Diagnostic Microbiology & Infectious Disease

Determination of capsulation status in Haemophilus influenzae by multiplex polymerase chain reaction

Kevin Lee Nelson, Arnold Lee Smith — 2009-11-23

Abstract: Since the introduction of Haemophilus influenzae type b conjugate vaccines, there have been concerns regarding the emergence of invasive non–type b strains. Serotyping of H. influenzae with commercially available reagents is subjective. Definitive characterization of the capsulation status can be performed by polymerase chain reaction (PCR) amplification of capsular genes. However, PCR amplification of the conserved export locus in the 2 known phylogenic lines of type b strains and detection of serotype conferring genes in each of the 6 serotypes require multiple assays. To rapidly screen multiple isolates, we devised a multiplex method using 15 primers, which produced a serotype-specific, distinct pattern of amplicons with reference-encapsulated H. influenzae. We applied this technique to a panel of 35 clinical isolates that had been serotyped as type a, c, d, e, or f by slide agglutination; 15 strains lacked capsular genes. Conversely, of 69 invasive isolates that were not serotypeable, all but 11 contained capsule genes. We conclude that this technique will be useful in screening recently isolated H. influenzae for capsulation status.

The epidemiology of travelers' diarrhea in Incirlik, Turkey: a region with a predominance of heat-stabile toxin producing enterotoxigenic Escherichia coli

2009-11-11

Abstract: This study evaluated travelers' diarrhea among US military personnel on short-term deployment to Incirlik Air Base, Turkey, from June through September 2002. Upon reporting for care for travelers' diarrhea, subjects were enrolled into the study and completed a series of questionnaires and provided stool specimens for pathogen identification and antimicrobial susceptibility testing. Fifty-three percent of the 202 participating subjects had a pathogen isolated from their stool. Enterotoxigenic Escherichia coli (ETEC) was the predominant pathogen (41%), followed by Campylobacter spp. (12%). The most common ETEC phenotype recovered was stable toxin (ST) CS6 (47% of all ETEC). Most (91.1%) of the cases presented with water diarrhea regardless of isolated pathogen. However, there were some differences in nongastrointestinal symptoms among subjects with Campylobacter spp. All illnesses were well managed with antibiotics with or without loperamide with a median time to the last unformed stool of 9 h (interquartile range, 1–32 h). We found no food or environmental factors associated with a differential risk of infection with a specific pathogen. Travelers' diarrhea among a US military population in and around Incirlik, Turkey, can commonly be attributed to ETEC and Campylobacter spp. The high proportion of ST-only–producing CS6 ETEC in this region highlights the pathogen's worldwide diversity. Future studies of travelers' diarrhea in this population should adapt more novel microbiologic techniques such as polymerase chain reaction and enhanced culture methods to increase the likelihood of identifying pathogenic E. coli.

Rapid detection and differentiation of the exfoliative toxin A-producing Staphylococcus aureus strains based on ϕETA prophage polymorphisms

2009-11-11

Abstract: The exfoliative toxin A (ETA) is encoded by the gene located on Staphylococcus aureus prophages. We have developed a single-reaction multiplex polymerase chain reaction (PCR) assay for rapid and specific detection of various ϕETA prophages of serogroup B responsible for dissemination of eta gene and ETA production in clinical strains. This PCR strategy enabled to classify the ETA-positive strains into 6 groups designated ETA-B1, ETA-B2, ETA-B3, ETA-B4, ETA-B5, and ETA-B6. The method was tested on a diverse set of 101 ETA and/or ETB-positive S. aureus strains isolated in 22 Czech maternity hospitals and 1 Slovak maternity hospital between 1998 and 2009. This novel PCR strategy is reliable in the rapid identification of yet undescribed ETA-converting B prophages and differentiation of the closely related ETA-positive strains, and it is a convenient tool for hospital epidermolytic infection control.

