Shotgun proteomics of detergent-solubilized proteins from Trypanosoma evansi.

Trypanosoma evansi, the causative agent of surra, is the most prevalent pathogenic salivarian trypanosome and affects the majority of domesticated and wild animals in endemic regions. This work aimed to analyze detergent-solubilized T. evansi proteins and identify potential diagnostic biomarkers for surra. Triton X-114-extracted membrane-enriched proteins (MEP) of T. evansi bloodstream forms were analyzed using a gel-free technique (LC-ESI-MS/MS). 247 proteins were identified following the MS analysis of three biological and technical replicates. Two of these proteins were predicted to have a GPI-anchor, 100 (40%) were predicted to have transmembrane domains, and 166 (67%) were predicted to be membrane-bound based on at least one of six features: location (WolfPSORT, DeepLoc-2.0, Protcomp-9.0), transmembrane, GPI, and gene ontology. It was predicted that 76 (30%) of proteins had membrane evidence. Typical membrane proteins for each organelle were identified, among them ISG families (64, 65, and 75 kDa), flagellar calcium-binding protein, 24 kDa calflagin, syntaxins and oligosaccharyltransferase some of which had previously been studied in other trypanosomatids. T. evansi lacks singletons and exclusive orthologous groups, whereas three distinct epitopes have been identified. Data are available via ProteomeXchange with identifier PXD040594. SIGNIFICANCE: Trypanosoma evansi is a highly prevalent parasite that induces a pathological condition known as "surra" in various species of ungulates across five continents. The infection gives rise to symptoms that are not pathognomonic, thereby posing challenges in its diagnosis and leading to substantial economic losses in the livestock industry. A significant challenge arises from the absence of a diagnostic test capable of distinguishing between Trypanosoma equiperdum and T. evansi, both of which are implicated in equine diseases. Therefore, there is a pressing need to conduct research on the biochemistry of the parasite in order to identify proteins that could potentially serve as targets for differential diagnosis or therapeutic interventions.

Exoproteomic analysis of two MLST clade 2 strains of Clostridioides difficile from Latin America reveal close similarities.

Clostridioides difficile BI/NAP1/ribotype 027 is an epidemic hypervirulent strain found worldwide, including in Latin America. We examined the genomes and exoproteomes of two multilocus sequence type (MLST) clade 2 C. difficile strains considered hypervirulent: ICC-45 (ribotype SLO231/UK[CE]821), isolated in Brazil, and NAP1/027/ST01 (LIBA5756), isolated during a 2010 outbreak in Costa Rica. C. difficile isolates were cultured and extracellular proteins were analyzed using high-performance liquid chromatography-tandem mass spectrometry. Genomic analysis revealed that these isolates shared most of the gene composition. Only 83 and 290 NAP1/027 genes were considered singletons in ICC-45 and NAP1/027, respectively. Exoproteome analysis revealed 197 proteins, of which 192 were similar in both strains. Only five proteins were exclusive to the ICC-45 strain. These proteins were involved with catalytic and binding functions and indirectly interacted with proteins related to pathogenicity. Most proteins, including TcdA, TcdB, flagellin subunit, and cell surface protein, were overrepresented in the ICC-45 strain; 14 proteins, including mature S-layer protein, were present in higher proportions in LIBA5756. Data are available via ProteomeXchange with identifier PXD026218. These data show close similarity between the genome and proteins in the supernatant of two strains with hypervirulent features isolated in Latin America and underscore the importance of epidemiological surveillance of the transmission and emergence of new strains.

MALDI-TOF MS: An alternative approach for ribotyping Clostridioides difficile isolates in Brazil.

