Identification of an atypical strain of toxoplasma gondii as the cause of a waterborne outbreak of toxoplasmosis in Santa Isabel do Ivai, Brazil.

Multilocus DNA sequencing has identified a nonarchetypal strain of Toxoplasma gondii as the causal agent of a waterborne outbreak in Brazil in 2001. The strain, isolated from a water supply epidemiologically linked to the outbreak, was virulent to mice, and it has previously been identified as BrI. Using a serologic assay that detects strain-specific antibodies, we found that 13 (65%) of 20 individuals who were immunoglobulin (Ig) M positive during the outbreak possessed the same serotype as mice infected with the purported epidemic strain. The remaining 7 individuals, plus additional IgM-negative, IgG-positive individuals, possessed 1 of 4 novel serotypes, the most common of which matched the serotype of mice infected with strains isolated from chickens foraging near the outbreak site. The latter strains likely reflect the genetic diversity of T. gondii circulating in highly endemic regions of Brazil. The serotyping assay proved a useful tool for identification of specific individuals infected with the outbreak agent.

Rapid antimicrobial susceptibility determination of uropathogens in clinical urine specimens by use of ATP bioluminescence.

We describe the first direct testing of the antimicrobial susceptibilities of bacterial pathogens in human clinical fluid samples by the use of ATP bioluminescence. We developed an ATP bioluminescence assay that eliminates somatic sources of ATP to selectively quantify the bacterial load in clinical urine specimens with a sensitivity of <1,000 CFU per milliliter. There was a log-log relationship between light emission and the numbers of CFU in clinical urine specimens. A clinical study was performed to evaluate the accuracy of the ATP bioluminescence assay for determination of the antimicrobial susceptibilities of uropathogens in clinical urine specimens tested in a blinded manner. ATP bioluminescent bacterial density quantitation was used to determine the inoculation volume in growth medium with and without antibiotics. After incubation at 37 degrees C for 120 min, the ATP bioluminescence assay was repeated to evaluate the uropathogen response to antibiotics. The ability of the ATP bioluminescence assay to discriminate between antimicrobial susceptibility and resistance was determined by comparison of the results obtained by the ATP bioluminescence assay with the results obtained by standard clinical microbiology methods. Receiver operator characteristic curves were used to determine the optimal threshold for discriminating between susceptibility and resistance. Susceptibility and resistance were correctly predicted in 87% and 95% of cases, respectively, for an overall unweighted accuracy of 91%, when the results were stratified by antibiotic. For samples in which the pathogen was susceptible, the accuracy improved to 95% when the results for samples with less than a 25-fold increase in the amount of bacterial ATP in the medium without antibiotics were excluded. These data indicate that a rapid bioluminescent antimicrobial susceptibility assay may be useful for the management of urinary tract infections.

Practical therapeutic application of the oxoid PBP2' latex agglutination test for the rapid identification of methicillin-resistant Staphylococcus aureus in blood cultures.

The Oxoid PBP2' latex agglutination test (OLA; Oxoid, Basingstoke, England) was evaluated in a controlled prospective study examining Staphylococcus aureus from 25 positive blood cultures. Subcultures of positive blood cultures with coagulase-positive, gram-positive cocci in clusters were batched, and the OLA was performed at the end of the working day, once growth was seen on the plate. Results were sent to the infectious disease pharmacist for therapy evaluation, and the 24-hour minimum inhibitory concentration (MIC) was confirmed the next day. Blood culture OLA results correlated 100% with oxacillin MIC results for the patient, and results were available in as little as 3 hours after the blood culture was positive. The mean time difference between the OLA and MIC reports was 19.4 hours. This test allowed same-day resistance marker reporting and was easily incorporated into the work flow of the clinical laboratory.

Ocular vaccinia following exposure to a smallpox vaccinee.

PURPOSE: To describe the presentation and management of the first identified case of ocular vaccinia infection associated with the current smallpox vaccination program. DESIGN: Case report. METHODS: Vaccinia virus was isolated by cell culture of a conjunctival swab. Direct staining with fluorescein isothiocyanate-labeled vaccinia antibody and polymerase chain reaction testing confirmed the diagnosis. RESULTS: In February 2003, a 26-year-old woman developed right preseptal cellulitis and blepharoconjunctivitis following contact with a vaccinated member of the military. The preseptal cellulitis resolved with antibacterial therapy, and the conjunctival infection was treated successfully with a 14-day course of topical trifluridine and a single dose of intravenous vaccinia immune globulin. CONCLUSIONS: To facilitate rapid diagnosis and appropriate treatment, clinicians must maintain a high index of suspicion for ocular smallpox vaccine-associated adverse reactions in vaccine recipients and their close contacts.

