Kinetic modeling of the biosorption of Cd2+ ions from aqueous solutions onto Eichhornia crassipes roots using potentiometry: low-cost alternative to conventional methods

This work presents the use of potentiometric measurements for kinetic studies of biosorption of Cd2+ ions from aqueous solutions on Eichhornia crassipes roots. The open circuit potential of the Cd/Cd2+ electrode of the first kind was measured during the bioadsorption process. The amount of Cd2+ ions accumulated was determined in real time. The data were fit to different models, with the pseudo-second-order model proving to be the best in describing the data. The advantages and limitations of the methodology proposed relative to the traditional method are discussed.

Evaluation of four molecular methods for the diagnosis of tuberculosis in pulmonary and blood samples from immunocompromised patients

The present study analysed the concordance among four different molecular diagnostic methods for tuberculosis (TB) in pulmonary and blood samples from immunocompromised patients. A total of 165 blood and 194 sputum samples were collected from 181 human immunodeficiency virus (HIV)-infected patients with upper respiratory complaints, regardless of suspicious for TB. The samples were submitted for smear microscopy, culture and molecular tests: a laboratory-developed conventional polymerase chain reaction (PCR) and real-time quantitative PCR (qPCR) and the Gen-Probe and Detect-TB Ampligenix kits. The samples were handled blindly by all the technicians involved, from sample processing to results analysis. For sputum, the sensitivity and specificity were 100% and 96.7% for qPCR, 81.8% and 94.5% for Gen-Probe and 100% and 66.3% for Detect-TB, respectively. qPCR presented the best concordance with sputum culture [kappa (k) = 0.864)], followed by Gen-Probe (k = 0.682). For blood samples, qPCR showed 100% sensitivity and 92.3% specificity, with a substantial correlation with sputum culture (k = 0.754) and with the qPCR results obtained from sputum of the corresponding patient (k = 0.630). Conventional PCR demonstrated the worst results for sputa and blood, with a sensitivity of 100% vs. 88.9% and a specificity of 46.3% vs. 32%, respectively. Commercial or laboratory-developed molecular assays can overcome the difficulties in the diagnosis of TB in paucibacillary patients using conventional methods available in most laboratories.

Novel methods for the encapsulation of meglumine antimoniate into liposomes

The antimonial drug, meglumine antimoniate, was successfully encapsulated in dehydration-rehydration vesicles and in freeze-dried empty liposomes (FDELs). High encapsulation efficiencies (from 28 to 58%) and low weight ratios of lipids to encapsulated antimony (from 1:0.15 to 1:0.3) were achieved. These formulations, contrary to those obtained by conventional methods, can be stored as intermediate lyophilized forms and reconstituted just before use. The efficacy of FDEL-encapsulated meglumine antimoniate was evaluated in hamsters experimentally infected with Leishmania chagasi. A significant reduction of liver parasite burdens was observed in animals treated with this preparation, when compared to control animals treated with empty liposomes. In contrast, free meglumine antimoniate was found to be inefficient when administered at a comparable dose of antimony. This novel liposome-based meglumine antimoniate formulation appears to be promising as a pharmaceutical product for the treatment of visceral leishmaniasis.

Evaluation of the PetrifilmTM and TEMPO® systems and the conventional method for counting microorganisms in pasteurized milk

New microbiological methods have been developed and commercialized, but their performance must be guaranteed. The aim of the present study was to evaluate the PetrifilmTM and TEMPO® systems compared to the conventional method for counting microorganisms in pasteurized milk. A total of 141 samples of pasteurized milk were analyzed by counting mesophilic aerobic, Coliforms at 35 ºC, Coliforms at 45 ºC, and Escherichia coli microorganisms. High correlation was found between the methods for counting Coliforms at 35 ºC, but low correlation was found for counting mesophilic aerobic, Coliforms at 45 ºC, and Escherichia coli. No significant statistical difference was found among the three methods for counting Coliforms at 35 ºC; however, the mean counts of mesophilic aerobic, Coliforms at 45 ºC, and Escherichia coli showed significant statistical difference. PetrifilmTM and TEMPO® systems had satisfactory results for Coliforms at 35 ºC in pasteurized milk but low performance for mesophilic aerobic, Coliforms at 45 ºC and Escherichia coli.

