Search

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.

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.

Background: Travelers' diarrhea (TD) is a significant problem for travelers. TD is treatable once it occurs, but few options for prevention exist. Probiotics have been studied for prevention or treatment of TD; however, very few combination probiotics have been studied. Therefore, the purpose of this study was to determine if prophylactic use of an oral synbiotic could reduce the risk of acquiring TD and reduce antibiotic use if TD occurred., Methods: Healthy subjects traveling to an area of the world with an increased risk of TD were eligible. All subjects received pre-travel counseling and were provided antibiotics and antidiarrheals (loperamide) for use only if TD developed. The subjects were blinded and randomized to take two capsules of placebo or oral synbiotic (a combination of two probiotics and a prebiotic) called Agri-King Synbiotic (AKSB) beginning 3 days prior to departure, daily while traveling, and for 7 days after return. All subjects kept symptom and medication diaries and submitted a stool sample for pathogen carriage within 7 days of return. The study was powered to detect a 50% reduction in the incidence of TD., Results: Of the 196 adults (over 18 years of age) enrolled in the study, 54.3% were female and 80.9% were younger than 60 years. The study randomized 94 people to the AKSB arm and 102 to placebo. The incidence of TD was 54.5% in the overall group with 55.3% in the AKSB arm and 53.9% in the placebo (p = 0.8864). Among the subjects who experienced diarrhea (n = 107) there was no significant difference in the proportion of subjects that took antibiotics versus those that did not take antibiotics (35% vs 29%, p = 0.68). AKSB was safe with no difference in toxicity between the two arms., Conclusions: The prophylactic oral synbiotic was safe but did not reduce the risk of developing TD among travelers, nor did it decrease the duration of TD or the use of antibiotics when TD occurred., (© 2013 International Society of Travel Medicine.)

Objective. We compared laboratory developed real-time PCR assays for detection of Mycoplasma hominis and for detection and differentiation of Ureaplasma urealyticum and parvum to culture using genitourinary specimens submitted for M. hominis and Ureaplasma culture. Methods. 283 genitourinary specimens received in the clinical bacteriology laboratory for M. hominis and Ureaplasma species culture were evaluated. Nucleic acids were extracted using the Total Nucleic Acid Kit on the MagNA Pure 2.0. 5 μL of the extracts were combined with 15 μL of each of the two master mixes. Assays were performed on the LightCycler 480 II system. Culture was performed using routine methods. Results.  M. hominis PCR detected 38/42 M. hominis culture-positive specimens, as well as 2 that were culture negative (sensitivity, 90.5%; specificity, 99.2%). Ureaplasma PCR detected 139/144 Ureaplasma culture-positive specimens, as well as 9 that were culture negative (sensitivity, 96.5%; specificity, 93.6%). Of the specimens that tested positive for Ureaplasma species, U. urealyticum alone was detected in 33, U. parvum alone in 109, and both in 6. Conclusion. The described PCR assays are rapid alternatives to culture for detection of M. hominis and Ureaplasma species, and, unlike culture, the Ureaplasma assay easily distinguishes U. urealyticum from parvum.

This article covers the laboratory diagnosis of infections that occur predominantly in the tropics. The discussion includes diagnosis of blood and tissue parasites, intestinal parasites, and tropical infections caused by fungi, bacteria, and mycobacteria. The laboratory performance of techniques for the identification of intestinal parasites and special requirements for the collection of specimens for virology testing are also discussed. Images demonstrating the characteristic features of selected tropical parasites and fungi are included for reference., (Copyright © 2012 Elsevier Inc. All rights reserved.)

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.

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.

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.

