Monitoring monkeypox virus in saliva and air samples in Spain: a cross-sectional study

Abstract

Background: The transmission of monkeypox virus occurs through direct contact, but transmission through saliva or exhaled droplets and aerosols has not yet been investigated. We aimed to assess the presence of monkeypox virus DNA and infectious virus in saliva samples and droplets and aerosols exhaled from patients infected with monkeypox virus.

Methods: We did a cross-sectional study in patients with monkeypox confirmed by PCR who attended two health centres in Madrid, Spain. For each patient, we collected samples of saliva, exhaled droplets within a mask, and aerosols captured by air filtration through newly developed nanofiber filters. We evaluated the presence of monkeypox virus in the samples by viral DNA detection by quantitative PCR (qPCR) and isolation of infectious viruses in cell cultures.

Findings: Between May 18 and July 15, 2022, 44 patients with symptomatic monkeypox attended two health centres in Madrid and were included in the study. All were cisgender men, with a median age of 35·0 years (IQR 11·3). We identified high loads of monkeypox virus DNA by qPCR in 35 (85%) of 41 saliva samples. Infectious monkeypox virus was recovered from 22 (67%) of 33 saliva samples positive for monkeypox virus DNA. We also found a significant association between the number of affected cutaneous areas or general symptoms and the viral load present in saliva samples. Droplets exhaled from patients with monkeypox, detected inside a mask, contained monkeypox virus DNA in 32 (71%) of 45 samples, with two of the 32 positive samples showing the presence of the infectious virus. Monkeypox virus DNA in aerosols, collected from the medical consultation room, were detected in 27 (64%) of 42 samples, despite patients wearing an FFP2 mask during the visit. Infectious virus was not recovered from aerosol samples. High levels of monkeypox virus DNA were identified in aerosols collected from a hospital isolation room housing a patient with monkeypox.

Interpretation: The identification of high viable monkeypox virus loads in saliva in most patients with monkeypox and the finding of monkeypox virus DNA in droplets and aerosols warrants further epidemiological studies to evaluate the potential relevance of the respiratory route of infection in the 2022 monkeypox virus outbreak.

Funding: EU, Consejo Superior de Investigaciones Científicas, and Ciberinfec.

Figure 1

Monkeypox virus detection in 45 samples from patients with confirmed infection Individual mean Ct values determined by a specific quantitative PCR for monkeypox virus DNA from every patient's saliva, mask filter, and air filter are shown. A mean Ct value of 35 was established as threshold for positivity (dotted line). Each sample was tested in triplicate, and at least two of three wells needed to be positive for the sample to be considered positive. Red colour indicates samples in which infectious viruses were detected after inoculation of cell monolayers. Ct=cycle threshold.

Figure 2

Infectious monkeypox virus detected in saliva samples from patients (A) Representative cytopathic effect in BSC-1 cells inoculated with a fraction of saliva sample from a patient and examined daily for cytopathic effect; images show viral spreading through the cell culture at 0, 3, and 5 days after infection. (B) Box and whisker plot showing the distribution of monkeypox virus loads from saliva samples containing (red) or not containing (blue) viable virus (n=41). (C, D) Identification of monkeypox virus particles (green arrows) in saliva samples by electron microscopy after negative staining; the inset shows the magnification of the indicated virion. (E) Monkeypox virus particle from a pustular lesion identified by electron microscopy after negative staining.

Figure 3

Monkeypox virus load determination from samples Estimated monkeypox virus genome copies per mL of saliva (A) and mask filter (B) or m³ of captured air (C) from every patient. We interpolated cycle thresholds in the calibration curve (range of linearity from 10⁶ to 1 copy per μL, with a slope of –3·10, intercept 35·82; R²=0·997 and amplification efficiency of 110%). Red bars indicate samples in which infectious monkeypox virus was detected. Dotted lines indicate the baseline of the assay in every sample; values below this line were considered negative. n=41 for saliva, n=45 for mask filters, and n=42 for air samples. Patient 12 was not confirmed positive for monkeypox virus infection by PCR of the cutaneous lesion, and was excluded from the study. *Not available.