An investigation of plasma collection, stabilization, and storage procedures for proteomic analysis of clinical samples

In order to evaluate the critical components of the process necessary to preserve clinical plasma samples collected at research sites for proteomic analysis, various collection and preservation protocols with controlled experimentation were evaluated. The presence of a protease inhibitor cocktail (PIC) included in the blood draw tube would stabilize the plasma proteins was hypothesized. To test this hypothesis, four plasma samples from each of 14 volunteers were collected. Samples were treated following a standard protocol that included PIC or were subjected to various processing treatments that included time, temperature, different anticoagulants, and the absence of PIC. Large format two dimensional-polyacrylamide gel electrophoresis (2D-PAGE) proteomic analysis and enzyme immunoassay (EIA) were used to detect differences between the treatment groups. A novel 2D-PAGE quality scoring method was developed to determine global differences in the treatment groups, wherein a rating scale questionnaire was used to quantify the quality of each 2D-PAGE gel. The data generated from EIAs, classical 2D-PAGE image analysis and 2D-PAGE quality scoring, each generated similar results. Inclusion of protease inhibitor cocktail in the sample tubes, provided stable and reliable human plasma samples that yielded reproducible results by proteomic analysis. When PIC was included, samples retained stability under less stringent processing, such that refrigeration for several hours before processing or one freeze-thaw cycle had little detrimental effect. We demonstrated that samples without PIC, from either heparin or ethylenediaminetetraacetic acid (EDTA) plasma tubes, gave results that varied significantly from the control samples. Also, even with PIC present in blood tubes, we found it was important to quickly decant the separated plasma from the cellular components found in the blood tubes following centrifugation, as prolonged exposure again yielded different results from the standard procedure.     

Improvement in cyst recovery and molecular detection of Giardia duodenalis from stool samples

Molecular Biology Reports - Molecular detection of Giardia duodenalis by polymerase chain reaction (PCR) is difficult in faecal samples due to inhibitors that contaminate DNA preparations, or due...     

DNA isolation from museum and type collection slides of microsporidia

DNA from 19 species of microsporidia was isolated and amplified from infected host tissue that were originally prepared between the years 1946 and 1996. The smears, on glass microscope slides, were either Giemsa-stained or unstained. Methanol-fixed, Giemsa-stained smears proved to be suitable for DNA isolation; DNA was amplified from only two of 14 unstained slides. The isolated DNA was successfully amplified in PCRs using small subunit and large subunit rDNA primers and sequenced. The high efficiency of DNA isolation demonstrates the usefulness of archival and type collection slides for some molecular biology and molecular taxonomy studies.     

Identification and characterization of microsporidia from fecal samples of HIV-positive patients from Lagos, Nigeria

Background

Microsporidia are obligate intracellular parasites that infect a broad range of vertebrates and invertebrates. They have been increasingly recognized as human pathogens in AIDS patients, mainly associated with a life-threatening chronic diarrhea and systemic disease. However, to date the global epidemiology of human microsporidiosis is poorly understood, and recent data suggest that the incidence of these pathogens is much higher than previously reported and may represent a neglected etiological agent of more common diseases indeed in immunocompetent individuals. To contribute to the knowledge of microsporidia molecular epidemiology in HIV-positive patients in Nigeria, the authors tested stool samples proceeding from patients with and without diarrhea.

Methodology/Principal Findings

Stool samples from 193 HIV-positive patients with and without diarrhea (67 and 126 respectively) from Lagos (Nigeria) were investigated for the presence of microsporidia and Cryptosporidium using Weber’s Chromotrope-based stain, Kinyoun stain, IFAT and PCR. The Weber stain showed 45 fecal samples (23.3%) with characteristic microsporidia spores, and a significant association of microsporidia with diarrhea was observed (O.R.  = 18.2; CI: 95%). A similar result was obtained using Kinyoun stain, showing 44 (31,8%) positive samples with structures morphologically compatible with Cryptosporidium sp, 14 (31.8%) of them with infection mixed with microsporidia. The characterization of microsporidia species by IFAT and PCR allowed identification of Enterocytozoon bieneusi, Encephalitozoon intestinalis and E. cuniculi in 5, 2 and 1 samples respectively. The partial sequencing of the ITS region of the rRNA genes showed that the three isolates of E.bieneusi studied are included in Group I, one of which bears the genotype B.

