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PubMedDate not supplied

[A nutrient medium for culturing fungi based on a flores chamomillae infusion. I. The nutrient capacity and capacities for selecting and recovering fungi from clinical samples of a medium with a flores chamomillae infusion].

The study suggests a new culture medium, of the Sabouraud type. The medium was tested for its nutritive and recovery properties of the fungi in clinical samples, in parallel to the classical medium Sabouraud with chloramphenicol (Sab. + C). The results obtained showed the following: the I.F.C. + C medium ensures optimum cultivation conditions of some fungi species, potentially pathogenic for man, has a high selectivity and the ability of making evident the fungi in different clinical samples at very near-related parameters practically identical with the parameters of the classical Sabouraud medium. These qualities, completed by the economical advantages and the availability of the raw material necessary for the preparation of the basic vegetal extract, recommend the use of the I.F.C. + C medium as a nutrient medium for isolation and cultivation of fungi.

Bacteria, Aerobic↗
PubMed2005

A methylation sensitive dot blot assay (MS-DBA) for the quantitative analysis of DNA methylation in clinical samples.

BACKGROUND: There is increasing interest in DNA methylation and in its implication in transcriptional gene silencing, a phenomenon commonly seen in human cancer. AIMS: To develop a new method that would allow quantitative DNA methylation analysis in a large range of clinical samples, independently of the processing protocol. METHODS: A methylation sensitive dot blot assay (MS-DBA) was developed, which is quantitative and combines bisulfite modification, PCR amplification using primers without CpG sites, and dot blot analysis with two probes specific for methylated and unmethylated DNA. RESULTS: The established method was used to study methylation of the hTERT, APC, and p16 promoter regions in microdissected, formalin fixed and paraffin wax embedded tissues. CONCLUSIONS: MS-DBA is a sensitive, specific, and quantitative approach to analyse DNA methylation in a variety of frozen or fixed tissues. Moreover, MS-DBA is rapid, easy to perform, and permits the screening of a large panel of samples in one experiment. Thus, MS-DBA can facilitate the routine analysis of DNA methylation in all types of clinical samples.

Adenocarcinoma↗
PubMed1998

Scripted imagery manipulations and smoking cue reactivity in a clinical sample of self-quitters.

The affectively valenced scripts used by S. Tiffany (1990) suggest that different scripts produce relatively equivalent levels of cue reactivity, although it is unclear if these laboratory findings generalize to clinical samples. In this study, cessation-motivated smokers were tested 7 days before they tried to quit smoking and were exposed to 3 audiotaped scripts that depicted different affectively valenced situations (neutral, positive, or negative). The latter 2 scripts also contained smoking cues. The findings using a clinical sample differed considerably from those using analogue laboratory samples across affective, cognitive, and physiological response measures. Reactivity to these standardized scripts failed to predict treatment outcome through a 30-day follow-up. The use of affectively valenced scripts beyond a laboratory sample is questioned.

Adult↗
PubMed1992

Polymerase chain reaction for the detection of Neisseria gonorrhoeae in clinical samples.

AIMS: To evaluate the use of a cppB gene derived polymerase chain reaction (PCR) assay for direct detection of Neisseria gonorrhoeae in clinical samples. METHODS: A PCR assay was performed on 33 N gonorrhoeae strains and 12 other Neisseria species and other normal genital flora to evaluate the specificity of the chosen cppB primers. The assay was subsequently evaluated with 52 clinical swab samples collected from China. RESULTS: An amplified product of 390 base pairs (bp) was observed with all the N gonorrhoeae strains, each of these products on digestion with the restriction enzyme MspI produced two bands of 250 bp and 140 bp respectively. This set of primers did not produce any amplified product of the expected length with the other non-gonococcal strains tested. For the 52 clinical swabs, 34 were culture positive and PCR successfully detected all these positives. In addition the PCR was positive for two swabs which were culture negative but positive for N gonorrhoeae antigens when tested with the ELISA method (Gonozyme). CONCLUSIONS: This PCR assay is a promising diagnostic tool for detection of gonococci directly from clinical swab samples. Further evaluation is necessary.

