Executive summary: a guide to utilization of the microbiology laboratory for diagnosis of infectious diseases: 2013 recommendations by the Infectious Diseases Society of America (IDSA) and the American Society for Microbiology (ASM)(a).

The critical role of the microbiology laboratory in infectious disease diagnosis calls for a close, positive working relationship between the physician and the microbiologists who provide enormous value to the health care team. This document, developed by both laboratory and clinical experts, provides information on which tests are valuable and in which contexts, and on tests that add little or no value for diagnostic decisions. Sections are divided into anatomic systems, including Bloodstream Infections and Infections of the Cardiovascular System, Central Nervous System Infections, Ocular Infections, Soft Tissue Infections of the Head and Neck, Upper Respiratory Infections, Lower Respiratory Tract infections, Infections of the Gastrointestinal Tract, Intraabdominal Infections, Bone and Joint Infections, Urinary Tract Infections, Genital Infections, and Skin and Soft Tissue Infections; or into etiologic agent groups, including Tickborne Infections, Viral Syndromes, and Blood and Tissue Parasite Infections. Each section contains introductory concepts, a summary of key points, and detailed tables that list suspected agents; the most reliable tests to order; the samples (and volumes) to collect in order of preference; specimen transport devices, procedures, times, and temperatures; and detailed notes on specific issues regarding the test methods, such as when tests are likely to require a specialized laboratory or have prolonged turnaround times. There is redundancy among the tables and sections, as many agents and assay choices overlap. The document is intended to serve as a reference to guide physicians in choosing tests that will aid them to diagnose infectious diseases in their patients.

A guide to utilization of the microbiology laboratory for diagnosis of infectious diseases: 2013 recommendations by the Infectious Diseases Society of America (IDSA) and the American Society for Microbiology (ASM)(a).

The critical role of the microbiology laboratory in infectious disease diagnosis calls for a close, positive working relationship between the physician and the microbiologists who provide enormous value to the health care team. This document, developed by both laboratory and clinical experts, provides information on which tests are valuable and in which contexts, and on tests that add little or no value for diagnostic decisions. Sections are divided into anatomic systems, including Bloodstream Infections and Infections of the Cardiovascular System, Central Nervous System Infections, Ocular Infections, Soft Tissue Infections of the Head and Neck, Upper Respiratory Infections, Lower Respiratory Tract infections, Infections of the Gastrointestinal Tract, Intraabdominal Infections, Bone and Joint Infections, Urinary Tract Infections, Genital Infections, and Skin and Soft Tissue Infections; or into etiologic agent groups, including Tickborne Infections, Viral Syndromes, and Blood and Tissue Parasite Infections. Each section contains introductory concepts, a summary of key points, and detailed tables that list suspected agents; the most reliable tests to order; the samples (and volumes) to collect in order of preference; specimen transport devices, procedures, times, and temperatures; and detailed notes on specific issues regarding the test methods, such as when tests are likely to require a specialized laboratory or have prolonged turnaround times. There is redundancy among the tables and sections, as many agents and assay choices overlap. The document is intended to serve as a reference to guide physicians in choosing tests that will aid them to diagnose infectious diseases in their patients.

An evaluation of repeat stool testing for Clostridium difficile infection by polymerase chain reaction.

