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PubMed2006

Development of a multiple internal control for clinical diagnostic real-time amplification assays.

A major pitfall in most published genomic amplification methods for the detection and identification of human pathogens is that they do not include an internal amplification control in order to achieve an acceptable level of confidence for the absence of false-negative results. By applying composite primer technology, a single multiple internal amplification control DNA molecule was constructed to detect and quantify the hepatitis B virus, human polyomavirus, Epstein-Barr virus, Toxoplasma gondii and cytomegalovirus using real-time PCR. The multiple internal amplification control contains all forward and reverse primer binding regions targeted in the five distinct duplex PCRs, but with a unique probe hybridization site. Multiple internal amplification control detection sensitivity, assessed by Probit analysis, was 58 copies per PCR, associated with an extremely wide dynamic range (8 log(10) units). Moreover, in testing 614 patient samples, PCR inhibition occurred at a frequency of 0-8.8%. Similar multiple internal amplification controls for quantitative PCR-based assays could be designed to accommodate any infectious profiles in a particular institution as they are easy to make and inexpensive.

Animals↗
PubMed1995

Pulmonary infiltrates in patients with haematologic malignancies: clinical usefulness of non-invasive bronchoscopic procedures.

In a prospective study 90 patients with haematologic malignancies (57 acute leukaemias, 6 Hodgkin's Diseases, 15 Non-Hodgkin Lymphomas, 12 other diseases), with fever exceeding 38.4 degrees C and newly developed pulmonary infiltrates underwent bronchoscopy obtaining bronchoalveolar lavage, bronchial washings and protected brush specimen (n = 71). Pneumonias due to gram-negative bacteria (n = 38) and fungi (n = 34) were most frequent. Bronchoscopic specimens yielded 226 isolates (2 different organisms/bronchoscopy on average). 112 organisms were finally regarded as causing pneumonia. Sensitivity of bronchoscopy in diagnosing infectious episodes was 66%, but only 4 out of 13 non-infectious pulmonary infiltrates could be identified. Bronchoscopy was most effective in the diagnosis of pneumocystis carinii and herpes virus pneumonia, whereas sensitivity and specificity of detecting fungal and bacterial pneumonia were low. Empirical antimicrobial therapy was verified by evaluation of bronchoscopic samples in 25 out of 90 cases. Empirical therapy was successfully changed according to the results of invasive samplings in 34 out of 90 cases. Early identification of causative pathogens had a significant impact on survival.

Adolescent↗
PubMed1998

CD4+ and CD8+ T-lymphocytopenia in a filly with Pneumocystis carinii pneumonia.

Decreased proportion of CD4+ and CD8+ T lymphocytes in peripheral blood likely contributed to susceptibility to Pneumocystis carinii in a foal. Cytological evaluation of bronchoalveolar lavage was required for identification of the pathogen and serial flow-cytometric analysis of peripheral blood lymphocytes documented transient low expression of CD4+ and CD8+ T lymphocytes. Although immunodeficiency is uncommon, it must be included in the differential diagnosis for patients suffering from chronic or opportunistic infections and may provide an indication for immunostimulant therapy.

Animals↗
PubMed2006

[Large-scale questionnaire surveillance concerning invasive infections with group C and G streptococci].

A large-scale questionnaire surveillance was conducted regarding the onset of invasive infections with beta-hemolytic group C (GCS) and group G (GGS) streptococci from clinical specimens that are normally aseptic and the backgrounds of these cases. The surveillance period of the questionnaire was 8 months from January to August 2005. Completed questionnaires were received from the clinical laboratories of 193 medical institutions. One hundred two clinical laboratories (52.8%) had isolated these beta-hemolytic streptococci. Of all the isolates, GCS and GGS accounted for 25 and 216 cases, respectively, or a ratio of almost 1:10. Isolates from blood cultures accounted for half the number of all isolates, followed by isolates from atretic pus or joint fluid. The isolates gradually became more prevalent from patients in their 40s, and peaked in patients in their 70s. The most prevalent disorder, described in 184 cases, was suppurative disease followed by (in descending order), bacteremia, sepsis, arthritis purulenta and cellulitis. A small number of patients had developed with streptococcal toxic shock syndrome, empyema or meningitis. Most of the patients had an underlying disease, such as diabetes mellitus, malignancy or cerebrovascular disease (in descending order). We conclude from the above findings that background factors in patients as well as identification of the pathogen should be made public when GCS or GGS is isolated from normally aseptic clinical specimens.

