Evaluation of a new multiplex PCR assay (ParaGENIE G-Amoeba Real-Time PCR kit) targeting Giardia intestinalis, Entamoeba histolytica and Entamoeba dispar/Entamoeba moshkovskii from stool specimens: evidence for the limited performances of microscopy-based approach for amoeba species identification.

OBJECTIVES: Besides the potential to identify a wide variety of gastrointestinal parasites, microscopy remains the reference standard in clinical microbiology for amoeba species identification and, especially when coupled with adhesin detection, to discriminate the pathogenic Entamoeba histolytica from its sister but non-pathogenic species Entamoeba dispar/Entamoeba moshkovskii. However, this approach is time-consuming, requires a high-level of expertise that can be jeopardized considering the low prevalence of gastrointestinal parasites in non-endemic countries. Here, we evaluated the CE-IVD-marked multiplex PCR (ParaGENIE G-Amoeba, Ademtech) targeting E. histolytica and E. dispar/E. moshkovskii and Giardia intestinalis. METHODS: This evaluation was performed blindly on a reference panel of 172 clinical stool samples collected prospectively from 12 laboratories and analysed using a standardized protocol relying on microscopy (and adhesin detection by ELISA for the detection of E. histolytica) including G. intestinalis (n = 37), various amoeba species (n = 55) including E. dispar (n = 15), E. histolytica (n = 5), as well as 17 other gastrointestinal parasites (n = 80), and negative samples (n = 37). RESULTS: This new multiplex PCR assay offers fast and reliable results with appropriate sensitivity and specificity for the detection of G. intestinalis and E. dispar/E. moshkovskii from stools (89.7%/96.9% and 95%/100%, respectively). Detection rate and specificity were greatly improved by the PCR assay, highlighting several samples misidentified by microscopy, including false-negative and false-positive results for both E. dispar/E. moshkovskii and E. histolytica. CONCLUSION: Given the clinical relevance of amoeba species identification, microbiologists should be aware of the limitations of using an algorithm relying on microscopy coupled with adhesin detection by ELISA.

Field application of mycorrhizal bio-inoculants affects the mineral uptake of a forage legume (Hedysarum coronarium L.) on a highly calcareous soil.

The efficiency of two mycorrhizal bio-inoculants on the mineral uptake during the growth stages of a Mediterranean forage legume sulla (Hedysarum coronarium L.) was studied in the field on a highly calcareous soil. The first inoculum (Mm) was made up of a mixture of native arbuscular mycorrhizal fungi (AMF) isolated from calcareous soils: Septoglomus constrictum, Funneliformis geosporum, Glomus fuegianum, Rhizophagus irregularis and Glomus sp. The second was a commercial inoculum (Mi) containing one AMF species: R. irregularis. Both mycorrhizal inoculants increased total and arbuscular colonization of sulla roots. Inoculation with Mm showed a positive effect on sulla shoot dry weight (SDW) when compared to Mi and non-inoculated plants (control). Mineral contents (P, Mg, Mn, Fe) were higher in the shoots of sulla plants cultivated on mycorrhiza-inoculated plots compared to non-inoculated ones. This enhancement was observed during the flowering stage for P, Mg and Mn and during the rosette stage for Fe. An increase in P content of 50 % in plants inoculated with Mm compared to non-inoculated ones may be explained by the induction of root alkaline and acid phosphatase activities. Higher efficiency of native AMF species adapted to calcareous soils opens the way towards the development of mycorrhiza bio-fertilizers targeted to improve sustainable fertilization management in such soils.

Identification of Leishmania at the species level with matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

Matrix-assisted laser desorption ionization time-of-flightMALDI-TOF mass spectrometry (MS) is now widely recognized as a powerful tool with which to identify bacteria and fungi at the species level, and sometimes in a rapid and accurate manner. We report herein an approach to identify, at the species level, Leishmania promastigotes from in vitro culture. We first constructed a reference database of spectra including the main Leishmania species known to cause human leishmaniasis. Then, the performance of the reference database in identifying Leishmania promastigotes was tested on a panel of 69 isolates obtained from patients. Our approach correctly identified 66 of the 69 isolates tested at the species level with log (score) values superior to 2. Two Leishmania isolates yielded non-interpretable MALDI-TOF MS patterns, owing to low log (score) values. Only one Leishmania isolate of Leishmania peruviana was misidentified as the closely related species Leishmania braziliensis, with a log (score) of 2.399. MALDI-TOF MS is a promising approach, providing rapid and accurate identification of Leishmania from in vitro culture at the species level.

Multicentric evaluation of a new real-time PCR assay for quantification of Cryptosporidium spp. and identification of Cryptosporidium parvum and Cryptosporidium hominis.

