Infectious Diseases: Selected Tests
Number: 1008
Table Of Contents
PolicyApplicable CPT / HCPCS / ICD-10 Codes
Background
References
Policy
Scope of Policy
This Clinical Policy Bulletin (CPB) addresses metagenomic next-generation sequencing (mNGS), multiplex immunoassay tests, molecular syndromic panels, and other tests for infectious diseases that do not fit in other CPBs.
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Medical Necessity
Aetna considers real-time quaking-induced conversion (RT-QuIC) medically necessary for evaluation of individuals with a rapidly progressive dementia suspected of having prion disease (e.g., Creutzfeldt-Jakob disease).
Aetna considers testing for syphilis using a non-treponemal test (e.g., Venereal Disease Research Laboratory (VDRL) or rapid plasma reagin (RPR) test) and/or a treponemal antibody detection test medically necessary for screening of all pregnant women, and persons who are at risk of syphilis infection, and for diagnostic testing of individuals with signs and symptoms of syphilis.
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Experimental, Investigational, or Unproven
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Metagenomic next-generation tests
Aetna considers the following metagenomic next-generation sequencing (mNGS) tests (not an all-inclusive list) as experimental, investigational, or unproven because of insufficient evidence in the peer-reviewed literature:
- IDbyDNA AlloID, Respiratory Pathogen ID/AMR Panel (RPIP), and Urinary Pathogen ID/AMR Panel (UPIP);
- Karius Test (mNGS of microbial cell-free-DNA) (see also, CPB 0650 - Polymerase Chain Reaction Testing: Selected Indications);
- Metagenomic next generation sequencing for central nervous system (CNS) infections (e.g., Johns Hopkins Metagenomic Next Generation Sequencing Assay for Infectious Disease Diagnostics, and Mayo Clinic Laboratories Metagenomic Sequencing, CSF, also known as the “MSCSF Test");
- MicroGenDX qPCR + NGS and MicroGenDX shotgun metagenomics tests;
- NeXGen Fungal/AFB NGS assay.
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Multiplex immunoassay, multiplex pathogen or multianalyte tests
Aetna considers the following multiplex immunoassay, multiplex pathogen, or multianalyte tests as experimental, investigational, or unproven for the diagnosis or management of infectious disease because the effectiveness of this approach has not been established:
- Accelerate PhenoTest BC Kit;
- Accelerate PhenoTest BC Kit AST Configuration;
- FebriDx Bacterial/Non-Bacterial Point-of-Care Assay;
- IntelliSep;
- MeMed BV.
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Other tests
Aetna considers bacterial typing, whole genome sequencing as experimental, investigational, or unproven for assisting in the workup of a potential outbreak by a single bacterial species or in the identification of a recurrent infection in an individual person because the effectiveness of this approach has not been established.
Aetna considers monocyte distribution width (measured by the Early Sepsis Indicator) experimental, investigational, or unproven for the diagnosis or management of infectious diseases (including sepsis) because the effectiveness of this approach has not been established.
Aetna considers ciprofloxacin resistance (gyrA S91F point mutation) testing and macrolide (clarithromycin) sensitivity (23S rRNA point mutation) testing experimental, investigational, or unproven because the effectiveness of these tests has not been established.
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Related Policies
Laboratory-related policies:
- CPB 0140 - Genetic Testing
- CPB 0215 - Lyme Disease and other Tick-Borne Diseases
- CPB 0227 - BRCA Testing, Prophylactic Mastectomy, and Prophylactic Oophorectomy
- CPB 0249 - Inflammatory Bowel Disease: Serologic Markers and Pharmacogenomic and Metabolic Assessment of Thiopurine Therapy
- CPB 0319 - RET Proto-Oncogene Testing
- CPB 0352 - Tumor Markers
- CPB 0433 - Chlamydia Trachomatis - Screening and Diagnosis
- CPB 0443 - Cervical Cancer Screening and Diagnosis
- CPB 0499 - Nonstandard Laboratory Test Panels
- CPB 0516 - Colorectal Cancer Screening
- CPB 0561 - Celiac Disease Laboratory Testing
- CPB 0643 - Diagnosis of Vaginitis
- CPB 0650 - Polymerase Chain Reaction Testing: Selected Indications
- CPB 0715 - Pharmacogenetic and Pharmacodynamic Testing
- CPB 0787 - Comparative Genomic Hybridization (CGH)
Background
Infectious diseases are caused by microscopic organisms (bacteria, viruses, fungi, and parasites) that penetrate the body’s protective barriers (e.g., skin, mucous membranes) and can result in symptoms ranging from mild to severe, including death. Timely and accurate diagnosis is an essential step in the management of infectious diseases. Although symptoms can indicate a disease, a laboratory test may be necessary to identify the specific microorganism causing the infection so that appropriate treatment can be prescribed. Metagenomic sequencing has been proposed as a laboratory method to diagnose infectious disease.
There are many different laboratory methods available on the market that are used to identify microorganisms. Microbiological approaches, such as culture and gram staining, are traditional methods for diagnosing infectious diseases. However, these approaches are considered labor-intensive and time-consuming. Furthermore, some microorganisms are difficult to culture or identify. Thus, newer approaches have been developed which use DNA sequencing technology to identify microbial agents.
Laboratory testing methods, such as nucleic acid amplification tests (NAATs), are used to extract/purify, amplify (copy) and detect genetic material [deoxyribonucleic acid (DNA) or ribonucleic acid (RNA)] in microorganisms, making the pathogen much easier to identify. The polymerase chain reaction (PCR) is an example of this type of test (Paul et al, 2020; Vazquez-Pertejo, 2020). See CPB 0650 - Polymerase Chain Reaction Testing: Selected Indications. Most culture-independent methods, such as PCR tests, require a priori knowledge of microorganisms that are suspected to be present within a clinical sample under investigation in order to detect them (Boers et al, 2019). PCR-based tests have been developed further into multiplex assays which allow for simultaneous detection of several biological agents. However, even multiplex (or panel-based) PCRs "can only identify predefined targets, so one must have suspect organisms or targets in mind in order to detect them" (Wang and Jean, 2021).
First-generation methods for determining the nucleotide sequence of DNA, such as Sanger sequencing, is a low-throughput method used to determine a portion of the nucleotide sequence of an individual's genome. This technique uses PCR amplification of genetic regions of interest followed by sequencing of PCR products (NIH/NCI, 2022). Sanger sequencing can be difficult to interpret when performed on complex or polymicrobial samples (Wang and Jean, 2021).
Next-generation sequencing (NGS)-based tests have emerged and present the "possibility of an agnostic diagnostic method capable of comprehensive detection of multiple pathogens simultaneously and directly from a patient sample" (Wang and Jean, 2021). Next-generation sequencing is a high-throughput, massively parallel, culture-free sequencing method that tests for an array of potential pathogens within a microbial sample simultaneously in a single sequencing run in order to determine a cause of disease (Boers et al, 2019). An advantage of NGS compared with PCR is that prior knowledge of the target organism(s), and thus target-specific primers, is not required. Major applications of NGS in clinical microbiology laboratories include: targeted NGS (tNGS), whole genome sequencing, and metagenomic NGS (mNGS) (Want and Jean, 2021).
Metagenomic Next-Generation Sequencing Tests
Metagenomic next-generation sequencing tests (mNGS), sometimes called "shotgun" sequencing, is an unbiased hypothesis-free diagnostic approach to the detection of pathogens. mNGS allows for thousands to billions of DNA fragments to be simultaneously and independently sequenced from a clinical sample which may contain mixed populations of microorganisms, and assigning these to their reference genomes to understand which microbes are present and in what proportions (Gu et al, 2019; Lee, 2019). "Clinical tests have been developed to detect the nucleic acids of microbes from various specimen types such as blood, joint fluid, and cerebrospinal fluid (CSF) to aid the diagnosis of various infections. A significant limitation of mNGS is that most of the nucleic acids in clinical samples are from the host, so the host genome dominates sequence reads. This can result in decreased analytical sensitivity for detection of pathogens present at relatively low burden" (Wang and Jean, 2019). Despite the potential of mNGS, major reservations "include the interpretation of findings (distinguishing contamination and colonization from true pathogens), selection and validation of databases used for analyses, and prediction (or lack thereof) of antimicrobial susceptibilities. A common perception is that mNGS is so incredibly sensitive that it will reveal a diagnosis when all other testing is negative. While mNGS may be analytically more sensitive than standard culturing methods in some cases, the necessary removal of vast amounts of human nucleic acid during sequencing preparation and (by computational methods) during the post-analytic process, can decrease the sensitivity in comparison to targeted PCR approaches for many organisms". Furthermore, "contamination of samples during specimen collection is a large concern given the increased analytical sensitivity of mNGS in comparison to standard culture methods, and there needs to be a validated quality-control process in place for steps from assessing reagent purity to measuring adequate genome coverage controls" (Lee, 2019).
IDbyDNA Tests
IDbyDNA, Inc. offers "syndromic Precision Metagenomics testing applications" to help identify various infectious disease indications. IDbyDNA utilizes the Explify software platform which integrates artificial intelligence (AI), knowledge from global experts, and proprietary reagents to assist laboratory professionals on obtaining rapid actionable infectious disease insights. The Explify software platform aims to provide ultra-rapid DNA search technology, AI-powered data interpretation, curated collections of millions of DNA sequences, comprehensive genotype-phenotype databases for antimicrobial resistance (AMR) prediction, and user-friendly software interfaces.
The Respiratory Pathogen ID/AMR Panel (RPIP) uses precision RNA and DNA sequencing, RPIP enrichment probes and automated Explify RPIP data analysis to deliver sensitive detection and quantification of over 280 respiratory pathogens causing pneumonia, and AMR information for 60 antibiotics and antivirals. RPIP can potentially identify over 180 bacteria including 13 mycobacteria and other slow growing pathogens, 50+ fungi, and 40+ viruses including full genome characterization of SARS-CoV-2 and influenza A/B viruses, and antibiotic and antiviral resistance information based on more than 2,000 genomic markers.
The Urinary Pathogen ID/AMR Panel (UPIP) uses mNGS to detect and quantify over 170 common, less common challenging-to-grow, and frequently missed uropathogens which can lead to recurrent or difficult to manage urinary tract infections. The comprehensive panel identifies more than 120 bacteria, 35 viruses, 14 fungi, 4 parasites, and antibiotic resistance information for 46 antibiotics based on more than 3,500 resistance markers.
The AlloID, by CareDX and powered by IDbyDNA's Explify software platform, aims to provide plasma-based precision metagenomics detection and quantification of viruses, bacteria, fungi, and parasites that are of particular concern for causing infections in patients with transplants. AlloID will also deliver genotyping information for transplant viruses, antiviral resistance profiling, and detection of multidrug-resistant bacteria.
Gaston et al (2022) state that NGS approaches hold the possibility of consolidating some or all diagnostic approaches for pathogen identification and characterization into a single assay. The authors evaluated the performance of the Respiratory Pathogen ID/AMR (RPIP) kit (Illumina, Inc.) with automated Explify bioinformatic analysis (IDbyDNA, Inc.), a targeted NGS workflow enriching specific pathogen sequences and antimicrobial resistance (AMR) markers, and a complementary untargeted metagenomic workflow with in-house bioinformatic analysis. Compared to a composite clinical standard consisting of provider-ordered microbiology testing, chart review, and orthogonal testing, both workflows demonstrated similar performances. The overall agreement for the RPIP targeted workflow was 65.6% (95% confidence interval, 59.2 to 71.5%), with a positive percent agreement (PPA) of 45.9% (36.8 to 55.2%) and a negative percent agreement (NPA) of 85.7% (78.1 to 91.5%). The overall accuracy for the metagenomic workflow was 67.1% (60.9 to 72.9%), with a PPA of 56.6% (47.3 to 65.5%) and an NPA of 77.2% (68.9 to 84.1%). The approaches revealed pathogens undetected by provider-ordered testing (Ureaplasma parvum, Tropheryma whipplei, severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2], rhinovirus, and cytomegalovirus [CMV]), although not all pathogens detected by provider-ordered testing were identified by the NGS workflows. The RPIP targeted workflow required more time and reagents for library preparation but streamlined bioinformatic analysis, whereas the metagenomic assay was less demanding technically but required complex bioinformatic analysis. The results from both workflows were interpreted utilizing standardized criteria, which is necessary to avoid reporting nonpathogenic organisms. The RPIP targeted workflow identified AMR markers associated with phenotypic resistance in some bacteria but incorrectly identified blaOXA genes in Pseudomonas aeruginosa as being associated with carbapenem resistance. The authors concluded that these workflows could serve as adjunctive testing with, but not as a replacement for, standard microbiology techniques. The authors note multiple limitations in this study. First, the patients in the study represented a heavily pretreated population, with many treated with antimicrobial agents in the 14 days prior to the acquisition of the BAL fluid specimens. It is possible that false-positive bacterial and fungal results represented organisms that could not be recovered in culture due to antimicrobial use. Second, BAL fluid was not collected using protected techniques, potentially allowing contamination of samples by oropharyngeal flora. Third, the same provider-ordered tests were not applied to all samples. Although this is representative of provider ordering practices, all pathogens may not have been identified if missed by NGS workflows and the lack of standardized testing. Finally, reporting of BAL fluid cultures is standardized in the Johns Hopkins clinical microbiology laboratory, but variability by technologists in the extent of workup for small quantities of bacterial isolates may have occurred.
Karius Test
The Karius Test for infectious disease uses next-generation sequencing (NGS) to detect microbial cell free DNA (cfDNA) in plasma from bacteria, DNA viruses, fungi and protozoa. Microbial cfDNA may be found in plasma when viable microorganisms are not detected in blood by other methods. The reported microorganism(s) may or may not be the cause of patient infection. Results should be interpreted within the context of clinical data, including medical history, physical findings, epidemiological factors, and other laboratory data (Karius, 2020). "The Karius Test can detect more than 1,000 bacteria, fungi, parasites, and select DNA viruses. Detected microorganisms are reported quantitatively as DNA molecules per microliter of plasma (MPM), and are compared to reference MPM ranges established in healthy, asymptomatic individuals" (Wang and Jean, 2019).
Camargo et al (2019) state that cell-free DNA (cfDNA) sequencing technology in diagnostic evaluation of infections in immunocompromised hosts is limited. The authors conducted an exploratory study using next-generation sequencing (NGS) for detection of microbial cfDNA in a cohort of 10 immunocompromised patients with febrile neutropenia, pneumonia or intra-abdominal infection. Pathogen identification by cfDNA NGS demonstrated positive agreement with conventional diagnostic laboratory methods in 7 (70%) cases, including patients with proven/probable invasive aspergillosis, Pneumocystis jirovecii pneumonia, Stenotrophomonas maltophilia bacteremia, Cytomegalovirus and Adenovirus viremia. NGS results were discordant in 3 (30%) cases including two patients with culture negative sepsis who had undergone hematopoietic stem cell transplant in whom cfDNA testing identified the etiological agent of sepsis; and one kidney transplant recipient with invasive aspergillosis who had received > 6 months of antifungal therapy prior to NGS testing. The authors concluded that these observations support the clinical utility of measurement of microbial cfDNA sequencing from peripheral blood for rapid noninvasive diagnosis of infections in immunocompromised hosts; however, larger studies are needed.
