Comparative evaluation of lateral flow assays to diagnose chronic Trypanosoma cruzi infection in Bolivia.

Bolivia has the highest incidence of Chagas disease (CD) worldwide. Caused by the parasite Trypanasoma cruzi, CD is generally a chronic condition. Diagnosis is logistically and financially challenging, requiring at least two different laboratory-based serological tests. Many CD cases are missed; in Bolivia it is estimated just 6% of individuals chronically infected with T. cruzi get diagnosed. Achieving control on the way to elimination of CD requires a radical simplification of the current CD testing pathways, to overcome the barriers to accessing CD treatment. We aimed to generate unbiased performance data of lateral flow assays (LFAs) for T. cruzi infection in Bolivia, to evaluate their usefulness for improving T. cruzi diagnosis rates in a precise and efficient manner. This retrospective, laboratory-based, diagnostic evaluation study sought to estimate the sensitivity/specificity of 10 commercially available LFAs for T. cruzi, using the current CD diagnostic algorithm employed in Bolivia as the reference test method. All tests were blinded at the study site and performed by three operators. In total, 470 serum samples were tested, including 221 and 249 characterized as CD-positive/-negative, respectively. The LFAs were scored according to their relative importance using a decision-tree-based algorithm, with the mean decrease in Gini index as the scoring metric. The estimates of sensitivities ranged from 62.2-97.7% (95% confidence interval (CI) lower bound 55.0-94.7%); for specificities the range was 78.6-100% (95% CI lower bound 72.0-97.5%); 5/10 and 6/10 tests had sensitivity >90% and specificity >95%, respectively. Four LFAs showed high values of both sensitivity (93-95%) and specificity (97-99%). The agreement between 6 LFAs and the reference tests was almost perfect (Kappa 0.83-0.94). Most LFAs evaluated thus showed performances comparable with current laboratory-based diagnostic methods.

Comparative evaluation of four rapid diagnostic tests that detect human Trypanosoma cruzi-specific antibodies to support diagnosis of Chagas Disease in urban population of Argentina.

BACKGROUND: Chagas disease (CD), caused by the parasite Trypanosoma cruzi, is the most important endemic anthropozoonosis in Argentina. Since 2010, the World Health Organization has highlighted the urgent need to validate diagnostic systems that allow rapid detection of T. cruzi, infection in primary healthcare centers. Serological rapid diagnostic tests (RDTs) for T. cruzi, infection could be used to improve case management, as RDTs do not require specialized laboratories or highly trained staff to use them. We aimed to generate unbiased performance data of RDTs in Argentina, to evaluate their usefulness for improving T. cruzi, diagnosis rates. METHODS AND PRINCIPAL FINDINGS: This is a retrospective, laboratory-based, diagnostic evaluation study to estimate the clinical sensitivity/specificity of four commercially available RDTs for T. cruzi, using the Chagas disease diagnostic algorithm currently used in Argentina as the reference standard. In total, 400 serum samples were tested, 200 from individuals with chronic T. cruzi infection and 200 from individuals not infected with T. cruzi. All results were registered as the agreement of at least two operators who were blinded to the reference standard results. The sensitivity estimates ranged from 92.5-100% (95% confidence interval (CI) lower bound 87.9-98.2%); for specificity, the range was 76-96% (95% CI lower bound 69.5-92.3%). Most RDTs evaluated showed performances comparable with the reference standard method, showing almost perfect concordance (Kappa 0.76-0.92). CONCLUSIONS: Our study demonstrates that, under controlled laboratory conditions, commercially available RDTs for CD have a performance comparable to the Argentinian diagnostic algorithm, which is based on laboratory-based serological tests. For the next stage of our work, the RDTs will be evaluated in real-world settings.

Metabolic and chromosomal changes in a Bacillus subtilis whiA mutant.

IMPORTANCE: WhiA is a conserved DNA-binding protein that influences cell division in many Gram-positive bacteria and, in B. subtilis, also chromosome segregation. How WhiA works in Bacillus subtilis is unknown. Here, we tested three hypothetical mechanisms using metabolomics, fatty acid analysis, and chromosome confirmation capture experiments. This revealed that WhiA does not influence cell division and chromosome segregation by modulating either central carbon metabolism or fatty acid composition. However, the inactivation of WhiA reduces short-range chromosome interactions. These findings provide new avenues to study the molecular mechanism of WhiA in the future.

Laboratory evaluation of eleven rapid diagnostic tests for serological diagnosis of Chagas disease in Colombia.

