Proceedings of the Clinical Microbiology Open 2023: discussions about pandemic preparedness.

The 4th Clinical Microbiology Open (CMO) took place in Carlsbad, California, on 10 and 11 February 2023. This event facilitated discussion between clinical and public health laboratory directors, government agencies, and industry representatives from the companies that make up ASM's Corporate Council. While many topics were discussed, much of the discussion focused on pandemic preparedness. There were four major questions addressed: (i) When is the perfect the enemy of good in pandemic testing? (ii) What other types of pathogens might cause another pandemic and how would this affect laboratory response? (iii) What research is needed to better understand the effectiveness of the pandemic response? (iv) What have we learned about the utility of self and at-home testing in future pandemics? This review serves as a summary of these discussions.

Clinical utility of multiplex PCR in the detection of pathogens from sterile body fluids.

Rapid identification of pathogens in normally sterile body fluid (NSBF) is essential for appropriate patient management, specifically antimicrobial therapy. Limited sensitivity and increased time to detection of traditional culture prompted us to evaluate additional testing to contribute to the diagnosis of infection. The purpose of this study was to evaluate the GenMark Dx ePlex Blood Culture Identification (BCID) Panels on positive body fluids inoculated into blood culture bottles for the detection of microorganisms. A total of 88 positive body fluids from blood culture bottles were analyzed using a Gram-Positive, Gram-Negative, and/or Fungal pathogen BCID Panel based on the Gram stain result. Each result was compared to routine culture performed from the positive bottle. When using culture as a reference standard, we found the ePlex multiplex panel performed with a positive percent agreement of 96.5% and a negative percent agreement of 99.8%. The use of multiplex PCR may be a useful supplement to routine culture for NSBF in blood culture bottles. IMPORTANCE: The identification of pathogens in normally sterile body fluid (NSBF) is performed using routine culture, the current gold standard. Limitations of this method include sensitivity and increased turnaround times which could potentially delay vital patient care, especially antimicrobial therapy. Adaptations of NSBF in blood culture bottles prompted us to consider the utility of additional methods to bridge the gap in diagnostic challenges for these life-threatening infections. Multiplex molecular panels have been manufactured for use with multiple specimen types including blood, cerebral spinal fluid, stool, and respiratory. Therefore, the purpose of this study was to evaluate the off-label use of ePlex Blood Culture Identification Panels on positive body fluids grown in blood culture bottles for the detection of microorganisms for research purposes.

In silico analysis of mutations associated with occult hepatitis B virus (HBV) infection in South Africa.

Occult hepatitis B virus (OBI) infection is defined by the presence of viral DNA in the liver and/or serum in absence of hepatitis B surface antigen (HBsAg). While multiple studies have identified mutations that are associated with OBI, only a small portion of these mutations have been functionally characterized in vitro. Using complementary in silico approaches, the effects of OBI-associated amino acid mutations on HBV protein function in HBV/HIV-positive ART-naïve South Africans were evaluated. Two OBI-associated mutations in the PreS1 region, one in the PreS2 region, and seven in the surface region of subgenotype A1 sequences were identified as deleterious. In subgenotype A2 sequences, 11 OBI-associated mutations in the PreS1 region, seven in the PreS2 region, and 31 in the surface region were identified as deleterious. In the polymerase region, 14 OBI-associated mutations in subgenotype A1 and 71 OBI-associated mutations in subgenotype A2 were identified as deleterious. This study utilized in silico approaches to characterize the likely impact of OBI-associated mutations on viral function, thereby identifying and prioritizing candidates and reducing the significant cost associated with functional studies that are essential for mechanistic studies of the OBI phenotype.

Utilizing BD MAX™ Enteric Bacterial Panel to Detect Stool Pathogens from Rectal Swabs.