Sequential changes of Legionella antigens and bacterial load in the lungs and urines of a mouse model of pneumonia

2010-03-01

Abstract: Legionella pneumophila is an important cause of community- and hospital-acquired pneumonia. In spite of the introduction of the urinary antigen detection method, Legionella pneumonia may be still underdiagnosed. We performed kinetic and quantitative analysis of diagnostic markers, such as bacterial loads, DNA assays, and antigen titers, in a 28-day time course murine model of L. pneumophila pneumonia. L. pneumophila replicated approximately 100-fold in the lungs of A/J mice in the first 48 h, and then became undetectable on day 14. Unexpectedly, pathogens other than L. pneumophila were consistently recovered from the lungs and livers at the acute phases, although those numbers were far below Legionella loads in the lungs. The peaks of specific antigen titer were observed on 48 h in the lungs, bronchoalveolar lavage (BAL) fluids, and urines and sustained positive even at 28 days after the infection. Especially, the lung homogenates and BAL fluids demonstrated 16 to 64 times higher levels of antigen titer than the urines by the end of observation. Legionella-specific DNA in the lungs was detected by polymerase chain reaction and loop-mediated isothermal amplification methods until 7 and 14 days after the infection, respectively. The inflammatory cytokines, such as tumor necrosis factor (TNF)-α, interleukin 6, and MIP-2, exhibited a peak on the acute phase, whereas the maximal production of high mobility group box 1 in the serum was observed on day 7. These results characterized the kinetic nature of diagnostic markers in L. pneumophila pneumonia. The present data suggested prolonged and compartmentalized deposition of antigen in the lungs, which may have an impact on the diagnosis of L. pneumophila pneumonia, especially in missed cases even after recovery from disease.

Evaluation of a real-time polymerase chain reaction assay for the laboratory diagnosis of giardiasis

2009-11-11

Abstract: A real-time polymerase chain reaction (PCR) assay was evaluated in comparison with the combination of conventional methods (microscopic examination and antigen detection assay) during the period 2006 to 2008 on 771 fecal samples belonging to 386 patients to assess its usefulness for an accurate laboratory diagnosis of giardiasis. The real-time PCR assay detected Giardia intestinalis DNA in 195 samples (106 patients), including 26 samples (21 patients) negative by the conventional assays. Among the 21 patients, in 8 cases, giardiasis was previously diagnosed also by conventional methods in additional samples of the same patients, whereas in 13, it would have been undiagnosed if real-time PCR assay was not used. The real-time PCR assay demonstrated a detection limit of 2 cysts per reaction and 100% specificity and sensitivity compared to conventional methods. A genotype analysis targeting the β-giardin gene allowed to identify 53 samples (23 patients) containing genotype A and 59 samples (45 patients) containing genotype B.

Detection of Histoplasma capsulatum DNA in human samples by real-time polymerase chain reaction

Stephane Simon, Vincent Veron, Rachida Boukhari, Denis Blanchet, Christine Aznar — 2010-03-01

Abstract: The main aim of our study was to determine the added value of real-time polymerase chain reaction (PCR) for the diagnosis of Histoplasma capsulatum in routine biologic practice. No amplification signal was observed with the 18 non-H. capsulatum strains used to test the specificity of the protocol. The sensitivity threshold of the real-time PCR assay was about 10 fg of H. capsulatum DNA per microliter, tested with a 10-fold serial dilution of the positive control. We analyzed 348 human samples submitted for the routine diagnosis of systemic mycosis. Real-time PCR using the TaqMan system was evaluated against direct microscopic examination and culture. Among the 341 samples without PCR inhibition (n = 7), 66 tested positive by culture, whereas 74 tested positive by real-time PCR. Sensitivity of the real-time PCR assay was estimated at 95.4% and specificity at 96.0% with respect to culture, widely considered to be the gold standard method; however, the molecular approach in fact produced better sensitivity and specificity results. Moreover, for the 38 samples that tested negative by direct examination but positive by culture, the culture method took a mean of 31 days longer than the PCR method to generate results. The protocol presented here may be very useful for improving routine histoplasmosis diagnosis.

Lichtheimia hongkongensis sp. nov., a novel Lichtheimia spp. associated with rhinocerebral, gastrointestinal, and cutaneous mucormycosis

2010-03-01

Abstract: Three thermotolerant “Absidia-like” isolates with unique morphologic characteristics, recovered from nasopharyngeal swab of a liver transplant recipient, gastric biopsy of a renal transplant recipient, and skin biopsy of a man with burn, respectively, were characterized. Microscopic examination showed nonseptate hyphae with highly branched sporangiophores. Uniquely, most side branches were circinate, and abundant pleomorphic giant cells with fingerlike projections were observed, characteristics absent from other Absidia/Lichtheimia spp. ITS1-5.8S–ITS2 rRNA gene cluster, partial EF1α gene, and partial β-actin gene sequencing showed that the 3 strains formed a distinct cluster, most closely related to, but distinct from, Lichtheimia corymbifera, Lichtheimia blakesleeana, and Lichtheimia hyalospora. Based on the morphologic and genotypic characteristics, we propose a new species, Lichtheimia hongkongensis sp. nov., to describe this fungus, which caused rhinocerebral, gastrointestinal, and cutaneous mucormycosis, respectively, in 3 patients. A significant proportion of L. corymbifera associated with mucormycosis reported may be L. hongkongensis.

