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Letter to the Editor| Volume 86, ISSUE 2, P154-225, February 2023

Role of multi-site sampling in the diagnosis of human Monkeypox

Published:December 12, 2022DOI:https://doi.org/10.1016/j.jinf.2022.12.010
Role of multi-site sampling in the diagnosis of human Monkeypox
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      Keywords

      Dear editor,
      We read with interest the manuscript by Li et al., in which the authors analysed the persistence of Monkeypox virus (MPXV) DNA in samples from different anatomical sites, providing a reference for the appropriate time for viral detection
      • Li Z.
      • Li X.X.
      • Chen Y.
      • Ruan Q.
      • Huang X.
      • Zhu G.
      • Sun J.
      Persistence of monkeypox virus DNA in clinical specimens.
      J Infect. 2022; 85 (DecEpub 2022 Oct 17. PMID: 36265827; PMCID: PMC9574605): 702-769https://doi.org/10.1016/j.jinf.2022.10.013
      . After its first identification in 1958, MPXV had a limited circulation outside endemic countries, restricting the possibility to study clinical and virological outcomes during infection; therefore, the ongoing global outbreak involving thousands of subjects provides an unprecedented opportunity to improve disease management and prevention.
      The World Health Organization (WHO) guidance on MPXV infection laboratory diagnosis recommends performing Real-Time Polymerase Chain Reaction (RT-PCR) on swab from lesions, exudates or crusts; in addition, it is suggested to collect and test additional specimens (i.e.: urine, semen, blood, rectal and genital swabs), to produce new evidences on disease progression and viral dynamics.

      Laboratory testing for the monkeypox virus: interim guidance, 23 May 2022. World Health Organization (WHO). 23 May 2022. https://apps.who.int/iris/handle/10665/354488 [Accessed on 21 Nov 2022].

