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1.
Am J Trop Med Hyg ; 95(6): 1424-1427, 2016 Dec 07.
Article in English | MEDLINE | ID: mdl-27928089

ABSTRACT

A 58-year-old Korean-born woman with a history of seizures and psychiatric issues was found dead at home. Autopsy was notable for large, calcified nodules that had nearly replaced her right temporal lobe. Histologic examination revealed the presence of Paragonimus eggs. This case demonstrates a rare manifestation of an aberrantly migrated lung fluke that resulted in epilepsy and sudden death years after the initial infection.


Subject(s)
Brain Diseases/parasitology , Paragonimiasis/pathology , Animals , Brain/parasitology , Brain/pathology , Death, Sudden , Fatal Outcome , Female , Humans , Middle Aged , Paragonimiasis/epidemiology , Paragonimus/isolation & purification
2.
Arch Pathol Lab Med ; 136(12): 1593-6, 2012 Dec.
Article in English | MEDLINE | ID: mdl-23194052

ABSTRACT

We present 2 cases of Cokeromyces recurvatus in routine, liquid-based Papanicolaou tests (ThinPrep). Patient 1 is a healthy, asymptomatic, 26-year-old woman with no pertinent past medical history. Patient 2 is a healthy, asymptomatic, 47-year-old woman with no pertinent past medical history. The Papanicolaou tests from both patients showed many fungal-like elements as globose, yeastlike forms measuring 10 to 30 µm in diameter with multiple, narrowly attached apparent "daughter" buds. This morphology was consistent with Paracoccidioides brasiliensis. However, broad-range fungal polymerase chain reaction and deoxyribonucleic acid sequence analysis performed with GenBank Basic Local Alignment Search Tool showed an exact match for C recurvatus. Our cases highlight the importance of molecular techniques to prevent misdiagnosis of C recurvatus as P brasiliensis, based on morphology alone. There have been 8 previously published cases of C recurvatus infection in humans, 3 of which were reported in the female genital tract.


Subject(s)
Cervix Uteri/microbiology , Mucorales/isolation & purification , Mucormycosis/diagnosis , Reproductive Tract Infections/diagnosis , Uterine Cervicitis/diagnosis , Adult , Cervix Uteri/pathology , Databases, Nucleic Acid , Diagnosis, Differential , Female , Humans , Middle Aged , Molecular Typing , Mucorales/classification , Mucorales/cytology , Mucormycosis/microbiology , Mucormycosis/pathology , Mycological Typing Techniques , Papanicolaou Test , Paracoccidioidomycosis/diagnosis , Reagent Kits, Diagnostic , Reproductive Tract Infections/microbiology , Reproductive Tract Infections/pathology , Uterine Cervicitis/microbiology , Uterine Cervicitis/pathology , Vaginal Smears
3.
J Clin Microbiol ; 50(1): 138-41, 2012 Jan.
Article in English | MEDLINE | ID: mdl-22031703

ABSTRACT

To investigate the performance of a nucleic acid amplification test (NAAT) for the diagnosis of Mycobacterium tuberculosis bacteremia, 5-ml aliquots of blood were inoculated into bioMérieux mycobacterial (MB) bottles and incubated, and 5-ml aliquots of blood were extracted and tested by real-time PCR. Of 25 samples from patients with M. tuberculosis bacteremia, 9 (36.0%) were positive and 1 (1.5%) of 66 control samples was positive by NAAT. The NAAT shows promise, but modifications should focus on improving sensitivity.


Subject(s)
Bacteremia/diagnosis , Bacteriological Techniques/methods , Blood/microbiology , Mycobacterium tuberculosis/isolation & purification , Real-Time Polymerase Chain Reaction/methods , Specimen Handling/methods , Tuberculosis/diagnosis , Adolescent , Adult , Aged , Aged, 80 and over , Bacteremia/microbiology , Humans , Middle Aged , Mycobacterium tuberculosis/genetics , Sensitivity and Specificity , Tuberculosis/microbiology , Young Adult
4.
Diagn Microbiol Infect Dis ; 68(1): 28-33, 2010 Sep.
Article in English | MEDLINE | ID: mdl-20727466

