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PubMed2005

Clinical management and outcome of childhood lung abscess: a 16-year experience.

In order to evaluate the clinical manifestations, management and outcome of childhood lung abscess, a retrospective chart review of 27 pediatric patients with International Classification of Diseases, Ninth Revision-Clinical Modification (ICD-9 CM) code of 503.1 (lung abscess) from August 1987 to August 2003 was conducted. Among the 27 patients (14 males and 13 females), 30% (8/27) were primary lung abscess and 70% (19/27) had underlying chronic diseases (secondary lung abscess). The predisposing factors of the primary group (n = 8) included 6 cases of respiratory tract infection, 1 with choking during swimming, and 1 with laceration wound. The underlying diseases in the secondary group (n = 19) included 10 cases of hematologic disorder (52%), 3 of congenital heart disease, 2 of central nervous system anomalies, and 1 each of hyperimmunoglobulin E syndrome, chronic lung disease, liver cirrhosis with fistula formation, and Swyer-James syndrome. Eleven patients (41%) underwent diagnostic tapping, including echo-guided aspiration (10 cases) and computed tomography-guided percutaneous needle aspiration (1 case). Positive yield rate from aspiration of lung abscess was 63.6% (7/11). Surgical intervention was performed in 8 (42%) of the secondary group and in 1 patient from the primary group. The pathogens were identified in 11 patients (41%): 5 with oral flora, 2 with Staphylococcus aureus plus other pathogens, 1 with S. aureus alone, 1 with Pseudomonas aeruginosa plus Proteus mirabilis, 1 with P. aeruginosa alone, and 1 with Aspergillus. The average duration of parenteral antibiotic use was 40 days. Five cases (18.5%) died due to poor control of the underlying diseases, and 4 of the patients (15%) had sequelae (2 with bronchiectasis and 2 with lung fibrosis). Seventy percent of lung abscess occurred in children with underlying medical conditions. Early percutaneous aspiration has an important role in identification of pathogens. Oral anaerobes and S. aureus are the core pathogens in primary lung abscess and gram-negative pathogens should also be considered in secondary lung abscess.

Adolescent↗
PubMed2000

Bacterial meningitis.

With nearly 8,000 cases in the United States per year, and 2,000 deaths annually, bacterial meningitis continues to be a significant source of morbidity and mortality. The principal pathogens are Neisseria meningitidis, Streptococcus pneumoniae, group B streptococci, and Hemophilus influenzae. In immunocompromised patients, Listeria monocytogenes is also an important pathogen. Rapid identification and evaluation of the patient with bacterial meningitis and prompt initiation of antibiotics are the cornerstones of therapy. Except in the rare patient with papilledema, focal neurologic symptoms, or a seizure, a lumbar puncture should be performed without delay, and antibiotic therapy should be administered promptly. Patients without a readily identifiable source of infection should be treated empirically with intravenous ceftriaxone. Ampicillin should also be administered in populations at increased risk for L. monocytogenes. The risk of meningitis in some populations can be reduced by administration of vaccines against selected pathogens such as N. meningitidis, S. pneumoniae, and H. influenzae.

Journal Article↗
PubMed2006

Identification of the emerging skin pathogen Corynebacterium amycolatum using PCR-amplification of the essential divIVA gene as a target.

The actinomycete Corynebacterium amycolatum is a saprophytic bacterium usually associated with the human skin, but it is at present considered an emergent pathogen as it is isolated from nosocomial settings from samples of immunosuppressed patients. The conventional method to distinguish C. amycolatum from closely related species is mainly based on phenotypic or chemotaxonomic studies. We developed a molecular method to identify rapidly C. amycolatum based on the use of different primers for amplification of the cell division divIVA gene using conventional or real-time PCR. This technique was used for the first time to distinguish C. amycolatum from the closely related Corynebacterium striatum, Corynebacterium minutissimum and Corynebacterium xerosis, without the requirement of further molecular analysis. The suitability of the identification method was tested on 51 clinical isolates belonging to the nonlipophilic fermentative group of corynebacteria (cluster C. striatum/C. amycolatum), which were accurately characterized by sequencing a 0.8 kb fragment of the 16S rRNA gene.

