ß-1,3-Glucan recognition by Acanthamoeba castellanii as a putative mechanism of amoeba-fungal interactions.

In this study, we conducted an in-depth analysis to characterize potential Acanthamoeba castellanii (Ac) proteins capable of recognizing fungal ß-1,3-glucans. Ac specifically anchors curdlan or laminarin, indicating the presence of surface ß-1,3-glucan-binding molecules. Using optical tweezers, strong adhesion of laminarin- or curdlan-coated beads to Ac was observed, highlighting their adhesive properties compared to controls (characteristic time τ of 46.9 and 43.9 s, respectively). Furthermore, Histoplasma capsulatum (Hc) G217B, possessing a ß-1,3-glucan outer layer, showed significant adhesion to Ac compared to a Hc G186 strain with an α-1,3-glucan outer layer (τ of 5.3 s vs τ 83.6 s). The addition of soluble ß-1,3-glucan substantially inhibited this adhesion, indicating the involvement of ß-1,3-glucan recognition. Biotinylated ß-1,3-glucan-binding proteins from Ac exhibited higher binding to Hc G217B, suggesting distinct recognition mechanisms for laminarin and curdlan, akin to macrophages. These observations hinted at the ß-1,3-glucan recognition pathway's role in fungal entrance and survival within phagocytes, supported by decreased fungal viability upon laminarin or curdlan addition in both phagocytes. Proteomic analysis identified several Ac proteins capable of binding ß-1,3-glucans, including those with lectin/glucanase superfamily domains, carbohydrate-binding domains, and glycosyl transferase and glycosyl hydrolase domains. Notably, some identified proteins were overexpressed upon curdlan/laminarin challenge and also demonstrated high affinity to ß-1,3-glucans. These findings underscore the complexity of binding via ß-1,3-glucan and suggest the existence of alternative fungal recognition pathways in Ac.IMPORTANCEAcanthamoeba castellanii (Ac) and macrophages both exhibit the remarkable ability to phagocytose various extracellular microorganisms in their respective environments. While substantial knowledge exists on this phenomenon for macrophages, the understanding of Ac's phagocytic mechanisms remains elusive. Recently, our group identified mannose-binding receptors on the surface of Ac that exhibit the capacity to bind/recognize fungi. However, the process was not entirely inhibited by soluble mannose, suggesting the possibility of other interactions. Herein, we describe the mechanism of ß-1,3-glucan binding by A. castellanii and its role in fungal phagocytosis and survival within trophozoites, also using macrophages as a model for comparison, as they possess a well-established mechanism involving the Dectin-1 receptor for ß-1,3-glucan recognition. These shed light on a potential parallel evolution of pathways involved in the recognition of fungal surface polysaccharides.

Prevalence of human cryptosporidiosis in the Americas: systematic review and meta-analysis.

Cryptosporidiosis is a disease caused by the Cryptosporidium spp parasite. As some species of Cryptosporidium have a wide host spectrum, the characterization of the pathogen at the species or genotype level is of great importance to define the sources of infection for humans and the potential for public health. This study investigated the diversity of the genus Cryptosporidium spp. in humans from all over the American continent and observed whether the method used to search for the parasite influenced the prevalence found in the Americas. This systematic review was carried out using the Pubmed, Science direct, Lilacs, Scielo, and Scopus databases with publications from January 1, 2010, to December 31, 2020. For data synthesis, the PRISMA flowchart was used and for the meta-analysis we used the MetaXL program. Of the selected publications, 57, 9 and 16 belonged to the region of South, Central and North America, respectively. The prevalence found for South, Central, and North America was 7%, 7%, and 8%, respectively, when analyzing publications that used only the microscopy method. When we analyzed the publications that used immunological and molecular methods, we found prevalences of 10%, 9%, and 21% for South, Central, and North America, respectively. The C. hominis subtype IbA10G2 was the most reported in the American continent, followed by subtype IeA11G3T3 and, for C. parvum, subtype IIaA15G2RI was the most reported. In conclusion, Cryptosporidium spp. is present throughout the American continent and its prevalence is higher when immunological and/or molecular methods are used, in addition to direct microscopic examination.

In vitro and in vivo efficacies of Dectin-1-Fc(IgG)(s) fusion proteins against invasive fungal infections.

