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

Heat Shock Response in Yeast Involves Changes in Both Transcription Rates and mRNA Stabilities

We have analyzed the heat stress response in the yeast Saccharomyces cerevisiae by determining mRNA levels and transcription rates for the whole transcriptome after a shift from 25°C to 37°C. Using an established mathematical algorithm, theoretical mRNA decay rates have also been calculated from the experimental data. We have verified the mathematical predictions for selected genes by determining their mRNA decay rates at different times during heat stress response using the regulatable tetO promoter. This study indicates that the yeast response to heat shock is not only due to changes in transcription rates, but also to changes in the mRNA stabilities. mRNA stability is affected in 62% of the yeast genes and it is particularly important in shaping the mRNA profile of the genes belonging to the environmental stress response. In most cases, changes in transcription rates and mRNA stabilities are homodirectional for both parameters, although some interesting cases of antagonist behavior are found. The statistical analysis of gene targets and sequence motifs within the clusters of genes with similar behaviors shows that both transcriptional and post-transcriptional regulons apparently contribute to the general heat stress response by means of transcriptional factors and RNA binding proteins

Increased Monocyte Turnover from Bone Marrow Correlates with Severity of SIV Encephalitis and CD163 Levels in Plasma

Cells of the myeloid lineage are significant targets for human immunodeficiency virus (HIV) in humans and simian immunodeficiency virus (SIV) in monkeys. Monocytes play critical roles in innate and adaptive immunity during inflammation. We hypothesize that specific subsets of monocytes expand with AIDS and drive central nervous system (CNS) disease. Additionally, there may be expansion of cells from the bone marrow through blood with subsequent macrophage accumulation in tissues driving pathogenesis. To identify monocytes that recently emigrated from bone marrow, we used 5-bromo-2′-deoxyuridine (BrdU) labeling in a longitudinal study of SIV-infected CD8+ T lymphocyte depleted macaques. Monocyte expansion and kinetics in blood was assessed and newly migrated monocyte/macrophages were identified within the CNS. Five animals developed rapid AIDS with differing severity of SIVE. The percentages of BrdU+ monocytes in these animals increased dramatically, early after infection, peaking at necropsy where the percentage of BrdU+ monocytes correlated with the severity of SIVE. Early analysis revealed changes in the percentages of BrdU+ monocytes between slow and rapid progressors as early as 8 days and consistently by 27 days post infection. Soluble CD163 (sCD163) in plasma correlated with the percentage of BrdU+ monocytes in blood, demonstrating a relationship between monocyte activation and expansion with disease. BrdU+ monocytes/macrophages were found within perivascular spaces and SIVE lesions. The majority (80–90%) of the BrdU+ cells were Mac387+ that were not productively infected. There was a minor population of CD68+BrdU+ cells (<10%), very few of which were infected (<1% of total BrdU+ cells). Our results suggest that an increased rate of monocyte recruitment from bone marrow into the blood correlates with rapid progression to AIDS, and the magnitude of BrdU+ monocytes correlates with the severity of SIVE

Hepatitis C infection: eligibility for antiviral therapies

peer reviewedBackground Current treatments of chronic hepatitis C virus (HCV) are effective, but expensive and susceptible to induce significant side effects. Objectives To evaluate the proportion of HCV patients who are eligible for a treatment. Methods In a database comprising 1726 viraemic HCV patients, the files of 299 patients who presented to the same hepatologist for an initial appointment between 1996 and 2003 were reviewed. Results Patients' characteristics were age 43.1 +/- 15.6 years, 53% male and 92% Caucasian. The main risk factors were transfusion (43%) and drug use (22%). Genotypes were mostly genotype 1 (66%), genotype 3 (12%) and genotype 2 (10%). These characteristics were not different from those of the whole series of 1726 patients. A total of 176 patients (59%) were not treated, the reasons for non-treatment being medical contraindications (34%), non-compliance (25%) and normal transaminases (24%). In addition, 17% of patients declined therapy despite being considered as eligible, mainly due to fear of adverse events. Medical contraindications were psychiatric (27%), age (22%), end-stage liver disease (15%), willingness for pregnancy (13%), cardiac contraindication (7%) and others (16%). Only 123 patients (41%) were treated. A sustained viral response was observed in 41%. The treatment was interrupted in 16% for adverse events. Conclusions The majority of HCV patients are not eligible for treatment. This implies that, with current therapies, only 17% of patients referred for chronic HCV become sustained responders. Some modifications of guidelines could extend the rate of treatment (patients with normal transaminases), but an important barrier remains the patients' and the doctors' fear of adverse events