Bacterial Proteins↗
PubMed2000

Rapid identification of bacteria in blood cultures by using fluorescently labeled oligonucleotide probes.

The applicability of whole-cell hybridization for the identification of pathogenic bacteria in blood from septic patients was examined. Oligonucleotide probes, fluorescently labeled with fluorescein isothiocyanate, directed against the variable regions of the 16S rRNAs of the following bacterial species and/or genera were used: Streptococcus spp., Enterococcus faecalis, Staphylococcus aureus, coagulase-negative staphylococci (CoNS), Escherichia coli, Pseudomonas aeruginosa, and the Enterobacteriaceae family. A probe specific for the rRNAs of almost all bacteria and its complementary, reversed counterpart was used as positive and negative control, respectively. The probes were used in conjunction with a fast and simple-to-use protocol for whole-cell hybridization. This protocol yields an identification after 25 to 45 min, depending on whether the bacterium is gram positive or gram negative. A total of 182 blood samples which tested positive in a blood culture machine were investigated. All probes except for the ones for S. aureus and the CoNS showed sensitivities and specificities of 1.000. It was concluded that whole-cell hybridization is well suited for the fast screening of septic blood containing streptococci and/or enterococci or gram-negative rods.

Bacteremia↗
PubMed2000

Mismatch cleavage detects pathogenic microorganisms.

When a DNA probe hybridizes a DNA target and generates a G/A mismatch in the probe-target DNA heteroduplex, the mismatch enzyme, mutY, will cut the A base at the site of the mismatch. This specific cleavage at the mismatched A on the known probe will reveal the complementary DNA sequences of the targets. This study shows mismatch cleavage assays identify the complementary sequence of cryptic plasmid target in the extract of chlamydia infected cells in culture. In addition, the specific cleavage at a single base permits differentiation of two sequences with one base difference. This was shown in differentiating the subtypes of human immunodeficiency virus (HIV) type 1. The addition of amines in the assays increases the sensitivity by freeing the target for recycling. The combined assay system of high sensitivity and demonstrated specificity allows further evaluation for direct identification of pathogenic microorganisms in patient samples.

Acetates↗
PubMed2004

Identification of emerging human-pathogenic Pythium insidiosum by serological and molecular assay-based methods.

Pythium insidiosum is a pathogen that causes disease in both animals and humans. Human infection is rare; however, when it does occur, most patients, especially those having underlying hemoglobinopathy syndromes, such as thalassemia, exhibit a severe form. We identified four isolates of P. insidiosum. Two were recovered from tissue biopsy specimens from thalassemic and leukemic patients, one was derived from brain tissue from a thalassemic patient, and another was isolated from a corneal ulcer from a fourth patient. Western blotting and an enzyme-linked immunosorbent assay (ELISA) were performed with a serum sample derived from one thalassemic patient. The methods used to identify the P. insidiosum isolates were based on morphology, nucleic acid sequencing, and a PCR assay. To confirm the identification, portions of the 18S rRNA genes of these four isolates were sequenced. The sequences were shown to be homologous to previously described P. insidiosum DNA sequences. In addition, PCR amplification of the internal transcribed spacer region specific for P. insidiosum was positive for all four isolates. The ELISA with the serum sample from the thalassemic patient gave a positive result from a serum dilution of 1:800. Finally, Western immunoblotting with this serum sample showed positive immunoglobulin G recognition for proteins of 110, 73, 56, 42 to 35, 30 to 28, 26, and 23 kDa. The results of this study show that both molecularly based diagnostic and serodiagnostic techniques are useful for the rapid identification of human pythiosis. The predominant antigens recognized by Western blotting may be useful in the development of a more sensitive and specific diagnostic tool for this disease.

Antigens↗
PubMed2000

[The niches and pathways of animal pathogens].