Fungal infections have increased in the last years, particularly associated to an increment in the number of immunocompromised individuals and the emergence of known or new resistant species, despite the difficulties in the often time-consuming diagnosis. The controversial efficacy of the currently available strategies for their clinical management, apart from their high toxicity and severe side effects, has renewed the interest in the research and development of new broad antifungal alternatives. These encompass vaccines and passive immunization strategies with monoclonal antibodies (mAbs), recognizing ubiquitous fungal targets, such as fungal cell wall ß-1,3-glucan polysaccharides, which could be used in early therapeutic intervention without the need for the diagnosis at species level. As additional alternatives, based on the Dectin-1 great affinity to ß-1,3-glucan, our group developed broad antibody-like Dectin1-Fc(IgG)(s) from distinct subclasses (IgG2a and IgG2b) and compared their antifungal in vitro and passive immunizations in vivo performances. Dectin1-Fc(IgG2a) and Dectin1-Fc(IgG2b) demonstrated high affinity to laminarin and the fungal cell wall by ELISA, flow cytometry, and microscopy. Both Dectin-1-Fc(IgG)(s) inhibited Histoplasma capsulatum and Cryptococcus neoformans growth in a dose-dependent fashion. For Candida albicans, such inhibitory effect was observed with concentrations as low as 0.098 and 0.049 µg/ml, respectively, which correlated with the impairment of the kinetics and lengths of germ tubes in comparison to controls. Previous opsonization with Dectin-1-Fc(IgG)(s) enhanced considerably the macrophage antifungal effector functions, increasing the fungi macrophages interactions and significantly reducing the intraphagosome fungal survival, as lower CFUs were observed. The administration of both Dectin1-Fc(IgG)(s) reduced the fungal burden and mortality in murine histoplasmosis and candidiasis models, in accordance with previous evaluations in aspergillosis model. These results altogether strongly suggested that therapeutic interventions with Dectin-1-Fc(IgG)(s) fusion proteins could directly impact the innate immunity and disease outcome in favor of the host, by direct neutralization, opsonization, phagocytosis, and fungal elimination, providing interesting information on the potential of these new strategies for the control of invasive fungal infections. LAY SUMMARY: Mycoses have increased worldwide, and new efficient therapeutics are needed. Passive immunizations targeting universally the fungal cell would allow early interventions without the species-level diagnosis. Lectins with affinity to carbohydrates could be used to engineer 'antibody-like' strategies.

Recognition of Cell Wall Mannosylated Components as a Conserved Feature for Fungal Entrance, Adaptation and Survival Within Trophozoites of Acanthamoeba castellanii and Murine Macrophages.

Acanthamoeba castellanii (Ac) is a species of free-living amoebae (FLAs) that has been widely applied as a model for the study of host-parasite interactions and characterization of environmental symbionts. The sharing of niches between Ac and potential pathogens, such as fungi, favors associations between these organisms. Through predatory behavior, Ac enhances fungal survival, dissemination, and virulence in their intracellular milieu, training these pathogens and granting subsequent success in events of infections to more evolved hosts. In recent studies, our group characterized the amoeboid mannose binding proteins (MBPs) as one of the main fungal recognition pathways. Similarly, mannose-binding lectins play a key role in activating antifungal responses by immune cells. Even in the face of similarities, the distinct impacts and degrees of affinity of fungal recognition for mannose receptors in amoeboid and animal hosts are poorly understood. In this work, we have identified high-affinity ligands for mannosylated fungal cell wall residues expressed on the surface of amoebas and macrophages and determined the relative importance of these pathways in the antifungal responses comparing both phagocytic models. Mannose-purified surface proteins (MPPs) from both phagocytes showed binding to isolated mannose/mannans and mannosylated fungal cell wall targets. Although macrophage MPPs had more intense binding when compared to the amoeba receptors, the inhibition of this pathway affects fungal internalization and survival in both phagocytes. Mass spectrometry identified several MPPs in both models, and in silico alignment showed highly conserved regions between spotted amoeboid receptors (MBP and MBP1) and immune receptors (Mrc1 and Mrc2) and potential molecular mimicry, pointing to a possible convergent evolution of pathogen recognition mechanisms.

Molecular characterization of Cryptosporidium spp. obtained from fecal samples of immunosuppressed patients from Brazil.

