Andre Dascal

Host and Pathogen Factors for Clostridium difficile Infection and Colonization

BACKGROUND Clostridium difficile infection is the leading cause of health care-associated diarrhea, and the bacterium can also be carried asymptomatically. The objective of this study was to identify host and bacterial factors associated with health care-associated acquisition of C. difficile infection and colonization. METHODS We conducted a 15-month prospective study in six Canadian hospitals in Quebec and Ontario. Demographic information, known risk factors, potential confounding factors, and weekly stool samples or rectal swabs were collected. Pulsed-field gel electrophoresis (PFGE) was performed on C. difficile isolates to determine the genotype. Levels of serum antibodies against C. difficile toxins A and B were measured. RESULTS A total of 4143 patients were included in the study; 117 (2.8%) and 123 (3.0%) had health care-associated C. difficile infection and colonization, respectively. Older age and use of antibiotics and proton-pump inhibitors were significantly associated with health care-associated C. difficile infection. Hospitalization in the previous 2 months; use of chemotherapy, proton-pump inhibitors, and H(2) blockers; and antibodies against toxin B were associated with health care-associated C. difficile colonization. Among patients with health care-associated C. difficile infection and those with colonization, 62.7% and 36.1%, respectively, had the North American PFGE type 1 (NAP1) strain. CONCLUSIONS In this study, health care-associated C. difficile infection and colonization were differentially associated with defined host and pathogen variables. The NAP1 strain was predominant among patients with C. difficile infection, whereas asymptomatic patients were more likely to be colonized with other strains. (Funded by the Consortium de Recherche sur le Clostridium difficile.).

Development of mupirocin resistance among methicillin-resistant Staphylococcus aureus after widespread use of nasal mupirocin ointment.

All methicillin-resistant Staphylococcus aureus (MRSA) strains isolated from colonized or infected patients in a 625-bed public teaching hospital during an epidemic, and for 3 years thereafter, underwent susceptibility testing to mupirocin. Mupirocin resistance among MRSA increased markedly over this period (1990, 2.7%; 1991, 8.0%; 1992, 61.5%; 1993, 65%) in association with increased use of mupirocin ointment as an adjunct to infection control measures.

A Portrait of the Geographic Dissemination of the Clostridium difficile North American Pulsed-Field Type 1 Strain and the Epidemiology of C. difficile-Associated Disease in Québec

BACKGROUND An increase in the incidence and severity of Clostridium difficile-associated disease in Québec and the United States has been associated with a hypervirulent strain referred to as North American pulsed-field type 1 (NAP1)/027. METHODS In 2005, a prospective study was conducted in 88 Québec hospitals, and 478 consecutive nosocomial isolates of C. difficile were obtained. The isolates were subjected to pulsed-field gel electrophoresis (PFGE) typing, antimicrobial susceptibility testing, and detection of binary toxin genes and tcdC gene deletion. Data on patient age and occurrence of complications were collected. RESULTS PFGE typing of 478 isolates of C. difficile yielded 61 PFGE profiles. Pulsovars A (57%), B (10%), and B1 (8%) were predominant. The PFGE profile of pulsovar A was identical to that of strain NAP1. It showed 67% relatedness with 15 other PFGE patterns, among which 11 had both binary toxin genes and a partial tcdC deletion but different antibiotic susceptibility profiles. Pulsovars B and B1 were identical to strain NAP2/ribotype 001. In hospitals showing a predominant clonal A or B-B1 PFGE pattern, incidence of C. difficile-associated disease was 2 and 1.3 times higher, respectively, than in hospitals without any predominant clonal PFGE pattern. Severe disease was twice as frequent among patients with strains possessing binary toxin genes and tcdC deletion than among patients with strains lacking these virulence factors. CONCLUSIONS This study helped to quantify the impact of strain NAP1 on the incidence and severity of C. difficile-associated disease in Québec in 2005. The identification of the geographic dissemination of this predominant strain may help to focus regional infection-control efforts.

Natural Killer Cell Function in Patients With Acquired Immunodeficiency Syndrome and Related Diseases

This review describes current knowledge of changes in natural killer (NK) cell function in acquired immunodeficiency syndrome (AIDS)‐related disorders, vis‐à‐vis associated abnormalities in NK cytolytic function, NK cell subset distribution, NK cytopathology, and lymphokine regulation. NK cells, which are closely associated with large granular lymphocytes, are spontaneously cytotoxic to tumor and virally infected targets. As such, they may play a role in natural resistance to human immunodeficiency virus type 1 (HIV‐1)‐associated disorders and other opportunistic infections. Yet, peripheral blood NK activity is frequently reduced in patients with HIV‐1‐induced disease. NK cells are heterogeneous both with respect to their expression of serologically defined membrane antigens and functional activity. In AIDS‐related syndromes, there appears to be a diminution of the NK pool (CD16+ cells) involved in cytolytic function, while there is an elevation of the NK pool that coexpresses NK (Leu 7+) and T (CD8+) cell markers, which show little or no involvement in cytolytic function. The impairment of in vitro NK function is not associated with a reduced frequency of lytic conjugates of effectors and target cells nor with the recycling capacity of these effector cells but rather is associated with defects in the NK cell lytic machinery following formation of such conjugates. NK cells in AIDS patients show an impairment in effector cell microtubule rearrangement following target cell interaction. The causes of NK cell dysfunction in AIDS‐related disorders remain unknown. NK cells do not appear to express the CD4 epitope of the HIV receptor, nor have they been demonstrated to be susceptible to infection by HIV‐1. There appears to be a preponderance of immature NK cells and a lymphokine imbalance in patients with HIV‐1 associated disease. Interleukin‐2 can partially restore diminished in vitro NK function. Elucidation of the involvement of the NK compartment in natural resistance to HIV‐1 merits further investigation.