Amino Acid Sequence↗
PubMed1999

Improved detection of rhinoviruses in clinical samples by using a newly developed nested reverse transcription-PCR assay.

This paper describes the development and evaluation of a new nested reverse transcription (RT)-PCR for the detection of rhinovirus in clinical samples. The nucleotide sequences of the 5' noncoding regions of 39 rhinoviruses were determined in order to map the most conserved subregions. We designed a set of rhinovirus-specific primers and probes directed to these subregions and developed a new nested RT-PCR. The new assay includes an optimal RNA extraction method and amplicon identification with probe hybridization to discriminate between rhinoviruses and the closely related enteroviruses. It proved to be highly sensitive and specific. When tested on a dilution series of cultured viruses, the new PCR protocol scored positive at 10- to 100-fold-higher dilutions than a previously used nested RT-PCR. When tested on a collection of clinical samples obtained from 1,070 acute respiratory disease patients who had consulted their general practitioners, the new assay demonstrated a rhinovirus in 24% of the specimens, including all culture-positive samples, whereas the previously used PCR assay or virus culture detected a rhinovirus in only 3.5 to 6% of the samples. This new assay should help determine the disease burden associated with rhinovirus infections.

Base Sequence↗
PubMed2000

A comparative study of immunocapture ELISA and RT-PCR for screening clinical samples from Southern Greece for human influenza virus types A and B.

An immunocapture (IC) ELISA and reverse transcriptase (RT)-PCR assays were evaluated as screening methods for the detection of influenza virus types A and B in clinical samples collected from individuals presenting with influenza-like symptoms in Southern Greece. Standard virus isolation in embryonated hens' eggs was taken as the reference method. According to the reference method, 25 (16.7%) of the 150 clinical samples examined were infected by influenza viruses - 19 type A (H3N2) and 6 type B. The sensitivity of immunocapture ELISA was 64% and that for RT-PCR was 100%. The specificity of IC ELISA was 98% and by RT-PCR 97%. The positive diagnostic value of IC ELISA was 94% and of RT-PCR 86%, whereas the negative diagnostic values for IC ELISA and PCR were 93% and 100%, respectively. These findings confirm that RT-PCR provides significantly increased sensitivity over IC ELISA and can be of value in the management of regional influenza screening surveys conducted by the national public health services.

Adolescent↗
PubMed2026

Molecular DNA enrichment methods for parasite genomic sequencing in clinical samples: a systematic review.

Parasitic diseases such as malaria, Chagas disease, leishmaniases, and helminthiases are major causes of sickness and death in low- and middle-income countries. The high genetic diversity of these pathogens affects virulence, immune evasion, and diagnostic accuracy. Although Whole Genome Sequencing (WGS) is a powerful tool for tracking genetic variants and drug resistance, low parasitemia and the predominance of host DNA limit its application to clinical samples. This study systematically reviewed molecular strategies to improve the recovery of parasite DNA from clinical samples, following PRISMA 2020 guidelines and registered in PROSPERO. Searches of PubMed, Scopus, Web of Science, and LILACS up to December 2025 identified 20 eligible studies, most of which focused on protozoa, particularly Plasmodium spp. The main approaches included hybridization capture, selective whole-genome amplification, host DNA depletion, and in silico enrichment via adaptive sampling. Overall, no single method is suitable for all parasites analyzed; the optimal approach depends on the pathogen, sample type, and research objective. The review emphasizes that parasite DNA enrichment is essential for enabling WGS in clinical settings, underscoring the need for protocol standardization and cost-effectiveness analyses to support public health genomic surveillance.

Adaptive sampling↗
PubMed1991

A simple method for diagnosing M. tuberculosis infection in clinical samples using PCR.

