Real-Time PCR in the early detection of invasive fungal infection in immunodeficient infants and children

Background: Crucial to the diagnosis and effective therapy of invasive fungal infection (IFI) in the immunodeficient is the early identification of the causative agent especially in patients who lack clinical evidence of the disease. The standard methods for the detection of fungi in clinical specimens are direct microscopy and mycological culture. Microscopy often lacks a satisfactory sensitivity, whereas diagnosis by mycological culture often requires a long growth period. Studies have demonstrated the feasibility of detecting molds and yeast in a single reaction using the universal fungal primer. Objective: Evaluation of the role of real-time PCR in the early detection of fungal infection in immunodeficient patients with suspected IFI, who lack clinical evidence of the disease. Methods: This study included 30 immunodeficiency patients suspected of having IFI; 9 with primary and 21 with secondary immunodeficiency. All patients had at least one host factor, but no clinical criteria according to the EORTC-MSG definition of IFI. Twenty seven had fever and 3 had bronchopneumonia, both not responding to broad spectrum antibiotics for 96 hrs. or more. Blood samples were cultured for fungi and were analyzed with real-time PCR using universal fungal primers. For positive samples of fungal infection, aspergillus-specific primers were used for detection of aspergillus. Results: Seventeen patients (56.7%) proved to have IFI. Blood culture detected Candida in 2 patients only, while PCR detected Candida in another 9 and Aspergillus in 6, thus 15/17 patients with IFI (88%) were missed by blood culture. Blood culture for IFI diagnosis had a very low sensitivity (12%) but had a 100% specificity and positive predictive value. The results PCR did not vary with gender, degree of fever, immunodeficiency type, clinical presentation or current intake of antifungal treatment. Patients with proven IFI showed significantly increased CRP levels as compared to those without infection. Conclusion: Real-time PCR proved superior to culture in early diagnosis of IFI in patients with immunodeficiency before the appearance of the characteristic clinical and imaging signs. Reliance on blood culture alone at that stage would result in missing most of the positive cases with consequent delay in the initiation of specific treatment. Keywords: Invasive fungal infection, immunodeficiency, blood culture, real-time PCR, candida, aspergillusEgypt J Pediatr Allergy Immunol 2012;10(2):67-7

Patterns of hepatocellular carcinoma incidence in Egypt from a population-based cancer registry

Hepatocellular carcinoma (HCC) is increasing worldwide, and is frequently attributed to rising rates of hepatitis C virus infection and interactions between viral and environmental risk factors. Because of Egypt's unique risk factor profile, we analyzed data from the Gharbiah Population-Based Cancer Registry for the period 1999–2003 to characterize demographic and geographic patterns of cases in this province. Methods:  We calculated age- and sex-specific and age- and sex-standardized HCC incidence rates for the eight districts in Gharbiah. We also compared rates from Gharbiah with the USA (US Surveillance Epidemiology and End Results [SEER] database). Results:  The analysis revealed a higher incidence in males than in females, significant geographic variations among districts, and a higher incidence in Gharbiah than that reported by SEER. Conclusion:  The findings of this study document the heterogeneous distribution of HCC at regional and international levels. This population-based registry offers the opportunity for careful representative studies of various etiologies, particularly infectious and/or environmental factors that may contribute to risk.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/75374/1/j.1872-034X.2007.00299.x.pd

Isolation and characterisation of human gingival margin-derived STRO-1/MACS+ and MACS− cell populations