Clostridioides difficile is an important organism causing healthcare-associated infections. It has been documented that specific strains caused multiple outbreaks globally, and patients infected with those strains are more likely to develop severe C. difficile infection (CDI). With the appearance of a variant strain, BI/NAP1 ribotype 027, responsible for several outbreaks and high mortality rates worldwide, the epidemiology of the CDI changed drastically in the United States, Europe, and some Latin American countries. Although the epidemic strain 027 was not yet detected in Brazil, there are ribotypes exclusively found in the country, such as, 131, 132, 133, 135, 142 and 143, which are responsible for outbreaks in Brazilian hospitals and nursing homes. Although PCR-ribotyping is the most used method in epidemiology studies of C. difficile, it is not available in Brazil. This study aimed to develop and validate an in-house database for detecting C. difficile ribotypes, usually involved in CDI in Brazilian hospitals, by using MALDI-TOF MS. A database with 19 different ribotypes, 13 with worldwide circulation and 6 Brazilian-restricted, was created based on 27 spectra readings of each ribotype. After BioNumerics analysis, neighbor-joining trees revealed that spectra were distributed in clusters according to ribotypes, showing that MALDI-TOF MS could discriminate all 19 ribotypes. Moreover, each ribotype showed a different profile with 42 biomarkers detected in total. Based on their intensity and occurrence, 13 biomarkers were chosen to compose ribotype-specific profiles, and in silico analysis showed that most of these biomarkers were uncharacterized proteins or well-conserved peptides, such as ribosomal proteins. A double-blind assessment using the 13 biomarkers correctly assigned the ribotype in 73% of the spectra analyzed, with 94%-100% of correct hits for 027 and for Brazilian ribotypes. Although further analyses are required, our results show that MALDI-TOF MS might be a reliable, fast and feasible alternative for epidemiological surveillance of C. difficile in Brazil.

Dynamics of protein synthesis in the initial steps of strobilation in the model cestode parasite Mesocestoides corti (syn. vogae).

Mesocestoides corti (syn. vogae) is a useful model for developmental studies of platyhelminth parasites of the Cestoda class, such as Taenia spp. or Echinococcus spp. It has been used in studies to characterize cestode strobilation, i.e. the development of larvae into adult worms. So far, little is known about the initial molecular events involved in cestode strobilation and, therefore, we carried out a study to characterize newly synthesized (NS) proteins upon strobilation induction. An approach based on bioorthogonal noncanonical amino acid tagging and mass spectrometry was used to label, isolate, identify, and quantify NS proteins in the initial steps of M. corti strobilation. Overall, 121 NS proteins were detected exclusively after induction of strobilation, including proteins related to development pathways, such as insulin and notch signaling. Metabolic changes that take place in the transition from the larval stage to adult worm were noted in special NS protein subsets related to developmental processes, such as focal adhesion, cell leading edge, and maintenance of location. The data shed light on mechanisms underlying early steps of cestode strobilation and enabled identification of possible developmental markers. We also consider the use of developmental responsive proteins as potential drug targets for developing novel anthelmintics. BIOLOGICAL SIGNIFICANCE: Larval cestodiases are life-threatening parasitic diseases that affect both man and domestic animals worldwide. Cestode parasites present complex life cycles, in which they undergo major morphological and physiological changes in the transition from one life-stage to the next. One of these transitions occurs during cestode strobilation, when the mostly undifferentiated and non-segmented larval or pre-adult form develops into a fully segmented and sexually differentiated (strobilated) adult worm. Although the proteomes of bona fide larvae and strobialted adults have been previously characterized for a few cestode species, little is still known about the dynamic of protein synthesis during the early steps of cestode strobilation. Now, the assessment of newly synthesized (NS) proteins within the first 48 h of strobilation the model cestode M. corti allowed to shed light on molecular mechanisms that are triggered by strobilation induction. The functional analyses of this repertoire of over a hundred NS proteins pointed out to changes in metabolism and activation of classical developmental signaling pathways in early strobilation. Many of the identified NS proteins may become valuable cestode developmental markers and their involvement in vital processes make them also good candidate targets for novel anthelmintic drugs.

Unraveling oxidative stress response in the cestode parasite Echinococcus granulosus.

Cystic hydatid disease (CHD) is a worldwide neglected zoonotic disease caused by Echinococcus granulosus. The parasite is well adapted to its host by producing protective molecules that modulate host immune response. An unexplored issue associated with the parasite's persistence in its host is how the organism can survive the oxidative stress resulting from parasite endogenous metabolism and host defenses. Here, we used hydrogen peroxide (H2O2) to induce oxidative stress in E. granulosus protoescoleces (PSCs) to identify molecular pathways and antioxidant responses during H2O2 exposure. Using proteomics, we identified 550 unique proteins; including 474 in H2O2-exposed PSCs (H-PSCs) samples and 515 in non-exposed PSCs (C-PSCs) samples. Larger amounts of antioxidant proteins, including GSTs and novel carbonyl detoxifying enzymes, such as aldo-keto reductase and carbonyl reductase, were detected after H2O2 exposure. Increased concentrations of caspase-3 and cathepsin-D proteases and components of the 26S proteasome were also detected in H-PSCs. Reduction of lamin-B and other caspase-substrate, such as filamin, in H-PSCs suggested that molecular events related to early apoptosis were also induced. We present data that describe proteins expressed in response to oxidative stress in a metazoan parasite, including novel antioxidant enzymes and targets with potential application to treatment and prevention of CHD.

