Direct Detection of Influenza A and B Viruses in Less Than 20 Minutes Using a Commercially Available Rapid PCR Assay.

We compared an FDA-cleared rapid (<20 min) PCR assay (Cobas Liat; Roche Diagnostics) to our routine influenza A and B real-time PCR assay (Simplexa Flu A/B & RSV Direct; Focus Diagnostics) using respiratory swabs (n = 197). The Cobas Liat influenza A and B assays demonstrated sensitivities of 99.2% (123/124) and 100% (23/23), respectively, while showing a specificity of 100% for each target.

Desulfovibrio legallii prosthetic shoulder joint infection and review of antimicrobial susceptibility and clinical characteristics of Desulfovibrio infections.

We describe a case of shoulder hemiarthroplasty infection with Desulfovibrio legallii. Antimicrobial susceptibilities of 36 Desulfovibrio isolates are presented. Metronidazole and carbapenems exhibited reliable activity, although piperacillin-tazobactam did not. Eleven previous cases of Desulfovibrio infection are reviewed; most arose from a gastrointestinal tract-related source.

Optimized pathogen detection with 30- compared to 20-milliliter blood culture draws.

Using data from 23,313 patients, we assessed whether two blood culture sets of three bottles per set would detect more pathogens than two sets of two bottles per set and achieve similar sensitivity to collecting three sets of two bottles per set. We also compared the yield of aerobic and anaerobic bottles. Thirty milliliters of blood was distributed to one anaerobic and two aerobic bottles. Among 26,855 collections of ≥ 60 ml within 30 min, 1,379 (5.1%) were positive for a pathogen not requiring detection in more than one set to be considered a pathogen, with 72 additional distinct pathogens detected using two 30-ml compared to two 20-ml sets of one aerobic and one anaerobic bottle (increased yield, 7.9%; 95% confidence interval [CI], 6.2 to 9.8%). For conditional pathogens requiring detection in at least two positive blood cultures for classification as pathogens (i.e., otherwise classified as contaminants), there were 162 positive detections with two 30-ml sets, of which 16 would not have been detected by two 20-ml sets (increased yield, 11.0% [95% CI, 6.4 to 17.2%]). Among 134 subjects who had three sets of 30 ml each within a 30-min interval, there was complete concordance between 60 ml of blood drawn in the first two sets of 30 ml and three 20-ml sets (P = 1.0). One aerobic bottle plus one anaerobic bottle yielded more pathogens than two aerobic bottles for organisms requiring a single (P < 0.001) and two (P = 0.04) positive sets to be defined as pathogens. In conclusion, we showed that collection of two aerobic and one anaerobic blood culture bottles per set results in improved yield compared to two bottles per set. We also confirmed that an anaerobic bottle should be included in blood culture sets.

Three-hour molecular detection of Campylobacter, Salmonella, Yersinia, and Shigella species in feces with accuracy as high as that of culture.

Campylobacter jejuni and Salmonella, Shigella, and Yersinia species (along with Shiga toxin-producing Escherichia coli) are the most common causes of acute bacterial diarrheal disease in the United States. Current detection techniques are time-consuming, limiting usefulness for patient care. We developed and validated a panel of rapid PCR assays for the detection and identification of C. jejuni, C. coli, Salmonella, and Yersinia species and Shigella and enteroinvasive E. coli in stool samples. A total of 392 archived stool specimens, previously cultured for enteric pathogens, were evaluated by PCR. Overall, 104 stool specimens had been culture positive (C. jejuni/coli [n = 51], Salmonella species [n = 42], Shigella species [n = 6], and Yersinia species [n = 5]). Compared to culture, the overall sensitivity and specificity of PCR detection of these organisms were 92 and 98% (96/104 and 283/288), respectively, from fresh or Cary Blair stool (P = 0.41); 87 and 98% (41/47 and 242/246), respectively, from fresh stool (P = 0.53); and 96 and 98% (55/57 and 41/42), respectively, from Cary Blair stool (P = 0.56). For individual genera, PCR was as sensitive as the culture method, with the exception of Salmonella culture using selenite enrichment for which PCR was less sensitive than culture from fresh, but not Cary Blair (P = 0.03 and 1.00, respectively) stools. This PCR assay panel for the rapid diagnosis of acute infectious bacterial diarrheal pathogens has a sensitivity and specificity equivalent to that of culture for stools in Cary Blair transport medium. Paired with reflexive culture of stools testing positive, this should provide an improvement in care for patients with acute infectious diarrheal disease.

