Molecular diagnostics of intestinal parasites in returning travellers

A new diagnostic strategy was assessed for the routine diagnosis of intestinal parasites in returning travellers and immigrants. Over a period of 13 months, unpreserved stool samples, patient characteristics and clinical data were collected from those attending a travel clinic. Stool samples were analysed on a daily basis by microscopic examination and antigen detection (i.e. care as usual), and compared with a weekly performed multiplex real-time polymerase chain reaction (PCR) analysis on Entamoeba histolytica, Giardia lamblia, Cryptosporidium and Strongyloides stercoralis. Microscopy and antigen assays of 2,591 stool samples showed E. histolytica, G. lamblia, Cryptosporidium and S. stercoralis in 0.3, 4.7, 0.5 and 0.1% of the cases, respectively. These detection rates were increased using real-time PCR to 0.5, 6.0, 1.3 and 0.8%, respectively. The prevalence of ten additional pathogenic parasite species identified with microscopy was, at most, 0.5%. A pre-selective decision tree based on travel history or gastro-intestinal complaints could not be made. With increased detection rates at a lower workload and the potential to extend with additional parasite targets combined with fully automated DNA isolation, molecular high-throughput screening could eventually replace microscopy to a large extent

A salting out and resin procedure for extracting Schistosoma mansoni DNA from human urine samples

Submitted by Nuzia Santos ([email protected]) on 2012-09-27T14:31:36Z No. of bitstreams: 1 36.2010.pdf: 789056 bytes, checksum: 0a4282ac34d4c6aef08223da45e0f126 (MD5)Made available in DSpace on 2012-09-27T14:31:36Z (GMT). No. of bitstreams: 1 36.2010.pdf: 789056 bytes, checksum: 0a4282ac34d4c6aef08223da45e0f126 (MD5) Previous issue date: 2010Fundação Oswaldo Cruz. Laboratório de Esquistossomose. Belo Horizonte, MG, BrasilFundação Oswaldo Cruz. Laboratório de Imunologia Celular e Molecular. Belo Horizonte, MG, BrasilFundação Oswaldo Cruz. Laboratório de Imunologia Celular e Molecular. Belo Horizonte, MG, Brasil/ Universidade Federal de Ouro Preto. Escola de Farmácia. Laboratório de Pesquisas Clínicas. Ouro Preto, MG, BraziBackground: In this paper a simple and cheap salting out and resin (InstaGene matrix® resin - BioRad) DNA extraction method from urine for PCR assays is introduced. The DNA of the fluke Schistosoma mansoni was chosen as the target since schistosomiasis lacks a suitable diagnostic tool which is sensitive enough to detect low worm burden. It is well known that the PCR technique provides high sensitivity and specificity in detecting parasite DNA. Therefore it is of paramount importance to take advantage of its excellent performance by providing a simple to handle and reliable DNA extraction procedure, which permits the diagnosis of the disease in easily obtainable urine samples. Findings: The description of the extraction procedure is given. This extraction procedure was tested for reproducibility and efficiency in artificially contaminated human urine samples. The reproducibility reached 100%, showing positive results in 5 assay repetitions of 5 tested samples each containing 20 ng DNA/5 ml. The efficiency of the extraction procedure was also evaluated in a serial dilution of the original 20 ng DNA/5 ml sample. Detectable DNA was extracted when it was at a concentration of 1.28 pg DNA/mL, revealing the high efficiency of this procedure. Conclusions: This methodology represents a promising tool for schistosomiasis diagnosis utilizing a bio-molecular technique in urine samples which is now ready to be tested under field conditions and may be applicable to the diagnosis of other parasitic disease

Multi-parallel qPCR provides increased sensitivity and diagnostic breadth for gastrointestinal parasites of humans: field-based inferences on the impact of mass deworming