Surface gene mutations of hepatitis B virus among high-risk patients with occult hepatitis B virus infection

2009-11-11

Abstract: Surface gene mutants of hepatitis B virus (HBV) have been reported in a variety of patient groups. Because of limited data regarding these mutations in patients with occult HBV infections; we aimed to determine these mutations among high-risk patients with occult HBV infection. The presence of HBV-DNA was determined in patients with isolated anti-HBc by real-time polymerase chain reaction (PCR). Then, surface gene region was amplified by nested PCR and mutations were analyzed after sequencing. The mutations that resulted in nonfunctional hepatitis B surface antigen (HBsAg) were insertion of single nucleotide in 2 cases, which causes frameshift and single-nucleotide replacement, and premature stop codons at Leu15 and Gly10 in the other 2 cases. Amino acid substitution at amino acid position 207(S207N) was found in the other isolates. Our study suggested that “a” region mutations did not play a major role in HBsAg detection, and other genetic and nongenetic factors may be responsible for failure to detect HBsAg by routine laboratory tests.

Trends in antibiotic susceptibility of bloodstream pathogens in hospitalized patients in France, 1996 to 2007

2009-11-11

Abstract: Nationwide surveys of antimicrobial susceptibility of bacteria isolated from bloodstream infections are required to fit empiric therapy to recent trends and detect emerging resistance. We report the results of a French national prospective survey based on the College of Bacteriology–Virology and Hygiene study group network performed each October during the 1996 to 2007 period, with focus on Enterobacteriaceae (7708 isolates) and Staphylococcus aureus (2271 isolates). The most relevant antimicrobial susceptibilities trends were i) a decrease in fluoroquinolones susceptibility among Enterobacteriaceae (96–90%, P < 0.0001) and Escherichia coli isolates (98–89%, P < 0.0001), respectively, ii) the slight but significant decrease in cefotaxime susceptibility among E. coli (P = 0.016), and iii) the significant increase in gentamicin susceptibility among S. aureus strains (P = 0.016). This survey reports antibiotic susceptibility of bloodstream pathogens in France. The empiric use of fluoroquinolones in severe infections should be cautiously monitored by thorough clinical and microbiologic follow-up.

Spectrum of activity, mutation rates, synergistic interactions, and the effects of pH and serum proteins for fusidic acid (CEM-102)

Douglas J. Biedenbach, Paul R. Rhomberg, Rodrigo E. Mendes, Ronald N. Jones — 2010-03-01

Abstract: Fusidic acid (CEM-102) is a steroidal antimicrobial agent with focused Gram-positive activity that acts by preventing bacterial protein synthesis via interacting with elongation factor G. A collection of 114 wild-type isolates (>80 species) was used to define the contemporary limits of fusidic acid spectrum against Gram-positive and Gram-negative species. Reference broth microdilution and anaerobic agar dilution methods were performed. Modifications of standardized test methods included adding 10% human serum and adjusting the medium pH to 5, 6, and 8. Synergy was assessed by the checkerboard method and time-kill studies. Mutational rates to resistance were determined at 4×, 8×, and 16× MIC. Against Gram-positive pathogens, fusidic acid MIC values ranged from 0.06 to 32 μg/mL with the greatest potency against Staphylococcus aureus, Corynebacterium spp., and Micrococcus luteus (MIC results, 0.25, ≤0.12, and ≤0.5 μg/mL, respectively). Enterococci and streptococci were less susceptible (MIC ranges, 2–8 and 16–32 μg/mL, respectively). Fusidic acid activity against Gram-negative species was more limited (all MIC values, ≥2 μg/mL) except for Empedobacter brevis, Moraxella catarrhalis and Neisseria meningitidis. A 4-fold increase in fusidic acid MIC results was observed when 10% serum was added to the broth. Decreasing medium pH to 5.0 to 6.0 negated the protein binding effects. Among the 8 antimicrobial combinations tested, gentamicin and rifampin enhanced the activity when combined with fusidic acid (no antagonism). Fusidic acid in vitro activity was most improved when combined with rifampin. Single-step mutational rates ranged from 1.2 × 10−6 for 4× MIC to 9.8 × 10−8 for 16× MIC. In conclusion, these in vitro results for fusidic acid tested against contemporary strains confirm a persisting antimicrobial spectrum, especially against staphylococci and some other Gram-positive species.