Conclusions/Significance

To our knowledge, this is the first report of microsporidia characterization in fecal samples from HIV-positive patients from Lagos, Nigeria. These results focus attention on the need to include microsporidial diagnosis in the management of HIV/AIDS infection in Nigeria, at the very least when other more common pathogens have not been detected.     

Identification of Encephalitozoon and Enterocytozoon (microsporidia) spores in stool and urine samples obtained from free-living South American Coatis (Nasua nasua)

This study emphasizes the importance of free-living coatis as a potential source of microsporidian infection for humans living in large cities. We found 19 (31.7%) positive results among 60 fecal samples analyzed by PCR-based analysis and the Gram-Chromotrope staining technique (11.7% were positive for Encephalitozoon cuniculi, 6.7% for E. intestinalis, 6.7% for E. hellem, and 6.7% for Enterocytozoon bieneusi). Only 5 (8.4%) urine samples tested positive for E. cuniculi as assessed by the two techniques.     

Long-term storage and safe retrieval of DNA from microorganisms for molecular analysis using FTA matrix cards

We assessed the potential use of Whatman FTA paper as a device for archiving and long-term storage of bacterial cell suspensions of over 400 bacterial strains representing 61 genera, the molecular applications of immobilised DNA on FTA paper, and tested its microbial inactivation properties. The FTA paper extracted bacterial DNA is of sufficiently high quality to successfully carryout the molecular detection of several key genes including 16S rRNA, esp (Enterococcus surface protein), Bft (Bacteroides fragilis enterotoxin) and por (porin protein) by PCR and for DNA fingerprinting by random amplified polymorphic DNA-PCR (RAPD-PCR). To test the long-term stability of the FTA immobilised DNA, 100 of the 400 archived bacterial samples were randomly selected following 3 years of storage at ambient temperature and PCR amplification was used to monitor its success. All of the 100 samples were successfully amplified using the 16S rDNA gene as a target and confirmed by DNA sequencing. Furthermore, the DNA was eluted into solution from the FTA cards using a new alkaline elution procedure for evaluation by real-time PCR-based assays. The viability of cells retained on the FTA cards varied among broad groups of bacteria. For the more fragile gram-negative species, no viable cells were retained even at high cell densities of between 10(7) and 10(8) colony forming units (cfu) ml(-1), and for the most robust species such as spore-formers and acid-fast bacteria, complete inactivation was achieved at cell densities ranging between 10(1) and 10(4) cfu ml(-1). The inactivation of bacterial cells on FTA cards suggest that this is a safe medium for the storage and transport of bacterial nucleic acids.     

The use of Endopep-MS for the detection of botulinum toxins A, B, E, and F in serum and stool samples

Botulinum neurotoxin (BoNT) causes the disease botulism, which can be lethal if untreated. Previous work in our laboratory focused on developing Endopep-MS, a mass spectrometric-based endopeptidase method for the detection and differentiation of BoNT serotypes. We have expanded this effort to include an antibody capture method to partially purify and concentrate BoNT from serum and stool extract samples for the Endopep-MS assay. Because complex matrices such as serum and stool contain abundant endogenous proteases, this technique was needed to remove most proteases from the sample while concentrating BoNT from a sample size of 100 to 500 microl to 20 microl. When this antibody capture method is combined with the Endopep-MS reaction, limits of detection in 500mul of spiked human serum are 10 mouse LD50 (20 mouse LD50/ml) for BoNT A, 0.5 mouse LD50 (1 mouse LD50/ml) for BoNT B, 0.1 mouse LD50 (0.2 mouse LD50/ml) for BoNT E, and 0.5 mouse LD50 (1 mouse LD50/ml) for BoNT F. The limits of detection in spiked stool extracts are somewhat higher due to the high-protease environment of stool extract that also requires use of protease inhibitors. The entire method can be performed in as short a time as 4 h.     