Cryptosporidium is a protozoan parasite responsible for gastroenteritis, especially in immunocompromised patients. Laboratory diagnosis of cryptosporidiosis relies on microscopy, antigen detection, and nucleic acid detection and analysis. Among the numerous molecular targets available, the 18S rRNA gene displays the best sensitivity and sequence variations between species and can be used for molecular typing assays. This paper presents a new real-time PCR assay for the detection and quantification of all Cryptosporidium species associated with the identification of Cryptosporidium hominis and Cryptosporidium parvum. The sensitivity and specificity of this new PCR assay were assessed on a multicentric basis, using well-characterized Cryptosporidium-positive and -negative human stool samples, and the efficiencies of nine extraction methods were comparatively assessed using Cryptosporidium-seeded stool samples and phosphate-buffered saline samples. A comparison of extraction yields showed that the most efficient extraction method was the Boom technique in association with mechanical grinding, and column extraction showed higher binding capacity than extraction methods based on magnetic silica. Our PCR assay was able to quantify at least 300 oocysts per gram of stool. Satisfactory reproducibility between laboratories was observed. The two main species causing human disease, Cryptosporidium hominis and Cryptosporidium parvum, were identified using a duplex real-time PCR assay with specific TaqMan minor-groove-binding ligand (MGB) probes for the same amplicon. To conclude, this one-step quantitative PCR is well suited to the routine diagnosis of cryptosporidiosis since practical conditions, including DNA extraction, quantification using well-defined standards, and identification of the two main species infecting humans, have been positively assessed.

A pilot study of the impact of an educational intervention aimed at improving medical record documentation.

INTRODUCTION: Studies have shown the importance of medical staff education in improving chart documentation and accuracy of medical coding. This study aimed to examine the effect of an educational intervention on recording medical diagnoses among a sample of medical residents based at Kashan University of Medical Sciences. METHODS: This pilot study was conducted in 2010 and involved 19 residents in different specialties (internal medicine, obstetrics and gynecology, and surgery). Guidelines for recording diagnostic information related to surgery, obstetrics and internal medicine were taught at a five-hour lecture. Five medical records from each resident from before and after the educational intervention were assessed using a checklist based on relevant diagnostic information related to each discipline. Data were analysed using a paired t-test and Wilcoxson signed rank test. RESULTS: There was no improvement in the quality and accuracy of the recording of obstetric diagnoses (type, place, outcome and complications of delivery) after the training. There was also no effect on the documentation of underlying causes and clinical manifestations of disease by internal medicine and surgery residents (p=0.285 and p=0.584, respectively). CONCLUSION: The single education session did not improve recording of diagnoses among residents. The gathering and recording of complete, accurate and high quality medical records requires interaction between the hospital management, health information management professionals and healthcare providers. It is therefore essential to develop a more sophisticated portfolio of strategies that involves these key stakeholders.

Baking soda and salt in bakeries of Mehrdasht (Najafabad), Isfahan, Iran: a survey on a typical rural population in a developing country.

BACKGROUND: Bread is a valuable source of proteins, minerals and calories. Baking soda prevents the absorption and digestion of bread and more salt used in production of bread also causes different diseases. This study was conducted to determine the amount of soda and salt in bakeries. METHOD: Cross-sectional descriptive study was carried out on 50 bakeries district during 2009. 400 samples were collected in four steps randomly. The standard PH < 6.2 indicative of no consumption of baking soda in bread and salt less than 2 g/100 g was considered as the reference. RESULTS: The PH less than 6.2 was seen in 91.5% of samples and analyzed by random effect analysis. In 64.5% of samples, the amount of salt was more than the standard. CONCLUSION: The amount of baking soda used in the bakeries was not high; bakers either had no enough knowledge about the amount of salt or had more other reasons. Drastic measures are recommended.

[Current epidemiological data on visceral leishmaniasis in Tunisia]. / Actualités épidémiologiques de la leishmaniose viscérale en Tunisie.

OBJECTIVE: Visceral leishmaniasis is an important health problem in Tunisia. The aim of this study was to update the epidemiological and clinical features of the disease. DESIGN: We performed a retrospective systematic sampling of epidemiological and clinical data collected from the medical records of 1,096 cases of visceral leishmaniasis diagnosed between 1996 and 2006 all over the country. RESULTS: The mean annual incidence of cases was 99.6 cases/year. The mean annual incidence rate was 1.04 cases/100,000 inhabitants, showing an important increase compared to former studies. As expected, children under 5 years (866 cases) were the most affected with a mean annual incidence rate of 9.6 cases/100,000 (p<0.001). The geographical distribution of cases revealed the spreading of the disease from the Northern parts of the country to the Central and even to Southern ones. Rural cases (65.3%) were significantly more numerous than urban ones (34.7%), p<0.001. The sex ratio was 1.03. The diagnostic delay (average of 54 days) was considerably shortened during the study period compared to previous reports, and explains the decrease of the lethality rate (2.9%). CONCLUSIONS: Visceral leishmaniasis has been present in central Tunisia since the early 1990 s. Its incidence and the distribution area have increased. This evolution is probably linked to the development of irrigation and agriculture favorable to the multiplication of vector sandflies and dogs reservoirs of Leishmania infantum.