Recently, gingival margin-derived stem/progenitor cells isolated via STRO-1/magnetic activated cell sorting (MACS) showed remarkable periodontal regenerative potential in vivo. As a second-stage investigation, the present study's aim was to perform in vitro characterisation and comparison of the stem/progenitor cell characteristics of sorted STRO-1-positive (MACS+) and STRO-1-negative (MACS−) cell populations from the human free gingival margin. Cells were isolated from the free gingiva using a minimally invasive technique and were magnetically sorted using anti-STRO-1 antibodies. Subsequently, the MACS+ and MACS− cell fractions were characterized by flow cytometry for expression of CD14, CD34, CD45, CD73, CD90, CD105, CD146/MUC18 and STRO-1. Colony-forming unit (CFU) and multilineage differentiation potential were assayed for both cell fractions. Mineralisation marker expression was examined using real-time polymerase chain reaction (PCR). MACS+ and MACS− cell fractions showed plastic adherence. MACS+ cells, in contrast to MACS− cells, showed all of the predefined mesenchymal stem/progenitor cell characteristics and a significantly higher number of CFUs (P<0.01). More than 95% of MACS+ cells expressed CD105, CD90 and CD73; lacked the haematopoietic markers CD45, CD34 and CD14, and expressed STRO-1 and CD146/MUC18. MACS− cells showed a different surface marker expression profile, with almost no expression of CD14 or STRO-1, and more than 95% of these cells expressed CD73, CD90 and CD146/MUC18, as well as the haematopoietic markers CD34 and CD45 and CD105. MACS+ cells could be differentiated along osteoblastic, adipocytic and chondroblastic lineages. In contrast, MACS− cells demonstrated slight osteogenic potential. Unstimulated MACS+ cells showed significantly higher expression of collagen I (P<0.05) and collagen III (P<0.01), whereas MACS− cells demonstrated higher expression of osteonectin (P<0.05; Mann–Whitney). The present study is the first to compare gingival MACS+ and MACS− cell populations demonstrating that MACS+ cells, in contrast to MACS− cells, harbour stem/progenitor cell characteristics. This study also validates the effectiveness of the STRO-1/MACS+ technique for the isolation of gingival stem/progenitor cells. Human free gingival margin-derived STRO-1/MACS+ cells are a unique renewable source of multipotent stem/progenitor cells

Female sex pheromone of the cone moth, dioryctria mendacella: Investigation of synergism between Type I and Type II pheromone components

Polyunsaturated hydrocarbons (Type II pheromone components) have been reported to be synergists for unsaturated acetates, alcohols or aldehydes (Type I components) in the sex pheromones of several species of Lepidoptera. However, there is some debate over whether the active components are the hydrocarbons themselves or more volatile degradation products. Extracts of pheromone glands of adult females of the cone moth, Dioryctria mendacella (Lepidoptera: Pyralidae), contain (Z,E)-9,11-tetradecadienyl acetate (ZE9,11-14:Ac) and at least ten times as much (Z,Z,Z,Z,Z)-3,6,9,12,15-pentacosapentaene (ZZZZZ3,6,9,12,15-25:H). The former elicits a strong electroantennogram response from males while no response could be recorded to the latter. In field trapping tests, both compounds were individually unattractive to male D. mendacella moths, but blends of the two compounds containing at least a 10:1 ratio of ZZZZZ3,6,9,12,15-25:H : ZE9,11-14:Ac were highly attractive. The relatively involatile hydrocarbon was shown to be released from the dispensers used and no significant degradation could be detected. Furthermore, blends of ZE9,11-14:Ac and analogs of ZZZZZ3,6,9,12,15-25:H with fewer carbons and/or double bonds that might be expected to produce similar degradation products to ZZZZZ3,6,9,12,15-25:H were unattractive. This indicated a specific response to the hydrocarbon itself, further substantiated by the observation that related hydrocarbons did not interfere with the activity of ZZZZZ3,6,9,12,15-25:H. Thus a three-step conversion of fish oil was used to produce a blend of unsaturated hydrocarbons containing ZZZZZ3,6,9,12,15-25:H as the major component, albeit only 30% of the total, and a blend of this material with ZE9,11-14:Ac was as attractive to male D. mendacella moths as blends with an equivalent amount of the purified material. This mixture of unsaturated hydrocarbons is much cheaper to produce than the pure pentaene, and may be useful in lures for other species using these compounds. Dioryctria mendacella is a major constraint to production of edible pine kernels throughout the Mediterranean region. Pheromone traps will provide a means to improve monitoring of seasonal flight patterns and changes in population abundance of this pest

A Brain-Computer Interface Based Attention Training Program for Treating Attention Deficit Hyperactivity Disorder

10.1371/journal.pone.0046692PLoS ONE710

Further evidence for association of hepatitis C infection with parenteral schistosomiasis treatment in Egypt

