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PubMed1992

Heparin-binding (fibroblast) growth factors are potential autocrine regulators of esophageal epithelial cell proliferation.

A serum-free culture system supplemented with neural tissue extract for normal human esophagus was applied to the culture of mouse esophageal epithelium. Similar to mouse mesenchyme and skin epithelium, esophageal epithelial lines (MEE) emerged after serial culture. The cells had an apparent unlimited life-span, but retained morphology and other characteristics of normal epithelial cells. A screen for growth factors that stimulated growth of MEE cells in the absence of neural extract revealed that epidermal growth factor (EGF) and heparin-binding (fibroblast) growth factors (HBGF) were most effective. An HBGF-like activity was apparent in extracts of rapidly proliferating, but not quiescent MEE cells at low or confluent densities. A cloned cell line (MEE/C8) was selected from MEE cell cultures in the absence of neural extract. MEE/C8 cells proliferated independent of either EGF or HBGF at rates equal to MEE cells, cell extracts exhibited HBGF-like activity at all stages of proliferation, and the cells formed invasive tumors in syngenic hosts. The HBGF-like activity present in extracts of tumorigenic MEE/C8 cells had properties similar to HBGF-1 (acidic fibroblast growth factor).

Animals↗
PubMed1994

Epitope masking of rat esophageal carcinoma tumor-associated antigen by certain coexisting glycolipid and phospholipid molecules: a potential mechanism for tumor cell escape from the host immune responses.

A monoclonal antibody (mAb-5G) produced against a tumorigenic rat esophageal cell line, B2T, was shown to react specifically with a unique glycolipid antigen expressed on the cell surface of tumorigenic and certain non-tumorigenic, immortalized rat esophageal cell lines [Cancer Immunol Immunother 36: 94 (1993)]. In enzyme-linked immunosorbent assay experiments, mAb-5G reacted with crude lipid extracts prepared from B2T cells cultured in vitro, but showed very little reactivity with crude lipid extracts prepared from the same cell line passaged once in vivo, unless the antigen was separated from other lipid components by column or thin-layer chromatography (TLC). When a secondary tissue-culture cell line was established from the above B2T tumor tissues and serially subcultured in vitro, the percentage of positively stained cells was increased significantly in immunofluorescence assay. It was also demonstrated that the amount of extractable antigen was increased as the cells were subcultured in vitro up to passage 15, and stabilized thereafter. These results indicate the presence of certain lipid components in crude lipid extracts from B2T cells grown in vivo that are capable of interfering with antigen-antibody binding. On TLC plates, these interfering lipids were identified as phosphatidylcholine, phosphatidylserine, sphingomyelin and gangliosides. The interfering lipids did not bind the antibody, rather they appeared to interfere with antigen accessibility. These lipid substances may modify tumor cell surface antigen(s), thus protecting the tumor cells from host immune destruction.

Animals↗
PubMed1991

Resolution limit of serial sections for 3D reconstruction of tubular cristae in rat liver mitochondria.

Following stereo, high resolution scanning electron microscopy (HRSEM) of various rat tissue mitochondria prepared by mild glutaraldehyde fixation followed by freeze cleavage and extraction of the cytosol (Lea and Hollenberg, 1989a), a new model for the ultrastructure of mitochondrial cristae has been developed (Lea and Hollenberg, 1989b). The cristae mitochondriales, previously considered in most cells to have a shelf like structure, were found in many cell types including hepatocytes by HRSEM to be tubes which spanned the mitochondrial matrix and were continuous with the inner mitochondrial membrane at both ends. Despite computer aided, serial, reconstruction of serial thin sections examined in the transmission electron microscope (TEM), tubular cristae in mitochondria have not been resolved entirely, even though circular cross sectional profiles of cristae have been observed in the TEM. The current study was undertaken to probe this deficiency and to understand its cause. In this study, the problems of inherent contrast and resolution loss in the digitized image have been reduced by using a computer based, background, extraction process analogous in the computer to the chemical cytosol extraction process used for HRSEM (Lea and Hollenberg, 1988; Hollenberg et al., 1989). Despite the use of this membrane enhancement algorithm, it was still not possible to reconstruct entire tubular cristae, as observed by HRSEM, even when the micrograph negatives were digitized and reprocessed (Lea and Hollenberg, 1989b). A comparison of serial section thickness deduced from the diffraction colour of the respective section (60 nm) to the measured diameter of tubular cristae profiles (30 nm), with membrane thickness of 10 nm, suggested that this size disparity is responsible for the apparent loss of resolution.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1998

A novel cartilage protein (CILP) present in the mid-zone of human articular cartilage increases with age.