Multicenter evaluation of the BD Phoenix Automated Microbiology System for antimicrobial susceptibility testing of Streptococcus species.

This multicenter study evaluated the BD Phoenix Automated Microbiology System STREP panel (BD Diagnostic Systems). Antimicrobial susceptibility testing (AST) with 13 agents was performed on 2,013 streptococci (938 Streptococcus pneumoniae isolates; 396 group B streptococci [GBS]; 369 viridans group streptococci [VGS]; 290 beta-hemolytic streptococcus groups A, C, and G; and 20 other streptococci) with the Phoenix system and a broth microdilution reference method. Clinical and challenge isolates were tested against cefepime, cefotaxime (CTX), ceftriaxone (CTR), clindamycin (CLI), erythromycin (ERY), gatifloxacin, levofloxacin, linezolid, meropenem, penicillin (PEN), tetracycline (TET), trimethoprim-sulfamethoxazole, and vancomycin. Clinical isolates with major errors or very major errors (VMEs) were retested in duplicate by both methods. The final results for clinical isolates showed the following trends. For all of the organism-antimicrobial agent combinations tested, categorical agreement (CA) was 92 to 100%, with one exception-VGS-PEN (87% CA; all errors were minor). For S. pneumoniae, there was one major error with CLI (0.1%) and one or two VMEs with CTX (4%), CTR (4.5%), ERY (0.9%), and TET (0.7%). For groups A, C, and G, the CA was 97 to 100% and the only VMEs were resolved by additional reference laboratory testing. For GBS, there was only one VME (TET, 0.3%) and D-zone testing of 23 isolates with CLI major errors (one isolate unavailable) revealed inducible CLI resistance. For VGS, the major error rates were 0 to 3% and VMEs occurred with seven agents (3.5 to 7.1%). The mean times required for organism groups to generate results ranged from 8.4 to 9.4 h. The Phoenix system provided reliable and rapid AST results for most of the organism-antimicrobial agent combinations tested.

Patient safety in the clinical laboratory: a longitudinal analysis of specimen identification errors.

CONTEXT: Patient safety is an increasingly visible and important mission for clinical laboratories. Attention to improving processes related to patient identification and specimen labeling is being paid by accreditation and regulatory organizations because errors in these areas that jeopardize patient safety are common and avoidable through improvement in the total testing process. OBJECTIVE: To assess patient identification and specimen labeling improvement after multiple implementation projects using longitudinal statistical tools. DESIGN: Specimen errors were categorized by a multidisciplinary health care team. Patient identification errors were grouped into 3 categories: (1) specimen/requisition mismatch, (2) unlabeled specimens, and (3) mislabeled specimens. Specimens with these types of identification errors were compared preimplementation and postimplementation for 3 patient safety projects: (1) reorganization of phlebotomy (4 months); (2) introduction of an electronic event reporting system (10 months); and (3) activation of an automated processing system (14 months) for a 24-month period, using trend analysis and Student t test statistics. RESULTS: Of 16,632 total specimen errors, mislabeled specimens, requisition mismatches, and unlabeled specimens represented 1.0%, 6.3%, and 4.6% of errors, respectively. Student t test showed a significant decrease in the most serious error, mislabeled specimens (P < .001) when compared to before implementation of the 3 patient safety projects. Trend analysis demonstrated decreases in all 3 error types for 26 months. CONCLUSIONS: Applying performance-improvement strategies that focus longitudinally on specimen labeling errors can significantly reduce errors, therefore improving patient safety. This is an important area in which laboratory professionals, working in interdisciplinary teams, can improve safety and outcomes of care.

Increased vancomycin MICs for Staphylococcus aureus clinical isolates from a university hospital during a 5-year period.

Staphylococcus aureus is one of the most commonly isolated organisms in nosocomial infections. While the prevalence of methicillin-resistant S. aureus (MRSA) continues to increase worldwide, there is concern about an increase in vancomycin MICs among S. aureus strains. The prevalence of MRSA and vancomycin MIC trends in S. aureus from patients in a university hospital were analyzed. Clinical Laboratory Standards Institute (CLSI, formerly NCCLS) reference broth microdilution MIC testing was performed on all clinically relevant S. aureus isolates from January 2000 through December 2004. A total of 6,003 S. aureus isolates were analyzed. No vancomycin-resistant S. aureus isolates were detected. One MRSA isolate had a vancomycin MIC of 8 mug/ml and was confirmed as vancomycin-intermediate S. aureus. Among the 6,002 remaining isolates, a shift in vancomycin MICs from

Clearance of Cellulosimicrobium cellulans bacteremia in a child without central venous catheter removal.