We assessed the prevalence of tcdC deletion-carrying Clostridium difficile using a stool polymerase chain reaction (PCR) assay that detects previously described 18- and 39-bp deletions (J. Clin. Microbiol. 2008;46:1996). We divided inpatients into 2 groups, those for whom the assay detected a deletion in tcdC and those for whom no deletion was detected. We compared risk factors (antibiotic use, hospitalization, nursing home stay, immunocompromise, age >65 years), complications (pseudomembranous colitis, toxic megacolon, colonic perforation, colectomy, and intensive care unit admission), duration of antibiotic treatment, and 30-day mortality between the groups. Forty-two of 141 patients had deletion-positive C. difficile. Prior nursing home stay and age >65 years were significantly more common in the deletion-positive group. Other risk factors, complications, antibiotic duration, and mortality did not differ significantly. Deletion-carrying C. difficile was commonly present but not associated with more severe disease and not markedly different in terms of risk factor profile. Severity of disease was relatively low, regardless of the presence or absence of a deletion.

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.

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.

We previously developed a real-time polymerase chain reaction (PCR) assay for detection of the four Plasmodium species that infect humans. Recent studies have shown that natural transmission of the simian parasite Plasmodium knowlesi to humans occurs frequently in Southeast Asia. We have expanded our PCR assay to include detection of P. knowlesi.

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.

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.

Transmission of toxoplasmosis via liver transplantation is extremely uncommon. Here we report the case of a 52-year-old male liver transplant recipient who on day 32 post transplant developed pneumonia followed by respiratory failure. Donor-transmitted toxoplasmosis was confirmed as the etiology by both serologic and molecular testing. We also review all previously published cases of toxoplasmosis in the English-language adult liver transplant literature.

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.

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.

Real-time PCR has revolutionized the way clinical microbiology laboratories diagnose many human microbial infections. This testing method combines PCR chemistry with fluorescent probe detection of amplified product in the same reaction vessel. In general, both PCR and amplified product detection are completed in an hour or less, which is considerably faster than conventional PCR detection methods. Real-time PCR assays provide sensitivity and specificity equivalent to that of conventional PCR combined with Southern blot analysis, and since amplification and detection steps are performed in the same closed vessel, the risk of releasing amplified nucleic acids into the environment is negligible. The combination of excellent sensitivity and specificity, low contamination risk, and speed has made real-time PCR technology an appealing alternative to culture- or immunoassay-based testing methods for diagnosing many infectious diseases. This review focuses on the application of real-time PCR in the clinical microbiology laboratory.

A real-time PCR method using the LightCycler (Roche Applied Science, Indianapolis, IN) was compared to a conventional PCR assay for the detection of Tropheryma whipplei in 321 clinical specimens. The LightCycler method had sensitivity and specificity comparable to the conventional method but required considerably less labor and time (3 h versus 3 to 5 days).

We compared the performance characteristics of a real-time PCR method, the LightCycler vanA/vanB detection assay (Roche Diagnostics Corporation, Indianapolis, Ind.) to that of Enterococcosel agar (BBL, Sparks, Md.) for direct detection of vancomycin-resistant enterococci (VRE) from 894 perianal stool swabs. For 421 of 894 swabs, the result for LightCycler PCR was compared to an Enterococcosel plate containing vancomycin at 6 microg/ml; for the remaining 473 swabs, the result for LightCycler PCR was compared to an Enterococcosel plate containing 8 microg/ml vancomycin. The LightCycler method produced considerably more positive results than either the Enterococcosel plate containing vancomycin at 6 microg/ml (n = 25 versus n = 11; sensitivity, 100%; specificity, 97%; positive predictive value [PPV], 42%; negative predictive value [NPV], 100%) or the Enterococcosel plate containing vancomycin at 8 microg/ml (n = 31 versus n = 10; sensitivity, 100%; specificity, 95%; PPV, 32%; NPV, 100%). When possible, additional testing, including culture, LightCycler PCR, and/or a conventional PCR method (PCR-restriction fragment length polymorphism assay), were performed on either the original specimens or original cultures or subsequent specimens for cases in which the original specimen was positive by LightCycler PCR but the Enterococcosel plate was negative. This additional testing demonstrated positive results for 7 of 14 (50%) evaluable discordant specimens which initially tested as LightCycler PCR positive but culture negative using the Enterococcosel plate containing vancomycin at 6 microg/ml and 12 of 17 (71%) evaluable discordant specimens which initially tested as LightCycler positive but culture negative using the Enterococcosel plate containing vancomycin at (8 microg/ml). These results demonstrate that the LightCycler VRE detection assay is considerably more sensitive than the standard culture method for detecting VRE directly from perianal swab specimens. The LightCycler assay also provides results much faster than culture (approximately 3.5 versus > or =72 h). The use of this test could have important implications for the effective control and prevention of nosocomial outbreaks of VRE.

