Utility evaluation of two molecular methods for Leptospira spp. typing in human serum samples.

Most of the available genotyping methods were applied and evaluated in Leptospira isolates and only few of them in a relevant sample size of blood specimens but not of sera. The objective of this study was to evaluate the utility of one partial 16S rRNA gene sequencing assay (16S rRNA) and an optimized. Multilocus sequence typing scheme (MLST) for Leptospira typing directly in serum samples. Confirmed leptospirosis patients (n = 228) from Argentina (2005-2016) were randomly included. Septicemic-phase serum samples (n = 228) were studied by two genotyping methods. Available immune-phase serum samples of the included patients (n = 159) were studied by MAT to compare serological and molecular results. In culture-proven cases (n = 8), genotyping results between clinical samples and isolates were compared. Typing success rate (TSR) was 21.9% for 16S rRNA and 11.4% for MLST (full allelic profile) and a positive trend in both TSR during the study period was observed. Two species (L. interrogans and L. borgpertesenii) were identified by both methods and MLST assigned 8 different STs. The probable serogroups identified by MLST were coincident with the presumptive infecting serogroups identified by MAT, but with different frequencies. The three serogroups (Canicola, Sejroe and Icterohaemorrhagiae) most frequently identified by MAT were also genotyped by MLST. Typing results via 16S rRNA and MLST in clinical samples and isolates of culture-proven cases, were consistent except for one case. Performance of partial 16S rRNA gene sequencing assay and the optimized MLST scheme directly in sera may increase and improve the knowledge about species and serogroups causing human leptospirosis, especially in countries with low rates of culture sample collection or Leptospira isolation.

Coupling ELISA to smartphones for POCT of chronic and congenital Chagas disease.

Chagas disease (CD) affects about 7 million people worldwide, presents a large prevalence in Latin America, and is growing in the rest of the world, where congenital CD is the main mode of transmission. Point-of-care testing (POCT) methods are increasingly required to ease early diagnostics and increase treatment success. This work presents the development and validation of a smartphone-integrated ELISA-based POCT system for the detection of both chronic and congenital CD. Expensive and bulky equipment used for ELISA in conventional laboratories was replaced as follows. A miniaturized device was fabricated for incubation of commercial ELISA plates, achieving ∼±1 °C uniformity and stability. The ELISA plate reader was replaced by smartphone camera and image processing, comprising algorithms to account for variability sources and spatial light non-uniformity; thus, additional hardware like a dark-box is not required. The agreement between samples classified with this novel reading method vs. ELISA plate reader was found to be 99.7% and 95.4% for chronic and congenital CD, respectively. Furthermore, a smartphone application was designed and implemented to guide the user during the assay, provide connectivity, and access databases, facilitating patient monitoring and health-policy making. The whole system is aimed to be used as a practical diagnostic tool in primary health care settings, as well as to facilitate patients' follow-up to provide better treatment. Concerning the technology itself, the proposed POCT platform is versatile enough to be readily adapted for the detection of other infectious diseases.

A free customizable tool for easy integration of microfluidics and smartphones.

The integration of smartphones and microfluidics is nowadays the best possible route to achieve effective point-of-need testing (PONT), a concept increasingly demanded in the fields of human health, agriculture, food safety, and environmental monitoring. Nevertheless, efforts are still required to integrally seize all the advantages of smartphones, as well as to share the developments in easily adoptable formats. For this purpose, here we present the free platform appuente that was designed for the easy integration of microfluidic chips, smartphones, and the cloud. It includes a mobile app for end users, which provides chip identification and tracking, guidance and control, processing, smart-imaging, result reporting and cloud and Internet of Things (IoT) integration. The platform also includes a web app for PONT developers, to easily customize their mobile apps and manage the data of administered tests. Three application examples were used to validate appuente: a dummy grayscale detector that mimics quantitative colorimetric tests, a root elongation assay for pesticide toxicity assessment, and a lateral flow immunoassay for leptospirosis detection. The platform openly offers fast prototyping of smartphone apps to the wide community of lab-on-a-chip developers, and also serves as a friendly framework for new techniques, IoT integration and further capabilities. Exploiting these advantages will certainly help to enlarge the use of PONT with real-time connectivity in the near future.