Nutrient sensing modulates malaria parasite virulence

The lifestyle of intracellular pathogens, such as malaria parasites, is intimately connected to that of their host, primarily for nutrient supply. Nutrients act not only as primary sources of energy but also as regulators of gene expression, metabolism and growth, through various signalling networks that enable cells to sense and adapt to varying environmental conditions. Canonical nutrient-sensing pathways are presumed to be absent from the causative agent of malaria, Plasmodium, thus raising the question of whether these parasites can sense and cope with fluctuations in host nutrient levels. Here we show that Plasmodium blood-stage parasites actively respond to host dietary calorie alterations through rearrangement of their transcriptome accompanied by substantial adjustment of their multiplication rate. A kinome analysis combined with chemical and genetic approaches identified KIN as a critical regulator that mediates sensing of nutrients and controls a transcriptional response to the host nutritional status. KIN shares homology with SNF1/AMPKα, and yeast complementation studies suggest that it is part of a functionally conserved cellular energy-sensing pathway. Overall, these findings reveal a key parasite nutrient-sensing mechanism that is critical for modulating parasite replication and virulence

Association of Six Single Nucleotide Polymorphisms with Gestational Diabetes Mellitus in a Chinese Population

To investigate whether the candidate genes that confer susceptibility to type 2 diabetes mellitus are also correlated with gestational diabetes mellitus (GDM) in pregnant Chinese women.In this study, 1764 unrelated pregnant women were recruited, of which 725 women had GDM and 1039 served as controls. Six single nucleotide polymorphisms (rs7754840 in CDKAL1, rs391300 in SRR, rs2383208 in CDKN2A/2B, rs4402960 in IGF2BP2, rs10830963 in MTNR1B, rs4607517 in GCK) were genotyped using TaqMan allelic discrimination assays. The genotype and allele distributions of each SNP between the GDM cases and controls and the combined effects of alleles for the risk of developing GDM were analyzed. We found that the rs4402960, rs2383208 and rs391300 were statistically associated with GDM (OR = 1.207, 95%CI = 1.029-1.417, p = 0.021; OR = 1.242, 95%CI = 1.077-1.432, p = 0.003; OR = 1.202, 95%CI = 1.020-1.416, P = 0.028, respectively). In addition, the effect was greater under a recessive model in rs391300 (OR = 1.820, 95%CI = 1.226-2.701, p = 0.003). Meanwhile, the joint effect of these three loci indicated an additive effect of multiple alleles on the risk of developing GDM with an OR of 1.196 per allele (p = 1.08×10(-4)). We also found that the risk alleles of rs2383208 (b = -0.085, p = 0.003), rs4402960 (b = -0.057, p = 0.046) and rs10830963 (b = -0.096, p = 0.001) were associated with HOMA-B, while rs7754840 was associated with decrease in insulin AUC during a 100 g OGTT given at the time of GDM diagnosis (b = -0.080, p = 0.007).Several risk alleles of type 2 diabetes were associated with GDM in pregnant Chinese women. The effects of these SNPs on GDM might be through the impairment of beta cell function and these risk loci contributed additively to the disease

GUCY2C Opposes Systemic Genotoxic Tumorigenesis by Regulating AKT-Dependent Intestinal Barrier Integrity

The barrier separating mucosal and systemic compartments comprises epithelial cells, annealed by tight junctions, limiting permeability. GUCY2C recently emerged as an intestinal tumor suppressor coordinating AKT1-dependent crypt-villus homeostasis. Here, the contribution of GUCY2C to barrier integrity opposing colitis and systemic tumorigenesis is defined. Mice deficient in GUCY2C (Gucy2c−/−) exhibited barrier hyperpermeability associated with reduced junctional proteins. Conversely, activation of GUCY2C in mice reduced barrier permeability associated with increased junctional proteins. Further, silencing GUCY2C exacerbated, while activation reduced, chemical barrier disruption and colitis. Moreover, eliminating GUCY2C amplified, while activation reduced, systemic oxidative DNA damage. This genotoxicity was associated with increased spontaneous and carcinogen-induced systemic tumorigenesis in Gucy2c−/− mice. GUCY2C regulated barrier integrity by repressing AKT1, associated with increased junction proteins occludin and claudin 4 in mice and Caco2 cells in vitro. Thus, GUCY2C defends the intestinal barrier, opposing colitis and systemic genotoxicity and tumorigenesis. The therapeutic potential of this observation is underscored by the emerging clinical development of oral GUCY2C ligands, which can be used for chemoprophylaxis in inflammatory bowel disease and cancer