Hogan et al (2021) state that mNGS of plasma cell-free DNA has emerged as an attractive diagnostic modality allowing broad-range pathogen detection, noninvasive sampling, and earlier diagnosis. However, little is known about its real-world clinical impact as used in routine practice. The authors performed a retrospective cohort study of all patients for whom plasma mNGS (Karius test) was performed for all indications at 5 United States institutions over 1.5 years. Comprehensive records review was performed, and standardized assessment of clinical impact of the mNGS based on the treating team's interpretation of Karius results and patient management was established. A total of 82 Karius tests were evaluated from 39 (47.6%) adults and 43 (52.4%) children and a total of 53 (64.6%) immunocompromised patients. The authors found that Karius positivity rate was 50 of 82 (61.0%), with 25 (50.0%) showing 2 or more organisms (range, 2-8). The Karius test results led to positive impact in 6 (7.3%), negative impact in 3 (3.7%), and no impact in 71 (86.6%), and was indeterminate in 2 (2.4%). Cases with positive Karius result and clinical impact involved bacteria and/or fungi but not DNA viruses or parasites. In 10 patients who underwent 16 additional repeated tests, only 1 was associated with clinical impact. The authors concluded that the real-world impact of the Karius test as currently used in routine clinical practice is limited. Further studies are needed to identify high-yield patient populations, define the complementary role of mNGS to conventional microbiological methods, and discern how best to integrate mNGS into current testing algorithms.
Shishido et al (2022) state that metagenomic next-generation sequencing of microbial cell-free DNA (mcfDNA) allows for non-invasive pathogen detection from plasma. However, there is little data describing the optimal role for this assay in real-world clinical decision making. The authors performed a single-center retrospective cohort study of 80 adult patients for whom a mcfDNA (Karius©) test was sent between May 2019 and February 2021. The most common reason for sending the assay was unknown microbiologic diagnosis (78%), followed by avoiding invasive procedures (14%). Categorical variables were reported using frequency and percentages. Mean ± standard deviation of age was reported and days of hospitalization reported using median and quartiles. Comparative analysis of categorical variables was conducted by the Fisher’s exact test or Chi-square test as appropriate, and days of hospitalization was compared using Mann–Whitney U test. Statistical tests were performed using SPSS (IBM SPSS Statistics for Windows, Version 27.0. Armonk, NY: IBM Corp) with p-values ≤ 0.05 as the significance threshold. The test had a positive impact in 34 (43%), a negative impact in 2 (3%), and uncertain or no impact in 44 (55%). A positive impact was observed in solid organ transplant recipients (SOTR, 71.4%, p = 0.003), sepsis (71.4%, p = 0.003), and those receiving antimicrobial agents for less than 7 days prior to mcfDNA testing (i.e., 61.8%, p = 0.004). Positive impact was driven primarily by de-escalation of antimicrobial therapy. The authors concluded that clinical impact of mcfDNA testing was highest in SOTR, patients with sepsis and patients who had been on antimicrobial therapy for less than 7 days. Positive impact was driven by de-escalation of antimicrobial therapy which may highlight a potential role for mcfDNA in the realm of stewardship. The authors note that as a descriptive retrospective study, the data were uncontrolled with a heterogenous patient population and lacked standard comparison to conventional testing. They were unable to incorporate patient outcomes (i.e., mortality) into the final analysis. Additionally, the retrospective nature of the study does not allow for control of the timing of testing and patient and disease characteristics, therefore there was considerable variability among factors and many underrepresented patient populations and diseases. Additionally, the small sample size and retrospective nature allow for only hypothesis-generating conclusions to be made.
Metagenomic Next-Generation Sequencing for CNS Infections
Zhang et al (2020) state that diagnostic value of metagenomic next-generation sequencing (mNGS), an emerging powerful platform, remains to be studied in CNS infections. The authors conducted a single-center prospective cohort study to compare mNGS with conventional methods including culture, smear and etc. The study included 248 suspected CNS infectious patients. The authors found that mNGS reported a 90% (9/10) sensitivity in culture-positive patients without empirical treatment and 66.67% (6/9) in empirically-treated patients. Detected an extra of 48 bacteria and fungi in culture-negative patients, mNGS provided a higher detection rate compared to culture in patients with (34.45% vs. 7.56%, McNemar test, p < 0.0083) or without empirical therapy (50.00% vs. 25.00%, McNemar test, p > 0.0083). Compared to conventional methods, positive percent agreement and negative percent agreement was 75.00% and 69.11% separately. The authors found that mNGS detection rate was significantly higher in patients with cerebrospinal fluid (CSF) WBC > 300 * 106/L, CSF protein greater than 500 mg/L or glucose ratio less than or equal to 0.3. mNGS sequencing read is correlated with CSF WBC, glucose ratio levels and clinical disease progression. The authors concluded that mNGS showed a satisfying diagnostic performance in CNS infections and had an overall superior detection rate to culture. mNGS may hold diagnostic advantages especially in empirically treated patients. CSF laboratory results were statistically relevant to mNGS detection rate, and mNGS could dynamically monitor disease progression. The authors noted limitations such as their study had a relatively small sample size of viral, fungal and parasitic CNS infections and therefore could not yet come to conclusions about the diagnostic value of mNGS in these groups. Second, they used preliminary data for analysis but still lack of health control simultaneously. Also, a bactec microbial detection system for CSF culture and novel optimized laboratory and statistical methods for mNGS could be applied to raise positivity. What’s more, RNA library preparations were conducted in a limited number of patients, which might neglect some neuro-invasive RNA viruses. Further, as the mNGS results may be easily influenced by many factors, the standards in their single center cross-section study should be thoroughly modified and tested before applying to other centers.
Xing et al (2020) conducted a prospective multicenter to assess the performance of metagenomic next-generation sequencing (mNGS) in the diagnosis of infectious encephalitis and meningitis. Cerebrospinal fluid samples from patients with viral encephalitis and/or meningitis, tuberculous meningitis, bacterial meningitis, fungal meningitis, and non-central nervous system (CNS) infections were subjected to mNGS. The study included a total of 213 patients with infectious and non-infectious CNS diseases. The authors found that the mNGS-positive detection rate of definite CNS infections was 57.0%. At a species-specific read number (SSRN) greater than or equal to 2, mNGS performance in the diagnosis of definite viral encephalitis and/or meningitis was optimal (area under the curve [AUC] = 0.659, 95% confidence interval [CI] = 0.566-0.751); the positivity rate was 42.6%. At a genus-specific read number greater than or equal to 1, mNGS performance in the diagnosis of tuberculous meningitis (definite or probable) was optimal (AUC=0.619, 95% CI=0.516-0.721); the positivity rate was 27.3%. At SSRNs greater than or equal to 5 or 10, the diagnostic performance was optimal for definite bacterial meningitis (AUC=0.846, 95% CI = 0.711-0.981); the sensitivity was 73.3%. The sensitivities of mNGS (at SSRN greater than or equal to 2) in the diagnosis of cryptococcal meningitis and cerebral aspergillosis were 76.92 and 80%, respectively. The authors concluded that mNGS of cerebrospinal fluid effectively identifies pathogens causing infectious CNS diseases. mNGS should be used in conjunction with conventional microbiological testing. However, the authors noted several limitations in this study. Firstly, RNA-Seq data were not tested in parallel with DNA sequencing, which might provide valuable complementary information. Furthermore, because DNA extraction efficiency is critical in terms of mNGS results, a comparison of the extraction efficiencies of the various kits must be performed in future studies. Finally, the sample size was relatively small, especially after stratification of patients according to the types of infections. The authors state that the new technology exhibits great potential; however, careful attention is needed with respect to DNA and RNA co-extraction methods, extraction efficiency, differentiation of colonization from infection, and method standardization.
Zhu et al (2022) state that it is not well-understood whether mNGS has comparable sensitivity to target-dependent nucleic acid test for pathogen identification. The authors evaluated 31 patients with chickenpox and neurological symptoms for screening of possible varicella-zoster virus (VZV) central nervous system (CNS) infection in a single-center hospital in China. Microbiological diagnosing of VZV cerebrospinal fluid (CSF) infection was performed on stored CSF samples using mNGS, quantitative and qualitative VZV-specific PCR assays, and VZV IgM antibodies test. The authors found that about 80.6% of the patients had normal CSF white blood cell counts (≤ 5 × 106/L). VZV IgM antibodies presented in 16.1% of the CSF samples, and nucleic acids were detectable in 16.1 and 9.7% using two different VZV-specific real-time PCR protocols. Intriguingly, maximal identification of VZV elements was achieved by CSF mNGS (p = 0.001 and p = 007; compared with qualitative PCR and VZV IgM antibody test, respectively), with sequence reads of VZV being reported in 51.6% (16/31) of the CSF samples. All VZV PCR positive samples were positive when analyzed by mNGS. Of note, human beta herpes virus 6A with clinical significance was unexpectedly detected in one CSF sample. The authors concluded that their study suggests that CSF mNGS may have higher sensitivity for VZV detection than CSF VZV PCR and antibody tests, and has the advantage of identifying unexpected pathogens. However, although the sensitivity of mNGS can be further increased by technical innovation, the specificity will continue to be a great concern. Clinical judgement of the treating physicians is very important for interpreting the results.
Mayo Clinic Laboratories Metagenomic Sequencing, CSF (also known as the MSCSF Test)
According to the Mayo Clinic Laboratories, the Metagenomic Next-Generation Sequencing test detects and identifies bacteria, DNA and RNA viruses, fungi, and parasites in the CSF using NGS. The target population is patients with suspected, but undiagnosed, CNS infection, which is a potentially life-threating condition that requires rapid diagnosis and clinical treatment. Infections of the CNS have broad pathogen etiology, including bacteria, fungi, viruses, and parasites. The breadth of causative agents challenges diagnostic test ordering and pathogen identification. Current clinical diagnostic methods, such as culture and specific-PCR assays, have limitations in the ability to detect non-viable organisms, or nucleic acids (NAs) that are not targeted by specific assays, respectively. An unbiased metagenomic sequencing approach may overcome diagnostic test limitations by interrogating microbiota without bias towards any specific microorganisms. Bioinformatic analysis of the resultant large sequencing dataset enables identification of a diversity of pathogens in this assay. The test can identify multiple pathogens in a single specimen if present. This test is not recommended as a test of cure because NAs may persist after successful treatment.
Rodino et al (2020) noted that shotgun metagenomic sequencing can detect NAs from bacteria, fungi, viruses, and/or parasites in clinical specimens; however, little data exist to guide its optimal application to clinical practice. In a retrospective study, these investigators examined results of shotgun metagenomic sequencing testing requested on CSF samples submitted to an outside reference laboratory from December 2017 through December 2019. Of the 53 samples from Mayo Clinic patients, 47 were requested by neurologists, with infectious diseases consultation in 23 cases. The majority of patients presented with difficult-to-diagnose sub-acute or chronic conditions. Positive results were reported for 9 (17 %) Mayo Clinic patient samples, with 6 interpreted as likely contamination. Potential pathogens reported included bunyavirus, human herpesvirus 7 (HHV-7), and enterovirus D-68, ultimately impacting care in 2 cases. Another 27 samples were submitted from Mayo Clinic Laboratories reference clients, with positive results reported for 3 (11 %): 2 with potential pathogens (West Nile virus and Toxoplasma gondii) and 1 with Streptococcus species with other bacteria below the reporting threshold (considered to represent contamination). Of 68 negative results, 10 included comments on decreased sensitivity due to high DNA background (n = 5), high RNA background (n = 1), insufficient RNA read depth (n = 3), or quality control (QC) failure with an external RNA control (n = 1). The overall positive-result rate was 15 % (12/80), with 58 % (7/12) of these interpreted as being inconsistent with the patient's clinical presentation. The authors concluded that overall, potential pathogens were found in a low percentage of cases, and positive results were often of unclear clinical significance. Testing was commonly employed in cases of diagnostic uncertainty and when immunotherapy was being considered.
Erdem et al (2021) stated that metagenomic NGS offers an unbiased approach for identifying viral pathogens in CSF of patients with meningoencephalitis of unknown etiology. In an 11-month, case-series study, these investigators examined the use of CSF mNGS to diagnose viral infections among pediatric hospitalized patients presenting with encephalitis or meningoencephalitis of unknown etiology – CSF from patients with known enterovirus meningitis were included as positive controls; and CSF from patients with primary intra-cranial hypertension were included to serve as controls without known infections. Cerebrospinal fluid mNGS was carried out for 37 patients. Among 27 patients with encephalitis or meningoencephalitis, 4 were later diagnosed with viral encephalitis, 6 had non-CNS infections with CNS manifestations, 6 had no positive diagnostic tests, and 11 were found to have a non-infectious diagnosis. Metagenomic NGS identified West Nile virus (WNV) in the CSF of 1 immunocompromised patient. Among the 4 patients with known enterovirus meningitis, mNGS correctly identified enteroviruses and characterized the viral genotype. No viral sequences were detected in the CSF of patients with primary intra-cranial hypertension. Metagenomic NGS also identified sequences of non-pathogenic torque Teno virus in CSF specimens from 13 patients. The authors concluded that the findings of this study showed viral detection by CSF mNGS only in 1 immunocompromised patient and did not offer a diagnostic advantage over conventional testing. Viral phylogenetic characterization by mNGS could be used in epidemiologic investigations of some viral pathogens, such as enteroviruses. The finding of torque Teno viruses in CSF by mNGS is of unknown significance, but may merit further investigations for a possible association with non-infectious CNS disorders.
Qu et al (2022) noted that it is widely acknowledged that CNS infection is a serious infectious disease accompanied by various complications; however, the accuracy of current detection methods is limited, resulting in delayed diagnosis and treatment. In recent years, mNGS has been increasingly adopted to improve the diagnostic yield. In a systematic review and meta-analysis, these researchers examined he value of mNGS in CNS infection diagnosis. Following the Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) 2022 guidelines, these investigators searched relevant articles published in 7 databases, including PubMed, Web of Science, and Cochrane Library, published from January 2014 to January 2022. High-quality articles related to mNGS applications in the CNS infection diagnosis were included. The comparison between mNGS and the gold standard of CNS infection, such as culture, PCR or serology, and microscopy, was carried out to obtain true positive (TP), true negative (TN), false positive (FP), and false negative (FN) values, which were extracted for sensitivity and specificity calculation. A total of 272 related studies were retrieved and strictly selected according to the inclusion and exclusion criteria; 12 studies were included for meta-analysis and the pooled sensitivity was 77 % (95 % CI: 70 % to 82 %, I2 = 39.69 %), and specificity was 96 % (95 % CI: 93 % to 98 %, I2 = 72.07 %). Although no significant heterogeneity in sensitivity was observed, a sub-group analysis was performed based on the pathogen, region, age, and sample pre-treatment method to ascertain potential confounders. The AUC of the summary ROC (SROC) of mNGS for CNS infection was 0.91 (95 % CI: 0.88 to 0.93). Besides, Deek's Funnel Plot Asymmetry Test indicated no publication bias in the included studies (p > 0.05). The authors concluded that overall, mNGS exhibited good sensitivity and specificity for diagnosing CNS infection and diagnostic performance during clinical application by assisting in identifying the pathogen. Moreover, these researchers stated that the effectiveness remained inconsistent, warranting subsequent studies for further performance improvement during its clinical application. These investigators stated that mNGS has great promise for clinical application. Furthermore, the most appropriate patient population should be identified and the entire process standardized from sample collection to data analysis to further improve its clinical effectiveness.