GOALS: To evaluate the yield of repeat stool polymerase chain reaction (PCR) testing in patients with suspected Clostridium difficile infection (CDI). BACKGROUND: CDI is a major challenge in health care due to its frequent occurrence and high associated costs. Enzyme immunoassay and PCR are commonly performed diagnostic tests for CDI. METHODS: Our microbiology laboratory database was queried from January 1, 2008 to June 30, 2010 for all patients who underwent PCR stool testing for suspected CDI. Data collected included age, sex, number of stool tests performed within a 14-day period after the first test, and location of patient (inpatient vs. outpatient). Analyses were performed using JMP version 9.0.1. RESULTS: PCR testing was performed in 15,515 patients. The median age was 58.3 years (range, 10 d to 104.3 y) and 46.2% of patients were women. Repeat testing was infrequent; 87.3% of patients had testing performed only once in a 14-day period. Increased age, male sex, and inpatient location were predictors of repeat testing. The median time between an initial test and the first repeat test was 5 days. After an initial negative test, the percentage of patients having a subsequent positive test was low (2.7% in 7 d and 3.2% in 14 d). The percentage of repeat tests that was positive within 7 days (2.9%) was lower than the percentage that was positive from day 8 to day 14 (4.8%, P=0.05). CONCLUSIONS: Repeat testing for C. difficile has a low yield, and patients with an initial negative test should not routinely be retested.

Actinobaculum bacteremia: a report of 12 cases.

Actinobaculum species are anaerobic Gram-positive rods that have previously been associated with urinary tract infection (UTI) in the elderly. We report 12 patients with Actinobaculum bacteremia. Only 40% of blood cultures were clinically considered significant by the treating physicians, but most patients were treated for UTI, suggesting a possible urinary source of bacteremia. Clinicians should be aware of the pathogenic potential of Actinobaculum spp.

Comparison of two commercial molecular assays to a laboratory-developed molecular assay for diagnosis of Clostridium difficile infection.

We compared two commercial PCR assays, the Prodesse ProGastro CD assay and the BD GeneOhm Cdiff assay, with a laboratory-developed Clostridium difficile toxin PCR assay with previously established performance characteristics. Results of all methods were in agreement for 333 (96%) of 346 stool specimens. No significant difference in performance among the assays was found (P values, >0.05).

Isolation of Robinsoniella peoriensis from four human specimens.

Robinsoniella peoriensis is a recently described anaerobic, spore-forming, Gram-positive bacillus originally recovered from swine manure. We report four human cases in which R. peoriensis was isolated from clinical samples.

Lessons learned from the anaerobe survey: historical perspective and review of the most recent data (2005-2007).

BACKGROUND: The rationale and lessons learned through the evolution of the National Survey for the Susceptibility of Bacteroides fragilis Group from its initiation in 1981 through 2007 are reviewed here. The survey was conceived in 1980 to track emerging antimicrobial resistance in Bacteroides species. METHODS: Data from the last 11 years of the survey (1997-2007), including 6574 isolates from 13 medical centers, were analyzed for in vitro antimicrobial resistance to both frequently used and newly developed anti-anaerobic agents. The minimum inhibitory concentrations of the antibiotics were determined using agar dilution in accordance with Clinical and Laboratory Standards Institute recommendations. RESULTS: The analyses revealed that the carbapenems (imipenem, meropenem, ertapenem, and doripenem) and piperacillin-tazobactam were the most active agents against these pathogens, with resistance rates of 0.9%-2.3%. In the most recent 3 years of the survey (2005-2007), resistance to some agents was shown to depend on the species, such as ampicillin-sulbactam against Bacteroides distasonis (20.6%) and tigecycline against Bacteroides uniformis and Bacteroides eggerthii ( approximately 7%). Very high resistance rates (>50%) were noted for moxifloxacin and trovafloxacin, particularly against Bacteroides vulgatus. During that period of study, non-B. fragilis Bacteroides species had >40% resistance to clindamycin. Metronidazole-resistant Bacteroides strains were also first reported during that period. CONCLUSIONS: In summary, resistance to antibiotics was greater among non-B. fragilis Bacteroides species than among B. fragilis and was especially greater among species with a low frequency of isolation, such as Bacteroides caccae and B. uniformis. The emergence of resistance among the non-B. fragilis Bacteroides species underscores the need for speciation of B. fragilis group isolates and for clinicians to be aware of associations between species and drug resistance.

Laboratory diagnosis of infections due to blood and tissue parasites.