BACKGROUND: Hepatitis C virus (HCV) infection and schistosomiasis are major public health problems in the Nile Delta of Egypt. To control schistosomiasis, mass treatment campaigns using tartar emetic injections were conducted in the 1960s through 1980s. Evidence suggests that inadequately sterilized needles used in these campaigns contributed to the transmission of HCV in the region. To corroborate this evidence, this study evaluates whether HCV infections clustered within houses in which household members had received parenteral treatment for schistosomiasis. METHODS: A serosurvey was conducted in a village in the Nile Delta and residents were questioned about prior treatment for schistosomiasis. Sera were evaluated for the presence of antibodies to HCV. The GEE2 approach was used to test for clustering of HCV infections, where correlation of HCV infections within household members who had been treated for schistosomiasis was the parameter of interest. RESULTS: A history of parenteral treatment for schistosomiasis was observed to cluster within households, OR for clustering: 2.44 (95% CI: 1.47–4.06). Overall, HCV seropositivity was 40% (321/796) and was observed to cluster within households that had members who had received parenteral treatment for schistosomiasis, OR for clustering: 1.76 (95% CI: 1.05–2.95). No such evidence for clustering was found in the remaining households. CONCLUSION: Clustering of HCV infections and receipt of parenteral treatment for schistosomiasis within the same households provides further evidence of an association between the schistosomiasis treatment campaigns and the high HCV seroprevalence rates currently observed in the Nile delta of Egypt

The Trypanosoma cruzi Sylvio X10 strain maxicircle sequence: the third musketeer

<p>Abstract</p> <p>Background</p> <p>Chagas disease has a diverse pathology caused by the parasite <it>Trypanosoma cruzi</it>, and is indigenous to Central and South America. A pronounced feature of the trypanosomes is the kinetoplast, which is comprised of catenated maxicircles and minicircles that provide the transcripts involved in uridine insertion/deletion RNA editing. <it>T. cruzi </it>exchange genetic material through a hybridization event. Extant strains are grouped into six discrete typing units by nuclear markers, and three clades, A, B, and C, based on maxicircle gene analysis. Clades A and B are the more closely related. Representative clade B and C maxicircles are known in their entirety, and portions of A, B, and C clades from multiple strains show intra-strain heterogeneity with the potential for maxicircle taxonomic markers that may correlate with clinical presentation.</p> <p>Results</p> <p>To perform a genome-wide analysis of the three maxicircle clades, the coding region of clade A representative strain Sylvio X10 (a.k.a. Silvio X10) was sequenced by PCR amplification of specific fragments followed by assembly and comparison with the known CL Brener and Esmeraldo maxicircle sequences. The clade A rRNA and protein coding region maintained synteny with clades B and C. Amino acid analysis of non-edited and 5'-edited genes for Sylvio X10 showed the anticipated gene sequences, with notable frameshifts in the non-edited regions of Cyb and ND4. Comparisons of genes that undergo extensive uridine insertion and deletion display a high number of insertion/deletion mutations that are likely permissible due to the post-transcriptional activity of RNA editing.</p> <p>Conclusion</p> <p>Phylogenetic analysis of the entire maxicircle coding region supports the closer evolutionary relationship of clade B to A, consistent with uniparental mitochondrial inheritance from a discrete typing unit TcI parental strain and studies on smaller fragments of the mitochondrial genome. Gene variance that can be corrected by RNA editing hints at an unusual depth for maxicircle taxonomic markers, which will aid in the ability to distinguish strains, their corresponding symptoms, and further our understanding of the <it>T. cruzi </it>population structure. The prevalence of apparently compromised coding regions outside of normally edited regions hints at undescribed but active mechanisms of genetic exchange.</p

Insecticide resistance in the sand fly, Phlebotomus papatasi from Khartoum State, Sudan