      In this regard, many authors detected MPXV DNA in human saliva, seminal fluid, urine, and faeces,
      • Peiró-Mestres A.
      • Fuertes I.
      • Camprubí-Ferrer D.
      • Marcos M.Á.
      • Vilella A.
      • Navarro M.
      • Rodriguez-Elena L.
      • Riera J.
      • Català A.
      • Martínez M.J.
      • Blanco J.L.
      Hospital Clinic de Barcelona Monkeypox Study Group. Frequent detection of monkeypox virus DNA in saliva, semen, and other clinical samples from 12 patients, Barcelona, Spain, May to June 2022.
      Eurosurveillance. 2022; 27 (JulPMID: 35837964; PMCID: PMC9284919)2200503https://doi.org/10.2807/1560-7917.ES.2022.27.28.2200503
      , 
      • Raccagni A.R.
      • Candela C.
      • Mileto D.
      • Canetti D.
      • Bruzzesi E.
      • Rizzo A.
      • Castagna A.
      • Nozza S.
      Monkeypox infection among men who have sex with men: PCR testing on seminal fluids.
      J Infect. 2022; 85 (NovEpub 2022 Jul 29. PMID: 35914609; PMCID: PMC9556608): 573-607https://doi.org/10.1016/j.jinf.2022.07.022
      , while Ouafi et al. compared oropharyngeal swab (OPS) and lesion sample (LS), concluding that OPS might not be useful for diagnosis.
      • Ouafi M.
      • Regueme A.
      • Alcaraz I.
      • Riviere P.
      • Bazus H.
      • Salmon-Rousseau A.
      • Cappeliez B.
      • Cartier N.
      • Guigon A.
      • Lazrek M.
      • Bocket L.
      • Faure E.
      • Robineau O.
      • Hober D.
      • Alidjinou E.K.
      Oropharyngeal samples versus lesion specimens at diagnosis in patients infected with monkeypox virus in Northern France.
      J Med Virol. 2022; (Nov 3Epub ahead of print. PMID: 36326021): e28276https://doi.org/10.1002/jmv.28276
      The aim of this study was to evaluate the role of different types of samples, in particular OPS, that might be collected in combination with LS to achieve the highest sensitivity for MPXV diagnosis.
      Study population included 625 subjects with clinical evidence of MPXV infection, attending Emergency Departments or Infectious Diseases Units of different hospitals in Lombardy from 25 May to 15 November 2022. The following specimens were collected and tested to detect the presence of MPXV: OPS, LS, anal (AS) and urethral (US) swabs, plasma (PL) and urine (U). OPS, LS, AS and US were collected by means of Universal Transport Medium swabs (UTM-RT®; COPAN Diagnostics, Italy). Sterile screw cap containers were used to collect urine, while blood samples were collected in the BD Vacutainer® K2EDTA (BD Diagnostics, USA) tube and centrifuged to obtain plasma.
      DNA was extracted using the QIAsymphony DSP Virus/Pathogen Kit on QIAsymphony SP instrument (QIAGEN, Germany). Samples were primarily screened by means RealStar Orthopoxvirus PCR Kit 1.0 (altona DIAGNOTICS, Germany), targeting variola virus and non-variola Orthopoxvirus species (Cowpox virus, Monkeypox virus, Raccoonpox virus, Camelpox virus, Vaccinia virus), confirming the presence of MPXV DNA with the species-specific Monkeypox Virus Real Time PCR Kit (Bioperfectus, Shanghai). Samples were considered positive with a Cycle threshold (Ct) value on RT-PCR ≤ 40.
      All laboratory analysis were performed in an Italian reference centre for MPX diagnosis.
      MPXV was detected in 46% (286/625) of patients, 99% (283/286) males with a median age of 38 years (IQR 33-44). LS showed the highest sensitivity for MPXV DNA, 93%. AS and OPS resulted positive in 79% and 69% of the cases. The highest viral load, reported as the lowest Ct value, was detected in LS, followed by AS, with median Ct values of 20 (IQR:17-24) and 24 (IQR:18-33), respectively. Urine resulted the less sensitive sample, considering a positive rate of 18%, despite the median Ct comparable to OPS (Table 1).
      Table 1Monkeypox virus detection in different types of samples.
      SamplenPositivePositivity rate (%)Median (IQR) Ct value
      LS2522359320 (17;24)
      OPS2781916929 (25;33)
      AS1651317924 (18;33)
      US53356631 (27;34)
      U94171828 (23;33)
      PL124856934 (31;35)
      Abbreviations: LS=lesion sample; OPS=oropharyngeal sample; AS=anal sample; US=urethral sample; U=urine; PL=plasma.
      For each type of sample, the median time of collection from symptoms onset was, in days: LS 6 (IQR: 0–21), OPS 6 (IQR: 0–23), AS 7 (IQR: 0–29), US 9 (IQR: 0–26), U 7 (IQR: 0–29) and 7 (IQR: 0–29) PL.