Microscopy remains the cornerstone of the laboratory diagnosis of infections due to blood and tissue parasites. Examination of thick and thin peripheral blood smears stained with Giemsa or other appropriate stains is used for detection and identification of species of Plasmodium, Babesia, Trypanosoma, Brugia, Mansonella, and Wuchereria. Even in the hands of well-trained technologists, diagnosis may be hampered by the sparseness of organisms on the slide and by the subjective nature of differentiating similar-appearing organisms. Microscopy and/or culture of ulcer, bone marrow, tissue aspirate, and biopsy samples are useful for the diagnosis of African trypanosomiasis, onchocerciasis, trichinosis, and leishmaniasis. Serologic assays are available for the diagnosis of a number of these infections, but none of these assays are sensitive or specific enough to be used on their own to establish a diagnosis. In particular, the use of assays for the diagnosis of infection with a particular helminth will often cross-react with antibodies to a different helminth. Very sensitive polymerase chain reaction assays have been developed for a number of these parasites and are available from the Centers for Disease Control and Prevention and from several referral laboratories.

Diagnostic performance of rapid diagnostic tests versus blood smears for malaria in US clinical practice.

BACKGROUND: Approximately 4 million US travelers to developing countries are ill enough to seek health care, with 1500 malaria cases reported in the United States annually. The diagnosis of malaria is frequently delayed because of the time required to prepare malaria blood films and lack of technical expertise. An easy, reliable rapid diagnostic test (RDT) with high sensitivity and negative predictive value (NPV), particularly for Plasmodium falciparum, would be clinically useful. The objective of this study was to determine the diagnostic performance of a RDT approved by the US Food and Drug Administration compared with traditional thick and thin blood smears for malaria diagnosis. METHODS: This prospective study tested 852 consecutive blood samples that underwent thick and thin smears and blinded malaria RDTs at 3 hospital laboratories during 2003-2006. Polymerase chain reaction verified positive test results and discordant results. RESULTS: Malaria was noted in 95 (11%) of the 852 samples. The RDT had superior performance than the standard Giemsa thick blood smear (p = .003). The RDT's sensitivity for all malaria was 97% (92 of 95 samples), compared with 85% (81 of 95) for the blood smear, and the RDT had a superior NPV of 99.6%, compared with 98.2% for the blood smear (p = .001). The P. falciparum performance was excellent, with 100% rapid test sensitivity, compared with only 88% (65 of 74) by blood smear (p = .003). CONCLUSIONS: This operational study demonstrates that the US Food and Drug Administration-approved RDT for malaria is superior to a single set of blood smears performed under routine US clinical laboratory conditions. The most valuable clinical role of the RDT is in the rapid diagnosis or the exclusion of P. falciparum malaria, which is particularly useful in outpatient settings when evaluating febrile travelers.

Rapid and sensitive detection of Shiga toxin-producing Escherichia coli from nonenriched stool specimens by real-time PCR in comparison to enzyme immunoassay and culture.

Shiga toxin (Stx)-producing Escherichia coli (STEC) bacteria are a frequent cause of food-borne gastroenteritis, hemorrhagic colitis, and hemolytic uremic syndrome. Because antimicrobial agents are generally contraindicated in patients infected with STEC, a sensitive and specific diagnostic test with rapid turnaround is essential. Current culture methods may fail to detect non-O157 STEC. We evaluated a Stx gene real-time PCR assay using hybridization probes and the LightCycler instrument with 204 prospectively collected stool specimens, which were also tested for Stx by enzyme immunoassay (EIA) (ProSpecT STEC; Remel, Lenexa, KS) and by culturing on chromogenic agar (Chromagar O157; BD BBL, Sparks, MD). In addition, 85 archived stool specimens previously tested for Stx (by EIA) and/or E. coli O157:H7 (by culture) were tested by PCR. Sample preparation for PCR included mixing the stool in sterile water and extraction of nucleic acid using the MagNA Pure LC instrument (Roche Diagnostics). The PCR assay had 100% sensitivity and specificity compared to EIA and culture for specimens collected prospectively (4 of 204 specimens were positive) and compared to culture and/or EIA for archival specimens (42 of 85 specimens were positive). Both the EIA and PCR produced positive results from a specimen containing an O103 serotype STEC in the prospective specimens, and the PCR test detected three positive specimens that contained nonviable STEC in the archived specimens. The PCR assay demonstrated 100% sensitivity and specificity compared to EIA and/or culture and more rapid turnaround than either EIA or culture.