Susceptibility to tigecycline of isolates from samples collected in hospitalized patients with secondary peritonitis undergoing surgery

2009-12-18

Abstract: Activity of tigecycline against nosocomial secondary peritonitis isolates collected along 18 months in 29 Spanish hospitals was tested by Etest in a central laboratory, considering Food and Drug Administration (FDA)/British Society for Antimicrobial Chemotherapy (BSAC)/European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints. A total of 600 facultative/aerobic isolates (392 Gram negative, 208 Gram positive) and 100 anaerobes were tested. None of the 220 Escherichia coli isolates was resistant to tigecycline (MIC50/MIC90 = 0.25/0.5 μg/mL), with 0.5% (FDA breakpoint) and 3.6% (BSAC/EUCAST breakpoint) intermediate strains. All Extended-spectrum beta-lactamase (ESBL)-producing E. coli isolates (15 strains), all Klebsiella pneumoniae, and Klebsiella oxytoca isolates (42 strains) were susceptible to tigecycline. No isolates resistant to tigecycline were found among Streptococcus viridans, Staphylococcus aureus, and Enterococcus faecium, but 18.9% of Enterococcus faecalis strains were intermediate following BSAC/EUCAST breakpoints. All (but 1) isolates of the Bacteroides fragilis group (n = 45) were tigecycline susceptible, as well as Gram-positive anaerobes. Tigecycline offers an adequate activity profile against isolates from secondary peritonitis when tested by Etest regardless of the breakpoints used for categorization.

Rapid identification of tuberculosis epididymo-orchitis by INNO-LiPA Rif TB and QuantiFERON-TB Gold In Tube tests: case report

Jolanta Paluch-Oleś, Agnieszka Magryś, Ewa Kot, Maria Kozioł-Montewka — 2009-11-09

Abstract: Diagnosis of extrapulmonary tuberculosis (TB) is often missed or delayed because of nonspecific clinical and laboratory findings. Novel detection methods, such as polymerase chain reaction and QuantiFERON®-TB Gold In Tube, can aid in the diagnosis of active extrapulmonary TB. Here, we demonstrate a case of epididymo-orchitis as the sole presentation of TB in a 32-year-old man.

Molecular identification of phaeohyphomycosis due to Alternaria infectoria in a patient with acute myeloid leukemia—a case report

2009-11-06

Abstract: We report the case of a 15-year old with acute myeloid leukemia who developed breakthrough invasive fungal rhinitis. The fungus was identified as Alternaria infectoria by polymerase chain reaction (PCR) and successfully treated by surgical excision and combination antifungal therapy, emphasizing the utility of fungal PCR in timely diagnosis of invasive fungal infections.

Fibrin ring granulomas in Rickettsia typhi infection

2010-01-13

Abstract: We describe a 71-year-old man hospitalized for fever and productive cough. Laboratory investigation showed anemia, thrombocytopenia, elevated transaminases, hyponatremia, and hypoalbuminemia. Computerized tomography of the abdomen, thorax, and sinuses, echocardiography, and a gallium scan did not reveal the source of the fever. The patient remained febrile despite courses of piperacillin–tazobactam/azithromycin and ceftriaxone/vancomycin. A bone marrow biopsy showed fibrin ring granulomas, and 2 rickettsial serologic panels were positive for Rickettsia typhi infection and negative for Q fever. The patient was given doxycycline, and the fever resolved within 48 h. We propose that fibrin ring granulomas also occur in murine typhus.