Cellulosimicrobium cellulans (formerly known as Oerskovia xanthineolytica) rarely causes human infection. Infections have been reported in immunocompromised hosts or in patients with foreign bodies, such as catheters, where treatment has generally involved removal of the foreign body. We report on a case in which the organism was isolated in multiple blood cultures from a 13-year-old male. After initial therapy failed, treatment with vancomycin and rifampin resulted in infection clearance without removal of the central venous catheter.

Isolation of a strictly anaerobic strain of Staphylococcus epidermidis.

Staphylococcus epidermidis is a well-characterized, nonfastidious, aerobic gram-positive coccus commonly isolated in the clinical microbiology laboratory. Although coagulase-negative staphylococci, including Staphylococcus epidermidis, are often considered a contaminant in the clinical laboratory, an increasing number of reports describe their pathogenesis, in particular in infections of prosthetic devices. This article describes the isolation of a strictly anaerobic strain of Staphylococcus epidermidis in pure culture from the site of an infected prosthetic hip. This isolate was unique in that it grew only under strictly anaerobic conditions. Initially, the isolate was thought to be a known anaerobic gram-positive coccus. However, certain key biochemical and antimicrobial tests performed as part of the standard laboratory identification procedure were not consistent with results expected for any known anaerobic gram-positive coccus; the isolate was catalase positive and metronidazole and penicillin resistant. This isolate was characterized by further biochemical analysis, antimicrobial testing, and nucleic acid sequencing. This paper presents the first documented isolation of a strictly anaerobic Staphylococcus epidermidis strain, confirmed by rpoB gene sequencing.

Use of electrochemical DNA biosensors for rapid molecular identification of uropathogens in clinical urine specimens.

We describe the first species-specific detection of bacterial pathogens in human clinical fluid samples using a microfabricated electrochemical sensor array. Each of the 16 sensors in the array consisted of three single-layer gold electrodes-working, reference, and auxiliary. Each of the working electrodes contained one representative from a library of capture probes, each specific for a clinically relevant bacterial urinary pathogen. The library included probes for Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa, Enterocococcus spp., and the Klebsiella-Enterobacter group. A bacterial 16S rRNA target derived from single-step bacterial lysis was hybridized both to the biotin-modified capture probe on the sensor surface and to a second, fluorescein-modified detector probe. Detection of the target-probe hybrids was achieved through binding of a horseradish peroxidase (HRP)-conjugated anti-fluorescein antibody to the detector probe. Amperometric measurement of the catalyzed HRP reaction was obtained at a fixed potential of -200 mV between the working and reference electrodes. Species-specific detection of as few as 2,600 uropathogenic bacteria in culture, inoculated urine, and clinical urine samples was achieved within 45 min from the beginning of sample processing. In a feasibility study of this amperometric detection system using blinded clinical urine specimens, the sensor array had 100% sensitivity for direct detection of gram-negative bacteria without nucleic acid purification or amplification. Identification was demonstrated for 98% of gram-negative bacteria for which species-specific probes were available. When combined with a microfluidics-based sample preparation module, the integrated system could serve as a point-of-care device for rapid diagnosis of urinary tract infections.

Sensitivity and specificity of a rapid rRNA gene probe assay for simultaneous identification of Staphylococcus aureus and detection of mecA.

rRNA gene sequences were used for identification and target adequacy controls in a DNA probe assay to identify isolates as Staphylococcus and, more specifically, as S. aureus within 1 hour. mecA status was simultaneously determined using a specific DNA probe. The target adequacy control guarded against false-negative mecA results.

Multicenter evaluation of BBL CHROMagar MRSA medium for direct detection of methicillin-resistant Staphylococcus aureus from surveillance cultures of the anterior nares.