ABSTRACT

The role of Panton-Valentine leukocidin (PVL) in methicillin-resistant Staphylococcus aureus (MRSA) infections is unclear. PVL has been long associated with soft tissue infections and necrotizing pneumonia, but inconsistently with other site infections or mortality. The retrospective cohort study explores the association between PVL and bacteremia in colonized medical intensive care unit (ICU) patients with surveillance isolates and blood cultures. A total of 840 patients were screened by nasal swab, with 266 patients found to be colonized and 46 with bacteremia. Colonization by PVL(+) MRSA increased the odds of bacteremia (odds ratio, 2.40; confidence interval, 1.23-4.57), and invasive infection developed earlier in these patients (relative risk, 0.44; confidence interval 0.25-0.85) compared to those colonized with PVL(0) MRSA. PVL was not associated with infections at other sites, length of ICU stay, or mortality. PVL decreases the time to bacteremia in colonized patients but does not otherwise contribute to disease course or clinical outcome.


Subject(s)
Bacteremia/epidemiology , Bacteremia/microbiology , Bacterial Toxins/metabolism , Exotoxins/metabolism , Intensive Care Units , Leukocidins/metabolism , Methicillin-Resistant Staphylococcus aureus/pathogenicity , Staphylococcal Infections/complications , Adult , Aged , Blood/microbiology , Cohort Studies , Culture Media , Female , Humans , Male , Methicillin-Resistant Staphylococcus aureus/isolation & purification , Middle Aged , Nasal Cavity/microbiology , Population Surveillance/methods , Staphylococcal Infections/microbiology
5.
Diagn Mol Pathol ; 19(2): 123-5, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20502190

ABSTRACT

We experienced significant problems while developing a PyrosequencingTM (Biotage, Uppsala, Sweden) assay to characterize the rifampin resistance-determining region of Mycobacteriumtuberculosis, a target with high guanosine-cytosine content. This paper describes the successful use of a modified pyrosequencing protocol through partial substitution of deoxyguanosine triphosphate with deoxyinosine triphosphate in the polymerase chain reaction.


Subject(s)
DNA, Bacterial/metabolism , Deoxyguanosine/metabolism , Inosine/analogs & derivatives , Pathology, Molecular/methods , Sequence Analysis, DNA/methods , Specimen Handling/methods , Bacterial Proteins/genetics , Base Composition , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Humans , Inosine/metabolism , Mycobacterium tuberculosis/genetics , Sweden
6.
Diagn Microbiol Infect Dis ; 67(3): 286-90, 2010 Jul.
Article in English | MEDLINE | ID: mdl-20462726
7.
J Orthop Res ; 28(9): 1245-51, 2010 Sep.
Article in English | MEDLINE | ID: mdl-20186836

ABSTRACT

One limitation to the use of the polymerase chain reaction (PCR) to identify orthopedic infections has been apparent false-positive results, possibly due to the detection of dead bacteria. We recently showed that the use of DNA-binding agent propidium monoazide (PMA) could distinguish viable from heat-inactivated bacteria, and, in this study, we investigated whether the same technique can be applied to bacteria killed by two antibiotics with distinctly different mechanisms of action, a test of greater clinical relevance than thermal inactivation. Staphylococcus aureus and S. epidermidis were inactivated by vancomycin and gentamicin and treated with PMA or left untreated before DNA extraction. The threshold cycle difference of antibiotic-treated bacteria with and without PMA pretreatment was investigated with PCR primers for the 16S rDNA and tuf genes. Our results indicated that PMA effectively inhibited detection by PCR of bacteria, which had been inactivated by either vancomycin or gentamicin. The effect was statistically significant at 24 h after treatment (C(t) difference consistently >3; p < 0.05) and after 10 days of treatment (C(t) difference >4; p < 0.01), when compared to viable cells (C(t) difference 1-2). Vancomycin had a stronger effect on the C(t) value than gentamicin, reflecting the different mechanism of action of each antibiotic. Techniques of this type may help reduce clinically false-positive PCR results caused by the detection of dead bacteria, and may be especially useful in patients who have received antibiotics, such as patients undergoing the second stage of a two-stage revision for infected arthroplasty.