Chen et al (2022) stated that early diagnosis of tuberculosis meningitis (TBM) remains a great challenge during clinical practice. The diagnostic effectiveness of CSF-based mycobacterial growth indicator tube (MGIT) culture, modified Ziehl-Neelsen (ZN) staining, Xpert MTB/RIF, and mNGS for TBM remained elusive. In a retrospective, multi-cohort study, these researchers examined the diagnostic performances for MGIT, modified ZN staining, Xpert MTB/RIF, and mNGS using CSF samples. A total of 216 adult patients with suspicious TBM enrolled in this trial. Uniform clinical case definition classified 88 (40.7 %) out of 216 patients as definite TBM, 5 (2.3 %) patients as probable TBM cases, and 24 (11.1 %) patients as possible TBM cases. The sensitivities of MGIT, modified ZN staining, Xpert MTB/RIF, and mNGS for TBM diagnosis against consensus uniform case definition for definite TBM were 25.0 %, 76.1 %, 73.9 %, and 84.1 %, respectively. NPVs were 66.0 %, 85.9 %, 84.8 %, and 90.1 %, respectively. The sensitivities of MGIT, modified ZN staining, Xpert MTB/RIF, and mNGS for TBM diagnosis against consensus uniform case definition for definite, probable, and possible TBM were 18.8 %, 57.3 %, 55.5 %, and 63.2 %, respectively. NPVs) were 51.0 %, 66.4 %, 65.6 %, and 69.7 %, respectively. mNGS combined with modified ZN stain and Xpert could cover TBM cases against a composite microbiological reference standard, yielding 100 % specificity and 100 % NPV. The authors concluded that mNGS detected TBM with higher sensitivity than Xpert, ZN staining and MGIT culture; however, mNGS could not be used as a rule-out test. mNGS combined with Xpert or modified ZN staining could enhance the sensitivity of diagnostic tests for TBM.
The authors stated that this study had 2 main drawbacks. First, this trial was unable to examine the diagnostic effectiveness of Xpert Ultra; further studies are needed to compare the Xpert Ultra to mNGS in term of TBM diagnosis using CSF samples. Second, these investigators did not include the analysis of the treatment history, which might hamper the sensitivities and effectiveness for Xpert and culture-based assay.
Xiang et al (2023) noted that the use of mNGS in the diagnosis of TBM remains uncertain. In a systematic review and meta-analysis, these investigators examined the diagnostic accuracy of mNGS for the early diagnosis of TBM. English (PubMed, Medline, Web of Science, Cochrane Library, and Embase) and Chinese (CNKI, Wanfang, and CBM) databases were searched for relevant studies examining the diagnostic accuracy of mNGS for TBM. Review Manager was used to evaluate the quality of the included studies, and Stata was used to conduct the statistical analysis. Of 495 relevant articles retrieved, 8 studies involving 693 participants (348 with and 345 without TBM) met the inclusion criteria and were included in the meta-analysis. The pooled sensitivity, specificity, PLR, NLR, diagnostic odds ratio (DOR), and AUC of the SROC of mNGS for diagnosing TBM were 62 % (95 % CI: 0.46 to 0.76), 99 % (95 % CI: 0.94 to 1.00), 139.08 (95 % CI: 8.54 to 2266), 0.38 (95 % CI: 0.25 to 0.58), 364.89 (95 % CI: 18.39 to 7239), and 0.97 (95 % CI: 0.95 to 0.98), respectively. The authors concluded that available evidence showed that mNGS has good specificity for the diagnosis of TBM; however, its sensitivity is moderate. The high requirements for laboratory infra-structure and high cost, make mNGS unsuitable for use as an initial test for TBM in the short-term. However, it should be used as an effective pathogen-screening method to diagnose patients with negative results to microbiological tests, failure of empirical therapy, and critical illness. Moreover, these researchers stated that due to the limited quality and quantity of the included studies, these conclusions need to be interpreted with caution. They stated that more high-quality prospective, large, multi-center studies are needed to confirm the diagnostic value of mNGS for TBM in a more comprehensive, systematic, scientific, and objective manner.
He et al (2024) stated that detecting pathogens in pediatric CNS infection (CNSI) is still a major challenge in medicine. In addition to conventional diagnostic patterns, mNGS shows great potential in pathogen detection. In a systematic review and meta-analysis, these investigators examined the diagnostic performance of mNGS in CSF in pediatric patients with CNSI. Related literature was searched in the Web of Science, PubMed, Embase, and Cochrane Library. These investigators screened the literature and extracted the data according to the selection criteria. The quality of included studies was assessed by the Quality Assessment of Diagnostic Accuracy Studies-2 (QUADAS-2) tool and the certainty of the evidence was measured by the Grading of Recommendations, Assessment, Development, and Evaluations (GRADE) score system. Then, the pooled sensitivity, specificity, PLR, NLR, DOR, AUC of the SROC were estimated in Stata Software and MetaDisc. Subgroup analyses were carried out to examine the potential factors that influence the diagnostic performance. A total of 10 studies were included in the meta-analysis. The combined sensitivity was 0.68 (95 % CI: 0.59 to 0.76, I2 = 66.77 %, p < 0.001), and the combined specificity was 0.89 (95 % CI: 0.80 to 0.95, I2 = 83.37 %, p < 0.001). The AUC of SROC was 0.85 (95 % CI: 0.81 to 0.87). The quality level of evidence assessed by the GRADE score system was low. The authors concluded that available evidence showed that mNGS presents a good diagnostic performance in pediatric CNSI. Moreover, these researchers stated that due to the limited quality and quantity of the included studies, more high-quality studies are needed to validate the above conclusion.
MicroGenDX Tests
MicroGenDX offers qPCR+NGS testing on a wide selection of specimen types for identifying mycobacteria, fungi, anaerobes, and microbes that can result in infectious disease. MicroGenDX combines multiplex quantitative (qPCR) with targeted 16S/ITS NGS and matches samples against a curated database of over 50,000 microbial species. MicroGenDX’s qPCR+NGS results identify all potentially pathogenic microbial taxa, along with their clinically relevant distribution in the sample, in 3.5 days. MicroGenDX is also able to provide shotgun metagenomics.
McDonald and colleagues (2017) conducted a prospective, randomized, open-label, controlled, head-to-head comparative phase II study of standard urine culture and sensitivity (C&S) versus DNA NGS testing (MicroGenDX test) for the diagnosis and treatment efficacy in patients with symptoms of acute cystitis based on short-term outcomes. A total of 44 patients and 22 control subjects completed the study, over 14 days. Patients were randomized to receive treatment based on culture results (Arm A, n=22) or treatment based on DNA NGS test results (Arm B, n=22). In total, 13 of 44 patients (30%) had positive urine culture results whereas 44 of 44 patients had positive DNA NGS results. Of the 22 control subjects, 5 had positive urine culture results and 21 of 22 had positive DNA NGS results. The five subjects with positive C&S and DNA NGS findings all had similar organisms. However, in three of these subjects, DNA NGS results reported two or more organisms in addition to the common one. On a head-to-head comparison, symptom scores were significantly better for those patients whose treatment was based on DNA NGS versus traditional C&S. Patients treated in Arm A, Subset 2, (culture-negative, DNA NGS-positive) improved with respect to symptom scores when they started treatment on day 8. The authors concluded that DNA NGS may help when diagnosing and treating symptoms of acute cystitis, especially when urine culture results are negative. However, the authors note that a significant limitation of the study is the small sample size, with low statistical power of the results. A larger study needs to be done with a bigger sample size to achieve more robust conclusions of this promising study.
Tarabichi and colleagues (2018) conducted a prospective, single-blinded study to assess the use of NGS (MicroGenDX) for detecting organisms in synovial fluid. Eighty-six anonymized samples of synovial fluid were obtained from patients undergoing aspiration of the hip or knee as part of the investigation of a periprosthetic infection. A panel of synovial fluid tests, including levels of C-reactive protein, human neutrophil elastase, total neutrophil count, alpha-defensin, and culture were performed prior to next-generation sequencing. Of these 86 samples, 30 were alpha-defensin-positive and culture-positive (Group I), 24 were alpha-defensin-positive and culture-negative (Group II) and 32 were alpha-defensin-negative and culture-negative (Group III). The authors found that among the 30 culture-positive samples in Group I, NGS detected at least 1 organism in 26 of the samples. In 25 (96%) of these, there was concordance between the bacteria detected in culture and the predominant organism detected by NGS sequencing. In another four samples with relatively low levels of inflammatory biomarkers, culture was positive but NGS was negative. A total of ten samples had a positive NGS result and a negative culture. In five of these, alpha-defensin was positive and the levels of inflammatory markers were high. In the other five, alpha-defensin was negative and the levels of inflammatory markers were low. While NGS detected several organisms in each sample, in most samples with a higher probability of infection, there was a predominant organism present, while in those presumed not to be infected, many organisms were identified with no predominant organism. The authors concluded that pathogens causing periprosthetic infection in both culture-positive and culture-negative samples of synovial fluid could be identified by NGS. The authors note that a limitation to the study may be the lack of clinical information about these patients as the samples were retrieved from an anonymized reservoir and therefore not able to determine whether the patients were infected or not. The study did not evaluate another molecular technique in parallel with NGS. The authors only tested synovial fluid, and hence a direct comparison to tissue or other samples could not be made. The authors state that their findings suggest that NGS holds great promise for the detection of potential pathogens from the synovial fluid of patients with a periprosthetic infection.
Haider et al (2019) state that high-throughput DNA sequencing of the paranasal sinus microbiome has potential in the diagnosis and treatment of sinusitis. The author conducted a case series chart review at a single tertiary care academic medical center to evaluate the use of high-throughput DNA sequencing to diagnose sinusitis of odontogenic origin. A chart review was performed of DNA sequencing results from the sinus aspirates obtained under endoscopic visualization in 142 patients with sinusitis. The identification of any potentially pathogenic bacteria associated with oral flora in a sample was classified as a positive result for sinusitis of odontogenic etiology. The sensitivity, specificity, and predictive values of using high-throughput DNA sequencing to diagnose sinusitis of odontogenic etiology were determined, with the patient's computed tomography sinus scan as the reference standard. On computed tomography scans, an odontogenic source was determined by the presence of a periapical lucency perforating the Schneiderian membrane. The authors found that 7 of the 142 patients enrolled in this study had an odontogenic source based on computed tomography scans. Relative to this reference standard, high-throughput DNA sequencing produced a sensitivity of 85.7% (95% CI, 42.1%-99.6%), a specificity of 81.5% (95% CI, 73.9%-87.6%), a positive predictive value of 19.4% (95% CI, 13.1%-27.7%), and a negative predictive value of 99.1% (95% CI, 94.7%-99.9%). The authors concluded that this study supports the use of high-throughput DNA sequencing in supplementing other methods of investigation for identifying an odontogenic etiology of sinusitis. The authors noted that there are several limitations noted in the current study. First, CT evidence of an odontogenic source was used as the diagnostic standard for determining odontogenic sinusitis. This may underestimate the number of cases of odontogenic sinusitis, since not all cases will manifest with apparent radiographic findings. This in turn might skew sensitivity and specificity calculations. Second, the retrospective design of this study limits the use of other criteria for the confirmatory diagnosis of odontogenic sinusitis. A prospective study addressing this question might better combine physical examination and dental consultation, with CT images, in establishing a gold standard for the diagnosis of odontogenic sinusitis. The authors state that despite these limitations, high-throughput DNA sequencing of sinus cavity purulence may offer a sensitive test for identifying patients who would benefit from additional investigation for odontogenic sinusitis, and that the results of this study lay the groundwork for future studies that examine the use of high-throughput DNA sequencing in the diagnosis and treatment of sinusitis.
Dixon and colleagues (2020) conducted an extensive review and analysis of the available literature on the topic of metagenomic sequencing in urological science. The search yielded a total of 406 results, and manual selection of appropriate papers was subsequently performed. Only one randomised clinical trial comparing metagenomic sequencing to standard culture and sensitivity in the arena of urinary tract infection was found. The authors concluded that their paper explores the limitations of traditional methods of culture and sensitivity and delves into the recent studies involving new high-throughput genomic technologies in urological basic and clinical research, demonstrating the advances made in the urinary microbiome in its entire spectrum of pathogens and the first attempts of clinical implementation in several areas of urology. Finally, this paper discusses the challenges that must be overcome for such technology to become widely used in clinical practice. The authors state that "although NGS is at an early stage of its development, its ability to quickly detect and identify the entire spectrum of microbes present within a sample with accuracy, and its capacity to predict phenotypic resistance patterns via genomic data proves its superiority to the slower, traditional methods of culture and sensitivity. However, at this point in time, there are still limitations in precisely defining leading pathogen(s) which can contribute to the development of UTI and sufficiently distinguish them from other contaminating or commensal strains. The implementation of NGS in clinical laboratories will certainly demand a great deal of careful thought to ensure patient confidentiality while simultaneously storing data in a manner which will optimize public benefit".
Goswami et al (2022) state that NGS technology, including 16S rRNA gene bacterial profiling, is an emerging diagnostic modality that is showing promise for detecting and precisely identifying a wide spectrum of microbial DNA present within a clinical sample. The authors conducted a prospective multicenter study to investigate the application of NGS pathogen detection to nonunion fracture. Samples were collected from 54 patients undergoing open surgical intervention for preexisting long-bone nonunion (n = 37) and control patients undergoing fixation of an acute fracture (n = 17). Intraoperative specimens were sent for dual culture and 16S rRNA gene-based microbial profiling using MicroGenDX NGS test. DNA was extracted and amplified via a PCR using forward and reverse primers flanking the 16S ribosomal rRNA gene. The amplified DNA was then sequenced on an Illumina MiSeq platform. Sequence reads were then denoised to remove short sequences and clustered into operational taxonomic units (OTUs). OTUs were then assigned taxonomy using a MicroGenDX curated taxonomic reference database. For clinical scoring of positive and negative samples by 16S rRNA gene sequencing, samples were required to pass amplification and pass an internally validated reporting threshold of at least more than 1000 classifiable microbial reads after quality control. This positive/negative score was used for evaluating utility against nonunion groups. In the study, patients were followed for a minimum of 6 months using comparative analyses aimed to determine whether microbial NGS diagnostics could discriminate between non-unions that healed during follow-up versus persistent nonunion. Among the 37 patients undergoing open surgical intervention, 22 had achieved union by 6 months and 15 were considered to have persistent non-unions. Of those that had persistent nonunion, 10 patients (67%) had a positive NGS test and 5 (33%) had a negative test. The authors concluded that positive NGS detection was significantly correlated with persistent non-union (p = 0.048), and suggest that the fracture-associated microbiome may be a significant risk factor for persistent nonunion. The authors note limitations to their study. The study included a small sample size and did not use other molecular techniques in parallel with NGS, thus no direct comparisons can be made between NGS and other molecular techniques studied. Furthermore, the study did not correlate the observed NGS signal with subsequent clinical outcomes and determine whether these pathogens indeed escape detection by culture and are implicated in subsequent failure to reach union. Thus, future work is needed to establish the optimal sampling methodology and bioinformatic quality control for reporting of clinical NGS data.
Multiplex Immunoassay Tests
Generally, the multiplex immunoassay platform applies technology to simultaneously measure multiple target analytes in a single biological sample. There are various types of multiplex immunoassays in use, or being developed, for identifying bacterial antigens in infectious disease.