BACKGROUND: Chagas disease is a public health challenge in Colombia, where only an estimated 1.2% of people at risk have accessed diagnosis, while less than 0.5% of affected people have obtained treatment. The development of simplified diagnostic algorithms would enable progress in access to diagnosis; however, the current diagnostic algorithm relies on at least two laboratory-based tests that require qualified personnel, processing equipment, and infrastructure, which are still generally lacking at the primary care level. Rapid diagnostic tests (RDTs) for Chagas disease could simplify diagnosis, but their performance in the epidemiological context of Colombia is not well known. METHODOLOGY: A retrospective analytical observational study of RDTs was performed to estimate the operational characteristics of 11 commercially available RDTs designed for in vitro detection of anti-T. cruzi IgG antibodies. The study was performed under controlled laboratory conditions using human serum samples. PRINCIPAL FINDINGS: Eleven RDTs were assessed, ten using 585 serum samples and one using 551 serum samples. Employing the current national diagnostic algorithm as a reference standard for serological diagnosis of chronic infection, the sensitivity of the assessed RDTs ranged from 75.5% to 99.0% (95% CI 70.5-100), while specificity ranged from 70.9% to 100% (95% CI 65.3-100). Most tests (7/11, 63.6%) had sensitivity above 90%, and almost all (10/11, 90.9%) had specificity above 90%. Five RDTs had both sensitivity and specificity above 90%. CONCLUSIONS/SIGNIFICANCE: The evaluation of these 11 commercially available RDTs under controlled laboratory conditions is a first step in the assessment of the diagnostic performance of RDTs in Colombia. As a next step, field studies will be conducted on available RDTs with sensitivity and specificity greater than 90% in this study, to evaluate performance in real world conditions, with the final goal to allow simplified diagnostic algorithms.

The CUIDA Chagas Project: towards the elimination of congenital transmission of Chagas disease in Bolivia, Brazil, Colombia, and Paraguay.

BACKGROUND: Mother-to-child transmission of Chagas disease (CD) has become a relevant problem in both endemic and non-endemic areas. METHODS: Description of the CUIDA Chagas Project - Communities United for Innovation, Development and Attention for Chagas disease'. RESULTS: Through innovative and strategic research, this project will provide improved diagnostic and treatment options as well as replicable implementation models that are adaptable to different contexts. CONCLUSIONS: By integrating test, treat and care actions for CD into primary health care practices, the burden of CD on people and health systems may be significantly reduced.

The CUIDA Chagas Project: towards the elimination of congenital transmission of Chagas disease in Bolivia, Brazil, Colombia, and Paraguay

ABSTRACT Background: Mother-to-child transmission of Chagas disease (CD) has become a relevant problem in both endemic and non-endemic areas. Methods: Description of the CUIDA Chagas Project - Communities United for Innovation, Development and Attention for Chagas disease'. Results: Through innovative and strategic research, this project will provide improved diagnostic and treatment options as well as replicable implementation models that are adaptable to different contexts. Conclusions: By integrating test, treat and care actions for CD into primary health care practices, the burden of CD on people and health systems may be significantly reduced.

The PASTA domains of Bacillus subtilis PBP2B strengthen the interaction of PBP2B with DivIB.

Bacterial cell division is mediated by a protein complex known as the divisome. Many protein-protein interactions in the divisome have been characterized. In this report, we analyse the role of the PASTA (Penicillin-binding protein And Serine Threonine kinase Associated) domains of Bacillus subtilis PBP2B. PBP2B itself is essential and cannot be deleted, but removing the PBP2B PASTA domains results in impaired cell division and a heat-sensitive phenotype. This resembles the deletion of divIB, a known interaction partner of PBP2B. Bacterial two-hybrid and co-immunoprecipitation analyses show that the interaction between PBP2B and DivIB is weakened when the PBP2B PASTA domains are removed. Combined, our results show that the PBP2B PASTA domains are required to strengthen the interaction between PBP2B and DivIB.

The Conserved DNA Binding Protein WhiA Influences Chromosome Segregation in Bacillus subtilis.

The DNA binding protein WhiA is conserved in Gram-positive bacteria and is present in the genetically simple cell wall-lacking mycoplasmas. The protein shows homology to eukaryotic homing endonucleases but lacks nuclease activity. WhiA was first characterized in streptomycetes, where it regulates the expression of key differentiation genes, including the cell division gene ftsZ, which is essential for sporulation. For Bacillus subtilis, it was shown that WhiA is essential when certain cell division genes are deleted. However, in B. subtilis, WhiA is not required for sporulation, and it does not seem to function as a transcription factor, despite its DNA binding activity. The exact function of B. subtilis WhiA remains elusive. We noticed that whiA mutants show an increased space between their nucleoids, and here, we describe the results of fluorescence microscopy, genetic, and transcriptional experiments to further investigate this phenomenon. It appeared that the deletion of whiA is synthetic lethal when either the DNA replication and segregation regulator ParB or the DNA replication inhibitor YabA is absent. However, WhiA does not seem to affect replication initiation. We found that a ΔwhiA mutant is highly sensitive for DNA-damaging agents. Further tests revealed that the deletion of parAB induces the SOS response, including the cell division inhibitor YneA. When yneA was inactivated, the viability of the synthetic lethal ΔwhiA ΔparAB mutant was restored. However, the nucleoid segregation phenotype remained. These findings underline the importance of WhiA for cell division and indicate that the protein also plays a role in DNA segregation.IMPORTANCE The conserved WhiA protein family can be found in most Gram-positive bacteria, including the genetically simple cell wall-lacking mycoplasmas, and these proteins play a role in cell division. WhiA has some homology with eukaryotic homing endonucleases but lacks nuclease activity. Because of its DNA binding activity, it is assumed that the protein functions as a transcription factor, but this is not the case in the model system B. subtilis The function of this protein in B. subtilis remains unclear. We noticed that a whiA mutant has a mild chromosome segregation defect. Further studies of this phenomenon provided new support for a functional role of WhiA in cell division and indicated that the protein is required for normal chromosome segregation.