Three enzyme immunoassays, Borrelia DotBlot G (GenBio, San Diego, Calif.), MarDx EIA (MarDx Diagnostics, Inc., Carlsbad, Calif.), and VIDAS (bioMérieux, St. Louis, Mo.) were compared for their ability to detect immunoglobulin G antibodies to Borrelia burgdorferi in 100 human serum samples. The "gold standard" positive result for each of these samples was determined by Western immunoblot analysis (MarDx Marblot Test System) (n = 99) or clinical signs and symptoms suggestive of Lyme disease with other laboratory results positive for B. burgdorferi (n = 1). Based on these criteria for a gold standard positive result, 29 of the 100 samples tested were considered true positives and 71 were considered true negatives. The following sensitivities and specificities were noted, respectively, for each method: Borrelia DotBlot G, 93 and 90%; MarDx, 100 and 35%; and VIDAS, 100 and 92%. Because of high sensitivity and specificity and ease of use, the VIDAS test is an appealing method, especially for laboratories that perform high volumes of tests. The high sensitivity but low specificity of the MarDx method compared to the VIDAS and Borrelia DotBlot G methods requires that Western blot confirmatory tests be performed frequently. The Borrelia DotBlot G method has acceptable specificity but appears to lack sensitivity when compared to the VIDAS and MarDx methods.

The prevalence of microsporidiosis is likely underestimated due to the labor-intensive, insensitive, and nonspecific clinical laboratory methods used for the diagnosis of this disease. A real-time PCR assay was designed to assess DNA extraction methods and to detect three Encephalitozoon species in feces. Modifications of the MagNA Pure LC DNA isolation kit protocol (Roche Applied Sciences, Indianapolis, Ind.) were compared by using the automated MagNA Pure LC instrument (Roche) and fecal specimens spiked with Encephalitozoon intestinalis spores. Extracted DNA was amplified by the LightCycler (Roche) PCR assay. Assay sensitivity, reproducibility, and efficiency were assessed by comparing threshold crossover values achieved with different extraction and storage conditions (fresh, refrigerated, frozen, and preserved specimens). Optimal extraction conditions were achieved by using a commercial buffer, tissue lysis buffer (Roche), as the specimen diluent. LightCycler PCR results were compared to those obtained from routine stool microscopy with trichrome blue stain. The lower limit of detection for the LightCycler PCR assay varied by storage conditions from 10(2) to 10(4) spores/ml of feces, a value which represented a significant improvement over that achieved by staining (> or =1.0 x 10(6) spores/ml). Melting temperature analysis of the amplicons allowed for the differentiation of three Encephalitozoon species (E. intestinalis, E. cuniculi, and E. hellem). The assay is readily adaptable to the clinical laboratory and represents the first real-time PCR assay designed to detect Encephalitozoon species.

A 5-laboratory study was performed that used the National Committee for Clinical Laboratory Standards (NCCLS) reference agar dilution method with 3 media formulations to determine whether the use of different media would affect minimum inhibitory concentration (MIC) results. Wilkins-Chalgren, Brucella-based blood agar (BRU), and Wilkins-Chalgren agar plus blood (WCB) and 6 antibiotics (clindamycin, cefoxitin, ceftizoxime, piperacillin, metronidazole, and trovafloxacin) were evaluated with 58 isolates. The MIC values were compared, and a significant correlation of >0.80 was demonstrated for all media and each antibiotic/organism group. The cumulative rate of errors for all antibiotics was 0.1%. These data indicate that a change in the NCCLS reference medium for testing of anaerobic bacteria susceptibility to either BRU or WCB will not affect the MIC results for the antibiotics and organisms evaluated.