MeMed BV
The MeMed BV (MeMed Diagnostics, Ltd.) is a blood test, used in conjunction with the MeMed Key analyzer, that simultaneously evaluates three independent immunoassays and quantitatively measures three host biomarkers (C-reactive protein [CRP], interferon γ-induced protein 10 [IP-10], and TNF-related apoptosis-inducing ligand [TRAIL]) to produce a host response score (ranging from 0 to 100) for differentiating between bacterial and viral infection.
In order to increase diagnostic accuracy and achieve better treatment guidance of infectious disease, Oved and colleagues (2015) developed and validated a promising signature that combines novel and traditional host-proteins for differentiating between bacterial and viral infections. The authors state that bacterial-induced host proteins such as procalcitonin, C-reactive protein (CRP), and Interleukin-6, are routinely used to support diagnosis of infection. However, their performance is negatively affected by inter-patient variability, including time from symptom onset, clinical syndrome, and pathogen species. The authors initially conducted a "bioinformatic screen to identify putative circulating host immune response proteins. The resulting 600 candidates were then quantitatively screened for diagnostic potential using blood samples from 1002 prospectively recruited patients with suspected acute infectious disease and controls with no apparent infection. For each patient, three independent physicians assigned a diagnosis based on comprehensive clinical and laboratory investigation including PCR for 21 pathogens yielding 319 bacterial, 334 viral, 112 control and 98 indeterminate diagnoses; 139 patients were excluded based on predetermined criteria". The authors report that the best performing host-protein was TNF-related apoptosis-inducing ligand (TRAIL) (area under the curve [AUC] of 0.89; 95% confidence interval [CI], 0.86 to 0.91), which was consistently up-regulated in viral infected patients. The authors "further developed a multi-protein signature using logistic-regression on half of the patients and validated it on the remaining half. The signature with the highest precision included both viral- and bacterial-induced proteins: TRAIL, Interferon gamma-induced protein-10, and CRP (AUC of 0.94; 95% CI, 0.92 to 0.96)". The authors report that the signature was superior to any of the individual proteins (p < 0.001), as well as routinely used clinical parameters and their combinations (p<0.001). The authors state that it remained robust across different physiological systems, times from symptom onset, and pathogens (AUCs 0.87-1.0). The authors conclude that "accurate differential diagnosis provided by this novel combination of viral- and bacterial-induced proteins has the potential to improve management of patients with acute infections and reduce antibiotic misuse". The authors do note a potential limitation of the study is the heterogeneity of the patient cohort (multiple clinical syndromes, pathogen species, and time from onset of symptoms). The diverse cohort makes it more challenging to control for confounding factors. Although they did not identify significant confounders, follow-up studies on homogenous subgroups are warranted. In addition, follow-up time course studies that assess whether the signature can predict response to treatment and patient prognosis are also warranted.
Eden et al (2016) describe a sub-study (of the Curiosity study) which evaluated the diagnostic accuracy of a novel host-biomarker assay (TRAIL, IP-10, and CRP) for discriminating bacterial and viral etiologies in a sub-population of the emergency department (ED). The reference standard was based on microbiological confirmation plus adjudication by an expert panel after review of all participant clinical, laboratory, radiological, microbiological and follow-up data. A “true diagnosis” required positive microbiological confirmation plus a unanimous expert panel, i.e., all three panel members independently assigned bacterial or viral etiology. The expert panel was blinded to the test result and test performers were blinded to the reference standard. Of the 744 participants that met the Curiosity study infectious disease inclusion criteria, 428 participants with suspected infections were recruited at the ED, of which 155 had a confirmed etiology (128 viral and 27 bacterial [or mixed co-infection]). The authors state that the combinatorial signature of all three biomarkers exhibited the greatest diagnostic accuracy, yielding a sensitivity of 96% [95% confidence interval: 78%, 100%] and specificity of 93% [87%, 97%], significantly better than the individual proteins. Furthermore, the signature outperformed routine lab parameters such as white blood cell count (sensitivity 56% [35%, 75%] and specificity 84% [77%, 90%]; cut-off 15,000 cells/ml) and absolute neutrophil count (sensitivity 59% [39%, 78%] and specificity 88% [81%, 93%]; cut-off 10,000 cells/ml). The authors concluded that the diagnostic performance data support that a host-biomarker signature comprising TRAIL, IP-10 and CRP represents a promising new tool for aiding ED clinicians in determining the bacterial versus viral etiology of infectious disease; however, future clinical studies are required to examine the usefulness of this host biomarker signature in safely decreasing unnecessary antibiotic prescription at the ED.
ImmunoXpert (MeMed) is a novel assay combining three proteins: TRAIL, IP-10, and CRP. Investigators, van Houten et al (2017), conducted a prospective, double-blind, international, multicenter study (OPPORTUNITY) to externally validate the diagnostic accuracy of this novel assay in differentiating between bacterial and viral infections and to compare this test with commonly used biomarkers in children with lower respiratory tract infection or clinical presentation of fever without source. The investigators recruited 777 children, of whom 577 were assessed. The investigators identified majority diagnosis when two of three panel members agreed on a diagnosis and unanimous diagnosis when all three panel members agreed on the diagnosis. The investigators calculated the diagnostic performance (i.e., sensitivity, specificity, positive predictive value, and negative predictive value) of the index test in differentiating between bacterial (index test positive) and viral (index test negative) infection by comparing the test classification with the reference standard outcome. The investigators found that the majority of the panel diagnosed 71 cases as bacterial infections and 435 as viral infections. In another 71 patients there was an inconclusive panel diagnosis. The assay distinguished bacterial from viral infections with a sensitivity of 86.7%, a specificity of 91.1%, a positive predictive value of 60.5%, and a negative predictive value of 97.8%. In the more clear cases with unanimous panel diagnosis (n=354), sensitivity was 87.8%, specificity 93.0%, positive predictive value 62.1%, and negative predictive value 98.3%. The investigators concluded that this diagnostic assay based on CRP, TRAIL, and IP-10 has the potential to reduce antibiotic misuse in young children.
A commentary by Esposito and Principi (2017) on the OPPORTUNITY study (van Houten et al, 2017) notes some study limitations that preclude its routine use in clinical practice, such as: the test requires advanced laboratory techniques not used outside a hospital setting, collected data was obtained from small sample size for which none had an underlying disease that might modify host response to infection, the definition of cause of infection used in studies that have tried to differentiate bacterial and viral infection varies, and respiratory infections are frequently classified on the basis of clinical and radiological findings and results of a microbiological assessment of nasopharyngeal swabs. The authors state that "it is well known that the investigation into upper respiratory secretions in children can be confounding and lead to the erroneous classification of a lower respiratory disease, and that bacteria and viruses can simply be carried and could have no association with the cause of a disease". The authors conclude that future studies using a larger study population with various characteristics are needed to confirm the results of host protein-based assays.
Srugo et al (2017) performed a double-blind, multicenter evaluation of a novel assay that integrates host-proteins (TRAIL, IP-10, and CRP) for differentiation between bacterial and viral disease in febrile children. The cohort included 361 pediatric patients, with 239 viral, 68 bacterial, and 54 indeterminate reference standard diagnoses. The reference standard diagnosis was based on predetermined criteria plus adjudication by experts blinded to assay results. Assay performers were blinded to the reference standard. Assay cutoffs were predefined. The authors found that the assay distinguished between bacterial and viral patients with 93.8% sensitivity (95% confidence interval: 87.8%-99.8%) and 89.8% specificity (85.6%-94.0%); 11.7% had an equivocal assay outcome. The assay outperformed CRP (cutoff 40 mg/L; sensitivity 88.2% [80.4%-96.1%], specificity 73.2% [67.6%-78.9%]) and procalcitonin testing (cutoff 0.5 ng/mL; sensitivity 63.1% [51.0%-75.1%], specificity 82.3% [77.1%-87.5%]). The authors concluded that their evaluation confirmed high assay performance in febrile children, and that the assay was significantly more accurate than CRP, procalcitonin, and routine laboratory parameters. However, additional studies are warranted to support its potential to improve antimicrobial treatment decisions.
A commentary by Kimberlin and Poole (2017) review the study conducted by Srugo et al (2017) on the novel assay that integrates host-proteins (TRAIL, IP-10, and CRP) for differentiation between bacterial and viral disease in febrile children (ImmunoXpert assay, MeMed Diagnostics, Ltd.). The authors point out that a number of confirmatory investigations are required, as the published studies that have assessed the ImmunoXpert assay have used specimens that were frozen at -80 degrees Celsius; thus, prospective trial designs to determine the performance characteristics of the test in a "more real-world manner", which includes use of refrigerated specimens, are warranted. Furthermore, the authors agree that the assay needs to also be assessed in the population of infants less than 3 months of age, as well as immunocompromised children, which has a need for improved diagnostics to drive decision-making in an evidence-based fashion and can reliably distinguish bacterial from viral infections. The authors state that "If the assay is validated in these future studies, performance of randomized trial designs that assess how knowledge of the assay result impacts clinical care should be considered, as has been done with influenza testing". The authors acknowledge that the work of Srugo et al substantially advances the opportunity to one day be able to more accurately assess bacterial infections.
Ashkenazi-Hoffnung and colleagues (2018) conducted a prospective observational study to compare the diagnostic performance of a host-protein signature (comprising of TRAIL, IP-10 and CRP) to other biomarkers for differentiating between bacterial and viral disease in children and adults with respiratory infection and fever without source. Comparator method was based on expert panel adjudication. Signature and biomarker cutoffs and prediction rules were predefined. Of 493 potentially eligible patients, 314 were assigned unanimous expert panel diagnosis and also had sufficient specimen volume. The resulting cohort comprised 175 (56%) viral and 139 (44%) bacterial infections. Signature sensitivity 93.5% (95% CI 89.1–97.9%), specificity 94.3% (95% CI 90.7–98.0%), or both were found to be significantly higher (all p values < 0.01) than for CRP, procalcitonin, interleukin-6, human neutrophil lipocalin, white blood cell count, absolute neutrophil count, and prediction rules. Signature identified as viral 50/57 viral patients prescribed antibiotics, suggesting potential to reduce antibiotic overuse by 88%. The authors state that the host-protein signature demonstrated superior diagnostic performance in differentiating viral from bacterial respiratory infections and fever without source; however, future utility studies are warranted to validate potential to reduce antibiotic overuse.
Stein et al (2018) state that a novel host-protein assay outperforms routine parameters for distinguishing between bacterial and viral lower respiratory tract infections. The authors compared the diagnostic accuracy of a new assay that combines 3 host-biomarkers (TRAIL, IP-10, CRP) with parameters in routine use to distinguish bacterial from viral lower respiratory tract infections (LRTIs). The study cohort included 184 potentially eligible pediatric and adult patients. Reference standard diagnosis was based on adjudication by an expert panel following comprehensive clinical and laboratory investigation (including respiratory PCRs). Experts were blinded to assay results and assay performers were blinded to reference standard outcomes. The evaluated cohort included 88 bacterial and 36 viral patients (23 did not fulfill inclusion criteria; 37 had indeterminate reference standard outcome). The authors state that the assay distinguished bacterial from viral LRTI patients with sensitivity of 0.93±0.06 and specificity of 0.91±0.09, outperforming routine parameters, including WBC, CRP and chest x-ray signs. The authors conclude that these findings support the assay's potential to help clinicians avoid missing bacterial LRTIs or overusing antibiotics. The authors state that a key study limitation is the lack of a broadly applicable reference (i.e., “gold”) standard that can reliably discriminate between these etiologies. Expert panel diagnosis is widely employed in the absence of a gold standard, as it is in this study. Other study limitations include the age heterogeneity and relatively small sample size.
Carlton et al (2021) conducted a systematic review to assess the diagnostic accuracy of biomarker combinations to rapidly differentiate between acute bacterial or viral respiratory tract infections (RTI) etiology at the point-of-care in order to guide antibiotic treatment. Twenty observational studies (3514 patients) were identified. Eighteen were judged at high risk of bias. For bacterial etiologies, sensitivity ranged from 61 to 100 percent and specificity from 18 to 96 percent. For viral etiologies, sensitivity ranged from 59 to 97 percent and specificity from 74 to 100 percent. Studies evaluating two commercial tests were meta-analyzed. For ImmunoXpert, the summary sensitivity and specificity were 85 percent (95% CI 75%-91%, k = 4) and 86 percent (95% CI 73%-93%, k = 4) for bacterial infections, and 90 percent (95% CI 79%-96%, k = 3) and 92 percent (95% CI 83%-96%, k = 3) for viral infections, respectively. FebriDx had pooled sensitivity and specificity of 84 percent (95% CI 75%-90%, k = 4) and 93 percent (95% CI 90%-95%, k = 4) for bacterial infections, and 87 percent (95% CI 72%-95%; k = 4) and 82 percent (95% CI 66%-86%, k = 4) for viral infections, respectively. The authors concluded that combination of biomarkers show potential clinical utility in discriminating the etiology of RTIs; however, there are limitations due to a high proportion of studies with high risk of bias, which preclude firm conclusions. The authors state "current research is overshadowed with bias and is insufficient to make recommendations, especially in primary care where the evidence is entirely lacking", and that "future research should aim to grow the evidence base in primary care and experimentally evaluate patient outcomes and cost-effectiveness".
Basharat and Horton (2021) author the Horizon Scan which provides an overview of emerging point-of-care tests for differentiating bacterial and viral infections to health care stakeholders in Canada. The report includes review of rapid molecular tests and immunoassays such as MeMed's ImmunoXpert. The report also describes the evidence about the diagnostic accuracy of certain tests and their effect on reducing antibiotic prescribing. The systematic review by Carlton et al (2021) is included in the report. The authors concluded that the "emerging evidence suggests that point-of-care tests could be effective tools as part of antibiotic stewardship programs, but further studies assessing specific devices in randomized controlled trials are recommended by researchers and health technology assessment agencies. Monitoring the continued development of devices and the testing landscape, especially in post-pandemic health care, will be important for decision-makers".
Hainrichson et al (2023) evaluated the analytical performance of a new point-of-need platform for rapid and accurate measurement of a host-protein score that differentiates between bacterial and viral infection. The system comprises a dedicated test cartridge (MeMed BV) and an analyzer (MeMed Key). In each run, three host proteins (TRAIL, IP-10 and CRP) are measured quantitatively and a combinational score (0–100) computed that indicates the likelihood of Bacterial versus Viral infection (BV score). Serum samples collected from patients with acute infection representing viral (0 ≤ score < 35), equivocal (35 ≤ score ≤ 65), or bacterial (65 < score ≤ 100) scores based on pre-defined score cutoffs were employed for the analytical evaluation studies as well as samples from healthy individuals. To assess reproducibility, triplicate runs were conducted at 3 different sites, on 2 analyzers per site over 5 non-consecutive days. Lower limit of quantitation (LLoQ) and analytical measurement range were established utilizing recombinant proteins. Sample stability was evaluated using patient samples representative of BV score range (0–100). The authors state that the MeMed Key and MeMed BV passed the acceptance criteria for each study. In the reproducibility study, TRAIL, IP-10 and CRP measurements ranged with coefficient of variation from 9.7 to 12.7%, 4.6 to 6.2% and 5.0 to 11.6%, respectively. LLoQ concentrations were established as 15 pg/mL, 100 pg/mL and 1 mg/L for TRAIL, IP-10 and CRP, respectively. The authors concluded that the analytical performance, along with diagnostic accuracy established in the Apollo clinical validation study (NCT04690569), supports that MeMed BV run on MeMed Key can serve as a tool to assist clinicians in differentiating between bacterial and viral infection. The authors note that a limitation of the LLoQ, hook effect and linearity studies determining the analytical measurement range was the need to employ recombinant samples. Another limitation is the relatively short amount of time (120 min) that TRAIL, IP-10 and CRP measurements were established as stable in unspun serum samples, which could constrain ease-of-use in some settings. Additional stability studies examining spun serum samples are warranted to further facilitate ease-of-use.
