An integrated lab-on-chip for rapid identification and simultaneous differentiation of tropical pathogens.

Tropical pathogens often cause febrile illnesses in humans and are responsible for considerable morbidity and mortality. The similarities in clinical symptoms provoked by these pathogens make diagnosis difficult. Thus, early, rapid and accurate diagnosis will be crucial in patient management and in the control of these diseases. In this study, a microfluidic lab-on-chip integrating multiplex molecular amplification and DNA microarray hybridization was developed for simultaneous detection and species differentiation of 26 globally important tropical pathogens. The analytical performance of the lab-on-chip for each pathogen ranged from 102 to 103 DNA or RNA copies. Assay performance was further verified with human whole blood spiked with Plasmodium falciparum and Chikungunya virus that yielded a range of detection from 200 to 4×105 parasites, and from 250 to 4×107 PFU respectively. This lab-on-chip was subsequently assessed and evaluated using 170 retrospective patient specimens in Singapore and Thailand. The lab-on-chip had a detection sensitivity of 83.1% and a specificity of 100% for P. falciparum; a sensitivity of 91.3% and a specificity of 99.3% for P. vivax; a positive 90.0% agreement and a specificity of 100% for Chikungunya virus; and a positive 85.0% agreement and a specificity of 100% for Dengue virus serotype 3 with reference methods conducted on the samples. Results suggested the practicality of an amplification microarray-based approach in a field setting for high-throughput detection and identification of tropical pathogens.

VereFlu™: an integrated multiplex RT-PCR and microarray assay for rapid detection and identification of human influenza A and B viruses using lab-on-chip technology.

Threatening sporadic outbreaks of avian influenza and the H1N1 pandemic of 2009 highlight the need for rapid and accurate detection and typing of influenza viruses. In this paper, we describe the validation of the VereFlu™ Lab-on-Chip Influenza Assay, which is based on the integration of two technologies: multiplex reverse transcription (RT)-PCR followed by microarray amplicon detection. This assay simultaneously detects five influenza virus subtypes, including the 2009 pandemic influenza A (H1N1), seasonal H1N1, H3N2, H5N1 and influenza B virus. The VereFlu™ assay was clinically validated in Singapore and compared against reference methods of real-time PCR, virus detection by immunofluorescence of cell cultures and sequencing. A sensitivity and specificity of 96.8% and 92.8%, respectively, was demonstrated for pandemic H1N1; 95.7% and 100%, respectively, for seasonal H1N1; 91.2% and 97.6%, respectively, for seasonal H3N2; 95.2% and 100%, respectively, for influenza B. Additional evaluations carried out at the World Health Organization (WHO) Collaborating Centre, Melbourne, Australia, confirmed that the test was able to reliably detect H5N1. This portable, fast time-to-answer (3 hours) device is particularly suited for diagnostic applications of detection, differentiation and identification of human influenza virus subtypes.

A critical role for lipophosphoglycan in proinflammatory responses of dendritic cells to Leishmania mexicana.

Recognition of pathogen-associated molecular patterns (PAMP) influences the response of dendritic cells (DC) and therefore development of innate and adaptive immunity. Different forms of Leishmania mexicana have distinct effects on DC, with promastigotes and amastigotes being activating and apparently neutral, respectively. We investigated whether stage-specific differences in surface composition might account for these distinct effects. Amastigotes and promastigotes lacking the lpg1 gene needed for lipophosphoglycan (LPG) biosynthesis could not activate DC in vitro. Genome-wide transcriptional profiling of DC infected with wild-type or mutant promastigotes or wild-type amastigotes revealed that wild-type promastigotes induce an inflammatory signature that is lacking in DC exposed to the other parasite forms. The proinflammatory response pattern was partly recovered by reconstitution of lpg1 expression in lpg1-/- parasites, and exposure to purified LPG increased the expression of MHC class II and CD86 on DC. Infection with wild-type but not lpg1-/- promastigotes increased the number of activated DC in draining lymph nodes, and this was correlated with lower early parasite burdens in wild-type-infected animals. These in vivo and in vitro results suggest an LPG-dependent activation of DC that contributes to host defense and agree with the notion that the parasites evolved under immune pressure to down-regulate PAMP expression in mammalian hosts.

A power law global error model for the identification of differentially expressed genes in microarray data.

BACKGROUND: High-density oligonucleotide microarray technology enables the discovery of genes that are transcriptionally modulated in different biological samples due to physiology, disease or intervention. Methods for the identification of these so-called "differentially expressed genes" (DEG) would largely benefit from a deeper knowledge of the intrinsic measurement variability. Though it is clear that variance of repeated measures is highly dependent on the average expression level of a given gene, there is still a lack of consensus on how signal reproducibility is linked to signal intensity. The aim of this study was to empirically model the variance versus mean dependence in microarray data to improve the performance of existing methods for identifying DEG. RESULTS: In the present work we used data generated by our lab as well as publicly available data sets to show that dispersion of repeated measures depends on location of the measures themselves following a power law. This enables us to construct a power law global error model (PLGEM) that is applicable to various Affymetrix GeneChip data sets. A new DEG identification method is therefore proposed, consisting of a statistic designed to make explicit use of model-derived measurement spread estimates and a resampling-based hypothesis testing algorithm. CONCLUSIONS: The new method provides a control of the false positive rate, a good sensitivity vs. specificity trade-off and consistent results with varying number of replicates and even using single samples.

A type I IFN-dependent pathway induced by Schistosoma mansoni eggs in mouse myeloid dendritic cells generates an inflammatory signature.

Schistosomes are helminth parasites that display a dual impact on the immune system of their hosts. Although the larval stage, also known as schistosomulum, appears to subvert the host defenses, the egg stage induces strong inflammatory reactions. Given the pivotal role of dendritic cells (DC) in initiating and regulating immune responses, we compared the distinct transcriptional programs induced in immature mouse DC by S. mansoni eggs or schistosomula. Although SLA abrogated the transcription of many genes implicated in DC functions, eggs caused myeloid DC to produce IFN-beta. Autocrine/paracrine signaling through the type I IFN receptor in response to eggs was necessary for the induction of known IFN-responsive genes and enhanced the synthesis of key inflammatory products. Taken as a whole, our data provide molecular insights into the immune evasion mechanism of schistosomula and suggest an unexpected role for type I IFN in the innate response to helminth eggs.