BACKGROUND: Although chronic morbidity in humans from soil transmitted helminth (STH) infections can be reduced by anthelmintic treatment, inconsistent diagnostic tools make it difficult to reliably measure the impact of deworming programs and often miss light helminth infections. METHODS: Cryopreserved stool samples from 796 people (aged 2-81 years) in four villages in Bungoma County, western Kenya, were assessed using multi-parallel qPCR for 8 parasites and compared to point-of-contact assessments of the same stools by the 2-stool 2-slide Kato-Katz (KK) method. All subjects were treated with albendazole and all Ascaris lumbricoides expelled post-treatment were collected. Three months later, samples from 633 of these people were re-assessed by both qPCR and KK, re-treated with albendazole and the expelled worms collected. RESULTS: Baseline prevalence by qPCR (n = 796) was 17 % for A. lumbricoides, 18 % for Necator americanus, 41 % for Giardia lamblia and 15% for Entamoeba histolytica. The prevalence was <1% for Trichuris trichiura, Ancylostoma duodenale, Strongyloides stercoralis and Cryptosporidium parvum. The sensitivity of qPCR was 98% for A. lumbricoides and N. americanus, whereas KK sensitivity was 70% and 32%, respectively. Furthermore, qPCR detected infections with T. trichiura and S. stercoralis that were missed by KK, and infections with G. lamblia and E. histolytica that cannot be detected by KK. Infection intensities measured by qPCR and by KK were correlated for A. lumbricoides (r = 0.83, p < 0.0001) and N. americanus (r = 0.55, p < 0.0001). The number of A. lumbricoides worms expelled was correlated (p < 0.0001) with both the KK (r = 0.63) and qPCR intensity measurements (r = 0.60). CONCLUSIONS: KK may be an inadequate tool for stool-based surveillance in areas where hookworm or Strongyloides are common or where intensity of helminth infection is low after repeated rounds of chemotherapy. Because deworming programs need to distinguish between populations where parasitic infection is controlled and those where further treatment is required, multi-parallel qPCR (or similar high throughput molecular diagnostics) may provide new and important diagnostic information

Confocal Laser Scanning Microscopy for Detection of Schistosoma mansoni Eggs in the Gut of Mice

Background: The gold standard for diagnosing Schistosoma mansoni infections is the detection of eggs from stool or biopsy specimens. The viability of collected eggs can be tested by the miracidium hatching procedure. Direct detection methods are often limited in patients with light or early infections, whereas serological tests and PCR methods fail to differentiate between an inactive and persistent infection and between schistosomal species. Recently, confocal laser scanning microscopy (CLSM) has been introduced as a diagnostic tool in several fields of medicine. In this study we evaluated CLSM for the detection of viable eggs of S. mansoni directly within the gut of infected mice. Methodology/Principal Findings: The confocal laser scanning microscope used in this study is based on the Heidelberg Retina Tomograph II scanning laser system in combination with the Rostock Cornea Module (image modality 1) or a rigid endoscope (image modality 2). Colon sections of five infected mice were examined with image modalities 1 and 2 for schistosomal eggs. Afterwards a biopsy specimen was taken from each colon section and examined by bright-field microscopy. Visualised eggs were counted and classified in terms of viability status. Conclusions/Significance: We were able to show that CLSM visualises eggs directly within the gut and permits discrimination of schistosomal species and determination of egg viability. Thus, CLSM may be a suitable non-invasive too

Quantitative Detection of Schistosoma japonicum Cercariae in Water by Real-Time PCR

In China alone, an estimated 30 million people are at risk of schistosomiasis, caused by the Schistosoma japonicum parasite. Disease has re-emerged in several regions that had previously attained transmission control, reinforcing the need for active surveillance. The environmental stage of the parasite is known to exhibit high spatial and temporal variability, and current detection techniques rely on a sentinel mouse method which has serious limitations in obtaining data in both time and space. Here we describe a real-time PCR assay to quantitatively detect S. japonicum cercariae in laboratory samples and in natural water that has been spiked with known numbers of S. japonicum. Multiple primers were designed and assessed, and the best performing set, along with a TaqMan probe, was used to quantify S. japonicum. The resulting assay was selective, with no amplification detected for Schistosoma mansoni, Schistosoma haematobium, avian schistosomes nor organisms present in non-endemic surface water samples. Repeated samples containing various concentrations of S. japonicum cercariae showed that the real-time PCR method had a strong linear correlation (R2 = 0.921) with light microscopy counts, and the detection limit was below the DNA equivalent of half of one cercaria. Various cercarial concentrations spiked in 1 liter of natural water followed by a filtration process produced positive detection from 93% of samples analyzed. The real-time PCR method performed well quantifying the relative concentrations of various spiked samples, although the absolute concentration estimates exhibited high variance across replicated samples. Overall, the method has the potential to be applied to environmental water samples to produce a rapid, reliable assay for cercarial location in endemic areas

Real-time PCR Demonstrates Ancylostoma duodenale Is a Key Factor in the Etiology of Severe Anemia and Iron Deficiency in Malawian Pre-school Children