Infectious diseases are not a relict of the past but a topical phenomenon determined by complex evolution of the currently existing and constantly changing microbial agents and their hosts. With regard to abundance of species within the microbial kingdom and rate of its changes and development, it is difficult to predict the role of the microbial factor in mortality of humans and animals. The study and generalization of sequential similarities of microbial virulence factors after the completion of genome sequencing of principal pathogens can play a positive role in this direction. At present, molecular-genetic methods allow us to study the phylogenetic relationships of microbes and categorize them according to new criteria. The efficient control of diseases caused by microbes requires knowledge on their physiological and ecological niche from which they penetrate, in various ways, into the host organisms and, under suitable conditions, induce mass diseases. This process has several stages and, in the recent period, it is increasingly affected by human activities. The knowledge on all participants in this process, i.e. the microbe and its niche, factors of virulence and pathways of their dissemination, requires a scientifically based surveillance. Abundance and variability is characteristic for both microbial kingdom and microbial niche. Some identification of pathogenic properties of microorganisms and factors affecting their movement from their niche to the recipients results in activation of old classical diseases (e.g. plague, cholera, tuberculosis etc.) or emerging of new, so far unknown infections diseases ("emerging inf. disease"--EID), caused for example by lentiviruses, oncoviruses, filiviruses, bartonella, borrelia etc. This has provided the basis for establishment of new medical trends and approaches, such as "Emergency medicine" or "Travel medicine", expressing their purpose by their names. The control of existing or proposed infectious diseases in the 21st century (in which majority of factors such as urbanization, environmental factors, evolution of the microbial kingdom, will contribute to the persistence or "emergence" of new diseases) will be affected by the input of new knowledge in the field of molecular biology, such as introduction of biosensors, genetic tests, microchips, new generation of DNA vaccines, enteric vaccines and antibodies produced by transgenic animal bioreactors or plants, "customized" vaccines assessed for individual genetic profiles, etc. (Tab. 5, Ref. 21.)

Animals↗
PubMed2006

FT-IR microspectroscopy for early identification of some clinically relevant pathogens.

AIMS: To investigate the potentials and limitations of Fourier transform-infrared (FT-IR) microspectroscopy as a tool to identify, at the level of microcolonies, pathogenic bacteria frequently isolated in the clinical environment. METHODS AND RESULTS: A total of 1570 FT-IR spectra from 164 gram-positive and gram-negative bacteria isolated from patients were recorded from 6 to 10-h old microcolonies of 50-150 microm size. A classification of 100% was obtained for the most frequent gram-positive bacteria, such as Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, and Enterococcus faecium at the species level. An average accuracy of about 80% was reached with Gram negative bacteria from the Enterobacteriaceae and Pseudomonaceae families; Enterobacter aerogenes, Enterobacter cloacae, Klebsiella spp., and Citrobacter koseri; and Proteus mirabilis and Escherichia coli. Results were comparable with FT-IR measurements on dried suspensions from 18-h cultures. CONCLUSIONS: Early identification of young microcolonies is feasible with FT-IR microscopy with a very high accuracy for gram-positive bacteria. Some improvement in the transfer of microcolonies is necessary to increase the accuracy for gram-negative bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: Combination of FT-IR microscopy and multivariate data analysis could be a complementary, rapid, and reliable tool for screening and discriminating, at species and subspecies level, micro-organisms of clinical, food-borne, or environmental origins.

Animals↗
PubMed2006

Hand rub consumption and hand hygiene compliance are not indicators of pathogen transmission in intensive care units.

