcra. This fraction was purified in only one step with a good yield for use in immunoassays. We suggest that the gp14Tcra antigen can be used for detecting anti-cysticercus antibodies in serum samples for epidemiological investigation purposes and also for diagnostic screening of NC patients.