Laboratory validation and clinical performance of a saliva‐based test for monkeypox virus

Abstract Improved diagnostic tests and accessibility are essential for controlling the outbreak of monkeypox. We describe a saliva‐based polymerase chain reaction (PCR) assay for monkeypox virus, in vitro test performance, and clinical implementation of that assay in Los Angeles, San Francisco, and Palm Springs, CA. Finally, using prespecified search terms, we conducted a systematic rapid review of PubMed and Web of Science online databases of studies reporting the performance of oral pharyngeal or saliva‐based tests for the monkeypox virus. The assay showed in silico inclusivity of 100% for 97 strains of monkeypox virus, with an analytic sensitivity of 250 copies/ml, and 100% agreement compared to known positive and negative specimens. Clinical testing identified 22 cases of monkeypox among 132 individuals (16.7%), of which 16 (72.7%) reported symptoms, 4 (18.2%) without a rash at the time of testing. Of an additional 18 patients with positive lesion tests, 16 (88.9%) had positive saliva tests. Our systematic review identified six studies; 100% of tests on oropharyngeal specimens from 23 patients agreed with the PCR test result of a lesion. Saliva‐based PCR tests are potential tools for case identification, and further evaluation of the performance of such tests is warranted.


| INTRODUCTION
With cases of monkeypox reported from 47 countries, the World Health Organization recently declared the current spread of the infection a global emergency. 1 Historically, the monkeypox virus has been endemic in tropical rainforest regions of Central and West Africa, with short-lived outbreaks driven by transmission through animal-to-human and human-to-human exposures. 2 However, the current outbreak is now spreading much more rapidly and pervasively than any previous outbreak, with a unique pattern of sexual transmission. [3][4][5][6] Such transmission has contributed to the disproportional burden of disease among gay, bisexual, and other men who have sex with men. 4 The US Centers for Disease Control and Prevention has stressed the need for timely diagnosis as a primary means for outbreak control, particularly in the absence of sufficient vaccine supplies. 7 Polymerase chain reaction (PCR) lesion testing was thought to be necessary, and the United States Food and Drug Administration has discouraged all testing apart from lesion swabs. 8 The US Centers for Disease Control and Prevention has given similar guidance. 9 Prior studies, however, suggest that viral DNA may be detected in saliva and oropharyngeal specimens. 2,10,11 One recent report noted that 100% (n = 12) of patients with monkeypox had positive saliva PCR tests. 10 Similar findings were reported among a study of seven individuals diagnosed with monkeypox in 2018 and 2019. 11 In both studies, many individuals had positive oropharyngeal tests early in the disease course. 10,11 Thus, via early case detection, saliva testing may provide the opportunity to limit infectiousness. We, therefore, assessed the performance of a newly-developed salivabased PCR assay. We further report the real-world implementation of that assay in clinical settings. replicates of a positive control within pooled negative saliva (oral saliva matrix) specimens, we report the limit of detection, defined as the lowest concentration providing a positive result for 100% of replicates. Using two known positive specimens as well as 20 known negative specimens, we report the in vitro agreement between those specimens and our assay. For measures of agreement, at least two different operators and instruments were used on three separate days at three different concentrations to assess reproducibility. Assay validation and clinical use was conducted in accordance with the United States Clinical Laboratory Improvement Act guidelines. 12 We reviewed deidentified patient records among individuals presenting for monkeypox virus testing at saliva collection sites in California, and, where available, concordance between saliva and lesion PCR tests (Monkeypox (Orthopox) DNA, PCR Test; Labcorp).
Cycle threshold values for lesion PCR results were only available for tests performed in Los Angeles. Advarra institutional review committee exempted the analysis of deidentified data from institutional review (Pro00065270).

| Literature review
Finally, we conducted a systematic rapid review of the literature on PubMed and Web of Science databases to assess the performance of saliva tests in comparison to PCR of lesion swabs. We used the following predefined search terms: "monkeypox" AND ("diagnosis" OR "diagnostic") AND ("saliva" OR "sputum" OR "throat" OR "pharyn*"). We further evaluated the references of all articles identified and searched preprint servers for forthcoming publications.
We included articles that reported the results of any oropharyngeal or saliva PCR tests for monkeypox in humans. We excluded review articles, studies among primates, and studies not in English. We then conducted a narrative review of the studies, reporting individual study-level summary data given the degree of heterogeneity within studies precluded a formal meta-analysis.

| RESULTS
The PCR saliva assay had an in silico inclusivity of 100% for all (n = 97) strains from the two different clades. The assay was specific to the orthopoxvirus genus, but not to the monkeypox virus, as the assay also detected cowpox and rabbitpox, but did not detect   One additional consideration beyond earlier detection is the concern that if the virus can be detected before lesion development, it may also be transmittable before lesion development.
Previous work has similarly suggested that viral shedding at various anatomic sites may contribute to transmission. Delayed viral detection may reflect protracted infectiousness. 11 In addition, from prior outbreaks of monkeypox human-to-human transmission through respiratory droplets has been suggested among a small subset of cases. 2 Two further benefits of saliva-based testing are worth considering. The current outbreak has consistently presented with anogenital lesions. 11,18,19 Rapid and accessible testing of anogenital lesions may be more challenging than saliva-based tests, given that patients will require privacy to collect anogenital specimens, in contrast to walk- call to action in the development and comparison of saliva-based specimen testing.

| CONCLUSION
We report the laboratory and clinical performance of a saliva-based PCR test for the monkeypox virus. Supplementing that report, we systematically reviewed the literature for all reports of saliva-based tests for the monkeypox virus. Our findings provide evidence that saliva-based tests may be a viable testing method for the monkeypox virus and may identify cases earlier than lesion-based tests, warranting further evaluation of saliva-based assays.

AUTHOR CONTRIBUTIONS
All authors contributed substantially to this study. Lao-Tzu Allan-