Active surveillance for methicillin-resistant Staphylococcus aureus (MRSA) is among the strategies recommended by the Society for Healthcare Epidemiology of America for control of nosocomial MRSA infections. Infection control and laboratory personnel desire rapid, sensitive, and inexpensive methods to enhance surveillance activities. A multicenter study was performed to evaluate a new selective and differential chromogenic medium, BBL CHROMagar MRSA (C-MRSA) medium (BD Diagnostics, Sparks, MD), which enables recovery and concomitant identification of MRSA strains directly from nasal swab specimens taken from the anterior nares. Specimens were inoculated to C-MRSA and Trypticase soy agar with 5% sheep blood agar (TSA II, BD Diagnostics). Mauve colonies on C-MRSA at 24 h and 48 h and suspicious colonies on TSA II were confirmed as Staphylococcus aureus by Gram stain morphology and a coagulase test. In addition, the results of C-MRSA were compared to results of susceptibility testing (five different methods) of S. aureus strains isolated on TSA II. A total of 2,015 specimens were inoculated to C-MRSA and TSA II. Three hundred fifty-four S. aureus isolates were recovered; 208 (59%) were oxacillin (methicillin) susceptible and 146 (41%) were oxacillin resistant (MRSA). On C-MRSA, 139/146 or 95.2% of MRSA isolates were recovered, whereas recovery on TSA II was 86.9% (127/146) (P = 0.0027). The overall specificity of C-MRSA was 99.7%. When C-MRSA was compared to each susceptibility testing method, the sensitivity and specificity, respectively, were as follows: oxacillin MIC by broth microdilution, 94.4% and 96.7%; oxacillin screen agar, 94.3% and 96.7%; PBP2' latex agglutination, 93.7% and 98.5%; cefoxitin disk diffusion, 95.0% and 98.1%; and mecA PCR, 95.1% and 98.1%. In this study, C-MRSA was superior to TSA II for recovery of MRSA from surveillance specimens obtained from the anterior nares and was comparable to conventional, rapid, and molecular susceptibility methods for the identification of MRSA isolates.

Conventional and molecular methods for verification of results obtained with BacT/Alert Nonvent blood culture bottles.

A strategy comparing molecular and conventional methods for verification of the BacT/Alert nonvent blood culture bottles (Organon Teknika, Durham, N.C.) was performed with seeded isolates. The bottles were evaluated with 12 common organisms from bloodstream infections. Overall, the bottles were equivalent as determined by conventional and molecular methods.

Legionella pneumophila serogroup 4 isolated from joint tissue.

We report the isolation of Legionella pneumophila serogroup 4 from synovial tissue obtained from an 80-year-old female with chronic swelling of her right metacarpophalangeal joint. Synovial tissue infections caused by L. pneumophila are rare. Interestingly, this isolate was recovered from chocolate agar after 5 days of incubation.

Application of an rRNA probe matrix for rapid identification of bacteria and fungi from routine blood cultures.

One of the most important functions of the clinical microbiology laboratory is the identification of the etiology of sepsis. For this study, aliquots from 405 positive blood cultures were tested against a unique array of DNA probes directed against rRNA subsequences of bacteria and fungi for identification. Another 280 samples that were negative after 5 days of incubation were also tested. Blood culture bottles were incubated in a BacT/Alert3D instrument. For the rRNA assay, a 0.4-ml aliquot was removed, and the cells were pelleted by centrifugation. The pellet was washed and frozen at -70 degrees C. Analysis of the pellet involved a lysis step and then the addition of samples to the reaction wells containing the probes in a microtiter plate format. Analysis was performed by using a hybridization protection assay. Results were taken through a series of deductive steps to obtain species, or in some cases genus, identification. Batch sample preparation required approximately 15 min, and sample analysis required another 60 min. Probe results were compared to conventional biochemical identifications. The probe test was negative for the 280 samples that were negative by the BacT/Alert 3D system and for another 21 samples that were false positive (the instrument signaled, but there was no growth). Microorganisms from the remaining 384 signal-positive samples included 60 Enterobacteriaceae, 10 Pseudomonas aeruginosa, 10 other gram-negative bacteria, 40 Staphylococcus aureus, 152 coagulase-negative staphylococci, 28 streptococci, 22 enterococci, 21 other gram-positive bacteria, 8 anaerobes, and 16 yeast organisms. Seventeen cultures were polymicrobial, and one was gram positive and culture negative. Discordance between probe and conventional identification results was noted for only 12 (1.75%) samples. This novel rapid molecular approach to the identification of bacteria and yeast in blood cultures was highly sensitive (100%) and specific (96%).

Bacteriostatic properties of biomatrices against common orthopaedic pathogens.