Prevalence of small intestine bacterial overgrowth diagnosed by quantitative culture of intestinal aspirate in celiac disease.

BACKGROUND AND AIM: A recent study using lactulose hydrogen-breath testing suggests that small intestine bacterial overgrowth (SIBO) is a common cause of nonresponsive celiac disease (CD). The prevalence of SIBO in CD diagnosed by quantitative culture of intestinal aspirate is unknown. The aim of this study is to evaluate the prevalence and significance of SIBO in CD based on the results of quantitative culture of intestinal aspirate. METHODS: We studied patients with CD in whom culture of intestinal aspirate was evaluated for the presence of anaerobes and aerobes. Bacterial overgrowth was diagnosed if culture demonstrated >10 colony forming units/mL. The causes of nonresponsive CD were investigated. RESULTS: We included 149 biopsy-confirmed CD patients. The intestinal aspirate was collected in 79 (53%) patients with nonresponsive CD, 47 (32%) as initial work-up for malabsorption, and in 23 (15%) asymptomatic treated CD. SIBO was diagnosed in 14 (9.3%). Nine (11%) with nonresponsive CD, 5 (11%) at initial work-up for malabsorption, and 0 in asymptomatic treated CD. Patients with a positive culture had evidence of worse malabsorption. A coexistent disorder was found in 67% of patients with both nonresponsive CD and bacterial overgrowth. CONCLUSIONS: The prevalence of SIBO diagnosed by quantitative culture of intestinal aspirate was 9.3% in patients with CD. Patients with symptomatic treated or untreated CD were affected. SIBO may coexist with other disorders associated with nonresponsive CD.

Comparison of real-time PCR for detection of the tcdC gene with four toxin immunoassays and culture in diagnosis of Clostridium difficile infection.

We have developed a rapid real-time PCR method using fluorescence resonance energy transfer probes and the LightCycler (Roche Diagnostics), which will detect the presence of the tcdC gene of Clostridium difficile in stool samples. Our PCR method also will identify the presence of base pair deletions, one of which (18 bp) has been associated with the "epidemic" toxin-hyperproducing strains. We compared the results of this PCR with those of three C. difficile toxin-detecting enzyme immunoassays (EIAs), an EIA for the detection of glutamate dehydrogenase (GDH), and culture of C. difficile. A total of 200 stool specimens were studied by the methods under comparison. C. difficile was isolated from 49 specimens by culture, and 44 of these were confirmed as containing one of the genes associated with toxin production ("toxigenic culture"). Using toxigenic culture as the "gold standard", the sensitivities, specificities, and positive and negative predictive values, respectively, of the assays were 48%, 98%, 88%, and 87% for the Premier toxin A and B test; 48%, 99%, 91%, and 87% for the ImmunoCard toxin A & B test; 48%, 84%, 46%, and 85% for the Xpect C. difficile toxin A/B test; 32%, 100%, 100%, and 84% for the Triage C. difficile panel (for toxin A); and 86%, 97%, 90%, and 96% for the LightCycler PCR. Thus, in comparison to the sensitivity of toxigenic culture, the sensitivities of the toxin immunoassays were unacceptably low, while the LightCycler real-time PCR assay for the detection of the tcdC gene of C. difficile is sensitive and specific.

Reemergence of anaerobic bacteremia.