Subject(s)
Azides , Propidium/analogs & derivatives , Reverse Transcriptase Polymerase Chain Reaction/methods , Staphylococcal Infections/diagnosis , Staphylococcus aureus/genetics , Staphylococcus epidermidis/genetics , Anti-Bacterial Agents/pharmacology , Cross-Linking Reagents/pharmacology , DNA, Bacterial/drug effects , DNA, Bacterial/genetics , Gentamicins/pharmacology , Hot Temperature , Humans , Microbial Viability , Microbiological Techniques , Orthopedic Procedures , Staphylococcal Infections/microbiology , Staphylococcus aureus/drug effects , Staphylococcus aureus/growth & development , Staphylococcus epidermidis/drug effects , Staphylococcus epidermidis/growth & development , Vancomycin/pharmacology
8.
J Clin Microbiol ; 47(12): 3985-90, 2009 Dec.
Article in English | MEDLINE | ID: mdl-19846642

ABSTRACT

After isoniazid and rifampin (rifampicin), the next pivotal drug class in Mycobacterium tuberculosis treatment is the fluoroquinolone class. Mutations in resistance-determining regions (RDR) of the rpoB, katG, and gyrA genes occur with frequencies of 97%, 50%, and 85% among M. tuberculosis isolates resistant to rifampin, isoniazid, and fluoroquinolones, respectively. Sequences are highly conserved, and certain mutations correlate well with phenotypic resistance. We developed a pyrosequencing assay to determine M. tuberculosis genotypic resistance to rifampin, isoniazid, and fluoroquinolones. We characterized 102 M. tuberculosis clinical isolates from the Philippines for susceptibility to rifampin, isoniazid, and ofloxacin by using the conventional submerged-disk proportion method and validated our pyrosequencing assay using these isolates. DNA was extracted and amplified by using PCR primers directed toward the RDR of the rpoB, katG, and gyrA genes, and pyrosequencing was performed on the extracts. The M. tuberculosis H37Rv strain (ATCC 25618) was used as the reference strain. The sensitivities and specificities of pyrosequencing were 96.7% and 97.3%, 63.8% and 100%, and 70.0% and 100% for the detection of resistance to rifampin, isoniazid, and ofloxacin, respectively. Pyrosequencing is thus a rapid and accurate method for detecting M. tuberculosis resistance to these three drugs.


Subject(s)
Antitubercular Agents/pharmacology , Drug Resistance, Bacterial , Isoniazid/pharmacology , Mycobacterium tuberculosis/drug effects , Ofloxacin/pharmacology , Rifampin/pharmacology , Sequence Analysis, DNA/methods , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Drug Resistance, Bacterial/genetics , Fluoroquinolones/pharmacology , Genotype , Humans , Microbial Sensitivity Tests , Mutation , Mycobacterium tuberculosis/genetics , Phenotype , Time Factors
9.
Diagn Microbiol Infect Dis ; 64(4): 445-7, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19631099

ABSTRACT

We compared 2 methods for determining Escherichia coli viability in vitro. A 16S rDNA polymerase chain reaction (PCR) assay detected bacteria irrespective of viability. A groEL mRNA reverse transcriptase PCR was positive for 72 h but later became negative. Detecting mRNA holds promise but is tedious, and groEL may not be the best target.


Subject(s)
Bacterial Infections/diagnosis , DNA, Bacterial/genetics , Diagnostic Errors , Microbial Viability , Polymerase Chain Reaction/methods , RNA, Bacterial/genetics , RNA, Messenger/genetics , Bacterial Infections/microbiology , Chaperonin 60/genetics , DEAD-box RNA Helicases , Escherichia coli/genetics , Escherichia coli Proteins/genetics , Polymerase Chain Reaction/standards , RNA, Ribosomal, 16S/genetics
10.
J Orthop Res ; 27(9): 1243-7, 2009 Sep.
Article in English | MEDLINE | ID: mdl-19322790

ABSTRACT

Molecular techniques, such as the polymerase chain reaction (PCR) have high sensitivity when used to diagnose infection, but may detect DNA, RNA, and proteins from dead, as well as viable, bacteria. Propidium monoazide (PMA) is a DNA binding agent, that has the ability to penetrate only dead cells with compromised membranes and has been used in conjunction with real-time PCR to distinguish intact from dead bacterial cells. In this study, intact, heat-inactivated (dead), and intact/dead admixed Staphylococcus aureus (S. aureus) and Staphylococcus epidermidis (S. epidermidis) were treated with PMA or left untreated before DNA extraction. We quantified levels of 16S rDNA and tuf gene by real-time quantitative PCR (qPCR), to test the ability of PMA to distinguish intact from dead bacteria. Our results indicated that PMA inhibited detection of dead bacteria, and the qPCR results reflected the number of intact bacteria without being impacted by the presence of the dead bacteria. This approach of combining qPCR with and without PMA treatment has promise to limit false-positive PCR results when used to diagnose infections, but needs to be further validated in clinical samples.


