Summary
Monkeypox virus (mpxv) is a DNA virus in the Orthopoxvirus genus which causes Mpox (previously monkeypox). Symptoms include fever, lymphadenopathy and vesicular lesions. There is limited evidence for the duration of mpxv infectivity. This study used cell culture as a proxy for infectivity. Clinical samples from four patients with Mpox were inoculated into African green monkey kidney (Vero E6) cells and monitored for cytopathic effects (CPE). From one patient, infectious mpxv was recovered 25 days after illness onset. Infectious virus was not isolated from samples with an Orthopoxvirus polymerase chain reaction (PCR) Ct value over 31.0, nor from urine.
Keywords
Introduction
Monkeypox virus (mpxv) is a member of the Orthopoxvirus genus, in the Poxviridae family of DNA viruses. Mpox is the clinical disease caused by mpxv and was first described in humans in the Democratic Republic of Congo in 1970. In the past decade, case detection has increased, most notably in Nigeria. In 2022, the World Health Organisation (WHO) declared a public health emergency of international concern in response to a global outbreak of Mpox in countries where the disease was not previously considered endemic, including many cases in Europe and North America. Symptoms of Mpox typically include a prodromal phase of fever, lymphadenopathy and myalgia followed by vesicular lesions.
Mpox is a classic zoonotic infection, but human-to-human transmission of mpxv is well documented, particularly in the current outbreak. Transmission is primarily through contact with infectious lesions, but transmission through fomites and via respiratory routes are also thought to occur [
[1]
,[2]
]. The duration of infectivity from different body sites in patients with varying severity of disease has not been fully characterized despite the large case numbers seen. In this study, virus isolation in cell culture was used as a proxy for infectivity to evaluate a variety of samples from patients with Mpox. Patients' samples ranged from acute presentation to recovery and prolonged shedding. We aimed to establish the duration of potential mpxv infectivity in patients.Methods
Prior to the current outbreak, seven patients were diagnosed with Mpox in the UK, between 2018 and 2021. These patients were cared for in specialist high-consequence infectious disease (HCID) centres. Their clinical progression has been previously described [
[3]
]. Initial diagnosis was made using pan-orthopoxvirus real-time polymerase chain reaction (PCR), and Mpox confirmed using a mpxv-specific real-time PCR. Once diagnosed, patients were clinically monitored with a pan-Orthopoxvirus deoxyribonucleic acid (DNA) PCR only. This assay was used clinically with a cut off for detection at cycle threshold (Ct) 40 and for positivity at Ct 38.This publication is an interim analysis of cell culture experiments from four of the seven patients described and comprises 54 longitudinal clinical samples. Forty samples comprised lesion swabs, throat swabs, plasma and urine, in which Orthopoxvirus DNA was detected by PCR. Twelve samples comprised throat, lesion, urine, serum and plasma from the same patients in which Orthopoxvirus DNA was not detected and were included as negative controls. Two additional stored serum samples that were not previously tested clinically were also included.
Aliquots of 100 μL of clinical sample were inoculated into African green monkey kidney (Vero E6) cells, termed passage 1 (P1). The P1 cultures were incubated for several days and monitored for cytopathic effects (CPE). See the Supplementary Data for images. P1 cultures were sampled on days 0 and 7 for virus inactivation, nucleic acid extraction and mpxv-specific PCR [
[4]
]. P2 cultures were established using supernatant from P1 cultures if infectious virus was not recovered. All work involving the manipulation of clinical samples and recovery of infectious virus was performed in a Containment Level 3 laboratory, within a Class III Microbiological Safety Cabinet (UKHSA, Porton Down).mpxv-specific PCR was performed for all P1 and P2 cultures to support CPE observations. A decrease in mean mpxv Ct values from day 0 to day 7 correlates with an increase in mpxv nucleic acid production and is indicative of mpxv replication. We have previously shown that it is sufficient to perform up to two sub-cultures (P1/P2) to recover infectious mpxv from environmental samples with high PCR Ct values [
[5]
].Ethical approval was granted by the NHS Research Ethics Committee (NHS REC).