Accelerate PhenoTest BC Kit and Accelerate PhenoTest BC Kit, AST Configuration
Accelerate Diagnostics, Inc. (Tucson, AZ) developed the Accelerate PhenoTest BC Kit, AST Configuration which is an FDA 510(k) cleared device. The Accelerate PhenoTest BC is a multiplexed in vitro diagnostic test utilizing both qualitative nucleic acid fluorescence in situ hybridization (FISH) identification and quantitative, antimicrobial susceptibility testing (AST) methods and should be used with the Accelerate Pheno system. The Accelerate Pheno Test BC Kit is capable of simultaneous detection and identification of multiple microbial targets followed by susceptibility testing of the appropriate detected bacterial organisms. The Accelerate PhenoTest BC Kit is performed directly on blood culture samples identified as positive by a continuous monitoring blood culture system. Results are intended to be interpreted in conjunction with Gram stain results (FDA, 2020).
FebriDx Bacterial / Non-bacterial Point of Care Assay
Lumos Diagnostics (Carlsbad, CA) developed the FebriDx Bacterial/Non-bacterial Point-of-Care Assay which is an FDA 510(k) cleared device. It is a qualitative visually read rapid immunoassay for the detection of human host response proteins, Myxovirus resistance protein A (MxA) and C-reactive protein (CRP) directly from fingerstick blood. FebriDx is indicated for use in patients aged 12-64 who present to urgent care or emergency care settings for evaluation of acute respiratory infection who have had symptoms for less than 7 days and within 3 days of fever onset (FDA, 2023).
FebriDx test results are to be used together with other clinical and diagnostic findings as an aid in the diagnosis of bacterial acute respiratory infection and differentiation from non-bacterial cause. The assessment of bacterial infection presence should always depend upon consideration of all available information and not just on the FebriDx test results. The test results are not intended to identify a specific pathogen or the severity of infection (FDA, 2023).
The Canadian Agency for Drugs and Technologies in Health (CADTH) Horizon Scan chapter on “An Overview of Emerging Point-of-Care Tests for Differentiating Bacterial and Viral Infections” (Basharat and Horton, 2021) commented that one small retrospective study reported that FebriDx was associated with reduced antibiotic use. However, in a systematic review, authors examined the diagnostic accuracy of immunoassay-based point-of-care (POC) tests and recommended more research, specifically randomized controlled trials, is required to assess the effectiveness of immunoassay-based POC tests.
IntelliSep
Cytovale, Inc. (San Francisco, CA) developed the IntelliSep test which is an FDA 510(k) cleared device. It is an in vitro diagnostic semi-quantitative test that assesses cellular host response by deformability cytometry of leukocyte biophysical properties and is intended for use in conjunction with clinical assessments and laboratory findings to assist in the early detection of sepsis with organ dysfunction evident within the first 3 days following testing. It is indicated for adult patients presenting with signs and symptoms of infection in the emergency department. The IntelliSep test is performed on an EDTA anticoagulated whole blood sample (FDA, 2022).
The test generates an IntelliSep Index value that lies within one of three discrete interpretation bands based on the probability of sepsis with organ dysfunction evident within the first three days following testing. The IntelliSep test represents the likelihood of the clinical syndrome of sepsis and should be used with other clinical information and clinical judgement. The test does not identify the infection-causing agent and should not be used by itself to determine the presence of sepsis (FDA, 2022).
Monocyte Distribution Width (Measured by the Early Sepsis Indicator)
Common signs of sepsis entails abdominal pain, confusion, coughing, fever, tachycardia, and tachypnea. It can result in septic shock, multiple-organ failure, and death. Sepsis is usually caused by bacterial infections but may be the result of other infections such as viruses, parasites, or fungi. C-reactive protein (CRP) and procalcitonin (PCT) are proteins produced in response to infection and/or inflammation. They are probably the 2 most widely used clinical tests to diagnose and manage patients with sepsis, with the exception of lactate. Monocyte distribution width (MDW; Beckman Coulter) is a new generation cell blood count (CBC) parameter providing a measure of monocyte anisocytosis. In the last decades, it has emerged as a biomarker of sepsis in the acute setting, especially emergency department (ED), and intensive care unit (ICU). Monocyte distribution width is a FDA-cleared hematological biomarker that aids in establishing the severity of infection and risk of sepsis in adult patients in the ED and ICU. MDW is a measure of increased morphological variability of monocytes in response to bacterial, viral or fungal infections. MDW is reported automatically as part of a routine CBC with Differential test on the DxH 900 and DxH 690T hematology analyzers using the Early Sepsis Indicator (ESId) application, which enables automatic reporting with no work-flow changes or need to order an additional test.
Agnello et al (2022) stated that monocyte distribution has recently emerged as a promising biomarker of sepsis, especially in acute setting, such as ED and ICU. In a systemic review and meta-analysis, these researchers examined the accuracy of MDW for early detection of patients with sepsis. Relevant publications were identified by a systematic literature search on PubMed and Google Scholar from inception to September 7, 2021. Studies were divided into 2 groups based on the sepsis criteria applied, namely sepsis-2 or sepsis-3. A total of 10 studies including 9,475 individuals, of whom 1,370 with sepsis (742 according to Sepsis-2 and 628 according to Sepsis-3), met the inclusion criteria for this meta-analysis. The pooled sensitivity and specificity were 0.789 and 0.777 for Sepsis-2 criteria, 0.838 and 0.704 for Sepsis-3 criteria, respectively. The authors concluded that MDW represented a reliable biomarker for sepsis screening.
Li et al (2022) noted that early diagnosis and treatment of patients with sepsis reduce mortality significantly. In terms of examining new diagnostic tools of sepsis, MDW, as part of the white blood cell (WBC) differential count, was first reported in 2017. MDW greater than 20 and abnormal WBC count together provided a satisfactory accuracy and was proposed as a novel diagnostic tool of sepsis. In a prospective, single-center study, these researchers compared MDW and PCT's diagnostic accuracy on sepsis in the ED. Laboratory examinations including CBC and differentiation count (DC), MDW, PCT were obtained while arriving at the ED. These investigators divided patients into non-infection, infection without systemic inflammatory response syndrome (SIRS), infection with SIRS, and sepsis-3 groups. This study's primary outcome was the sensitivity and specificity of MDW, PCT, and MDW + WBC in differentiating septic and non-septic patients. Furthermore, the cut-off value for MDW was established to maximize sensitivity at an optimal level of specificity. From May 2019 to September 2020, a total of 402 patients were enrolled for data analysis. Patient number in each group was: non-infection 64 (15.9 %), infection without SIRS 82 (20.4 %), infection with SIRS 202 (50.2 %), sepsis-3 15 (7.6 %). The AUC of MDW, PCT, and MDW + WBC to predict infection with SIRS was 0.753, 0.704, and 0.784, respectively (p < 0.01). The sensitivity, specificity, PPV, and NPV of MDW using 20 as the cut-off were 86.4 %, 54.2 %, 76.4 %, and 70 %, compared to 32.9 %, 88 %, 82.5 %, and 43.4 % using 0.5 ng/ml as the PCT cut-off value. On combing MDW and WBC count, the sensitivity and NPV further increased to 93.4 % and 80.3 %, respectively. In terms of predicting sepsis-3, the AUC of MDW, PCT, and MDW + WBC was 0.72, 0.73, and 0.70, respectively. MDW, using 20 as cut-off, exhibited sensitivity, specificity, PPV, and NPV of 90.6 %, 37.1 %, 18.7 %, and 96.1 %, respectively, compared to 49.1 %, 78.6 %, 26.8 %, and 90.6 % when 0.5 ng/m PCT was used as cut-off. The authors concluded that MDW was a more sensitive biomarker than PCT in predicting infection-related SIRS and sepsis-3 in the ED. MDW of less than 20 demonstrated a higher NPV to exclude sepsis-3. Combining MDW and WBC count further improved the accuracy in predicting infection with SIRS but not sepsis-3.
The authors stated that this study had several drawbacks. First, this trial was a single-center study in only 1 ED. The results might not be generalizable to all EDs. These researchers might need a multi-center study to validate these findings. Second, these researchers enrolled patients only during the working hours. There might be potential selection bias. Third, these investigators enrolled patients with specific symptoms instead of consecutive patients in the ED and excluded those without laboratory testing. Nonetheless, they believed that it best fits the ED practice model. Fourth, PCT might be falsely positive in patients with malignancy. The authors found that the performance of PCT diminished in patients with malignancy; however, further investigation is mandated to address this issue. Fifth most of the documented pathogens were Gram-negative bacteria. Lipopolysaccharide (LPS) is one of the most important bacterial components involved in monocyte activation. The role of MDW in Gram-positive or fungal infection needs further study in the future.
Liu et al (2023) noted that MDW is a quantitative measurement of monocyte anisocytosis and has been proposed as an efficient marker for early sepsis detection. These researchers examined the prognostic potential of MDW in septic patients. A total of 252 adult septic patients were enrolled. Demographic, clinical, and laboratory finding including MDW and traditional inflammatory biomarkers detected at 3 time-points (day 1, day 3, and day 6) after admission were collected and compared between 28-day survivors and non-survivors. Receiver operating characteristic (ROC) curves, Kaplan-Meier survival curve, and Cox regression analyses were carried out to evaluate and compare their predictive values. Group-based trajectory modeling was used to identify MDW trajectory endotypes. Basic characteristics and 28-day outcomes were compared between the trajectories. ROC curve analysis showed that MDW levels measured on day 3 after admission (D3-MDW) had moderate prognostic value and was independently associated with 28-day mortality in patients with sepsis. A D3-MDW value of 26.20 allowed discrimination between survivors and non-survivors with a sensitivity of 77.8 % and a specificity of 67.6 %. However, the prognostic accuracy of D3-MDW was diminished in immune-compromised patients and patients who already received antibiotics before admission. Group-based trajectory modeling indicated that excessively elevated and delayed decreased MDW levels during the 1st week after admission inversely correlated with prognosis. The authors concluded that the MDW values detected on day 3 after admission and its kinetic change might be potential markers for predicting short-term outcome in adult septic patients.
In a meta-analysis, Motawea et al (2023) to examined if PCT and MDW could be used as accurate diagnostic markers for sepsis. These investigators searched PubMed, WOS, and SCOPUS databases. Inclusion criteria were any observational or clinical trials that compared MDW with PCT as diagnostic markers in a patient with sepsis. Case reports, editorials, conference abstracts, and animal studies were excluded. RevMan software [5.4] was used to perform the meta-analysis. After the complete screening, 5 observational studies were included in the meta-analysis. The total number of patients included in the meta-analysis in the sepsis group was 565 and 781 in the control group. The pooled analysis between the sepsis group and controls showed a statistically significant association between sepsis and increased levels of MDW and PCT (MD = 3.94, 95 % CI: 2.53 to 5.36, p-value < 0.00001) and (MD = 9.29, 95 % CI: 0.67 to 17.91, p-value = 0.03), respectively. Moreover, the subgroup analysis showed that the p-value of MDW levels (< 0.00001) was more significant than the p-value of PCT levels = 0.03, the p-value between the 2 subgroups (< 0.00001). Furthermore, the overall ROC Area for MDW (0.790) > the overall ROC Area for PCT (0.760). The authors concluded that the findings of this study demonstrated a statistically significant association between sepsis and increased MDW and PCT levels compared with controls, and the overall ROC Area for MDW was higher than the overall ROC Area for PCT, indicating that the diagnostic accuracy of MDW was higher than PCT. These researchers stated that MDW can be used as a diagnostic marker for sepsis patients in the ED. Moreover, these investigators stated that multi-center studies are needed to validate these findings.
In a pilot study, Encabo et al (2023) examined the diagnostic performance of MDW as a biomarker for sepsis diagnosis in severe patients attended in the ED for different conditions and not only infections. These researchers carried out an observational study in a consecutive prospective cohort including severe patients attending the ED with different conditions. MDW and other biomarkers were determined from samples obtained during the first care of patients. The diagnostic performance of the different biomarkers was determined based on the final diagnosis at patient discharge. A total of 102 patients, with a mean age of 76.7 (SD 16.5) years were included, 53 being (51.9 %) men. Among the patients included, 65 (63.7 %) had an infectious disease while the remaining had other different conditions. A MDW cut-off of 20.115 provided the best accuracy to identify infected patients, with a sensitivity of 89.2 (95 % CI: 79.4 to 94.7), a specificity of 89.2 (95 % CI: 75.3 to 95.7), a positive predictive value (PPV) of 93.5 (95 % CI: 84.6 to 97.5), a negative predictive value (NPV) of 82.5 % (95 % CI: 68.0 to 91.3), a positive likelihood ratio (PLR) of 8.25 (3.26 to 20.91), and a negative likelihood ratio (NLR) of 0.12 (0.06 to 0.24). The area under the ROC curve for infection according to MDW was 0.943 (95 % CI: 0.897 to 0.989; p < 0.001). The authors concluded that a MDW of greater than 20.115 may be associated with infection and could aid in distinguishing between infected and non-infected patients in severe patients. Moreover, these researchers stated that these findings must be confirmed in new studies due to the limited patient sample included.
The authors stated that this study had several drawbacks. First, only 102 patients were included, which could influence some results. The odds ratio (OR) of MDW for infection was 1.83 (0.93 to 3.58), which meant there was no association between exposure and outcome. Nevertheless, a statistical trend to significance was observed (p = 0.008) and the low OR may be a consequence of the limited sample size of this trial. Second, this was a mono-centric study that precluded the generalization of the results. However, it was carried out in a university hospital, the characteristics of which have been previously described and were similar to the majority of European university hospitals. Third, its retrospective nature may limit the applications of some conclusions. These researchers stated that clinical trials are mandatory to change medical approaches; however, this was only a pilot study that may be useful to design new studies along the same line and sample calculation.
Furthermore, UpToDate reviews on “Evaluation and management of suspected sepsis and septic shock in adults” (Schmidt et al, 2024), “Sepsis syndromes in adults: Epidemiology, definitions, clinical presentation, diagnosis, and prognosis” (Neviere, 2024), and “Sepsis in children: Definitions, epidemiology, clinical manifestations, and diagnosis” (Pomerantz and Weiss, 2024) do not mention monocyte distribution width / Early Sepsis Indicator as a management option.