BACKGROUND: The BD MAX™ Enteric Bacterial Panel (BDM-EBP) is designed and FDA-cleared to detect Salmonella, Shigella, Campylobacter, and Shiga toxin genes stx1/2 from stool samples. However, rectal swabs, which are not FDA-cleared for clinical testing with the BDM-EBP, are common specimens received from pediatric patients for enteric pathogen testing. The purpose of this study was to evaluate the ability of the BDM-EBP to detect stool pathogens from rectal swabs. METHODS: Routine cultures, Shiga toxin testing, and molecular testing with BDM-EBP were performed on 272 sequential rectal swabs collected from August 2015 to December 2015. Discrepant test results were resolved using Verigene® Enteric Pathogens Nucleic Acid Test (EP). 36 challenge samples (13 Salmonella spp., 3 Shigella spp., 10 Campylobacter spp., and 10 Shiga toxin positive Escherichia coli) were tested using reference strains (American Type Culture Collection) and previous patient isolates diluted to103-104 cfu/ml in saline then added to Sample Buffer Tube (SBT) with negative stool matrix delivered via a swab. Limit of detection testing was performed by serial 10 fold dilutions in saline then added to SBT with negative stool matrix provided via a swab. RESULTS: A total of 272 rectal swab specimens were evaluated and 89 were positive by culture and/or MAX EBP. All discrepant results were BDM-EBP positive and culture negative. 21 of 31 (68%) of the apparent false positive BDM-EBP discrepant results resolved as positive with Nanosphere's Verigene® EP. After resolution of the discordant results, the Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) are as follows for each target: Salmonella (n = 4) 100%, PPA and 100%, NPA; Shigella (n = 79) 100%, PPA and 95.3%, NPA; Campylobacter (n = 4) 100%, PPA and 99.6%, NPA; and Shiga toxin producing organisms (n = 2) 100%, PPA and 100%, NPA. 8.8% of the patient samples did not initially yield a result on the BDM-System. Upon repeat, half of the problematic samples resolved, and 4.4% of the total specimen tested did not yield a result. All organisms in the challenge samples were detected. Limits of detection for BDM-EBP testing of rectal swabs were as follows (in cfu/ml in SBT): Salmonella-1.44 × 102; Shigella-5.10 × 100; Campylobacter-1.51 × 101; and Shiga Toxin-1.13 ×103. CONCLUSION: Rectal swabs are acceptable samples for detecting Salmonella, Shigella, Campylobacter, and Shiga toxin using BDM-EBP.

Functional analysis of 'a' determinant mutations associated with occult HBV in HIV-positive South Africans.

Occult hepatitis B is defined by the presence of hepatitis B virus (HBV) DNA in the absence of hepatitis B surface antigen (HBsAg). Occult HBV is associated with the development of hepatocellular carcinoma, reactivation during immune suppression, and virus transmission. Viral mutations contribute significantly to the occult HBV phenotype. Mutations in the 'a' determinant of HBsAg are of particular interest, as these mutations are associated with immune escape, vaccine escape and diagnostic failure. We examined the effects of selected occult HBV-associated mutations identified in a population of HIV-positive South Africans on HBsAg production in vitro. Mutations were inserted into two different chronic HBV backbones and transfected into a hepatocyte-derived cell line. HBsAg levels were quantified by enzyme-linked immunosorbent assay (ELISA), while the detectability of mutant HBsAg was determined using an HA-tagged HBsAg expression system. Of the seven mutations analysed, four (S132P, C138Y, N146D and C147Y) resulted in decreased HBsAg expression in one viral background but not in the second viral background. One mutation (N146D) led to a decrease in HBsAg detected as compared to HA-tag, indicating that this mutation compromises the ability of the ELISA to detect HBsAg. The contribution of occult-associated mutations to the HBsAg-negative phenotype of occult HBV cannot be determined adequately by testing the effect of the mutation in a single viral background, and rigorous analysis of these mutations is required.

Extraction of total nucleic acids from bacterial isolates using the bioMérieux NucliSENS easyMAG total nucleic acid extractor.

The BioMerieux NucliSENS easyMAG total nucleic acid extractor was evaluated for use on bacterial isolates in the clinical microbiology laboratory. Forty eight isolates were extracted, yielding quantifiable amounts of DNA for all isolates. The easyMAG is appropriate for DNA extraction from bacterial isolates and will be incorporated in the clinical laboratory.

Mutations associated with occult hepatitis B in HIV-positive South Africans.

Occult hepatitis B is characterized by the absence of hepatitis B surface antigen (HBsAg) but the presence of HBV DNA. Because diagnosis of hepatitis B virus (HBV) typically includes HBsAg detection, occult HBV remains largely undiagnosed. Occult HBV is associated with increased risk of hepatocellular carcinoma, reactivation to chronic HBV during immune suppression, and transmission during blood transfusion and liver transplant. The mechanisms leading to occult HBV infection are unclear, although viral mutations are likely a significant factor. In this study, sera from 394 HIV-positive South Africans were tested for HBV DNA and HBsAg. For patients with detectable HBV DNA, the overlapping surface and polymerase open reading frames (ORFs) were sequenced. Occult-associated mutations-those mutations found exclusively in individuals with occult HBV infection but not in individuals with chronic HBV infection from the same cohort or GenBank references-were identified. Ninety patients (22.8%) had detectable HBV DNA. Of these, 37 had detectable HBsAg, while 53 lacked detectable surface antigen. The surface and polymerase ORFs were cloned successfully for 19 patients with chronic HBV and 30 patients with occult HBV. In total, 235 occult-associated mutations were identified. Ten occult-associated mutations were identified in more than one patient. Additionally, 15 amino acid positions had two distinct occult-associated mutations at the same residue. Occult-associated mutations were common and present in all regions of the surface and polymerase ORFs. Further study is underway to determine the effects of these mutations on viral replication and surface antigen expression in vitro.

