John Keiser

Single-Molecule Long-Read 16S Sequencing To Characterize the Lung Microbiome from Mechanically Ventilated Patients with Suspected Pneumonia

ABSTRACT In critically ill patients, the development of pneumonia results in significant morbidity and mortality and additional health care costs. The accurate and rapid identification of the microbial pathogens in patients with pulmonary infections might lead to targeted antimicrobial therapy with potentially fewer adverse effects and lower costs. Major advances in next-generation sequencing (NGS) allow culture-independent identification of pathogens. The present study used NGS of essentially full-length PCR-amplified 16S ribosomal DNA from the bronchial aspirates of intubated patients with suspected pneumonia. The results from 61 patients demonstrated that sufficient DNA was obtained from 72% of samples, 44% of which (27 samples) yielded PCR amplimers suitable for NGS. Out of the 27 sequenced samples, only 20 had bacterial culture growth, while the microbiological and NGS identification of bacteria coincided in 17 (85%) of these samples. Despite the lack of bacterial growth in 7 samples that yielded amplimers and were sequenced, the NGS identified a number of bacterial species in these samples. Overall, a significant diversity of bacterial species was identified from the same genus as the predominant cultured pathogens. The numbers of NGS-identifiable bacterial genera were consistently higher than identified by standard microbiological methods. As technical advances reduce the processing and sequencing times, NGS-based methods will ultimately be able to provide clinicians with rapid, precise, culture-independent identification of bacterial, fungal, and viral pathogens and their antimicrobial sensitivity profiles.

A Cost-Effective Interdisciplinary Approach to Microbiologic Send-Out Test Use

CONTEXT Use of reference laboratories for selected laboratory testing (send-out tests) represents a significant source of laboratory costs. As the use of more complex molecular analyses becomes common in the United States, strategies to reduce costs in the clinical laboratory must evolve in order to provide high-value, cost-effective medicine. OBJECTIVE To report a strategy that employs clinical pathology house staff and key hospital clinicians in the effective use of microbiologic send-out testing. DESIGN The George Washington University Hospital is a 370-bed academic hospital in Washington, DC. In 2012 all requisitions for microbiologic send-out tests were screened by the clinical pathology house staff prior to final dispensation. Tests with questionable utility were brought to the attention of ordering clinicians through the use of interdisciplinary rounds and direct face-to-face consultation. RESULTS Screening resulted in a cancellation rate of 38% of send-out tests, with proportional cost savings. Nucleic acid tests represented most of the tests screened and the largest percentage of cost saved through screening. Following consultation, requested send-out tests were most often canceled because of a lack of clinical indication. CONCLUSIONS Direct face-to-face consultation with ordering physicians is an effective, interdisciplinary approach to managing the use of send-out testing in the microbiology laboratory.

INDUCTION OF MRSA BIOFILM BY LOW-DOSE β-LACTAM ANTIBIOTICS: SPECIFICITY, PREVALENCE AND DOSE-RESPONSE EFFECTS

Methicillin-resistant Staphylococcus aureus (MRSA) is a leading cause of hospital- and community-associated infections. The formation of adherent clusters of cells known as biofilms is an important virulence factor in MRSA pathogenesis. Previous studies showed that subminimal inhibitory (sub-MIC) concentrations of methicillin induce biofilm formation in the community-associated MRSA strain LAC. In this study we measured the ability sub-MIC concentrations of eight other β-lactam antibiotics and six non-β-lactam antibiotics to induce LAC biofilm. All eight β-lactam antibiotics, but none of the non-β-lactam antibiotics, induced LAC biofilm. The dose-response effects of the eight β-lactam antibiotics on LAC biofilm varied from biphasic and bimodal to near-linear. We also found that sub-MIC methicillin induced biofilm in 33 out of 39 additional MRSA clinical isolates, which also exhibited biphasic, bimodal and linear dose-response curves. The amount of biofilm formation induced by sub-MIC methicillin was inversely proportional to the susceptibility of each strain to methicillin. Our results demonstrate that induction of biofilm by sub-MIC antibiotics is a common phenotype among MRSA clinical strains and is specific for β-lactam antibiotics. These findings may have relevance to the use of β-lactam antibiotics in clinical and agricultural settings.