Beta-lactam and fluoroquinolone combination antibiotic therapy for bacteremia caused by gram-negative bacilli.

The role of combination antibiotic therapy with a beta-lactam and a fluoroquinolone for bacteremia caused by gram-negative bacilli, to our knowledge, has not been previously described. Much of the previous study of combination therapy has included beta-lactams and aminoglycosides. We conducted a large retrospective cohort study to evaluate 28-day all-cause mortality in patients with monomicrobial bacteremia due to aerobic gram-negative bacilli who received either a combination of beta-lactams and fluoroquinolones or beta-lactam monotherapy. We enrolled adult patients admitted to Mayo Clinic hospitals from 1 January 2001 to 31 October 2006 in the study. After stratification of patients by Pitt bacteremia scores, we used Cox regression models to estimate the hazard ratios (HR) for 28-day all-cause mortality after adjusting for the propensity to receive combination therapy. We identified 398 and 304 unique patients with bacteremia caused by gram-negative bacilli who received single and combination antibiotic therapy, respectively. In less severely ill patients with Pitt bacteremia scores of <4, combination therapy was associated with lower 28-day mortality than single therapy (4.2% [9 of 214] versus 8.8% [28 of 319]; adjusted HR, 0.44; 95% confidence interval [CI], 0.20 to 0.98; P = 0.044). In critically ill patients with Pitt bacteremia scores of >or=4, there was no difference in 28-day mortality between combination and single therapy (25.6% [23 of 90] versus 27.8% [22 of 79]; adjusted HR, 0.87; 95% CI, 0.47 to 1.62; P = 0.660). These findings were consistent for 14-day all-cause mortality. In this large cohort, we found for the first time that combination therapy with beta-lactams and fluoroquinolones was associated with a reduction in 28-day all-cause mortality among less severely ill patients with bacteremia caused by gram-negative bacilli.

Percent positive rate of Lyme real-time polymerase chain reaction in blood, cerebrospinal fluid, synovial fluid, and tissue.

We performed a retrospective review of 23,777 specimens tested by our Lyme real-time polymerase chain reaction assay to determine the percent positive rates by specimen source. The percent positive rates were highest in synovial fluid (6.4%) and tissue (6.5%), and lowest in blood (0.1%) and cerebrospinal fluid (0.09%).

Acanthamoeba keratitis: the persistence of cases following a multistate outbreak.

PURPOSE: To describe the trend of Acanthamoeba keratitis case reports following an outbreak and the recall of a multipurpose contact lens disinfection solution. Acanthamoeba keratitis is a serious eye infection caused by the free-living amoeba Acanthamoeba that primarily affects contact lens users. METHODS: A convenience sample of 13 ophthalmology centers and laboratories in the USA, provided annual numbers of Acanthamoeba keratitis cases diagnosed between 1999-2009 and monthly numbers of cases diagnosed between 2007-2009. Data on ophthalmic preparations of anti-Acanthamoeba therapies were collected from a national compounding pharmacy. RESULTS: Data from sentinel site ophthalmology centers and laboratories revealed that the yearly number of cases gradually increased from 22 in 1999 to 43 in 2003, with a marked increase beginning in 2004 (93 cases) that continued through 2007 (170 cases; p < 0.0001). The outbreak identified from these sentinel sites resulted in the recall of a contact lens disinfecting solution. There was a statistically significant (p ≤ 0.0001) decrease in monthly cases reported from 28 cases in June 2007 (following the recall) to seven cases in June 2008, followed by an increase (p = 0.0004) in reported cases thereafter; cases have remained higher than pre-outbreak levels. A similar trend was seen in prescriptions for Acanthamoeba keratitis chemotherapy. Cases were significantly more likely to be reported during summer than during other seasons. CONCLUSION: The persistently elevated number of reported cases supports the need to understand the risk factors and environmental exposures associated with Acanthamoeba keratitis. Further prevention efforts are needed to reduce the number of cases occurring among contact lens wearers.

