ABSTRACT Serodiagnosis of strongyloidiasis is usually performed by ELISA for the detection of IgG antibodies due to its high sensitivity and practicality, but its main limitation is a constant source of S. stercoralis antigens. The use of S. venezuelensis as a heterologous source of antigens has facilitated several published studies on the serodiagnosis and epidemiology of human strongyloidiasis. The main objective of this study was to evaluate the diagnostic accuracy of surface cuticle antigens of infective larvae of S. venezuelensis extracted with CTAB detergent (L3-CTAB) in comparison with soluble somatic extracts (L3-SSE) using a panel of sera from immunocompetent and immunocompromised individuals, at three different cut-offs. ROC curve analysis showed that L3-CTAB had an AUC of 0.9926. At the first cut-off value (OD 450 nm = 0.214), sensitivity and specificity were 100% and 90.11%, respectively, with a diagnostic accuracy of 0.93. At a second cut-off value (OD 450 nm = 0.286), sensitivity and specificity were 70% and 100%, respectively, with a diagnostic accuracy of 0.91. However, at an alternative third cut-off value (OD 450 nm = 0.589), sensitivity and specificity were 95% and 97.8%, respectively, with a diagnostic accuracy of 0.97. Using L3-CTAB as an antigenic source, the seropositivity rate in immunocompromised patients was 28.13% (9/32) whereas a seropositivity rate of 34.38% (11/32) was found when L3-SSE was used in ELISA. Therefore, the L3-CTAB is simple and practical to obtain and was found to be highly sensitive and specific.

ABSTRACT The Western-blotting technique was applied to identify antigenic fractions of excretory-secretory Toxocara canis antigen recognized by IgG antibodies throughout an experimental infection in mice challenged by different inocula. Mice were inoculated with 5, 50 and 500 embryonated eggs and serum samples were collected 15, 30, 60, 90 and 120 days post-infection. Serum samples were analyzed using an excretory-secretory Toxocara antigen. Antibodies recognized antigenic fractions from 30 to 90 kDa. The protein fraction of 30-35 kDa was the most frequently recognized regardless of the size of inoculum and the stage of infection represented by the different collection times, but the antigenic recognition was more evident in groups infected with 50 and 500 eggs. This study presents an antigenic panel of the excretory-secretory antigen of T. canis and suggests that the 30-35 kDa antigenic fraction is a promising marker of the infection and should be further explored in future studies on experimental toxocariasis.

Abstract Objective: The association between diabetes and Strongyloides infection remains controversial. This study aimed to detect Strongyloides stercoralis DNA in the feces of patients with Diabetes Mellitus type 2 (DM2). Methods: Fecal samples were analyzed via the Lutz, Rugai, and agar plate culture methods. PCR amplification was performed using two targets (PCR-genus and PCR-species) located on the S. stercoralis 18S ribosomal. Results: The positivity for S. stercoralis using parasitological methods was 1.1%. PCR-genus (14.13%) demonstrated a higher positivity than PCR-species (9.78%). Conclusion: The results confirm the greater positivity of the molecular diagnosis in relation to parasitological methods, reinforcing its use as an additional tool for the diagnosis of S. stercoralis infection in patients with DM2 living in endemic areas for this helminthiasis. HIGHLIGHTS Positivity for strongyloidiasis in coproscopic exam was low in diabetic patients. PCR is more sensitive for detecting S. stercoralis infection in diabetic patients. Molecular diagnosis is an important tool for the detection of S. stercoralis.

Abstract Introduction Hematopoietic Stem Cell Transplant (HSCT) has been successfully used as standard therapy for hematological disorders. After conditioning therapy, patients undergoing allogeneic HSCT, present three different phases of engraftment: early pre-engraftment, early post-engraftment, and late engraftment. Severe complications are associated with morbidity, mortality, and malignancies in these phases, which include effects on the oral cavity. Objectives The changes in the salivary composition after HSCT may contribute to identifying relevant proteins that could map differences among the phases of diseases, driven for personalized diagnostics and therapy. Methods Unstimulated whole saliva was collected from patients submitted to HSCT. The samples were submitted to trypsin digestion for a Mass spectrometry analysis. MaxQuant processed the Data analysis, and the relevant expressed proteins were subjected to pathway and network analyses. Results Differences were observed in the most identified proteins, specifically in proteins involved with the regulation of body fluid levels and the mucosal immune response. The heatmap showed a list of proteins exclusively expressed during the different phases of HSCT: HBB, KNG1, HSPA, FGB, APOA1, PFN1, PRTN3, TMSB4X, YWHAZ, CAP1, ACTN1, CLU and ALDOA. Bioinformatics analysis implicated pathways involved in protein processing in the endoplasmic reticulum, complement and coagulation cascades, apoptosis signaling, and cholesterol metabolism. Conclusion The compositional changes in saliva reflected the three phases of HSCT and demonstrated the usefulness of proteomics and computational approaches as a revolutionary field in diagnostic methods. (HSCT disorders engraftment preengraftment, preengraftment pre engraftment, pre-engraftment postengraftment, postengraftment post post-engraftment morbidity mortality cavity diseases analyses response HBB KNG1 KNG HSPA FGB APOA1 APOA PFN1 PFN PRTN3 PRTN TMSB4X TMSBX TMSB X YWHAZ CAP1 CAP ACTN1 ACTN ALDOA reticulum cascades signaling metabolism methods