Adolescent↗
PubMed2000

Application of 5'-nuclease PCR for quantitative detection of Listeria monocytogenes in pure cultures, water, skim milk, and unpasteurized whole milk.

PCR techniques have significantly improved the detection and identification of bacterial pathogens. Countless adaptations and applications have been described, including quantitative PCR and the latest innovation, real-time PCR. In real-time PCR, e.g., the 5'-nuclease chemistry renders the automated and direct detection and quantification of PCR products possible (P. M. Holland et al., Proc. Natl. Acad. Sci. USA 88:7276-7280, 1991). We present an assay for the quantitative detection of Listeria monocytogenes based on the 5'-nuclease PCR using a 113-bp amplicon from the listeriolysin O gene (hlyA) as the target. The assay was positive for all isolates of L. monocytogenes tested (65 isolates including the type strain) and negative for all other Listeria strains (16 isolates from five species tested) and several other bacteria (18 species tested). The application of 5'-nuclease PCR in diagnostics requires a quantitative sample preparation step. Several magnetic bead-based strategies were evaluated, since these systems are simple and relatively easy to automate. The combination of nonspecific binding of bacteria to paramagnetic beads, with subsequent DNA purification by use of the same beads, gave the most satisfactory result. The detection limit was approximately 6 to 60 CFU, quantification was linear over at least 7 log units, and the method could be completed within 3 h. In conclusion, a complete quantitative method for L. monocytogenes in water and in skimmed and raw milk was developed.

Animals↗
PubMed2003

Targeting single-nucleotide polymorphisms in the 18S rRNA gene to differentiate Cyclospora species from Eimeria species by multiplex PCR.

Cyclospora cayetanensis is a coccidian parasite that causes protracted diarrheal illness in humans. C. cayetanensis is the only species of this genus thus far associated with human illness, although Cyclospora species from other primates have been named. The current method to detect the parasite uses a nested PCR assay to amplify a 294-bp region of the small subunit rRNA gene, followed by restriction fragment length polymorphism (RFLP) or DNA sequence analysis. Since the amplicons generated from C. cayetanensis and Eimeria species are the same size, the latter step is required to distinguish between these different species. The current PCR-RFLP protocol, however, cannot distinguish between C. cayetanensis and these new isolates. The differential identification of such pathogenic and nonpathogenic parasites is essential in assessing the risks to human health from microorganisms that may be potential contaminants in food and water sources. Therefore, to expand the utility of PCR to detect and identify these parasites in a multiplex assay, a series of genus- and species-specific forward primers were designed that are able to distinguish sites of limited sequence heterogeneity in the target gene. The most effective of these unique primers were those that identified single-nucleotide polymorphisms (SNPs) at the 3' end of the primer. Under more stringent annealing and elongation conditions, these SNP primers were able to differentiate between C. cayetanensis, nonhuman primate species of Cyclospora, and Eimeria species. As a diagnostic tool, the SNP PCR protocol described here presents a more rapid and sensitive alternative to the currently available PCR-RFLP detection method. In addition, the specificity of these diagnostic primers removes the uncertainty that can be associated with analyses of foods or environmental sources suspected of harboring potential human parasitic pathogens.

Animals↗
PubMed1974

Presumptive identification of group A, B, and D streptococci.

A battery of five tests was used for presumptive identification of the pathogenic streptococci. The non-serological methods included determination of hemolysis for all strains, bacitracin susceptibility for group A streptococci, hippurate hydrolysis by group B streptococci, and bile-esculin reaction for group D streptococci. Enterococcal group D streptococci were differentiated from non-enterococcal group D streptococci by 6.5% NaCl tolerance. Two other categories of streptococci resulted: beta-hemolytic streptococci non-groups A, B, or D; and alpha- or nonhemolytic streptococci, not enterococci, not further identified (viridans streptococci). The tests were used as a battery and not as single entities. In this manner more than 99% of the group A, 99% of the group B, 81% of the beta-hemolytic streptococci non-group A, B, or D, 99% of the group D enterococci, 97% of the group D non-enterococci, and 94% of the viridans streptococci were correctly identified.