A multilaboratory study compared the growth of 30 fastidious anaerobes, using 5 different agar media: Wilkins-Chalgren (WC), WC with either whole or laked sheep blood, and Brucella supplemented with vitamin K(1) and hemin and either laked or whole sheep blood. The media were compared for quality and quantity of growth. Experiments were conducted either entirely in an anaerobic chamber or inoculated in ambient air with anaerobic incubation. The results showed that (1) any medium plus whole or laked blood was better than unsupplemented WC, (2) whole blood and laked blood additives gave similar results, (3) supplemented Brucella with whole or laked blood was superior to WC and WC with whole or laked blood, and (4) anaerobic and aerobic inoculation with anaerobic incubation gave similar results. Brucella agar supplemented with whole or laked blood supports the growth of fastidious anaerobic species better than the WC agars do.

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.

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.

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.

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.

Detection of Giardia and Cryptosporidium in clinical stool specimens using the ColorPAC and ProSpecT enzyme immunoassays revealed 98.7% agreement for Giardia detection and 98.1% agreement for Cryptosporidium detection. Sensitivities were uniformly 100%. The specificities of the ColorPAC immunoassay for Giardia and Cryptosporidium detection were 100 and 99.5%, respectively, and those for the ProSpecT assay were 98.4 and 98.6%, respectively. The false-positive reactions with the ProSpecT assay occurred with specimens that were grossly bloody.

Both serologic and molecular assays are useful in the diagnosis of viral hepatitis. They may detect early infections before other signs of disease appear, differentiate acute from chronic infections, and detect persistence of viremia or verify development of immunity. Molecular assays may also be used to monitor responses to antiviral therapy, and in the future, be a primary method to screen blood and organ donors (NAT). EIA serologies are used to diagnose acute HAV infections or establish immune status. Similar immunoassays are used to detect HBV infections, verify persistence of antigenemia and degree of infectivity, and indicate immunity (including the response to vaccination). HBV molecular assays can shorten the diagnostic window period, verify persistence of viremia, including monitoring response to antiviral therapy, and be useful in NAT screening of donors. Molecular assays play a major role in HCV diagnosis where serologic tests can document past or present infection but cannot differentiate one from the other. A variety of molecular tests can be used as sensitive (and early) detectors of viremia (and serve as confirmatory tests for positive serologies and as donor NAT methods), document its persistence as an indicator of chronic infection, and monitor responses to antiviral therapy. Both qualitative and quantitative molecular assays are available, and their efficient use requires familiarity with the sensitivity and dynamic ranges of each method.

We evaluated two automated systems, MagNA Pure (Roche Molecular Biochemicals, Indianapolis, Ind.) and BioRobot 9604 (Qiagen, Inc., Chatsworth, Calif.) as effective replacements for the manual IsoQuick method (Orca Research, Inc., Bothell, Wash.) for extraction of herpes simplex virus (HSV) DNA from dermal and genital tract specimens prior to analysis by LightCycler PCR. Of 198 specimens (152 genital, 46 dermal), 92 (46.2%) were positive for HSV DNA by LightCycler PCR after automated extraction of specimens with either the MagNA Pure or BioRobot 9604 instrument. The manual IsoQuick method yielded HSV DNA (total n = 95) from three additional specimens that were negative by the automated method (P = 0.25, sign test). Although the mean numbers of LightCycler PCR cycles required to reach positivity differed statistically significantly among all three of the methods of extraction, the estimated means differed by no more than 1.5 cycles (P < 0.05). Seventy (76%) of the 92 specimens that were LightCycler PCR positive by all three extraction methods were also positive by shell vial cell culture assay. HSV DNA was detected by a lower LightCycler PCR cycle number (26.1 cycles) in specimens culture positive for the virus than in culture-negative samples (33.3 cycles) (P < 0.0001). The manual IsoQuick and automated MagNA Pure and BioRobot 9604 methods provide standardized, reproducible extraction of HSV DNA for LightCycler PCR. The decision to implement a manual versus an automated procedure depends on factors such as costs related to the number of specimens processed rather than on the minimal differences in the technical efficiency of extraction of nucleic acids among these methods.