ABSTRACT Blastocystis sp. is an enteric protist commonly found in human fecal samples. In Brazil, few studies have been developed, but none of them has explored the presence of Blastocystis in patients with diabetes mellitus. We evaluated the occurrence and molecular identification of Blastocystis sp. among patients with diabetes mellitus in the Midwest region, Goias State, Brazil. Genomic DNA was obtained from 175 fecal samples (99 from the diabetic group and 76 from the control group). PCR was performed using pan-Blastocystis primers from the SSU-rDNA gene. Microscopic examination revealed positivity of 12.1% and 7.9% for Blastocystis in diabetics and in controls, respectively. Amplification of Blastocystis DNA was observed in 34.4% (34 of 99) and 30.3% (23 of 76) from the diabetic and control groups, respectively. Phylogenetic analyses and BLAST searches revealed six subtypes among Blastocystis isolates in the diabetic group, represented by ST1 (38.2%), ST2 (11.8%), ST3 (35.3%), ST6 (2.9%), ST7 (2.9%) and ST8 (8.8%). In the control group, ST1 (21.8%), ST2 (21.8%), ST3 (43.5%), ST6 (4.4%) and ST8 (8.7%) were identified. This study is the first report regarding the occurrence and subtypes distribution of Blastocystis in patients with diabetes mellitus in Brazil. The results reinforce the potential risk of Blastocystis infection in patients with diabetes, in addition, it contributes to the understanding of the genetic diversity of this enigmatic organism.

The present study aimed to evaluate the occurrence of Blastocystis sp. in Brazilian studies over a period of years (2000-2020), as well as point out relevant aspects of this enigmatic organism. We performed a literature search using six sources of international databases. The data were divided into diagnostic by parasitological and molecular techniques, and relevant aspects. After applying the inclusion and exclusion criteria, 52 studies were included in the final analysis. The occurrence of Blastocystis sp. in Brazil ranged from 0.5% to 86.6%, as determined using parasitological techniques. The highest occurrence was in the North (27.3%) and the lowest, in the Midwest region (13.4%). In Brazil, most studies have employed molecular techniques and are concentrated in the Southeast region. The Blastocystis sp. subtype ST3 had the highest average positivity, followed by ST1 and ST2. These findings represent a panorama that reflects the reality of Brazil; thus, we believe that the effectiveness of parasitological diagnosis should be considered with regard to making an appropriate choice of technique for detecting Blastocystis sp. Additionally, we emphasize the importance of further studies in the context of molecular epidemiology with regard to this genus. Blastocystis sp. is not well understood yet, and very little information regarding this genus is available; hence, further research regarding this genus is urgently needed.

ABSTRACT Approximately 240 million people worldwide are infected by Schistosoma. In Brazil, one of the main intermediate hosts of this parasite is Biomphalaria glabrata snails. The early detection of larval stages in intermediate hosts is an important challenge to public health, but it also represents an opportunity as a new alternative to indicate earlier natural infections before cercariae differentiation and emergence. In this context, we demonstrated that PCR amplification of a 28S gene fragment from the parasite does demonstrate S. mansoni infection in snails 14 days post infection. This conventional polymerase chain reaction amplified clear bands and was able to detect parasitic infection in the intermediate host B. glabrata under experimental conditions. However, we reinforce that this approach requires deeper investigations and further comparisons to confirm its specificity and sensitivity in earlier time points after miracidia infection. This approach has relevant potential as an effective molecular-based strategy for the monitoring of schistosomiasis transmission.