A novel, somewhat basic noncollagenous protein was purified from guanidine hydrochloride extracts of human articular cartilage using cesium chloride density gradient centrifugation, followed by ion-exchange chromatography at pH 5, and gel filtration on two serially coupled columns of Superose 6 and Superdex 200. The protein of 91.5 kDa contains a single polypeptide chain substituted with N-linked oligosaccharides. It appeared unique to cartilage as studied by enzyme-linked immunosorbent assay and immunoblots of various tissue extracts. Its concentration in articular cartilages showed some variability with age being lower in young individuals. It represents a chondrocyte product, since it is synthesized by articular chondrocytes in explant cultures. Interestingly, the distribution of the protein in the articular cartilage provides important information on the nature of chondrocytes at different compartments in the tissue. Thus, chondrocytes in the middle/deeper layers of the tissue in particular, appeared to have produced the protein and deposited it in the interterritorial matrix. The protein was neither seen in the superficial nor in the deepest regions of the articular cartilage. Based on its immunolocalization we have named this protein CILP (cartilage intermediate layer protein).

Adolescent↗
PubMed1990

Localization and characterization of neuropeptide Y in the brain of Microcebus murinus (Primate, Lemurian).

The distribution of neuropeptide Y (NPY) in the brain of the lemur Microcebus murinus was determined by immunocytochemistry with the aid of a highly specific antiserum against synthetic porcine NPY. When compared with previous immunohistochemical data obtained in primates and other mammalian species, the localization of NPY-immunoreactive (IR) structures in the Microcebus murinus brain revealed particular features. (1) Numerous NPY-IR perikarya and a dense network of IR nerve terminals were found in the supraoptic and suprachiasmatic nuclei, respectively. The occurrence of NPY-IR perikarya in the supraoptic nucleus, also reported in the squirrel monkey, seems to be specific to primates. In the squirrel monkey, the suprachiasmatic nucleus exhibits only a moderate innervation, whereas in humans it appears totally devoid of NPY-IR fibers. (2) IR perikarya and axon processes were observed in many upper brainstem areas, in particular in the interpeduncular, raphe pontine, dorsal tegmental, parabrachial, and dorsal raphe nuclei, in the locus coeruleus, the nucleus of the solitary tract, and the reticular formation; in this latter area, the occurrence of two categories of NPY-IR neurons was demonstrated on the basis of their morphology and localization, suggesting that they may play distinct roles. (3) NPY-IR nerve processes could be traced over a long distance. (4) For the first time, numerous NPY-IR terminals were observed close to the lumen of the various cerebral ventricles. The immunoreactive NPY-like peptide was characterized by combining high performance liquid chromatography (HPLC) analysis and radioimmunoassay quantification. The dilution curves obtained with synthetic porcine NPY and serial dilutions of occipital cortex, paraventricular and supraoptic hypothalamus, posterior hypothalamus, medulla oblongata, or preoptic area extracts were parallel. The highest amounts of NPY were measured in the hypothalamus and telencephalon. HPLC analysis resolved a single peak of NPY-like immunoreactivity that exhibited the same retention time as synthetic porcine NPY. The distribution of NPY in the lemurian brain is discussed with respect to phylogeny and putative functions.

Animals↗
PubMed1983

Inhibition by acyclovir of herpes simplex virus type 2 morphologically transformed cell growth in tissue culture and tumor-bearing animals.

Rat embryo fibroblasts (REF) morphologically transformed by herpes simplex virus type 2 (HSV-2) and tumor-derived cells were tested for ability to grow in the presence of 9-(2-hydroxyethoxymethyl) guanine (acyclovir). Results indicated that the effective dose of acyclovir (ACV) required to inhibit HSV-2-transformed and tumor-derived cell growth by 50% (ED50) compared to mock-treated control cells averaged 15 to 75 micrograms/ml. In contrast, the ED50 of acyclovir was more than HEp-2 cells. HSV-2-transformed and tumor-derived cells after both low (less than 30) and high (greater than 30) serial passages expressed detectable levels of the virus-coded thymidine kinase (TK) measured in cell extracts by serum neutralization assay. HSV-2-transformed or tumor-derived cells converted two- to ten-fold more acyclovir to phosphorylated forms than nontransformed REF cells. Preliminary data showed that the drug inhibited tumor development in newborn syngeneic rats inoculated with HSV-2-transformed cells. The inhibitory activity of acyclovir and presence of low levels of HSV-2 TK activity appeared to correlate.