Species identification of Mycobacterium tuberculosis remains a cumbersome process. We have developed a simple method for treating clinical samples which permits direct polymerase chain reaction (PCR) amplification of mycobacterial target DNA without organic extraction. Samples were boiled for 30 min in TE-Triton, then were subjected to 30 cycles of amplification using primers derived from the repetitive clone pMTb4 developed by Patel and coworkers (1990; Journal of Clinical Microbiology 28, 513). Specificity of amplification was confirmed by hybridization with a specific probe labelled non-isotopically. In a model system consisting of cultured bacilli, this system routinely allows detection of a single organism in a sample. In preliminary studies examining applicability of this method to 96 clinical samples, 74 were positive by both PCR and mycobacterial culture, and eight were negative by both methods. Fourteen samples were negative by culture and positive by PCR, and none were positive by culture and negative by PCR. These results suggest that the PCR method may provide a sensitive alternative to conventional species-specific diagnostic methods, and that non-isotopic labelling can be used to detect hybridization in this assay.

Base Sequence↗
PubMed2002

Detection of encephalomyocarditis virus in clinical samples by immunomagnetic separation and one-step RT-PCR.

A method of immunomagnetic separation and one-step reverse transcription-polymerase chain reaction (RT-PCR) was developed for the detection of Encephalomyocarditis virus (EMCV). EMCV was captured from sample on magnetic beads with homologous monoclonal antibody and then heat denatured. The heated beads were used directly in one-step RT-PCR reaction to amplify a 285-bp PCR fragment at the 3' end of the genomic region that encodes the viral polymerase. This method detected as little as 3.5 TCID(50) of EMCV from infected cell culture. It was shown with this method that the sensitivity of RT-PCR increased when applied for the detection of EMCV added to fecal extract. Using this protocol EMCV was detected from heart homogenate samples containing less than 100 TCID(50)/ml. The amplified product was sequenced to ensure specificity. The immunomagnetic-RT/PCR procedure described here should be useful for the rapid, specific and sensitive detection of EMCV in clinical samples. This technique is rapid, reliable and can be readily adapted to detect EMCV from other clinical samples.

Animals↗
PubMed2001

Genotypic characterization of Toxoplasma gondii strains associated with human toxoplasmosis in Spain: direct analysis from clinical samples.

Genetic analysis of the SAG2 locus was performed to determine the prevalence of the different genotypes of Toxoplasma gondii (strain types I, II, and III) associated with human toxoplasmosis in Spain. This determination was made directly from primary clinical samples, obviating the previous process of isolation in mice or cell culture. A total of 34 isolates of T. gondii, collected from immunocompromised patients and congenital infection cases, were analyzed. Restriction fragment length polymorphism in PCR-amplified SAG2 products was used to group strains into one of the three genotypes of T. gondii. Complete characterization of the SAG2 gene was successful in 76.5% of the cases, demonstrating the feasibility of direct genotype analysis from clinical samples of different origins. Strains of T. gondii type II were the most prevalent in immunocompromised patients, with 52% of cases, while strains of type I were present in 75% of the congenital infection cases. These data differ from previous reports that show type II strains to be mostly associated with all kinds of human toxoplasmosis. These differences might be an effect of selection in the process of culture and isolation of the samples performed by other researchers prior to strain characterization.

Animals↗
PubMed2006

Microarrays as validation strategies in clinical samples: tissue and protein microarrays.

The widespread use of DNA microarrays has led to the discovery of many genes whose expression profile may have significant clinical relevance. The translation of this data to the bedside requires that gene expression be validated as protein expression, and that annotated clinical samples be available for correlative and quantitative studies to assess clinical context and usefulness of putative biomarkers. We review two microarray platforms developed to facilitate the clinical validation of candidate biomarkers: tissue microarrays and reverse-phase protein microarrays. Tissue microarrays are arrays of core biopsies obtained from paraffin-embedded tissues, which can be assayed for histologically-specific protein expression by immunohistochemistry. Reverse-phase protein microarrays consist of arrays of cell lysates or, more recently, plasma or serum samples, which can be assayed for protein quantity and for the presence of post-translational modifications such as phosphorylation. Although these platforms are limited by the availability of validated antibodies, both enable the preservation of precious clinical samples as well as experimental standardization in a high-throughput manner proper to microarray technologies. While tissue microarrays are rapidly becoming a mainstay of translational research, reverse-phase protein microarrays require further technical refinements and validation prior to their widespread adoption by research laboratories.