<p>Abstract</p> <p>Background</p> <p><it>Phlebotomus papatasi </it>the vector of cutaneous leishmaniasis (CL) is the most widely spread sand fly in Sudan. No data has previously been collected on insecticide susceptibility and/or resistance of this vector, and a first study to establish a baseline data is reported here.</p> <p>Methods</p> <p>Sand flies were collected from Surogia village, (Khartoum State), Rahad Game Reserve (eastern Sudan) and White Nile area (Central Sudan) using light traps. Sand flies were reared in the Tropical Medicine Research Institute laboratory. The insecticide susceptibility status of first progeny (F1) of <it>P. papatasi </it>of each population was tested using WHO insecticide kits. Also, <it>P. papatasi </it>specimens from Surogia village and Rahad Game Reserve were assayed for activities of enzyme systems involved in insecticide resistance (acetylcholinesterase (AChE), non-specific carboxylesterases (EST), glutathione-S-transferases (GSTs) and cytochrome p450 monooxygenases (Cyt p450).</p> <p>Results</p> <p>Populations of <it>P. papatasi </it>from White Nile and Rahad Game Reserve were sensitive to dichlorodiphenyltrichloroethane (DDT), permethrin, malathion, and propoxur. However, the <it>P. papatasi </it>population from Surogia village was sensitive to DDT and permethrin but highly resistant to malathion and propoxur. Furthermore, <it>P. papatasi </it>of Surogia village had significantly higher insecticide detoxification enzyme activity than of those of Rahad Game Reserve. The sand fly population in Surogia displayed high AChE activity and only three specimens had elevated levels for EST and GST.</p> <p>Conclusions</p> <p>The study provided evidence for malathion and propoxur resistance in the sand fly population of Surogia village, which probably resulted from anti-malarial control activities carried out in the area during the past 50 years.</p

Analyses of 32 Loci Clarify Phylogenetic Relationships among Trypanosoma cruzi Lineages and Support a Single Hybridization prior to Human Contact

Trypanosoma cruzi is the protozoan parasite that causes Chagas disease, a major health problem in Latin America. The genetic diversity of this parasite has been traditionally divided in two major groups: T. cruzi I and II, which can be further divided in six major genetic subdivisions (subgroups TcI-TcVI). T. cruzi I and II seem to differ in important biological characteristics, and are thought to represent a natural division relevant for epidemiological studies and development of prophylaxis. Having a correct reconstruction of the evolutionary history of T. cruzi is essential for understanding the potential connection between the genetic and phenotypic variability of T. cruzi with the different manifestations of Chagas disease. Here we present results from a comprehensive phylogenetic analysis of T. cruzi using more than 26 Kb of aligned sequence data. We show strong evidence that T. cruzi II (TcII-VI) is not a natural evolutionary group but a paraphyletic lineage and that all major lineages of T. cruzi evolved recently (<3 million years ago [mya]). Furthermore, the sequence data is consistent with one major hybridization event having occurred in this species recently (< 1 mya) but well before T. cruzi entered in contact with humans in South America

The steady-state transcriptome of the four major life-cycle stages of Trypanosoma cruzi

<p>Abstract</p> <p>Background</p> <p>Chronic chagasic cardiomyopathy is a debilitating and frequently fatal outcome of human infection with the protozoan parasite, <it>Trypanosoma cruzi</it>. Microarray analysis of gene expression during the <it>T. cruzi </it>life-cycle could be a valuable means of identifying drug and vaccine targets based on their appropriate expression patterns, but results from previous microarray studies in <it>T. cruzi </it>and related kinetoplastid parasites have suggested that the transcript abundances of most genes in these organisms do not vary significantly between life-cycle stages.</p> <p>Results</p> <p>In this study, we used whole genome, oligonucleotide microarrays to globally determine the extent to which <it>T. cruzi </it>regulates mRNA relative abundances over the course of its complete life-cycle. In contrast to previous microarray studies in kinetoplastids, we observed that relative transcript abundances for over 50% of the genes detected on the <it>T. cruzi </it>microarrays were significantly regulated during the <it>T. cruzi </it>life-cycle. The significant regulation of 25 of these genes was confirmed by quantitative reverse-transcriptase PCR (qRT-PCR). The <it>T. cruzi </it>transcriptome also mirrored published protein expression data for several functional groups. Among the differentially regulated genes were members of paralog clusters, nearly 10% of which showed divergent expression patterns between cluster members.</p> <p>Conclusion</p> <p>Taken together, these data support the conclusion that transcript abundance is an important level of gene expression regulation in <it>T. cruzi</it>. Thus, microarray analysis is a valuable screening tool for identifying stage-regulated <it>T. cruzi </it>genes and metabolic pathways.</p