Molecular characterisation of hepatitis B virus in HIV-1 subtype C infected patients in Botswana.

BACKGROUND: Hepatitis B virus (HBV) is a major global health problem especially in sub-Saharan Africa and in East Asia. Ten hepatitis B virus genotypes have been described that differ by geographic distribution, disease progression, and response to treatment. Escape mutations within the surface open reading frame (ORF) affect HBV antigenicity leading to failures in diagnosis, vaccine and hepatitis B immunoglobulin therapy. However, the molecular characteristics of HBV in Botswana, a highly endemic country, are unknown. We describe the molecular characteristics of HBV and prevalence of escape mutants among HIV/HBV coinfected individuals Botswana. METHODS: DNA was extracted from archived plasma samples from 81 HIV/HBV co-infected participants from various clinical studies at the Botswana Harvard AIDS Institute Partnership. A 415 base pair (bp) fragment of the polymerase gene was amplified by semi-nested PCR. In a subset of samples, a 2100 bp fragment was amplified. The PCR product was genotyped using Big Dye sequencing chemistry and the sequences were analysed for genotypes and mutations. RESULTS: Of the 81 samples included, 70 (86 %) samples were successfully genotyped. Genotype A was found in 56 (80 %) participants, D in 13 (18.6 %), and 1 (1.4 %) was genotype E. Escape mutations previously linked with failure of diagnosis or escaping active vaccination and passive immunoglobulin therapy were detected in 12 (17.1 %) participants at positions 100, 119, 122, 123, 124, 126, 129, 130, 133, 134 and 140 of the S ORF. Genotypes and escape mutations were not significantly associated with aspartate aminotransferase (AST), alanine aminotransferase (ALT) and AST platelet ratio index (APRI). CONCLUSION: Genotypes A, D and E were found in this cohort of HIV coinfected patients in Botswana, consistent with the findings from the sub-Saharan Africa region. Some escape mutations which have previously been associated with diagnosis failure, escaping vaccine and immunoglobulin therapy were also observed and are important in guiding future policies related to vaccine implementation, therapeutic guidelines, and diagnostic guidelines. They are also important for identifying patients who are at an increased risk of disease progression and to choose optimal therapy. Future research should focus on determining the clinical significance of the different HBV genotypes and mutations found in this population.

Evidence of distinct populations of hepatitis C virus in the liver and plasma of patients co-infected with HIV and HCV.

Viral diversity is an important predictor of hepatitis C virus (HCV) treatment response and may influence viral pathogenesis. HIV influences HCV variability in the plasma; however, limited data on viral variability are available from distinct tissue/cell compartments in patients co-infected with HIV and HCV. Thus, this exploratory study evaluated diversity of the hypervariable region 1 (HVR1) of HCV in the plasma and liver for 14 patients co-infected with HIV and HCV. Median intra-patient genetic distances and entropy values were similar in the plasma and liver compartments. Positive immune selection pressure was observed in the plasma for five individuals and in the liver for three individuals. Statistical evidence supporting viral compartmentalization was found in five individuals. Linear regression identified ALT (P = 0.0104) and AST (P = 0.0130) as predictors of viral compartmentalization. A total of 12 signature amino acids that distinguish liver from plasma E1/HVR1 were identified. One signature amino acid was shared by at least two individuals. These findings suggest that HCV compartmentalization is relatively common among patients co-infected with HIV and HCV. These data also imply that evaluating viral diversity, including drug resistance patterns, in the serum/plasma only may not adequately represent viruses replicating with in the liver and, thus, deserves careful consideration in future studies.

Preanalytical Challenges of Molecular Microbiology Tests.

As infectious disease diagnostics increasingly incorporates molecular techniques, there are unique preanalytical concerns that must be considered. First, noninvasive specimen types that may be inadequate for culture-based diagnostics may be acceptable when using molecular tests. Second, specimen containers must be evaluated for the presence of substances that may interfere with amplification or sequencing reactions. Finally, the capacity of transport, storage, and processing conditions to maintain nucleic acid integrity and avoid contamination must be assessed. This review explores these issues and the effects they may have on result quality.

Application of matrix-assisted laser desorption ionization-time of flight mass spectrometry for identification of the fastidious pediatric pathogens Aggregatibacter, Eikenella, Haemophilus, and Kingella.