In vitro surveys of antimicrobial resistance among clinically important anaerobes are an important source of information that can be used for clinical decisions in the choice of empiric antimicrobial therapy. This study surveyed the susceptibilities of 556 clinical anaerobic isolates from four large medical centers using a broth microdilution method. Piperacillin-tazobactam was the only antimicrobial agent to which all the isolates were susceptible. Similarly, imipenem, meropenem, and metronidazole were highly active (resistance, <0.5%), whereas the lowest susceptibility rates were noted for penicillin G, ciprofloxacin, and clindamycin. For most antibiotics, blood isolates were less susceptible than isolates from intra-abdominal, obstetric-gynecologic, and other sources. All isolates of the Bacteroides fragilis group were susceptible to piperacillin-tazobactam and metronidazole, while resistance to imipenem and meropenem was low (<2%). For these same isolates, resistance rates (intermediate and resistant MICs) to ampicillin-sulbactam, cefoxitin, trovafloxacin, and clindamycin were 11, 8, 7, and 29%, respectively. Among the individual species of the B. fragilis group, the highest resistance rates were noted among the following organism-drug combinations: for clindamycin, Bacteroides distasonis and Bacteroides ovatus; for cefoxitin, Bacteroides thetaiotaomicron, B. distasonis, and Bacteroides uniformis; for ampicillin-sulbactam, B. distasonis, B. ovatus, and B. uniformis; and for trovafloxacin, Bacteroides vulgatus. For the carbapenens, imipenem resistance was noted among B. fragilis and meropenem resistance was seen among B. fragilis, B. vulgatus, and B. uniformis. With few exceptions all antimicrobial agents were highly active against isolates of Prevotella, Fusobacterium, Porphyromonas, and Peptostreptococcus. These data further establish and confirm that clinically important anaerobes can vary widely in their antimicrobial susceptibilities. Fortunately most antimicrobial agents were active against the test isolates. However, concern is warranted for what appears to be a significant increases in resistance to ampicillin-sulbactam and clindamycin.

Several important developments have occurred in recent years in the chemotherapy for and prophylaxis of parasitic infections. Although mefloquine is clearly the most effective agent for prevention of chloroquine-resistant falciparum malaria, its use has been compromised by side effects, both real and imagined. Well-designed studies have shown that side effects occur no more frequently with low-dose mefloquine than with chloroquine. Use of mefloquine in pregnant women has not been associated with birth defects, but the incidence of stillbirths may be increased. Malarone is a new agent that combines atovaquone and proguanil, and it may be as effective as mefloquine; however, it is not yet available in the United States. Several newer agents have appeared in response to the development of multidrug resistant Plasmodium falciparum, especially in Southeast Asia. Halofantrine is available for the treatment of mild to moderate malaria due to P. falciparum and for P. vivax infections. Because of severe toxic effects, use of halofantrine should be restricted to only those unusual and rare situations in which other agents cannot be used. Artemisinin (an extract of the Chinese herbal remedy qinghaosu) and two derivatives, artesunate and artemether, are active against multidrug resistant P. falciparum and are widely used in Asia in oral, parenteral, and rectal forms. The antibacterial azithromycin in combination with atovaquone or quinine has now been reported to treat babesiosis effectively in experimental animals and in a few patients. Azithromycin in combination with paromomycin has also shown promise in the treatment of cryptosporidiosis (and toxoplasmosis when combined with pyrimethamine) in patients with the acquired immunodeficiency syndrome (AIDS). Albendazole is currently the only systemic agent available for treatment of microsporidiosis, an infection primarily of patients with AIDS. In addition, albendazole and ivermectin have emerged as effective broad-spectrum antihelminthics, with albendazole becoming the drug of choice for hydatid disease (echinococcosis), neurocysticercosis, and most intestinal nematode infections (except strongyloidiasis and trichuriasis). Liposomal amphotericin B is the first drug approved by the Food and Drug Administration for the treatment of visceral leishmaniasis.