Bacitracin↗
PubMed1989

Single primer pair for amplifying segments of distinct Shiga-like-toxin genes by polymerase chain reaction.

By using a single synthetic oligonucleotide primer pair in the polymerase chain reaction, we amplified specific Shiga-like-toxin (SLT) gene segments from DNAs of 20 clinical Escherichia coli isolates, irrespective of whether they produce SLT-I, SLT-II, or heretofore uncategorized SLTs. These segments were not detectable in any of 20 nontoxigenic E. coli strains. The primers deduced from a conserved region among SLT genes are so-called degenerate-sequence primers; i.e., they contain intentionally introduced sequence ambiguities to overcome minor sequence variations within different SLT genes. In direct gel hybridization with genomic DNA, both primers recognized SLT-I and SLT-II DNA sequences. Amplified sequences of target DNA obtained by polymerase chain reaction were visualized after gel electrophoresis by ethidium bromide staining, and definitive identification of the amplification product as an SLT gene segment was achieved by hybridization to SLT-I- and SLT-II-specific 20-base oligonucleotide probes complementary to a portion of the amplified sequences but not to the primers. The detecting oligonucleotide probes shared only 30% base homology and were shown to recognize specifically SLT-I or SLT-II sequences within genomic DNA. Moreover, they were used to distinguish whether the amplified sequence originated from SLT-I or SLT-II genes. The PCR system with the primers described here is a powerful technique to amplify SLT sequences in E. coli strains that produce serologically distinct SLTs and will facilitate identification of these pathogens, particularly among a multitude of nonpathogenic E. coli strains.

Animals↗
PubMed1990

Endocarditis associated with Comamonas acidovorans.

A case of endocarditis caused by Comamonas acidovorans (Pseudomonas acidovorans) in a 42-year-old intravenous-drug abuser is described. This article appears to be the first detailed report of the isolation of this organism from a systemic clinical infection and its identification as a pathogen.

Adult↗
PubMed1990

Amplification of bacterial 16S ribosomal DNA with polymerase chain reaction.

The sequence of small-subunit rRNA varies in an orderly manner across phylogenetic lines and contains segments that are conserved at the species, genus, or kingdom level. By directing oligonucleotide primers at sequences conserved throughout the eubacterial kingdom, we amplified bacterial 16S ribosomal DNA sequences with the polymerase chain reaction. Priming sites were located at the extreme 5' end, the extreme 3' end, and the center of 16S ribosomal DNA. The isolates tested with these primers included members of the genera Staphylococcus, Coxiella, Rickettsia, Clostridium, Neisseria, Mycobacterium, Bilophila, Eubacterium, Fusobacterium, and Lactobacillus and the family Enterobacteriaceae. Initially, the yields from the reactions were erratic because the primers were self-complementary at the 3' ends. Revised primers that were not self-complementary gave more reproducible results. With the latter primers, 0.4 pg of Escherichia coli DNA consistently gave a visible band after amplification. This method should be useful for increasing the amounts of bacterial 16S ribosomal DNA sequences for the purposes of sequencing and probing. It should have a broad range of applications, including the detection and identification of known pathogens that are difficult to culture. This approach may make it possible to identify new, nonculturable bacterial pathogens.

Base Sequence↗
PubMed1994

Use of induced sputum specimens for microbiologic diagnosis of infections due to organisms other than Pneumocystis carinii.