Intracellular changes of a swine tracheal cell line infected with a Mycoplasma hyopneumoniae pathogenic strain.

Mycoplasma hyopneumoniae is the etiological agent of enzootic pneumonia (EP), a widespread disease that causes major economic losses to the pig industry. The swine host response plays an important role in the outcome of M. hyopneumoniae infections. The whole proteome of newborn pig trachea (NPTr) epithelial cells infected with the M. hyopneumoniae pathogenic strain 7448 was analyzed using an LC-MS/MS approach to shed light on intracellular processes triggered in response to the pathogen. Overall, 853 swine protein species were identified, 156 of which were differentially represented in response to M. hyopneumoniae 7448 infection in comparison with non-infected control cells. These differentially represented proteins were categorized by function. Fifty-seven of them were assigned to the immune system and/or response to stimulus functional subcategories. Comparative expression analysis of these immune-related proteins in NPTr cells infected with attenuated or non-pathogenic mycoplasmas (M. hyopneumoniae J strain and M. flocculare, respectively) revealed proteins whose abundance was altered only in response to the pathogenic M. hyopneumoniae 7448 strain. Among these proteins, calcium homeostasis and endoplasmic reticulum stress-related biomarkers were detected, providing evidence of molecular mechanisms that might lead to swine cell apoptosis.

Lessons from the reestablishment of Public Health Laboratory activities in Puerto Rico after Hurricane Maria.

Public Health Laboratories (PHLs) in Puerto Rico did not escape the devastation caused by Hurricane Maria. We implemented a quality management system (QMS) approach to systematically reestablish laboratory testing, after evaluating structural and functional damage. PHLs were inoperable immediately after the storm. Our QMS-based approach began in October 2017, ended in May 2018, and resulted in the reestablishment of 92% of baseline laboratory testing capacity. Here, we share lessons learned from the historic recovery of the largest United States' jurisdiction to lose its PHL capacity, and provide broadly applicable tools for other jurisdictions to enhance preparedness for public health emergencies.

Differential responses to stress of two Mycoplasma hyopneumoniae strains.

Mycoplasma hyopneumoniae is a respiratory pathogen, causing porcine enzootic pneumonia. To survive in the porcine respiratory tract, M. hyopneumoniae must cope with both oxidative and heat stress imposed by the host. To get insights into M. hyopneumoniae stress responses and pathogenicity mechanisms, the protein profiles of two M. hyopneumoniae strains, pathogenic 7448 strain and non-pathogenic strain J, were surveyed under oxidative (OS) or heat (HS) stress. M. hyopneumoniae strains were submitted to OS (0.5% hydrogen peroxide) or HS (temperature shifts to 42 °C) conditions and protein profiling was carried out by LC-MS/MS and label-free quantitative analyses. Data are available via ProteomeXchange with identifier PXD012742. Qualitative and quantitative differences involving 40-60 M. hyopneumoniae proteins were observed for both strains when comparing bacteria exposed to OS or HS to non-treated controls. However, no differences in abundance were found in proteins classically related to stress responses, as peroxidases and chaperones, suggesting that these proteins would be constitutively present in both strains in the tested conditions. Interestingly, under stress conditions, more virulence-related proteins were detected in M. hyopneumoniae 7448 differentially represented proteins than in M. hyopneumoniae J, suggesting that stress may trigger a differential response of the corresponding genes, shared by both strains.

Differential secretome profiling of a swine tracheal cell line infected with mycoplasmas of the swine respiratory tract.

