Mercedes Marín

Nocardiosis at the turn of the century.

Nocardia species is an uncommon pathogen that affects both immunosuppressed and immunocompetent patients. The clinical and microbiologic spectrum of nocardiosis has changed recently due to the widespread use of cotrimoxazole prophylaxis, the emergence of new types of immunosuppressed patients, and the improved identification of isolates using molecular techniques. Nocardia asteroides was traditionally considered the predominant organism, and prophylaxis with cotrimoxazole was considered almost universally protective. We conducted the current study to determine the incidence of nocardiosis and its microbiologic and clinical characteristics in a general hospital over the last 12 years. We reviewed the clinical records of all patients in whom Nocardia species was isolated from clinical specimens between 1995 and 2006. Nocardia isolates were identified by standard procedures and by 5&vprime; end 16S rRNA gene polymerase chain reaction (PCR) and sequencing. Susceptibility to cotrimoxazole, minocycline, imipenem, linezolid, and amikacin was determined by the broth microdilution method following the guidelines of the Clinical and Laboratory Standards Institute. The incidence of Nocardia infections did not increase significantly during the study period (0.39/100,000 inhabitants in 1995-1998 and 0.55/100,000 inhabitants in 2003-2006). Nocardia was recovered from 43 patients. Six were considered to be colonized. The colonizing species were N. farcinica, N. nova, and N. asteroides. All colonized patients had severe underlying pulmonary conditions and were treated with antimicrobials (6 patients) or corticosteroids (4 patients). Invasive nocardiosis was diagnosed in 37 patients (86.5% were men, and their mean age was 55.8 ± 17.3 yr). The most common underlying condition in our institution was human immunodeficiency virus (HIV) infection (10 patients; 27%), followed by chronic obstructive pulmonary disease (8 patients; 21.6%), autoimmune diseases (8 patients; 21.6%), solid organ transplantation (7 patients; 18.9%), and cancer (4 patients; 10.8%). The most important risk factor for nocardiosis was corticosteroid administration (23 patients; 62.2%). Nocardiosis affected the lungs in 26 cases (70.3%), the skin in 3 cases (8.1%), and the central nervous system in 2 cases (5.4%). It was disseminated in 5 cases (13.5%) and caused otomastoiditis in 1 (2.7%). The species identified were N. cyriacigeorgica (32.4%), N. farcinica (24.3%), N. otitidiscaviarum (10.8%), N. veterana (8.1%), N. nova (5.4%), N. abscessus (5.4%), N. asiatica (2.7%), N. beijingensis (2.7%), N. brasiliensis (2.7%), N. carnea (2.7%), and Nocardia species (2.7%). Linezolid and amikacin were uniformly active against all the isolates, whereas 29.7% of isolates showed intermediate susceptibility to minocycline (minimum inhibitory concentration = 2 mg/L), 10.8% were resistant to cotrimoxazole, and 5.4% were resistant to imipenem. Nocardiosis occurred while the patients were on cotrimoxazole prophylaxis in 8 cases (21.6%). The strains isolated from these patients were susceptible to cotrimoxazole in 5 cases (62.5%) and resistant in 3 (37.5%). Overall, 13 patients died (35.1%); related mortality was 21.6% (8 patients). We conclude that HIV infection has become the most common underlying condition for invasive nocardiosis in our institution, followed by chronic lung disease. Previous use of corticosteroids was the main risk factor and was present in more than half the patients. New species of Nocardia have been identified, and administration of cotrimoxazole prophylaxis should no longer be considered highly reliable protection against nocardiosis. Larger studies of nocardiosis are required to better identify risk factors associated with mortality, and alternative and more effective methods of prevention must be developed. Abbreviations: AIDS = acquired immunodeficiency syndrome, CLSI = Clinical and Laboratory Standards Institute, CMV = cytomegalovirus, CNS = central nervous system, COPD = chronic obstructive pulmonary disease, CT = computed tomography, HAART = highly active antiretroviral therapy, HIV = human immunodeficiency virus, MIC = minimum inhibitory concentration, PCR = polymerase chain reaction.