ABSTRACT Blastocystis sp. is a protist commonly found in stool samples of humans and animals. Biological and genetic factors of this organism remain controversial. The present study aimed to develop and implement the Blastocystis in vitro culture of Brazilian human isolates for routine use. The fecal isolates (n = 20) were maintained in our laboratory by several passages in Pavlova's medium. Cultures were monitored every 72 h by light microscopy. Genomic DNA was extracted to identify the subtypes (STs). In most isolates, the vacuolar form was prevalent. The amoeboid, granular and cystic forms were observed during in vitro cultivation. STs 1, 2, 3, 4 and 7 were identified. Our preliminary results show the generation time and forms present in the in vitro culture of Blastocystis subtypes isolated from Brazilian human isolates. Therefore, we emphasize the use of in vitro culture as a tool in future studies for the better understanding of the biological aspects of Blastocystis sp.

ABSTRACT In experimental infection with Strongyloides venezuelensis, the acute and recovery phases can be distinguished, unlike human infections caused by Strongyloides stercoralis. The objective of this study was to evaluate the production of anti-Strongyloides IgG antibodies and the recognition of immunogenic protein bands during the acute and the recovery phases in rats experimentally infected with S. venezuelensis. Rats were infected subcutaneously with 400 or 4,000 S. venezuelensis infective larvae. The acute phase was characterized by elimination of a large number of eggs in the faeces on days 6-14 post infection; the recovery phase was characterized by the resolution of the infection between days 30 and 35 post infection. Differences in IgG levels were observed in the acute and the recovery phases. Different antigenic fractions were recognized in both phases of infection. It is concluded that proteins within the 30-40 kDa range are immunoreactive markers for both the acute and the recovery phases in rats experimentally infected with S. venezuelensis, particularly using membrane antigen.

BALB/c mice were inoculated with 5-500 Toxocara canis infective eggs, and bled at 15-120 days post infection (dpi) to evaluate the dynamics of IgG antibody response and larvae distribution. Positive results were observed in all occasions for every inoculum, and a direct proportional relationship between antibody detection and the parasitic load was observed. In samples collected at 60 dpi, detection of IgG was more intense, especially with the 50 and 500 egg doses; also, a correlation between antibody level and egg count was observed with these two inocula. At 120 dpi, a decrease in antibody titer was observed for all groups; and at the end of the experiment, larvae were recovered from carcass, liver and brain. In the liver, larvae were only found in mice inoculated with 500 T. canis eggs. In carcasses, these were recovered in all groups, and the group inoculated with 50 eggs showed the highest percentage of larvae in the brain.

SUMMARY Strongyloidiasis is a potentially serious infection in immunocompromised patients. Thus, the availability of sensitive and specific diagnostic methods is desirable, especially in the context of immunosuppressed patients in whom the diagnosis and treatment of strongyloidiasis is of utmost importance. In this study, serological and molecular tools were used to diagnose Strongyloides stercoralis infections in immunosuppressed patients. Serum and stool samples were obtained from 52 patients. Stool samples were first analyzed by Lutz, Rugai, and Agar plate culture methods, and then by a quantitative real time polymerase chain reaction (qPCR). Serum samples were evaluated by an enzyme-linked immunosorbent assay (ELISA) using a soluble (AS) or a membrane fractions antigen (AM) obtained from alkaline solutions of the filariform larvae of Strongyloides venezuelensis. Of the 52 immunosuppressed patients, three (5.8%) were positive for S. stercoralis by parasitological methods, compared to two patients (3.8%) and one patient (1.9%) who were detected by ELISA using the AS and the AM antigens, respectively. S. stercoralis DNA was amplified in seven (13.5%) stool samples by qPCR. These results suggest the utility of qPCR as an alternative diagnostic tool for the diagnosis of S. stercoralis infection in immunocompromised patients, considering the possible severity of this helminthiasis in this group of patients.