BACKGROUND: During 1974-1988, the incidence of anaerobic bacteremia at the Mayo Clinic (Rochester, MN) decreased. This trend occurred nationally, prompting calls for discontinuation of routine anaerobic blood cultures. However, recently, the sites of anaerobic infection have been shown not to be as predictable as once thought, and since 1993, the incidence of anaerobic bacteremia has increased significantly in our medical center. METHODS: Records from the Mayo Clinic Division of Clinical Microbiology were used to tabulate the number of cases of anaerobic bacteremia in patients at the clinic for the 12-year period from 1993 through 2004. Medical records for patients with anaerobic bacteremia were reviewed from the periods of 1993-1994 and 2004 to identify differences between these 2 patient populations with different rates of bacteremia. RESULTS: The mean incidence of anaerobic bacteremias increased from 53 cases per year during 1993-1996 to 75 cases per year during 1997-2000 to 91 cases per year during 2001-2004 (an overall increase of 74%). The total number of cases of anaerobic bacteremia per 100,000 patient-days increased by 74% (P<.001). The number of anaerobic blood cultures per 1000 cultures performed increased by 30% (P=.002). Organisms from the Bacteroides fragilis group, other species of Bacteroides, and Clostridium species were most commonly isolated. CONCLUSIONS: Anaerobic bacteremia has reemerged as a significant clinical problem. Although there are probably multiple reasons for this change, the increasing number of patients with complex underlying diseases makes the clinical context for anaerobic infections less predictable than it once was. Anaerobic blood cultures should be routinely performed in medical centers with a patient population similar to ours.

Evaluation of malaria screening in newly arrived refugees to the United States by microscopy and rapid antigen capture enzyme assay.

Before an empiric malaria treatment program, >60% of Liberian refugees had malaria on arrival to Minnesota. We compared microscopy with rapid antigen testing for detecting asymptomatic parasitemia. Nine of 103 (8.7%) had malaria by polymerase chain reaction (blood smear and rapid testing had a sensitivity of 22%). The empiric treatment program has decreased the rate of imported asymptomatic malaria. Blood film and rapid antigen testing are poor screening tests.

Evaluation of a real-time polymerase chain reaction assay for the diagnosis of malaria in patients from Thailand.

We compared the diagnosis of malaria in 297 patients from Thailand by a real-time polymerase chain reaction (PCR) assay using the LightCycler with conventional microscopy using Giemsa-stained thick and thin blood films. The PCR assay can be completed in one hour and has the potential to detect and identify four species of Plasmodium in a single reaction by use of melting temperature curve analysis (however, we did not detect Plasmodium ovale in this study). Blood was collected, stored, and transported on IsoCode STIX, which provide a stable matrix for the archiving and rapid simple extraction of DNA. A genus-specific primer set corresponding to the 18S ribosomal RNA was used to amplify the target sequence. Fluorescence resonance energy technology hybridization probes were designed for P. falciparum over a region containing basepair mismatches, which allowed differentiation of the other Plasmodium species. The PCR results correlated with the microscopic results in 282 (95%) of 297 patient specimens. Most of these were single-species infections caused by P. vivax (150) and P. falciparum (120), along with 5 P. malariae, 2 mixed infections (P. falciparum and P. vivax), and 5 negative specimens. No negative microscopy specimens were positive by PCR (100% specificity for detection of any Plasmodium). The 15 discrepant results could not be resolved, but given the subjective nature of microscopy and the analytical objectivity of the PCR, the PCR results may be correct. The ability of the PCR method to detect mixed infections or to detect P. ovale could not be determined in this study. Within the limitations of initial equipment costs, this real-time PCR assay is a rapid, accurate, and efficient method for the specific diagnosis of malaria. It may have application in clinical laboratories, as well as in epidemiologic studies and antimalarial efficacy trials.

Detection of vaccinia virus, herpes simplex virus, varicella-zoster virus, and Bacillus anthracis DNA by LightCycler polymerase chain reaction after autoclaving: implications for biosafety of bioterrorism agents.