Metronidazole Resistance in Clostridium difficile Is Heterogeneous

ABSTRACT At our institution, the prevalence of clinical isolates of Clostridium difficile with resistance to metronidazole is 6.3%. We observed that initial metronidazole MICs of 16 to 64 mg/liter against toxigenic, primary fresh C. difficile isolates, as determined by agar dilution, decreased to 0.125 mg/liter after the isolates were thawed. In this study, we examined the possibility of heterogeneous or inducible resistance. Totals of 14 metronidazole-resistant and 10 metronidazole-susceptible clinical isolates of toxigenic C. difficile were studied. The isolates were investigated for the presence of nim genes by PCR. After the isolates were thawed, susceptibility testing was done by agar dilution, by disc diffusion using a 5-μg metronidazole disc, and by the Etest method. An experiment for determining the effect of prolonged exposure to metronidazole was applied to all resistant isolates and to susceptible control strains. None of the isolates presented the nim genes. All initially metronidazole-resistant C. difficile isolates became susceptible after thawing; however, they presented slow-growing subpopulations within the inhibition zones of both the disk and the Etest strip. All metronidazole-susceptible isolates remained homogeneously susceptible by both methods. After prolonged exposure in vitro to metronidazole, no zone of inhibition was found around the 5-μg disk in any of the metronidazole-resistant isolates, and the MICs as determined by the Etest method ranged from 0.125 to >256 mg/liter, with colonies growing inside the inhibition zone. Our results indicate that (i) resistance to metronidazole was not due to the presence of nim genes, (ii) resistance to metronidazole in toxigenic C. difficile isolates is heterogeneous, and (iii) prolonged exposure to metronidazole can select for in vitro resistance. We recommend routine performance of the disk diffusion method (5-μg metronidazole disk) with primary fresh C. difficile isolates in order to ensure that metronidazole-heteroresistant populations do not go undetected.

Successful Outcome of Scedosporium apiospermum Disseminated Infection Treated with Voriconazole in a Patient Receiving Corticosteroid Therapy

A disseminated Scedosporium apiospermum infection was diagnosed in a woman with severe asthma and treated with corticosteroids. This fungi is resistant to fluconazole and amphotericin B. The infection was refractory to itraconazole, but responded successfully to voriconazole. A review of the literature is provided.

Molecular diagnosis of infective endocarditis by real-time broad-range polymerase chain reaction (PCR) and sequencing directly from heart valve tissue

Traditionally, infective endocarditis (IE) has been microbiologically diagnosed by blood cultures or serology. However, conventional microbiologic methods do not always provide an etiologic diagnosis. We conducted the current study to evaluate the usefulness of a universal real-time polymerase chain reaction (PCR) of the 16S rRNA gene followed by sequencing for the diagnosis of IE in explanted heart valve tissue (HV) as part of the routine of a clinical microbiology laboratory, and to compare it with conventional culture of blood or HV. We prospectively analyzed 177 HV samples by universal PCR and sequencing: 48 were from 35 patients with definite IE and 129 were from 120 patients without IE. Specific PCR tests were used when necessary to confirm broad-range PCR results. For the 35 patients with IE, all of the HV samples except for 2 from the same patient gave positive PCR results. The microorganisms identified matched those isolated by blood culture in 31 cases. The other 3 patients had negative blood culture IE, but PCR made possible the detection of Tropheryma whipplei, Bartonella quintana, and Streptococcus gallolyticus. For the negative control group, universal PCR was completely negative in 123 of the 129 samples. Sensitivity, specificity, and negative and positive predictive values of this real-time PCR method were 96%, 95.3%, 98.4%, and 88.5%, respectively, for the diagnosis of IE, using the Duke criteria to define IE and using blood culture results to identify etiologic microorganisms. Conventional HV culture correlated poorly with blood cultures and molecular techniques, and frequently represented tissue contamination resulting from valve handling. Our universal PCR method has proved to be more sensitive, specific, and rapid than conventional culture methods, and should therefore be included as a new major Duke criterion for the diagnosis of IE. According to the results of the current study, this technique should be used to supplement blood and HV culture. Conventional HV cultures are frequently responsible for false-positive and false-negative results, and are not always useful to establish the etiology of IE. Abbreviations: HV = heart valve tissue, IE = infective endocarditis, PCR = polymerase chain reaction.

Methicillin-susceptible Staphylococcus aureus endocarditis isolates are associated with clonal complex 30 genotype and a distinct repertoire of enterotoxins and adhesins.

BACKGROUND Using multinational collections of methicillin-susceptible Staphylococcus aureus (MSSA) isolates from infective endocarditis (IE) and soft tissue infections (STIs), we sought to (1) validate the finding that S. aureus in clonal complex (CC) 30 is associated with hematogenous complications and (2) test the hypothesis that specific genetic characteristics in S. aureus are associated with infection severity. METHODS IE and STI isolates from 2 cohorts were frequency matched by geographic origin. Isolates underwent spa typing to infer CC and multiplex polymerase chain reaction for presence of virulence genes. RESULTS 114 isolate pairs were genotyped. IE isolates were more likely to be CC30 (19.5% vs 6.2%; P = .005) and to contain 3 adhesins (clfB, cna, map/eap; P < .0001 for all) and 5 enterotoxins (tst, sea, sed, see, and sei; P ≤ .005 for all). CC30 isolates were more likely to contain cna, tst, sea, see, seg, and chp (P < .05 for all). CONCLUSIONS MSSA IE isolates were significantly more likely to be CC30 and to possess a distinct repertoire of virulence genes than MSSA STI isolates from the same region. The genetic basis of this association requires further study.