Subject(s)
Microbial Viability , Microbiological Techniques/methods , Reverse Transcriptase Polymerase Chain Reaction/methods , Staphylococcal Infections/diagnosis , Staphylococcus aureus/genetics , Staphylococcus epidermidis/genetics , Azides , DNA, Bacterial/genetics , Humans , Propidium/analogs & derivatives , Staphylococcal Infections/microbiology
11.
Int J Infect Dis ; 13(6): e463-6, 2009 Nov.
Article in English | MEDLINE | ID: mdl-19269872

ABSTRACT

Salmonella septic arthritis is rare. Our objective was to identify bacterial species from joint fluid using a broad-range real-time PCR and pyrosequencing technique. We describe a case of bilateral Salmonella enterica serotype Enteritidis infection of right and left total knee arthroplasties. DNA was extracted from the joint fluid of the left knee, amplified by PCR, and the amplicons were evaluated by pyrosequencing. The patient was treated with ciprofloxacin, and the polyethylene liners were replaced in both knees. The results of pyrosequencing detected a Salmonella species. To the best of our knowledge, this is the first report describing the detection of Salmonella in joint fluid by universal PCR followed by pyrosequencing.


Subject(s)
Arthroplasty, Replacement, Knee/adverse effects , Polymerase Chain Reaction/methods , Prosthesis-Related Infections/diagnosis , Salmonella Infections/diagnosis , Salmonella enteritidis/classification , Salmonella enteritidis/isolation & purification , Sequence Analysis, DNA/methods , Aged , Female , Humans , Joint Prosthesis/microbiology , Knee Joint/microbiology , Prosthesis-Related Infections/microbiology , Salmonella Infections/microbiology , Salmonella enteritidis/genetics
12.
Clin Orthop Relat Res ; 467(5): 1360-4, 2009 May.
Article in English | MEDLINE | ID: mdl-18989729

ABSTRACT

Bacteria such as staphylococci commonly encountered in orthopaedic infections form biofilms and adhere to bone implants and cements. Various methods to disrupt the biofilm and enhance bacterial detection have been reported. We will describe the effectiveness of vortexing and sonication to improve the detection of biofilm-formative bacteria from polymethylmethacrylate by conventional quantitative bacterial culture and real-time quantitative PCR. We used a single biofilm-formative Staphylococcus aureus strain and 20 polymethylmethacrylate coupons as an in vitro biofilm model; four coupons were used for each of two control groups or three experimental sonication times (1, 5, and 30 minutes). Vortexing the cement without sonication increased the yield of adherent bacteria to a considerable extent. The combination of vortexing and sonication further enhanced the yield regardless of the duration of sonication. Quantitative conventional cultures correlated with quantitative PCR assay. The combination of vortexing and sonication to disrupt the bacterial biofilm followed by quantitative PCR and/or culture seems to be a sensitive method for detecting bacteria adherent to bone cement.


Subject(s)
Bacterial Typing Techniques , Biofilms , Bone Cements/analysis , Polymethyl Methacrylate/analysis , Sonication , Staphylococcus aureus/isolation & purification , Bacterial Adhesion , Colony Count, Microbial , Pilot Projects , Polymerase Chain Reaction , Ribotyping , Staphylococcus aureus/classification , Staphylococcus aureus/genetics , Staphylococcus aureus/growth & development , Time Factors
13.
Surg Neurol ; 72(1): 74-9; discussion 79, 2009 Jul.
Article in English | MEDLINE | ID: mdl-18514285