Identification of Leishmania chagasi from skin in Leishmania/HIV co-infection: a case report

A case of HIV/Leishmania co-infection presenting both visceral and cutaneous manifestations is reported. Leishmania infection was confirmed by conventional methods (parasitological approach and serology) and by PCR. Leishmania chagasi isolated from the skin lesion was characterized by enzyme electrophoresis and by restriction fragment length polymorphism of the internal transcribed spacer of the ribosomal gene.

Single-tube nested PCR assay with in-house DNA extraction for Mycobacterium tuberculosis detection in blood and urine

Abstract: INTRODUCTION : Molecular analyses are auxiliary tools for detecting Koch's bacilli in clinical specimens from patients with suspected tuberculosis (TB). However, there are still no efficient diagnostic tests that combine high sensitivity and specificity and yield rapid results in the detection of TB. This study evaluated single-tube nested polymerase chain reaction (STNPCR) as a molecular diagnostic test with low risk of cross contamination for detecting Mycobacterium tuberculosis in clinical samples. METHODS: Mycobacterium tuberculosis deoxyribonucleic acid (DNA) was detected in blood and urine samples by STNPCR followed by agarose gel electrophoresis. In this system, reaction tubes were not opened between the two stages of PCR (simple and nested). RESULTS: STNPCR demonstrated good accuracy in clinical samples with no cross contamination between microtubes. Sensitivity in blood and urine, analyzed in parallel, was 35%-62% for pulmonary and 41%-72% for extrapulmonary TB. The specificity of STNPCR was 100% in most analyses, depending on the type of clinical sample (blood or urine) and clinical form of disease (pulmonary or extrapulmonary). CONCLUSIONS: STNPCR was effective in detecting TB, especially the extrapulmonary form for which sensitivity was higher, and had the advantage of less invasive sample collection from patients for whom a spontaneous sputum sample was unavailable. With low risk of cross contamination, the STNPCR can be used as an adjunct to conventional methods for diagnosing TB.

The vacuum assisted closure of complex wounds: report of 3 cases

Treatment of wounds using conventional methods is frequently limited by inadequate local wound conditions, or by a poor systemic clinical situation. Vacuum system may promote faster granulation tissue formation, remove excessive exudate, increase blood flow in the wound, and attract the borders of the wound to the center, reducing its dimension. We present 3 cases of patients with difficult wounds, due to bad local conditions, or poor clinical situation, in whom we used a vacuum system to prepare the wound for the surgical closure. One patient had a pressure ulcer, another had a diabetic foot ulcer, and the third one had an open foot stump. In the 3 cases a significant improvement of the wound conditions was achieved after 7 to 8 days, allowing successful surgical treatment with flap or skin grafts.

Polymerase chain reaction with two molecular targets in mucosal leishmaniasis' diagnosis: a validation study

We validated the polymerase chain reaction (PCR) with a composite reference standard in 61 patients clinically suspected of having mucosal leishmaniasis, 36 of which were cases and 25 were non-cases according to this reference standard. Patient classification and test application were carried out independently by two blind observers. One pair of primers was used to amplify a fragment of 120 bp in the conserved region of kDNA and another pair was used to amplify the internal transcript spacers (ITS) rDNA. PCR showed 68.6% (95% CI 59.2-72.6) sensitivity and 92% (95% CI 78.9-97.7) specificity; positive likelihood ratio: 8.6 (95% CI 2.8-31.3) and negative likelihood ratio: 0.3 (95% CI 0.3-0.5), when kDNA molecular target was amplified. The test performed better on sensitivity using this target compared to the ITS rDNA molecular target which showed 40% (95% CI 31.5-42.3) sensitivity and 96% (95% CI 84.1-99.3) specificity; positive likelihood ratio: 10 (95% CI 2.0-58.8) and negative likelihood ratio: 0.6 (95% CI 0.6-0.8). The inter-observer agreement was excellent for both tests. Based upon results obtained and due to low performance of conventional methods for diagnosing mucosal leishmaniasis, we consider PCR with kDNA as molecular target is a useful diagnostic test and the ITS rDNA molecular target is useful when the aim is to identify species.

