Efficient and easy-to-use DNA extraction and purification methods are critical in implementing PCR-based diagnosis of pathogens. In order to optimize the routine clinical laboratory diagnosis of ...eukaryotic enteric pathogens, we compare, via quantitative PCR cycle threshold (Ct) values, the efficiency of two DNA extraction kits: the semi-automated EZ1
(Qiagen) and the manual QIAamp
DNA Stool Mini Kit (Qiagen), on six protozoa: Blastocystis spp., Cryptosporidium parvum/hominis, Cyclospora cayetanensis, Dientamoeba fragilis, Giardia intestinalis and Cystoisospora belli and one microsporidia: Enterocytozoon bieneusi.
Whereas EZ1
(Qiagen) and QIAamp
DNA Stool Mini Kit (Qiagen) yielded similar performances for the detection of Cryptosporidium spp. and D. fragilis, significant lower Ct values (p < 0.002) pointed out a better performance of EZ1
on the five remaining pathogens. DNA extraction using the semi-automated EZ1
procedure was faster and as efficient as the manual procedure in the seven eukaryotic enteric pathogens tested. This procedure is suitable for DNA extraction from stools in both clinical laboratory diagnosis and epidemiological study settings.
Zoonotic visceral leishmaniasis (VL) is endemic in the Mediterranean basin. However, large-scale comparative analyses of the commercial kits for the serological diagnosis of this neglected disease ...are lacking. This study compared the performances of four enzyme-linked immunosorbent assays (ELISA) and two immunochromatographic tests (ICT) as screening tests for the serodiagnosis of human VL in the Mediterranean region.
Serum samples from 319 patients living in France, Tunisia or Morocco were tested using two ICT (IT LEISH and TruQuick LEISH IgG/IgM Meridian) and four ELISA reagents (NovaLisa Leishmania infantum IgG, Bordier Leishmania infantum, Ridascreen Leishmania IgG, and Vircell Leishmania). The population with proven VL (n = 181) included 65 immunocompromised patients. Significantly higher percentages of false-negative results were obtained with all assays in immunocompromised patients, compared with the immunocompetent population. In the whole population, sensitivity and specificity ranged from 80.7% to 93.9% and from 95.7% to 100%, respectively. The maximum accuracy was observed with the Bordier and Vircell ELISA kits (96.2%), and the lowest accuracy with Ridascreen reagent (88.7%). New thresholds of positivity are proposed for the Bordier, Vircell and NovaLisa ELISA kits to achieve 95% sensitivity with the highest possible specificity. Western blot (WB), used as a confirmation method, showed 100% sensitivity and identified 10.1% of asymptomatic carriers among the control population from the South of France.
This is the first study that compared commercially available kits for VL serodiagnosis in the endemic region of the Mediterranean basin. It provides specific information about the tests' performance to help clinicians and biologists to select the right assay for VL screening.
Human and animal fungal pathogens are a growing threat worldwide leading to emerging infections and creating new risks for established ones. There is a growing need for a rapid and accurate ...identification of pathogens to enable early diagnosis and targeted antifungal therapy. Morphological and biochemical identification methods are time-consuming and require trained experts. Alternatively, molecular methods, such as DNA barcoding, a powerful and easy tool for rapid monophasic identification, offer a practical approach for species identification and less demanding in terms of taxonomical expertise. However, its wide-spread use is still limited by a lack of quality-controlled reference databases and the evolving recognition and definition of new fungal species/complexes. An international consortium of medical mycology laboratories was formed aiming to establish a quality controlled ITS database under the umbrella of the ISHAM working group on “DNA barcoding of human and animal pathogenic fungi.” A new database, containing 2800 ITS sequences representing 421 fungal species, providing the medical community with a freely accessible tool at http://www.isham.org/ and http://its.mycologylab.org/ to rapidly and reliably identify most agents of mycoses, was established. The generated sequences included in the new database were used to evaluate the variation and overall utility of the ITS region for the identification of pathogenic fungi at intra-and interspecies level. The average intraspecies variation ranged from 0 to 2.25%. This highlighted selected pathogenic fungal species, such as the dermatophytes and emerging yeast, for which additional molecular methods/genetic markers are required for their reliable identification from clinical and veterinary specimens.
