key: cord-0887045-s59kooqs
authors: Koike, Masami; Hayashi, Shujiro; Baba, Akiko; Koike, Saiko; Gonmori, Takeshi; Kaminaga, Tomoko; Ishii, Eisuke; Ishikawa, Satoko; Hamasaki, Yoichiro; Igawa, Ken
title: PCR-based diagnosis of Sporothrix infection using DNA from paraffin-embedded skin specimens in previously undiagnosed cases
date: 2021-05-14
journal: Eur J Dermatol
DOI: 10.1684/ejd.2020.3862
sha: 136ee62ffccf342e178fa6a0ea795aca660a8974
doc_id: 887045
cord_uid: s59kooqs

nan

Definitive diagnosis of dermatomycosis requires identification based on culture of focal eruption samples. However, it is occasionally necessary to also consider culture-negative cases and cases without culture. PCR may aid in the diagnosis of such cases. However, although nested-PCR (involving two rounds of PCR amplification) is highly sensitive, it is liable to contamination, which may affect the interpretation of results [1] . Histopathological study of the first skin biopsy (haematoxylin and eosin staining). In the sample from the first biopsy, acanthosis, pseudo-cancerous proliferation between epidermis to dermis (arrow), and inflammatory cells (lymphocytes, histiocytes, neutrophils, and giant cells) are present in the dermis (×100) (B), and nuclear division is observed in a proportion of the proliferating epidermal cells (×400) (C). In the entire sample from the second surgery, inflammatory granulation tissue and giant cells (arrow), suggesting a fungal infection, are shown in (D) (×100) and (E) (×400). Fungal elements (arrow) are apparent with Periodic acid-Schiff (F; arrow) (×400) and Grocott's methenamine silver (G; arrow) (×400). H) Representative PCR-positive results for 7/21 patients (sample No.1 refers to the presented case), whose specimens were not submitted for culture or culture-negative, and who were suspected to have cutaneous sporotrichosis based on histopathological evaluation; the presence of Sporothrix is indicated by the presence of the 152-bp fragment (arrow).

We previously reported identification of Sporothrix based on extraction of DNA from formalin-fixed and paraffinembedded (FFPE) tissues using nested PCR [1] . The sensitivity and specificity of this method was 100% and 98.7%, respectively (sample size: culture-positive n=52, controls n=79). Herein, we present a patient whose biopsy was not submitted for culture because the skin lesion was a suspected cutaneous neoplastic lesion based on clinical and histopathological investigation, however, sporotrichosis was subsequently detected by PCR. An 85-year-old man was referred to our department because of an irregularly raised, crusted mass on the back of his right hand ( figure 1A) . The lesion was reported to have gradually increased in size over about two months and measured 3.5 cm. A histopathological study of the skin biopsy at the first visit revealed acanthosis, pseudo-cancerous proliferation between epidermis to dermis, and inflammatory cells (lymphocytes, histiocytes, neutrophils, and giant cells) in the dermis ( figure 1B) . Nuclear division was observed in a proportion of the proliferating epidermal cells ( figure 1C) .

The clinical and initial pathological findings suggested a highly differentiated squamous cell carcinoma-like lesion. The entire lesion was excised and the skin was surgically grafted. A specimen was not sent for culture. However, histopathological examination of the entire sample showed inflammatory granulation tissue and giant cells, findings suspicious of fungal infection, with no evidence of squamous cell carcinoma ( figure 1D, E) . Staining with Periodic acid-Schiff (PAS) and Grocott's methenamine silver (GMS)

showed fungal elements ( figure 1F, G) . Therefore, our PCR method for Sporothrix was performed using the FFPE sample retrieved at the second surgery, and a positive result was obtained. The patient received additional treatment with an antifungal agent for one month after which there was no recurrence. Cutaneous sporotrichosis is a deep dermatomycosis caused by organisms of the Sporothrix schenckii complex [2] , and is known to occur frequently on the face of children and extremities of the elderly [3, 4] . The gold standard for diagnosis involves the isolation of the fungus based on a culture test. Because these fungi are not detected superficially on the skin, definitive diagnosis requires culture of skin biopsy samples. However, cultures occasionally return false-negative results [2] . In addition, skin biopsy is invasive, requiring local anaesthesia, and is especially difficult to perform multiple times on the face in children. Moreover, it is occasionally challenging to distinguish these lesions from other infections and cutaneous neoplasms [5] , therefore cases exist in which a culture has not been submitted or the disease is not suspected prior to skin biopsy. We collected 21 biopsy specimens from patients at Dokkyo Medical University (1979 to 2019), who were suspected to have cutaneous sporotrichosis based on histopathological evaluation (haematoxylin and eosin, PAS and GMA), but for whom cultures were not obtained or were negative. PCR of DNA samples obtained from FFPE samples revealed positive results in 81.3% (17/21) of the patients (seven samples are presented in figure 1H ).

Other ancillary diagnostic tools for cutaneous sporotrichosis include a sporotrichin skin test, microscopic examination after applying potassium hydroxide and a histopathological test. However, these tests do not always provide positive results [4] . A method for extracting DNA from fresh samples and performing PCR for early diagnosis has also been reported [6, 7] , however, in such cases, a suspicion of cutaneous sporotrichosis is required before surgery or skin biopsy in order to obtain a sample for culture testing.

Cutaneous sporotrichosis occasionally relapses [3] , in particular, in patients for whom culture was not performed and definitive diagnosis was not made. In these situations, patients require a clinical diagnosis from testable samples, thus the PCR method from FFPE samples could aid diagnosis. However, false positives due to contamination may occur [1] , therefore diagnosis should be considered along with comprehensive clinical findings and other tests in addition to PCR. [2, 3] , but documenting such cases is crucial for the management of the disease.

Here we report a case of SLE/systemic sclerosis (Ssc) overlap syndrome in which a long-term successful response to belimumab was demonstrated.

The patient was a 31-year-old Japanese female with SLE/Ssc overlap syndrome. She was diagnosed with SLE at age 18 due to joint pain, a high fever, stomatitis, anaemia, high serum IgG, and serum anti-double-stranded (ds) DNA and anti-Sm antibodies, and had been treated with prednisolone (PSL) at 5 mg/day and azathioprine (AZA) at 50-100 mg/day. In her 20s, she was diagnosed with SSc based on serum anti-Scl70 antibody level

Diagnostic value of a nested polymerase chain reaction for diagnosing cutaneous sporotrichosis from paraffin-embedded skin tissue

Taxonomy and phylogeny of new wood-and soil-inhabiting Sporothrix species in the Ophiostoma stenoceras-Sporothrix schenckii complex

Sporothrix schenckii and Sporotrichosis

Sporotrichosis: from KOH to Molecular Biology

Differential identification of Sporothrix spp. and Leishmania spp. by conventional PC and qPCR in multiplex format

Detection of Sporothrix schenckii in clinical samples by a nested PCR assay

Fast diagnosis of sporotrichosis caused by Sporothrix globosa, Sporothrix schenckii, and Sporothrix brasiliensis based on multiplex real-time PCR