Aqueous extract of Terminalia arjuna prevents carbon tetrachloride induced hepatic and renal disorders

BACKGROUND: Carbon tetrachloride (CCl(4)) is a well-known hepatotoxin and exposure to this chemical is known to induce oxidative stress and causes liver injury by the formation of free radicals. Acute and chronic renal damage are also very common pathophysiologic disturbances caused by CCl(4). The present study has been conducted to evaluate the protective role of the aqueous extract of the bark of Termnalia arjuna (TA), an important Indian medicinal plant widely used in the preparation of ayurvedic formulations, on CCl(4 )induced oxidative stress and resultant dysfunction in the livers and kidneys of mice. METHODS: Animals were pretreated with the aqueous extract of TA (50 mg/kg body weight) for one week and then challenged with CCl(4 )(1 ml/kg body weight) in liquid paraffin (1:1, v/v) for 2 days. Serum marker enzymes, namely, glutamate pyruvate transaminase (GPT) and alkaline phosphatase (ALP) were estimated in the sera of all study groups. Antioxidant status in both the liver and kidney tissues were estimated by determining the activities of the antioxidative enzymes, superoxide dismutase (SOD), catalase (CAT) and glutathione-S-transferase (GST); as well as by determining the levels of thiobarbutaric acid reactive substances (TBARS) and reduced glutathione (GSH). In addition, free radical scavenging activity of the extract was determined from its DPPH radical quenching ability. RESULTS: Results showed that CCl(4 )caused a marked rise in serum levels of GPT and ALP. TBARS level was also increased significantly whereas GSH, SOD, CAT and GST levels were decreased in the liver and kidney tissue homogenates of CCl(4 )treated mice. Aqueous extract of TA successfully prevented the alterations of these effects in the experimental animals. Data also showed that the extract possessed strong free radical scavenging activity comparable to that of vitamin C. CONCLUSION: Our study demonstrated that the aqueous extract of the bark of TA could protect the liver and kidney tissues against CCl(4)-induced oxidative stress probably by increasing antioxidative defense activities

Sex, Subdivision, and Domestic Dispersal of Trypanosoma cruzi Lineage I in Southern Ecuador

Trypanosoma cruzi is transmitted by blood sucking insects known as triatomines. This protozoan parasite commonly infects wild and domestic mammals in South and Central America. However, triatomines also transmit the parasite to people, and human infection with T. cruzi is known as Chagas disease, a major public health concern in Latin America. Understanding the complex dynamics of parasite spread between wild and domestic environments is essential to design effective control measures to prevent the spread of Chagas disease. Here we describe T. cruzi genetic diversity and population dynamics in southern Ecuador. Our findings indicate that the parasite circulates in two largely independent cycles: one corresponding to the sylvatic environment and one related to the domestic/peridomestic environment. Furthermore, our data indicate that human activity might promote parasite dispersal among communties. This information is the key for the design of control programmes in Southern Ecuador. Finally, we have encountered evidence of a sexual reproductive mode in the domestic T. cruzi population, which constitutes a new and intriguing finding with regards to the biology of this parasite

Refined human artificial chromosome vectors for gene therapy and animal transgenesis

Human artificial chromosomes (HACs) have several advantages as gene therapy vectors, including stable episomal maintenance, and the ability to carry large gene inserts. We previously developed HAC vectors from the normal human chromosomes using a chromosome engineering technique. However, endogenous genes were remained in these HACs, limiting their therapeutic applications. In this study, we refined a HAC vector without endogenous genes from human chromosome 21 in homologous recombination-proficient chicken DT40 cells. The HAC was physically characterized using a transformation-associated recombination (TAR) cloning strategy followed by sequencing of TAR-bacterial artificial chromosome clones. No endogenous genes were remained in the HAC. We demonstrated that any desired gene can be cloned into the HAC using the Cre-loxP system in Chinese hamster ovary cells, or a homologous recombination system in DT40 cells. The HAC can be efficiently transferred to other type of cells including mouse ES cells via microcell-mediated chromosome transfer. The transferred HAC was stably maintained in vitro and in vivo. Furthermore, tumor cells containing a HAC carrying the suicide gene, herpes simplex virus thymidine kinase (HSV-TK), were selectively killed by ganciclovir in vitro and in vivo. Thus, this novel HAC vector may be useful not only for gene and cell therapy, but also for animal transgenesis