BACKGROUND: Cryptosporidium spp. are pathogenic protozoans that play an important role in developing diseases in the elderly, children, and immunosuppressed individuals. METHODS: The objective of this study was to detect and genetically characterize Cryptosporidium spp. in kidney transplanted patients (n = 97 samples; group 1) and immunosuppressed individuals from an outpatient clinic suspected of having Cryptosporidium infection (n = 53 samples; group 2). All fecal samples were analyzed by parasitological stool examination, immunochromatographic test, and real-time polymerase chain reaction (real-time PCR). Cryptosporidium-positive samples were tested using nested PCR for the gp60 gene, followed by sequencing for subtype determination. RESULTS: Parasitological examination was negative in all Group 1, and positive in four Group 2 samples. Real-time PCR revealed Cryptosporidium in 13 samples: four in Group 1 (three C. hominis and one C. parvum) and nine in Group 2 (seven C. hominis, one C. parvum, and one mixed C. hominis/C. parvum). The immunochromatographic test was reactive in 11 samples (four in Group 1 and seven in Group 2). All 11 C. hominis isolates were identified as subtype IbA10G2 and one C. parvum as subtype IIbA15G2R1. All C. hominis belonged to subtype IbA10G2, which is recognized as the most prevalent and pathogenic subtype. CONCLUSIONS: This study showed, for the first time, that the presence of Cryptosporidium subtypes is considered more virulent in Brazilian transplanted kidney patients.

Prevalence of human cryptosporidiosis in the Americas: systematic review and meta-analysis

ABSTRACT Cryptosporidiosis is a disease caused by the Cryptosporidium spp parasite. As some species of Cryptosporidium have a wide host spectrum, the characterization of the pathogen at the species or genotype level is of great importance to define the sources of infection for humans and the potential for public health. This study investigated the diversity of the genus Cryptosporidium spp. in humans from all over the American continent and observed whether the method used to search for the parasite influenced the prevalence found in the Americas. This systematic review was carried out using the Pubmed, Science direct, Lilacs, Scielo, and Scopus databases with publications from January 1, 2010, to December 31, 2020. For data synthesis, the PRISMA flowchart was used and for the meta-analysis we used the MetaXL program. Of the selected publications, 57, 9 and 16 belonged to the region of South, Central and North America, respectively. The prevalence found for South, Central, and North America was 7%, 7%, and 8%, respectively, when analyzing publications that used only the microscopy method. When we analyzed the publications that used immunological and molecular methods, we found prevalences of 10%, 9%, and 21% for South, Central, and North America, respectively. The C. hominis subtype IbA10G2 was the most reported in the American continent, followed by subtype IeA11G3T3 and, for C. parvum, subtype IIaA15G2RI was the most reported. In conclusion, Cryptosporidium spp. is present throughout the American continent and its prevalence is higher when immunological and/or molecular methods are used, in addition to direct microscopic examination.

Case Report: Diagnosis and Assessment of Cure Approaches for Acute Schistosomiasis in Pre-School Children.

Acute schistosomiasis (AS) manifests with a broad spectrum of clinical features in pediatric populations. Diagnosis may be difficult in the absence of detectable numbers of eggs. As a result, new approaches may be required to achieve an accurate diagnosis. Optimal praziquantel (PZQ) treatment regimen for young children is debatable. Also, the post-treatment response is still poorly evaluated due to the lack of reliable markers. A group of 6 children (a toddler and 5 pre-school children) and one pre-adolescent were investigated for AS clinical manifestations and followed-up for two years after treatment. Ova detection was performed by Kato-Katz (KK) and presence of Schistosoma mansoni DNA was assessed by real-time PCR (rt-PCR) in stool samples. IgG and IgE anti-Schistosoma levels and urinary antigen were detected by ELISA and point-of-care circulating cathodic antigen (POC-CCA) testing in serum and urine, respectively. AS clinical symptoms were present in 5/7 (71.4%) of the infected children, and hypereosinophilia was detected in all of them. Ova detection and serology were positive in only 3/7 (44.9%) and 4/7 (57.1%), respectively. However, real-time PCR (rt-PCR) showed the presence of Schistosoma DNA in 6/7 (85.7%) of the cases, and urinary antigen was detected in all infected children. The long-term follow-up after treatment with three doses of PZQ (80mg/kg/dose), showed high cure rates (CR) as demonstrated by the DNA-based assay as well as reduced levels of side effects. CR based on urinary antigen detection ranged from 28.6 to 100%, being the highest CR due to double testing the 2-year post-treatment samples. The results suggest that high dose and repeated treatment with PZQ might be effective for AS in young children. Also, new laboratory markers should be considered to diagnosis and monitor the drug response.

Use of Cerebrospinal Fluid for the Diagnosis of Neuroinvasive Dengue, Zika, and Chikungunya: A 19-year systematic review.