Congenital chagas disease: Development and assessment of a specific IgM capture-based assay for diagnosis of transmission.

Transplacental transmission by Trypanosoma cruzi (T. cruzi) infection can be effectively treated if parasiticide drugs are administered as early as possible during childhood. Furthermore, an ideal situation would be to diagnose the infection near birth in order to avoid the loss of patients during the subsequent follow-up. These situation are desirable due to the maximum benefit of drugs in early stages which, consequently, implies a relevant contribution to eliminate mother-to-child transmission. However, available techniques for that purpose have limitations as being operator-dependent (microhematocrit), require several months follow-up (IgG detection) or specialized laboratories (PCR). In this study we propose to detect specific IgM antibodies (Ab) by developing a capture-based ELISA employing an improved antigen (Ag) to diagnose the transplacental transmission of T. cruzi, and in consequence, to enhance access to effective treatment. Firstly, a new chimera Ag (CP4) was obtained from the fusion of CP1 and CP3 protein, carrying FRA, SAPA, MAP, TSSAII/V/VI and TcD Ag from T. cruzi. Then, we optimized the assay by capturing IgM Ab with a polyclonal anti-IgM Ab and evaluating three Ag formulations to detect specific IgM bound. The formulations were formed as follows: i) F1: CP1 and CP3; ii) F2: CP1, CP3, B13 and P2ß; iii) F3: by CP4. Detection of Ab-binding Ag was carried out using an anti-His Ab since all Ag were expressed with a His-tag. The evaluation panel consisted of sera from vertically infected children under 1-year-old (6 younger than 15 days, 7 older) and samples from non-infected children of women with chronic Chagas Disease. The ELISA assay employing CP4 showed better performance with notable high sensitivity and specificity (92.3% and 93.9%, respectively). Positive and negative likelihood ratios of the test (15.2 and 0.082) suggest its potential clinical relevance in term of post-test probability of infection. In conclution, we developed a standardized and non-operator dependent test to detect specific anti-T. cruzi IgM Ab. Although increased sample size is needed for its validation, our results indicate that this capture-based technique employing CP4 Ag can certainly improve the diagnosis of connatal infection.

Survey of potential factors involved in the low frequency of CP5 and CP8 expression in Staphylococcus aureus isolates from mastitis of dairy cattle from Argentina, Chile, and Uruguay.

Staphylococcus aureus produces capsular polysaccharides (CPs) both in vivo and under defined culture conditions being serotypes 5 and 8 the most prevalent. S. aureus isolates that fail to produce CP5 or CP8 are defined as non-typeable (NT). Loss of capsule expression, however, may lead to S. aureus persistence in a chronically infected host. The prevalence of NT strains of S. aureus isolated from bovine mastitis varies according to the geographic origin of the strain. The aims of this work were to detect phenotypically and genotypically the capsular profile of 144 S. aureus isolated from bovine mastitis in Argentina, Chile, and Uruguay and explore the factors that are considered to be associated with capsule expression as presence of IS257, IScap, and agr typing of non-related collection. The detection of the IS257, IScap, cap genes, and agr typing was performed using PCR. The detection and quantification of capsular polysaccharide production were performed by ELISA assays. We found that 96% of the S. aureus isolates investigated carried cap5(8) genes but over 75% of strains do not express capsule in the three countries studied. However, only 6 isolates from Argentina carried the IScap element that totally suppressed the expression of the capsule, suggesting that other factors could influence on CP expression. Moreover, the agrI/NT association was statistically significant suggesting that this profile is a phenomenon observed not only in other parts of the world but also in our region.