Acyclovir↗
PubMed2004

Recombinant tropomyosin from Penaeus aztecus (rPen a 1) for measurement of specific immunoglobulin E antibodies relevant in food allergy to crustaceans and other invertebrates.

Immunoglobulin E (IgE)-mediated food allergy to crustaceans and mollusks is relatively common and affected individuals typically react to a range of different species. The only known major allergen of shrimp was first described over 20 years ago and later identified as the muscle protein tropomyosin. This protein may be useful as a defined and relevant diagnostic marker for allergic sensitization to invertebrate foods. In order to generate an assay reagent suitable for this purpose, tropomyosin from the shrimp Penaeus aztecus (Pen a 1) was produced as a recombinant protein in Escherichia coli and characterized with respect to IgE antibody binding properties in comparison to natural shrimp tropomyosin. Hexahistidine-tagged rPen a 1 accumulated as a predominantly soluble protein in the E. coli expression host and a two-step chromatographic procedure provided a high yield of pure and homogeneous protein. rPen a 1 displayed chromatographic and folding characteristics similar to those of purified natural shrimp tropomyosin. Serum preincubation with serial protein dilutions revealed similar capacity of recombinant and natural tropomyosin to compete with immobilized shrimp extract for IgE binding. rPen a 1 was further shown to extensively and specifically compete for IgE binding to extracts of other crustacean species, house dust mite and German cockroach.

Allergens↗
PubMed1993

Measurement of gene expression by multiplex competitive polymerase chain reaction.

We have developed a polymerase chain reaction (PCR)-based method to measure glutathione peroxidase (GSH-Px) mRNA levels. Expression was measured by multiplex competitive PCR amplification of (a) cDNA from GSH-Px and the "housekeeping" gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and (b) two internal standards consisting of single-base mutants of GSH-Px and GAPDH cDNA that cause either a loss (GSH-Px) or a gain (GAPDH) of an EcoRI restriction endonuclease recognition site. RNA extracted from a human papillomavirus-immortalized human bronchial epithelial cell line (BEP2D) was reverse transcribed. Serial dilutions of cDNA were PCR amplified in the presence of GSH-Px and GAPDH primers and quantified amounts of mutated internal standards. The amplified DNA was restriction digested with EcoRI and electrophoresed on an agarose gel stained with ethidium bromide, separating native from mutated products. Densitometry was performed to quantitate the bands. Our studies demonstrate that this technique measures the relative expression of GSH-Px to GAPDH precisely and reproducibly for studies done with the same master mixture and dilution of internal standards. Ratios of relative gene expression varied less than 25% from the mean. This technique will be useful to measure changes in gene expression, particularly when the amount of study sample is limited or the level of gene expression is low.

Base Sequence↗
PubMed1988

Identification and quantification of kynurenic acid in human brain tissue.

Serial ion-exchange and high-performance liquid chromatography separations were employed for the tissue extraction and purification of kynurenic acid (KYNA). Subsequently, the compound isolated from postmortem human brain tissue was unequivocally identified as KYNA by nuclear magnetic resonance and mass spectrometric analyses. Regional distribution analyses revealed the highest concentration of KYNA (1.58 +/- 0.43 pmol/mg tissue) in the caudate nucleus with lower levels in the thalamus, globus pallidus, hippocampus, parietal cortex and frontal cortex. Of the brain structures examined, the lowest concentration of KYNA (0.14 +/- 0.02 pmol/mg tissue) was found in the cerebellum.

Aged↗
PubMed1985

Underestimation of specific immunoglobulin E by microtiter plate enzyme-linked immunosorbent assays.

