Linoj Samuel

Evaluation of a Microarray-Based Assay for Rapid Identification of Gram-Positive Organisms and Resistance Markers in Positive Blood Cultures

ABSTRACT Rapid identification of pathogens directly from positive blood cultures can play a major role in reducing patient mortality rates. We evaluated the performance of the Verigene Gram-Positive Blood Culture (BC-GP) assay (Nanosphere Inc., Northbrook, IL) for detection of commonly isolated Gram-positive organisms as well as associated resistance markers from positive blood cultures. Positive blood cultures (VersaTREK; Trek Diagnostic Systems, Independence, OH) from 203 patients with Gram-positive organism infections were analyzed using the BC-GP assay within 12 h for the detection of 12 different organisms, including staphylococci, streptococci, and enterococci, as well as for the presence of 3 resistance markers (mecA, vanA, and vanB). Results were compared to those of routine laboratory methods for identification and susceptibility testing. For identification of organisms and detection of resistance markers in 178 monomicrobial positive blood cultures, the BC-GP assay showed 94% and 97% concordance, respectively, with routine methods. After 25 polymicrobial cultures were included, the results showed 92% and 96% agreement for identification and resistance markers, respectively, for a total of 203 positive cultures. In 6/25 polymicrobial cultures, at least 1 isolate was not detected. Concordance levels for detection of major pathogens such Staphylococcus aureus (n = 45) and enterococci (n = 19) were 98% and 95%, respectively. Agreement levels for detection of resistance markers such as mecA and vanA/B were 92% and 100%, respectively. The BC-GP assay is capable of providing rapid identification of Gram-positive cocci as well as detection of resistance markers directly from positive blood cultures at least 24 to 48 h earlier than conventional methods.

Identification of Gram-Negative Bacteria and Genetic Resistance Determinants from Positive Blood Culture Broths by Use of the Verigene Gram-Negative Blood Culture Multiplex Microarray-Based Molecular Assay

ABSTRACT Bloodstream infection is a serious condition associated with significant morbidity and mortality. The outcome of these infections can be positively affected by the early implementation of effective antibiotic therapy based on the identification of the infecting organism and genetic markers associated with antibiotic resistance. In this study, we evaluated the microarray-based Verigene Gram-negative blood culture (BC-GN) assay in the identification of 8 genus or species targets and 6 genetic resistance determinants in positive blood culture broths. A total of 1,847 blood cultures containing Gram-negative organisms were tested using the BC-GN assay. This comprised 729 prospective fresh, 781 prospective or retrospective frozen, and 337 simulated cultures representing 7 types of aerobic culture media. The results were compared to those with standard bacterial culture and biochemical identification with nucleic acid sequence confirmation of the resistance determinants. Among monomicrobial cultures, the positive percent agreement (PPA) of the BC-GN assay with the reference method was as follows; Escherichia coli, 100%; Klebsiella pneumoniae, 92.9%; Klebsiella oxytoca, 95.5%; Enterobacter spp., 99.3%; Pseudomonas aeruginosa, 98.9%; Proteus spp., 100%; Acinetobacter spp., 98.4%; and Citrobacter spp., 100%. All organism identification targets demonstrated >99.5% negative percent agreement (NPA) with the reference method. Of note, 25/26 cultures containing K. pneumoniae that were reported as not detected by the BC-GN assay were subsequently identified as Klebsiella variicola. The PPA for identification of resistance determinants was as follows; bla CTX-M, 98.9%; bla KPC, 100%; bla NDM, 96.2%; bla OXA, 94.3%; bla VIM, 100%; and bla IMP, 100%. All resistance determinant targets demonstrated >99.9% NPA. Among polymicrobial specimens, the BC-GN assay correctly identified at least one organism in 95.4% of the broths and correctly identified all organisms present in 54.5% of the broths. The sample-to-result processing and automated reading of the detection microarray results enables results within 2 h of culture positivity.

Evaluation of a Real-Time PCR Assay for the Detection of the Klebsiella pneumoniae Carbapenemase Genes in Microbiological Samples in Comparison With the Modified Hodge Test

Transfer of the bla(KPC) genes encoding the Klebsiella pneumoniae carbapenemase (KPC) are increasingly responsible for emerging carbapenem resistance. The modified Hodge test (MHT) is recommended for the detection of KPC. We compared MHT with a real-time polymerase chain reaction (PCR) assay targeting common subtypes of bla(KPC), using previously described forward and reverse primer sequences. The PCR product was detected using SYBR Green (Applied Biosystems, Foster City, CA) and confirmed by melt curve analysis. PCR was positive in 96% (52/54) of isolates that were MHT+, 90% (28/31) of MHT- isolates were PCR-, and the results were strongly correlated (P = .0001; Fisher exact test). The PCR assay is a sensitive, specific, and rapid test for detecting bla(KPC) genes. It could help optimize patient care by reducing the time taken to institute appropriate antimicrobial therapy and so help improve patient outcomes.