Resistance to antiretroviral drugs in patients with primary HIV-1 infection

The widespread use of antiretroviral agents (ARVs) and the growing occurrence of HIV strains resistant to these drugs have given rise to serious concerns regarding the transmission of resistant viruses to newly infected persons. Plasma viral RNA from 80 individuals newly infected between 1997 and 1999 was genotyped by automated sequencing to analyze the profile of viruses resistant to nucleoside and non-nucleoside reverse transcriptase inhibitors (NRTIs and NNRTIs) and to protease inhibitors (PIs). The prevalence of mutations that conferred primary resistance to PIs (L10I, D30Y, V82A, L90M) was 15% of the cohort. RT genotypic variants, associated with high-level resistance to ARVs, were observed in 21% of individuals, including NRTI, NNRTI and multidrug (MDR) resistance in 6, 5, and 10% of cases, respectively. The phenotypic susceptibility of viral isolates to ARVs was also assayed and showed transmission of high-level resistance to ZDV, 3TC, and PIs in those individuals with MDR. The transmission of drug-resistant HIV genotypic variants is a serious problem that merits further attention by public health officials, virologists, and clinicians.

Growth curves of Staphylococcus aureus, Candida albicans, and Moraxella osloensis in propofol and other media

Propofol, 2,6 diisopropylphenol, in an emulsion formulation (Diprivan), has been associated with postsurgical infections caused by Staphylococcus aureus, Moraxella osloensis and Candida albicans. These organisms were individually inoculated into each of the following media: (1) the emulsion preparation of propofol, (2) Intralipid 10%, (3) pure 2,6 diisopropylphenol, and (4) trypticase soy broth (TSB). The organisms were incubated and subcultured hourly for eight hours at room temperature. Propofol supported the growth of all three organisms, but for S. aureus and M. osloenis, the growth rate was slower in propofol than in TSB (P < 0.05). There was no difference between the growth rate of any organism in propofol than in Intralipid 10%. The authors conclude that propofol, in the emulsion formulation, supports bacterial growth and, therefore, must be prepared for administration in an aseptic manner. Also, by administering propofol soon after preparation, the risk of introduction of a significant inoculum to the patient will be reduced.RésuméLutilisation du propofol, ou 2–6 diisopropylphénol, sous forme d’émulsion (Diprivan) a récemment éte associée à des infections postopératoires à Staphylococcus aureus, Moraxella osloensis et Candida albicans. Dans la présente étude, ces trois micro-organismes sont placés séparément dans chacun des milieux de cultures suivants: 1) propofol en émulsion, 2) Intralipide 10%, 3) 2–6 diisoprophylphénol pur et 4) bouillon trypticase soja (BTS). Ces milieux colonisés sont incubés pendant huit heures à la température ambiante. Des cultures de chacun des milieux sont répétées à chaque heure. Les résultats des cultures démontrent que le propofol permet la croissance de chacun des trois organismes. Cependant, la croissance du S. aureus et du M. osloensis est plus lente dans le propofol que dans le BTS (P < 0,05). La croissance des trois organismes dans le propofol est comparable à celle retrouvée dans l’Intralipide 10%. Le 2–6 diisopropylphénol pur ne permet aucune croissance des organismes. Les auteurs concluent que le propofol sous forme d’émulsion est un milieu de culture potentiel pour différents micro-organismes et que toute manipulation de ce produit pour utilisation clinique doit se faire de façon aseptique. De plus, on recommande d’administrer le propofol peu de temps apres l’aspiration dans une seringue afin de diminuer le risque d’infection chez les patients qui doivent recevoir ce médicament.