INTRODUCTION: Cerebrospinal fluid analysis contributes to the diagnosis and neuropathogenesis of neuroinvasive arboviruses. Neurological complications caused by dengue, Zika, and chikungunya infections have high clinical relevance because of their high potential to cause death or neurological deficits. We aimed to evaluate the use of cerebrospinal fluid assays for diagnostic support in neurological disorders associated with dengue, chikungunya, and Zika infections. METHODS: A systematic review was carried out by searching the electronic databases LILACS, PubMed, Scopus, and Embase for articles written in English, Portuguese, or Spanish in the last 19 years. Published studies were reviewed using the terms "dengue," "Zika", "chikungunya", alone or in combination with "cerebrospinal fluid" in the period from 2000 to 2019. RESULTS: A total of 98,060 studies were identified; of these, 1.1% (1,041 studies, 58,478 cases) used cerebrospinal fluid assays for neurological investigations. The most frequent neurological disorders included encephalitis (41.4%), congenital syndromes (17%), and microcephaly associated with Zika virus infections (8.9%). Neuroinvasive disorders were confirmed in 8.03% of 58,478 cases by specific cerebrospinal fluid analyses. The main methods used were IgM-specific antibodies (66%) and reverse transcription-polymerase chain reaction (10%). The largest number of scientific papers (29%) originated from Brazil, followed by India (18.4%) and the United States (14.4%). CONCLUSIONS: Although cerebrospinal fluid analysis is of great importance for increasing neurological diagnostic accuracy and contributes to the early diagnosis of neuroinvasive dengue, chikungunya, and Zika infections, it is underused in routine laboratory investigations worldwide.

Description of optochin-resistant Streptococcus pneumoniae due to an uncommon mutation in the atpA gene and comparison with previously identified atpC mutants from Brazil.

Optochin susceptibility testing is a major assay used for presumptive identification of Streptococcus pneumoniae. Still, atypical optochin-resistant (Optr) pneumococci have been reported and this phenotype has been attributed to nucleotide substitutions in the genes coding for the F0F1ATPase. While substitutions in the atpC gene (c-subunit of ATPase) are more common and better characterized, data on mutations in the atpA (a-subunit) are still limited. We have characterized five Optr isolates presenting alterations in the atpA (Trp206Cys in four isolates and Trp206Ser in one isolate), constituting the first report of such mutations in Brazil. Most of the Optr isolates consisted of heterogeneous populations. Except for Opt MICs and the nucleotide changes in the atpA gene, Optr and Opts subpopulations originating from the same culture had identical characteristics. In addition, we compared phenotypic and genetic characteristics of these atpA mutants with those of atpC mutants previously identified in Brazil. No structural alterations were detected among predicted proteins, regardless of mutations in the coding gene, suggesting that, despite the occurrence of mutations, protein structures tend to be highly conserved, ensuring their functionalities. Phylogenetic analysis revealed that atypical Optr strains are true pneumococci and Opt resistance does not represent any apparent selective advantage for clinical isolates.

Neuroinvasive chikungunya in a liver transplant recipient.

The influence of chronic immunosuppression on the course of chikungunya virus (CHIKV) infection in recipients of solid organ transplantation (SOT) is still unsettled. Scarce data suggest that the course of CHIKV infection is generally benign in this population. In addition, the occurrence of severe atypical manifestations associated with CHIKV has not been well documented among SOT recipients. In this report, we describe a 64-year-old male liver transplant recipient who was admitted with fever, headache, arthralgia, left palpebral ptosis, mydriasis, and right hemiparesis. Cranial magnetic resonance imaging did not reveal any alteration suggestive of acute infection. Nevertheless, CHIKV was detected in the cerebrospinal fluid (CSF) with a real-time reverse transcriptase assay. Other PCR assays carried out in CSF were negative for HSV-1 and 2, cytomegalovirus, dengue virus (DENV), and Zika virus (ZIKV). CHIKV viremia was also detected while PCR assays for ZIKV and DENV in the blood were negative. ZIKV viruria was simultaneously present in this case. All neurologic manifestations waned within 2 weeks after the onset. This report shows that chikungunya must be considered in the differential diagnosis of acute neurologic disease in SOT recipients who live in or have recently traveled to endemic areas.

Use of Cerebrospinal Fluid for the Diagnosis of Neuroinvasive Dengue, Zika, and Chikungunya: A 19-year systematic review