Chorioamnionitis and Neonatal Sepsis from Community-associated MRSA

To the Editor: Chorioamnionitis is a common cause of maternal and neonatal illness and death (1), but chorioamnionitis attributed to Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA), is reported infrequently (2–5). In the context of the rising incidence of community-associated MRSA (CA-MRSA) infections (6), we report an apparent case of CA-MRSA chorioamnionitis. The patient, a 31-year-old woman with polycystic ovary syndrome and hypothyroidism, had 1 prior pregnancy but no viable offspring. After a clomiphene-assisted conception, routine ultrasound at 21 weeks’ gestation identified a shortened cervix (5 mm). The patient declined amniocentesis for cerclage and was treated with pelvic rest and vaginal progesterone. Five days later, she arrived at the emergency department with foul-smelling vaginal discharge. At this time, the patient was afebrile and hemodynamically stable, had no abdominal pain, and had a leukocyte count of 9.5 × 103 cells/mm3. Premature rupture of membranes was diagnosed, and the patient was admitted and administered intravenous ampicillin and azithromycin. Nine days into treatment, at 23 weeks’ gestation, 210 hours after membrane rupture, a 415-g live-born girl was delivered spontaneously in footling breech with Apgar scores of 1 (1 min) and 5 (5 min). During admission, the mother was never febrile and did not complain of abdominal tenderness or chills. The highest leukocyte count was 12.4 × 103 cells/mm3. The mother was discharged the day after delivery without further complications. At 6-week follow-up, she remained well, with no signs of infection. Pathologic examination of the placenta demonstrated focal acute funisitis, acute chorioamnionitis with fetal surface acute arteritis and acute deciduitis. Cultures from the maternal and fetal sides of the placenta grew predominantly MRSA and rare colonies of methicillin-susceptible S. aureus. The MRSA antimicrobial drug profile, including trimethoprim/sulfamethoxazole and clindamycin susceptibility, was characteristic of CA-MRSA (6). The neonate, who died on day 16, was culture-positive for CA-MRSA from blood, 2 umbilical swabs, and a tracheal aspirate. The antibiogram of these isolates was identical to the placental cultures, including absence of inducible clindamycin resistance. Postmortem examination showed hemorrhagic necrotizing pneumonia and gram-negative bacilli. Culture of lung tissue grew Escherichia coli. Isolates from the placenta and neonate were identified phenotypically, without molecular testing. Maternal complications of chorioamnionitis include endometritis, bacteremia, hemorrhage, and cesarean delivery (1). Clinically, chorioamnionitis can be diagnosed by maternal fever (>38°C) and 2 of the following: maternal leukocytosis (>15 × 103cells/mm3), maternal tachycardia (>100 bpm), fetal tachycardia (>160 bpm), uterine tenderness, and foul-smelling amniotic fluid (1). This patient had none of these signs, except foul-smelling amniotic fluid, and fetal tachycardia was absent. In this case, chorioamnionitis was diagnosed by histology. Amniotic fluid cultures from pregnancies complicated by chorioamnionitis have shown multiple organisms from the vaginal flora, such as Streptococcus agalactiae, Gardnerella vaginalis, Mycoplasma hominis, Ureaplasma urealyticum, anaerobes, and E. coli (1). Chorioamnionitis associated with S. aureus is uncommon (2,3), and MRSA chorioamnionitis is rare (4,5). The first 2 reports of MRSA chorioamnionitis appeared in 1998 (4) and 2002 (5). In both instances, the patients worked in the healthcare industry, and the authors considered the MRSA to have been nosocomial strains. The patient in our report was a restaurant manager, had no prior recorded hospital admissions, and was not previously known to be colonized by MRSA. CA-MRSA strains are epidemiologically and clonally unrelated to hospital-associated MRSA (HA-MRSA) strains and can be differentiated by the presence of staphylococcal cassette chromosome mec type IV and the absence of multidrug resistance seen with HA-MRSA (6). Recently, the incidence of CA-MRSA infections increased in community settings, including outbreaks in settings in which CA-MRSA is endemic, with manifestations ranging from skin and soft tissue infections to necrotizing pneumonia (6). Genital colonization with MRSA recently has been reported with a frequency of 0.5%–3.5% in pregnant women (7,8). In 1 study, most (93%) of these isolates were CA-MRSA (7). Eckhardt et al. described a patient with chorioamnionitis in whom CA-MRSA bacteremia developed (9). However, this descriptor was used to specify multidrug-resistant MRSA not acquired in a hospital. Moreover, neither placental nor amniotic fluid cultures were described. Laibl et al. reported 2 patients with CA-MRSA infections in whom chorioamnionitis developed (10). Again, placental and amniotic fluid culture results were not reported, nor was chorioamnionitis listed as an infection caused by CA-MRSA in their cohort. However, these latter 2 patients might represent additional cases of CA-MRSA chorioamnionitis. Although the incidence of CA-MRSA infections continues to increase, CA-MRSA chorioamnionitis appears to remain rare. Nevertheless, the prevalence of MRSA genital colonization among pregnant women creates an opportunity for this agent to cause ascending gestational infection. This finding is meaningful because recommended empirical antimicrobial drug treatments may not cover CA-MRSA, increasing the likelihood of infectious complications (1). However, culture results when available can provide therapeutic guidance. We hope this report raises awareness of the possibility of CA-MRSA chorioamnionitis and encourages reports from other authors so this entity can be better established, characterized, and monitored.