Effect of Genome Position on Heterologous Gene Expression in Bacillus subtilis: An Unbiased Analysis.

A fixed gene copy number is important for the in silico construction of engineered synthetic networks. However, the copy number of integrated genes depends on their genomic location. This gene dosage effect is rarely addressed in synthetic biology. Two studies in Escherichia coli presented conflicting data on the impact of gene dosage. Here, we investigate how genome location and gene orientation influences expression in Bacillus subtilis. An important difference with the E. coli studies is that we used an unbiased genome integration approach mediated by random transposon insertion. We found that there is a strong gene dosage effect in fast growing B. subtilis cells, which can amount to a 5-fold difference in gene expression. In contrast, gene orientation with respect to DNA replication direction does not influence gene expression. Our study shows that gene dosage should be taken into account when designing synthetic circuits in B. subtilis and presumably other bacteria.

Neotropical Andes hot springs harbor diverse and distinct planktonic microbial communities.

Microbial explorations of hot springs have led to remarkable discoveries and improved our understanding of life under extreme conditions. The Andean Mountains harbor diverse habitats, including an extensive chain of geothermal heated water sources. In this study, we describe and compare the planktonic microbial communities present in five high-mountain hot springs with distinct geochemical characteristics, at varying altitudes and geographical locations in the Colombian Andes. The diversity and structure of the microbial communities were assessed by pyrosequencing the V5 - V6 region of the 16S rRNA gene. The planktonic communities varied in terms of diversity indexes and were dominated by the bacterial phyla Proteobacteria, Aquificae, Chloroflexi, Cyanobacteria, Firmicutes, Nitrospirae, and Thermotogae, with site-specific bacterial taxa also observed in some cases. Statistical analyses showed that these microbial communities were distinct from one another and that they clustered in a manner consistent with physicochemical parameters of the environment sampled. Multivariate analysis suggested that pH and sulfate were among the main variables influencing population structure and diversity. The results show that despite their geographical proximity and some shared geochemical characteristics, there were few shared operational taxonomic units (OTUs) and that community structure was influenced mainly by environmental factors that have resulted in different microbial populations.

Genes para proteorodopsinas en aguas termales de los Andes colombianos / Genes for proteorhodopsins in hot springs in the Colombian Andes

La identificación de genes para proteorodopsinas en aguas termales amplía el rango de ecosistemas en los cuales se encuentran estos genes, los que codifican para bombas de protones y pueden estar implicados en la productividad de estas comunidades microbianas acuáticas.

Genes para proteorodopsinas en aguas termales de los Andes colombianos / Genes for proteorhodopsins in hot springs in the Colombian Andes

La identificación de genes para proteorodopsinas en aguas termales amplía el rango de ecosistemas en los cuales se encuentran estos genes, los que codifican para bombas de protones y pueden estar implicados en la productividad de estas comunidades microbianas acuáticas. (AU)

Proteorhodopsin-like genes present in thermoacidophilic high-mountain microbial communities.

Proteorhodopsin (PR) sequences were PCR amplified from three Andean acidic hot spring samples. These sequences were similar to freshwater and marine PRs and they contained residues indicative of proton-pumping activity and of proteins that absorb green light; these findings suggest that PRs might contribute to cellular metabolism in these habitats.

In-depth characterization via complementing culture-independent approaches of the microbial community in an acidic hot spring of the Colombian Andes.

The microbial community of a Colombian high mountain hot spring, El Coquito, was analyzed using three different culture-independent assessments of 16S ribosomal RNA genes: clone libraries, pyrosequencing of the V5-V6 hypervariable region, and microarray. This acidic spring had a diverse community composed mainly of Bacteria that shared characteristics with those from other hot springs and extreme acidic environments. The microbial community was dominated by Proteobacteria, Firmicutes, and Planctomycetes and contained chemotrophic bacteria potentially involved in cycling of ferrous and sulfur-containing minerals and phototrophic organisms, most of which were eukaryotic micro-algae. Despite the presence of a large proportion of novel, unclassified sequences, the taxonomic profiles obtained with each strategy showed similarities at higher taxonomic levels. However, some groups, such as Spirochaetes and Aquificae, were identified using only one methodology, and more taxa were detected with the gene array, which also shared more groups with the pyrosequencing data. Overall, the combined use of different approaches provided a broader view of the microbial community in this acidic hot spring.