Hookworm infections are a major cause of childhood anemia and iron deficiency. Two hookworm species exist of which Ancylostoma duodenale is the less common, yet causing more blood loss than Necator americanus. Although species differentiation and quantification are both of clinical importance, these are often not performed as the technique is complex and laborious using microscopy. Multiplex real-time PCR is a novel diagnostic tool which allows hookworm species differentiation and infection quantification. We applied this test in 830 stool samples of Malawian children with and without severe anemia. The prevalence of hookworm infections was high. A. duodenale was unexpectedly more prevalent than N. americanus. A. duodenale infections were associated with increased risk for severe anemia and iron deficiency, both of which increased with infection load. The study identifies a need for the quantitative screening of species-specific hookworm infections, which readily can be achieved by real-time-PCR. A. duodenale was independently associated with severe anemia and iron deficiency in our study population

Interleukin-10 enhances the intestinal epithelial barrier in the presence of corticosteroids through p38 MAPK activity in Caco-2 monolayers : a possible mechanism for steroid responsiveness in ulcerative colitis

Altres ajuts: 2012 Spanish Gastroenterological Association i CIBER G0034Glucocorticosteroids are the first line therapy for moderate-severe flare-ups of ulcerative colitis. Despite that, up to 60% of patients do not respond adequately to steroid treatment. Previously, we reported that low IL-10 mRNA levels in intestine are associated with a poor response to glucocorticoids in active Crohn's disease. Here, we test whether IL-10 can favour the response to glucocorticoids by improving the TNFα-induced intestinal barrier damage (assessed by transepithelial electrical resistance) in Caco-2 monolayers, and their possible implications on glucocorticoid responsiveness in active ulcerative colitis. We show that the association of IL-10 and glucocorticoids improves the integrity of TNFα-treated Caco-2 cells and that p38 MAPK plays a key role. In vitro, IL-10 facilitates the nuclear translocation of p38 MAPK-phosphorylated thereby modulating glucocorticoids-receptor-α, IL-10-receptor-α and desmoglein-2 expression. In glucocorticoids-refractory patients, p38 MAPK phosphorylation and membrane desmoglein-2 expression are reduced in colonic epithelial cells. These results suggest that p38 MAPK-mediated synergism between IL-10 and glucocorticoids improves desmosome straightness contributing to the recovery of intestinal epithelium and reducing luminal antigens contact with lamina propria in ulcerative colitis. This study highlights the link between the intestinal epithelium in glucocorticoids-response in ulcerative colitis

A protease-based biosensor for the detection of schistosome cercariae

Parasitic diseases affect millions of people worldwide, causing debilitating illnesses and death. Rapid and cost-effective approaches to detect parasites are needed, especially in resource-limited settings. A common signature of parasitic diseases is the release of specific proteases by the parasites at multiple stages during their life cycles. To this end, we engineered several modular Escherichia coli and Bacillus subtilis whole-cell-based biosensors which incorporate an interchangeable protease recognition motif into their designs. Herein, we describe how several of our engineered biosensors have been applied to detect the presence and activity of elastase, an enzyme released by the cercarial larvae stage of Schistosoma mansoni. Collectively, S. mansoni and several other schistosomes are responsible for the infection of an estimated 200 million people worldwide. Since our biosensors are maintained in lyophilised cells, they could be applied for the detection of S. mansoni and other parasites in settings without reliable cold chain access

Diagnostic Accuracy and Applicability of a PCR System for the Detection of Schistosoma mansoni DNA in Human Urine Samples from an Endemic Area

Schistosomiasis caused by Schistosoma mansoni, one of the most neglected human parasitoses in Latin America and Africa, is routinely confirmed by microscopic visualization of eggs in stool. The main limitation of this diagnostic approach is its lack of sensitivity in detecting individual low worm burdens and consequently data on infection rates in low transmission settings are little reliable. According to the scientific literature, PCR assays are characterized by high sensitivity and specificity in detecting parasite DNA in biological samples. A simple and cost effective extraction method for DNA of Schistosoma mansoni from urine samples in combination with a conventional PCR assay was developed and applied in an endemic area. This urine based PCR system was tested for diagnostic accuracy among a population of a small village in an endemic area, comparing it to a reference test composed of three different parasitological techniques. The diagnostic parameters revealed a sensitivity of 100%, a specificity of 91.20%, positive and negative predictive values of 86.25% and 100%, respectively, and a test accuracy of 94.33%. Further statistical analysis showed a k index of 0.8806, indicating an excellent agreement between the reference test and the PCR system. Data obtained from the mouse model indicate the infection can be detected one week after cercariae penetration, opening a new perspective for early detection and patient management during this stage of the disease. The data indicate that this innovative PCR system provides a simple to handle and robust diagnostic tool for the detection of S. mansoni DNA from urine samples and a promising approach to overcome the diagnostic obstacles in low transmission settings. Furthermore the principals of this molecular technique, based on the examination of human urine samples may be useful for the diagnosis of other neglected tropical diseases that can be detected by trans-renal DNA