Leptospirosis humana en Argentina: un esquema de análisis, 2014 / Human Leptospirosis in Argentina: An Analysis Scheme, 2014

INTRODUCCIÓN: En general, sólo existen estimaciones sobre el número de casos de leptospirosis en las Américas. La fuente más común son los Ministerios de Salud, tanto nacionales como provinciales, que proporcionan datos útiles sobre tendencias de incidencia de la leptospirosis, identificación de brotes y efectos de intervenciones gubernamentales. OBJETIVOS: Conocer la incidencia de casos de leptospirosis detectados por laboratorio durante 2014 en Argentina y generar un esquema de análisis de bases de datos de diferentes organismos nacionales para que sea repetido y difundido anualmente. MÉTODOS: Se analizaron las bases de datos del Sistema de Vigilancia Laboratorial (SIVILA)y del Instituto Nacional de Enfermedades Respiratorias (INER), teniendo en cuenta los casos notificados en 2014, según la fecha de inicio de síntomas. RESULTADOS: La incidencia de casos confirmados de leptospirosis en 2014 fue de 0,32/100 000 habitantes, mayormente detectados en las provincias de Santa Fe, Buenos Aires y Entre Ríos. Los serogrupos circulantes más prevalentes fueron Icterohaemorrhagiae, Sejroe y Canicola. CONCLUSIONES: Se resalta la utilidad de este tipo de estudios, que incentivan la búsqueda de casos, notificación y vigilancia de la leptospirosis, tanto para mejorar el conocimiento de la incidencia de la enfermedad y su distribución como para orientar las acciones hacia los lugares de mayor riesgo del país.

INTRODUCTION: In general, there are only estimates of the number of cases in the Americas. This data is commonly obtained by both national and provincial Ministries of Health which, despite data limitations, provide useful information on leptospirosis incidence trends, outbreak identification and effects of government interventions. OBJECTIVES: To know the incidence of leptospirosis cases detected by laboratory during 2014 in Argentina, and to generate an analysis scheme of databases from different national organisms to be repeated and diffused annually. METHODS: The SIVILA and INER databases were analyzed, taking into account the cases reported in 2014, according to the date of onset of symptoms. RESULTS: The incidence of confirmed leptospirosis cases in 2014 was 0.32/100 000 persons, mostly detected in Santa Fe, Buenos Aires and Entre Ríos provinces. The most prevalent circulating serogroups were Icterohaemorrhagiae, Sejroe and Canicola. CONCLUSIONS: These studies motivate the suspicion, notification and surveillance of leptospirosis and are useful both to improve the knowledge of the incidence of cases and their distribution, as well as to guide actions towards the most risky places in the country.

Development and assessment of a new cage-like particle adjuvant.

OBJECTIVES: To obtain and assess stable cage-like particles with low surface charge density, which can be prepared using a standardized, economic and scalable method. METHODS: To form these nanoparticles, the lipid composition and proportion as well the method were modified in relation to cage-like particles previously described elsewhere. Bovine albumin was used to compare ISPA performance with that of other adjuvants in mice and to assess stability. Adjuvant efficacy was analysed using a mouse model of Trypanosoma cruzi infection, which shows protection against an intracellular infection that needs a strong cellular response. KEY FINDINGS: The new particles were better in terms of level, kinetics and profile of humoral responses than Freund Adjuvant, aluminium hydroxide and Montanide TM ISA 206; they also tended to improve ISCOMATRIX™ performance. Particle size and adjuvant performance were conserved during the 6-month period assessed after preparation. In the model of Trypanosoma cruzi infection, mice immunized with ISPA and trans-sialidase developed high protection. CONCLUSIONS: The obtained nanoparticles were stable and outperformed the other assessed adjuvants in joining together the capacity of most adjuvants to enhance the immune response against specific antigen, to reduce the number of doses, to homogenize the response between individuals and to reach a balanced TH1/TH2 response.

Assessment of the potential utility of different regions of Streptococcus uberis adhesion molecule (SUAM) for mastitis subunit vaccine development.

Streptococcus uberis is one of the most prevalent pathogens causing clinical and subclinical mastitis worldwide. Among bacterial factors involved in intramammary infections caused by this organism, S. uberis adhesion molecule (SUAM) is one of the main virulence factors identified. This molecule is involved in S. uberis internalization to mammary epithelial cells through lactoferrin (Lf) binding. The objective of this study was to evaluate SUAM properties as a potential subunit vaccine component for prevention of S. uberis mastitis. B epitope prediction analysis of SUAM sequence was used to identify potentially immunogenic regions. Since these regions were detected all along the gene, this criterion did not allow selecting a specific region as a potential immunogen. Hence, four fractions of SUAM (-1fr, 2fr, 3fr and 4fr), comprising most of the protein, were cloned and expressed. Every fraction elicited a humoral immune response in mice as predicted by bioinformatics analysis. SUAM-1fr generated antibodies with the highest recognition ability towards SUAM native protein. Moreover, antibodies against SUAM-1fr produced the highest proportion of internalization inhibition of S. uberis to mammary epithelial cells. In conclusion, SUAM immunogenic and functionally relevant regions were identified and allowed to propose SUAM-1fr as a potential candidate for a subunit vaccine for S. uberis mastitis prevention.