The objective of this study was to investigate whether nosocomial infection (NI) rates, hand hygiene compliance rates and the amount of alcohol-based hand rub used for hand disinfection are useful indicators of pathogen transmission in intensive care units (ICUs), and whether they could be helpful in identifying infection control problems. All isolates of 10 of the most frequent pathogens from patients who were hospitalized in an ICU for >48 h were genotyped to identify transmission episodes in five ICUs. The incidence of transmission was correlated with hand hygiene compliance, hand rub consumption and NI rates. The incidence of transmission episodes varied between 2.8 and 6.8 in the five ICUs. The NI rate was 8.6-22.5 per 1000 patient-days, hand hygiene compliance was 30-47% and hand rub consumption was 57-102 L per 1000 patient-days. There was no correlation between the incidence of transmission episodes and hand rub consumption or hand hygiene compliance. The correlation between transmission rates and NI rates was 0.4 (P = 0.5), and with the exclusion of one ICU, it was 1 (P < 0.01). The incidence of NI is a relatively good indicator for the identification of pathogen transmissions, but hand rub consumption and hand hygiene compliance, at least with the relatively low level of compliance found in this study, are not indicators of pathogen transmission.

Anti-Infective Agents, Local↗
PubMed2002

[Detection of mycobacterium bovis BCG Tokyo in sputum from a tuberculosis patient and administrative action in public health].

PURPOSE: We encountered a tuberculosis patient with the BCG strain in his sputum. Based on this experience, we investigated ideal surveillance and quality assurance for bacteriological examination as well as cooperation between medical organizations and public administrative organizations such as public health centers for prompt and appropriate measures against tuberculosis. METHODS: Acid-fast bacilli isolated in the laboratory of a hospital were analyzed by restriction fragment length polymorphism (RFLP) and molecular epidemiological data for pathogen were provided to the public health center in charge. RESULTS: DNA of acid-fast bacilli isolated from sputum of a tuberculosis patient showed a BCG like pattern on RFLP analysis using two IS6110 and PGRS (polymorphic GC-rich sequence) probes. Additional investigation revealed that the patient had been treated with BCG immuno-therapy against bladder cancer. Accordingly, it was suggested that this was the origin of the BCG strain in the sputum. Therefore, the patient was re-examined clinically, and further contact tracing by the public health center was stopped. CONCLUSIONS: This study indicates that adequate surveillance and quality assurance in bacteriological examination as well as effective exchange of information between medical organization and public health centers are very important for prompt and appropriate measures against tuberculosis. Further, it provided strong support for identification of pathogens of the molecular level by RFLP analysis.

Aged↗
PubMed1990

[Clinical evaluation of aztreonam in neonatal infections].

Serum concentration, urinary excretion and clinical application of aztreonam (AZT) were studied as follows: 1. Serum concentrations of AZT 1 hour after intravenous injection were 21.0 micrograms/ml in 1 case administered with approximately 10 mg/kg drug and 44.2 micrograms/ml on the average for 7 cases given approximately 20 mg/kg, indicating that serum concentrations are dose-dependent. Average serum half-life in 3 mature babies was 4.75 hours and that in 4 premature babies was 6.59 hours thus T 1/2 was longer in the latter. T 1/2 of 64 days of age newborn was 3.80 hours. Urinary recovery rates in 2 cases examined were 52.1 and 51.9%. 2. Daily dosages of AZT 39.9-63.3 mg/kg were intravenously administered to 10 newborns and prematures b.i.d. or t.i.d., 5 cases of which received AZT alone and the other 5 received AZT in combination with ampicillin (ABPC). Of the above 10 cases, AZT was given to 8 cases for treatment and to the other 2 cases for prophylaxis. Excluding 2 unascertainable cases, AZT showed good or better effectiveness in all the 6 cases in the treatment group, i.e., sepsis 1, suspected sepsis 1 and urinary tract infection 4 cases. All the identified pathogens (Escherichia coli 2 strains, Klebsiella pneumoniae 1 strain and Enterobacter 2 strains) were eliminated by the treatment. No onset of infection was observed in either of the 2 cases with prophylaxis. One of them was administered with AZT for 52 days consecutively but neither side effect nor abnormal laboratory test value was observed. 3. Side effect was not observed at all. One case each of minor degree of platelet increase and GOT elevation was recorded as an abnormal test value. The elevated GOT value continued to be high even after the completion of the administration and it was presumed to be due to the primary disease, heart failure. 4. As results of the above studies, AZT was considered to be effective and safe for neonatal infections caused by Gram-negative bacteria. It may be safer to initiate the treatment with AZT and ABPC in combination than with AZT alone before the identification of pathogen and to change the therapy to single administration of either AZT or ABPC when the pathogens are identified. With respect to method of administration, AZT 20 mg/kg 2 or 3 times a day appeared to show expected efficacy for the newborns with in 7 days after birth.