Mycoplasma hyopneumoniae and Mycoplasma flocculare are genetically similar. However, M. hyopneumoniae causes porcine enzootic pneumonia, while M. flocculare is a commensal bacterium. M. hyopneumoniae and M. flocculare do not penetrate their host cells, and secreted proteins are important for bacterium-host interplay. Thus, the secretomes of a swine trachea cell line (NPTr) infected with M. hyopneumoniae 7448 (a pathogenic strain), M. hyopneumoniae J (a non-pathogenic strain) and M. flocculare were compared to shed light in bacterium-host interactions. Medium from the cultures was collected, and secreted proteins were identified by a LC-MS/MS. Overall numbers of identified host and bacterial proteins were, respectively, 488 and 58, for NPTr/M. hyopneumoniae 7448; 371 and 67, for NPTr/M. hyopneumoniae J; and 203 and 81, for NPTr/M. flocculare. The swine cells revealed different secretion profiles in response to the infection with each M. hyopneumoniae strain or with M. flocculare. DAMPs and extracellular proteasome proteins, secreted in response to cell injury and death, were secreted by NPTr cells infected with M. hyopneumoniae 7448. All three mycoplasmas secreted virulence factors during NPTr infection, but M. hyopneumoniae 7448 secreted higher number of adhesins and hypothetical proteins, that may be related with pathogenicity. SIGNIFICANCE: The enzootic pneumonia caused by mycoplasmas of swine respiratory tract has economic loss consequences in pig industry due to antibiotic costs and pig weight loss. However, some genetically similar mycoplasmas are pathogenic while others, such as Mycoplasma hyopneumoniae and Mycoplasma flocculare, are non-pathogenic. Here, we conducted an infection assay between swine cells and pathogenic and non-pathogenic mycoplasmas to decipher secreted proteins during host-pathogen interaction. Mycoplasma response to cell infection was also observed. Our study provided new insights on secretion profile of swine cells in response to the infection with pathogenic and non-pathogenic mycoplasmas. It was possible to observe that pathogenic M. hyopneumoniae 7448 secreted known virulence factors and swine cells responded by inducing cell death. Otherwise, M. hyopneumoniae J and M. flocculare, non-pathogenic mycoplasmas, secreted a different profile of virulence factors in response to swine cells. Consequently, swine cells altered their secretome profile, but the changes were not sufficient to cause disease.

Comparative proteomics of two Mycoplasma hyopneumoniae strains and Mycoplasma flocculare identified potential porcine enzootic pneumonia determinants.

Mycoplasma hyopneumoniae and Mycoplasma flocculare are genetically similar bacteria, which coinhabit the porcine respiratory tract. These mycoplasmas share most of the known virulence factors, but, while M. hyopneumoniae causes porcine enzootic pneumonia (PEP), M. flocculare is a commensal species. To identify potential PEP determinants and provide novel insights on mycoplasma-host interactions, the whole cell proteomes of two M. hyopneumoniae strains, one pathogenic (7448) and other non-pathogenic (J), and M. flocculare were compared. A cell fractioning approach combined with mass spectrometry (LC-MS/MS) proteomics was used to analyze cytoplasmic and surface-enriched protein fractions. Average detection of ~ 50% of the predicted proteomes of M. hyopneumoniae 7448 and J, and M. flocculare was achieved. Many of the identified proteins were differentially represented in M. hyopneumoniae 7448 in comparison to M. hyopneumoniae J and M. flocculare, including potential PEP determinants, such as adhesins, proteases, and redox-balancing proteins, among others. The LC-MS/MS data also provided experimental validation for several genes previously regarded as hypothetical for all analyzed mycoplasmas, including some coding for proteins bearing virulence-related functional domains. The comprehensive proteome profiling of two M. hyopneumoniae strains and M. flocculare provided tens of novel candidates to PEP determinants or virulence factors, beyond those classically described.

Comparative proteomics of the larval and adult stages of the model cestode parasite Mesocestoides corti.