RESUMO O objetivo deste estudo foi avaliar seis frações antigênicas diferentes de fêmeas parasitas de Strongyloides venezuelensis para o diagnóstico sorológico da estrongiloidíase humana. As frações solúveis e de membrana de fêmeas parasitas de S. venezuelensis foram preparadas em solução salina tamponada (SSF e SMF, respectivamente), Tris-HCl (TSF e TMF, respectivamente) e tampão alcalino (ASF e AMF, respectivamente). As amostras de soro obtidas de pacientes com estrongiloidíase, com outras parasitoses e indivíduos saudáveis, foram analisadas pelo ensaio imunoenzimático (ELISA). As frações solúveis SSF, TSF e ASF apresentaram sensibilidade de 85,0%, 75,0%, e 80,0% e especificidade de 93,1%, 93,1% e 87,5%, respectivamente. As frações de membrana, SMF, TMF e AMF demonstraram sensibilidade de 80,0%, 75,0%, e 85,0%, e especificidade de 95,8%, 90,3% e 91,7%, respectivamente. Em conclusão os presentes resultados sugerem que as frações obtidas a partir de fêmeas parasitas, especialmente o SSF e SMF, podem ser utilizadas como fonte alternativa de antígenos no imunodiagnóstico da estrongiloidiase humana.

SUMMARY The aim of this study was to evaluate six different antigenic fractions from Strongyloides venezuelensis parasitic females for the immunodiagnosis of human strongyloidiasis. Soluble and membrane fractions from S. venezuelensis parasitic females were prepared in phosphate-buffered saline (SSF and SMF, respectively), Tris-HCl (TSF and TMF, respectively), and an alkaline buffer (ASF and AMF, respectively). Serum samples obtained from patients with strongyloidiasis or, other parasitic diseases, and healthy individuals were analyzed by enzyme-linked immunosorbent assay (ELISA). Soluble fractions SSF, TSF, and ASF showed 85.0%, 75.0%, and 80.0% sensitivity and 93.1%, 93.1%, and 87.5% specificity, respectively. Membrane fractions SMF, TMF, and AMF showed 80.0%, 75.0%, and 85.0% sensitivity, and 95.8%, 90.3%, and 91.7% specificity, respectively. In conclusion, the present results suggest that the fractions obtained from parasitic females, especially the SSF and SMF, could be used as alternative antigen sources in the serodiagnosis of human strongyloidiasis.

This study aimed to evaluate the use of conventional polymerase chain reaction (cPCR) and real-time quantitative PCR (qPCR) in the diagnosis of human strongyloidiasis from stool samples in tropical areas. Stool samples were collected from individuals and were determined to be positive for Strongyloides stercoralis (group I), negative for S. stercoralis (group II) and positive for other enteroparasite species (group III). DNA specific to S. stercoralis was found in 76.7% of group I samples by cPCR and in 90% of group I samples by qPCR. The results show that molecular methods can be used as alternative tools for detecting S. stercoralis in human stool samples in tropical areas.

Strongyloides venezuelensis é um nematódeo parasita de roedores, frequentemente usado como antígeno heterólogo para o diagnóstico imunológico da estrongiloidíase humana. O objetivo deste estudo foi avaliar frações de membrana de S. venezuelensis para o imunodiagnóstico da estrongiloidíase humana. Para tanto, frações solúveis e de membrana foram obtidas em solução salina fosfato (SS e MS) e Tris-HCl (ST e MT) de larvas filarioides de S. venezuelensis. Amostras de soro de 92 indivíduos, sendo 20 com estrongiloidíase (Grupo I); 32 com outras parasitoses (Grupo II), e 40 indivíduos saudáveis (Grupo III), foram analisadas pelo teste Imunoenzimático (ELISA). As frações solúveis (SS e ST) apresentaram 90,0% e 88,9%, enquanto que as frações de membrana (MS e MT) demonstraram 95,0% e 94,4%, de sensibilidade e especificidade, respectivamente. Os resultados obtidos permitem indicar as frações de membranas como antígeno alternativo para o diagnóstico da estrongiloidíase humana.

Strongyloides venezuelensis is a parasitic nematode of rodents frequently used to obtain heterologous antigens for the immunological diagnosis of human strongyloidiasis. The aim of this study was to evaluate membrane fractions from S. venezuelensis for human strongyloidiasis immunodiagnosis. Soluble and membrane fractions were obtained in phosphate saline (SS and SM) and Tris-HCl (TS and TM) from filariform larvae of S. venezuelensis. Ninety-two serum samples (n = 92) were obtained from 20 strongyloidiasis patients (Group I), 32 from patients with other parasitic diseases (Group II), and 40 from healthy individuals (Group III), and were analyzed by enzyme-linked immunosorbent assay (ELISA). Soluble fractions (SS and TS) showed 90.0% sensitivity and 88.9% specificity, whereas the membrane fractions (SM and TM) showed 95.0% sensitivity and 94.4% specificity. The present results suggest the possible use of membrane fractions of S. venezuelensis as an alternative antigen for human strongyloidiasis immunodiagnosis.