The accuracy of matrix-assisted laser desorption-ionization time of flight mass spectrometry (MALDI-TOF MS) in the identification of Haemophilus, Aggregatibacter, Cardiobacterium, Eikenella, and Kingella (HACEK) species was compared to that of phenotypic methods (Remel RapID and Vitek 2). Overall, Vitek MS correctly identified more isolates, incorrectly identified fewer isolates, and failed to identify fewer isolates than both phenotypic methods.

Utility and Recommendations for the Use of Multiplex Molecular Gastrointestinal Pathogen Panels.

BACKGROUND: Many molecular gastrointestinal pathogen panels (GIPs) are Food and Drug Administration (FDA) cleared but it is still unclear how to best utilize these new diagnostic tools. GIPs are highly sensitive and specific, simultaneously detect multiple pathogens in one reaction, and can shorten the overall time of diagnosis for infectious gastroenteritis but are also expensive with relatively poor insurance reimbursement. CONTENT: In this review, we take a comprehensive approach to discuss issues with utilization of GIPs from a physician perspective, and implementation from a laboratory perspective. The information presented is to assist physicians in deciding on appropriate use of GIPs in diagnostic algorithms for their patients, and to provide information to laboratories that may be considering the addition of these powerful diagnostic assays to their test menu. Some of the important topics discussed are inpatient vs outpatient use, the appropriate panel size and organisms to include, interpretation of results, laboratory validation, and reimbursement. SUMMARY: The information in this review provides clear guidance to both clinicians and laboratories in deciding the best use of GIPs for a specific patient population. While this technology provides many benefits over traditional methods, it can also complicate result interpretation and comes with a high cost, which necessitates the need for use recommendations.

Viral pathogen detection using multiplex gastrointestinal molecular panels: The pros and cons of viral target inclusion.

Diagnosis of gastrointestinal infections has been revolutionized by the development of in vitro diagnostic (IVD) multiplex molecular panels for the detection of viral nucleic acids. In addition to a high degree of accuracy, these panels are commercially available and relatively simple to perform in the clinical laboratory. However, use of these panels must be carefully considered owing to the laboratory costs of the test, limited reimbursement, and potential for overuse. In this review from the Pan American Society for Clinical Virology, we focus on the viral components of GI multiplex panels (GIPs), presenting a brief overview of pathogens included on most panels and a discussion of advantages and challenges of the inclusion of viral targets on GIPs that should be considered before implementation in the clinical laboratory.

Clinical features and laboratory diagnosis of emerging arthropod-transmitted viruses: A Report from the Pan American Society for Clinical Virology Clinical Practice Committee.

Arthropod-borne viruses (arboviruses) are an increasing global threat due to their ability to cause human disease and their expanding geographical distribution. They circulate in nature between arthropod vectors and vertebrate hosts. Infection of susceptible human hosts leads to harmful developmental and neurological manifestations. Arboviruses have caused recent outbreaks with significant public health implications, such as the Zika virus outbreak in the western hemisphere which caused fetal abnormalities in some infected pregnant women, or Eastern Equine Encephalitis which caused 15 deaths in 2019. This review discusses several arboviral infections and their clinical manifestations while highlighting the importance of laboratory diagnostics to detect infections and current attempts at vaccine development. The ability to accurately diagnose an arbovirus infection is critical for initiating a timely response to infections in order to improve patient outcomes.

Combined Molecular and Phenotypic Antimicrobial Susceptibility Testing Is Beneficial in Detection of ESBL and AmpC Beta-Lactamase Producing Isolates of Enterobacteriaceae in Pediatric Patients with Bloodstream Infections.

Objectives: The aim was to assess the potential advantage of combined genotypic testing with phenotypic antimicrobial susceptibility testing (AST) to detect AmpC ß-lactamases (AmpC) and extended-spectrum ß-lactamases (ESBL) producing Enterobacteriaceae isolated from blood cultures in a pediatric population. Materials and Methods: All first-time Enterobacteriaceae isolates recovered from blood cultures of pediatric patients at the Cincinnati Children's Hospital Medical Center between January 2017 and December 2018 were evaluated. The Check-MDR CT103XL ß-lactamase assay was used to determine the presence of AmpC and ESBL, while AST was performed using the VITEK 2 platform. Phenotypic ESBL resistance was defined by resistance to either ceftriaxone or ceftazidime using Clinical and Laboratory Standards Institute breakpoints, while combined cefoxitin resistance with ceftriaxone or ceftazidime resistance was used to detect AmpCs (as per European Committee on Antimicrobial Susceptibility Testing standards). Results: Overall, there were 170 isolates. Genotypically, 21 (12.4%) had AmpC and 18 (10.6%) had ESBL genes detected. Phenotypically, 11 (6.5%) isolates were AmpC and 26 (15.3%) were ESBL producing organisms. Genotypic testing identified an additional 14 AmpC and two ESBL isolates that failed to meet phenotypic criteria. Conclusions: Using combined genotypic and phenotypic methods to detect AmpC and ESBL producing organisms increased the identification of resistant organism and provided potentially clinical relevant data to guide the treatment of resistant organisms.