      Fifteen patients, despite the presence of skin lesions, resulted negative for MPXV DNA on LS. In 6 of them viral DNA was detected only in the OPS, in 4 it was detected in OPS and AS; 1 patient resulted positive in OPS and PL; 4 patients had detectable DNA only in AS whose only 2 of them had lesions in the anal area (Table 2).
      Table 2Detection of MPXV DNA in patients resulted negative for viral DNA in lesion sample. For each positive sample is reported in brackets ( ) the Real-Time PCR Ct value.
      PatientLSOPSASUSUPL
      patient 1negpos (29)nananegpos (33)
      patient 2negpos (33)nananana
      patient 3negpos (28)pos (31)nanegneg
      patient 4negnegpos (37)negnaneg
      patient 5negnegpos (29)nanegneg
      patient 6negpos (34)pos (21)nanegna
      patient 7negpos (35)pos (18)nanana
      patient 8negnegpos (37)nanegneg
      patient 9negpos (38)negnanegneg
      patient 10negpos (32)nananegneg
      patient 11negpos (36)negnanegneg
      patient 12negnegpos (17)nanana
      patient 13negpos (34)negnanegneg
      patient 14negpos (35)pos (24)negnapos (34)
      patient 15negpos (35)negnanana
      Abbreviations: LS=lesion sample; OPS=oropharyngeal sample; AS=anal sample; US=urethral sample; U=urine; PL=plasma; neg=sample negative for MPXV DNA; pos=sample positive for MPXV DNA; na=data not available.
      The present study reports the results of molecular analysis performed on 966 samples from 625 patients suspected for MPX, in five different biological matrices at the time of diagnosis. As showed by other authors, viral DNA can be detected in several specimens:
      • Peiró-Mestres A.
      • Fuertes I.
      • Camprubí-Ferrer D.
      • Marcos M.Á.
      • Vilella A.
      • Navarro M.
      • Rodriguez-Elena L.
      • Riera J.
      • Català A.
      • Martínez M.J.
      • Blanco J.L.
      Hospital Clinic de Barcelona Monkeypox Study Group. Frequent detection of monkeypox virus DNA in saliva, semen, and other clinical samples from 12 patients, Barcelona, Spain, May to June 2022.
      Eurosurveillance. 2022; 27 (JulPMID: 35837964; PMCID: PMC9284919)2200503https://doi.org/10.2807/1560-7917.ES.2022.27.28.2200503
      similarly, in this analysis viral DNA was detected in plasma (69%), urine (18%), and anal swabs (79%); in addition, 66% of urethral swabs were positive.
      Data confirm that lesion material is the most sensitive specimen for MPXV DNA detection, showing the highest positivity rate (93%) and lowest Ct value at RT-PCR. A similar data about positivity rate (97%) was observed in a recent large study on MPX cases across non endemic countries.
      • Thornhill J.P.
      • Barkati S.
      • Walmsley S.
      • Rockstroh J.
      • Antinori A.
      • Harrison L.B.
      • Palich R.
      • Nori A.
      • Reeves I.
      • Habibi M.S.
      • Apea V.
      • Boesecke C.
      • Vandekerckhove L.
      • Yakubovsky M.
      • Sendagorta E.
      • Blanco J.L.
      • Florence E.
      • Moschese D.
      • Maltez F.M.
      • Goorhuis A.
      • Pourcher V.
      • Migaud P.
      • Noe S.
      • Pintado C.
      • Maggi F.
      • Hansen A.E.
      • Hoffmann C.
      • Lezama J.I.
      • Mussini C.
      • Cattelan A.
      • Makofane K.
      • Tan D.
      • Nozza S.
      • Nemeth J.
      • Klein M.B.
      Orkin CM; SHARE-net clinical group. Monkeypox Virus infection in humans across 16 Countries - April-June 2022.
      N Engl J Med. 2022; 387 (Aug 25Epub 2022 Jul 21. PMID: 35866746): 679-691https://doi.org/10.1056/NEJMoa2207323
      The presence of viable virions in anal and urethral sites has also been demonstrated through isolation in cell culture.
      • Moschese D.
      • Pozza G.
      • Mileto D.
      • Giacomelli A.
      • Cutrera M.
      • Cossu M.V.
      • Matone M.
      • Beltrami M.
      • Salari F.
      • Antinori S.
      • Lombardi A.
      • Rizzardini G.
      Isolation of viable monkeypox virus from anal and urethral swabs, Italy, May to July 2022.
      Eurosurveillance. 2022; 27 (SepPMID: 36082684; PMCID: PMC9461308)2200675https://doi.org/10.2807/1560-7917.ES.2022.27.36.2200675
      Our data confirm the utility of these samples for the detection of MPXV for diagnostic purposes: AS showed higher positivity rate and viral load in comparison to US, and its collection is much less invasive. Urine does not represent a valid choice; despite the easy procedure of collection, it showed the lowest positivity rate. Considering the lower viral load in plasma and the invasive procedure for blood collection, plasma sample might not represent a suitable specimen for MPX diagnosis.
      According to WHO, clinicians are encouraged to collect OPS in patients suspected with MPX.