Recently, a treponema-specific immunoglobulin G (IgG) enzyme immunoassay (EIA), the CAPTIA Syphilis-G (Trinity Biotech, Jamestown, N.Y.), has become available as a diagnostic test for syphilis. A total of 89 stored sera previously tested by the fluorescent treponemal antibody absorption (FTA-ABS) IgG assay were evaluated by the CAPTIA EIA. The FTA-ABS IgG procedure was performed by technologists unblinded to results of rapid plasmid reagin (RPR) testing of the same specimens. Borderline CAPTIA-positive samples (antibody indices of >/=0.650 and 0.900, the sample was considered positive. Thirteen of 89 (15%) samples had discrepant results. Compared to the FTA-ABS assay, the CAPTIA EIA had a sensitivity and specificity and positive and negative predictive values of 70.7, 97.9, 96.7, and 79.7%, respectively. In another analysis, discrepancies between results were resolved by repeated FTA-ABS testing (technologists were blinded to previous RPR results) and patient chart reviews. Seven CAPTIA-negative samples which were previously interpreted (unblinded) as minimally reactive by the FTA method were subsequently interpreted (blinded) as nonreactive. One other discrepant sample (CAPTIA negative and FTA-ABS positive [at an intensity of 3+], unblinded) was FTA negative with repeated testing (blinded). For the five remaining discrepant samples, chart reviews indicated that one patient (CAPTIA negative and FTA-ABS positive [minimally reactive], blinded) had possible syphilis. These five samples were also evaluated and found to be negative by another treponema-specific test, the Treponema pallidum microhemagglutination assay. Therefore, after repeated testing and chart reviews, 2 of the 89 (2%) samples had discrepant results; the adjusted sensitivity, specificity, and positive and negative predictive values were 96.7, 98.3, 96.7, and 98.3%, respectively. This study demonstrates that the CAPTIA IgG EIA is a reliable method for syphilis testing and that personnel performing tests which require subjective interpretation, like the FTA-ABS test, may be biased by RPR test results.

Although rarely encountered in the United States, urinary tract schistosomiasis occurs commonly in many countries in the eastern hemisphere. Travel and immigration may contribute to imported cases of schistosomiasis. Excessive morbidity and increased mortality, including the development of urinary-tract squamous-cell carcinoma, are associated with untreated Schistosoma haematobium infection. Therefore, in the appropriate clinical context, all efforts should be made to rule out infectious and readily treatable causes of chronic hematuria. The presence of characteristic eggs in the urinary sediment is the usual means of diagnosing a S. haematobium infection. Additionally, the small and less commonly encountered miracidium stage of S. haematobium may also be present in the urine, which is another means of diagnosing urinary tract schistosomiasis. The present report describes a case in which a miracidium was detected in a fresh, unstained urine specimen. As detection of miracidia can be made in specimens also processed by routine cytologic methods, it behooves cytologists to be aware of this entity for the diagnosis of schistosomiasis.