Mesocestoides corti is a widely used model for the study of cestode biology, and its transition from the larval tetrathyridium (TT) stage to the strobilated, adult worm (ST) stage can be induced and followed in vitro. Here, a proteomic approach was used to describe and compare M. corti TT and ST protein repertories. Overall, 571 proteins were identified, 238 proteins in TT samples and 333 proteins in ST samples. Among the identified proteins, 207 proteins were shared by TTs and STs, while 157 were stage-specific, being 31 exclusive from TTs, and 126 from STs. Functional annotation revealed fundamental metabolic differences between the TT and the ST stages. TTs perform functions related mainly to basic metabolism, responsible for growth and vegetative development by asexual reproduction. STs, in contrast, perform a wider range of functions, including macromolecule biosynthetic processes, gene expression and control pathways, which may be associated to its proglottization/segmentation, sexual differentiation and more complex physiology. Furthermore, the generated results provided an extensive list of cestode proteins of interest for functional studies in M. corti. Many of these proteins are novel candidate diagnostic antigens, and/or potential targets for the development of new and more effective antihelminthic drugs. BIOLOGICAL SIGNIFICANCE: Cestodiases are parasitic diseases with serious impact on human and animal health. Efforts to develop more effective strategies for diagnosis, treatment or control of cestodiases are impaired by the still limited knowledge on many aspects of cestode biology, including the complex developmental processes that occur in the life cycles of these parasites. Mesocestoides corti is a good experimental model to study the transition from the larval to the adult stage, called strobilation, which occur in typical cestode life-cycles. The performed proteomics approach provided large-scale identification and quantification of M. corti proteins. Many stage-specific or differentially expressed proteins were detected in the larval tetrathyridium (TT) stage and in the strobilated, adult worm (ST) stage. Functional comparative analyses of the described protein repertoires shed light on function and processes associated to specific features of both stages, such as less differentiation and asexual reproduction in TTs, and proglottization/segmentation and sexual differentiation in ST. Moreover, many of the identified stage-specific proteins are useful as cestode developmental markers, and are potential targets for development of novel diagnostic methods and therapeutic drugs for cestodiases.

Ribotypes associated with Clostridium difficile outbreaks in Brazil display distinct surface protein profiles.

Clostridium difficile is a spore-forming anaerobic intestinal pathogen that causes Clostridium difficile infection (CDI). C. difficile is the leading cause of toxin-mediated nosocomial antibiotic-associated diarrhea. The pathogenesis of CDI is attributed to two major virulence factors, TcdA and TcdB toxins, that cause the symptomatic infection. C. difficile also expresses a number of key proteins, including cell wall proteins (CWPs). S-layer proteins (SLPs) are CWPs that form a paracrystalline surface array that coats the surface of the bacterium. SLPs have a role in C. difficile binding to the gastrointestinal tract, but their importance in virulence need to be better elucidated. Here, we describe bottom-up proteomics analysis of surface-enriched proteins fractions obtained through glycine extraction of five C. difficile clinical isolates from Brazil using gel-based and gel-free approaches. We were able to identify approximately 250 proteins for each strain, among them SlpA, Cwp2, Cwp6, CwpV and Cwp84. Identified CWPs presented different amino acid coverage, which might suggest differences in post-translational modifications. Proteomic analysis of SLPs from ribotype 133, agent of C. difficile outbreaks in Brazil, revealed unique proteins and provided additional information towards in depth characterization of the strains causing CDI in Brazil.

Secretomes of Mycoplasma hyopneumoniae and Mycoplasma flocculare reveal differences associated to pathogenesis.

Mycoplasma hyopneumoniae and Mycoplasma flocculare cohabit the porcine respiratory tract. However, M. hyopneumoniae causes the porcine enzootic pneumonia, while M. flocculare is a commensal bacterium. Comparative analyses demonstrated high similarity between these species, which includes the sharing of all predicted virulence factors. Nevertheless, studies related to soluble secretomes of mycoplasmas were little known, although they are important for bacterial-host interactions. The aim of this study was to perform a comparative analysis between the soluble secreted proteins repertoires of the pathogenic Mycoplasma hyopneumoniae and its closely related commensal Mycoplasma flocculare. For that, bacteria were cultured in medium with reduced serum concentration and secreted proteins were identified by a LC-MS/MS proteomics approach. Altogether, 62 and 26 proteins were identified as secreted by M. hyopneumoniae and M. flocculare, respectively, being just seven proteins shared between these bacteria. In M. hyopneumoniae secretome, 15 proteins described as virulence factors were found; while four putative virulence factors were identified in M. flocculare secretome. For the first time, clear differences related to virulence were found between these species, helping to elucidate the pathogenic nature of M. hyopneumoniae to swine hosts. BIOLOGICAL SIGNIFICANCE: For the first time, the secretomes of two porcine respiratory mycoplasmas, namely the pathogenic M. hyopneumoniae and the commensal M. flocculare were compared. The presented results revealed previously unknown differences between these two genetically related species, some of which are associated to the M. hyopneumoniae ability to cause porcine enzootic pneumonia.

The Trypanosoma rangeli trypomastigote surfaceome reveals novel proteins and targets for specific diagnosis.