Children living with chronic illnesses are offered formulations based on manufacturer and distributor research. The aim of this study is to better understand the perspectives of children and their ...caregivers in accepting Lopinavir/ritonavir (LPV/r) formulations.
362 participants were recruited from two pediatric HIV/AIDS clinics in Mbeya and Mwanza, Tanzania, from December 2021 to May 2022. A translated questionnaire was piloted and validated at both clinics, followed by the implementation of a cross-sectional study.
169 participants (47.1%) reported general difficulties in swallowing, regardless of formulation, while 34.3% and 38.5% reported vomiting tablets and syrups, respectively. Statistical significance is shown to support that children can swallow medications if they can eat stiffened porridge (Ugali). This correlated with the lower incidence of younger children being able to swallow compared to older children (above six years of age). Children older than six years preferred taking tablets (independent of daily dosage) better than other formulations. Significantly, older children who attend school were associated with high odds of swallowing medicine (AOR = 3.06, 95%CI; 1.32-7.05); however, age was not found to be statistically related to ease of administration for Lopinavir/Ritonavir in this study.
Lopinavir/Ritonavir tablets remain the most accepted formulation among children and adolescents with HIV/AIDS. This study highlights the impact of various factors affecting the acceptability of pediatric formulation, suggesting that children younger than six years, unable to eat Ugali and not attending schools may be most vulnerable regarding their ability to accept Lopinavir/Ritonavir formulations. Further studies are needed to assess the acceptability of other medications in chronically ill children.
Cu2ZnSnSe4 (CZTSe) nanoparticles (NPs) are successfully synthesized via cost‐effective hot‐injection process and morphologically modified by biphasic ligand exchange method. X‐ray diffraction (XRD) ...results indicate kesterite phase for the as‐prepared CZTSe NPs. Scherrer equation is used to evaluate the average crystallite size (≈30 nm). Scanning electron microscopy (SEM) images reveal that the interconnected nanoflakes structure combines to produce hierarchical CZTSe nanoflower. Fourier‐transform infrared spectroscopy (FTIR) results confirm the replacement of native long‐chain ligand over the surface of NPs with inorganic salts. The optical studies reveal that the CZTSe NPs have a large absorbance over the visible region with optimum bandgap value of 1.23 eV. The photocatalytic studies confirm the high efficiency of CZTSe NPs in degrading the methylene blue (MB) dye within 120 min under visible light irradiation. The intermediate products formed during the degradation of MB have been analyzed by electrospray ionization mass spectrometry (ESI‐MS). The photocatalytic degradation mechanism is discussed.
Cu2ZnSnSe4 (CZTSe) nanoparticles are synthesized in nanoflake morphology using hot injection method and the surfaces are modified with ligand exchange process. The photocatalytic analysis on methylene blue (MB) solution under visible light illumination showed over 70% degradation of MB within 120 min. Electrospray ionization mass spectrometry results elucidate the MB dye degradation mechanism with CZTSe nanoflakes as photocatalysts.
During the last 20 years, molecular biology techniques have propelled the diagnosis of parasitic diseases into a new era, as regards assay speed, sensitivity, and parasite characterization. However, ...DNA extraction remains a critical step and should be adapted for diagnostic and epidemiological studies. The aim of this report was to document the constraints associated with DNA extraction for the diagnosis of parasitic diseases and illustrate the adaptation of an automated extraction system, NucliSENS easyMAG, to these constraints, with a critical analysis of system performance. Proteinase K digestion of samples is unnecessary with the exception of solid tissue preparation. Mechanically grinding samples prior to cell lysis enhances the DNA extraction rate of fungal cells. The effect of host-derived nucleic acids on the extraction efficiency of parasite DNA varies with sample host cell density. The optimal cell number for precise parasite quantification ranges from 10 to 100,000 cells. Using the NucliSENS easyMAG technique, the co-extraction of inhibitors is reduced, with an exception for whole blood, which requires supplementary extraction steps to eliminate inhibitors.
