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Review 1: "Laboratory Validation and Clinical Implementation of Human Monkeypox Saliva-Based Tests"

Reviewers found this study to be potentially informative to not informative, highlighting several gaps in the study design and reporting of results that render it difficult to draw meaningful conclusions from the preprint without major clarification or further research.

Published onSep 07, 2022
Review 1: "Laboratory Validation and Clinical Implementation of Human Monkeypox Saliva-Based Tests"
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key-enterThis Pub is a Review of
Laboratory Validation and Clinical Implementation of Human Monkeypox Saliva-Based Tests

AbstractBackgroundImproved diagnostic capabilities and accessibility are essential for controlling the outbreak of Human Monkeypox.MethodsWe describe a saliva-based polymerase chain reaction (PCR) assay for Human Monkeypox, in vitro test performance, and clinical implementation of that assay at three testing sites in Los Angeles. Finally, using pre-specified 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 Human Monkeypox.ResultsLaboratory evaluation of the assay showed in silico inclusivity of 100% for 97 strains of Human Monkeypox, with an analytic sensitivity of 250 copies/mL, and 100% agreement compared to known positive and negative specimens. Clinical testing identified 22 cases of Human Monkeypox among 132 individuals (16.7%). Of those 22 cases, 16 (72.7%) reported symptoms, 4 (18.2%) without a rash at the time of testing. Our systematic rapid review identified 6 studies; 23 patients had tests performed on oropharyngeal specimens 100% agreed with the PCR test result of a lesion swab.ConclusionSaliva-based PCR tests are potential tools for outbreak control, and further evaluation of the performance of such tests is warranted.

RR:C19 Evidence Scale rating by reviewer:

Not informative. The flaws in the data and methods in this study are sufficiently serious that they do not substantially justify the claims made. It is not possible to say whether the results and conclusions would match that of the hypothetical ideal study. The study should not be considered as evidence by decision-makers.



This manuscript aims to present the validation of saliva-based tests to detect Human Monkeypox and is complemented with a review on the available bibliography related to oral pharyngeal or saliva test for Human Monkeypox.

The authors divide the manuscript into two parts, one regarding the validation of the method and the other regarding a critical review of published results on the use of saliva or oropharyngeal samples to detect Monkeypox. However, the critical review is very limited, having been carried out using PubMed and Web of Science databases and, not being in fact a meta-analysis, due to the limitations invoked by the authors, is therefore nothing more than the review normally performed on any scientific article to support, compare and discuss results. No deadline is displayed for the search performed. The manuscript is more of a preliminary report that can be used to write the final paper as relevant details are omitted or relegated to the supplementary material. No details on the PCR conditions used and threshold for positivity are given in the manuscript. The experimental results are presented in a basic way (2 tables) and it is not clear whether there is any relationship between the results obtained and the clinical/epidemiological characterization data of the study participants, since there is no statistical treatment of the data.

It is not clear how the positive samples used for the calculation of the limit of detection (LOD) were prepared and if the PCR was performed in the presence of saliva or if the DNA was extracted from saliva previously spiked. This is relevant as saliva is a matrix that can affect the PCR performance and might also influence the reproducibility of the results. If the DNA has been extracted from saliva it is also not clear how the authors guaranteed the amount they claim to test for the LOD determination.

Although saliva is a convenient and easily obtainable specimen, its use in molecular tests for the detection of viral nucleic acids has been cautiously reported due to the potential existence of factors that can influence the results and also the stability of the genetic material in this matrix. Thus, details related to the collection, transport and processing of the sample are in fact relevant as they can influence the results obtained. In this sense, the manuscript is sparse in details or even confusing, and it is not possible to understand how much time elapsed from collection to analysis and how the samples were maintained. The initial processing of saliva is incompletely and confusingly described as it is not possible to understand what the authors mean by “specimens were washed”.

In terms of specificity, authors refer that the test is specific to orthopoxvirus but not specifically to Human Monkeypox but do not present any results, despite stating that analytical specificity was determined. In fact, there is some data on Supplementary material regarding the specificity tests but not presented in an adequate format to be published. Authors have detected a positive patient without any clinical symptoms but do not discuss the possibility of a false positive. No confirmation of the PCR amplicons sequence is presented. 

Despite the authors claim that “accurate and precise estimate of saliva-based test performance in comparison to PCR of lesion swabs is urgently needed”, no comparison of this type of results is presented since saliva and lesion swabs samples were not collected in parallel, although many of the studied patients had them.

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