ABSTRACT

BACKGROUND: Nocardia brain abscess carries a higher morbidity and mortality rate than other bacterial cerebral abscesses, with reported mortality rates of 55% and 20% in immunocompromised and immunocompetent patients, respectively. To prevent a delay in diagnosis and treatment, an aggressive therapeutic approach is required. In the present study, a rapid and accurate molecular diagnostic approach using pyrosequencing (PS), a semiautomated molecular genotyping method of nucleotide sequencing-by-synthesis, was performed. CASE DESCRIPTION: A 53-year-old man developed word-finding difficulties, followed by confusion and disorientation. On examination, the patient had a mixed aphasia; the receptive component was greater than the expressive component. The remainder of his neurologic examination findings was normal. Gadolinium-enhanced magnetic resonance imaging of the brain revealed a 2.0-cm multilobular, partially cystic, peripheral-enhancing mass in the posterior left temporal-parietal region with significant vasogenic edema and localized mass effect. A detailed laboratory investigation revealed that this patient was immunocompetent. An awake left posterior temporal-parietal craniotomy with cortical motor and speech mapping, using frameless stereotactic image guidance and intraoperative real-time ultrasound, was performed. Frozen section was consistent with cerebral abscess and methenamine silver staining revealed many beaded, thin-branching gram-positive bacilli. Colonies suspicious for Nocardia sp were seen within 2 days, and PCR followed by pyrosequencing (PS) identified Nocardia farcinica. CONCLUSIONS: We report a nocardial cerebral abscess mimicking a metastatic brain tumor, and we demonstrate that PS technology can be used for the accurate and rapid identification of N farcinica isolated from a brain abscess -- facilitating a rapid diagnosis and successful, durable treatment.


Subject(s)
Brain Abscess/diagnosis , Brain Abscess/microbiology , Diagnostic Techniques, Neurological , Nocardia Infections/complications , Nocardia Infections/diagnosis , Nocardia/genetics , Anti-Bacterial Agents/adverse effects , Anti-Bacterial Agents/therapeutic use , Brain/microbiology , Brain/pathology , Brain/surgery , Brain Abscess/surgery , Brain Neoplasms/diagnosis , Brain Neoplasms/secondary , Carcinoma/diagnosis , Carcinoma/secondary , DNA, Bacterial/analysis , DNA, Bacterial/genetics , Diagnosis, Differential , Drug Hypersensitivity , Epilepsy/chemically induced , Genotype , Humans , Immunocompetence , Lung Neoplasms/pathology , Magnetic Resonance Imaging , Male , Middle Aged , Molecular Biology/methods , Neurosurgical Procedures , Nocardia Infections/drug therapy , Treatment Outcome
14.
Am J Clin Pathol ; 130(2): 284-9, 2008 Aug.
Article in English | MEDLINE | ID: mdl-18628099

ABSTRACT

Salmonella and Shigella species are routinely sought in stool specimens submitted for culture. It is a common practice to screen lactose-negative colonies by using triple sugar iron agar, lysine iron agar, and Christensen urea agar to determine if further identification is necessary. We designed and evaluated a novel combination of media, which are layered in a single tube, for screening isolates suspected to possibly represent Salmonella or Shigella. We tested this media combination with 106 Salmonella, 56 Shigella, and 56 other gram-negative bacilli. All Salmonella and Shigella isolates tested were appropriately characterized as possible Salmonella or Shigella by using an algorithm developed for use with this media combination. Similarly, 53 (95%) of 56 other gram-negative bacilli were appropriately screened as non -Salmonella and non -Shigella isolates. This unique media combination provides the most important biochemical reactions needed to screen for Salmonella and Shigella in a single-tube format, which decreases labor by two thirds (ie, 1 tube is inoculated vs 3).


Subject(s)
Bacteriological Techniques , Culture Media , Salmonella/isolation & purification , Shigella/isolation & purification , Agar , Algorithms , Gram-Negative Aerobic Bacteria/isolation & purification
15.
Diagn Microbiol Infect Dis ; 58(3): 315-23, 2007 Jul.
Article in English | MEDLINE | ID: mdl-17382507

ABSTRACT

To rapidly identify Mycobacterium and Nocardia spp. without costly probes, we had implemented capillary electrophoresis (CE) in polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) analysis to analyze their 65-kDa heat shock protein (hsp65) gene. The PCR-RFLP analysis with CE (PRACE) involved only one restriction enzyme, HaeIII, and a single electrophoretic separation less than 10 min. Full-range (10-200 bp) RFLP patterns of 12 less common Mycobacterium and 7 Nocardia spp. were investigated. A good agreement was observed between the sizes of restriction fragments resolved by CE and the real sizes deduced from sequence analysis. Including hsp65 gene patterns of 12 Mycobacterium spp. published earlier, differentiation was distinct among 24 Mycobacterium and 7 Nocardia spp. Some closely related species exhibiting similar biochemical characteristics could be well discriminated by an extra HaeIII digestion site. Thus, PRACE offers a nonprobe alternative for rapid identification of various cultured Mycobacterium and Nocardia to the species level.