We developed an ELISA by use of monoclonal anti-IgE to measure allergen-specific IgE. We measured perennial ryegrass (PRG) specific immunoglobulin E (sIgE) with conventional single incubations and with the recently reported transfer method to decrease interference in the assay. Paired sera in 10 patients before and during immunotherapy with PRG extract were analyzed for PRG sIgE with single serum incubations and with the sum of five serial serum incubations. A standardized reference serum and patient sera were incubated 12 hours and then transferred to a second well, incubated 12 hours more, and transferred again. The process was repeated until five pairs of wells had been incubated with each serum dilution. Color was developed, and optical density was measured. PRG sIgE was calculated by interpolation from the reference curve. Evidence of interference was inferred when less PRG sIgE was detected by single incubation than by transfer. Inhibition of 71.3% occurred in the sera drawn during immunotherapy. Inhibition of 32.9% was found before immunotherapy. Also, a late peaking of the well-to-well curve of optical density was observed in nine of 10 sera drawn during immunotherapy (the exception had not reached full strength), whereas all of the sera drawn before immunotherapy had normal curves. This suggests that sIgE determinations performed in microtiter plate format with single incubations will usually underestimate the sIgE level. This drawback needs to be kept in mind by the clinician interpreting the results of these assays.

Enzyme-Linked Immunosorbent Assay↗
PubMed1982

An in vitro assay for K-papovavirus of mice.

Primary cultures of mouse embryo cells were inoculated with K virus, a murine papovavirus, and were examined for cytopathic effect (CPE) of for the development of fluorescent antibody staining specific for K virus V antigen. CPE was not observed. However, numerous cells in infected cultures exhibited positive nuclear fluorescence, and the presence of papovavirus virions was demonstrated by electron microscopy. Extracts from infected cultures produced typical K virus pneumonia in newborn mice. Inoculation of cultures with serial dilutions of virus demonstrated that these cells provide a fluorescent antibody assay for K virus equal in sensitivity to animal inoculation methods. Although specific K virus fluorescence was also detected in cultures of fetal mouse endocardial cells, livers, placentas, and brains, positive cells were much less abundant in these cultures than in cultures of mouse embryo cells. The mouse embryo culture assay described in the present paper represents the first method of measuring K virus infectivity in vitro.

Animals↗
PubMed2005

Development of a polymerase chain reaction method for diagnosis of Babesia ovis infection in sheep and goats.

In this study, a pair of oligonucleotide primers were designed according to the nucleotide sequence of the small subunit ribosomal RNA (ssu rRNA) gene of Babesia ovis isolated from sheep in eastern Turkey. The primers were used to detect parasite DNA from blood samples of B. ovis-infected sheep and goats by polymerase chain reaction (PCR). A 549-bp DNA fragment was specifically amplified from blood samples from sheep and goats, naturally infected with B. ovis. No PCR products resulted from Babesia motasi, T. ovis, Theileria sp. OT1, Theileria sp. OT3, T. lestoquardi, B. canis, B. microti,T. annulata or normal sheep leucocytes DNA using these specific primers. B. ovis-infected erythrocytes with 1% parasitemia were subjected to 10-fold serial dilutions (from 10(-1) to 10(-9)) using an uninfected sheep erythrocytes, and DNA was extracted from each diluted sample for testing the sensitivity of the PCR. The PCR was sensitive enough to detect parasite DNA from the dilution of 10(-5) with 0.00001% parasitemia. This is more sensitive than examining 200 fields under light microscopy. In addition, 98 field samples collected from small ruminanats in eastern Turkey were tested for B. ovis infection. Four samples were positive Babesia spp. in blood smears, 21 samples were positive for B. ovis DNA by PCR. These results indicate that the PCR provides a useful diagnostic tool for the detection of B. ovis infection in sheep and goats.

Animals↗
PubMed1998

A North Carolina macular dystrophy phenotype in a Belizean family maps to the MCDR1 locus.

PURPOSE: To describe the clinical findings of an autosomal dominant macular dystrophy in a family of Mayan Indian ancestry in Belize, Central America, and to determine its molecular genetic relationship with the original North Carolinian family. METHODS: We performed comprehensive ophthalmic examinations on 56 members of a single family living in Chicago, Illinois, and Belize, Central America. Fundus photography and fluorescein angiography were performed on 17 affected subjects and six affected family members were serially examined over a 12-year period. Blood was collected from 26 individuals, and DNA was extracted for genotyping. Two-point linkage, multipoint linkage, and haplotype analysis was performed. RESULTS: In 17 affected individuals, the clinical features were consistent with the diagnosis of North Carolina macular dystrophy. Multipoint linkage analysis generated a peak lod score of 5.6 in the MCDR1 region. The haplotype associated with the disease was, however, different from that of the original North Carolinian family. CONCLUSIONS: This family has an autosomal dominant macular dystrophy that is clinically indistinguishable from North Carolina macular dystrophy (MCDR1). Our findings indicate that the mutated gene in this Belizean family maps precisely to the same region as that of the North Carolina macular dystrophy (MCDR1) locus. This study provides evidence that MCDR1 occurs in various ethnic groups and that there is no evidence of genetic heterogeneity.