Ampicillin↗
PubMed2005

Pathogenic mutations and polymorphisms in the lipoprotein receptor-related protein 5 reveal a new biological pathway for the control of bone mass.

PURPOSE OF REVIEW: This review summarizes recent findings concerning the genomic variations of the lipoprotein receptor-related protein 5 (LPR5) in relation to bone biology. RECENT FINDINGS: Mutations in the LRP5 gene causing high bone mass (HBM) and osteoporosis-pseudoglioma (OPPG) underscored the role of the Wnt-LRP5 canonical signaling on bone formation. Additional LRP5 activating mutations have been identified in a variety of sclerosing bone dysplasias, improving the diagnostic classification of these disorders. Association of polymorphisms in LRP5 with bone mineral density indicate that LRP5 genetic variation contribute to the risk of osteoporosis. Transgenic mice carrying the LRP5 HBM mutation have improved bone biomechanical properties, and the molecular mechanisms by which this mutation exerts its effects have been clarified. A number of KO mice have shown the complex effects of the Wnt-LRP5 pathway on bone mass and skeletal morphology. In vitro studies indicate that osteoblasts produce a variety of Wnts, the LRP5 co-receptor frizzled (Fzd), as well as LRP5 and Wnt inhibitors, i.e. dickkopf (Dkk1) and frizzled-related proteins (Sfrps), respectively, and delineate the role of these molecules in regulating the commitment of mesenchymal stem cells along the osteoblastic lineage. SUMMARY: Identification of pathogenic mutations and allelic variations in LRP5 has improved our understanding of the physiology of bone mass acquisition and the pathophysiology of several bone diseases, including osteoporosis. Understanding how complex interactions between agonistic and inhibitory factors in the Wnt-LRP5 canonical pathway influence osteoblast functions has the potential of providing new anabolic treatments for osteoporosis.

Alleles↗
PubMed2005

Bacterial protein microarrays for identification of new potential diagnostic markers for Neisseria meningitidis infections.

Neisseria meningitidis is the most common cause of meningitis and causes epidemic outbreaks. One trait of N. meningitidis, which is associated with most of the currently recognized virulence determinants, is the presence of phase-variable genes that are suspected to enhance its ability to cause an invasive disease. To detect the immune responses to phase-variable expressed proteins, we applied protein microarray technology for the screening of meningitis patient sera. We amplified all 102 known phase-variable genes from N. meningitidis serogroup B strain MC58 by polymerase chain reaction and subcloned them for expression in Escherichia coli. With this approach, we were able to express and purify 67 recombinant proteins representing 66% of the annotated genes. These were spotted robotically onto coated glass slides to generate protein microarrays, which were screened using 20 sera of patients suffering from meningitis, as well as healthy controls. From these screening experiments, 47 proteins emerged as immunogenic, exhibiting a variable degree of seroreactivity with some of the patient sera. Nine proteins elicited an immune response in more than three patients, with one of them, the phase-variable opacity protein OpaV (NMB0442), showing responses in 11 patient sera. This is the first time that protein microarray technology has been applied for the investigation of genetic phase variation in pathogens. The identification of disease-specific proteins is a significant target in biomedical research, as such proteins may have medical, diagnostic, and commercial potential as disease markers.

Bacterial Proteins↗
PubMed2000

Identification of genes required for chronic persistence of Brucella abortus in mice.