Subject(s)
Bacterial Proteins/genetics , Chaperonins/genetics , Electrophoresis, Capillary/methods , Mycobacterium/classification , Nocardia/classification , Polymorphism, Restriction Fragment Length , Chaperonin 60 , DNA, Bacterial/genetics , Deoxyribonucleases, Type II Site-Specific , Mycobacterium/genetics , Nocardia/genetics , Polymerase Chain Reaction
16.
Clin Orthop Relat Res ; 457: 210-3, 2007 Apr.
Article in English | MEDLINE | ID: mdl-17195819

ABSTRACT

Biofilms are complex microenvironments produced by microorganisms on surfaces. Ultrasonication disrupts biofilms and may make the microorganism or its DNA available for detection. We determined whether ultrasonication could affect our ability to detect bacteria adherent to a metal substrate. A biofilm-formative Staphylococcus aureus strain was used for an in vitro implant infection model (biofilm-formative condition). We used quantitative culture and real time-polymerase chain reaction to determine the influence of different durations of ultrasound on bacterial adherence and viability. Sonication for 1 minute increased the yield of bacteria. Sonication longer than 5 minutes led to fewer bacterial colonies by conventional culture but not by polymerase chain reaction. This suggests short periods of sonication help release bacteria from the metal substrate by disrupting the biofilm, but longer periods of sonication lyse bacteria prohibiting their detection in microbiologic cultures. A relatively short duration of sonication may be desirable for maximizing detection of biofilm-formative bacteria around implants by culture or polymerase chain reaction.


Subject(s)
Biofilms , Prostheses and Implants/microbiology , Sonication , Staphylococcus aureus/isolation & purification , Colony Count, Microbial , DNA, Bacterial/analysis , Microbial Viability , Pilot Projects , Polymerase Chain Reaction , Stainless Steel , Staphylococcus aureus/genetics , Staphylococcus aureus/growth & development , Time Factors
17.
Arch Pathol Lab Med ; 130(7): 1053-6, 2006 Jul.
Article in English | MEDLINE | ID: mdl-16831035

ABSTRACT

We identified Mycobacterium tuberculosis DNA using real-time polymerase chain reaction on a specimen from an osteolytic lesion of a femoral condyle, in which the frozen section demonstrated granulomas. The process was much more rapid than is possible with culture. The rapid detection of M tuberculosis and the concomitant exclusion of granulomatous disease caused by nontuberculous mycobacteria or systemic fungi are necessary to appropriately treat skeletal tuberculosis. The detection and identification of M tuberculosis by culture may require several weeks using traditional methods. The real-time polymerase chain reaction method used has been shown to be rapid and reliable, and is able to detect and differentiate both tuberculous and nontuberculous mycobacteria. Real-time polymerase chain reaction may become a diagnostic standard for the evaluation of clinical specimens for the presence of mycobacteria; this case demonstrates the potential utility of this assay for the rapid diagnosis of skeletal tuberculosis.


Subject(s)
DNA, Bacterial/analysis , Mycobacterium tuberculosis/isolation & purification , Reverse Transcriptase Polymerase Chain Reaction/methods , Tuberculosis, Osteoarticular/diagnosis , Antitubercular Agents/therapeutic use , Bacterial Typing Techniques , Drug Therapy, Combination , Ethambutol/therapeutic use , Female , Femur/microbiology , Femur/pathology , Humans , Isoniazid/therapeutic use , Middle Aged , Mycobacterium tuberculosis/genetics , Osteolysis/microbiology , Osteolysis/pathology , Pyrazinamide/therapeutic use , Rifampin/therapeutic use , Tuberculosis, Osteoarticular/drug therapy , Tuberculosis, Osteoarticular/microbiology
18.
J Orthop Res ; 24(8): 1641-9, 2006 Aug.
Article in English | MEDLINE | ID: mdl-16788984