Isolation and clinical sample typing of human leptospirosis cases in Argentina.

Leptospira typing is carried out using isolated strains. Because of difficulties in obtaining them, direct identification of infective Leptospira in clinical samples is a high priority. Multilocus sequence typing (MLST) proved highly discriminatory for seven pathogenic species of Leptospira, allowing isolate characterization and robust assignment to species, in addition to phylogenetic evidence for the relatedness between species. In this study we characterized Leptospira strains circulating in Argentina, using typing methods applied to human clinical samples and isolates. Phylogenetic studies based on 16S ribosomal RNA gene sequences enabled typing of 8 isolates (6 Leptospira interrogans, one Leptospira wolffii and one Leptospira broomii) and 58 out of 85 (68.2%) clinical samples (55 L. interrogans, 2 Leptospira meyeri, and one Leptospira kirschneri). MLST results for the L. interrogans isolates indicated that five were probably Canicola serogroup (ST37) and one was probably Icterohaemorrhagiae serogroup (ST17). Eleven clinical samples (21.6%), provided MLST interpretable data: five were probably Pyrogenes serogroup (ST13), four Sejroe (ST20), one Autumnalis (ST22) and one Canicola (ST37). To the best of our knowledge this study is the first report of the use of an MLST typing scheme with seven loci to identify Leptospira directly from clinical samples in Argentina. The use of clinical samples presents the advantage of the possibility of knowing the infecting strain without resorting to isolates. This study also allowed, for the first time, the characterization of isolates of intermediate pathogenicity species (L. wolffii and L. broomii) from symptomatic patients.

Preparation and characterization of a Staphylococcus aureus capsular polysaccharide-protein conjugate prepared by a low cost technique: a proof-of-concept study.

Staphylococcus aureus is a worldwide distributed pathogen that produces several diseases in many species and is the major cause of mastitis in dairy cows. S. aureus capsular polysaccharide 5 (CP5) has been widely proposed as a vaccine candidate since it is expressed in a high proportion of isolates from intramammary infections and is able to induce opsonophagocytic antibodies. However, to reach immunological properties, polysaccharides need to be coupled to carrier proteins. The aim of this study was to evaluate a conjugation method employing p-benzoquinone (PBQ), which was not previously reported for the development of vaccine components. Purified S. aureus CP5 was coupled to human serum albumin (HSA) with high efficiency, reaching a rate PS/protein of 0.5. Mice groups were immunized at days 0, 14, 28, and 42, with the conjugate (CP5-HSAPBQ), free CP5, or PBS, formulated with incomplete Freund adjuvant, and after 3 months, they were challenged with free CP5 to evaluate the memory response. IgG and IgM isotypes were measured on serum samples all along the experiment, and IgG subclasses were determined to analyze the humoral profile. In contrast to the response obtained with free CP5, CP5-HSAPBQ induced IgG titers of 1/238,900 after three doses and a memory response was observed after the challenge. Results indicate that immunization with CP5-HSAPBQ effectively induce a T-dependent immune response against CP5. Moreover, besides IgG2a was the main subtype obtained, the joint production of specific IgG1, IgG2b, and IgG3 types indicated a balanced humoral response. As p-benzoquinone conjugation of CPs to proteins is far less expensive and straightforward than other methods commonly used in vaccine preparations, the robust humoral response obtained using this method points out that this can be an interesting alternative to prepare S. aureus CP5 conjugate vaccines.

ß1-selective adrenoceptor antagonists increase plasma levels of anti-p2ß antibodies and decrease cardiac involvement in chronic progressive Chagas heart disease.