The optimal diagnostic approach to pneumonia provides a rapid microbiologic identification of pulmonary pathogens by the least invasive means. The technique of sputum induction has been useful in the evaluation of patients with Pneumocystis carinii or mycobacterial pneumonia. It is not known whether induced sputum samples are preferable for the detection of pathogens other than P. carinii or mycobacteria. Microbiologic yields were evaluated from identically processed induced and conventional sputum samples collected from 509 consecutive patients. No statistically significant differences were found between the microbiologic yields of induced and spontaneous sputum samples. Bacterial pathogens were isolated in 19.6% of induced and 23.5% of routine specimens. Mycobacteria were cultured from 13.1% of routine and 9.4% of induced specimens. Non-Candida albicans fungi grew from 24% of routine and 20% of induced specimens. The process of sputum induction with aerosolized hypertonic saline did not alter either the purulence or the bacterial quantitation of Gram-stained sputum specimens. Sputum induction has been useful for the cytologic diagnosis of malignancy, for the diagnosis of pneumocystosis and tuberculosis, and in patients unable to spontaneously produce sputum samples. The use of induced sputum samples for the diagnosis of other infections may not be necessary when routine sputum specimens are available.

Bacteria↗
PubMed1996

Diagnostic antigenemia tests for penicilliosis marneffei.

Disseminated penicilliosis marneffei is an emerging opportunistic mycosis seen in severely immunocompromised human immunodeficiency virus (HIV)-infected patients and is caused by the dimorphic fungus Penicillium marneffei. Early diagnosis and treatment improve clinical outcome. Proper diagnosis is complicated by nonspecific signs and symptoms and by difficulties in histologic recognition and species identification of the pathogen. Since no established immunodiagnostic methods for penicilliosis marneffei are available, we attempted to develop separate immunodiffusion tests to detect P. marneffei antigens and antibodies in patient serum specimens and a latex agglutination test for antigenemia. Antigens consisted of 2-week-old fission arthroconidial filtrates produced in Pine's broth at 37 degrees C. Rabbit antisera were prepared against the 10 x -concentrated filtrate antigens. Studies were carried out with 17 serum specimens from HIV-seropositive adult Thai patients with penicilliosis marneffei and 15 control serum specimens from Thai persons free of HIV and P. marneffei infection. The immunodiffusion tests detected P.marneffei antigenemia in 10 (58.8%) of 17 patients, whereas the latex agglutination test detected antigenemia in 13 (76.5%) of the 17 patients. Antibody was demonstrated in only 2 of the 17 patient sera. All of the tests appeared to be highly specific, since none were positive with sera from 15 Thai control patients, six serum samples containing cryptococcal antigen, or six urine specimens positive for Histoplasma polysaccharide antigens.

AIDS-Related Opportunistic Infections↗
PubMed2001

Differentiation of Candida dubliniensis from Candida albicans on staib agar and caffeic acid-ferric citrate agar.

The methods currently available for the identification of the pathogenic yeast Candida dubliniensis all have disadvantages in that they are time-consuming, expensive, and/or, in some cases, unreliable. In a recent study (P. Staib and J. Morschhäuser, Mycoses 42:521-524; 1999) of 14 C. dubliniensis and 11 C. albicans isolates, it was suggested that the ability of C. dubliniensis to produce rough colonies and chlamydospores (chlamydoconidia) on Staib agar (SA) provided a simple means of differentiating it from its close relative C. albicans. In the present investigation, we examined the colony morphology and chlamydospore production of 130 C. dubliniensis and 166 C. albicans isolates on SA and on the related defined medium caffeic acid-ferric citrate agar (CAF). All of the C. dubliniensis and C. albicans isolates produced chlamydospores on the control medium, i.e., rice-agar-Tween agar. However, while none of the C. albicans isolates produced chlamydospores on either SA or CAF, 85.4 and 83.8% of the C. dubliniensis isolates produced chlamydospores on SA and CAF, respectively. All of the C. albicans isolates grew as smooth, shiny colonies on SA after 48 to 72 h of incubation at 30 degrees C, while 97.7% of the C. dubliniensis isolates grew as rough colonies, many (65%) with a hyphal fringe. In contrast, 87.4% of the C. albicans and 93.8% of the C. dubliniensis isolates yielded rough colonies on CAF. Although the results of this study confirm that SA is a good medium for distinguishing between C. dubliniensis and C. albicans, we believe that discrimination between these two species is best achieved on the basis of colony morphology rather than chlamydospore production.

Agar↗
PubMed2005

Evaluation of PCR testing of ethanol-fixed nasal swab specimens as an augmented surveillance strategy for influenza virus and adenovirus identification.