Results
The patients have been described previously [
[3]
] and included a diversity of disease severity (see Table I). None of the patients had underlying HIV. The sampling frame for these patients ranged from one day to 68 days. The patients and the culture results from their samples will be described in turn.Table ITiming of sample in relation to day of illness, correlated with Orthopox Ct value at diagnosis and Monkeypox virus Ct value following Passage 1 and Passage 2
| Approximate day of illness | Symptomatic lesions | Treatment | Sample type | Site of lesion | Diagnostic result orthopox mean Ct | Passage 1 mean mpx | Passage 2 mean mpx | Day post infection when CPE was observed | Infectious virus recovered | |||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| D0 | D7 | D0 | D7 | |||||||||
| Patient 1 | 7 | Y | Day of Brincidofovir 200 mg (one dose) | Plasma | N/A | 33.8 | UD | 39.0 | UD | UD | X | N |
| 10 | Y | Post-treatment | Plasma | N/A | UD | UD | UD | UD | UD | X | N | |
| 10 | Y | Post-treatment | Urine | N/A | 35.4 | 39.1 | UD | UD | 39.4 | X | N | |
| 12 | Y | Post-treatment | Plasma | N/A | UD | UD | UD | 37.92 | UD | X | N | |
| 12 | Y | Post-treatment | Urine | N/A | UD | UD | UD | UD | UD | X | N | |
| Patient 2 | 2 | Y | Pre-treatment | Plasma | N/A | 33 | UD | UD | 38.12 | UD | X | N |
| 7 | Y | Pre-treatment | Plasma | N/A | 30.1 | 36.9 | UD | UD | 38.66 | X | N | |
| 7 | Y | Pre-treatment | Urine | N/A | UD | UD | UD | UD | UD | X | N | |
| 8 | Y | 1 day post-Brincidofivir | Plasma | N/A | 30.3 | 38.8 | 39.5 | UD | 38.18 | X | N | |
| 8 | Y | 1 day post-Brincidofivir | Urine | N/A | 29.9 | 39.0 | UD | UD | 38.32 | X | N | |
| Patient 3 | 3 | Y | Pre-treatment | Plasma (spun) | N/A | 35.4 | 37.9 | UD | UD | UD | X | N |
| 3 | Y | Pre-treatment | Urine | N/A | 28.8 | UD | UD | 38.3 | 38.5 | X | N | |
| 3 | Y | Pre-treatment | Throat swab | Throat | 34.7 | UD | UD | UD | UD | X | N | |
| 3 | Y | Pre-treatment | Swab | Nose | 23.9 | 29.8 | 18.9 | . | . | 3 | Y | |
| 3 | Y | Pre-treatment | Swab | Arm | 29.6 | 36.0 | 23.3 | . | . | 3 | Y | |
| 3 | Y | Pre-treatment | Swab | Chin | 31 | 34.9 | 22.1 | . | . | 3 | Y | |
| 3 | Y | Pre-treatment | Serum (spun) | N/A | Not tested | 39.2 | UD | UD | UD | X | N | |
| Patient 4 | 11 | Y | None | Swab | Unknown | 33.4 | UD | 38.5 | UD | UD | x | N |
| 11 | Y | None | Swab | Unknown | 22.9 | 28.3 | 19.8 | . | . | 3 | Y | |
| 11 | Y | None | Swab | Unknown | 26.2 | 31.0 | 24.9 | . | . | 3 | Y | |
| 11 | Y | None | Urine | N/a | 31.1 | UD | 39.21 | UD | UD | x | N | |
| 11 | Y | None | Swab | Unknown | 30.6 | 36.8 | 30.8 | . | . | 6 | Y | |
| 11 | Y | None | Serum | N/A | Not tested | 37.86 | UD | UD | UD | x | N | |
| 11 | Y | None | Plasma | N/A | 35.8 | UD | 38.2 | UD | UD | X | N | |
| 13 | Y | None | Urine | N/A | 30.5 | 37.8 | UD | 38.1 | UD | X | N | |
| 13 | Y | None | Throat swab | Throat | UD | UD | UD | UD | UD | X | N | |
| 13 | Y | None | Swab | Unknown | 18.8 | 24.4 | 16.2 | . | . | 3 | Y | |
| 13 | Y | None | Swab | Unknown | 28.4 | 34.3 | 31.7 | 34.99 | 20.94 | x | Y | |
| 13 | Y | None | Swab | Groin | 20.8 | 28 | 21.4 | . | . | 3 | Y | |
| 18 | Y | None | Urine | N/A | 35.3 | UD | UD | UD | 38.55 | X | N | |
| 18 | Y | None | Swab | Unknown | 26.4 | 32.4 | 36.3 | 37.45 | UD | X | N | |
| 18 | Y | None | Swab | Hand | 36.6 | 37 | UD | 38.2 | UD | X | N | |
| 18 | Y | None | Swab | Groin | 25.4 | 32.9 | 26.9 | . | . | 6 | Y | |
| 18 | Y | None | Swab | Throat | 36.7 | 37.5 | UD | UD | 39.1 | X | N | |
| 25 | Y | None | Urine | N/A | 35.3 | 38.3 | UD | 38.06 | UD | X | N | |
| 25 | Y | None | Swab | Face | 31.8 | 38.7 | UD | UD | UD | X | N | |
| 25 | Y | None | Throat swab | Throat | UD | UD | UD | UD | UD | X | N | |
| 25 | Y | None | Swab | Palm | 33 | UD | UD | UD | 38.33 | X | N | |
| 25 | Y | None | Swab | Penis | 25.3 | 33.5 | 29 | . | . | X | Y | |
| 27 | Y | None | Urine | N/A | UD | UD | UD | X | N | |||
| 27 | Y | None | Swab | Face | 35.3 | UD | UD | X | N | |||
| 27 | Y | None | Swab | Penis | 29.5 | 36.69 | UD | X | N | |||