In Vitro Activity of Ramoplanin against Clostridium difficile, Including Strains with Reduced Susceptibility to Vancomycin or with Resistance to Metronidazole

ABSTRACT We evaluated the in vitro activity of ramoplanin, an antimicrobial compound that inhibits cell wall synthesis by acting at the level of lipid intermediate formation, against Clostridium difficile. We included strains with reduced susceptibilities to vancomycin (vancomycin-intermediate [Vani] strains) or with resistance to metronidazole (Mtzr), in order to assess the potential utility of ramoplanin for the treatment of C. difficile-associated diarrhea. We tested the activity of ramoplanin against a total of 105 nonduplicate clinical isolates of toxigenic C. difficile, including 8 Vani isolates and 6 Mtzr isolates, obtained from our laboratory. Ramoplanin was active against all strains tested at concentrations ranging from 0.03 to 0.5 μg/ml (MICs at which 50 and 90% of isolates were inhibited, 0.25 μg/ml; geometric mean MIC, 0.22 μg/ml). All isolates, independently of their levels of susceptibility to vancomycin or metronidazole, were considered susceptible to ramoplanin (MICs, ≤0.5 μg/ml).

Role of Universal 16S rRNA Gene PCR and Sequencing in Diagnosis of Prosthetic Joint Infection

ABSTRACT The etiological diagnosis of prosthetic joint infection (PJI) requires the isolation of microorganisms from periprosthetic samples. Microbiological cultures often yield false-positive and false-negative results. 16S rRNA gene PCR combined with sequencing (16SPCR) has proven useful for diagnosing various infections. We performed a prospective study to compare the utility of this approach with that of culture to diagnose PJI using intraoperative periprosthetic samples. We analyzed 176 samples from 40 patients with PJI and 321 samples from 82 noninfected patients using conventional culture and 16SPCR. Three statistical studies were undertaken following a previously validated mathematical model: sample-to-sample analysis, calculation of the number of samples to be studied, and calculation of the number of positive samples necessary to diagnose PJI. When only the number of positive samples is taken into consideration, a 16SPCR-positive result in one sample has good specificity and positive predictive value for PJI (specificity, 96.3%; positive predictive value, 91.7%; and likelihood ratio [LR], 22), while 3 positive cultures with the same microorganism are necessary to achieve similar specificity. The best combination of results for 16SPCR was observed when 5 samples were studied and the same microorganism was detected in 2 of them (sensitivity, 94%; specificity, 100%; and LR, 69.62). The results for 5 samples with 2 positive cultures were 96% and 82%, respectively, and the likelihood ratio was 1.06. 16SPCR is more specific and has a better positive predictive value than culture for diagnosis of PJI. A positive 16SPCR result is largely suggestive of PJI, even when few samples are analyzed; however, culture is generally more sensitive.

Campylobacter bacteremia: clinical characteristics, incidence, and outcome over 23 years.