Performance of lateral flow device and galactomannan for the detection of Aspergillus species in bronchoalveolar fluid of patients at risk for invasive pulmonary aspergillosis.

Early diagnosis of invasive pulmonary aspergillosis (IPA) remains difficult due to the variable performance of the tests used. We compared the performance characteristics of Aspergillus lateral flow device (LFD) in bronchoalveolar lavage (BAL) vs. BAL‐galactomannan (GM), for the diagnosis of IPA. 311 BAL specimens were prospectively collected from patients who underwent bronchoscopy from January to May 2013. Patients at risk for IPA were divided into haematological malignancy (HEM) and non‐HEM groups: solid organ transplants (SOT) (lung transplant (LT) and non‐LT SOT); chronic steroid use (CSU); solid tumour (STU) and others. We identified 96 patients at risk for IPA; 89 patients (93%) were in the non‐HEM groups: SOT 57 (LT, 46, non‐LT SOT, 11); CSU 21; STU 6, other 5. Only three patients met criteria for IA (two probable; one possible). Overall sensitivity (SS) was 66% for both and specificity (SP) was 94% vs. 52% for LFD and GM respectively. LFD and GM performance was similar in the HEM group (SS 100% for both and SP 83% vs. 100% respectively). LFD performance was better than GM among non‐HEM SOT patients (P = 0.02). Most false‐positive GM results occurred in the SOT group (50.8%), especially among LT patients (56.5%). LFD performance was superior with an overall SP of 95.6% in SOT (P < 0.002) and 97% in LT patients (P = 0.0008). LFD is a rapid and simple test that can be performed on BAL to rule out IPA.

Transmission of infection to liver transplant recipients from donors with infective endocarditis: lessons learned

Donors not meeting standard criteria, such as those with bacteremia, are now being used in response to the increasing need for organs for transplantation. Recommended strategies to prevent the occurrence of donor‐derived bacteremia include the use of directed antibiotic prophylaxis. However, this approach does not eliminate the risk of infection transmission. Similarly, the management of organ recipients from donors with infective endocarditis (IE) remains uncharacterized. We report 2 cases of donor‐derived bacterial infections in liver transplant recipients despite pathogen‐specific antibiotic prophylaxis. In both instances, the donors had documented IE treated with appropriate antimicrobial therapy and clearance of bacteremia. Recipients had very distinctive clinical outcomes likely related to pathogen virulence and the extent of donor infection. Persistent infection in the transplanted liver should be suspected in organ recipients of a liver from donors with IE, despite the absence of bacteremia at the time of death and organ procurement. For eradication, recipients may require prolonged pathogen‐directed antimicrobial therapy, such as is used for endovascular infections. Prompt recognition of donors with IE, appropriate notification, and prolonged antibiotic prophylaxis are key to reducing the risk of such donor‐derived infections.

Outcomes of Aminopenicillin Therapy for Vancomycin-Resistant Enterococcal Urinary Tract Infections.