Sympatric distribution and sharing of hosts and antigens by Trypanosoma rangeli and Trypanosoma cruzi, the etiological agent of Chagas' disease, often incur in misdiagnosis and improper epidemiological inferences. Many secreted and surface proteins (SP) have been described as important antigens shared by these species. This work describes the T. rangeli surfaceome obtained by gel-free (LC-ESI-MS/MS) and gel-based (GeLC-ESI-MS/MS) proteomic approaches, and immunoblotting analyses and the comparison of these SP with T. cruzi. A total of 138 T. rangeli proteins and 343 T. cruzi proteins were obtained, among which, 42 and 157 proteins were exclusively identified in T. rangeli or T. cruzi trypomastigotes, respectively. Immunoblotting assays using sera from experimentally infected mice revealed a distinct band pattern for each species. MS/MS analysis of T. rangeli exclusive bands revealed two unique GP63-related proteins and flagellar calcium-binding protein. Also, a ~32kDa band composed of 12 distinct proteins was exclusively recognized by anti-T. cruzi serum. This highly sensitive proteomic assessment of surface proteins characterized the T. rangeli surfaceome, revealing several differences and similarities between these two parasites. The study reports new T. rangeli-specific proteins with promising use in differential diagnosis from T. cruzi. BIOLOGICAL SIGNIFICANCE: In this manuscript, we report the first proteomic analysis of the T. rangeli surface (surfaceome), a non-pathogenic parasite occurring in sympatry with T. cruzi, the etiological agent of Chagas disease. This comparative proteomic analysis was performed using high-throughput in-gel and gel-free proteomic approaches combined with immunoblotting, allowing us to identify new T. rangeli-specific proteins with promising use in differential serodiagnosis, among several other protein not previously reported for this taxon. Additionally, cross-recognition assays showed that T. cruzi surface proteins were recognized by heterologous serum (anti-T. rangeli) that strengthens the possibility of misdiagnosis of Chagas disease in humans and other mammals. Thus, this work provides new insights to understand the serological cross-reactivity between T. cruzi and T. rangeli, as well as, the identification of targets for specific T. rangeli diagnosis as revealed by the comparative surfaceome analysis. We strongly believe that this research is of importance to the readers of Journal of Proteomics since it provides new potential markers for diagnosis of both T. cruzi and T. rangeli parasites increasing the spectrum of specific targets for unambiguous diagnosis of T. rangeli and T. cruzi infections, besides describing new approaches to assess the trypanosomatids proteome.

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.

Prevalence of intestinal microsporidiosis in human immunodeficiency virus-infected patients with diarrhea in major United States cities.

To determine the prevalence of intestinal microsporidiosis in HIV-infected patients, we performed a prospective study of HIV-infected patients with diarrheal illnesses in three US hospitals and examined an observational database of HIV-infected patients in 10 US cities. Among 737 specimens from the three hospitals, results were positive for 11 (prevalence 1.5%); seven (64%) acquired HIV through male-to-male sexual contact, two (18%) through male-to-male sexual contact and injection drug use, and one (9%) through heterosexual contact; one (9%) had an undetermined mode of transmission. Median CD4 count within six months of diagnosis of microsporidiosis was 33 cells/microL (range 3 to 319 cells/microL). For the national observational database (n = 24,098), the overall prevalence of microsporidiosis was 0.16%. Prevalence of microsporidiosis among HIV-infected patients with diarrheal disease is low, and microsporidiosis is most often diagnosed in patients with very low CD4+ cell counts. Testing for microsporidia appears to be indicated, especially for patients with very low CD4+ cell counts.

Prevalence of intestinal microsporidiosis in Human Immunodeficiency Virus-infected patients with diarrhea in major United States cities

To determine the prevalence of intestinal microsporidiosis in HIV-infected patients, we performed a prospective study of HIV-infected patients with diarrheal illnesses in three US hospitals and examined an observational database of HIV-infected patients in 10 US cities. Among 737 specimens from the three hospitals, results were positive for 11 (prevalence 1.5 percent); seven (64 percent) acquired HIV through male-to-male sexual contact, two (18 percent) through male-to-male sexual contact and injection drug use, and one (9 percent) through heterosexual contact; one (9 percent) had an undetermined mode of transmission. Median CD4 count within six months of diagnosis of microsporidiosis was 33 cells/µL (range 3 to 319 cells/µL). For the national observational database (n = 24,098), the overall prevalence of microsporidiosis was 0.16 percent. Prevalence of microsporidiosis among HIV-infected patients with diarrheal disease is low, and microsporidiosis is most often diagnosed in patients with very low CD4+ cell counts. Testing for microsporidia appears to be indicated, especially for patients with very low CD4+ cell counts.