The genetic basis for chronic persistence of Brucella abortus in lymphoid organs of mice, cows, and humans is currently unknown. We identified B. abortus genes involved in chronic infection, by assessing the ability of 178 signature-tagged mutants to establish and maintain persistent infection in mice. Each mutant was screened for its ability to colonize the spleens of mice at 2 and 8 weeks after inoculation. Comparison of the results from both time points identified two groups of mutants attenuated for chronic infection in mice. The first group was not recovered at either 2 or 8 weeks postinfection and was therefore defective in establishing infection. Mutants in this group carried transposon insertions in genes involved in lipopolysaccharide biosynthesis (wbkA), in aromatic amino acid biosynthesis, and in type IV secretion (virB1 and virB10). The second group, which was recovered at wild-type levels 2 weeks postinfection but not 8 weeks postinfection was able to establish infection but was unable to maintain chronic infection. One mutant in this group carried a transposon insertion in a gene with homology to gcvB of Mycobacterium tuberculosis, encoding glycine dehydrogenase, an enzyme whose activity is increased during the state of nonreplicating persistence. These results suggest that some mechanisms for long-term persistence may be shared among chronic intracellular pathogens. Furthermore, identification of two groups of genes, those required for initiating infection and those required only for long-term persistence, suggests that B. abortus uses distinct sets of virulence determinants to establish and maintain chronic infection in mice.

Animals↗
PubMed2000

Isolation and characterization of two pathogen- and salicylic acid-induced genes encoding WRKY DNA-binding proteins from tobacco.

A pathogen- and salicylic acid (SA)-induced DNA-binding activity has been recently identified in tobacco that is related to a previously identified class of WRKY DNA-binding proteins. To identify members of the WRKY gene family associated with this DNA-binding activity, we have attempted to isolate those WRKY genes that are induced by pathogen infection. Using a domain-specific differential display procedure, we have isolated two tobacco WRKY genes, tWRKY3 and tWRKY4, that are rapidly induced in resistant tobacco plants after infection by tobacco mosaic virus (TMV). Both tWRK3 and tWRKY4 encode proteins with a single WRKY domain that contain the conserved WRKYGQK sequence. Unlike other isolated WRKY proteins that contain the Cys2His2 zinc motif, tWRKY3 and tWRKY4 appear to contain the Cys2HisCys zinc motif. Nonetheless, both tWRKY3 and tWRKY4 are capable of binding DNA molecules with the W-box (TTGAC) element recognized by other WRKY proteins. Expression of the tWRKY3 and tWRKY4 genes could be rapidly induced not only by TMV infection but also by SA or its biologically active analogues that are capable of inducing pathogenesis-related genes and enhanced resistance. Interestingly, induction of both genes by TMV infection was still observed in resistant tobacco plants expressing the bacterial salicylate hydroxylase gene (nahG), although the levels of induction appeared to be reduced. Identification of pathogen- and SA-induced genes encoding WRKY DNA-binding proteins should facilitate future studies on the regulation and functions of this novel group of DNA-binding proteins.

Amino Acid Sequence↗
PubMed1997

Immunohistologic identification of Aspergillus spp. and other hyaline fungi by using polyclonal fluorescent antibodies.

Isolation and identification of pathogenic Aspergillus and Fusarium spp. from clinical materials provide the most accurate means for establishing a diagnosis of infections by these molds. Such efforts, however, are not always successful. Histologic diagnosis also has its limitations. In vivo the hyphae of Aspergillus and Fusarium spp. are very similar and their in situ manifestations are not pathognomonic. To improve the histologic diagnosis of infections by Aspergillus and Fusarium species, we developed polyclonal fluorescent-antibody reagents to Aspergillus fumigatus and Fusarium solani and evaluated their diagnostic utilities. Our studies revealed that A. fumigatus and F. solani share epitopes not only with one another but also with other Aspergillus and Fusarium spp. as well as with Paecilomyces lilacinus and Pseudallescheria boydii. Adsorption of the A. fumigatus conjugate with cells of Fusarium proliferatum and F. solani and F. solani antiserum with cells of Aspergillus flavus resulted in reagents that distinguished Aspergillus spp. from Fusarium spp. but that still cross-stained P. lilacinus and P. boydii. Adjunctive use of a specific P. boydii conjugate enabled the identification of Aspergillus spp., Fusarium spp., P. lilacinus, and P. boydii in formalin-fixed tissue sections from 19 humans with culture-proven cases of mycotic infection.