ABSTRACT Vancomycin-resistant urinary tract infections are often challenging to treat. This retrospective cohort study compared outcomes between patients treated for vancomycin-resistant enterococcal urinary tract infection with an aminopenicillin and those treated with a non-β-lactam antibiotic. Inpatients treated with an enterococcus-active agent for their first symptomatic vancomycin-resistant enterococcal urinary tract infection between 1 January 2012 and 31 December 2013 were considered for inclusion. Patients with colonization, on hospice, or receiving comfort care only were excluded. The primary endpoint of clinical cure was defined as resolution of clinical symptoms, or symptom improvement to the extent that no additional antibacterial drug therapy was necessary, and lack of microbiologic persistence. Secondary endpoints of 30-day readmission or retreatment and 30-day all-cause mortality were also compared. A total of 316 urinary isolates were screened, and 61 patients with symptomatic urinary tract infection were included. Twenty (35%) of the 57 isolates tested were ampicillin susceptible. Thirty-one patients received an aminopenicillin, and 30 received a non-β-lactam. Rates of clinical cure for aminopenicillin versus non-β-lactam treatment were 26/31 (83.9%) and 22/30 (73.3%) (P = 0.315), respectively. Rates of 30-day readmission (6/31, or 19.4%, versus 9/30, or 30%, respectively; P = 0.334), 30-day retreatment (4/31, or 12.9%, versus 4/30, 13.3%, respectively; P = 0.960), and 30-day all-cause mortality (2/31, or 6.5%, versus 1/30, or 3.3%, respectively; P = 0.573) were also not significantly different between groups. Aminopenicillins may be a viable option for treating vancomycin-resistant urinary tract infection regardless of the organisms ampicillin susceptibility. Prospective validation with larger cohorts of patients should be considered.

Comparison of time to positivity of the VersaTREK® REDOX 80-mL and the REDOX EZ draw 40-mL blood culture bottles for common bacterial bloodstream pathogens

The VersaTREK(®) microbial detection system offers 2 media formulations, an aerobic and an anaerobic bottle available in a 40-mL direct draw format and an 80-mL format. The 40-mL EZ Draw(®) bottle can be inoculated with a maximum volume of 5 mL, while the REDOX 80-mL bottle accommodates a 10-mL volume. The effect of volume of blood inoculum on time to positivity (TTP) has not been clearly established with these bottle types. This study utilized simulated blood cultures seeded with clinically relevant microorganisms in human blood to evaluate the impact of inoculum volume and organism load on TTP for the 2 bottle types. For 13/15 organisms, the EZ Draw bottle flagged positive earlier than the REDOX 80-mL bottles. The lower volume of blood inoculum did not negatively impact TTP using the EZ Draw blood culture bottles as compared to REDOX 80-mL bottles.

Characterization of Salmonella Isangi possessing a CTX-M15 ESBL associated with an outbreak in a US Hospital

Over an approximately 50-day period in 2015, an outbreak of CTX-M-15 extended spectrum β-lactamase-(ESBL)-possessing Salmonella Isangi occurred among 19 adult surgical patients and one healthcare worker (HCW) at a large urban tertiary care hospital in the United States. A total of 45 S. Isangi isolates were isolated from stool (35), blood (4), urine (3), respiratory (2), and wound (1) cultures. Phenotypically, all but three isolates demonstrated resistance to ampicillin, ampicillin/sulbactam, ceftriaxone, and cefepime, and one isolate was resistant to ertapenem. Genotypically, a single CTX-M-15 ESBL was identified in all but three isolates by real-time PCR. Interestingly, two of the CTX-M-15 negative, susceptible isolates were isolated from a single patient who initially had a CTX-M positive, resistant strain. Isolates were clonally related, including both resistant and susceptible strains, as confirmed by pulse field gel electrophoresis (PFGE). This is the first case of a novel Salmonella outbreak at this hospital, and we believe it to be the first case of an S. Isangi serotype outbreak in the United States.

Stewardship opportunities in viral pneumonia: Why not the immunocompromised?

Antimicrobial management of viral pneumonia has proven to be a challenge in hospitalized immunocompromised patients. A host of factors contribute to the dilemma, such as diagnostic uncertainty, lack of organism identification, and clinical status of the patient. Respiratory virus panel (RVP) use was compared between 131 immunocompromised patients who received send‐out (n = 56) vs in‐house (n = 75) testing. Antimicrobial optimization interventions consisted of antiviral addition/discontinuation, antibiotic discontinuation/de‐escalation, or modification of immunosuppressive regimen. After implementation of an in‐house test with audit and feedback, turnaround time of the RVP was reduced from 46.7 to 5.5 hours (P < .001) and time to intervention was reduced from 52.1 to 13.9 hours (P < .001), yet the frequency of antimicrobial optimization interventions was unchanged (30.7% vs 35.7%). Differences were not observed in duration of empiric antibiotic therapy or length of stay. The overall discontinuation rate for patients tested with a RVP was low (4.6%), and those with positive RVP (n = 43) had antibiotics stopped in 14% of cases. Bacterial pneumonia coinfection was confirmed in 2 patients. Further systematic efforts should be taken to reduce antibiotic use in viral pneumonia and identify the major barriers in the immunocompromised population.