Viewpoint <\/p>\n\n\n\n
August 22, 2022<\/p>\n\n\n\n
The prime directive in diagnosing neuroinfectious disease is to answer 3 deceptively simple questions: (1) Is the patient infected? (2) If so, with what? (3) How do we treat the infection? Our ability to answer the first 2 questions has been substantially improved by the emergence of multiplex approaches to diagnosis of central nervous system (CNS) infections, as exemplified by metagenomic next-generation sequencing (mNGS). Eight years ago, the first publications of proof-of-concept case reports were just appearing, establishing that mNGS could lead to actionable diagnosis with clear treatment implications, as in a case involving neuroleptospirosis in an immunocompromised child.1<\/a><\/sup> The passage of time has enabled a more mature assessment of the role and limitations of mNGS in neurodiagnostics. The prospective Precision Diagnosis of Acute Infectious Diseases (PDAID) Study2<\/a><\/sup>enrolled 204 patients with idiopathic encephalitis (64%), meningitis (34%), or myelitis (2%) from 8 hospitals. Of the 58 CNS infections ultimately diagnosed, 13 (22%) were only diagnosed using mNGS, including cases of St Louis encephalitis virus, hepatitis E virus, and Streptococcus agalactiae<\/em>; 8 of these diagnoses affected clinical care. Overall, mNGS was highly concordant with other cerebrospinal fluid (CSF) direct detection tests (ie, pathogen-specific polymerase chain reaction [PCR], culture, and antigen testing); nevertheless, 26 additional infections (45%) diagnosed with conventional testing were missed by mNGS.<\/a><\/p>\n\n\n\n Categories of mNGS misses included cases in which (1) standard diagnosis was based on serologic testing rather than nucleic acid amplification (n\u2009=\u200911); (2) the infection was present in biological sources other than CSF (n\u2009=\u20097); or (3) the CSF \u201ctiter\u201d of the pathogen, and hence its nucleic acid load, was low (n\u2009=\u20098), including cases due to Mycobacterium <\/em>species, Cryptococcus neoformans<\/em>, Staphylococcus aureus<\/em>, cytomegalovirus, and herpes simplex virus type 2 (HSV-2). In other words, 18 of the 26 CSF infections missed by mNGS were not detectable in the CSF by any means. Similar results emerged from a study of 68 patients with known (65%) or suspected (35%) encephalitis in a series from Mass General Brigham.3<\/a><\/sup> In 37 of these cases (57%), an infectious etiology was ultimately diagnosed. mNGS missed infection in 2 of 12 cases (1 case patient was HSV-2 positive, 1 was HIV-1 positive) in which standard CSF monoplex PCR testing was more sensitive than mNGS, presumably because of low nucleic acid load. mNGS also missed infection in 16 of 25 cases in which diagnosis was ultimately made by serologic testing or testing of blood rather than CSF.<\/a><\/p>\n\n\n\n Because mNGS is an unbiased technique that results in amplification of any nucleic acid sequences in CSF, the potential for false-positive results due to amplification of trace levels of environmental contaminants in reagents or other materials has also been repeatedly noted; interpretation, especially of unusual or exotic potential pathogens, requires caution and, ideally, independent validation. In a recent Mayo Clinic retrospective review4<\/a><\/sup> of 80 CSF samples from patients with suspected subacute or chronic CNS infection seen at Mayo Clinic or with CSF samples referred for mNGS, there were 12 mNGS positive test results; however, 6 of them were believed to be false-positive as a result of contamination. Importantly, 4 of the 6 patients had normal CSF cell counts, and an additional patient had only 7 cells\/\u03bcL.<\/a><\/p>\n\n\n\n So where does this leave the current state of mNGS testing for CNS infection? First, as exemplified in the early case reports, mNGS may be especially valuable as adjunctive testing in immunocompromised patients, in whom the list of candidate pathogens is often large and for many of which no approved monoplex PCR tests are available. Second, the testing complements rather than replaces many standard diagnostics, especially in cases in which pathogen load may be low, cases in which serologic testing is more sensitive than nucleic acid amplification for infection diagnosis, or cases involving abscesses, in which infection may be compartmentalized. Third, except in immunocompromised patients, the pretest probability of a CNS infection is low in CSF samples with a normal cell count; as a result, the proportion of false-positive detections by mNGS (or other neurodiagnostic tests) dramatically increases.