Real-Time Quaking-Induced Conversion (RT-QuIC)
Weijie et al (2024) noted that recently, the investigation of CSF biomarkers for diagnosing human prion diseases (HPD) has gained significant attention. Reproducibility and accuracy are critical in biomarker research, especially in the measurement of total tau (T-tau) protein, which is a crucial diagnostic marker. Given the global impact of the coronavirus disease pandemic, the frequency of measuring this protein using one of the world's fully automated assays, chemiluminescent enzyme immunoassay (CLEA), has increased. Currently, the diagnosis and monitoring of neurological diseases mainly rely on traditional methods; however, their accuracy and responsiveness are limited. There is limited knowledge of the accuracy of CLEA in tau measurements. These investigators measured T-tau protein using CLEA and elucidated its merits and limitations. They randomly selected 60 patients with rapidly progressive dementia, using ELISA and CLEA analysis of CSF specimens. Furthermore, these researchers employed Western blotting to detect the presence of 14-3-3 protein and used real-time quaking-induced conversion (RT-QuIC) assays to analyze the same set of samples. Additionally, they examined the correlation coefficient between ELISA and CLEA results in a subset of 60 samples. Moreover, using CLEA, the authors evaluated the diurnal reproducibility, storage stability, dilutability, and freeze-thaw effects in 3 selected samples. In 172 patients, 172 samples were extracted, with each patient providing only 1 sample, and a total of 88 (35 men and 53 women) tested positive for HPD in the RT-QuIC assay. In contrast, all CSF samples from the remaining 84 patients without HPD (50 men and 34 women) tested negative in the RT-QuIC assay. Both ELISA and CLEA showed perfect sensitivity and specificity (100 %) in measuring T-tau protein levels. Furthermore, ELISA and CLEA were similar in terms of measurement sensitivity and marginal effect of detection extrema. CLEA analysis exhibited instability for certain samples with T-tau protein levels exceeding 2,000 pg/ml, resulting in low reproducibility during dilution analysis. The authors concluded that these findings showed that CLEA outperformed ELISA in terms of diurnal reproducibility, storage stability, and freeze-thaw effects. Moreover, ELISA showed superior performance in the dilution assay. These investigators stated that these results underscored the robustness and clinical utility of CLEA for analyzing CSF samples as a valuable diagnostic tool for HPDs, warranting its inclusion in the comprehensive evaluation of patients presenting with HPD.
Ghazanfar et al (2024) stated that sporadic Creutzfeldt-Jakob disease (SCJD) is a rare neurodegenerative disease with a very low prevalence; its etiology is theorized to be genetic. Modern laboratory techniques, such as the RT-QuIC assay, have allowed clinicians to diagnose CJD with greater sensitivity and specificity. Previously, the diagnosis rested primarily on a post-mortem brain biopsy. Although advancements in laboratory techniques have allowed earlier diagnosis of CJD, the treatment is still supportive. Research is still ongoing for a curative treatment, but so far, the fatality rate remains at 100 %. Early vague symptoms of CJD delay the diagnosis further, as multiple pathologies need to be ruled out before consideration of the diagnosis of CJD.
Thomas et al (2024) noted that variant CJD (vCJD) is a devastating disease caused by transmission of bovine spongiform encephalopathy (BSE) to humans. Although vCJD cases are now rare, evidence from appendix surveys suggested that a small proportion of the U.K. population may be infected without showing signs of disease. These "silent" carriers could present a risk of iatrogenic vCJD transmission via medical procedures or blood/organ donation, and currently there are no validated tests to identify infected asymptomatic individuals using easily accessible samples. To address this issue, these researchers examined the performance of 3 blood-based assays in a blinded study, using longitudinal sample series from a well-established large animal model of vCJD. The assays rely on amplification of misfolded prion protein (PrPSc; a marker of prion infection), and include RT-QuIC, and 2 versions of protein misfolding cyclic amplification (PMCA). Although diagnostic sensitivity was higher for both PMCA assays (100 %) than RT-QuIC (61 %), all 3 assays detected prion infection in blood samples collected 26 months before the onset of clinical signs, and gave no false positive results. Parallel estimation of blood prion infectivity titers in a sensitive transgenic mouse line showed positive correlation of infectivity with PrPSc detection by the assays, suggesting that they are suitable for detection of asymptomatic vCJD infection in the human population. The authors concluded that this study represented the largest comparison to-date of pre-clinical prion detection in blood samples from a relevant animal model. The outcomes will guide efforts to improve early detection of HPD and reduce infection risks in humans.
Furthermore, an UpToDate review on “Creutzfeldt-Jakob disease” (Appleby and Cohen, 2024) states that “Real-time quaking-induced conversion -- Real-time quaking-induced conversion (RT-QuIC) is an assay in which disease-associated prion protein (PrPSc) initiates a conformational transition in recombinant prion protein (recPrP), resulting in the formation of amyloid that can be monitored in real time. In one series with a validation cohort, the sensitivity and specificity of RT-QuIC were 87 to 91 % and 98 to 100 %, respectively. In a United States sample, sensitivity and specificity of RT-QuIC were 92 to 95 % and 98.5 to 100 %, respectively. Diagnostic sensitivity is higher in common sCJD molecular subtypes (e.g., MM1 and MV1) and lower in less common subtypes (e.g., VV1, MM2-C, and sFI). The National Prion Disease Pathology Surveillance Center based at Case Western Reserve University is the only clinical laboratory in the United States that performs RT-QuIC. CSF samples submitted to the Center also undergo 14-3-3 and tau analyses. In one preliminary study that included 31 patients with CJD, RT-QuIC testing of olfactory epithelium obtained from nasal brushings was more sensitive (97 versus 77 %) and similarly specific (100 %) when compared with CSF testing; however, RT-QuIC on nasal brushings is not performed clinically in the United States”. Moreover, this UTD review lists neuropsychiatric disorder with a positive RT-QuIC test as 1 of the Centers for Disease Control and Prevention (CDC) outline 2 criteria for probable sCJD.
Testing for Syphilis
The U.S. Preventive Services Task Force (USPSTF, 2018) provided the following recommendation regarding “Screening of syphilis infection in pregnant Women”:
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The USPSTF recommends early screening for syphilis infection in all pregnant women. (Grade A)
The USPSTF (2022) provided the following recommendation regarding “Screening of syphilis infection in nonpregnant adolescents and adults”:
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The USPSTF recommends screening for syphilis infection in persons who are at increased risk for infection. (Grade A)
The Center for Disease Control and Prevention (CDC) recommends at least annual screening for syphilis in sexually active men who have sex with men (MSM), with confirmatory testing for individuals with reactive serology. The CDC recommends that persons with HIV infection who are sexually active be screened at the 1st HIV evaluation and at least annually thereafter. Men who have sex with men and persons with HIV infection may benefit from more frequent screening (e.g., every 3 to 6months) based on individual risk behaviors and local epidemiology. The CDC also recommends opt out syphilis screening in correctional facilities based on the local area and institutional prevalence (Workowski et al, 2021).
Furthermore, the Center for Disease Control and Prevention (CDC, 2015) recommended screening for syphilis infection in all pregnant women at their 1st prenatal visit.
Ciprofloxacin Resistance (gyrA S91F Point Mutation) Testing
Pasquali et al (2007) examined the molecular basis of nalidixic acid and ciprofloxacin resistance in Helicobacter pullorum by sequencing the gyrA gene of H. pullorum CIP 104787T. Furthermore, 9 isolates (2 susceptible to ciprofloxacin and resistant to nalidixic acid, 3 susceptible and 4 resistant to both antibiotics) were selected from 44 poultry isolates and the nucleotide sequences of their quinolone resistance-determining regions (QRDRs) were compared. The 2,490 bp gyrA gene showed an open reading frame encoding a polypeptide of 829 amino acids (AAs). The deduced AA sequence of gyrA showed 72 % or greater identity to Helicobacter hepaticus, Helicobacter pylori, and Wolinella succinogenes. Moreover, 98 % or greater AA sequence identity was found comparing the QRDR of the H. pullorum type strain with the QRDRs of the afore-mentioned bacterial species. All ciprofloxacin-resistant poultry isolates showed an ACA-->ATA (Thr-->Ile) substitution at codon 84 of gyrA, corresponding to codons 86, 87 and 83 of Campylobacter jejuni, H. pylori and Escherichia coli gyrA genes, respectively. This substitution was functionally confirmed to be associated with the ciprofloxacin-resistant phenotype of poultry isolates. The authors concluded that this was the 1st report describing the complete 2,490 bp nucleotide sequence of H. pullorum gyrA and confirming the involvement of the Thr84Ile substitution of GyrA in ciprofloxacin resistance of H. pullorum.
Hadad et al (2021) noted that accurate molecular assays for prediction of anti-microbial resistance (AMR)/susceptibility in Neisseria gonorrhoeae (Ng) could offer individualized treatment of gonorrhea and enhanced AMR surveillance. These researchers examined the new ResistancePlus GC assay and the GC 23S 2611 (beta) assay (SpeeDx), for prediction of resistance/susceptibility to ciprofloxacin and azithromycin, respectively. A total of 967 whole-genome-sequenced Ng isolates from 20 European countries, 143 Ng-positive (37 with paired Ng isolates) and 167 Ng-negative clinical Aptima Combo 2 (AC2) samples, and 143 non-gonococcal Neisseria isolates and closely related species were examined with both SpeeDx assays. The sensitivity and specificity of the ResistancePlus GC assay to detect Ng in AC2 samples were 98.6 % and 100 %, respectively. ResistancePlus GC showed 100 % sensitivity and specificity for GyrA S91 WT/S91F detection, and 99.8 % sensitivity and specificity in predicting phenotypic ciprofloxacin resistance. The sensitivity and specificity of the GC 23S 2611 (beta) assay for Ng detection in AC2 samples were 95.8 % and 100 %, respectively. GC 23S 2611 (beta) showed 100 % sensitivity and 99.9 % specificity for 23S rRNA C2611 WT/C2611T detection, and 64.3 % sensitivity and 99.9 % specificity for predicting phenotypic azithromycin resistance. Cross-reactions with non-gonococcal Neisseria species were observed with both assays; however, the analysis software solved most cross-reactions. The authors concluded that the new SpeeDx ResistancePlus GC assay performed well in the detection of Ng and AMR determinants, especially in urogenital samples. The GC 23S 2611 (beta) assay performed relatively well; however, its sensitivity, especially for predicting phenotypic azithromycin resistance, was suboptimal and further optimizations are needed, including detection of additional macrolide resistance determinant(s).
Shariati et al (2022) stated that for approximately 30 years, the fluoroquinolone (FQ) antibiotic ciprofloxacin has been used for the treatment of various diseases, including chronic otorrhea, endocarditis, lower respiratory tract, gastro-intestinal (GI), skin and soft tissue, and urinary tract infections (UTIs). Ciprofloxacin's main mode of action is to stop DNA replication by blocking the A subunit of DNA gyrase and having an extra impact on the substances in cell walls. Available in intravenous (IV) and oral formulations, ciprofloxacin reaches therapeutic concentrations in the majority of tissues and bodily fluids with a low possibility for side effects. Despite the outstanding qualities of this antibiotic, Salmonella typhi, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa have all shown an increase in ciprofloxacin resistance over time. The rise of infections that are resistant to ciprofloxacin revealed that new pharmacological synergisms and derivatives are needed. To this end, ciprofloxacin may be more effective against the biofilm community of microorganisms and multi-drug resistant isolates when combined with a variety of anti-bacterial agents, such as antibiotics from various classes, nanoparticles, natural products, bacteriophages, and photodynamic therapy (PDT). The authors focused on the resistance mechanisms of bacteria against ciprofloxacin and new approaches for enhancing its effectiveness.
These researchers noted that mechanisms of quinolone resistance in H. pylori are point mutations in the quinolone resistance-determining region (QRDR) of the gyrA gene encoding the A subunit of DNA gyrase. The AA substitutions observed in clinical strains occur mainly at positions 91 and 87, which correspond to positions 87 and 83 in Escherichia coli numbering. No gyrB mutation has been described as being involved in quinolone resistance. Active efflux pumps have been described but do not yet appear to be competitive for mediating antibiotic, and especially quinolone, resistance. Genome sequencing revealed that H. pylori lacks the topoisomerase IV parC and parE genes as in other rare genomes of Campylobacter jejuni, Mycobacterium tuberculosis, and Treponema pallidum (GenBank accession numbers NC002163, NC00092, and NC00919, respectively). Consequently, DNA gyrase is the unique target for quinolones in H. pylori.
Rubin et al (2023) stated that the etiological bacterial agent of gonorrhea, Neisseria gonorrhoeae, has become resistant to each of the 1st-line antibiotics used to treat it, including ciprofloxacin. One diagnostic approach to identify ciprofloxacin-susceptible isolates is to determine codon 91 in the gene encoding the A subunit of DNA gyrase, gyrA, where coding for the wild-type serine (gyrA91S) is associated with ciprofloxacin susceptibility and phenylalanine (gyrA91F) with resistance. These investigators examined the possibility of diagnostic escape from gyrA susceptibility testing. They used bacterial genetics to introduce pair-wise substitutions in GyrA positions 91 (S or F) and 95 (D, G, or N), which is a second site in GyrA associated with ciprofloxacin resistance, into 5 clinical isolates of N gonorrhoeae. All 5 isolates encoded GyrA S91F, an additional substitution in GyrA at position 95, substitutions in ParC that are known to cause an increased minimum inhibitory concentration (MIC) to ciprofloxacin, and GyrB 429D, which is associated with susceptibility to zoliflodacin (a spiropyrimidinetrione-class antibiotic in phase-III clinical trials for the treatment of gonorrhea). These researchers evolved these isolates to evaluate for the existence of pathways to ciprofloxacin resistance (MIC greater than or equal to 1 μg/ml) and measured MICs for ciprofloxacin and zoliflodacin. In parallel, these investigators searched metagenomic data for 11 355 N gonorrhoeae clinical isolates with reported ciprofloxacin MICs that were publicly available from the European Nucleotide Archive for strains that would be identified as susceptible by gyrA codon 91-based assays. Three clinical isolates of N gonorrhoeae with substitutions in GyrA position 95 associated with resistance (G or N) maintained intermediate ciprofloxacin MICs (0.125 to 0.5 μg/mL), which has been associated with treatment failure, despite reversion of GyrA position 91 from phenylalanine to serine. From an in-silico analysis of the 11,355 genomes from N gonorrhoeae clinical isolates, these researchers identified 30 isolates with gyrA codon 91 encoding a serine and a ciprofloxacin resistance-associated mutation at codon 95. The reported MICs for these isolates varied from 0.023 μg/ml to 0.25 μg/ml, including 4 with intermediate ciprofloxacin MICs (associated with substantially increased risk of treatment failure). Lastly, through experimental evolution, 1 clinical isolate of N gonorrhoeae bearing GyrA 91S acquired ciprofloxacin resistance via mutations in the gene encoding for the B subunit of DNA gyrase (gyrB) that also conferred reduced susceptibility to zoliflodacin (i.e., MIC greater than or equal to 2 μg/ml). The authors concluded that diagnostic escape from gyrA codon 91 diagnostics could occur via either reversion of the gyrA allele or expansion of circulating lineages. N gonorrhoeae genomic surveillance efforts might benefit from including gyrB, given its potential for contributing to ciprofloxacin and zoliflodacin resistance, and diagnostic strategies that reduce the likelihood of escape, such as the incorporation of multiple target sites, should be investigated. These researchers stated that diagnostics that guide antibiotic therapy could have unintended consequences, including novel resistance determinants and antibiotic cross-resistance.