Animals↗
PubMed2001

Burkholderia fungorum sp. nov. and Burkholderia caledonica sp. nov., two new species isolated from the environment, animals and human clinical samples.

A polyphasic taxonomic study that included DNA-DNA hybridizations, DNA base ratio determinations, 16S rDNA sequence analyses, whole-cell protein and fatty acid analyses and an extensive biochemical characterization was performed on 16 strains isolated from the environment, animals and human clinical samples. The isolates belonged to the genus Burkholderia, were phylogenetically closely related to Burkholderia graminis, Burkholderia caribensis and Burkholderia phenazinium and had G+C contents between 61.9 and 62.2 mol%. Seven strains isolated from the rhizosphere were assigned to Burkholderia caledonica sp. nov. [type strain LMG 19076T (= CCUG 42236T)]. Nine strains isolated from the environment, animals and human clinical samples were assigned to Burkholderia fungorum sp. nov. [type strain LMG 16225T (= CCUG 31961T)]. Differential tests for B. graminis, B. caribensis, B. phenazinium, B. caledonica and B. fungorum include the following: assimilation of trehalose, citrate, DL-norleucine, adipate and sucrose; nitrate reduction; growth in the presence of 0.5% NaCl; and beta-galactosidase activity.

Animals↗
PubMed2004

The Eating Disorder Inventory-2 in Japanese clinical and non-clinical samples: psychometric properties and cross-cultural implications.

OBJECTIVE: To determine the usefulness of and cross-cultural differences in the Japanese version of the eating disorder inventory-2 (EDI-2). METHOD: A Japanese version of the EDI-2 was administered to a consecutive series (1995-2001) of 91 Japanese eating disordered inpatients and 119 matched non-clinical controls. Scores were compared with each other and with those of Canadian data previously reported. Internal consistencies were also examined. RESULTS: All subscales (except Ascetism) reached sufficient internal consistencies. The patients' mean scores were significantly higher than controls on most subscales. Restricting anorexics (AN-R) scored higher than controls on body dissatisfaction (BD) and Social Insecurity (SI) subscales. Purging bulimics (BN-P) scored higher than controls on most subscales, except for maturity fears (MF) and SI subscales. Scores of binge-eating/purging type anorexics (AN-B/P) were higher than controls on all subscales. AN-B/P and BN-P scored higher than AN-R on bulimia (B) subscale. Comparison with Canadian subjects showed that Japanese control subjects scored significantly higher on the MF, ineffectiveness (I), impulse regulation (IR), SI subscales, and lower on the perfectionism (P) subscale. DISCUSSION: The EDI-2 is useful and reliable in understanding Japanese clinical and non-clinical samples. High MF and low P subscales in our samples confirm previous findings in Chinese samples suggesting meaningful cultural differences in the emphasis placed on family bonds and individual accomplishments. In addition, high I, IR, and SI scores in Japanese non-clinical samples may reflect recent problems in Japanese culture.

Adult↗
PubMed1999

A sensitive, non-radioactive quantitative method for measuring IL-4 and IL-4delta2 mRNA in unstimulated cells from multiple clinical samples, using nested RT-PCR.

Measurements of IL-4 mRNA directly from clinical samples are technically difficult as IL-4 is a low copy number cytokine. Moreover, most existing studies involving RT-PCR are confused by the use of primers which simultaneously amplify cDNA of IL-4 and its splice-variant (IL-4delta2). We describe a sensitive nested RT-PCR method to quantify mRNA expression of IL-4 and IL-4delta2 separately. It involves a simple method of generating cRNA standards without cloning. The use of external synthetic RNA standards, for which we validate that amplification kinetics are equivalent to the target, obviates the need for multiple sample dilutions. The assay is sensitive enough to measure IL-4 and IL-4delta2 mRNA expression in unstimulated PBMCs of normal subjects, and the reproducibility and throughput make this assay suitable for use in clinical studies with multiple samples.

Alternative Splicing↗
PubMed2003

Identification and differentiation of Leishmania species in clinical samples by PCR amplification of the miniexon sequence and subsequent restriction fragment length polymorphism analysis.

