We conducted a retrospective observational study to analyze demographic characteristics, symptom onset, specimen types, qPCR results, and cycle threshold (Ct) values of suspected MPXV cases in South Korea referred to the Korea Disease Control and Prevention Agency (KDCA). Clinical specimens were collected by trained healthcare personnel and included skin lesions, upper respiratory tract swab specimens, blood, and rectal swab specimens. We categorized skin lesions by type (crusted, vesicular, papular, pustular, or unknown) and site (anogenital, nonanogenital, or unknown), including the anus, perineum, penis, and vulva. We grouped samples by days since symptom onset (0–1, 2–3, 4–10, and >10 days). We considered Ct values >40 or undetectable negative and Ct values <40 positive.

We extracted viral DNA from 200 µL of clinical specimens by using the QIAamp Blood Mini Kit (QIAGEN, https://www.qiagen.com) following the manufacturer’s protocol. qPCR targeted F3L and A39R genes by using the following primers and probes: F3L forward 5′-CATCTATTATAGCATCAGCATCAGA-3′, reverse 5′-GATACTCCTCCTCGTTGGTCTAC-3′, probe 5′-FAM-TGTAGGCCGTGTATCAGCATCCATT-IBFQ-3′; A39R forward 5′-TGGGATAACGAATCCAATGTCA-3′, reverse 5′-GCGTGCTTCCAGCAACACT-3′, probe 5′-FAM-AGCGCCTAGCACAGAACACATTTACGA-IBFQ-3.

For group comparisons, we used χ² or Fisher exact test for categorical variables and the Kruskal-Wallis test or analysis of variance for Ct value comparisons. We conducted analyses by using SPSS Statistics 29.0.10 (IBM, https://www.ibm.com) and GraphPad Prism version 8 (https://www.graphpad.com). This study was approved by the KDCA institutional review board (approval no. 2020-03-01-P-A), which waived written informed consent.

During August 28, 2022–October 3, 2023, the KDCA received 1,668 specimens from 379 patients with suspected mpox. After we excluded 244 patients (1,056 specimens) because of missing clinical data (999 samples from 231 patients) and unclear onset times (57 samples from 13 patients), we analyzed 612 specimens from 135 patients. Most (97.7%) specimens were from men. Median patient age was 31.7 (interquartile range [IQR] 27.7–37.1) years, and median collection time was 6 (IQR 2.0–8.0) days after onset. Specimens were 362 skin lesions, 109 upper respiratory tract swab specimens, 135 blood samples, and 6 rectal swab specimens; 445 samples were PCR-positive.

Figure 1

Figure 1. Comparison of Ct values of real-time PCR at each lesion and time point of each specimen after symptom onset in patients with mpox, South Korea, 2022–2023. Horizontal lines within boxes...

MPXV DNA was more frequently detected from skin lesions (92.8%, n = 336/362) and rectal swab specimens (100%, n = 6/6) than from upper respiratory tract (68.8%, n = 75/109) and blood samples (28.2%, n = 38/135) (Table 1; Appendix Figure 1). Median Ct values were significantly lower for skin lesions (median 22.01, IQR 19.25–28.19) and rectal swab specimens (median 20.19, IQR 18.85–24.23) than in the upper respiratory tract (median 33.61, IQR 29.09–40.00) and blood samples (median 40.00, IQR 38.61–40.00; p<0.001); we found no significant difference between skin lesions and rectal swab specimens (p = 0.36) (Table 1; Figure 1). Among skin lesions, crusted types demonstrated the highest positivity rate (93.3%, n = 70/75) and the lowest median Ct value (median 20.63, IQR 18.30–24.48), significantly lower than for other lesion types (median 23.33, IQR 19.74–30.00; p<0.001). Anogenital lesions had lower Ct values (median 20.78, IQR 17.91–25.65) than lesions at other sites (median 22.82, IQR 19.91–29.94; p = 0.018). Positivity rates (p = 0.556) and Ct values (p = 0.351) did not differ significantly between oropharyngeal and nasopharyngeal specimens; however, oropharyngeal samples tended toward higher positivity (64.3%) and lower Ct values (median 34.37, IQR 29.78–40.00) compared with nasopharyngeal samples (50.0%, n = 2/4; median Ct 37.14, IQR 32.45–40.00), despite the limited sample size.

Figure 2

Figure 2. Monkeypox viral loads given as Ct values in patients with mpox, according to sampled sites and time of onset, South Korea, 2022–2023. Blue dots indicate Ct results for each sample....

Positivity and Ct values varied over time after symptom onset (Table 2; Figure 2). The Ct values of the skin lesions changed significantly (p<0.001), whereas positivity remained high (≈90%) for up to 60 days (p = 0.142). Upper respiratory tract specimens showed no significant changes in positivity (p = 0.738) or Ct values (p = 0.369), with peak positivity (75%) on day 1, lasting until day 30. Blood samples had low positivity (28.2%) and no great variation over time, peaking in the first 3 days (32%), and were detectable for ≈20 days.

Mpox is primarily transmitted through direct contact with body fluids, respiratory droplets, or contaminated surfaces (6). Increasing evidence indicates that sexual contact is the dominant transmission route during outbreaks, particularly among men who have sex with men (7). This evidence is supported by frequent anogenital lesions and the detection of culturable viruses in saliva, the nasopharynx, and semen (7). The high positivity and low Ct values observed in anogenital lesions in our study further support that finding.

After inoculation, MPXV replicates in the skin, spreads to the lymph nodes, and causes viremia and systemic dissemination (8). Skin lesions progress through macules, papules, vesicles or pustules, and crusts (6). Crusted lesions have historically been regarded as less infectious, but accumulating evidence indicates that viral DNA and infectious virus might persist until crust detachment (9,10). Consistent with that persistence of infectious virus, we observed high MPXV DNA levels in crusted lesions, indicating its potential infectivity. MPXV also demonstrates strong tropism for keratinocytes. Skin specimens, particularly anogenital lesions and rectal swab specimens, exhibit higher viral loads than blood or respiratory specimens, underscoring the central role of the skin in viral replication and direct contact transmission (11,12).

Ct values in skin lesions decreased significantly over time, consistent with reports from China demonstration high viral DNA detection for up to 3 weeks (13). Upper respiratory tract samples peaked in positivity 1 day after symptom onset and then declined, similar to the oropharyngeal patterns reported in China (13). Blood samples demonstrated low positivity, which increased slightly in the first 3 days and then declined, consistent with reports of plasma and serum viral DNA peaking before day 10 (13). Our findings support evidence that skin and rectal swab specimens yield higher viral loads than blood or respiratory swab specimens (14,15).

The limitations of our study include incomplete clinical data and a small number of rectal swabs. Many patients relied on referral records that lacked detailed lesion or location data. Prospective studies are necessary to clarify the links between symptoms, severity, duration, and outcomes.

Our study of MPXV clade IIb infection during the 2022–2023 outbreak in Korea found that crusted and anogenital skin lesions and rectal swab specimens demonstrated the highest PCR positivity, followed by upper respiratory tract and blood samples. Peak positivity occurred around day 1 in the respiratory tract and on day 3 in blood. The Ct values differed significantly by lesion type and anatomic location, highlighting the importance of targeted specimen collection and the appropriate sampling timing in MPXV clade IIb infection.