      Laboratory testing for the monkeypox virus: interim guidance, 23 May 2022. World Health Organization (WHO). 23 May 2022. https://apps.who.int/iris/handle/10665/354488 [Accessed on 21 Nov 2022].

      Mai et al. highlighted the importance of oral cavity examination, given the growing knowledge of oral involvement.
      • Mai A.S.
      • Tan E.K.
      Oropharyngeal involvement of monkeypox: key considerations.
      J Med Virol. 2022; (Oct 13Epub ahead of print. PMID: 36229423)https://doi.org/10.1002/jmv.28217
      Screening the oropharynx for MPXV DNA is indicated by many authors as useful to assess the risk of transmission through respiratory secretions and oral sex;

      CDC Monkeypox Response: Transmission. Centers for Disease Control and Prevention (CDC). 9 Jun 2022. https://www.cdc.gov/media/releases/2022/0509-monkeypox-transmission.html (Accessed on 21 Nov 2022).

      , 
      • Brown K.
      • Leggat P.A.
      Human Monkeypox: current state of knowledge and implications for the future.
      Trop Med Infect Dis. 2016; 1 (Dec 20PMID: 30270859; PMCID: PMC6082047): 8https://doi.org/10.3390/tropicalmed1010008
      although intuitively feasible, very few reported cases of MPX transmission are accountable to this spreading route. Possibly, one explanation for this, is the higher Ct values observed. Ouafi et al. detected viral DNA in 55 OPS out of 61, with a median Ct value of 29.2 (IQR: 24.4–33.9); since those patients had other typical lesions with higher viral load, they concluded that OPS in this population might not be very useful.
      • Ouafi M.
      • Regueme A.
      • Alcaraz I.
      • Riviere P.
      • Bazus H.
      • Salmon-Rousseau A.
      • Cappeliez B.
      • Cartier N.
      • Guigon A.
      • Lazrek M.
      • Bocket L.
      • Faure E.
      • Robineau O.
      • Hober D.
      • Alidjinou E.K.
      Oropharyngeal samples versus lesion specimens at diagnosis in patients infected with monkeypox virus in Northern France.
      J Med Virol. 2022; (Nov 3Epub ahead of print. PMID: 36326021): e28276https://doi.org/10.1002/jmv.28276
      In our study, we found that OPS was positive for MPXV DNA in 69% (191/278) of the cases with a median Ct value of 29 (IQR: 25–33). Amongst MPX patients, although lesion swab resulted negative in 15 cases, the diagnosis was confirmed in 11 patients due to the presence of OPS alone or in combination with AS/PL samples while in 4 patients on AS alone. Thus, in our population, non-lesion sampling was crucial for MPX diagnosis in patients with PCR negative-LS.
      Despite only few MPX cases with PCR-negative LS took advantage of OPS and AS testing, these data support on a large scale the importance of collecting and testing samples from different anatomical sites, including oropharynx and anus, in combination with LS in highly suspected patients, to maximize MPXV detection and improve diagnostic performances.

      Ethical considerations

      All data were anonymised.

      Funding

      This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

      CRediT authorship contribution statement

      Alberto Rizzo: Writing – original draft, Writing – review & editing, Investigation. Davide Mileto: Writing – review & editing. Davide Moschese: Writing – review & editing, Conceptualization. Caterina Candela: Conceptualization, Writing – review & editing. Alessandro Mancon: Writing – review & editing. Andrea Giacomelli: Conceptualization, Writing – review & editing. Angelo Roberto Raccagni: Conceptualization, Writing – review & editing. Federica Salari: Writing – original draft, Investigation, Writing – review & editing. Maria Vittoria Cossu: Conceptualization, Writing – review & editing. Valeria Micheli: Writing – review & editing. Antonella Castagna: Writing – review & editing, Supervision. Giuliano Rizzardini: Writing – review & editing, Supervision. Alessandra Lombardi: Writing – review & editing, Supervision. Silvia Nozza: Writing – review & editing, Conceptualization. Maria Rita Gismondo: Writing – review & editing, Supervision.

      Declaration of Competing Interest

      The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

      References

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        • Li X.X.
        • Chen Y.
        • Ruan Q.
        • Huang X.
        • Zhu G.
        • Sun J.
        Persistence of monkeypox virus DNA in clinical specimens.
        J Infect. 2022; 85 (DecEpub 2022 Oct 17. PMID: 36265827; PMCID: PMC9574605): 702-769https://doi.org/10.1016/j.jinf.2022.10.013
        View in Article

      2. Laboratory testing for the monkeypox virus: interim guidance, 23 May 2022. World Health Organization (WHO). 23 May 2022. https://apps.who.int/iris/handle/10665/354488 [Accessed on 21 Nov 2022].