Viral culture isolation has been widely accepted as the "gold standard" for laboratory confirmation of viral infection; however, it requires ultralow temperature specimen storage. Storage of specimens in ethanol at room temperature could expand our ability to conduct active surveillance and retrospective screenings of viruses with rapid and inexpensive real-time PCR tests, including isolates from remote regions where freezing specimens for culture is not feasible. Molecular methods allow for rapid identification of viral pathogens without the need to maintain viability. We hypothesized that ethanol, while inactivating viruses, can preserve DNA and RNA for PCR-based methods. To evaluate the use of ethanol-stored specimens for augmenting surveillance for detection of influenza viruses A and B and adenoviruses (AdV), paired nasal swab specimens were collected from 384 recruits with febrile respiratory illness at Fort Jackson, S.C., in a 2-year study. One swab was stored at ambient temperature in 100% ethanol for up to 6 months, and the other swab was stored at -70 degrees C in viral medium. For viral detection, frozen specimens were cultured for a variety of respiratory viruses, and ethanol-fixed specimens were tested with TaqMan (TM) probe and LightCycler SYBR green (SG) melting curve assays with at least two different PCR targets for each virus. The sensitivities of the TM and SG assays on specimens stored in ethanol for 1 month were 75% and 58% for influenza A, 89% and 67% for influenza B, and 93 to 98% and 57% for AdV, respectively. Lower specificities of the real-time assays corresponded to the increased detection of PCR-positive but culture-negative specimens. Influenza virus RNA was detected as well or better after 6 months of storage in ethanol.

Adenovirus Infections, Human↗
PubMed2005

Clinical use of 16S rRNA gene sequencing to identify Mycoplasma felis and M. gateae associated with feline ulcerative keratitis.

Routine bacterial cultures of corneal scrapings from seven cats with either ulcerative feline keratitis, keratomalacia, or both yielded colonies which were identified by 16S rRNA gene sequencing as Mycoplasma felis (six cases) and Mycoplasma gateae (one case). Identification of the pathogens allowed the use of less empirical and more organism-specific therapy.

Animals↗
PubMed2006

Newer approaches to the diagnosis of early onset neonatal sepsis.

Accurate and timely diagnosis of early onset neonatal sepsis remains challenging to the clinician and the laboratory. A test with a rapid turnaround time with 100% sensitivity, rather than high specificity, which allows accurate diagnosis and appropriate antimicrobial treatment or which allows antibiotics to be safely withheld in non-infected infants, is desirable. Many potential markers (acute phase reactants, cell surface markers, cytokines) are not routinely available to the laboratory, and most likely combinations of markers will ensure greater diagnostic accuracy. In the future, molecular biology techniques offer the prospect of rapid identification of both pathogens and antimicrobial resistance markers.

Acute-Phase Reaction↗
PubMed1989

Chronic orbital inflammatory disease: parasitisation of orbital leucocytes by mollicute-like organisms.

Chronic orbital inflammatory disease (COID) is usually considered non-infectious and idiopathic. Treatment is empirical, palliative, and may not prevent disease progression. COID occurs in isolation or in association with various systemic diseases. Exophthalmos may be an important presenting sign. Vasculitis, lymphoid infiltrates, and granulomas are common. Mollicute-like organisms (MLO) parasitising and destroying vitreous leucocytes are often found to cause human chronic uveitis when an appropriate search is made. Inoculation of these MLO into mouse eyelids produced chronic uveitis and exophthalmic orbital inflammatory disease. Mollicutes are cell wall deficient bacteria. Extracellular mollicutes cause human and animal diseases characterised by lymphoid infiltrates, immunosuppression, and autoantibody production. Intracellular morphologically similar bacteria are non-cultivable pathogens termed MLO. Identification is based on direct detection in diseased cells by transmission electron microscopy. MLO are cytopathogenic and detection is aided by the alterations they produce. MLO replace the cytoplasm, destroy the organelles, and alter the nucleus. This results in cell proliferation, destruction, and dysfunction. MLO parasitise lymphocytes, monocytes, and polymorphonuclear leucocytes. This report describes orbital leucocytes parasitised by MLO in three patients with isolated COID. Rifampicin treatment of MLO disease is discussed.

Adolescent↗