Clinical significance and antimicrobial susceptibilities of Aerococcus sanguinicola and Aerococcus urinae.

A retrospective chart review was performed on 92 patients from whom 118 isolates of Aerococcus sanguinicola (n = 52) or Aerococcus urinae (n = 66) were obtained from urine cultures between October 2007 and June 2008 to assess clinical presentation and antimicrobial susceptibilities. The mean patient age was 82 (range 24-101) years. The majority was female (76% and 87% for A. sanguinicola and A. urinae, respectively) and institutionalized (61% and 60%, respectively). The majority of male patients had underlying prostatic disease (55% and 63%, respectively). Thirty-one of 46 patients with A. sanguinicola and 45 of 57 patients with A. urinae isolated from the urine had a clinical diagnosis of urinary tract infection. One subject had A. sanguinicola isolated from blood cultures. A. sanguinicola and A. urinae had low ceftriaxone, penicillin, and vancomycin MICs. MICs to erythromycin and levofloxacin were ≥0.5 and >4 µg/mL in 41% and 78% of A. sanguinicola and 17% and 23% of A. urinae isolates, respectively. In conclusion, A. sanguinicola and A. urinae are not infrequent causes of urinary tract infection and most A. sanguinicola isolates have elevated MICs to levofloxacin.

Small intestinal bacterial overgrowth: histopathologic features and clinical correlates in an underrecognized entity.

CONTEXT: Small intestinal bacterial overgrowth (SIBO) is a common cause of chronic diarrhea and malabsorption. Morphologic changes associated with this condition have not, to our knowledge, been studied in detail. OBJECTIVE: To better characterize the histopathologic changes associated with SIBO by comparing the clinicopathologic features of patients with SIBO (duodenal aspirate cultures with > or =10(5) colony-forming units [CFUs]/mL) to controls with cultures found to be negative (<10(5) CFUs/mL). DESIGN: We included 67 consecutive patients with SIBO and 55 controls in the series. Each duodenal biopsy was assessed for the following features: villous to crypt ratio, intraepithelial lymphocytosis, crypt apoptoses, basal plasmacytosis, cryptitis/villitis, peptic duodenitis, erosions/ulcers, eosinophilia, and absence of goblet and Paneth cells; and correlated with clinical features and culture results. RESULTS: Decreased villous to crypt ratio (<3ratio1) was more frequent in SIBO than controls (24% versus 7%; P = .01). Duodenal biopsies from patients with SIBO were slightly less likely to be judged within reference range than were controls (52% versus 64%; P = .27). There were no significant differences in any of the other histologic features. Clinically, patients in the SIBO group were older than the age of controls (mean, 60 years versus 52 years; P = .02), and they were more likely to have one of the known predisposing factors for bacterial overgrowth (66% versus 36%; P = .002). Other clinical features, including presenting symptoms, were similar. CONCLUSIONS: Villous blunting is the only feature more common to SIBO than to controls. More than half of biopsies from SIBO patients are histologically unremarkable. Therefore, SIBO needs to be considered as a potential etiology for gastrointestinal symptoms even when duodenal biopsies are found to be normal.

Bronchoscopy in ventilator-associated pneumonia: agreement of calibrated loop and serial dilution.

RATIONALE: Although the serial dilution technique for quantitative culture of bronchoalveolar fluid is considered to be the gold standard for the diagnosis of ventilator-associated pneumonia, it is more labor intensive than the calibrated loop technique. OBJECTIVE: We sought to determine the agreement between the calibrated loop and serial dilution techniques in the diagnosis of ventilator-associated pneumonia. METHODS: We prospectively measured bacterial colony counts by the serial dilution and calibrated loop techniques in 121 bronchoalveolar lavage samples of 104 patients with suspected ventilator-associated pneumonia. MEASUREMENTS AND MAIN RESULTS: At the time of bronchoscopy, patients had received mechanical ventilation for a median of 8 d. Patients were receiving antibiotics when 90 of the 121 (74.4%) bronchoalveolar samples were obtained. The colony counts of 13 bacterial isolates were too numerous to count by the calibrated loop technique; by serial dilution technique, their counts ranged from 4.70 to 6.74 log10 cfu/ml. Fifty other bacteria had paired colony counts measured by each of the two techniques: the bias (95% confidence interval) between the two techniques was -0.380 (-0.665 to -0.095) log10 cfu/ml, with precision of 1.002 log10 cfu/ml and 95% limits of agreement of -2.344 to 1.584 log10 cfu/ml. Using the threshold of 4 log10 cfu/ml as a criterion for the diagnosis of ventilator-associated pneumonia, there was discordance only for one bacterial organism between the two techniques. CONCLUSIONS: The calibrated loop technique can be used for the diagnosis of ventilator-associated pneumonia using bronchoalveolar lavage fluid.