Evaluation of chromogenic media for the isolation of vancomycin-resistant enterococci from stool samples

Aims:  This study sought to evaluate the performance of two chromogenic media designed for the isolation of vancomycin-resistant enterococci (VRE) and compare them with a traditional bile-esculin medium for the isolation of VRE from stool samples.
Methods and Results:  A total of 285 stool samples were inoculated onto Chromogenic VRE Agar (AES VRE agar; AES Chemunex), chromID VRE (bioMérieux) and VRE Agar (Oxoid) both directly and also following broth enrichment. In total 18 strains of vancomycin-resistant Enterococcus faecium were recovered, including 17 harbouring the vanA gene and one with vanB . On direct culture, the sensitivity of the three media was 66·7%, 77·8% and 44·4% and after broth enrichment 66·7%, 83·3% and 77·8% using AES VRE Agar, chromID VRE and Oxoid VRE Agar respectively.
Conclusions:  All three media are useful tools for the isolation of VRE from stool samples. AES VRE Agar and bioMérieux chromID VRE are easier to use than Oxoid VRE Agar due to diffusion of black coloration from the latter.
Significance and Impact of the Study:  This is the first study to evaluate the performance of AES VRE Agar and the first to compare two media containing synthetic chromogens for the isolation of VRE.     

Detection and characterization of Shigella species isolated from food and human stool samples in Nabeul, Tunisia, by molecular methods and culture techniques

Aims: This study was designed to isolate Shigella spp. strains from food and stool samples by a combination of PCR and culture methods and characterize their serotypes, antibiotic resistance profiles, virulence genes and pulsed‐field gel electrophoresis (PFGE) patterns to investigate possible clonal relationships amongst strains circulating. Methods and Results: Six Shigella spp. strains were isolated from 280 food samples against 16 Shigella isolates from 236 stool samples of symptomatic patients and asymptomatic food handlers during the period from January 2007 to December 2009 in Public Health Regional Laboratory of Nabeul. The detection of ipaH, ipaBCD, ial, ShET‐1 and ShET‐2 was performed by a PCR technique with specific primers. Conclusions: The use of PCR technique improved the rate of detecting Shigella in stool samples from 6·7 to 14% and in food samples from 2·1 to 8·6%. Percentage of Shigella isolates and ipaH‐specific PCR demonstrated a marked pattern of seasonality, increasing in summer and fall seasons for human and food isolates. Amongst the environmental strains, 50% of isolates were invasive. However, for the 16 clinical strains isolated, nine were found to be positive for both ial and ipaBCD gene and 11 were found to produce ShET‐1 and/or ShET‐2. XbaI PFGE analysis revealed the presence of a predominant clone amongst Shigella sonnei strains recovered from different sources circulating in Nabeul, Tunisia, throughout the years 2007–2009. Significance and Impact of the Study: This study demonstrated the existence of Shigella in food samples and dispersion of different virulence genes amongst these isolates, which appear to constitute an environmental source of epidemic spread. The clonal relationships amongst strains isolated from food elements and human stools indicate the incrimination of different kinds of foods as vehicle of transmission of Shigella, which are usually escaped from detection by traditional culture methods.     

Preservation of Giardia cysts in stool samples for subsequent PCR analysis

Genotyping of Giardia duodenalis cysts in faecal samples has become a regularly employed tool by researchers investigating different aspects of the epidemiology and pathology of Giardia infection in human and animal populations. However, such investigations are often limited to some extent by lack of PCR amplification from a proportion of the samples, and this often seems to be associated with the storage medium used for the samples. Various different storage media have been used in different studies, but investigation of which storage media are most appropriate and which may compromise subsequent PCR investigations has not been systematically explored to date.In this study, 4 different, commonly used storage media were investigated for their effects over time on subsequent PCR amplification of DNA from Giardia cysts in stool samples. Microscopic examination of the samples and real-time PCR were used to investigate 7 different samples over a period of 3 months. Our findings indicate that storage in ethanol or potassium dichromate at 4 °C gave the best results and, that if immunomagnetic separation was used prior to PCR (as may be appropriate for samples with low cyst numbers), then storage in potassium dichromate gave the best results.     

Comparison of three concentration methods for the recovery of canine intestinal parasites from stool samples

The aim of present study was to compare the efficiency of a commercial assay and two conventional methods for fecal concentration in detecting canine gastrointestinal parasites. Fecal samples from 254 dogs were processed by centrifugation-sedimentation (CS), centrifugation-flotation (CF) and a commercial assay for fecal concentration (TF-test®). The following parasites were detected: Ancylostoma (37.8%), Giardia (16.9%), Toxocara canis (8.7%), Trichuris vulpis (7.1%), Isospora (3.5%), and Sarcocystis (2.7%). The calculated analytical sensitivity indicated that CF was more accurate (P < 0.01) in detecting Ancylostoma, T. canis, T. vulpis and Giardia infections. However, CF showed significantly higher sensitivity only for Ancylostoma, compared to the other two methods. The kappa index value of diagnostic agreement between TF-test and CF was high for T. canis (83%) and moderate for Giardia (72%) and Ancylostoma (63%). The advantages and limitations of each method were assessed for individual diagnosis and epidemiological investigation.     