Abstract INTRODUCTION: Cerebrospinal fluid analysis contributes to the diagnosis and neuropathogenesis of neuroinvasive arboviruses. Neurological complications caused by dengue, Zika, and chikungunya infections have high clinical relevance because of their high potential to cause death or neurological deficits. We aimed to evaluate the use of cerebrospinal fluid assays for diagnostic support in neurological disorders associated with dengue, chikungunya, and Zika infections. METHODS: A systematic review was carried out by searching the electronic databases LILACS, PubMed, Scopus, and Embase for articles written in English, Portuguese, or Spanish in the last 19 years. Published studies were reviewed using the terms "dengue," "Zika", "chikungunya", alone or in combination with "cerebrospinal fluid" in the period from 2000 to 2019. RESULTS: A total of 98,060 studies were identified; of these, 1.1% (1,041 studies, 58,478 cases) used cerebrospinal fluid assays for neurological investigations. The most frequent neurological disorders included encephalitis (41.4%), congenital syndromes (17%), and microcephaly associated with Zika virus infections (8.9%). Neuroinvasive disorders were confirmed in 8.03% of 58,478 cases by specific cerebrospinal fluid analyses. The main methods used were IgM-specific antibodies (66%) and reverse transcription-polymerase chain reaction (10%). The largest number of scientific papers (29%) originated from Brazil, followed by India (18.4%) and the United States (14.4%). CONCLUSIONS: Although cerebrospinal fluid analysis is of great importance for increasing neurological diagnostic accuracy and contributes to the early diagnosis of neuroinvasive dengue, chikungunya, and Zika infections, it is underused in routine laboratory investigations worldwide.

Protective Efficacy of Lectin-Fc(IgG) Fusion Proteins In Vitro and in a Pulmonary Aspergillosis In Vivo Model.

Aspergillosis cases by Aspergillus fumigatus have increased, along with fungal resistance to antifungals, urging the development of new therapies. Passive immunization targeting common fungal antigens, such as chitin and ß-glucans, are promising and would eliminate the need of species-level diagnosis, thereby expediting the therapeutic intervention. However, these polysaccharides are poorly immunogenic. To overcome this drawback, we developed the lectin-Fc(IgG) fusion proteins, Dectin1-Fc(IgG2a), Dectin1-Fc(IgG2b) and wheat germ agglutinin (WGA)-Fc(IgG2a), based on their affinity to ß-1,3-glucan and chitooligomers, respectively. The WGA-Fc(IgG2a) previously demonstrated antifungal activity against Histoplasma capsulatum, Cryptococcus neoformans and Candida albicans. In the present work, we evaluated the antifungal properties of these lectin-Fc(s) against A. fumigatus. Lectin-Fc(IgG)(s) bound in a dose-dependent manner to germinating conidia and this binding increased upon conidia germination. Both lectin-Fc(IgG)(s) displayed in vitro antifungal effects, such as inhibition of conidia germination, a reduced length of germ tubes and a diminished biofilm formation. Lectin-Fc(IgG)(s) also enhanced complement deposition on conidia and macrophage effector functions, such as increased phagocytosis and killing of fungi. Finally, administration of the Dectin-1-Fc(IgG2b) and WGA-Fc(IgG2a) protected mice infected with A. fumigatus, with a 20% survival and a doubled life-span of the infected mice, which was correlated to a fungal burden reduction in lungs and brains of treated animals. These results confirm the potential of lectin-Fc(IgGs)(s) as a broad-spectrum antifungal therapeutic.

Cerebrospinal fluid challenges for the diagnosis of herpes simplex infection in the central nervous system.

Herpes simplex virus (HSV) is a cause of a severe disease of the central nervous system (CNS) in humans. The demonstration of specific antibodies in the cerebrospinal fluid (CSF) may contribute to the retrospective neurological diagnosis. However, the commercial immunological tests for HSV infection are for use in serum samples. OBJECTIVE: The aim of the present study was to adapt a commercial kit anti-HSV IgG used for serum samples to be performed with a CSF sample. METHODS: Forty CSF specimens from 38 patients with suspected CNS HSV infection were serially diluted for detecting anti-HSV IgG by enzyme immunoassay (EIA). The same samples were also analyzed with the polymerase chain reaction (PCR). RESULTS: The sensitivity of EIA test for HSV was 5% (dilution 1:40) and 65% (dilution 1:2) in CSF, and HSV DNA PCR was 15%. The combined analysis of EIA (dilution 1:2) and PCR increased the sensitivity up to 72.5%. The inflammatory CSF was associated with positive HSV PCR. CONCLUSIONS: We demonstrated the importance to adapt serological anti-HSV IgG EIA test for CSF assays to increase the accuracy of the analysis, considering the low concentration of specific antibodies in CSF.