Nasal Colonization among Premature Infants Treated with Nasal Continuous Positive Airway Pressure

We examined the relationship between the use of nasal continuous positive airway pressure (CPAP) and nasal colonization among low-birth-weight (LBW) infants. We prospectively cultured the nares of LBW infants on admission and weekly until hospital discharge. The modality of respiratory support during each culture was recorded. Bivariate and multivariate analyses were conducted to test the relationship between CPAP and nasal colonization. Analyses were repeated after stratifying infants into three birth-weight categories: 1500 to 2499 g, 1000 to 1499 g, and < 1000 g. In total, 766 nasal cultures were obtained from 167 infants. Nasal colonization with gram-negative bacilli was increased with the use of CPAP in all birth-weight categories ( P < 0.05) and with vaginal delivery in infants weighing < 1000 g and 1500 to 2499 g ( P = 0.04 and P = 0.02, respectively). Nasal colonization with any potential pathogen increased with the use of CPAP in all birth-weight categories ( P < 0.001), with the presence of chorioamnionitis in infants < 1000 g ( P = 0.055) and at younger gestational age in infants 1000 to 1499 g ( P = 0.0026). Caucasian infants 1500 to 2499 g had less colonization than infants of other races ( P = 0.01). Nasal CPAP is associated with increased colonization with gram-negative bacilli.

Characteristics and Severity of Disease among 100 Cases of Imported Malaria Seen at a U.S. University Hospital, 2000–2017

Malaria acquired in endemic areas poses a substantial risk to travelers arriving in or returning to the United States. Timely diagnosis and recognition of severe illness are crucial; however, many U.S.-based clinicians lack familiarity with this disease. We conducted a retrospective review of 100 cases of malaria in adults seen at a single urban university hospital during 2000-2017. Descriptive and analytical statistics were calculated, including logistic regression modeling case severity. Most of the patients presented with Plasmodium falciparum (76%), most commonly after travel from sub-Saharan Africa (94%). Prior malaria experience was common (50%), but adherence to a prophylactic regimen was exceedingly rare (4%). Twenty-one patients had severe malaria, including 10 with cerebral malaria. Severity was predicted by high parasitemia, bandemia, hypoglycemia, and hypotension at the time of presentation. In 24 patients, the initial treatment regimen was changed, usually because of the appearance of clinical deterioration or drug toxicity. One patient required intravenous artesunate. All patients survived, although one suffered fetal loss. Among 30 patients initially evaluated at other institutions, 43% had been treated for an alternative diagnosis. The most common reasons for transfer of patients to our hospital were inadequate facilities and lack of expertise with malaria. There needs to be increased awareness among U.S.-based travelers and clinicians regarding malaria as a potentially lethal condition, emphasizing the use of appropriate prophylaxis. Our simple model of disease severity could serve frontline physicians when deciding which patients should be admitted to the intensive care unit or transferred for higher level care.

Should Polymerase Chain Reaction–Based Assays Be Used in All Patients With Suspected Clostridium difficile Colitis?