These investigators stated that as an in-vitro study, this study had drawbacks for translation to in-vivo clinical N gonorrhoeae infections. First, these researchers used a kanamycin resistance cassette for co-selection and worked with paired isogenic strains that were kanamycin-resistant. The kanamycin cassette used in this trial was a kanamycin phosphotransferase; thus, it acted through drug inactivation -- a mechanism that is probably non-overlapping with ciprofloxacin resistance. Second, because strains expressing the kanamycin cassette had similar MICs to parental isolates with the same gyrase mutations, the authors believed that the co-selected kanamycin cassette at most minimally affected the ciprofloxacin MIC profile. These MICs, however, were at the upper range of detection; therefore, they could not rule out the possibility that the kanamycin cassette had effects on ciprofloxacin MIC or pathways to ciprofloxacin resistance. Third, although the gyrB mutants described in this study could be isolated in the laboratory, their in-vivo viability is unknown. Fourth, the method of sequential passaging on ciprofloxacin used to identify these mutations might not replicate the evolutionary pressure applied by antibiotic in the in-vivo setting. Fifth, as only 30 clinical isolates of the 11,355 bore GyrA 91S and substitutions in GyrA position 95 associated with non-susceptibility, most of the sequenced isolates at present would probably be correctly identified by gyrA codon 91-based diagnostics. This was consistent with the 100 % effectiveness reported for using ciprofloxacin in the context of infections with N gonorrhoeae isolates with GyrA 91S.11.
Balduck et al (2024) noted that tolerance enables bacteria to survive intermittent antibiotic exposure without an increase in anti-microbial susceptibility. In this study, these investigators examined the presence of tolerance to 3 anti-microbials, ceftriaxone, azithromycin and ciprofloxacin, in clinical isolates as well as the World Health Organization (WHO) reference panel of Neisseria gonorrhoeae. They employed the modified tolerance disk (TD test) to evaluate tolerance to ceftriaxone, azithromycin and ciprofloxacin in 14 WHO reference strains and 62 N. gonorrhoeae clinical isolates -- evenly divided between anorectal and urogenital infections. The isolates underwent a 3-step incubation process wherein the isolates were exposed to an antibiotic disk for 20 hours of incubation (Step I), followed by the replacement of the antibiotic disk with a nutrient disk for over-night incubation (Step II), and additional over-night incubation with extra nutrients (Step III). A total of 4 of the 62 clinical anorectal isolates and none of the urogenital isolates exhibited tolerance to azithromycin (p = 0.033). Tolerance to ceftriaxone and ciprofloxacin was observed in 8 and 4 isolates, respectively, with no difference between infection sites. Tolerance was also detected in 8 (K, M, N, O, P, U, V, W) out of the 14 WHO reference strains, with varying patterns of tolerance to ceftriaxone (n = 8), ciprofloxacin (n = 2), and azithromycin (n = 1). The authors concluded that the findings of this study identified ceftriaxone, azithromycin, and ciprofloxacin tolerance in clinical and WHO reference N. gonorrhoeae isolates. Azithromycin tolerance was more common in anorectal than urogenital infections.
The authors stated that the drawbacks of this trial included the use of only TD tests for the detection of tolerance; other techniques, such as MDK99 killing curves, could have provided useful complementary information. Furthermore, no genotyping or transcriptomics was carried out on the obtained tolerant colonies, as this was beyond the scope of this study. However, these researchers recently conducted omics on tolerant colonies that will create a better understanding of the mutations associated with ceftriaxone tolerance in N. gonorrhoeae. Although these investigators tested all the clinical isolates in triplicate, they did not rerun the TD- tests on a separate occasion to examine the reproducibility of these findings. They only examined if ceftriaxone-tolerant isolates could accelerate the emergence of ciprofloxacin resistance and did not include other anti-microbial combinations. Lasty, the authors did not have an explanation for why there was no difference in the prevalence of ciprofloxacin tolerance between anatomical sites. Moreover, these researchers stated that this was the 1st in-vitro study to detect tolerance to azithromycin and ciprofloxacin in clinical isolates of N. gonorrhoeae. This study established a difference in the prevalence of tolerance to azithromycin based on the infection site. Additionally, these investigators employed a large sample size (the biggest to-date), carried out the experiment in triplicate and conducted the investigation blinded to infection sites. These researchers stated that future studies are needed to confirm these finding of differences in tolerance by the site of infection (including the oropharynx) and to examine the clinical consequences (such as differences in infectivity) and epidemiological consequences (such as the probability of AMR emerging).
Furthermore, an UpToDate review on “Fluoroquinolones” (Hooper, 2024) states that “Resistance to quinolones may occur via mutations in bacterial chromosomal genes or via acquisition of resistance genes on plasmids … Mutations in chromosomal genes occur in genes that encode the subunits of DNA gyrase and topoisomerase IV (altered target mechanism) … Major plasmid-mediated resistance mechanisms include Qnr proteins, which protect DNA gyrase and topoisomerase IV from quinolone activity … Plasmid-mediated resistance mechanisms typically confer low-level resistance. However, high-level resistance can result when plasmid-mediated mechanisms accumulate or co-occur with chromosomal mutations. The likelihood of developing resistance is believed to be related to the intensity and duration of antibiotic therapy. As an example, ≥ 5 days of fluoroquinolone exposure was associated with significant resistance in an in vitro model. Plasmid-mediated resistance mechanisms can confer resistance to other antimicrobial classes directly or because they are linked to other drug-resistance genes encoded on the same plasmid … Fluoroquinolone resistance is relatively uncommon among S. pneumoniae, H. influenzae, and M. catarrhalis”. Moreover, this UTD review does not mention gyrA S91F point mutation testing for determination of ciprofloxacin resistance.
Macrolide (Clarithromycin) Sensitivity (23S rRNA Point Mutation) Testing
Samanic et al (2023) stated that point mutations in the 23S rRNA, gyrA, and gyrB genes can confer resistance to clarithromycin (CAM) and levofloxacin (LVX) by altering target sites or protein structure; thus, lowering the effectiveness of standard antibiotics in the treatment of H. pylori infections. Considering the confirmed primary CAM and LVX resistance in H. pylori infected patients from southern Croatia, these researchers carried out a molecular genetic analysis of 3 target genes (23S rRNA, gyrA, and gyrB) by PCR and sequencing, together with computational molecular docking analysis. In the CAM-resistant isolates, the mutation sites in the 23S rRNA gene were A2142C, A2142G, and A2143G. Furthermore, the mutations D91G and D91N in GyrA and N481E and R484K in GyrB were associated with resistance to LVX. Molecular docking analyses showed that mutant H. pylori strains with resistance-related mutations exhibited a lower susceptibility to CAM and LVX compared with wild-type strains due to significant differences in non-covalent interactions (e.g., hydrogen bonds, ionic interactions) resulting in de-stabilized antibiotic-protein binding, ultimately leading to antibiotic resistance. The authors concluded their preliminary investigation on H. pylori antibiotic resistance is a 1st step toward a deeper understanding of this issue. A comprehensive research approach would provide insights into the complex interplay of patient characteristics, bacterial genetics, and antibiotic resistance at the atomic level. Molecular docking analysis revealed that point mutations alter the binding interactions between targets and antibiotics. These researchers stated that extensive investigations including molecular dynamics could shed light on the dynamical properties of the target/antibiotic complex and provide atomic explanations of the observed resistance. Finally, such all-encompassing research could revolutionize the treatment of H. pylori infections by supporting the development of targeted therapies and providing information for public health policy to curb the rise of antibiotic-resistant strains.
Lyu et al (2023) noted that detection of mutations in 1 or a couple of genes may not provide enough data or cover all the genomic DNA variance related to antibiotic resistance of H. pylori to CAM and LVX. These researchers carried out whole genome sequencing to examine novel antibiotic resistance-related genes to increase predictive accuracy for future targeted sequencing tests. Gastric mucosal biopsies were taken during upper endoscopy in 27 H. pylori-infected patients. According to culture-based anti-bacterial susceptibility test, H. pylori strains were divided into 3 groups, with 9 strains in each group: CAM single-drug resistance (group C), LVX single-drug resistance (group L), and strains sensitive to all anti-bacterial drugs (group S). Based on whole genome sequencing with group S being the control, group C and group L group-specific single nucleotide variants and AA mutations were screened, and potential candidate genes related to CAM and LVX resistance were identified. The median age of study subjects was 35 years (inter-quartile range [IQR]: 31 to 40), and 17 (63.0 %) were men. All 9 CAM-resistant strains had A2143G mutations in 23S rRNA, while none of 9 sensitive strains had the mutation; 6 of 9 strains in group L and 6 of 9 strains in group S had 87th or 91st mutation in gyrA. After comparing sequencing data of strains among the 3 groups, these investigators identified 5 mutated positions belonging to 4 genes related to CAM resistance, and 31 mutated positions belonging to 20 genes related to LVX resistance. Novel genetic mutations were detected for CAM resistance (including fliJ and clpX) and LVX resistance (including fliJ, cheA, hemE, Val360Ile, and HP0568). Missense mutations in fliJ and cheA gene were mainly involved in chemotaxis and flagellar motility to facilitate bacterial escape of antibiotics, while the functions of other novel gene mutations underpinning antibiotic resistance remain to be investigated. The authors concluded that whole genome sequencing detected potential novel genetic mutations conferring resistance of H. pylori to CAM and LVX including fliJ and cheA. Moreover, these researchers stated that further studies are needed to confirm the role of these novel gene mutations to increase predictive accuracy of future targeted sequencing tests, in particular LVX resistance, to treat H. pylori infection in a personalized approach.
The authors stated that this study had 2 main drawbacks. First, these researchers did not have the treatment outcome data to correlate with the predictive accuracy of the new genes identified. Second, the small sample size and the average sequencing coverage of 90 % to 91 % did not allow these investigators to reach a definite conclusion regarding the role of the novel gene mutations in antibiotic resistance. Nevertheless, the fact that these findings on the correlation of the known A2143G mutation of the 23S rRNA gene with CAM resistance helped to support the potential role of whole genome sequencing in identification of other novel potential candidate mutations. These investigators stated that further prospective, multi-center studies with larger samples and treatment outcome data are needed to confirm these findings.
Hu et al (2023) stated that the rates of antibiotic resistance of H. pylori are increasing, and the patterns of resistance are region and population specific. In a single-center study, these researchers examined the antibiotic resistance pattern of H. pylori, and compared short-read- and long-read-based whole-genome sequencing for identifying the genotypes. Resistance rates of 38.5 %, 61.5 %, 27.9 %, and 13.5 % against CAM, metronidazole (MTZ), LVX, and amoxicillin (AMX) were determined, respectively, while no strain was resistant to tetracycline (TCY) or furazolidone (FZD). Single nucleotide variations (SNVs) in the 23S rRNA and GyrA/B genes revealed by Illumina short-read sequencing showed good diagnostic abilities for CAM and LVX resistance, respectively. Nanopore long-read sequencing also showed a good efficiency in elucidating SNVs in the 23S rRNA gene and, therefore, a good ability to detect CAM resistance. The 2 technologies displayed good consistency in discovering SNVs and shared 76 % of SNVs detected in the rRNA gene. Taking Sanger sequencing as the gold standard, Illumina short-read sequencing demonstrated a slightly higher accuracy for discovering SNVs than Nanopore sequencing. There were 2 copies of the rRNA gene in the genome of H. pylori, and these investigators found that the 2 copies were not the same in at least 26 % of the strains tested, indicating their heterozygous status. In particular, 3 strains harboring a 2143G/A heterozygous status in the 23S rRNA gene, which is the most important site for CAM resistance, were found. The authors concluded that these findings provided evidence for an empirical 1st-line treatment for H. pylori eradication in clinical settings. Moreover, these researchers showed that Nanopore sequencing is a potential tool for predicting CAM resistance.
Tran et al (2024) noted that the management of H. pylori in Vietnam is becoming progressively more difficult as a consequence of increasing antibiotic resistance, especially to CAM and LVX. In Vietnam, the selection of an H. pylori eradication regimen is primarily based on empirical evidence. However, molecular analysis aimed at identifying H. pylori antibiotic-resistant genotypes is a promising method in antibiotic susceptibility testing. These investigators examined the rates of genotypic H. pylori resistance to CAM and LVX by means of DNA strip technology. They carried out DNA-strip technology-based testing on 112 patients with H. pylori-positive gastro-duodenal diseases for detection of 23S rRNA and gyrA mutations. H. pylori genotypic resistance to CAM and LVX was observed in 81.3 % and 53.6 % of the patients, respectively, and dual resistance was observed in 48.2 %. The 23S rRNA A2142G and A2143G mutations accounted for 1.8 % and 79.5 % of cases, respectively. The gyrA N87K, D91N, D91G, and D91Y mutations were present in 37.5 %, 11.6 %, 5.4 %, and 5.4 % of patients, respectively. All 4 gyrA mutations were observed in both the naive as well as failed pharmacotherapy patients. These researchers further found an association between the 23S rRNA A2143G mutation and a history of CAM use as well as between the gyrA N87K mutation and a history of LVX use. The authors found a very high prevalence of H. pylori resistance to CAM and LVX; and dual resistance to these antibiotics in Vietnam. These researchers stated that the use of molecular assays is feasible and may improve the management of H. pylori infection in Vietnam.
Hasanuzzaman et al (2024) stated that H. pylori is a pathogenic bacterium associated with various GI diseases, including chronic gastritis, peptic ulcers, mucosa-associated lymphoid tissue lymphoma, and gastric cancer. The increasing rates of H. pylori antibiotic resistance and the emergence of multidrug-resistant strains pose significant challenges to its treatment. In a comprehensive review, these researchers examined the mechanisms underlying the resistance of H. pylori to commonly used antibiotics and the clinical implications of antibiotic resistance. Furthermore, potential strategies for overcoming antibiotic resistance were discussed. These approaches aim to improve the therapeutic outcomes of H. pylori infections while minimizing the development of antibiotic resistance. The continuous evolution of treatment perspectives and ongoing research in this field are crucial for effectively combating this challenging infection.
These investigators stated that CAM, a macrolide antibiotic, is often employed in the front-line regimen to eradicate H. pylori. Clarithromycin has pharmacokinetic advantages over other macrolides, including increased oral bioavailability, higher plasma concentration, and longer elimination half-life. Concomitant administration with acid-suppressive agents also increases its stability in acidic environments. Clarithromycin exerts anti-microbial effects by binding to the peptidyl transferase loop of domain V in the 23S ribosomal RNA (rRNA) in the bacterial ribosomal subunit 50S. Mutations in domain V of the 23S rRNA gene of H. pylori, A2142G/C and A2143G, could result in reduced binding affinity of the antibiotic agent, making it less effective at inhibiting bacterial growth. Other point mutations have also been reported in the 23S rRNA in H. pylori isolates. Furthermore, studies using experimentally-induced resistant phenotype for CAM found that mutations in rpl22 and infB genes had synergistic effects with mutations in the 23S rRNA genes, resulting in higher MIC of CAM. Another relevant mechanism for CAM resistance is attributed to the efflux pump system. Moreover, these researchers stated that the role of novel mutations and specific function of efflux pump system in the development of CAM resistance in clinical isolates should be further clarified.
Furthermore, an UpToDate review on “Azithromycin and clarithromycin” (Graziani, 2024) states that “Two main mechanisms of acquired macrolide resistance have been described: Methylases encoded by the erm (erythromycin ribosome methylase) genes (ermA, ermB, ermC) alter the macrolide-binding site on the bacterial ribosomal RNA, usually conferring a high degree of macrolide resistance as well as resistance to clindamycin. Active macrolide efflux pumps, encoded by the mef (macrolide efflux) msrA and msrB genes, confer a low to moderate degree of macrolide resistance. These mechanisms are responsible for erythromycin resistance in most gram-positive cocci (e.g., Staphylococcus aureus, S. pneumoniae, other streptococci). In contrast with the mechanisms of acquired resistance, intrinsic resistance exhibited by Enterobacterales, Pseudomonas spp, and Acinetobacter spp is due to decreased permeability of the outer cell envelope”. Moreover, this UTD review does not mention 23S rRNA point mutation testing for determination of clarithromycin resistance.