Durant les vingt dernières années, l’avènement des techniques de biologie moléculaire a révolutionné le diagnostic des maladies parasitaires sur le plan de la rapidité, de la sensibilité et du typage des parasites. Cependant l’extraction des acides nucléiques reste une étape critique qui doit être adaptée au diagnostic et à l’épidémiologie. L’objectif de ce travail est d’illustrer les contraintes liées à l’extraction d’ADN en vue du diagnostic des parasitoses et de proposer les adaptations nécessaires à l’utilisation d’un système automatisé, NucliSENS easyMAG, en procédant à une analyse critique de ses performances. Ce système permet d’éviter le prétraitement par la protéinase K à l’exception des tissus solides. Le broyage mécanique avant la lyse cellulaire améliore l’extraction de l’ADN des cellules fongiques. L’effet des acides nucléiques dérivés de l’hôte sur le rendement d’extraction de l’ADN du parasite varie en fonction de la densité en cellules de l’échantillon de l’hôte. Le nombre optimal de cellules pour une quantification précise des parasites varie de 10 à 100,000 cellules. En utilisant la technique NucliSENS easyMAG, la co-extraction des inhibiteurs est réduite, avec une exception pour le sang total, qui nécessite des étapes d’extraction supplémentaires pour éliminer les inhibiteurs.
Child eating behaviors play an important role in nutrient intake, ultimately affecting child growth and later outcomes in adulthood. The study assessed the effects of iron-folic acid and zinc ...supplementation on child temperament and child eating behaviors in rural Nepal. Children (N = 569) aged 4-17 months in Sarlahi district, southern Nepal were randomized to receive daily supplements of placebo, iron-folic acid, zinc, or zinc plus iron-folic acid and followed for approximately 1 year. At baseline and four follow-up visits mothers completed questionnaires including information on demographic characteristics and child temperament and eating behaviors. The main effects of zinc and iron-folic acid supplementation on temperament and eating behaviors were assessed through crude and adjusted differences in mean cumulative score changes between visits 1 and 5. The adjusted rate-of-change for these outcomes was modeled using generalized estimating equations. Mean changes in temperament scores and in eating behavior scores between visits 1 and 5 were not significant in either the zinc or non-zinc group. Children in the iron-folic acid group increased temperament scores by 0.37 points over 5 visits (95% CI 0.02, 0.7), which was not significant after adjustment. Neither the adjusted rate-of-change in temperament scores between zinc and non-zinc (β = -0.03, 95% CI -0.3, 0.2) or iron-folic acid and non-iron-folic acid (β = 0.08, 95% CI -0.2, 0.3) were significantly different. Adjusted rate of change analysis showed no significant difference between zinc and non-zinc (β = -0.14, 95% CI -0.3, 0.04) or between iron and non-iron eating behavior scores (β = -0.11, 95% CI -0.3, 0.1). Only among children with iron-deficiency anemia at baseline was there a significant decrease in eating behavior score, indicating better eating behaviors, when supplemented with zinc (β = -0.3, 95% CI -0.6, -0.01), Ultimately, this effect of zinc on eating behaviors was the only effect we observed after approximately one year of micronutrient supplementation.
ClinicalTrials.gov NCT00109551.
Key Clinical Message
Images of parasitic forms of Leishmania infantum are typical in the hands of a skilled expert but should be known by biologists of Hematology Department. In an endemic region, ...the diagnosis of visceral leishmaniasis (VL) must be considered because of its potential role in accelerating hematological malignancy.
Images of parasitic forms of Leishmania infantum are typical in the hands of a skilled expert but should be known by biologists of Hematology Department. In an endemic region, the diagnosis of visceral leishmaniasis (VL) must be considered because of its potential role in accelerating hematological malignancy.
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