BACKGROUND: Studies indicate that antibodies cross-reacting with cardiac ß1 adrenergic receptors are likely to play a role in the development of chronic Chagas heart disease (CCHD). In parallel, clinical trials have shown that ß1 antagonist drugs exert beneficial effects in the prognosis of patients with CCHD. In a group of patients with CCHD undergoing therapy with ß1-blockers, we have now evaluated the levels of anti-p2ß antibodies and the severity of CCHD. METHODS: We performed a cross-sectional study in Trypanosoma cruzi seropositive patients categorized according to a standard CCHD classification. All individuals were subjected to a complete clinical examination. RESULTS: There was no association between CCHD stages, electrocardiographic conduction disturbances, and echocardiogram pathological signs with the levels of autoantibodies. However, when patients were analyzed according to selective cardio-ß1-blocker therapy, those receiving treatment had higher levels of anti-p2ß. Patients from CCHD stage III treated with combined therapy of cardio-ß1-selective blockers, enalapril, and statins, presented decreased cardiac involvement and lower score of risk of mortality than individuals from the same group who were not treated. CONCLUSIONS: Our results suggest that selective cardio-ß1-blockers might modify the autoantibody anti-p2ß levels, and that combined therapy in patients with stage III CCHD might be associated with lower cardiac involvement and risk score of mortality in patients with heart failure. Longitudinal studies will help to ascertain the proper role of ß1-blockers in the immunopathological processes underlying chronic Chagas disease.

Immune response of heifers against a Staphylococcus aureus CP5 whole cell and lysate vaccine formulated with ISCOM Matrix adjuvant.

Staphylococcus aureus is the most frequently isolated pathogen from bovine intramammary infections worldwide. Commercially available vaccines for mastitis control are composed either of S. aureus lysates or whole-cells formulated with traditional adjuvants. We recently showed the ability of a S. aureus CP5 whole-cell vaccine adjuvanted with ISCOM Matrix to increase specific antibodies production in blood and milk, improving opsonic capacity, compared with the same vaccine formulated with Al(OH)3. However, there is no information about the use of ISCOM Matrix for the formulation of bacterial lysates. The aim of this study was to characterize the innate and humoral immune responses induced by a S. aureus CP5 whole-cell or lysate vaccine, formulated with ISCOM Matrix after immunization of pregnant heifers. Both immunogens stimulated strong humoral immune responses in blood and milk, raising antibodies that increased opsonic capacity. Lysate formulation generated a higher and longer lasting antibody titer and stimulated a higher expression of regulatory and pro-inflammatory cytokines compared with the whole-cell vaccine.

Comparison of recombinant Trypanosoma cruzi peptide mixtures versus multiepitope chimeric proteins as sensitizing antigens for immunodiagnosis.

The aim of this work was to determine the best strategy to display antigens (Ags) on immunochemical devices to improve test selectivity and sensitivity. We comparatively evaluated five Trypanosoma cruzi antigenic recombinant peptides, chose the three more sensitive ones, built up chimeras bearing these selected Ags, and systematically compared by enzyme-linked immunosorbent assay the performance of the assortments of those peptides with that of the multiepitope constructions bearing all those peptides lineally fused. The better-performing Ags that were compared included peptides homologous to the previously described T. cruzi flagellar repetitive Ag (here named RP1), shed acute-phase Ag (RP2), B13 (RP5), and the chimeric recombinant proteins CP1 and CP2, bearing repetitions of RP1-RP2 and RP1-RP2-RP5, respectively. The diagnostic performances of these Ags were assessed for discrimination efficiency by the formula +OD/cutoff value (where +OD is the mean optical density value of the positive serum samples tested), in comparison with each other either alone, in mixtures, or as peptide-fused chimeras and with total parasite homogenate (TPH). The discrimination efficiency values obtained for CP1 and CP2 were 25% and 52% higher, respectively, than those of their individual-Ag mixtures. CP2 was the only Ag that showed enhanced discrimination efficiency between Chagas' disease-positive and -negative samples, compared with TPH. This study highlights the convenience of performing immunochemical assays using hybrid, single-molecule, chimeric Ags instead of peptide mixtures. CP2 preliminary tests rendered 98.6% sensitivity when evaluated with a 141-Chagas' disease-positive serum sample panel and 99.4% specificity when assessed with a 164-Chagas' disease-negative serum sample panel containing 15 samples from individuals infected with Leishmania spp.