| 27 | Y | None | Swab | Palm | 33.4 | UD | UD | X | N | |||
| 28 | Y | None | Swab | Unknown | 33.9 | UD | 38.05 | X | N | |||
| 28 | Y | None | Swab | Penis | 28.7 | 38.89 | UD | X | N | |||
| 28 | Y | None | Urine | N/A | UD | UD | UD | X | N | |||
| 28 | Y | None | Swab | Face | 35.3 | 37.98 | UD | X | N | |||
| 28 | Y | None | Swab | Penis | 34 | UD | UD | X | N | |||
| 38 | Y | None | Swab | Hand | UD | UD | UD | X | N | |||
| 38 | Y | None | Swab | Palm | UD | UD | UD | X | N | |||
| 60 | Y | None | Swab | Genital | UD | UD | 38.3 | X | N | |||
| 75 | Y | None | Swab | Palm | 33.9 | 38.06 | UD | X | N | |||
| 78 | Y | None | Throat swab | Throat | UD | UD | UD | X | N | |||
| 78 | Y | Treatment | Swab | Groin | 36.4 | UD | 38.95 | X | N | |||
Samples whereby replication competent virus was identified are highlighted in bold. If passage two is highlighted in grey, it has not been performed as passage one was confirmative. For mpxv PCR assay, 38.0 was used as the cut off for genuine positivity. CPE, cytopathic effects; N, no; UD, undetected; X, not observed; Y, yes. Bold indicates isolation of virus (i.e. positive results).
Patient 1 was a male aged 30–40 years. He acquired Mpox in Nigeria and had a prodromal illness of fever and night sweats. He had over 150 lesions during his illness and was in hospital for 26 days. He had ulcerated inguinal lesions with a prolonged period of detectable Orthopoxvirus DNA from lesion swabs. He was kept in hospital until his lesions had healed completely. His diagnostic sample was not available for inclusion in this study but five follow-up samples (plasma and urine), starting one week after the initial lesions appeared and on the day of his Brincidofovir treatment were included. Of the five samples tested, Orthopoxvirus DNA was not detected by PCR in three. These were included as negative controls and no infectious virus was recovered from P1 and P2 cultures. The two samples where Orthopoxvirus DNA had been detected on initial PCR did not yield infectious virus from either culture. (Table I).
Patient 2 was a 30- to 40-year-old male who acquired his infection in Nigeria. He had initial fever and groin swelling, followed by over 100 lesions including a thigh abscess which was drained surgically. He had five samples of urine and plasma ranging from day 2 to day 8 of illness and spanning treatment with Brincidofovir. Four samples were Orthopoxvirus DNA positive by the diagnostic assay; none of the samples resulted in isolation of infectious virus.
Patient 3 was from a female case of UK-acquired Mpox. She was vaccinated with Modified vaccinia Ankara (MVA) six days post-exposure (12 days pre-illness) and developed 24 h of coryzal illness followed by approximately 32 lesions. She spent 35 days in hospital before being discharged when fully recovered. Seven samples from a single time point at the start of her illness have been analysed and are included in this report; six were Orthopoxvirus DNA positive by diagnostic PCR, and one was not analysed at the time of diagnosis. Of the seven samples, four (plasma, serum, urine and throat) did not result in infectious virus isolation (Table I). Analysis of P1 cultures by mpxv-specific PCR showed that three lesion samples had decreased Ct values from day 0 to day 7, demonstrating infectious virus was recovered. CPE was detected on day 3 (Table I).
Patient 4 was a male aged 40–50 years. He was infected with mpxv in Nigeria and had experienced a prodromal illness of fever and headache followed by the development of over 100 lesions. He has had 37 clinical samples over a 68-day period during which no antiviral treatment was administered. These samples were taken from 11 days into his illness, and all were taken when the patient was symptomatic. On diagnostic PCR, no Orthopoxvirus DNA was detected from eight samples (throat swabs at days 13, 25 and 78, lesion swabs, two from day 38 and day 60, and urine from day 27 and 28) and one sample (serum from day 1) was not tested. Of the nine samples inoculated into Vero E6 cells, no infectious virus was recovered from either P1 or P2 cultures (Table I).
