Diagnostic Microbiology & Infectious Disease

Reevaluation of the Premier Clostridium difficile toxin A and B immunoassay with comparison to glutamate dehydrogenase common antigen testing evaluating Bartels cytotoxin and Prodesse ProGastro™ Cd polymerase chain reaction as confirmatory procedures

2010-02-01

Abstract: Enzyme immunoassays are currently the most common tests used in the clinical laboratory for the detection of Clostridium difficile toxins; however, significant problems with their performance have recently been described. We prospectively reevaluated the Meridian Premier C. difficile toxin A/B assay with direct comparison to a 2-step algorithm that screened for C. difficile common antigen and compared cytotoxin and real-time polymerase chain reaction (PCR) as confirmatory procedures. The Premier assay lacked sufficient sensitivity, missing 25% of true-positive samples. PCR was the most sensitive method and the only procedure that allowed same day testing and reporting.

A rapid, simple, and sensitive loop-mediated isothermal amplification method to detect toxigenic Vibrio cholerae in rectal swab samples

2009-10-08

Abstract: Loop-mediated isothermal amplification (LAMP) method was designed for clinical diagnosis of Vibrio cholerae carrying the ctxA gene. The detection limits of the method were 5 fg of purified genomic DNA/reaction and 0.54 CFU/reaction. The method was applied to rectal swab samples from cholera patients and healthy volunteers (19 subjects each) and yielded the same results as the “gold standard” culture method, while the polymerase chain reaction-based method failed to detect V. cholerae in 8 of the positive samples. Direct application of this LAMP method without precultivation enabled the rapid detection of 5 asymptomatic carriers from rectal swabs of 21 household contacts of cholera patients. This LAMP method could be a sensitive, specific, inexpensive, and rapid detection tool for V. cholerae carrying the ctxA gene in the clinical laboratory and in the field.

Development of a microarray for identification of pathogenic Clostridium spp.

2009-11-02

Abstract: In recent years, Clostridium spp. have rapidly reemerged as human and animal pathogens. The detection and identification of pathogenic Clostridium spp. is therefore critical for clinical diagnosis and antimicrobial therapy. Traditional diagnostic techniques for clostridia are laborious, are time consuming, and may adversely affect the therapeutic outcome. In this study, we developed an oligonucleotide diagnostic microarray for pathogenic Clostridium spp. The microarray specificity was tested against 65 Clostridium isolates. The applicability of this microarray in a clinical setting was assessed with the use of mock stool samples. The microarray was successful in discriminating at least 4 species with the limit of detection as low as 104 CFU/mL. In addition, the pattern of virulence and antibiotic resistance genes of tested strains were determined through the microarrays. This approach demonstrates the high-throughput detection and identification of Clostridium spp. and provides advantages over traditional methods. Microarray-based techniques are promising applications for clinical diagnosis and epidemiologic investigations.

Evaluation of direct method of drug susceptibility testing of Mycobacterium tuberculosis to rifampicin and isoniazid by nitrate reductase assay in a national reference laboratory

2009-10-19

Abstract: The aim of this study was to evaluate a simple, rapid, and inexpensive colorimetric nitrate reductase assay (NRA) for direct drug susceptibility testing (DST) of Mycobacterium tuberculosis against rifampicin (RIF) and isoniazid (INH). A total of 118 smear-positive specimens were processed from patients on antituberculosis treatment. A comparison was made between the direct NRA of DST with the direct proportion method and with the internationally accepted indirect 1% proportion method as the “gold standard”. The sensitivity and specificity of the direct NRA and indirect proportion method were 94% and 98%, and 100% and 98% for RIF and INH, respectively. Excellent agreement was found between the 2 tests with κ values of 0.92 and 0.98, and P value was less than 0.001 for RIF and INH. The results in most cases were available in 14 days (turnaround time). The direct NRA is a rapid, accurate, simple, and inexpensive method to determine multidrug resistance from sputum. Direct NRA may become an appropriate alternative method, especially for the resource poor settings.