Evaluation of Whatman FTA cards for the preservation of yellow fever virus RNA for use in molecular diagnostics

Yellow fever virus (YFV) is a flavivirus that frequently causes outbreaks of hemorrhagic fever in Africa and South America and is considered a reemerging public health threat. Accurate diagnosis of yellow fever (YF) disease is critical as one confirmed case constitutes an outbreak and may trigger a mass vaccination campaign. Highly sensitive and specific molecular diagnostics have been developed; however, these assays require maintenance of cold-chain during transport of specimens to prevent the degradation of viral RNA prior to testing. Such cold-chain requirements are difficult to meet in some regions. In this study, we investigated Whatman FTA cards as an alternative stabilization method of YFV RNA for use in molecular diagnosis. Using contrived specimens, linear regression analysis showed that RNA detection from a single 6mm FTA card punch was significantly less sensitive than traditional RNA extraction; however, pooling RNA extracted from two FTA punches significantly lowered the limit of detection to be equal to that of the traditional RNA extraction gold standard. In experiments addressing the ability of FTA card methodology to stabilize YFV RNA at variable temperature, RNA could be detected for more than two weeks following storage at 25°C. Even more promising, YFV RNA was detectable on cards held at 37°C from two days to over two weeks depending on viral input. FTA cards were also shown to stabilize YFV RNA at high humidity if cards were desiccated prior to inoculation. These results support that FTA cards could be cost effective and easy to use in molecular diagnosis of YF, preserving viral RNA to allow for positive diagnoses in situations where maintaining cold-chain is not feasible.     

Routine identification of Campylobacter jejuni and Campylobacter coli from human stool samples

Correct identification of Campylobacter jejuni and Campylobacter coli isolates to the species or subspecies level is a cumbersome but nevertheless important task for a routine diagnostic laboratory. The widely used biochemical tests might be often misleading while more sophisticated phenotypic or genotypic methods are not generally available. This investigation was performed to assess the performance of common biochemical identification in comparison with species-specific PCR and gas liquid chromatography of whole cell fatty acid extracts (GLC). A total of 150 consecutive isolates from human stool samples were investigated (134 C. jejuni ssp. jejuni, 14 C. coli, two Helicobacter pullorum). From these 144, 145 and 149 isolates were correctly identified by biochemistry, GLC and PCR, respectively. Biochemical identification of all C. jejuni isolates was confirmed by PCR. GLC detected both H. pullorum strains but misidentified two C. coli strains as C. jejuni and one C. jejuni strain as C. coli. No single method can be defined as 'gold standard' for identification of C. jejuni and C. coli but a combination of techniques is needed. Therefore a stepwise identification scheme starting with biochemical reactions is suggested. All results other than C. jejuni should be confirmed by further methods. For indoxyl acetate-positive isolates species-specific PCR is recommended while GLC seems to be advantageous in indoxyl acetate-negative isolates.     

An improved simple technique for the collection of blood samples from rats and mice

The technique of blood collection from the lateral caudal vein has been improved. The method requires only moderate skill and no anaesthesia is necessary. Collection of blood samples causes little trauma and can be repeated at frequent (8 h) intervals thus making the method particularly suitable for pharmacokinetic and hormonal studies. Blood samples are uncontaminated by tissue fluids. Large volumes can be obtained, in mice up to 1.5 ml and in Wistar rats over 4 ml.     