Antibodies, Fungal↗
PubMed1996

Amebic keratitis in a wearer of disposable contact lenses due to a mixed Vahlkampfia and Hartmannella infection.

PURPOSE: To support the hypothesis that Acanthamoeba is not a unique cause of amebic keratitis, we report a case of amebic keratitis in which viable Acanthamoeba could not be isolated from corneal tissue. Vahlkampfia and Hartmannella, two other genera of free-living ameba, were isolated, however, using prolonged culture. METHODS: A 24-year-old wearer of soft contact lenses had keratitis. Extensive histologic and microbiologic investigations were performed on corneal scrape, biopsy, and keratoplasty tissue. Contact lenses, storage case, and the home water supply, where contact lens hygiene was practiced, were examined for the presence of micro-organisms. RESULTS: No viruses, pathogenic bacteria, or fungi were detected from corneal tissue samples. Amebae were observed using light and electron microscopy, but these could not be unequivocally classified using immunocytochemical staining. Viable Vahlkampfia and Hartmannella, but no Acanthamoeba, were isolated from the corneal biopsy sample. Indirect immunofluorescence with a range of polyclonal rabbit antisera raised against axenically cultivated stains of the three amebal genera was unhelpful because of cross-reactivity. A diverse range of micro-organisms was present within the storage case, including the three amebal species. Amebic cysts also were associated with the contact lens. CONCLUSION: A mixed non-Acanthamoeba amebic keratitis has been identified in a wearer of soft contact lenses where lack of storage case hygiene provided the opportunity for the free-living protozoa Vahlkampfia and Hartmannella to be introduced to the ocular surface. When Acanthamoeba-like keratitis occurs, but where Acanthamoeba cannot be isolated using conventional laboratory culture methods, alternate means should be used to identify other amebae that may be present. Polyclonal immunofluorescent antibody staining was unreliable for generic identification of pathogenic free-living amebae in corneal tissue.

Adult↗
PubMed2005

Detection and genotyping of Mycobacterium species from clinical isolates and specimens by oligonucleotide array.

Identification of pathogenic Mycobacterium species is important for a successful diagnosis of mycobacteriosis. The purpose of this study was to develop an oligonucleotide array which could detect and differentiate mycobacteria to the species level by using the internal transcribed spacer (ITS) sequence. Using a genus-specific probe and 20 species-specific probes including two M. avium-intracellulare complex (MAC)-specific probes, we have developed an ITS-based oligonucleotide array for the rapid and reliable detection and discrimination of M. tuberculosis, MAC, M. fortuitum, M. chelonae, M. abscessus, M. kansasii, M. gordonae, M. scrofulaceum, M. szulgai, M. vaccae, M. xenopi, M. terrae, M. flavescens, M. smegmatis, M. malmoense, M. simiae, M. marinum, M. ulcerans, M. gastri, and M. leprae. All mycobacteria were hybridized with a genus-specific probe (PAN-03) for detection of the genus Mycobacterium. Mycobacterial species were expected to show a unique hybridization pattern with species-specific probes, except for M. marinum and M. ulcerans, which were not differentiated by ITS-based probe. Among the species-specific probes, two kinds of species-specific probes were designed for MAC in which there were many subspecies. The performance of the oligonucleotide array assay was demonstrated by using 46 reference strains, 149 clinical isolates, and 155 clinical specimens. The complete procedure (DNA extraction, PCR, DNA hybridization, and scanning) was carried out in 4.5 h. Our results indicated that the oligonucleotide array is useful for the identification and discrimination of mycobacteria from clinical isolates and specimens in an ordinary clinical laboratory.

Bacterial Typing Techniques↗