Clinical characteristics and comparison of longitudinal qPCR results from different specimen types in a cohort of ambulatory and hospitalized patients infected with monkeypox virus

Abstract

Background: The ongoing monkeypox virus outbreak includes at least 7553 confirmed cases in previously non-endemic countries worldwide as of July 2022. Clinical presentation has been reported as highly variable, sometimes lacking classically described systemic symptoms, and only small numbers of cutaneous lesions in most patients. The aim of this study was to compare clinical data with longitudinal qPCR results from lesion swabs, oropharyngeal swabs and blood in a well characterized patient cohort.

Methods: 16 male patients (5 hospitalized, 11 outpatients) were included in the study cohort and serial testing for monkeypox virus-DNA carried out in various materials throughout the course of disease. Laboratory analysis included quantitative PCR, next-generation sequencing, immunofluorescence tests and virus isolation in cell culture.

Results: All patients were male, between age 20 and 60, and self-identified as men having sex with men. Two had a known HIV infection, coinciding with an increased number of lesions and viral DNA detectable in blood. In initial- and serial testing, lesion swabs yielded viral DNA-loads at, or above 10⁶ cp/ml and only declined during the third week. Oropharyngeal swabs featured lower viral loads and returned repeatedly negative in some cases. Viral culture was successful only from lesion swabs but not from oropharyngeal swabs or plasma.

Discussion: The data presented underscore the reliability of lesion swabs for monkeypox virus-detection, even in later stages of the disease. Oropharyngeal swabs and blood samples alone carry the risk of false negative results, but may hold value in pre-/asymptomatic cases or viral load monitoring, respectively.

Keywords: Clinical cohort; Clinical specimens; Monkeypox virus; Viral load kinetics; qPCR.

Fig. 1

Viral DNA-load time courses were plotted for hospitalized patients with available serial measurements. A) Swabs from cutaneous lesions were taken according to established procedures; however, the exact location where swabs were taken has not been recorded. Also, swabbing procedures may entail opening a fresh lesion, which will then crust over. The indicated viral loads represent generic lesion swabs from the respective patient, not necessarily from the same lesion. (1st week: median 3.31E+07 cp/ml, range 2.19E+07 – 3.95E+07 cp/ml; 2nd week: median 3.04E+06 cp/ml, range 2.11E+05 – 5.48E+05 cp/ml). Graphs B) and C) represent oropharyngeal swabs (1st week: median 8.44E+04 cp/ml, range 6.93E+04 – 7.31E+05 cp/ml; 2nd week: median 4.04E+03 cp/ml, range 0 – 6.75E+06 cp/ml; 3rd week: median 0 cp/ml, range 0 – 2.00E+04 cp/ml) and EDTA plasma-samples (1st week: median 5.85E+02 cp/ml, range 1.58E+02 – 1.05E+03 cp/ml; 2nd week: median 7.80E+00 cp/ml, range 0 – 1.20E+03 cp/ml; 3rd week: single sample, 2.37E+01 cp/ml) respectively.

Fig. 2

A) Viral DNA-loads of different specimen types are plotted for patient 1. gray area represents their stay in the hospital. Red asterisk (*) represents a sample with successful isolation of infections virus, whereas black asterisks (*) represent unsuccessful attempts at viral culture. B) The evolution of an exemplary pustula is displayed over the same timeframe.

Fig. 3

A) phylogenetic analysis of MPXV virus sequences related to the 2022 global outbreak. Sequences used here were obtained from NCBI (as of July 21, 2022). Color coding represents the individual clades with clade B.1 containing the outbreak related sequences. B) Section of the phylogenetic tree shown in A. The sequences reconstructed from the lesion of patients 1, 4–6 of this study are marked with an arrow, MPXV/Germany/2022/HH-LIV00, MPXV/Germany/2022/HH-LIV004, MPXV/Germany/2022/HH-LIV005, MPXV/Germany/2022/HH-LIV006.). The color code represents the country from which the sequences were provided in NCBI.