Adolescent↗
PubMed2002

High-throughput SELEX SAGE method for quantitative modeling of transcription-factor binding sites.

The ability to determine the location and relative strength of all transcription-factor binding sites in a genome is important both for a comprehensive understanding of gene regulation and for effective promoter engineering in biotechnological applications. Here we present a bioinformatically driven experimental method to accurately define the DNA-binding sequence specificity of transcription factors. A generalized profile was used as a predictive quantitative model for binding sites, and its parameters were estimated from in vitro-selected ligands using standard hidden Markov model training algorithms. Computer simulations showed that several thousand low- to medium-affinity sequences are required to generate a profile of desired accuracy. To produce data on this scale, we applied high-throughput genomics methods to the biochemical problem addressed here. A method combining systematic evolution of ligands by exponential enrichment (SELEX) and serial analysis of gene expression (SAGE) protocols was coupled to an automated quality-controlled sequence extraction procedure based on Phred quality scores. This allowed the sequencing of a database of more than 10,000 potential DNA ligands for the CTF/NFI transcription factor. The resulting binding-site model defines the sequence specificity of this protein with a high degree of accuracy not achieved earlier and thereby makes it possible to identify previously unknown regulatory sequences in genomic DNA. A covariance analysis of the selected sites revealed non-independent base preferences at different nucleotide positions, providing insight into the binding mechanism.

Base Sequence↗
PubMed1992

Metabolic derangement in viable periinfarct tissue in the course of acute ischaemic infarction: a multitracer positron emission tomography (PET) study.

We used a multitracer positron emission tomography (PET) approach to assess metabolic changes in infarcted and periinfarct tissue in acute ischaemic stroke. 16 patients were studied within 6-48 hours (mean, 23 h) after onset of symptoms from a first hemispheric stroke and again 13-25 days later (mean, 15.6 days). Regional cerebral metabolic rates of oxygen (CMRO2) and glucose (CMRGlc), blood flow (CBF) and blood volume (CBV) were measured and oxygen extraction (OEF) as well as glucose extraction (GEF) and microvascular transit time were calculated. PET images were three-dimensionally aligned using serial CT or MRI scans. Regions of interest on the side of the infarction were individually compared to contralateral mirror regions. In the infarction core CBF, CMRO2 and CMRGlc were significantly lower than on the contralateral side and did not change during time. In the periinfarct regions there was a decreased CMRO2 with progressive deterioration over time while CBF slightly increased. Only in a few ischaemic regions with initially increased OEF oxygen metabolism was preserved during the course of time.

Blood Volume↗
PubMedDate not supplied

Characterization of a rapid and sensitive enzyme immunoassay (EIA) for progesterone applied to conditioned cell culture media.

Progesterone accumulation in conditioned media is a frequently employed endpoint for in vitro cell culture of steroidogenic cells. Although radioimmunoassay (RIA) has been the predominant method for measurement of progesterone, a number of nonradiometric immunoassays have been described but they have not been applied to conditioned media. Here, we report the characterization of a microtitre plate enzyme immunoassay (EIA) for determination of progesterone in conditioned media. The EIA has a sensitivity of 0.3 pg per well with intraassay and interassay coefficients of variation of 7.3 and 10.2%, respectively. The specificity of the EIA is no different than that of a comparable RIA showing crossreactivities of less than 0.1% for other steroids except 5 alpha-pregnan-3,20-dione (47%) and 11 alpha-hydroxyprogesterone (18%). Progesterone levels from conditioned cell culture media of either rat or human granulosa cell cultures measured by both EIA and RIA were in close agreement (r = 0.96) and serial dilutions of culture samples in the EIA were parallel to those of the standards. Also, extraction of culture media prior to EIA was found not to be necessary. Thus, this EIA is a highly sensitive and specific assay that provides a rapid, simple, inexpensive, and non-radiometric alternative to radioimmunoassay for measurement of progesterone in conditioned cell culture media.

