Evaluation of the Performance and Clinical Impact of a Rapid Phenotypic Susceptibility Testing Method Directly from Positive Blood Culture at a Pediatric Hospital.

Bloodstream infection poses a significant medical emergency that necessitates timely administration of appropriate antibiotics. Standard laboratory workup for antimicrobial susceptibility testing (AST) involves subculture of organisms from positive blood bottles followed by testing using broth microdilution; however, this process can take several days. The Accelerate Pheno Blood Culture panel (Pheno) provides rapid phenotypic testing of selected Gram-negative organisms directly from positive blood cultures. This has the potential to shorten the AST process to several hours and impact time to antimicrobial optimization and subsequent clinical outcomes; however, these metrics have not been assessed in pediatric populations. We retrospectively compared two patient cohorts with blood cultures positive for on-panel Gram-negative organisms: 82 cases tested by conventional AST methods, and 80 cases postintervention at our pediatric hospital. Susceptibility testing from the Pheno yielded 91.5% categorical agreement with a broth microdilution-based reference method with 7.4% minor error, 1.1% major error, and 0.1% very major error rates. The median time from blood culture positivity to AST decreased from 20.0 h to 9.7 h (P < 0.001), leading to an overall decrease in time from blood culture positivity to change in therapy from 36.0 h to 25.0 h (P < 0.001). There was no observed change in length of stay or 30-day mortality. Median duration on meropenem decreased from 64.8 h to 31.6 h (P = 0.04). We conclude the Pheno had accurate performance and that implementation allowed for faster AST reporting, improved time to optimal therapy, and decreased duration on meropenem in children.

Cell division is antagonized by the activity of peptidoglycan endopeptidases that promote cell elongation.

A peptidoglycan (PG) cell wall composed of glycans crosslinked by short peptides surrounds most bacteria and protects them against osmotic rupture. In Escherichia coli, cell elongation requires crosslink cleavage by PG endopeptidases to make space for the incorporation of new PG material throughout the cell cylinder. Cell division, on the contrary, requires the localized synthesis and remodeling of new PG at midcell by the divisome. Little is known about the factors that modulate transitions between these two modes of PG biogenesis. In a transposon-insertion sequencing screen to identify mutants synthetically lethal with a defect in the division protein FtsP, we discovered that mutants impaired for cell division are sensitive to elevated activity of the endopeptidases. Increased endopeptidase activity in these cells was shown to interfere with the assembly of mature divisomes, and conversely, inactivation of MepS was found to suppress the lethality of mutations in essential division genes. Overall, our results are consistent with a model in which the cell elongation and division systems are in competition with one another and that control of PG endopeptidase activity represents an important point of regulation influencing the transition from elongation to the division mode of PG biogenesis.

Saliva Is a Promising Alternative Specimen for the Detection of SARS-CoV-2 in Children and Adults.

Testing efforts for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been burdened by the scarcity of testing materials and personal protective equipment for health care workers. The simple and painless process of saliva collection allows for widespread testing, but enthusiasm is hampered by variable performance compared to that of nasopharyngeal swab (NPS) samples. We prospectively collected paired NPS and saliva samples from a total of 300 unique adult and pediatric patients. SARS-CoV-2 RNA was detected in 32.2% (97/300) of the individuals using the TaqPath COVID-19 Combo kit (Thermo Fisher). Performance of saliva and NPS was compared against the total number of positives regardless of specimen type. The overall concordances for saliva and NPS were 91.0% (273/300) and 94.7% (284/300), respectively. The values for positive percent agreement (PPA) for saliva and NPS were 81.4% (79/97) and 89.7% (87/97), respectively. Saliva yielded detection of 10 positive cases that were negative by NPS. For symptomatic and asymptomatic pediatric patients not previously diagnosed with COVID-19, the performances of saliva and NPS were comparable (PPA, 82.4% versus 85.3%). The overall values for PPA for adults were 83.3% and 90.7% for saliva and NPS, respectively, with saliva yielding detection of 4 fewer cases than NPS. However, saliva performance for symptomatic adults was identical to NPS performance (PPA of 93.8%). With lower cost and self-collection capabilities, saliva can be an appropriate sample choice alternative to NPS for detection of SARS-CoV-2 in children and adults.