Purification and biochemica characterisation of endoplasmic reticulum α 1,2-mannosidase from Sporothrix schenckiil

Alpha 1,2-mannosidases from glycosyl hydrolase family 47 participate in N-glycan biosynthesis. In filamentous fungi and mammalian cells, α1,2-mannosidases are present in the endoplasmic reticulum (ER) and Golgi complex and are required to generate complex N-glycans. However, lower eukaryotes such Saccharomyces cerevisiae contain only one α1,2-mannosidase in the lumen of the ER and synthesise high-mannose N-glycans. Little is known about the N-glycan structure and the enzyme machinery involved in the synthesis of these oligosaccharides in the dimorphic fungus Sporothrix schenckii. Here, a membrane-bound α-mannosidase from S. schenckii was solubilised using a high-temperature procedure and purified by conventional methods of protein isolation. Analytical zymograms revealed a polypeptide of 75 kDa to be responsible for enzyme activity and this purified protein was recognised by anti-α1,2-mannosidase antibodies. The enzyme hydrolysed Man9GlcNAc2 into Man8GlcNAc2 isomer B and was inhibited preferentially by 1-deoxymannojirimycin. This α1,2-mannosidase was localised in the ER, with the catalytic domain within the lumen of this compartment. These properties are consistent with an ER-localised α1,2-mannosidase of glycosyl hydrolase family 47. Our results also suggested that in contrast to other filamentous fungi, S. schenckii lacks Golgi α1,2-mannosidases and therefore, the processing of N-glycans by α1,2-mannosidases is similar to that present in lower eukaryotes.

Evaluation of polymerase chain reaction in the routine diagnosis for tegumentary leishmaniasis in a referral centre

The present study investigated the diagnostic value of polymerase chain reaction (PCR) performed in parallel to conventional methods at an American tegumentary leishmaniasis (ATL) referral centre for diagnosis. Accuracy parameters for PCR were calculated using 130 patients with confirmed ATL (ATL group), 15 patients established with other diseases and 23 patients with a lesion suggestive of ATL, but without parasitological confirmation (NDEF group). PCR showed 92.3% sensitivity, 93.3% specificity, a 99.2% positive predictive value and a 13.84 positive likelihood ratio. In the NDEF group, PCR confirmed ATL in 13 of the 23 patients, seven of whom responded to leishmaniasis treatment and six who presented spontaneous healing of the lesion. PCR should be included in the routine diagnostic procedures for ATL, especially for cases found to be negative by conventional methods.

Detection of rifampin-resistant genotypes in Mycobacterium tuberculosis by reverse hybridization assay

We used a colorimetric reverse dot blot hybridization (CRDH) assay to detect the presence of mutations in a specific region of the rpoB gene, associated with rifampin (RIF) resistance, in a panel of 156 DNAs extracted from 103 RIF-sensitive and 53 RIF-resistant cultures of Mycobacterium tuberculosis. When compared with the antimicrobial susceptibility test (AST), the sensitivity and specificity of the CRDH were 92.3% and 98.1%, respectively. When compared with sequencing, the sensitivity and specificity of the CRDH were 90.6% and 100%, respectively. To evaluate the performance of the assay directly in clinical specimens, 30 samples from tuberculosis patients were used. For these samples, the results of the CRDH were 100% consistent with the results of the AST and sequencing. These results indicate that the rate of concordance of the CRDH is high when compared to conventional methods and sequencing data. The CRDH can be successfully applied when a rapid test is required for the identification of RIF resistance in M. tuberculosis.

Validation of an immunochromatographic assay kit for the identification of the Mycobacterium tuberculosis complex

The performance of the immunochromatographic assay, SD BIOLINE TB Ag MPT64 RAPID®, was evaluated in Madagascar. Using mouse anti-MPT64 monoclonal antibodies for rapid discrimination between the Mycobacterium tuberculosis complex and nontuberculous mycobacteria, the kit was tested on mycobacteria and other pathogens using conventional methods as the gold standard. The results presented here indicate that this kit has excellent sensitivity (100%) and specificity (100%) compared to standard biochemical detection and can be easily used for the rapid identification of M. tuberculosis complex.