We recently developed a new PCR-restriction fragment length polymorphism (RFLP)-based assay using the miniexon sequence from the genus Leishmania. Here we report the application of this new genotyping method to naturally infected clinical samples for the differentiation of New and Old World Leishmania species. Of the newly developed assay and four currently applied diagnostic tests (i.e., in vitro cultivation, serology, and two other molecular assays using either the small subunit-internal transcribed spacer sequence or a repetitive genomic sequence), the miniexon assay showed the highest sensitivity, 89.7%, compared to 70.6, 57.1, 51.7, and 79.3%, respectively. Species differentiation was robust and reliable compared with that by two other Leishmania genotyping techniques. The assay provides a valuable tool for the identification of Leishmania directly from clinical samples and enables determination of the infecting species by a facile technique with high discrimination power. Since Leishmania causes a broad spectrum of diseases distinguished by different parasite and host factors, detection and characterization of the infecting species is crucial for the confirmation of a diagnosis as well as the establishment of the clinical prognosis and the initiation of an adequate therapeutic approach. The miniexon PCR-RFLP assay will facilitate such determination and might improve diagnosis and treatment of leishmaniasis.

Animals↗
PubMed2006

Novel phylogenetic studies of genomic sequence fragments derived from uncultured microbe mixtures in environmental and clinical samples.

A self-organizing map (SOM) was developed as a novel bioinformatics strategy for phylogenetic classification of sequence fragments obtained from pooled genome samples of uncultured microbes in environmental and clinical samples. This phylogenetic classification was possible without either orthologous sequence sets or sequence alignments. We first constructed SOMs for tetranucleotide frequencies in 210,000 5 kb sequence fragments obtained from 1502 prokaryotes for which at least 10 kb of genomic sequence has been deposited in public DNA databases. The sequences could be classified primarily according to phylogenetic groups without information regarding the species. We used the SOM method to classify sequence fragments derived from environmental samples of the Sargasso Sea and of an acidophilic biofilm growing in acid mine drainage. Phylogenetic diversity of the environmental sequences was effectively visualized on a single map. Sequences that were derived from a single genome but cloned independently could be reassociated in silico. G + C% has been used for a long period as a fundamental parameter for phylogenetic classification of microbes, but the G + C% is apparently too simple a parameter to differentiate a wide variety of known species. Oligonucleotide frequency can be used to distinguish the species because oligonucleotide frequencies vary significantly among their genomes.

Algorithms↗
PubMed1998

Detection of infectious bursal disease virus of poultry in clinical samples by RT-PCR.

The RT-PCR technique was adopted to amplify variable region of VP2 gene of infectious bursal disease virus using three different sets of primers. These primers could generate products of 643, 474 and 552 bp sizes. The authenticity of the amplicons was confirmed by their size in agarose gel, restriction enzyme digestion and by nested PCR. Out of total five clinical samples tested, IBD viral genomic RNA could be detected in four by RT-PCR. Restriction enzyme digestion of PCR products with StuI could differentiate clinical samples from an intermediate vaccine strain currently being used in India.

Animals↗
PubMed2003

Short report: quantification of leishmaniavirus RNA in clinical samples and its possible role in pathogenesis.

Leishmaniavirus (LRV) is a double-stranded RNA virus that infects the protozoa Leishmania and has been identified in numerous strains of Leishmania braziliensis and L. braziliensis guyanensis. In general, the species of Leishmania dictates disease manifestation except in the case of L. braziliensis, which is capable of causing either cutaneous or mucocutaneous leishmaniasis. We wanted to determine 1) the quantity of LRV RNA present in a clinical sample and 2) if infection with LRV was associated with a specific disease manifestation. A real-time reverse transcriptase-polymerase chain reaction assay was used to assay clinical samples for the presence of LRV. Of 47 samples tested, 12 positive samples were obtained from patients with cutaneous lesions, lesions in the process of scarring, and cutaneous scars. This is the first study to examine the prevalence of LRV RNA within a small cohort from Brazil.

Adolescent↗