OBJECTIVE: To determine whether autoclaving suspensions of vaccinia virus, herpes simplex virus (HSV), varicella-zoster virus (VZV), and Bacillus anthracis inactivate infectivity of these agents but allow detection of target DNA by LightCycler polymerase chain reaction (PCR). MATERIAL AND METHODS: Swabs were inserted into tubes containing serial 10-fold dilutions (10(-1) to 10(-5); 500 microL; 6 samples per dilution) of vaccinia virus, HSV, VZV, or a single suspension of 10(8) colony-forming units of B anthracis (2 samples). One half of the samples were autoclaved, and the remainder were not. An aliquot of each not autoclaved sample served as a control for infectivity. RESULTS: Autoclaving swabs saturated with suspensions of vaccinia virus, HSV, or VZV eliminated the infectivity of these agents; however, DNA was detectable in most autoclaved samples in dilutions of 10(-1) to 10(-4) by LightCycler PCR. All not autoclaved specimens were detected by culture (infectivity) except for VZV and, in most dilutions of 10(-1) to 10(-3), by assay of target DNA by LightCycler PCR. Similarly positive results were obtained for PCR assessment of sporulated B anthracis. CONCLUSIONS: Standard autoclaving procedures eliminated the infectivity of viruses (and B anthracis), but target DNA was often retained for detection by LightCycler PCR. Current recommendations indicate that the laboratory diagnosis of smallpox virus infection be performed only within Biosafety Level 4 facilities. We suggest that, in addition to the requirement for immediate coordination with public health officials, the federal government consider expanding the existing guidelines for processing these specimens to encourage immediate collection, autoclaving, and testing by LightCycler PCR to differentiate smallpox virus from other dermal pathogens such as HSV and VZV by specific qualified laboratories.

Application of rapid-cycle real-time polymerase chain reaction for the detection of microbial pathogens: the Mayo-Roche Rapid Anthrax Test.

Rapid-cycle real-time polymerase chain reaction has immediate and important implications for diagnostic testing in the clinical microbiology laboratory. In our experience this novel testing method has outstanding performance characteristics. The sensitivities for detecting microorganisms frequently exceed standard culture-based assays, and the time required to complete the assays is considerably shorter than that required for culture-based assays. We describe the principle of real-time polymerase chain reaction and present clinical applications, including the detection of Bacillus anthracis, the causative agent of anthrax. This latter test is commercially available as the result of a collaborative venture between Mayo Clinic and Roche Applied Science, hence the designation The Mayo-Roche Rapid Anthrax Test.

Comparison of line probe assay and DNA sequencing of 5' untranslated region for genotyping hepatitis C virus: description of novel line probe patterns.

We compared a commercial line probe assay (INNO-LiPA HCV II, Innogenetics, N.V., Ghent, Belgium, distributed by Bayer Diagnostics) to an in-house 5' untranslated region direct DNA sequencing method for genotyping hepatitis C virus (HCV). Initial evaluation demonstrated that the INNO-LiPA HCV II assay and sequencing assay assigned the same genotype for 110/132 (83.3%) patient specimens (98 subtype and 12 genotype only identifications). Following the initial evaluation, the INNO-LiPA HCV II assay was used routinely to genotype HCV from patient specimens submitted to our laboratory for genotyping (n = 1,739). During this second part of the study, novel line probe patterns have been noted and interpreted using the in-house direct sequencing assay. Reactivity at bands 1, 2, 3, 4, 5 and 8 (n = 4) or 1, 2, 3, 4, 6 and 7 (n = 2) represented HCV genotype 1. Reactivity at bands 1, 2, 5 and 9 (n = 1) represented HCV genotype 2. Reactivity at bands 1, 2, 5, 9 and 16 (n = 1) represented HCV genotype 4. Reactivity at bands 1, 2, 5, 9, 10, 11 (weak band) and 12 (n = 118) most likely represented HCV genotype 2b. This information should be of use to INNO-LiPA HCV II assay users.