正常人粪便中酪酸梭菌的分离与分子鉴定

【摘 要】 目的 肠道益生菌在人体内发挥重要的生理作用,探明肠道益生菌群的作用对人体有着十分重要的意义。运用TSN培养基,从健康正常人的粪便中分离培养、筛选出1株肠道菌株。方法 从健康正常人粪便中分离与纯化肠道细菌,获得一株具有特殊作用的细菌,通过对该菌的细菌培养、菌落形态观察、革兰染色镜检、分离纯化培养、生化试验并结合16S rDNA方法进行了鉴定。结果 该菌为革兰阳性梭状芽胞杆菌,属于梭菌属,分离培养出的1株菌株为酪酸梭菌亚种JMY1。结论 本实验为进一步研究酪酸梭菌的作用及其是否对结肠癌发挥作用奠定了实验基础并提供了一定的理论依据。     

Dipstick test for rapid diagnosis of Shigella dysenteriae 1 in bacterial cultures and its potential use on stool samples

Background

We describe a test for rapid detection of S. dysenteriae 1 in bacterial cultures and in stools, at the bedside of patients.

Methodology/Principal Findings

The test is based on the detection of S. dysenteriae 1 lipopolysaccharide (LPS) using serotype 1-specific monoclonal antibodies coupled to gold particles and displayed on a one-step immunochromatographic dipstick. A concentration as low as 15 ng/ml of LPS was detected in distilled water and in reconstituted stools in 10 minutes. In distilled water and in reconstituted stools, an unequivocal positive reaction was obtained with 1.6×10⁶ CFU/ml and 4.9×10⁶ CFU/ml of S. dysenteriae 1, respectively. Optimal conditions to read the test have been determined to limit the risk of ambiguous results due to appearance of a faint yellow test band in some negative samples. The specificity was 100% when tested with a battery of Shigella and unrelated strains in culture. When tested on 328 clinical samples in India, Vietnam, Senegal and France by laboratory technicians and in Democratic Republic of Congo by a field technician, the specificity (312/316) was 98.7% (95% CI:96.6–99.6%) and the sensitivity (11/12) was 91.7% (95% CI:59.8–99.6%). Stool cultures and the immunochromatographic test showed concordant results in 98.4 % of cases (323/328) in comparative studies. Positive and negative predictive values were 73.3% (95% CI:44.8–91.1%) and 99.7% (95% CI:98–100%).

Conclusion

The initial findings presented here for a simple dipstick-based test to diagnose S. dysenteriae 1 demonstrates its promising potential to become a powerful tool for case management and epidemiological surveys.     

Evaluation of procedures for the collection, processing, and analysis of biomolecules from low-biomass surfaces

To comprehensively assess microbial diversity and abundance via molecular-analysis-based methods, procedures for sample collection, processing, and analysis were evaluated in depth. A model microbial community (MMC) of known composition, representative of a typical low-biomass surface sample, was used to examine the effects of variables in sampling matrices, target cell density/molecule concentration, and cryogenic storage on the overall efficacy of the sampling regimen. The MMC used in this study comprised 11 distinct species of bacterial, archaeal, and fungal lineages associated with either spacecraft or clean-room surfaces. A known cellular density of MMC was deposited onto stainless steel coupons, and after drying, a variety of sampling devices were used to recover cells and biomolecules. The biomolecules and cells/spores recovered from each collection device were assessed by cultivable and microscopic enumeration, and quantitative and species-specific PCR assays. rRNA gene-based quantitative PCR analysis showed that cotton swabs were superior to nylon-flocked swabs for sampling of small surface areas, and for larger surfaces, biological sampling kits significantly outperformed polyester wipes. Species-specific PCR revealed differential recovery of certain species dependent upon the sampling device employed. The results of this study empower current and future molecular-analysis-based microbial sampling and processing methodologies.     

The use of molecular markers for germplasm management in a French olive collection

With more than 100 accessions, the CBNMP olive collection includes a major part of the French germplasm. We used molecular markers to characterise all accessions and to study genetic relationships between cultivars. Firstly, 497 olive trees were genotyped using 32 RAPD markers. We identified 114 RAPD profiles and detected several cases of mislabelling, synonymy and homonymy. Secondly, for each RAPD profile, one tree was analysed using mtDNA RFLPs to determine the cytoplasmic lineage of each cultivar and using five nuclear SSR loci. French germplasm displayed ME1, MOM and MCK mitotypes with ME1 prevailing (84%). Based on SSR markers, we revealed a slight differentiation between French cultivars growing in the West and the East side of the Rh?ne Valley. This study allowed us to construct a molecular data-base for the reference collection and to analyse genetic diversity for further prospecting, and for introducing new olive accessions.