Utility of a combination of RD1 and RD2 antigens as a diagnostic marker for tuberculosis

2009-10-16

Abstract: We evaluated the diagnostic potential of a cocktail of 4 antigens encoded by regions of difference (RD) 1 and 2 of Mycobacterium tuberculosis, that is, early secretory antigenic target-6, culture filtrate protein-10 (CFP-10), CFP-21, and mycobacterial protein from species tuberculosis-64 (MPT-64) on the basis of antigen and antibody detection by enzyme-linked immunosorbent assay. Parallel detection of antigens and antibodies in the serum samples of pulmonary tuberculosis (PTB) patients resulted in higher sensitivity as compared to either of the single tests in both smear-positive (90%) and smear-negative (60%) PTB patients. In addition, combined detection of antigens and antibodies in the fluids of extrapulmonary tuberculosis (EPTB) patients could detect >90% of the patients with high specificity. These results demonstrate the ability of the combination of antigen and antibody detection assays based on the cocktail of RD antigens to diagnose a substantial number of PTB and EPTB cases with high specificity.

Evaluation of rapid diagnostic tests for malaria case management in Gabon

2009-10-21

Abstract: A laboratory-confirmed diagnosis is the basis of malaria case management. Rapid diagnostic tests (RDTs) create new opportunities for improved care in endemic areas. Diagnostic performance of OptiMAL-IT® and Acon® was assessed in comparison with microscopy at 2 sites in Gabon. Between February 2008 and January 2009, 2125 febrile children under 11 years old were diagnosed using microscopy and RDTs. Plasmodial infection was detected more frequently using Acon® (27%) and OptiMAL-IT® (27%) compared to microscopy (20%) (P < 0.01). Among the samples diagnosed positive by OptiMAL-IT®, 78% were infected by Plasmodium falciparum, whereas 99% of positive blood smears were P. falciparum infections, 0.5% Plasmodium malariae, and 0.5% Plasmodium ovale. Both RDTs had similar sensitivity (Se) (94.0%; 95% confidence interval [CI], 92–96), which varied depending on the site. When parasite density was >100 p/μL, the Se of the 2 tests was >98% (95% CI, 96–100). Likewise, the negative predictive values were high and comparable (>98%). Overtreatment with antimalarial drugs was 12%. These tests should be considered as a good alternative to microscopy, allowing not only an efficient and rapid diagnosis of malaria in primary health facilities but also to aid in promoting changes for antimalarial prescription behavior.

Imported malaria in children: incidence and risk factors for severity

2009-09-30

Abstract: To assess the incidence of imported malaria in children and to determine the frequency of delayed diagnosis and risk factors for severe malaria, we performed a retrospective multicenter cohort study in the northern region of France and included all children with a positive test for malaria from 2000 to 2006. The incidence of imported malaria in children <18 years, the frequency of a delayed diagnosis, and the risk factors for severe malaria were determined. The study identified 133 children with imported malaria. The mean incidence of this disease was 1.9/100 000 children <18 years (95% confidence interval [CI], 1.6–2.2). Detailed data were available for 120 children. Disease was considered severe in 19% of cases. The diagnosis was delayed (≥1 day after the first medical contact) in 31% of cases, and this delay was the only independent risk factor identified for severe imported malaria in children (adjusted odds ratio, 3.2; 95% CI, 1.2–8.8; P = 0.02).

Leishmania spp. identification by polymerase chain reaction–restriction fragment length polymorphism analysis and its applications in French Guiana

Stéphane Simon, Vincent Veron, Bernard Carme — 2009-09-27

Abstract: Leishmania (Viannia) guyanensis was for many years the only species commonly identified in French Guiana, but precise species identifications were quite rare. We describe a new restriction fragment length polymorphism–polymerase chain reaction technique using a 615-bp fragment of the RNA polymerase II gene and 2 restriction enzymes, TspRI and HgaI. Seven reference strains (Leishmania (Leishmania) amazonensis, Leishmania (Viannia) lainsoni, Leishmania (Viannia) braziliensis, L. (V.) guyanensis, Leishmania (Viannia) naiffi, Leishmania (Leishmania) major, Leishmania (Leishmania) infantum) and 112 clinical samples from positive lesions were used for the development of the technique. The rates of positive species identification were 85.7% for punch skin biopsy specimens, 93.1% for positive Giemsa-stained smears, and 100% for positive culture supernatants. In the framework of cutaneous leishmaniasis species surveillance for the 2006 to 2008 period, parasite identification was carried out for 199 samples from different patients. The prevalence of the various Leishmania spp. was 84.4% for L. (V.) guyanensis, 8.0% for L. (V.) braziliensis, 5.0% for L. (L.) amazonensis, and 2.6% for L. (V.) lainsoni. L. (V.) braziliensis seems to be locally an emerging pathogen.