C lostridium difficile antibiotic-associated colitis (CDAD) is one of the most serious complications of antibiotic use. The United States estimates suggest that C. difficile infection affects 7178 inpatients on any given day and causes the deaths of approximately 300 patients per day. Being able to rapidly and accurately identify those with CDAD is paramount in stopping the spread of C. difficile infection in hospitals. Nucleic acid amplification tests forC. difficile are being widely developed for diagnostic purposes because they are consideredmore sensitive than standard enzymelinked immunosorbent assay–based assays. Since toxin B is required for C. difficile virulence, toxin B gene has become the primary target for commercial C. difficile polymerase chain reaction (PCR) tests. The Cepheid Xpert C. difficile assay is a PCR assay that detects expression of the C. difficile toxin B gene. The Xpert C. difficile toxin B assay has a sensitivity of 93.5% and a specificity of 94.0% when compared with C. difficile culture. The currentC. difficile testing algorithm atGeorgeWashington University Hospital is to start with the C. Diff Quik Chek Complete assay, which tests for the glutamate dehydrogenase (GDH) antigen and toxin A and B production and reflex to the Xpert C. difficile PCR assay for discordant results. If both GDH and toxins are positive, then CDAD is confirmed. If both are negative, then CDAD is ruled out. If results are discordant, the Xpert C. difficile PCR assay is performed with a positive result confirming CDAD and a negative result ruling it out. It has been suggested that PCR assays should become the standard test for all C. difficile testing. However, the Xpert C. difficile assay is twice as expensive as the C. Diff Quik Chek Complete, and it is not clear that this approach would be more cost-effective. We tested 240 stool samples submitted to GeorgeWashington University Hospital micro laboratory from August to November 2014 for C. difficile testing using both the C. Diff Quik Chek Complete and the Xpert C. difficile assays. The cost of using the C. Diff Quik Chek with reflex to C. difficile PCR was compared with universal C. difficile PCR testing. The level of leukocytosis was compared between different groups. Thirty-six patients tested positive for CDAD; 10 patients were GDH, toxin, and PCR (triple) positive and 26 patients were GDH and PCR positive. Two hundred three patients tested negative, 163 were triple negative, and 18 were only GDH positive. One patient tested GDH and toxin negative, but PCR positive possibly representing C. difficile colonization. The mean white blood

Late-onset group B streptococcus infection in a preterm neonate

Early-onset group B streptococcus (GBS) is transmitted vertically from the birth canal of colonized mothers. The mode of transmission of late-onset GBS is less clear, although it is believed to reflect a delayed infection after early colonization from either vertical or horizontal transmission. We present a premature female infant whose mother was GBS negative at the time of delivery but later had GBS bacteriuria in postpartum. The infant had an initial blood culture and multiple weekly nasal surveillance cultures that were all negative for GBS. The infant’s nasal culture grew GBS after 25 days, 1 day after she had signs of sepsis and her blood grew GBS. This is the first case in preterm infants to demonstrate the horizontal transmission of late-onset GBS bacteremia without the need for early-onset colonization.

Diagnostic Challenges in the Identification of Rothia aeria Bacteremia in a Patient With Relapsing Acute Myeloid Leukemia

Rothia aeria is a dental colonizer known to cause disease in immunocompromised patients, especially thosewith oral-dental pathology. We describe a case of Rothia aeria bacteremia in a patient receiving chemotherapy for acute myeloid leukemia that was misidentified as R. dentocariosa and R. mucilaginosa and describe the diagnostic challenges of identifying this organism at a species level using both standard methods and newer diagnostic techniques.

146Review of Empiric Echinocandin Therapy for Candidemia

146. Review of Empiric Echinocandin Therapy for Candidemia Cristina Amado, MD; Paul Blair, MD; Marc Siegel, MD, Medicine; John Keiser, MD; Infectious Diseases, George Washington University, Alexandria, VA; Internal Medicine, George Washington University, Washington, DC; Medical Faculty Associates/George Washington University Medical Center, Washington, DC; George Washington University Hospital, Washington, DC

Positive Blood Culture Results After Plasmodium falciparum Diagnosis

We describe a case of falsely positive blood culture results due to the presence of Plasmodium falciparum in the cultured blood specimen. Although awareness among health care professionals of this phenomenon has been reported in the literature, we discovered that attending physicians and house staff at our institution were largely unaware of this phenomenon. Similarly, laboratory staff and technologists therein had little experience identifying this organism on Gram-stained blood smears. We believe that this report will serve as a useful reminder to some readers and will broaden the differential diagnoses generated by others. * WBCs : white blood cells RBCs : red blood cells