Para determinar a prevalência de microsporidiose intestinal em pacientes infectados pelo HIV foi realizado um estudo prospectivo em três hospitais dos Estados Unidos da América do Norte (EUA) e analizada uma base de dados nacional composta de dados coletados de pacientes infectados pelo HIV em 10 cidades dos EUA. De um total de 737 amostras de fezes de pacientes infectados pelo HIV que apresentavam diarréia, amostras de 11 pacientes (prevalência de 1,5 por cento) foram positivas para microsporídios. Todos os positivos eram do sexo masculino e, entre eles, sete (64 por cento) pacientes adquiriram a infecção pelo HIV através de relação homossexual, dois (18 por cento) através de relação sexual e drogas injetáveis e um (9 por cento) através de contato heterosexual, enquanto que em um paciente o modo de transmissão do HIV não foi determinado. A contagem média de linfócitos CD4 realizada até seis meses do diagnóstico de microsporidiose foi de 33 células/microlitro (3 a 319 células/microlitro). A análise da base de dados nacional (n = 24.098) mostrou uma prevalência de microsporidiose de 0,16 por cento. A prevalência de microsporidiose em pacientes HIV-positivos com diarréia é baixa. Entretando, como a microsporidiose é mais frequentemente diagnosticada em pacientes com contagens de CD4 muito baixas, a indicação de pesquisa de microsporídios é justificada, especialmente para estes pacientes.

The epidemiology of intestinal microsporidiosis in patients with HIV/AIDS in Lima, Peru.

We studied microsporidiosis in human immunodeficiency virus-positive patients in 2 Lima hospitals. Of 2652 patients, 66% were male, 6% received antiretroviral therapy (ART), and the median CD4 lymphocyte count was 131 cells/microL. Sixty-seven patients (3%) had microsporidiosis; stool specimens from 56 were identified as having Enterocytozoon bieneusi of 10 different genotypes. The 2 most common genotypes, Peru-1 and Peru-2, were not associated with significant increases in chronic diarrhea; other genotypes were associated with a 4-fold increased risk. Risk factors for E. bieneusi infection segregated by genotype: contact with duck or chicken droppings and lack of running water, flush toilet, or garbage collection with genotype Peru-1 and watermelon consumption with other genotypes. Shortened survival was associated with low CD4 lymphocyte count (P<.0001), no ART (P<.0001), and cryptosporidiosis (P=.004) but not with microsporidiosis (P=.48). Our data suggest the possibility of zoonotic E. bieneusi transmission and an association with poor sanitary conditions.

[Occurrence of Cryptosporidium sp in fecal samples of children less than 10 years old with clinical indication of Rotavirus]. / Ocorrência de Cryptosporidium sp em amostras fecais de crianças, menores de 10 anos de idade, com indicação clínica de Rotavírus.

The presence of oocysts of Cryptosporidium sp was investigated in 485 fecal samples of children with clinical indication of Rotavirus. No significant differences were observed between Cryptosporidium sp. and rotavirus occurrence and fecal consistency. Cryptosporidium sp also should be performed in the laboratory diagnosis of diarrheic episodes in children.

Analysis of four human microsporidian isolates by MALDI-TOF mass spectrometry.

Spores of four species of microsporidia isolated from humans were analyzed by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) and specific biomarkers were found for each. The microsporidia analyzed included three species, Encephalitozoon cuniculi, Encephalitozoon hellem, and Encephalitozoon intestinalis and the fourth organism is the recently described Brachiola algerae. Whole spores, spore shells, and soluble fractions were applied directly to the MALDI target without further purification steps. MALDI-TOF MS analysis of both whole spores and soluble fractions of the four isolates revealed a group of unique, characteristic, and reproducible spectral markers in the mass range of 2,000-8,000 Da. Statistical analysis of the averaged centroided masses uncovered two distinct sets of unique peptides or biomarkers, one originated from whole spores and the other from soluble fractions, that can differentiate the four microsporidian species studied. MALDI-TOF MS analysis of whole organisms is a rapid, sensitive, and specific option to characterize microsporidian isolates and has the potential for several applications in parasitology.