A variant of hepatitis E virus (HEV), designated HEV US-1, was identified in a hepatitis patient in the United States (US); the patient had no history of travel to areas where HEV is endemic. Nucleotide sequences were obtained from the 5' end of open reading frame (ORF) 1 (1418 nt), the 3' end of ORF1 (1359 nt), the entire ORF2 and ORF3 regions, and the 3'-untranslated region (2127 nt). The HEV US-1 strain is significantly divergent from other human HEV isolates with nucleotide identities ranging from 76.8 to 77.5%. Phylogenetic analyses indicate that HEV US-1 and a recently discovered HEV variant from swine may represent separate isolates of a new strain of HEV, significantly divergent from the Mexican and Burmese strains. Synthetic peptides derived from the carboxyl amino acids of ORF2 and ORF3 were shown to be useful for detecting exposure to HEV. In addition, IgM class antibodies directed against HEV US-1 synthetic peptides were detected in the US patient infected with HEV US-1, but were absent using synthetic peptides from the Burmese or Mexican strains of HEV. A preferential reactivity to HEV US-1 specific peptides has lead to the identification of a second isolate of this virus also from a patient with acute hepatitis from the US. The discovery of these HEV variants may be important in understanding the worldwide distribution of HEV infection.

Objective: To report the first case of acute hepatitis E by a novel isolate acquired in the United States and confirmed by nucleotide sequencing., Material and Methods: We describe the clinical manifestations and the results of associated laboratory studies in a man who was found to have acute hepatitis E infection., Results: A 62-year-old man was hospitalized because of fever, abdominal pain, and jaundice. After an initial evaluation did not provide a cause, his serum was found to be positive for IgG anti-hepatitis E virus (HEV) by three antibody assays. Serum was also positive for HEV RNA by reverse transcriptase polymerase chain reaction (PCR). Sequencing results from the PCR products demonstrated substantial differences at the nucleotide level between this strain and the known Mexican and Burmese strains., Conclusion: On the basis of this initial report, HEV should be considered an etiologic agent in patients with acute non-ABC hepatitis in the United States.

Because anaerobic bacteria cause significant human infections that require specific therapy and because anaerobes are resistant to certain antimicrobials, the isolation, identification, and determination of antimicrobial susceptibilities are as important for these bacteria as they are for other pathogenic bacteria. Anaerobic culturing can be made cost-efficient by strict adherence to several principles, including the selective culturing of only appropriate general specimens that are uncontaminated by normal flora (this can be achieved by educating physicians and nurses in recognizing likely sources of anaerobic infection); rapid transport of specimens and use of appropriate transport system; use of a system of rejection and notification when inappropriate or when multiple specimens have been received; and use of a logical algorithm for determining the degree of isolation and identification to be performed, according to the numbers and types of organisms present. Testing of all significant gram-negative organisms for the production of beta-lactamases can provide an early indication of antimicrobial susceptibility, and actual testing limited to screening of three or four drugs can be performed on selected isolates by using a rapid and simple method such as the Etest (AB BIODISK, Solna, Sweden). Although the number of anaerobic bacteremias has declined since the 1970s, this number has plateaued in recent years, and these infections are life-threatening. Routine culturing of blood for anaerobes is still indicated in many institutions because of the unpredictable clinical sources of some bacteremias and the improved yields of both anaerobes and some streptococci when anaerobic blood culture systems are used.

The results for 281,797 blood culture sets of specimens collected from adult patients at the Mayo Clinic over an approximately 8-year period (1 November 1984 through 30 November 1992) were analyzed in order to determine whether there were differences in the types of microorganisms isolated over this time and to assess the usefulness of anaerobic culturing of blood. Each blood culture set consisted of two aerobic blood cultures (Septi-Chek [Becton Dickinson, Sparks, MD] and Isolator [Wampole Laboratories, Cranbury, NJ]) and one anaerobic culture (nonvented tryptic or trypticase soy broth [NVTSB; Difco Laboratories, Detroit, or Becton Dickinson]). The relative frequency of isolation of aerobic and facultatively anaerobic gram-positive bacteria and obligately anaerobic bacteria increased over the second half of the 1984-1992 surveillance period. The value of the NVTSB anaerobic blood culture was demonstrated for diagnosing bloodstream infections caused by certain facultatively anaerobic bacteria in addition to obligately anaerobic bacteria and supported the inclusion of the NVTSB anaerobic blood culture as a standard part of the three-component blood culture set used at this institution.