A Burkholderia thailandensis Acyl-Homoserine Lactone-Independent Orphan LuxR Homolog That Activates Production of the Cytotoxin Malleilactone.

UNLABELLED: Burkholderia thailandensis has three acyl-homoserine lactone (AHL) LuxR-LuxI quorum-sensing circuits and two orphan LuxR homologs. Orphans are LuxR-type transcription factors that do not have cognate LuxI-type AHL synthases. One of the orphans, MalR, is genetically linked to the mal gene cluster, which encodes enzymes required for production of the cytotoxic polyketide malleilactone. Under normal laboratory conditions the mal gene cluster is silent; however, antibiotics like trimethoprim induce mal transcription. We show that trimethoprim-dependent induction of the mal genes requires MalR. MalR has all of the conserved amino acid residues characteristic of AHL-responsive LuxR homologs, but in B. thailandensis, MalR activation of malleilactone synthesis genes is not responsive to AHLs. MalR can activate transcription from the mal promoter in E. coli without addition of AHLs or trimethoprim. Expression of malR in B. thailandensis is induced by trimethoprim. Our data indicate that MalR binds to a lux box-like element in the mal promoter and activates transcription of the mal genes in an AHL-independent manner. Antibiotics like trimethoprim appear to activate mal gene expression indirectly by somehow activating malR expression. MalR activation of the mal genes represents an example of a LuxR homolog that is not a receptor for an AHL quorum-sensing signal. Our evidence is consistent with the idea that mal gene activation depends solely on sufficient transcription of the malR gene. IMPORTANCE: LuxR proteins are transcription factors that are typically activated by acyl-homoserine lactone (AHL) signals. We demonstrate that a conserved LuxR family protein, MalR, activates genes independently of AHLs. MalR is required for transcription of genes coding for synthesis of the cytotoxic polyketide malleilactone. These genes are not expressed when cells are grown under normal laboratory conditions. In laboratory culture, MalR induction of malleilactone requires certain antibiotics, such as trimethoprim, which increase malR expression by an unknown mechanism. At sufficient levels of malR expression, MalR functions independently of any external signal. Our findings show that MalR is an activator of the silent malleilactone biosynthesis genes and that MalR functions independently of AHLs.

Re-examining the classification of abdominal pseudocysts as surgical site infections through review of broad-range polymerase chain reaction results.

BACKGROUND: The Hydrocephalus Clinical Research Network-quality group (HCRNq) historically defined all abdominal pseudocysts associated with a ventriculoperitoneal shunt as a surgical site infection regardless of culture result. METHODS: We retrospectively reviewed broad-range polymerase chain reaction (BRPCR) results sent between January 2017 and July 2023 from abdominal pseudocyst fluid sent from hospitals around the country to a reference laboratory to help further characterize these collections. RESULTS: A total of 19 samples were tested via BRPCR between 1/2017 and 7/2023. Two (10.5 %) had organisms identified; one with Staphylococcus epidermidis and one with Candida parapsilosis. No fastidious organisms that would be expected to not grow with typical culture techniques were identified. CONCLUSIONS: Few abdominal pseudocysts had organisms identified by BRPCR, suggesting that not all pseudocysts are due to infectious causes. Consideration should be given to alternate causes of pseudocyst development when cultures are negative.

SARS-CoV-2 Variants from Long-Term, Persistently Infected Immunocompromised Patients Have Altered Syncytia Formation, Temperature-Dependent Replication, and Serum Neutralizing Antibody Escape.