<\/a><\/p>\n\n\n\n When the nucleic acid content of CSF is very low, there is an overrepresentation of environmental contaminating bacterial sequences in the mNGS data, increasing the likelihood of false-positive results. New analytic approaches may reduce the risk of these false-positives.5<\/a><\/sup> For example, if a potential environmental contaminant is detected, an analysis of the host gene expression data generated by the mNGS assay could confirm that the host response is not consistent with the response seen in bona fide CNS infections.6<\/a><\/sup> Similarly, if no infection is identified in a patient with a clinical phenotype that overlaps with viral and autoimmune encephalitis, a host RNA-sequencing analysis could confirm that the patient\u2019s host profile is consistent with autoimmune encephalitis rather than viral infection.<\/a><\/p>\n\n\n\n What does the future hold? There have been new and innovative methods for enhancing pathogen detection.7<\/a><\/sup>CRISPR-Cas systems, using CRISPR-Cas13\u2013 or CRISPR-Cas12a\u2013based techniques, have been developed for direct detection of pathogen sequences in clinical samples without the need to extract DNA or RNA or perform any amplification or sequencing steps. These techniques can rapidly generate a fluorescent signal with no computational analysis required and are conducive to diagnostic testing in low-resource settings.7<\/a><\/sup> Serologic testing of CSF facilitates diagnosis for some pathogens for which nucleic acid amplification is comparatively insensitive, as exemplified by neuroinvasive West Nile virus and, potentially, SARS-CoV-2 infections. Several research approaches are now available that allow CSF to be probed for antibodies directed against large libraries of peptide fragments generated from available nucleic acid sequences from huge numbers of potential human pathogens. This approach has provided strong evidence for the etiologic role of viruses in diseases in which mNGS and conventional PCR testing on CSF have produced negative results.8<\/a><\/sup>-10<\/a><\/sup> It is likely that in the future of neuroinfectious disease, diagnosis will depend on a trifecta of evaluations, including mNGS or related techniques to detect nucleic acid, unbiased screening for antibodies to virtually all known human pathogens, and analysis of gene expression to search for unique host gene expression profiles suggestive of autoimmune or specific categories of infections. The complexity of some of these technologies and the sophistication of interpretation of results will require us to develop equally robust neuroinfectious disease stewardship and consultation programs that help clinicians select, interpret, and respond to test results.<\/p>\n","protected":false},"excerpt":{"rendered":" Viewpoint August 22, 2022 The Current Status of N […]<\/p>\n","protected":false},"author":3,"featured_media":0,"comment_status":"closed","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":[],"categories":[24,23],"tags":[],"_links":{"self":[{"href":"https:\/\/csccm.org.cn\/index.php?rest_route=\/wp\/v2\/posts\/23255"}],"collection":[{"href":"https:\/\/csccm.org.cn\/index.php?rest_route=\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/csccm.org.cn\/index.php?rest_route=\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/csccm.org.cn\/index.php?rest_route=\/wp\/v2\/users\/3"}],"replies":[{"embeddable":true,"href":"https:\/\/csccm.org.cn\/index.php?rest_route=%2Fwp%2Fv2%2Fcomments&post=23255"}],"version-history":[{"count":1,"href":"https:\/\/csccm.org.cn\/index.php?rest_route=\/wp\/v2\/posts\/23255\/revisions"}],"predecessor-version":[{"id":23256,"href":"https:\/\/csccm.org.cn\/index.php?rest_route=\/wp\/v2\/posts\/23255\/revisions\/23256"}],"wp:attachment":[{"href":"https:\/\/csccm.org.cn\/index.php?rest_route=%2Fwp%2Fv2%2Fmedia&parent=23255"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/csccm.org.cn\/index.php?rest_route=%2Fwp%2Fv2%2Fcategories&post=23255"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/csccm.org.cn\/index.php?rest_route=%2Fwp%2Fv2%2Ftags&post=23255"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}