Cerebrospinal fluid challenges for the diagnosis of herpes simplex infection in the central nervous system / Desafios do exame do líquido cefalorraquidiano para o diagnóstico de infecção por herpes simplex no sistema nervoso central

Abstract Herpes simplex virus (HSV) is a cause of a severe disease of the central nervous system (CNS) in humans. The demonstration of specific antibodies in the cerebrospinal fluid (CSF) may contribute to the retrospective neurological diagnosis. However, the commercial immunological tests for HSV infection are for use in serum samples. Objective: The aim of the present study was to adapt a commercial kit anti-HSV IgG used for serum samples to be performed with a CSF sample. Methods: Forty CSF specimens from 38 patients with suspected CNS HSV infection were serially diluted for detecting anti-HSV IgG by enzyme immunoassay (EIA). The same samples were also analyzed with the polymerase chain reaction (PCR). Results: The sensitivity of EIA test for HSV was 5% (dilution 1:40) and 65% (dilution 1:2) in CSF, and HSV DNA PCR was 15%. The combined analysis of EIA (dilution 1:2) and PCR increased the sensitivity up to 72.5%. The inflammatory CSF was associated with positive HSV PCR. Conclusions: We demonstrated the importance to adapt serological anti-HSV IgG EIA test for CSF assays to increase the accuracy of the analysis, considering the low concentration of specific antibodies in CSF.

Resumo O vírus herpes simples (HSV) é um dos agentes causadores de uma doença grave no sistema nervoso central (SNC) em humanos. A detecção de anticorpos específicos no líquido cefalorraquidiano (LCR) pode contribuir para o diagnóstico neurológico retrospectivo. Entretanto, os testes imunológicos comerciais são para uso em amostras de soro. Objetivo: Adaptar um kit comercial sorológico anti-HSV IgG para ser utilizado no de LCR. Metodos: Quarenta amostras de LCR de 38 pacientes com suspeita de infecção por HSV no SNC foram diluídas pesquisa de anticorpos anti-HSV IgG pelo método imunoenzimático (EIA). Além disso, as mesmas amostras também foram analisadas por reação em cadeia da polimerase (PCR). Resultados: A sensibilidade do teste EIA para o HSV consistiu em 5% (diluição 1:40) e 65% (diluição 1:2) no LCR, e o PCR do DNA do HSV, 15%. A análise combinada de EIA (diluição 1:2) e PCR aumentou a sensibilidade para 72,5%. Houve associação entre presença do LCR inflamatório e PCR positiva para HSV. Conclusões: Demonstramos a importância na adaptação previa do teste sorológico anti-HSV IgG EIA para ensaios do no LCR, a fim de aumentar a acuracia da análise, considerando a baixa concentração de anticorpos específicos no LCR.

Dengue and chikungunya infection in neurologic disorders from endemic areas in Brazil.

OBJECTIVE: To detect the frequency of dengue virus (DENV), Chikungunya virus (CHIKV), and Zika virus (ZIKV) in adult patients with suspected viral infection of the CNS or postinfectious syndromes living in the state of Rio de Janeiro, Brazil. METHODS: DENV, CHIKV, and ZIKV RNA by reverse transcription PCR (RT-PCR) and specific IgM antibodies were investigated in 47 CSF and serum samples of 36 adult patients suspected with viral infection or postinfectious neurologic diseases. In addition, intrathecal synthesis of anti-DENV and anti-CHIKV IgG antibodies was also evaluated using a specific antibody index. RESULTS: Of the total group, neuroinvasive arbovirus was confirmed in 31% (11/36) of the cases: 6 (55%) by RT-PCR in CSF and/or serum, 1 (9%) by RT-PCR in CSF and/or serum and specific IgM in CSF, and 4 (36%) by specific IgM in CSF. Five cases had DENV infection, and 6 patients were positive for CHIKV. No sample amplified for ZIKV. In addition, 3 of 7 (42%) tested cases had intrathecal synthesis of DENV or CHIKV antibodies. The neurologic complications included encephalitis (7), Guillain-Barré syndrome (2), optic neuritis (1), neuromyelitis optica spectrum disorder (1), polyneuropathy, (1) and myelitis (1). CONCLUSION: DENV and CHIKV are a frequent cause of emerging and reemerging infections. It increases the number of cases with neurologic complications worldwide. We demonstrated that the combined use of molecular and immunologic tests in CSF/serum might support more widely the diagnosis of neurologic disorders caused by arbovirus in endemic areas. The detection of intrathecal synthesis of specific IgG antibodies may be promising for the retrospective diagnosis of neuroinvasive disorders caused by arbovirus.

Histoplasma capsulatum Glycans From Distinct Genotypes Share Structural and Serological Similarities to Cryptococcus neoformans Glucuronoxylomannan.