Use of the Roche LightCycler Strep B assay for detection of group B Streptococcus from vaginal and rectal swabs.

The results for a real-time PCR assay, using the LightCycler Strep B analyte-specific reagents (Roche Diagnostics Corporation, Indianapolis, Ind.), were compared to a direct plate method combined with a broth enrichment culture method for detection of group B streptococcus colonization in pregnant women. Two separate evaluations were conducted using two different automated nucleic extraction instruments, the MagNA Pure LC instrument (Roche Diagnostics Corporation) and the lower-capacity MagNA Pure Compact instrument (Roche Diagnostics Corporation). The sensitivities, specificities, and positive and negative predictive values for the different evaluation methods were as follow: for the LightCycler Strep B assay with MagNA Pure LC, 100, 97, 90, and 100%, respectively; for the LightCycler Strep B assay with MagNA Pure Compact, 92.5, 99, 97, and 97.5%, respectively. The LightCycler Strep B assay combined with either MagNA Pure LC or MagNA Pure Compact extraction is a suitable method for detecting group B streptococcus colonization in pregnant women. An advantage of the LightCycler assay over culture is the considerably reduced turnaround time for results.

Multiplex LightCycler PCR assay for detection and differentiation of Bordetella pertussis and Bordetella parapertussis in nasopharyngeal specimens.

A rapid real-time multiplex PCR assay for detecting and differentiating Bordetella pertussis and Bordetella parapertussis in nasopharyngeal swabs was developed. This assay (LC-PCR-IS) targets the insertion sequences IS481 and IS1001 of B. pertussis and B. parapertussis, respectively, and is performed using the LightCycler (Roche Molecular Biochemicals, Indianapolis, Ind.). The analytical sensitivity is less than one organism per reaction. Results for Bordetella culture and/or direct fluorescent antibody testing and a second LightCycler PCR assay (target, pertussis toxin gene) were compared to results of the LC-PCR-IS assay for 111 nasopharyngeal swabs submitted for pertussis testing. Of the specimens, 12 were positive (9 B. pertussis and 3 B. parapertussis) and 68 specimens were negative by all methods. Three other specimens were positive for B. pertussis by at least two of the methods (including the LC-PCR-IS assay), and another 28 specimens were positive for B. pertussis by the LC-PCR-IS assay only. No specimens were negative by the LC-PCR-IS assay and positive by the other methods. A conventional PCR method (target, IS481) was also compared to the LC-PCR-IS assay for a different group of nasopharyngeal swab specimens (n = 96): 44 specimens were positive and 41 specimens were negative for B. pertussis with both PCR methods. Nine specimens were positive for B. pertussis by the LC-PCR-IS assay and negative by the conventional PCR assay, and two specimens were positive for B. pertussis by the conventional PCR assay and negative by the LC-PCR-IS assay. Positivity of the two assays was not significantly different (P = 0.0654). The insertion sequence IS481 is also present in Bordetella holmesii; specimens containing B. holmesii may yield false-positive results. The LC-PCR-IS assay takes approximately 45 min to complete post-nucleic acid extraction, compared to 24 h for the conventional PCR assay previously used in our laboratory. The LC-PCR-IS assay is easier to perform than the conventional PCR assay, and the closed system decreases the chance of contamination. All of these characteristics represent a significant improvement in the detection of B. pertussis and B. parapertussis in nasopharyngeal specimens.