ABSTRACT

We have developed a combined real-time PCR and pyrosequencing assay that successfully differentiated the vast majority of gram-positive and gram-negative bacteria when bacterial isolates were tested. The purpose of this study was to evaluate this assay on clinical specimens obtained from orthopedic surgeries, and to prospectively compare the results of "molecular Gram stain" with culture and conventional direct Gram stain. Forty-five surgical specimens were obtained from patients who underwent orthopedic surgery procedures. The DNA was extracted and a set of broad-range PCR primers that targeted a part of the 16S rDNA gene was used for pan-bacterial PCR. The amplicons were submitted for pyrosequencing and the resulting molecular Gram stain characteristics were recorded. Culture and direct Gram staining were performed using standard methods for all cases. Surgical specimens were reviewed histologically for all cases that had a discrepancy between culture and molecular results. There was an 86.7% (39/45) agreement between the traditional and molecular methods. In 12/14 (85.7%) culture-proven cases of bacterial infection, molecular Gram stain characteristics were in agreement with the culture results, while the conventional Gram stain result was in agreement only for five cases (35.7%). In the 31 culture negative cases, 27 cases were also PCR negative, whereas 4 were PCR positive. Three of these were characterized as gram negative and one as gram positive by this molecular method. Molecular determination of the Gram stain characteristics of bacteria that cause orthopedic infections may be achieved, in most instances, by this method. Further studies are necessary to understand the clinical importance of PCR-positive/culture-negative results.


Subject(s)
Gram-Positive Bacterial Infections/diagnosis , Osteitis/diagnosis , Reverse Transcriptase Polymerase Chain Reaction/methods , Staphylococcal Infections/diagnosis , Staphylococcus/genetics , Base Sequence , Candidiasis/diagnosis , Corynebacterium Infections/diagnosis , DNA, Bacterial/analysis , Gentian Violet , Humans , Microbiological Techniques , Molecular Sequence Data , Osteitis/microbiology , Phenazines , Proteus Infections/diagnosis , RNA, Ribosomal, 16S/genetics , Sequence Analysis, DNA/methods , Staphylococcus/isolation & purification
19.
Diagn Mol Pathol ; 15(2): 70-3, 2006 Jun.
Article in English | MEDLINE | ID: mdl-16778586

ABSTRACT

Progressive multifocal leukoencephalopathy (PML) is a demyelinating disease of the central nervous system caused by the JC polyoma virus. Electron microscopy and immunohistochemistry are the traditional methods of confirming the presence of the virus in brain biopsies from these patients. We studied the brain biopsies from 7 patients with PML and 6 patients without PML with chromogenic in situ hybridization (CISH) for the JC polyoma virus using a commercially available probe. The biopsies from the patients with the PML cases were proven to contain the JC polyoma virus by traditional and molecular methods. The CISH findings were compared with the known state of infection. All (7/7) of the biopsies from patients with PML were positive for the presence of polyoma virus by CISH, whereas the biopsies from patients without PML were uniformly negative. CISH seems to be a useful tool for the detection of the JC virus in brain biopsies from patients with PML, and is more accessible because a commercial probe is available.


Subject(s)
Brain/virology , JC Virus/genetics , JC Virus/isolation & purification , Leukoencephalopathy, Progressive Multifocal/virology , Case-Control Studies , Chromogenic Compounds , Humans , In Situ Hybridization/methods , JC Virus/pathogenicity , Leukoencephalopathy, Progressive Multifocal/diagnosis , Polymerase Chain Reaction
20.
J Clin Microbiol ; 43(12): 6144-6, 2005 Dec.
Article in English | MEDLINE | ID: mdl-16333115

ABSTRACT

We evaluated the Roche LightCycler Staphylococcus M(GRADE) kits to differentiate between Staphylococcus aureus and coagulase-negative staphylococci in blood cultures growing clusters of gram-positive cocci. Testing 100 bottles (36 containing S. aureus), the assay was 100% sensitive and 98.44% specific for S. aureus and 100% sensitive and specific for coagulase-negative staphylococci.


Subject(s)
Blood/microbiology , Culture Media , Gram-Positive Cocci/classification , Gram-Positive Cocci/growth & development , Reagent Kits, Diagnostic , Bacterial Typing Techniques , Coagulase/metabolism , DNA, Bacterial/analysis , Gram-Positive Cocci/isolation & purification , Humans , Polymerase Chain Reaction/methods , Sensitivity and Specificity , Species Specificity , Staphylococcus/classification , Staphylococcus/enzymology , Staphylococcus/growth & development , Staphylococcus aureus/classification , Staphylococcus aureus/growth & development , Staphylococcus aureus/isolation & purification
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