Effect of oxygen limitation on the in vitro activity of levofloxacin and other antibiotics administered by the aerosol route against Pseudomonas aeruginosa from cystic fibrosis patients

2009-10-15

Abstract: Studies have demonstrated that thickened mucous layers in the lungs of cystic fibrosis (CF) patients contain areas of low oxygen tension. These microaerophilic environments may reduce the activity of aerosol antibiotics used in the management of chronic infection in CF. The aim of this study was to compare the MICs of levofloxacin, tobramycin, amikacin, and aztreonam against Pseudomonas aeruginosa under reference and anaerobic conditions and evaluate the in vitro pharmacodynamics of levofloxacin under aerobic and hypoxic testing conditions. The MICs for 114 isolates of P. aeruginosa from CF patients were determined in cation-adjusted Mueller Hinton broth alone or supplemented with 1% potassium nitrate for anaerobic testing. Levofloxacin time–kill curves were performed under aerobic and hypoxic conditions using strains of P. aeruginosa with elevated efflux pump overexpression and/or target mutations. The MICs of nonmucoid or mucoid P. aeruginosa isolates to levofloxacin incubated under aerobic and anaerobic conditions were similar. In contrast, anaerobic incubation resulted in higher MICs for tobramycin, amikacin, and aztreonam among nonmucoid or mucoid isolates, with ≥4-fold increase in MICs for over 40% of the isolates. Time–kill curves performed in aerobic and hypoxic environments with levofloxacin concentrations attained in CF sputum demonstrated similar activity, approaching a maximum bactericidal effect within 10 min of exposure. Together, these results indicate that the activity of some antibiotics against P. aeruginosa is significantly reduced under conditions relevant to the CF lung environment. In contrast, levofloxacin maintains activity against P. aeruginosa under anaerobic or hypoxic conditions similar to those found in CF microaerophilic environments.

In vitro activity of tigecycline against 2423 clinical isolates and comparison of the available interpretation breakpoints

2009-10-16

Abstract: MICs to tigecycline and 12 antimicrobials were performed by microdilution method, against 2423 nonduplicate pathogens recently isolated in 17 Greek hospitals. The Food and Drug Administration (FDA) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria were used comparatively for interpretation of tigecycline MICs. Tigecycline exhibited potent in vitro activity against the majority of the isolates tested. (MIC90 values of 0.5, 1, 2, 0.125, 1, 0.25, 0.125, and 1 mg/L were observed for Escherichia coli, Klebsiella pneumoniae, Enterobacter spp., Moraxella catarrhalis, Acinetobacter spp., Staphylococcus aureus, Enterococcus spp., and Streptococcus pneumoniae isolates, respectively.) Tigecycline activity was the same, irrespective of the resistance profile to other antimicrobials (Gram-negative pathogens susceptible or resistant to imipenem, Enterococcus spp., S. aureus, or S. pneumoniae isolates, susceptible or resistant to vancomycin, methicillin or penicillin, respectively). Interpretation using EUCAST and FDA breakpoints differed among isolates of K. pneumoniae and Enterobacter spp. having tigecycline MICs of 2 to 4 mg/L.In conclusion, tigecycline exhibited potent activity against pathogens recently isolated in a region that experiences high antimicrobial resistance rates. Indications that the available criteria might categorize differently tigecycline susceptibility status in K. pneumoniae and Enterobacter spp. isolates were also detected.

Distribution of blaOXA-carrying imipenem-resistant Acinetobacter spp. in 3 hospitals in Taiwan

Han-Yueh Kuo, Chih-Man Yang, Ming-Feng Lin, Wen-Lin Cheng, Ni Tien, Ming-Li Liou — 2009-10-16