Among the three recently described GB viruses (GBV-A, GBV-B, and GBV-C), only GBV-C has been linked to cryptogenic hepatitis in man. Because of the limited utility of currently available research tests to determine antibody response to GBV-C proteins, the prevalence of GBV-C RNA in human sera was studied using reverse transcription-polymerase chain reaction (RT-PCR). The prevalence of GBV-C is higher among volunteer blood donors with elevated serum alanine aminotransferase (ALT) levels (3.9%) than among volunteer blood donors with normal ALT levels (0.8%). Higher rates were also noted among commercial blood donors (12.9%) and intravenous drug users (16.0%). GBV-C was frequently detected in residents of West Africa, where the prevalence was > 10% in most age groups. Approximately 20% of patients diagnosed with either acute or chronic hepatitis C virus (HCV) were found to be positive for GBV-C RNA. In addition, GBV-C RNA sequences were detected in individuals diagnosed with non-A-E hepatitis, with clinical courses ranging from mild disease to fulminant hepatitis. Fourteen of sixteen subjects with or without clinically apparent hepatitis were positive for GBV-C RNA more than 1 year after the initial positive result.

The ESP 80A aerobic blood culture of the ESP automated blood culture system (Difco Laboratories. Detroit, Mich.) was compared with two manual aerobic blood culture systems, the Isolator (Wampole Laboratories, Cranbury, N.J.) and the Septi-Chek (Becton Dickinson, Cockeysville, Md.) systems, for the detection of bloodstream microorganisms from 5,845 blood samples for culture collected from adult patients with suspected septicemia. The bottles were incubated for 7 days, and the sediment from the Isolator tube was inoculated onto solid medium and this medium was incubated for 72 h. A total of 609 microorganisms were recovered from 546 blood cultures. There was no statistically significant difference in the total recovery of microorganisms for the ESP 80A system when compared with that for the Septi-Chek system (P = 0.083); however, the Isolator system recovered significantly more microorganisms overall than either the ESP 80A (P < 0.001) or the Septi-Chek (P < 0.001) system. When assessing individual probable pathogens, the Isolator system detected statistically significantly more Staphylococcus aureus and Candida spp. than either the ESP 80A or the Septi-Chek system (P < 0.05). Similarly, the Isolator system detected statistically significantly more bloodstream infections (septic episodes) caused by S. aureus and Candida spp. than either the ESP 80A or the Septi-Chek system (P < 0.05). In blood culture sets which produced growth of the same probable pathogens in the ESP 80A and the Isolator systems, there was no statistically significant difference in the median times to detection for all pathogens combined (P = 0.067). However, a similar comparison showed the Isolator and the ESP 80A systems to have statistically significantly shorter median detection times for all pathogens combined (P < 0.001) when they were independently compared with the Septi-Chek system. The ESP 80A system had 29 (0.5%) false-positive signals. The ESP system required less processing time than the Isolator system and eliminates the hands-on time for the detection of positive cultures required by the manual systems.

We compared the susceptibility test results of 220 anaerobes against 14 antimicrobials using the Etest (AB Biodisk, Solna, Sweden) with those using the National Committee for Clinical Laboratory Standards (NCCLS) standard agar dilution method (Wadsworth version). The Etest medium was brucella blood (whole) agar and the inoculum size was equivalent to a no. 1 McFarland standard. Thirty-six percent of Etest results were unreadable after 24 h of anaerobic incubation compared to only 5% after 48 h. Also, there were more results with categorical agreement with the NCCLS method after 48 h (97.9%) than at 24 h (89%) and more very major errors (VMEs) at 24 h (22% of resistant organisms) than at 48 h (3.2%). VMEs and major errors occurred most frequently with clindamycin, ceftriaxone, and trospectomycin (which should not be used with the Etest) and involved the Bacteroides fragilis group and/or Clostridium most commonly. The Etest is simple to perform and is a generally reliable method that is optimally read after 48 h of incubation. It should be an acceptable alternative to the agar dilution standard, although results with certain organism-antimicrobial combinations should be read very conservatively because of the frequency of VMEs.