        View in Article
        • Peiró-Mestres A.
        • Fuertes I.
        • Camprubí-Ferrer D.
        • Marcos M.Á.
        • Vilella A.
        • Navarro M.
        • Rodriguez-Elena L.
        • Riera J.
        • Català A.
        • Martínez M.J.
        • Blanco J.L.
        Hospital Clinic de Barcelona Monkeypox Study Group. Frequent detection of monkeypox virus DNA in saliva, semen, and other clinical samples from 12 patients, Barcelona, Spain, May to June 2022.
        Eurosurveillance. 2022; 27 (JulPMID: 35837964; PMCID: PMC9284919)2200503https://doi.org/10.2807/1560-7917.ES.2022.27.28.2200503
        View in Article
        • Raccagni A.R.
        • Candela C.
        • Mileto D.
        • Canetti D.
        • Bruzzesi E.
        • Rizzo A.
        • Castagna A.
        • Nozza S.
        Monkeypox infection among men who have sex with men: PCR testing on seminal fluids.
        J Infect. 2022; 85 (NovEpub 2022 Jul 29. PMID: 35914609; PMCID: PMC9556608): 573-607https://doi.org/10.1016/j.jinf.2022.07.022
        View in Article
        • Ouafi M.
        • Regueme A.
        • Alcaraz I.
        • Riviere P.
        • Bazus H.
        • Salmon-Rousseau A.
        • Cappeliez B.
        • Cartier N.
        • Guigon A.
        • Lazrek M.
        • Bocket L.
        • Faure E.
        • Robineau O.
        • Hober D.
        • Alidjinou E.K.
        Oropharyngeal samples versus lesion specimens at diagnosis in patients infected with monkeypox virus in Northern France.
        J Med Virol. 2022; (Nov 3Epub ahead of print. PMID: 36326021): e28276https://doi.org/10.1002/jmv.28276
        View in Article
        • Thornhill J.P.
        • Barkati S.
        • Walmsley S.
        • Rockstroh J.
        • Antinori A.
        • Harrison L.B.
        • Palich R.
        • Nori A.
        • Reeves I.
        • Habibi M.S.
        • Apea V.
        • Boesecke C.
        • Vandekerckhove L.
        • Yakubovsky M.
        • Sendagorta E.
        • Blanco J.L.
        • Florence E.
        • Moschese D.
        • Maltez F.M.
        • Goorhuis A.
        • Pourcher V.
        • Migaud P.
        • Noe S.
        • Pintado C.
        • Maggi F.
        • Hansen A.E.
        • Hoffmann C.
        • Lezama J.I.
        • Mussini C.
        • Cattelan A.
        • Makofane K.
        • Tan D.
        • Nozza S.
        • Nemeth J.
        • Klein M.B.
        Orkin CM; SHARE-net clinical group. Monkeypox Virus infection in humans across 16 Countries - April-June 2022.
        N Engl J Med. 2022; 387 (Aug 25Epub 2022 Jul 21. PMID: 35866746): 679-691https://doi.org/10.1056/NEJMoa2207323
        View in Article
        • Moschese D.
        • Pozza G.
        • Mileto D.
        • Giacomelli A.
        • Cutrera M.
        • Cossu M.V.
        • Matone M.
        • Beltrami M.
        • Salari F.
        • Antinori S.
        • Lombardi A.
        • Rizzardini G.
        Isolation of viable monkeypox virus from anal and urethral swabs, Italy, May to July 2022.
        Eurosurveillance. 2022; 27 (SepPMID: 36082684; PMCID: PMC9461308)2200675https://doi.org/10.2807/1560-7917.ES.2022.27.36.2200675
        View in Article
        • Mai A.S.
        • Tan E.K.
        Oropharyngeal involvement of monkeypox: key considerations.
        J Med Virol. 2022; (Oct 13Epub ahead of print. PMID: 36229423)https://doi.org/10.1002/jmv.28217
        View in Article

      9. CDC Monkeypox Response: Transmission. Centers for Disease Control and Prevention (CDC). 9 Jun 2022. https://www.cdc.gov/media/releases/2022/0509-monkeypox-transmission.html (Accessed on 21 Nov 2022).

        View in Article
        • Brown K.
        • Leggat P.A.
        Human Monkeypox: current state of knowledge and implications for the future.
        Trop Med Infect Dis. 2016; 1 (Dec 20PMID: 30270859; PMCID: PMC6082047): 8https://doi.org/10.3390/tropicalmed1010008
        View in Article

      Article info

      Publication history

      Published online: December 12, 2022
      Accepted: December 9, 2022

      Identification

      DOI: https://doi.org/10.1016/j.jinf.2022.12.010

      Copyright

      © 2022 The British Infection Association. Published by Elsevier Ltd. All rights reserved.

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