Campylobacter is a very rare cause of bloodstream infection, although it has been found relatively frequently in patients infected with human immunodeficiency virus (HIV). The impact of highly active antiretroviral therapy (HAART) and new forms of immunosuppression on the incidence of Campylobacter bacteremia has not been sufficiently assessed. In this study we analyzed the incidence and microbiologic and clinical characteristics of Campylobacter bacteremia over 23 years. We reviewed the clinical records of all patients who had Campylobacter bacteremia from 1985 to 2007. Available strains were reidentified using universal polymerase chain reaction (PCR). During the study period, there were 71 episodes of Campylobacter bacteremia in 63 patients (0.24% of all bloodstream infections), and the incidence remained stable (mean, 0.06/1000 admissions per year and 0.47/100,000 inhabitants per year). Median age was 52 years (interquartile range, 31.25-72.5 yr), and 82% of patients were male. The underlying conditions included liver disease (21/64, 32.8%), HIV infection (15/64, 23.4%), malignancy (7/64, 10.9%), solid organ transplantation (2/64, 3%), hypogammaglobulinemia (10/64, 15.6%), and other (18/64, 31.2%). Twelve patients shared more than 1 underlying condition. Campylobacter bacteremia was community acquired in 81% of the episodes. The origin of the bloodstream infection was abdominal (43.5%), primary (26%), or extraintestinal (31%: respiratory 15%, cellulitis 4.8%, urinary 8%, other 3%). C jejuni was recovered in 66% of cases, C fetus in 19%, and C coli in 12%. Universal PCR was performed on 14 available strains. Molecular and conventional identification matched in 8 isolates. In contrast, molecular methods classified as C fetus (n = 2) and C jejuni (n = 1) 3 strains formerly identified only to genus level as Campylobacter species. In another 3 isolates, molecular identification was not consistent with the phenotypic identification (C fetus identified as C jejuni). Complications appeared in 23.9% of patients. Quinolone resistance was observed in 50% of the isolates. Only 37.8% of patients received appropriate empirical therapy. Mortality was 16.4%, although it was higher in HIV-infected patients than uninfected patients (33% vs. 10%; p = 0.04), in cases of hospital-acquired Campylobacter bacteremia compared with community-acquired cases (38.5% vs. 9.4%; p = 0.02), and in the presence of complications compared with patients without complications (100% vs. 0%; p < 0.001). The incidence of recurrence was 5% (3 patients with humoral immunodeficiency). There was a higher proportion of HIV-infected patients among patients with Campylobacter bacteremia in the pre-HAART era (1985-1996) than in the HAART era (1997-2007)-27.5% (11/40) vs. 14.3% (4/28)-although the difference was not statistically significant. Debilitating diseases such as chronic obstructive pulmonary disease emerged as predisposing conditions in the HAART era (0% before HAART era vs. 14.3% in HAART era; p = 0.032). Campylobacter bacteremia is no longer a significant disease of HIV-positive patients on HAART, but often affects other immunocompromised patients as well. Campylobacter bacteremia has an extraintestinal origin in as many as 31% of cases, and humoral immunodeficiency must be sought in patients with recurrent episodes. Quinolones should not be considered for empirical therapy. Abbreviations: AIDS = acquired immunodeficiency syndrome, ARIMA = autoregressive integrated moving average test, BSI = bloodstream infection, COPD = chronic obstructive pulmonary disease, HAART = highly active antiretroviral therapy, HIV = human immunodeficiency virus, IQR = interquartile range, PCR = polymerase chain reaction, rRNA = ribosomal ribonucleic acid.

Comparison of Three Commercial Methods for Rapid Detection of Clostridium difficile Toxins A and B from Fecal Specimens

ABSTRACT Three rapid enzyme immunoassays (X/pect Clostridium difficile Toxin A/B test, Wampole Tox A/B Quik Chek, and ImmunoCard Toxins A&B) were compared for the diagnosis of Clostridium difficile infection. Of the 367 stool specimens tested, 102 (27.8%) were positive for toxigenic C. difficile when a combination of direct cytotoxicity assay and cytotoxic culture was used as the gold standard. Sensitivity/specificity values were 49.0%/95.8%, 54.9%/95.5%, and 66.7%/95.1%, respectively. The median times to test five stool specimens were 28, 30, and 24 min, respectively. The ImmunoCard test was the quickest and most sensitive test of the three enzyme immunoassays evaluated.

The undiagnosed cases of Clostridium difficile infection in a whole nation: where is the problem?

Underdiagnosis of Clostridium difficile infection (CDI) because of lack of clinical suspicion or the use of non-sensitive diagnostic techniques is a known problem whose real magnitude has not yet been quantified. In order to estimate the extent of this underdiagnosis, we performed C. difficile cultures on all unformed stool specimens sent-irrespective of the type of request-to a series of laboratories in Spain on a single day. The specimens were cultured, and isolates were characterized at a central reference laboratory. A total of 807 specimens from 730 patients aged ≥ 2 years were selected from 118 laboratories covering 75.4% of the Spanish population. The estimated rate of hospital-acquired CDI was 2.4 episodes per 1000 admissions or 3.8 episodes per 10,000 patient-days. Only half of the episodes occurred in patients hospitalized for >2 days. Two of every three episodes went undiagnosed or were misdiagnosed, owing to non-sensitive diagnostic tests (19.0%) or lack of clinical suspicion and request (47.6%; mostly young people or non-hospitalized patients). The main ribotypes were 014/020 (20.5%), 001 (18.2%), and 126/078 (18.2%). No ribotype 027 strains were detected. Strains were fully susceptible to metronidazole and vancomycin. CDI was underdiagnosed in diarrhoeic stools in a high proportion of episodes, owing to the use of non-sensitive techniques or lack of clinical suspicion, particularly in people aged <65 years or patients with community-acquired diarrhoea. C. difficile toxins should be routinely sought in unformed stools of any origin sent for microbiological diagnosis. The ribotype 027 clone has not yet disseminated in Spain.