The cell wall is a ubiquitous structure in the fungal kingdom, with some features varying depending on the species. Additional external structures can be present, such as the capsule of Cryptococcus neoformans (Cn), its major virulence factor, mainly composed of glucuronoxylomannan (GXM), with anti-phagocytic and anti-inflammatory properties. The literature shows that other cryptococcal species and even more evolutionarily distant species, such as the Trichosporon asahii, T. mucoides, and Paracoccidioides brasiliensis can produce GXM-like polysaccharides displaying serological reactivity to GXM-specific monoclonal antibodies (mAbs), and these complex polysaccharides have similar composition and anti-phagocytic properties to cryptococcal GXM. Previously, we demonstrated that the fungus Histoplasma capsulatum (Hc) incorporates, surface/secreted GXM of Cn and the surface accumulation of the polysaccharide enhances Hc virulence in vitro and in vivo. In this work, we characterized the ability of Hc to produce cellular-attached (C-gly-Hc) and secreted (E-gly) glycans with reactivity to GXM mAbs. These C-gly-Hc are readily incorporated on the surface of acapsular Cn cap59; however, in contrast to Cn GXM, C-gly-Hc had no xylose and glucuronic acid in its composition. Mapping of recognized Cn GXM synthesis/export proteins confirmed the presence of orthologs in the Hc database. Evaluation of C-gly and E-gly of Hc from strains of distinct monophyletic clades showed serological reactivity to GXM mAbs, despite slight differences in their molecular dimensions. These C-gly-Hc and E-gly-Hc also reacted with sera of cryptococcosis patients. In turn, sera from histoplasmosis patients recognized Cn glycans, suggesting immunogenicity and the presence of cross-reacting antibodies. Additionally, C-gly-Hc and E-gly-Hc coated Cn cap59 were more resistant to phagocytosis and macrophage killing. C-gly-Hc and E-gly-Hc coated Cn cap59 were also able to kill larvae of Galleria mellonella. These GXM-like Hc glycans, as well as those produced by other pathogenic fungi, may also be important during host-pathogen interactions, and factors associated with their regulation are potentially important targets for the management of histoplasmosis.

The Advances in Molecular and New Point-of-Care (POC) Diagnosis of Schistosomiasis Pre- and Post-praziquantel Use: In the Pursuit of More Reliable Approaches for Low Endemic and Non-endemic Areas.

Like soil-transmitted helminth infections, schistosomiasis is an important neglected tropical disease (NTD) related to poverty with a major impact on public health in developing countries. Diagnosis of active infection is crucial for surveillance of controlled or post-elimination schistosomiasis areas. In addition, the use of conventional diagnostic tools in non-exposed populations (such as travelers) results in misdiagnoses in the prepatent period of infection. Also, the accuracy of standard tests applied in low-endemicity areas (LEAs) decreases after several rounds of treatment. We aimed to determine whether it would be necessary to replace schistosomiasis conventional diagnostic tests such as parasitological methods in LEAs. Also, we evaluate the use of new tools in non-endemic areas. Reliable, cheap and easy-to-use diagnostic tools are needed to respond to the demands of a new era of elimination and eradication of schistosomiasis. To this end, molecular diagnosis-including nucleic acid-based assays (loop-mediated isothermal amplification, polymerase chain reaction) and circulating cathodic and anodic antigen detection tests have become promising strategies. In this review, we attempt to address the use of alternative diagnostic tests for active infection detection and drug-monitoring after specific schistosomiasis treatment.

New insights into the detection and molecular characterization of Cryptosporidium with emphasis in Brazilian studies: a review.

Cryptosporidium spp. is a pathogenic protozoan present in the gastrointestinal tract of several hosts. This protozoan was originally classified as within the Coccidia Class and has recently been reclassified to gregarine based on studies that observed the evolutionary phases from the process of excision and sequencing of the 18S rRNA gene. Molecular biology techniques have become diagnostic tools and have also been used to understand the epidemiology of Cryptosporidium spp., since several species of this genus are very similar morphologically and morphometrically. Molecular techniques have been used in the identification of parasites, at the species and subtypes levels and to study disease transmission. The laboratory diagnosis of human cryptosporidiosis can be made by parasite detection methods, such as optical microscopy, antigens or genetic material detection, as well as serum antibodies raised to Cryptosporidium spp. Molecular methods were developed and allowed, not only an extensive revision of the taxonomy, but also an improvement in the laboratory diagnosis. In Brazil, there are few reports of Cryptosporidium spp. outbreaks in humans and all of them took place in nurseries. A few epidemiological studies developed in Brazil have used molecular methods for the detection of Cryptosporidium spp., as well as genotyping studies of their species and subtypes. The use of real-time PCR, together with microscopy and immunochromatography techniques, would result in a more precise diagnosis of cryptosporidiosis. The analysis of genotypes, subtypes and clonality of Cryptosporidium could be useful to understand and define the prognosis and severity of infections.