Plasmid-mediated carbapenem-hydrolyzing enzyme KPC-2 and ArmA 16S rRNA methylase conferring high-level aminoglycoside resistance in carbapenem-resistant Enterobacter cloacae in China

Qiong Wu, Qingzhong Liu, Lizhong Han, Jingyong Sun, Yuxing Ni — 2009-11-11

Abstract: The emergence and spread of carbapenem-resistant Enterobacteriaceae in the world are a major concern. We investigated 5 isolates of Enterobacter cloacae that were resistant to all clinically available antimicrobial agents, except polymyxin B. The MICs of imipenem and aminoglycosides were >32 and >256 mg/L, respectively. All of the isolates produced 5 extended-spectrum beta-lactamase (ESBLs) with pIs of 5.4 (TEM-1), 6.7 (KPC-2), 8.2 (SHV-12), 8.4 (CTX-M-14), and ArmA 16S rRNA methylase. blaKPC-2 was located on a large nonconjugative plasmid, whereas armA was located on another conjugative plasmid. Although carbapenem-resistant Enterobacteriaceae remains rare, the emergence of this group of organism merits monitoring.

Antimicrobial activity of daptomycin tested against Staphylococcus aureus with vancomycin MIC of 2 μg/mL isolated in the United States and European hospitals (2006–2008)

Helio S. Sader, Holly K. Becker, Gary J. Moet, Ronald N. Jones — 2010-03-01

Abstract: We evaluated the activity of daptomycin (minimum inhibitory [MIC] and bactericidal [MBC] concentration) against Staphylococcus aureus strains with elevated (2 μg/mL) vancomycin MIC values. A total of 410 contemporary clinical S. aureus isolates (282 from the United States and 128 from Europe) with vancomycin MIC values of 2 μg/mL were tested by reference broth microdilution method. Vancomycin MBC and the presence of vancomycin-heteroresistant population (heterogeneous vancomycin-intermediate S. aureus [hVISA]) were evaluated in 31 randomly selected strains. Overall, 97.3% of isolates were susceptible to daptomycin (MIC90, 0.5 μg/mL). Daptomycin exhibited potent bactericidal activity with MBC values at the MIC concentration (74.2%) or 1 log2 dilution above the MIC (25.8%). In contrast, vancomycin MBC was ≥32 μg/mL in 12.9% of strains tolerance, and 25.8% of strains tested positive for hVISA (AB BIODISK GRD Etest, Solna, Sweden). In conclusion, S. aureus strains with vancomycin MIC of 2 μg/mL showed high rates of hVISA and vancomycin tolerance. Daptomycin retained potent bactericidal activity against S. aureus with decreased susceptibility to vancomycin.

Epidemiology of pediatric community-acquired bloodstream infections in a children hospital in Paris, France, 2001 to 2008

2010-03-01

Abstract: In 2001 to 2008, we documented 483 cases of pediatric community-acquired bacteremia mostly because of Streptococcus agalactiae (<4 days), Escherichia coli (4 days to 3 months), pneumococci (3 months to 5 years), and Staphylococcus aureus (>5 years). Pneumococcal conjugate vaccination affected the serotype distribution of pneumococcal bacteremia but not its frequency. Serotype 19A represented 12% and 22% of pneumococci in the prevaccine and vaccine periods, respectively.

Antimicrobial susceptibility of multidrug-resistant Streptococcus pneumoniae strains with penicillin MICs of 8 to 32 mg/L

2009-11-27

Abstract: The in vitro activity of 22 antibiotics (including novobiocin) and β-lactam/gentamicin combinations was assessed against 11 multidrug-resistant pneumococcal strains. Among orally administered drugs, only telithromycin, levofloxacin, and linezolid were active against all isolates, but their use is not indicated in pediatrics. Novobiocin could be a potential therapeutic alternative.

Extent of Interlaboratory discrepancies for polyclonal Histoplasma antigen Enzyme imunoassay (EIA) cannot be determined without a large split-sample study (reply)

David S. McKinsey, Joel McKinsey, Noelle Northcutt, Juan Sarria — 2009-11-27

We appreciate the clarification by regarding their Associated Regional and University Pathologists (ARUP) Histoplasma antigen assay that utilizes reagents provided by Immuno-Mycologics, Inc., Norman, OK. However, we differ with a number of opinions expressed in their letter.

Erratum to “Selection of colistin-resistant Acinetobacter baumannii isolates in postneurosurgical meningitis in an intensive care unit with high presence of heteroresistance to colistin” [Diagn Microbiol Infect Dis 65 (2009) 188–191]

2010-03-01

After publication of the abovementioned article, it was discovered that surnames and given names of authors were reversed. Names should be as follows: Carlos Hernan Rodriguez, Karina Bombicino, Gabriela Granados, Marcela Nastro, Carlos Vay, and Angela Famiglietti.