NeXGen Fungal/AFB NGS Assay
NeXGen Fungal/AFB NGS assay is a hybrid-capture NGS-based technique that combines the specificity of target capture with the high-throughput capabilities of NGS offering advantages such as comprehensive pathogen detection, increased sensitivity and specificity, as well as simultaneous detection of multiple pathogens. The NeXGen Fungal/AFB NGS assay is designed for identifying infectious disease including invasive fungal disease (IFD) and acid-fast bacteria (AFB). Developers of the NeXGen Fungal/AFB NGS assay believe that testing of IFD and AFB using NGS has the potential to improve patient outcomes by enabling early and accurate diagnosis, guiding appropriate therapy, as well as monitoring treatment response.
Cai et al (2024) stated that the pathogens causing unexplained pneumonia in both HIV-infected or HIV-uninfected patients are likely to be complex. In a retrospective study, these researchers characterized the etiology of pneumonia in HIV-infected and HIV-uninfected patients using broncho-alveolar lavage fluid (BALF) analysis with metagenomic NGS (mNGS) and X-pert MTB/RIF. Between January 2022 and May2024, 141 HIV-infected and 104 HIV-uninfected patients admitted to Nanjing Second Hospital with pneumonia were included. BALF samples were collected and analyzed using mNGS to detect bacteria, fungi, viruses, tuberculosis (TB) and non-tuberculous mycobacteria (NTM), and X-pert for TB detection. Clinical data including CD4 T-cell counts, comorbidities, and ART status were collected and analyzed. HIV-uninfected patients were found to be older and exhibited a higher prevalence of co-morbidities compared to HIV-infected patients. Despite higher median CD4 T-cell counts in HIV-uninfected individuals (412 cells/μL versus 31 cells/μL in HIV-infected), TB detection rates using X-pert and mNGS were lower than anticipated, especially in HIV-infected patients. Mixed-pathogen infections were significantly more prevalent in HIV-infected patients, especially those with lower CD4 T-cell counts. The authors concluded that ART use showed variable impacts on pathogen diversity, with longer treatment durations associated with reduced infection complexity but persistent immunodeficiency in some cases. In patients with pneumonia, whether HIV-infected or HIV-uninfected, pathogens often exhibit complexity, underscoring the critical role of timely mNGS and X-pert analysis of BALF for early pathogen detection. Moreover, these researchers stated that while challenges persist in distinguishing pathogenic infections from colonization using mNGS, experienced clinical judgment remains invaluable in clinical decision-making. These investigators stated that future studies should address limitations such as small sample sizes, especially in the HIV-uninfected group, to mitigate inter-group biases and enhance the generalizability of findings.
Liu et al (2024) noted that the emergence of mNGS may provide a promising tool for early and comprehensive identification of the causative pathogen in community-acquired pneumonia (CAP). These researchers examined the etiological diagnostic value of mNGS in suspected CAP. A total of 555 broncho-alveolar lavage fluid (BALF) samples were collected for pathogen detection by mNGS from 541 patients with suspected CAP. The clinical value was assessed based on infection diagnosis and treatment guidance. The diagnostic performance for pathogen identification by mNGS and sputum culture and for tuberculosis (TB) by mNGS and X-pert MTB/RIF were compared. To examine the potential for treatment guidance, these investigators analyzed the treatment regimen of patients with suspected CAP, including imaging changes of lung following empirical anti-bacterial therapy, intensified regimen, anti-fungal treatment, and a 1-year follow up for patients with unconfirmed diagnosis and non-improvement imaging after anti-infective treatment and patients with high suspicion of TB or non-tuberculous mycobacteria (NTM) infection who were transferred to the Wuhan Pulmonary Hospital for further diagnosis and even anti-mycobacterium therapy. Of the 516 BALF samples that were analyzed by both mNGS and sputum culture, the positivity rate of mNGS was significantly higher than that of sputum culture (79.1 % versus 11.4 %, p = 0.001). A total of 48 samples from patients with confirmed TB were analyzed by both mNGS and X-pert MTB/RIF, and the sensitivity of mNGS for the diagnosis of active TB was significantly lower than that of X-pert MTB/RIF (64.6 % versus 85.4 %, p = 0.031). Of the 106 pathogen-negative cases, 48 were eventually considered non-infectious diseases, with a NPV of 45.3 %. Of the 381 pathogen-positive cases, 311 were ultimately diagnosed as CAP, with a PPV of 81.6 %. A total of 487 patients were included in the evaluation of the therapeutic effect, and 67.1 % improved with initial empirical antibiotic treatment. Of the 163 patients in which bacteria were detected, 77.9 % improved with anti-bacterial therapy; of the 85 patients in which fungi were detected, 12.9 % achieved remission after anti-fungal therapy. The authors concluded that mNGS had unique advantages in the detection of suspected CAP pathogens; however, mNGS was not superior to X-pert MTB/RIF for the diagnosis of TB. Furthermore, mNGS was not necessary as a routine test for all patients admitted with suspected CAP. In addition, when fungi were detected by mNGS, anti-fungal therapy should be cautious. These researchers stated that prospective studies with larger sample sizes are needed to further examine the etiological diagnostic value of mNGS for patients with suspected CAP. These researchers stated that this trial had 2 main drawbacks. First, viruses were not included in the pathogens detected by mNGS. Second, the tracking time of lung nodules was insufficient, and all were considered to be inflammatory nodules with or without inert lung cancer, which warrants further investigations.
Lu et al (2024) noted that the contribution of the lung microbiota to pneumonia in children of varying severity remains poorly understood. In a retrospective, observational study, these investigators used mNGS technology to examine the characteristics of lung microbiota and their association with disease severity. This trial examined BALF mNGS data of 92 children diagnosed with pneumonia between January 2021 and July 2022. A comparative analysis of the lung microbiota was carried out between the severe pneumonia (SP) (n = 44) and non-severe pneumonia (NSP) (n = 48) groups. Compared to conventional microbiological tests (CMT), mNGS had a higher positivity rate in etiology detection (68 % versus 100 %). In the NSP group, the predominant type of infection was Mycoplasma pneumoniae single infection, whereas in the SP group, the main type involved a combination of M pneumoniae and bacterial infection. The top 3 identified microbial taxa in both the groups were M pneumoniae, Rothia mucilaginosa, and Schaalia odontolyticus. Although there were no significant differences in the α and β diversity of the lung microbiota between the SP and NSP groups, the abundance of M pneumoniae was higher in the SP group (p = 0.053). Spearman analysis revealed a highly significant positive correlation between the abundance of Prevotella melaninogenica and M pneumoniae (p < 0.001). The authors concluded that this analysis identified an association between M pneumoniae infections and disease severity. This study provided a foundation for a better understanding of the pathogenesis of pediatric pneumonia and the relationship between micro-organisms.
These researchers stated that by means of BALF mNGS, they identified the pathogens responsible for the pulmonary infections in children. Furthermore, these researchers examined the differences in microbial composition among the various groups based on the information provided by mNGS. Despite the potential interference from colonizers, BALF has significant advantages over other respiratory samples for pathogen detection. However, obtaining BALF samples from healthy children as controls is challenging. This limitation hindered the investigation of the pathogenic and commensal species in the lung microbiota of children with pneumonia. Moreover, the relatively small number of enrolled patients may have introduced a bias into the results. Another drawback was the relatively high detection rate of M pneumoniae, which reduced the diversity of patient cohorts. These investigators stated that further large-scale investigations are needed to attain a more comprehensive understanding of the association between microbiome and disease severity, which will facilitate the use of mNGS to determine the etiology and control disease progression.
Xiang et al (2024) stated that reports on the use of mNGS in adult patients with severe pneumonia following cardiac surgery remain limited. In a retrospective, single-center study, these investigators examined the clinical outcomes of mNGS analysis of BALF in adult patients who had undergone cardiac surgery. Samples were collected from patients in the surgical ICU (SICU) of Sichuan Provincial People's Hospital between January 2019 and March 2024. Upon diagnosis of severe pneumonia, BALF was obtained via bronchoscopy within 24 hours. The mNGS group was composed of patients tested using mNGS and conventional microbiological tests. BALF was detected only by the conventional microbiological test (CMT) method in the CMT group, which involved examining bacterial and fungal smears and cultures at least. These researchers reviewed a total of 4,064 cardiac surgeries, and based on the inclusion criteria, a total of 113 adult patients with severe pneumonia after cardiac surgery were included in this study. The overall positive rate detected by mNGS was significantly higher than that of the culture method (98 % versus 58 %, p < 0.0001). After receipt of the microbiological results, the mNGS group exhibited a higher incidence of antibiotic adjustments in comparison to the CMT group (p = 0.0021). After adjusting the treatment plan based on microbial testing results, the mNGS group showed an improvement in ventilator-free days within 28 days (p = 0.0475), with a shorter duration of invasive ventilation compared to the CMT group (p = 0.0208). The detection of mNGS could significantly improve the Acute Physiology and Chronic Health Evaluation-II (APACHE II) score (p = 0.0161) and Sequential Organ Failure Assessment (SOFA) score (p = 0.0076) on the 7th day after admission to the SICU. In this study, the mNGS group showed signs of having a positive impact on the length of stay in ICU (median of 9 days, IQR: 7 to 10 days versus median of 10 days, IQR: 8 to 13.75 days, p = 0.0538), hospital length of stay (LOS; median of 20 days, IQR: 17 to 28 days versus median of 25 days, IQR: 18 to 29 days, p = 0.1558), mortality in 28 days (19 % versus 20 %, p = 0.8794), in-hospital mortality (19 % versus 22 %, p = 0.7123); however, statistical analysis did not confirm these differences to be significant. The authors concluded that mNGS could serve as a valuable complement to conventional diagnostic approaches in adult patients with severe pneumonia following cardiac surgery, potentially improving diagnostic accuracy and resulting in more precise and timely interventions, with significant potential to inform clinical decision-making and enhance patient outcomes. Moreover, these researchers stated that further investigations should include a larger sample size, involving multi-center, prospective, and controlled studies, which will enhance the understanding of the impact of mNGS on the medium or long-term survival rate, economic implications as well as health resource utilization in this specific patient population.
The authors stated that this study had several drawbacks. First, the retrospective design of this trial introduced potential biases, especially in patient selection for mNGS testing. The higher initial testing costs associated with mNGS meant that it was more likely to be used in patients with more severe clinical presentations resulting in baseline disparities between the patient groups undergoing mNGS and those receiving CMT, potentially confounding the comparison of outcomes between these groups. Second, this trial was carried out at a single center and focused specifically on a relatively narrow patient population -- individuals who developed severe pneumonia after cardiac surgery. This specificity, while valuable for targeted insights, resulted in a smaller sample size. The limited sample size posed challenges to the statistical robustness of these findings, necessitating a cautious interpretation of the data.
Men et al (2025) noted that acute lower respiratory tract infections (ALRIs) is a leading cause of child mortality globally. In a retrospective study, these researchers evaluated the role of mNGS in identifying ALRIs pathogens and examined the lung microbiota's role in disease severity and clinical outcomes. This study examined the association between lung microbiota and ALRIs outcomes in children, exploring its potential as a prognostic biomarker. These investigators analyzed mNGS data from BALF of 83 pediatric ALRIs patients from 2019 to 2023. Microbial diversity and relative abundances of specific taxa were compared between survivor and non-survivor groups, as well as between varying severity levels. LEfSe was used to identify key biomarkers related to survival and disease severity. Among the 83 patients, 68 survived and 15 died. Patients were also divided into a low severity group (n = 38) and a moderate-to-very-high severity group (n = 45) according to mPIRO score at admission. Significant differences in beta diversity were observed between the survival groups and across different severity levels. Prevotella, Haemophilus and Veillonella exhibited higher abundances in both the survivor and low severity groups, suggesting their potential as predictors of better outcomes. Conversely, Enterococcus and Acinetobacter baumannii were more prevalent in the non-survivor and moderate-to-very-high severity groups. Furthermore, Streptococcus pneumoniae and Streptococcus mitis showed increased abundances in survivors. LEfSe further showed that these micro-organisms may predict outcomes and severity in ALRIs. The authors concluded that the findings of this study underscored the complex relationship between lung microbiota and ALRIs, with specific microbial profiles associated with disease severity and clinical outcomes. Moreover, these researchers stated that future studies with large, more balanced samples and prospective designs are needed to validate these findings and provide a more comprehensive understanding of the microbiome’s role in ALRIs.
The authors stated that this study had several drawbacks. First, a significant imbalance existed between the groups, with only 15 non-survivors compared to 68 survivors, which reduced statistical power and weakened the conclusions. The longer pediatric ICU (PICU) stays in the non-survivor group suggested more severe underlying conditions, complicating the interpretation of microbiome differences. Although these investigators re-analyzed the data based on the mPIRO score at admission, the retrospective design still posed limitations. Second, these researchers did not account for confounding factors such as antibiotic exposure, different types of ALRIs, and variable timing of sample collection, all of which may influence the results. Third, these investigators only extracted DNA from the samples for analysis of bacteria in this research, meaning that DNA viruses, RNA viruses and fungi were not included in the scope of analysis, which could be detected by other molecular methods otherwise]. Fourth, due to the invasive procedure and financial burden of bronchoscopy, it is challenging to collect multiple BALF samples from patients during infection; thus, the authors could not track the dynamic changes in the microbiome of patients over the course of the disease consequently. Given the significantly higher detection rate of mNGS compared to traditional microbiological testing, and the absence of specific species information for many commensal bacteria in this retrospective study, the authors did not compare mNGS results with traditional findings, although future research should include such comparisons to enhance mNGS interpretability. Fifth, the association between lung microbes and ALRIs can be complex and multi-directional, and this study did not analyze non-linear associations.
Furthermore, UpToDate reviews on “Diagnosis of invasive aspergillosis” (Patterson, 2025), “Fungal rhinosinusitis” (Cox and Perfect, 2025), and “Diagnosis of pulmonary tuberculosis in adults” (Bernardo, 2025) do not mention next generation sequencing as a diagnostic tool.
Glossary of Terms
| Term | Definition |
|---|---|
| Metagenomics | Molecular tool used to analyze DNA acquired from environmental samples, in order to study the community of microorganisms present, without the necessity of obtaining pure cultures (Ghosh et al, 2019) |
| Metagenomic next-generation sequencing (mNGS) |
|
| Next-generation sequencing (NGS) | Any of several high-throughput (or massively parallel) sequencing methods whereby thousands to billions of nucleic acid fragments can be simultaneously and independently sequenced (Lee, 2019; Gu et al, 2019) |
| Shotgun sequencing | Laboratory technique for determining the DNA sequence of an organism’s genome. The method involves randomly breaking up the genome into small DNA fragments that are sequenced individually. A computer program looks for overlaps in the DNA sequences, using them to reassemble the fragments in their correct order to reconstitute the genome (NIH/NHGRI, 2022) |
| Immunoassay |
Bioanalytical methods in which the quantitation of the analyte depends on the reaction of an antigen (analyte) and an antibody (Darwish, 2006); Bioanalytical method that measures the presence or concentration of analytes ranging from small molecules to macromolecules in a solution through the use of an antibody or an antigen as a biorecognition agent (Ju et al, 2017). |
References
The above policy is based on the following references:
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