SARS-CoV-2 infection of immunocompromised individuals often leads to prolonged detection of viral RNA and infectious virus in nasal specimens, presumably due to the lack of induction of an appropriate adaptive immune response. Mutations identified in virus sequences obtained from persistently infected patients bear signatures of immune evasion and have some overlap with sequences present in variants of concern. We characterized virus isolates obtained greater than 100 days after the initial COVID-19 diagnosis from two COVID-19 patients undergoing immunosuppressive cancer therapy, wand compared them to an isolate from the start of the infection. Isolates from an individual who never mounted an antibody response specific to SARS-CoV-2 despite the administration of convalescent plasma showed slight reductions in plaque size and some showed temperature-dependent replication attenuation on human nasal epithelial cell culture compared to the virus that initiated infection. An isolate from another patient-who did mount a SARS-CoV-2 IgM response-showed temperature-dependent changes in plaque size as well as increased syncytia formation and escape from serum-neutralizing antibodies. Our results indicate that not all virus isolates from immunocompromised COVID-19 patients display clear signs of phenotypic change, but increased attention should be paid to monitoring virus evolution in this patient population.

Descriptive and molecular epidemiology of leishmaniasis diagnosed from clinical samples in the United States, 2021-2022.

Leishmaniasis is a rare disease in the United States, with an estimated annual incidence of dozens of cases occurring primarily in travelers, migrants, and military personnel. True disease incidence is unknown, since leishmaniasis is not a nationally notifiable condition. Here, we describe the results of molecular leishmaniasis over a 1-year interval (September 2021 to August 2022) when our laboratory served as the primary national reference laboratory for molecular diagnosis of civilian leishmaniasis. We tested 218 specimens submitted from 36 states yielding 94 of the 186 (50.5%) positive cases with species or species complex-level identification and 18 novel mini-exon alleles. Most species belonged to subgenus Viannia (75.6%) and associated with cutaneous or mucocutaneous disease. Cases were associated with recent travel (18.1%), travel timing unspecified (7.4%), migration (7.4%), remote travel (2.1%), military (1.1%), or unknown history (63.8%). These data illustrate the clinical utility of molecular testing for leishmaniasis and provide unique insight into disease epidemiology. IMPORTANCE: Leishmaniasis is a disfiguring, neglected parasitic infection endemic to the Southern United States and the Americas. Despite significant populations at risk-travelers, military and foreign service members, and migrating persons-the epidemiology of the disease in the United States is poorly understood. Moreover, few clinical laboratories in the United States can test for the disease. Here, we present results from 1 year of testing for this disease at a major reference laboratory. These findings are particularly relevant because they coincide with a temporary "pause" on all clinical testing at the CDC. Our findings suggest at least several hundred cases occur each year in the United States. In particular, mucosal leishmaniasis may be more common than previously reported. We also highlight greater genetic diversity in Leishmania species endemic to the Americas than has been previously sampled, with implications for diagnostic specificity.

Rapid Antigen Assays for SARS-CoV-2: Promise and Peril.

Though rapid antigen tests have historically problematic performance characteristics for the diagnosis of respiratory viral infections such as influenza, they have attained an unprecedented level of use in the context of the COVID-19 pandemic. Ease of use and scalability of rapid antigen tests has facilitated a democratization and scale of testing beyond anything reasonably achievable by traditional laboratory-based testing. In this chapter, we discuss the performance characteristics of rapid antigen testing for SARS-CoV-2 detection and their application to non-traditional uses beyond clinical diagnostic testing.

Increased viral variants in children and young adults with impaired humoral immunity and persistent SARS-CoV-2 infection: A consecutive case series.

BACKGROUND: There is increasing concern that persistent infection of SARS-CoV-2 within immunocompromised hosts could serve as a reservoir for mutation accumulation and subsequent emergence of novel strains with the potential to evade immune responses. METHODS: We describe three patients with acute lymphoblastic leukemia who were persistently positive for SARS-CoV-2 by real-time polymerase chain reaction. Viral viability from longitudinally-collected specimens was assessed. Whole-genome sequencing and serological studies were performed to measure viral evolution and evidence of immune escape. FINDINGS: We found compelling evidence of ongoing replication and infectivity for up to 162 days from initial positive by subgenomic RNA, single-stranded RNA, and viral culture analysis. Our results reveal a broad spectrum of infectivity, host immune responses, and accumulation of mutations, some with the potential for immune escape. INTERPRETATION: Our results highlight the potential need to reassess infection control precautions in the management and care of immunocompromised patients. Routine surveillance of mutations and evaluation of their potential impact on viral transmission and immune escape should be considered.