Fourteen-Genome Comparison Identifies DNA Markers for Severe-Disease-Associated Strains of Clostridium difficile

ABSTRACT Clostridium difficile is a common cause of infectious diarrhea in hospitalized patients. A severe and increased incidence of C. difficile infection (CDI) is associated predominantly with the NAP1 strain; however, the existence of other severe-disease-associated (SDA) strains and the extensive genetic diversity across C. difficile complicate reliable detection and diagnosis. Comparative genome analysis of 14 sequenced genomes, including those of a subset of NAP1 isolates, allowed the assessment of genetic diversity within and between strain types to identify DNA markers that are associated with severe disease. Comparative genome analysis of 14 isolates, including five publicly available strains, revealed that C. difficile has a core genome of 3.4 Mb, comprising ∼3,000 genes. Analysis of the core genome identified candidate DNA markers that were subsequently evaluated using a multistrain panel of 177 isolates, representing more than 50 pulsovars and 8 toxinotypes. A subset of 117 isolates from the panel had associated patient data that allowed assessment of an association between the DNA markers and severe CDI. We identified 20 candidate DNA markers for species-wide detection and 10,683 single nucleotide polymorphisms (SNPs) associated with the predominant SDA strain (NAP1). A species-wide detection candidate marker, the sspA gene, was found to be the same across 177 sequenced isolates and lacked significant similarity to those of other species. Candidate SNPs in genes CD1269 and CD1265 were found to associate more closely with disease severity than currently used diagnostic markers, as they were also present in the toxin A-negative and B-positive (A-B+) strain types. The genetic markers identified illustrate the potential of comparative genomics for the discovery of diagnostic DNA-based targets that are species specific or associated with multiple SDA strains.

Predictors of asymptomatic Clostridium difficile colonization on hospital admission

BACKGROUND Clostridium difficile (CD) is the leading cause of health care-associated diarrhea and can result in asymptomatic carriage. Rates of asymptomatic CD colonization on hospital admission range from 1.4%-21%. The objective of this study was to evaluate host and bacterial factors associated with colonization on admission. METHODS The Consortium de recherche québécois sur le Clostridium difficile study provided data for analysis, including demographic information, known risk factors, and potential confounding factors, prospectively collected for 5,232 patients from 6 hospitals in Quebec and Ontario over 15 months from 2006-2007. Stool or rectal swabs were obtained for culture on admission. Pulsed-field gel electrophoresis was performed on the isolates. The presence of antibody against CD toxins A and B was measured. RESULTS There were 212 (4.05%) patients colonized with CD on admission, and 5,020 patients were not colonized with CD. Multivariate logistic regression analysis showed that hospitalization within the last 12 months, use of corticosteroids, prior CD infection, and presence of antibody against toxin B were associated with colonization on admission. Of patients colonized on admission, 79.4% had non-NAP1, non-NAP2 strains. CONCLUSION There are identifiable risk factors among asymptomatic CD carriers that could serve in their detection and provide a basis for targeted screening.

Highly diversified multiply drug-resistant HIV-1 quasispecies in PBMCs: a case report

BackgroundAlthough drug resistance is a major challenge in HIV therapy, the effect of drug resistance mutations on HIV evolution in vivo is not well understood. We have now investigated genetic heterogeneity in HIV-1 by performing drug resistance genotyping of the PR-RT regions of viruses derived from plasma and peripheral blood mononuclear cells (PBMCs) of a single patient who had failed multiple regimens of anti-retroviral therapy.ResultsPatterns of drug resistance mutations showed that the viral populations in PBMCs were more heterogeneous than in plasma. Extensive analysis of HIV from infected PBMCs in this patient showed that high-level diversity existed among 109 cloned PR-RT sequences and that the majority of mutations were related to drug resistance. Moreover, the PBMCs included archival species that reflected the treatment history of the patient while those in plasma were mainly related to the most recent treatment. Some of the proviral clones contained single or multiple mutations in various combinations. Approximately eighteen percent of the proviral clones derived from infected PBMCs were defective, i.e. 5.5% contained single nucleotide deletions (frameshift mutations) and 12.8% encoded in-frame stop codons (nonsense mutations). Amino acid substitutions in PR and the polymerase region of RT occurred in 12–15% of cases but were much less frequent in the RNase H region of RT, which might not have been under drug selection pressure.ConclusionSelective drug pressure can yield multiple drug-resistant quasispecies that include archival and replication-incompetent species in PBMC reservoirs.

Evidence of viremia in 2 cases of severe pandemic influenza A H1N1/09

The recent pandemic of the 2009 pandemic influenza A (H1N1) infrequently caused severe disease. We describe 2 cases of 2009 H1N1 influenza with rapid progression resulting in respiratory failure and need for prolonged intensive care support. Real-time polymerase chain reaction amplification for influenza A (using a Centers for Disease Control and Prevention protocol) and the 2009 H1N1 influenza (using an in-house protocol) was performed on serial respiratory and serum specimens from both patients collected over 3 weeks. Both patients repeatedly demonstrated 2009 H1N1 influenza in respiratory specimens. Evidence of influenza A viremia was also detected in both cases, although it was confirmed as 2009 H1N1 influenza in only one. The presence of viremia in cases of severe 2009 H1N1 influenza has potential prognostic and therapeutic implications. Detection of viremia may be useful as a predictive marker for severe disease. Antiviral agents with low serum levels may be ineffective if administered to patients with influenza viremia.