Infectious virus was recovered from eight samples. In two samples, CPE was not observed (Ct 25.3 and 28.4; Table I). A fall in Ct value over time confirmed presence of infectious virus in these samples.
Infectious virus was recovered from a penile swab sample taken 25 days after the onset of illness. The first swab samples sent for this patient were not labelled with a body location, thus the exact correlation with previous samples from 11 days into his illness is not possible. Of the three lesion swabs, one throat swab and one urine screened from day 25 of his illness, infectious virus was recovered from only the penile swab.
The patient was unwell for 11 days prior to the first samples being taken and after 25 days of illness, infectious virus was still isolated. After 28 days of illness, the diagnostic PCR remained positive but infectious virus was not recovered from the clinical samples. These were lesion swabs from an unknown region, two penile swabs and a face swab (Cts 33.9, 28.7, 35.3, 34.0, respectively).
Across the four patients, infectious virus was isolated from 11 samples, all of which were swabs with Orthopoxvirus DNA detected by PCR and Ct values in the range 18.8–31.0.
Discussion
The extent of human mpxv infectivity in infected individuals over time is under ongoing evaluation. In this study, we demonstrated detection of infectious mpxv in a penile lesion swab 25 days after the onset of illness in a patient with prolonged clinical disease. A recent study showed a maximum duration of 15 days for infectious virus in patients not requiring hospitalization [
[6]
]. Ours is the first study to perform longitudinal experiments to recover infectious virus in a patient cohort that includes those with severe disease.Additionally, the role of genomic variation of mpxv on disease phenotype is not yet well characterized. The patients in this study were all diagnosed prior to the 2022 outbreak, in which the B.1 lineage of mpxv was dominant [
[7]
]. The mpxv isolates in this study will be of a different, non-B1 lineage and it is currently unclear the extent to which disease phenotype characteristics are generalizable across genomic variants. These findings have relevant infection control implications.Limitations in the study include the number of samples available and the lack of variety in sample type from some patients. These were clinician-collected samples and are paired with detailed clinical progress described previously [
[3]
]. Although clinical samples are often not treated as carefully as research samples, the samples included in this study have not knowingly had repeated freeze–thaw cycles, and were maintained at -70 °C, protecting the integrity of the samples for cell culture experiments.The infectious dose of mpxv is not known and may vary by site of exposure (keratinized epithelium vs mucous membrane), inoculating dose (high vs low viral load) and carrier (semen vs lesion fluid vs urine). The findings presented contribute to some of these unknowns. To date, we have not recovered infectious virus from a sample where the diagnostic Orthopoxvirus PCR Ct value was greater than 31.0, nor from urine. Although mpxv culture is not standardized, this result is consistent with others including Suner [
[6]
], who did not isolate infectious virus with a mpxv PCR Ct of more than 26.0, Paran [[8]
], who did not isolate above a Ct of 35.0 and Hernaez whose limit was Ct 30 [[9]
]. Notably, Ct is not a standardized measure and varies between laboratories and platforms. Lack of detection of infectious virus from urine is key for infection control since patients have demonstrated shedding of mpxv DNA in urine for prolonged duration; 73 days in the case of Patient 4 [[3]
]; additional studies are required. This study used clinical samples collected to inform clinical care and decisions around stopping isolation. Prior to the 2022 outbreak, patients with Mpox were managed in isolation in HCID units with prolonged time to mpxv DNA clearance by PCR. In clinical settings, detection of DNA by PCR is used because culture is a lengthy, expensive process, requiring significant laboratory infrastructure. The identification of a cut-off Ct value above which one can expect patients not to be infectious (31.0), using this pan-orthopox PCR assay, is useful for clinicians managing patients, as well as for public health advisors co-ordinating individual patient isolation and writing national policy.Acknowledgements
We would like to thank Elizabeth Truelove from the Rare and Imported Pathogens Laboratory (RIPL) for providing us with the stored samples. We would also like to thank the International Severe Acute Respiratory and emerging Infection Consortium (ISARIC) and all those who contributed to the care of the patient, including the high consequence infectious disease (HCID) network. Finally, we thank Hugh Adler et al. who described these patients clinical care in the 2022 Lancet paper.
Conflict of interest statement
The authors have no conflicts of interest to declare.
Funding sources
This work was funded by a Healthcare Infection Society (HIS) Small Research Grant (reference SRG/2022/03/002) to Catherine Houlihan and a Microbiology Society High Containment Microbiology Grant.
Appendix A. Supplementary data
The following are the Supplementary data to this article.
- Multimedia component 1
References
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Article info
Publication history
Published online: March 21, 2023
Accepted:
March 14,
2023
Received:
January 3,
2023
Identification
Copyright
Crown Copyright © 2023 Published by Elsevier Ltd on behalf of The Healthcare Infection Society. All rights reserved.