Persistent SARS-CoV-2 infection and increasing viral variants in children and young adults with impaired humoral immunity.

Background: There is increasing concern that persistent infection of SARS-CoV-2 within immunocompromised hosts could serve as a reservoir for mutation accumulation and subsequent emergence of novel strains with the potential to evade immune responses. Methods: We describe three patients with acute lymphoblastic leukemia who were persistently positive for SARS-CoV-2 by real-time polymerase chain reaction. Viral viability from longitudinally-collected specimens was assessed. Whole-genome sequencing and serological studies were performed to measure viral evolution and evidence of immune escape. Findings: We found compelling evidence of ongoing replication and infectivity for up to 162 days from initial positive by subgenomic RNA, single-stranded RNA, and viral culture analysis. Our results reveal a broad spectrum of infectivity, host immune responses, and accumulation of mutations, some with the potential for immune escape. Interpretation: Our results highlight the need to reassess infection control precautions in the management and care of immunocompromised patients. Routine surveillance of mutations and evaluation of their potential impact on viral transmission and immune escape should be considered. Funding: The work was partially funded by The Saban Research Institute at Children's Hospital Los Angeles intramural support for COVID-19 Directed Research (X.G. and J.D.B.), the Johns Hopkins Center of Excellence in Influenza Research and Surveillance HHSN272201400007C (A.P.), NIH/NIAID R01AI127877 (S.D.B.), NIH/NIAID R01AI130398 (S.D.B.), NIH 1U54CA260517 (S.D.B.), an endowment to S.D.B. from the Crown Family Foundation, an Early Postdoc.Mobility Fellowship Stipend to O.F.W. from the Swiss National Science Foundation (SNSF), and a Coulter COVID-19 Rapid Response Award to S.D.B. L.G. is a SHARE Research Fellow in Pediatric Hematology-Oncology.

Bactobolin A binds to a site on the 70S ribosome distinct from previously seen antibiotics.

The ribosome is the target of a large number of antibiotics. Here, we report a 3.4-Å-resolution crystal structure of bactobolin A bound to 70S ribosome-tRNA complex. The antibiotic binds at a previously unseen site in the 50S subunit and displaces tRNA bound at the P-site. It thus likely has a similar mechanism of action as blasticidin S despite binding to a different site. The structure also rationalizes previously identified resistance mutations.

Bactobolin resistance is conferred by mutations in the L2 ribosomal protein.

UNLABELLED: Burkholderia thailandensis produces a family of polyketide-peptide molecules called bactobolins, some of which are potent antibiotics. We found that growth of B. thailandensis at 30°C versus that at 37°C resulted in increased production of bactobolins. We purified the three most abundant bactobolins and determined their activities against a battery of bacteria and mouse fibroblasts. Two of the three compounds showed strong activities against both bacteria and fibroblasts. The third analog was much less potent in both assays. These results suggested that the target of bactobolins might be conserved across bacteria and mammalian cells. To learn about the mechanism of bactobolin activity, we isolated four spontaneous bactobolin-resistant Bacillus subtilis mutants. We used genomic sequencing technology to show that each of the four resistant variants had mutations in rplB, which codes for the 50S ribosome-associated L2 protein. Ectopic expression of a mutant rplB gene in wild-type B. subtilis conferred bactobolin resistance. Finally, the L2 mutations did not confer resistance to other antibiotics known to interfere with ribosome function. Our data indicate that bactobolins target the L2 protein or a nearby site and that this is not the target of other antibiotics. We presume that the mammalian target of bactobolins involves the eukaryotic homolog of L2 (L8e). IMPORTANCE: Currently available antibiotics target surprisingly few cellular functions, and there is a need to identify novel antibiotic targets. We have been interested in the Burkholderia thailandensis bactobolins, and we sought to learn about the target of bactobolin activity by mapping spontaneous resistance mutations in the bactobolin-sensitive Bacillus subtilis. Our results indicate that the bactobolin target is the 50S ribosome-associated L2 protein or a region of the ribosome affected by L2. Bactobolin-resistant mutants are not resistant to other known ribosome inhibitors. Our evidence indicates that bactobolins interact with a novel antibiotic target.