Tissue-engineered grafts for tissue regeneration include either mature or progenitor cells seeded onto biomatrices that provide shape and support for developing tissue. Popular biomaterials used in orthopaedic surgery include collagen type I, hyaluronic acid, hydroxyapatite, and polylactic polyglycolic acid (PLGA). Biomatrices with bacteriostatic properties may be beneficial in promoting tissue-engineered graft survival in patients susceptible to infection. We evaluated the bacteriostatic effects of these biomaterials on the growth of the four most common orthopaedic bacterial pathogens: Staphylococcus aureus, Staphylococcus epidermidis, beta-hemolytic Streptococcus, and Pseudomonas aeruginosa. Hyaluronic acid demonstrated the largest bacteriostatic effect on these pathogens by inhibiting bacterial growth by an average of 76.8% (p = 0.0005). Hydroxyapatite and collagen inhibited growth on average by 49.7% (p = 0.011) and 37.5% (p = 0.102), respectively. PLGA exhibited the least bacteriostasis with an average inhibition of 9.8% (NS) and actually accelerated the growth of beta-hemolytic Streptococcus and P. aeruginosa.

Multicenter study to evaluate the OptiMAL test for rapid diagnosis of malaria in U.S. hospitals.

More than 1,000 cases of malaria are diagnosed each year in the United States. Reported numbers, however, may be artificially low because many clinicians fail to consider the diagnosis on presentation, U.S. hospital laboratory technologists have very limited experience in detecting and identifying malaria parasites, and reporting of malaria to state health departments is sporadic in many states. In this study, a rapid malaria diagnostic test, the OptiMAL test (DiaMed; under license from Flow Inc., Portland, Oreg.) was evaluated in six U.S. hospitals and compared with results of microscopy. The OptiMAL test is a 15-min rapid immunochromatographic test that both identifies and differentiates Plasmodium falciparum from non-P. falciparum malaria parasites on the basis of the detection of parasite lactate dehydrogenase in a drop of patient blood. A total of 216 specimens from patients suspected of having malaria were tested. Results indicated that 43 samples (20%) were positive for malaria parasites by microscopy (32 P. falciparum, 11 non-P. falciparum) while 42 (19%) were positive by OptiMAL (31 P. falciparum, 11 non-P. falciparum). The sensitivity of the OptiMAL test was 98%; its specificity was 100%, with positive and negative predictive values of 100 and 99%, respectively. Participating hospital physicians and laboratory directors independently reported that the OptiMAL rapid malaria test was accurate, easy to use, and well accepted by those working in their diagnostic laboratories. The overall conclusion was that integration of the OptiMAL rapid malaria test into the U.S. health care infrastructure would provide an important and easy-to-use tool for the timely diagnosis of malaria.

Parenteral infusions as culture media from a viewpoint of nosocomial bacteremia / Infusiones parenterales como medios de cultivo desde el punto de vista de las bacteremias nosocomiales

Objetivo. Estudiar los patrones de crecimiento de microorganismos seleccionados en soluciones parenterales comunes, evaluando sus implicaciones para las bacteriemias nosocomiales. Diseño. Se cultivaron, secuencialmente y a temperatura ambiente, suspensiones microbianas de aproximadamente 300 UFC/mL en soluciones parenterales de tres fabricantes. En la etapa inicial se estudiaron 11 aislamientos clínicos bacterianos y uno de Candida. En otra fase, ocho bacilos gramnegativos fueron estudiados a diferentes rangos de pH. Finalmente, la variabilidad entre las especies se probó en 39 aislamientos. Resultados. Los ocho bacilos gramnegativos desarrollaron in Ringer-lactato. Sólo Klebsiella pneumoniae y Serratia marcescens desarrollaron en dextrosa al 5 por ciento en agua. Ningún microorganismo desarrolló en solución salina isotónica o en mixta (dextrosa al 5 por ciento en salina 0.9 por ciento). En ninguna solución desarrollaron los cocos grampositivos ni Candida. No se observaron cambios significativos en el crecimiento después de modificar el pH de las soluciones. Se observó una importante variación de crecimiento dentro de cada especie. Conclusiones. La solución Ringer-lactato es un buen medio de cultivo para bacilos gramnegativos y la dextrosa al 5 por ciento en agua es un medio pobre, excepto para algunas bacterias de la tribu Klebsiellae. Recomendamos apego estricto a los estándares de enfermería para aplicar infusiones intravenosas, y evitar el uso de soluciones con nutrientes, cuando sea posible.