Abstract: We investigated the molecular epidemiology and OXA-type carbapenemase genes of 83 imipenem-resistant Acinetobacter spp. collected from 2 university hospitals (hospitals A and B) and a regional hospital (hospital C) during 2007 in Taiwan. Genotyping by pulsed-field gel electrophoresis identified 51 pulsotypes. None of the pulsotypes established predominance throughout the 3 hospitals. Multiplex polymerase chain reaction of blaOXA genes showed that 100% (18/18), 91%(31/34), and 100% (31/31) of the Acinetobacter spp. collected from hospital A, B, and C, respectively, possessed blaOXA-51–like genes. None of the strains carrying blaOXA-23–like and blaOXA-24–like genes were found in hospital A. The coexistences of blaOXA-51–like/blaOXA-23–like and blaOXA-51–like/blaOXA-24–like genes detected in hospitals B and C were 26% (9/34) and 12% (4/34) and 58% (18/31) and 3% (1/31), respectively. Among blaOXA-23–like gene-carrying isolates collected from hospitals, clonal spread of strains carrying the blaOXA-23 gene was detected in the regional hospital but not the other 2 university hospitals. The results suggest that interhospital dissemination of imipenem-resistant Acinetobacter spp. was not found in these hospitals. The increasing percentage of OXA-23 in OXA-type carbapenemases in Acinetobacter spp. from the regional hospitals to medical centers deserves further attention in Taiwan.

Lack of galactomannan reactivity in dematiaceous molds recovered from cancer patients with phaeohyphomycosis

Ronen Ben-Ami, P. Rocco LaSala, Russell E. Lewis, Dimitrios P. Kontoyiannis — 2009-10-16

Abstract: We determined the in vitro galactomannan reactivity of isolates recovered from 17 patients with invasive phaeohyphomycosis, 4 of whom had positive galactomannan antigenemia. All isolates were nonreactive using the galactomannan immunoassay. Galactomannan antigenemia in patients with phaeohyphomycosis should raise the possibility of concomitant invasive aspergillosis.

Direct detection of Streptococcus pneumoniae in positive blood cultures by real-time polymerase chain reaction

2010-02-01

Abstract: We developed a real-time polymerase chain reaction specific for Streptococcus pneumoniae to be applied directly from blood culture bottles without previous DNA extraction step. For the 128 blood culture bottles tested, the assay had 94% and 98.4% sensitivity and specificity, respectively. This assay provides rapid and accurate identification of this pathogen.

Emergence of multidrug-resistant Salmonella enterica serovar Typhi with reduced susceptibility to fluoroquinolones in Cambodia

2009-10-05

Abstract: From December 2006 to April 2009, we conducted an etiology study among Cambodian patients presenting with acute fever of unknown origin. Salmonella enterica serovar Typhi was detected in 0.9% (41/4985) blood cultures. Antimicrobial susceptibility testing showed decreased susceptibility to ampicillin (56% resistant; MIC90, >256 μg/mL), chloramphenicol (56% resistant; MIC90, >256 μg/mL), trimethoprim/sulfamethoxazole (56% resistant; MIC90, >256 μg/mL), nalidixic acid (81% resistant; MIC90, not defined), ciprofloxacin (0% resistant; MIC90, 0.5 μg/mL), and ceftriaxone (0% resistant; MIC90, 0.094 μg/mL). Multidrug resistance, defined as antimicrobial resistance to ampicillin, chloramphenicol, and trimethoprim/sulfamethoxazole, was found in 56% of the isolates, and 80% had reduced susceptibility to ciprofloxacin (defined as MIC ≥0.12 μg/mL).

CTX-M and ampC β-lactamases contributing to increased prevalence of ceftriaxone-resistant Escherichia coli in Changi General Hospital, Singapore

Thean Yen Tan, Lily Siew Yong Ng, Jie He, Li Yang Hsu — 2009-10-05

Abstract: Data from an 800-bed hospital showed an increase in the incidence density of ceftriaxone-resistant Escherichia coli, from 13.1 to 21.7 per 100 000 inpatient days over a 4-year period. Detailed testing performed on 54 E. coli isolates showed blaCTX-M extended-spectrum β-lactamase (ESBL) genes (n = 37) and blaCMY-like ampC genes (n = 15). Typing data showed only limited clonal transmission in isolates with ESBL.

Rapid identification of Propionibacterium acnes from blood cultures by fluorescence in situ hybridization

Sven Poppert, Melanie Riecker, Andreas Essig — 2009-10-08

Abstract: A newly designed probe for rapid identification of Propionibacterium acnes by fluorescence in situ hybridization was evaluated using 111 isolates from subculture and showed 100% sensitivity and specificity. A sensitivity of 95% and a specificity of 100% were achieved with direct application on 55 blood cultures containing Gram-positive rods.