Multiplex polymerase chain reaction to identify and determine the toxigenicity of Corynebacterium spp with zoonotic potential and an overview of human and animal infections

Corynebacterium diphtheriae, Corynebacterium ulcerans and Corynebacterium pseudotuberculosis constitute a group of potentially toxigenic microorganisms that are related to different infectious processes in animal and human hosts. Currently, there is a lack of information on the prevalence of disease caused by these pathogens, which is partially due to a reduction in the frequency of routine laboratory testing. In this study, a multiplex polymerase chain reaction (mPCR) assay that can simultaneously identify and determine the toxigenicity of these corynebacterial species with zoonotic potential was developed. This assay uses five primer pairs targeting the following genes: rpoB (Corynebacterium spp), 16S rRNA (C. ulcerans and C. pseudotuberculosis), pld (C. pseudotuberculosis), dtxR (C. diphtheriae) and tox [diphtheria toxin (DT) ]. In addition to describing this assay, we review the literature regarding the diseases caused by these pathogens. Of the 213 coryneform strains tested, the mPCR results for all toxigenic and non-toxigenic strains of C . diphtheriae, C. ulcerans and C. pseudotuberculosis were in 100% agreement with the results of standard biochemical tests and PCR-DT. As an alternative to conventional methods, due to its advantages of specificity and speed, the mPCR assay used in this study may successfully be applied for the diagnosis of human and/or animal diseases caused by potentially toxigenic corynebacterial species.

Optimisation of an asymmetric polymerase chain reaction assay for the amplification of single-stranded DNA from Wuchereria bancrofti for electrochemical detection

Single-stranded DNA (ssDNA) is a prerequisite for electrochemical sensor-based detection of parasite DNA and other diagnostic applications. To achieve this detection, an asymmetric polymerase chain reaction method was optimised. This method facilitates amplification of ssDNA from the human lymphatic filarial parasite Wuchereria bancrofti. This procedure produced ssDNA fragments of 188 bp in a single step when primer pairs (forward and reverse) were used at a 100:1 molar ratio in the presence of double-stranded template DNA. The ssDNA thus produced was suitable for immobilisation as probe onto the surface of an Indium tin oxide electrode and hybridisation in a system for sequence-specific electrochemical detection of W. bancrofti. The hybridisation of the ssDNA probe and target ssDNA led to considerable decreases in both the anodic and the cathodic currents of the system's redox couple compared with the unhybridised DNA and could be detected via cyclic voltammetry. This method is reproducible and avoids many of the difficulties encountered by conventional methods of filarial parasite DNA detection; thus, it has potential in xenomonitoring.

Spore communities of arbuscular mycorrhizal fungi and mycorrhizal associations in different ecosystems, south Australia

Communities of arbuscular mycorrhizal fungi (AMF) were surveyed in different South Australian ecosystems. The soil was wet-sieved for spore extraction, followed by the determination of presence and abundance of AMF species as well as the percentage of root colonization. Mycorrhizal associations were common and there was substantial fungal diversity in different ecosystems. Spores were most abundant in the permanent pasture system and less abundant under continuous wheat. The incidence of mycorrhizal associations in different plant species and the occurrence of Arum and Paris type colonization generally conformed with previous information. Spores of seventeen AMF were verified throughout seasonal changes in 1996 and 1997 in the permanent pasture and on four host species (Lolium perenne, Plantago lanceolata, Sorghum sp. and Trifolium subterraneum) , set up with the same soils under greenhouse conditions. Glomus mosseae was the dominant spore type at all sampling times and in all trap cultures. Mycorrhizal diversity was significantly affected by different sampling times in trap cultures but not in field-collected soil. P. lanceolata, Sorghum sp. and T. subterraneum as hosts for trap cultures showed no differences in richness and diversity of AMF spores that developed in association with their roots. Abundance and diversity were lowest, however, in association with L. perenne , particularly in December 1996. Results show that the combination of spore identification from field-collected soil and trap cultures is essential to study population and diversity of AMF. The study provides baseline data for ongoing monitoring of mycorrhizal populations using conventional methods and material for the determination of the symbiotic effectiveness of AMF key members.