Animals↗
PubMed1996

Peripheral blood tyrosinase messenger RNA detection and survival in malignant melanoma.

BACKGROUND: The most widely accepted criteria for the evaluation of prognosis of malignant melanoma are histopathologic and clinical presentation. No currently available laboratory tests provide additional prognostic information. It has recently been suggested that reverse transcription and polymerase chain reaction (RT-PCR)-based detection of tyrosinase messenger RNA (mRNA) in peripheral blood might be useful in the early detection of circulating tumor cells, since tyrosinase is thought to be a melanocyte-specific marker. PURPOSE: To further evaluate the clinical relevance of this potential marker, we examined peripheral blood samples from patients with malignant melanoma in different stages of disease for the presence of tyrosinase mRNA. METHODS: Total cellular RNA was extracted from heparinized peripheral blood cells from 64 patients with malignant melanoma, from five healthy control subjects, and from four patients with other cancers using the RNAzol A method. For analysis of tyrosinase mRNA, RT-PCR was performed as previously described by Smith et al.; the sensitivity of this assay was tested using RNA extracted from human melanoma cells (SK-mel 1 and SK-mel 3 cell lines) serially diluted with peripheral blood obtained from healthy control subjects. Two additional human melanoma cell lines (SK-mel 30 and RPMI-7951) served as positive controls for RT-PCR detection of tyrosinase mRNA. Overall patient survival curves were constructed using Kaplan-Meier estimates. RESULTS: Tyrosinase mRNA was detected by RT-PCR assay of all four of the established melanoma cell lines tested. Nine of the 64 patients with malignant melanoma were found to have detectable tyrosinase mRNA in their peripheral blood cells (tyrosinase-positive patients). The 16 patients with localized primary melanoma did not have detectable tyrosinase mRNA in their peripheral blood cells. Among the 48 patients with metastatic disease, all 27 patients who exhibited no evidence of disease progression were tyrosinase negative. Notably, all nine tyrosinase-positive patients had visceral metastases and were found to exhibit disease progression at the time of the sampling. Four of the nine tyrosinase-positive patients were also found to test negative at times without evidence of progressive disease; one patient became negative after achieving stable disease and three became positive for tyrosinase transcripts on disease progression. The probability of survival from time of sampling was significantly lower in the nine tyrosinase-positive patients when tested versus the 23 patients with comparable disease but without detectable tyrosinase mRNA (two-sided; P < or = .05). CONCLUSIONS: The results of this study demonstrate that the detection of tyrosinase mRNA in cells in the peripheral blood by RT-PCR may be a useful prognostic marker for predicting tumor progression and poor clinical outcome in patients with malignant melanoma.

Adult↗
PubMed2001

Quantitative detection of Streptococcus pneumoniae in nasopharyngeal secretions by real-time PCR.

Streptococcus pneumoniae is an important cause of community-acquired pneumonia. However, in this setting the diagnostic sensitivity of blood cultures is below 30%. Since during such infections changes in the amounts of S. pneumoniae may also occur in the upper respiratory tract, quantification of these bacteria in nasopharnygeal secretions (NPSs) may offer a suitable diagnostic approach. Real-time PCR offers a sensitive, efficient, and routinely reproducible approach to quantification. Using primers and a fluorescent probe specific for the pneumolysin gene, we were able to detect DNA from serial dilutions of S. pneumoniae cells in which the quantities of DNA ranged from the amounts extracted from 1 to 10(6) cells. No difference was noted when the same DNA was mixed with DNA extracted from NPSs shown to be deficient of S. pneumoniae following culture, suggesting that this bacterium can be detected and accurately quantitated in clinical samples. DNAs from Haemophilus influenzae, Moraxella catarrhalis, or alpha-hemolytic streptococci other than S. pneumoniae were not amplified or were only weakly amplified when there were > or =10(6) cells per reaction mixture. When the assay was applied to NPSs from patients with respiratory tract infections, the assay performed with a sensitivity of 100% and a specificity of up to 96% compared to the culture results. The numbers of S. pneumoniae organisms detected by real-time PCR correlated with the numbers detected by semiquantitative cultures. A real-time PCR that targeted the pneumolysin gene provided a sensitive and reliable means for routine rapid detection and quantification of S. pneumoniae present in NPSs. This assay may serve as a tool to study changes in the amounts of S. pneumoniae during lower respiratory tract infections.

Bacterial Proteins↗