Unravelling the interactions of the environmental host Acanthamoeba castellanii with fungi through the recognition by mannose-binding proteins.

Free-living amoebae (FLAs) are major reservoirs for a variety of bacteria, viruses, and fungi. The most studied mycophagic FLA, Acanthamoeba castellanii (Ac), is a potential environmental host for endemic fungal pathogens such as Cryptococcus spp., Histoplasma capsulatum, Blastomyces dermatitides, and Sporothrix schenckii. However, the mechanisms involved in this interaction are poorly understood. The aim of this work was to characterize the molecular instances that enable Ac to interact with and ingest fungal pathogens, a process that could lead to selection and maintenance of possible virulence factors. The interaction of Ac with a variety of fungal pathogens was analysed in a multifactorial evaluation that included the role of multiplicity of infection over time. Fungal binding to Ac surface by living image consisted of a quick process, and fungal initial extrusion (vomocytosis) was detected from 15 to 80 min depending on the organism. When these fungi were cocultured with the amoeba, only Candida albicans and Cryptococcus neoformans were able to grow, whereas Paracoccidioides brasiliensis and Sporothrix brasiliensis displayed unchanged viability. Yeasts of H. capsulatum and Saccharomyces cerevisiae were rapidly killed by Ac; however, some cells remained viable after 48 hr. To evaluate changes in fungal virulence upon cocultivation with Ac, recovered yeasts were used to infect Galleria mellonella, and in all instances, they killed the larvae faster than control yeasts. Surface biotinylated extracts of Ac exhibited intense fungal binding by FACS and fluorescence microscopy. Binding was also intense to mannose, and mass spectrometry identified Ac proteins with affinity to fungal surfaces including two putative transmembrane mannose-binding proteins (MBP, L8WXW7 and MBP1, Q6J288). Consistent with interactions with such mannose-binding proteins, Ac-fungi interactions were inhibited by mannose. These MBPs may be involved in fungal recognition by amoeba and promotes interactions that allow the emergence and maintenance of fungal virulence for animals.

Performance of an Ultra-Sensitive Assay Targeting the Circulating Anodic Antigen (CAA) for Detection of Schistosoma mansoni Infection in a Low Endemic Area in Brazil.

Techniques with high sensitivity and specificity are required for an accurate diagnosis in low-transmission settings, where the conventional parasitological methods are insensitive. We determined the accuracy of an up-converting phosphor-lateral flow circulating anodic antigen (UCP-LF CAA) assay in urine and serum for Schistosoma mansoni diagnosis in low-prevalence settings in Ceará, Brazil, before and after praziquantel treatment. Clinical samples of a total of 258 individuals were investigated by UCP-LF CAA, point-of-care-circulating cathodic antigen (POC-CCA), soluble worm antigen preparation (SWAP)-ELISA and Kato-Katz (KK); a selection of 128 stools by real-time PCR technique. Three and 6-weeks after treatment, samples were collected and evaluated by detection Schistosoma circulating antigens (CAA and CCA). The UCP-LF CAA assays detected 80 positives (31%) with urine and 82 positives (31.8%) with serum. The urine POC-CCA and serum SWAP-ELISA assays detected 30 (11.6%) and 107 (40.7%) positives, respectively. The Kato-Katz technique revealed only 4 positive stool samples (1.6%). Among the 128 individuals with complete data records, 19 cases were identified by PCR (14.8%); Sensitivities and specificities of the UCP-LF CAA assays, determined versus a combined reference standard based on CCA/KK/PCR positivity, ranged from 60-68% to 68-77%, respectively. In addition only for comparative purposes, sensitivities of the different assays were determined vs. a comparative reference based on CAA/KK/PCR positivity, showing the highest sensitivity for the urine CAA assay (80%), followed by the serum CAA (70.9%), SWAP-ELISA (43.6%), PCR (34.5%), POC-CCA (29.1%), whilst triplicate Kato-Katz thick smears had a very low sensitivity (3.6%). CAA concentrations were higher in serum than in urine and were significantly correlated. There was a significant decrease in urine and serum CAA levels 3 and 6-weeks after treatment. The UCP-LF CAA assays revealed 33 and 28 S. mansoni-infected patients at the 3- and 6-week post-treatment follow-up, respectively. The UCP-LF CAA assays show high sensitivity for the diagnosis of S. mansoni in low-endemicity settings. It detects a considerably higher number of infections than microscopy, POC-CCA or PCR. Also it shows to be very useful for evaluating cure rates after treatment. Hence, the UCP-LF CAA assay is a robust and promising diagnostic approach in low-transmission settings.