Association of systemic antimicrobials with the expression of beta-lactamases in bacteria cultured from urological patients.

BACKGROUND: Patients with abnormalities of the genitourinary tract are at high risk for infections with antimicrobial-resistant pathogens. METHODS: All urine cultures ordered by members of the Division of Urology from four quarterly one-week periods were included. All gram-negative bacilli isolated were analyzed using the Check-Points Check-MDR CT103XL assay to identify the presence of genes associated with resistance to beta-lactam antibiotics. Association between the days of antibiotics and the presence of an ESBL-producing organism was determined. RESULTS: One hundred eleven positive cultures were included in this analysis, of which 5 (4.5%) contained ESBL-producing species. Days of systemic antibiotics within 30 days of urine culture was associated with an increased risk of isolating an ESBL-producing pathogen. CONCLUSION: The overall prevalence of ESBL-producing organisms is low in this cohort. The number of days of systemic antibiotics within 30 days of a urine culture was significantly associated with increased risk of isolating an ESBL-producing organism.

Frequency of Hepatitis B Virus Resistance Mutations in Women Using Tenofovir Gel as Pre-Exposure Prophylaxis.

Intermittent use of a single antiretroviral agent in the presence of a replicating virus could potentially increase the development of antiviral resistance. The pericoital, before-and-after sex, dosing regimen used in the Centre for the AIDS Programme of Research in South Africa (CAPRISA) 004 tenofovir gel trial meant that women who were infected with hepatitis B virus (HBV) were exposed intermittently to tenofovir during their participation. The impact of this dosing regimen on HBV resistance was assessed by amplification of the HBV polymerase region from 37 stored plasma samples of women who were HBV surface antigen positive. All samples belonged to HBV genotype A. None of the known tenofovir resistance mutations (M240V/I, L180M, A194T, V214A, N238T) were identified in any individuals. While it is reassuring that no resistance mutations were found among women using topical tenofovir, the rapidly expanding access to oral tenofovir-containing HIV pre-exposure prophylaxis (PrEP), with higher systemic exposure to the drug, makes monitoring for potential HBV drug resistance important.

Accelerating Initiation of Adequate Antimicrobial Therapy Using Real-Time Decision Support and Microarray Testing.

INTRODUCTION: Bloodstream infections (BSI) represent a common cause of sepsis and mortality in children. Early and adequate empirical antimicrobial therapy is a critical component of successful treatment of BSI. Rapid PCR-based diagnostic technologies, such as nucleic acid microarrays, can decrease the time needed to identify pathogens and antimicrobial resistance and have the potential to ensure patients are started on adequate antibiotics as early as possible. However, without appropriate processes to support timely and targeted interpretation of these results, these advantages may not be realized in practice. METHODS: Our Antimicrobial Stewardship Program (ASP) implemented a quality improvement initiative using the Institute for Healthcare Improvement's Model for Improvement to decrease the time between a nucleic acid microarray result for Gram-positive bacteremia and the time a patient was placed on adequate antimicrobial therapy. The primary effective intervention was a near real-time notification system to the managing physicians of inadequate antimicrobial therapy via a call from the ASP team. RESULTS: Following the intervention, the average time to adequate antimicrobial therapy in patients with Gram-positive BSI and inadequate coverage decreased from 38 hours with the nucleic acid microarray result alone to 4.7 hours when results were combined with an ASP clinical decision support intervention, an 87% reduction. CONCLUSIONS: The positive effects of rapid-detection technologies to improve patient care are enhanced when combined with clinical decision support tools that can target inadequate antimicrobial treatments in near real time.

Performance of the check-points check-MDR CT103XL assay utilizing the CDC/FDA antimicrobial resistance isolate bank.

The Check-MDR CT103XL beta-lactamase assay was validated for use in the clinical microbiology laboratory using two CDC-FDA Antimicrobial Resistant Isolate Bank panels (133 gram-negative bacilli known beta-lactamase genes). The CT103XL detected most reported resistance genes (123 of 136 genes) and additionally identified several resistance genes not reported by the CDC. Discrepant results were confirmed via whole genome sequencing.