Occurrence and genotyping using automated repetitive-sequence–based PCR of methicillin-resistant Staphylococcus aureus ST398 in Southeast Austria

2009-10-15

Abstract: In this retrospective study, the occurrence and genetic relatedness of methicillin-resistant Staphylococcus aureus (MRSA) ST398 in Austrian MRSA patients was investigated. From 2002 to 2008, 14 MRSA ST398 were detected. First occurrence of MRSA ST398 was already found in 2004. Spa ribotyping assigned 12 isolates to spa type t011 and 1 each to spa type t034 and spa type t1451. Isolated MRSA ST398 was nontypeable by pulsed-field gel electrophoresis (NT-MRSA) using restriction enzyme SmaI; therefore, genotyping was performed using automated repetitive-sequence–based PCR (rep-PCR) on the DiversiLab system. Rep-PCR results assigned 10 (71%) of the 14 MRSA ST398 into 1 cluster with a similarity >95%; there was 1 cluster consisting of 2 isolates with a similarity >99% and 2 unique MRSA ST398 isolates. In conclusion, MRSA ST398 was continuously detected in Southeast Austria; first in 2004 with up to 5 MRSA ST398 isolates in 2008. Automated rep-PCR proved as a reliable technique in determining genetic relatedness of NT-MRSA ST398 and demonstrates clonal spread of MRSA ST398 in the investigated region.

Genotypes, superantigen gene profiles, and presence of exfoliative toxin genes in clinical methicillin-susceptible Staphylococcus aureus isolates

2009-10-15

Abstract: We compared genotype and virulence gene profiles for strains from carriers with autologous invasive infection (n = 56), nasal isolates from matched carriers (n = 108), and invasive strains from noncarriers (n = 34). Superantigen gene profiles and presence of exfoliative toxin genes A and D were associated with clonal complex rather than with invasive disease.

Molecular epidemiology of a Streptococcus pneumoniae serotype 1 outbreak in a Tunisian jail

2009-10-05

Abstract: The microbiologic investigations of an outbreak implicating 17 inmates demonstrated the spread of a pneumococcal serotype 1 clone not only in this jail but also in the area of Tunis. This clone, which is fully susceptible to antibiotics but not included in the heptavalent conjugate vaccine, should be closely monitored.

A novel OXA-10–like β-lactamase is present in different Enterobacteriaceae

Ayelén Porto, Juan Ayala, Gabriel Gutkind, José Di Conza — 2009-10-16

Abstract: OXA 101, a novel OXA-10 like enzyme, was found forming part of a class 1 integron located in a conjugative plasmid in three different species of Enterobacteriaceae. This β-lactamase is related to OXA-35 and OXA-56 and displays a narrow substrate hydrolysis profile.

The effect of fecal turbidity on norovirus detection by reverse transcriptase polymerase chain reaction

Kristie J. Witlox, Theo Karapanagiotidis, Leesa D. Bruggink, John A. Marshall — 2008-10-23

Abstract: A 10-min fecal preparation results in greater specimen turbidity than a 45-min protocol, but reverse transcriptase polymerase chain reaction (RT-PCR) norovirus test sensitivity is essentially the same. Feces processed so that particle size does not exceed approximately 560 nm do not display greater norovirus RT-PCR inhibitory effects than those that have undergone greater purification.

Extent of interlaboratory discrepancies for polyclonal Histoplasma antigen enzyme immunoassay cannot be determined without a large split-sample study

Joann L. Cloud, Kimberly E. Hanson, Sean K. Bauman, Edward R. Ashwood — 2009-11-09

We read with interest the article by and we would like the opportunity to comment. It is necessary to clarify that Immuno-Mycologics, Inc. (IMMY, Norman, OK) does not perform laboratory testing, as implied in the article. IMMY produces standardized reagents for purchase by clinical laboratories that perform testing. ARUP Laboratories, Salt Lake City, UT, purchases reagents from IMMY and, after validating the performance of the reagents in the ARUP reagent laboratory, performs Histoplasma antigen testing at ARUP Laboratories. The key reagent in both the ARUP assay and the test developed by MiraVista Diagnostics (Indianapolis, IN) is a polyclonal antibody generated from rabbits immunized with Histoplasma antigens (). Polyclonal antibodies have the limitation of not detecting a specific antigenic epitope but rather a myriad of antigenic epitopes. The identity of the actual target molecule or molecules that are present in biologic samples from patients with histoplasmosis is not known and may vary over the course of the disease. Monoclonal antibodies have been produced and studied in the past but appear to lack sensitivity ().