Investigation of human herpesvirus 8 & Leishmania species in malignant skin tumours, psoriasis, actinic keratoses, & seborrheic keratoses: A single-center experience from Ankara, Turkey

Materials & Methods

This study was conducted at the department of Microbiology, Ankara Etlik City Hospital, a tertiary care centre located in Ankara, Turkey. It included 275 individuals aged 18 yr and older who were treated at the hospital’s Dermatology Clinic between November 1, 2022, and June 23, 2023, for the evaluation of various skin diseases, from whom skin biopsy samples were obtained. This retrospective observational study was approved by the Clinical Research Ethics Committee of Ankara Etlik City Hospital, Turkey. The study was conducted on archived FFPE skin biopsy samples, without any direct involvement or intervention on patients. All procedures were performed in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments.

Results

The ages of the 275 participants enrolled in the study ranged from 18 to 93 yr (median: 65 yr [Interquartile range (IQR) 49-65]), and 53.8% (n=148) were male. The median age of the men was 66 yr (IQR 54-74), while that of the women was 63 yr (IQR 47-74) (P=0.302).

The distribution of the participants in the study group (n=254) according to their diagnoses was as follows: 43.3 per cent had malignant skin tumours (n=119), 13.5 per cent had psoriasis (n=37), 16.7 per cent had AK (n=46), and 18.9 per cent had SK (n=52). Those diagnosed with chronic dermatitis, considered as the control group, constituted 7.6 per cent (n=21) of the study.

PCR analysis did not detect HHV-8 DNA in any of the samples. On the other hand, Leishmania spp. DNA was detected in 8.4 per cent of all samples (n=23). However, no positivity results were observed in the control group (P=0.387). The Ct values of Leishmania spp. positive samples ranged from 16 to 30. The positivity rate was 9.4 per cent (2/127) in women and 7.4 per cent (11/148) in men (P=0.547).

Based on the diagnosis, Leishmania spp. PCR positivity was most frequently detected in psoriasis (32.4%), followed by AK (8.7%), malignant skin tumours (4.2%), and SK (3.8%).

When the Leishmania spp. positivity rate in patients diagnosed with psoriasis was analysed in relation to the control group, a statistically significant difference was observed (P=0.002) (Table I).

Although the positivity rate in SCC (7.3%), a type of malignant skin tumour, was higher than that in BCC (1.6%), this difference was not statistically significant (P=0.180).

The distribution of Leishmania spp. positivity rates according to diagnoses is presented in the figure.

Close

Figure.

Distribution of Leishmania spp. positivity rates according to diagnoses.

Export to PPT

When considering only the Leishmania spp.-positive cases, 11 out of 23 (48%) were male. Lesions were predominantly located on the head and extremities. In three participants, the lesions were atypically located (scalp, palm, and infraorbital region). A total of 34.8 per cent of the lesions were plaque-like (squamous and/or erythematous). The histopathological diagnosis with the highest positivity by PCR was psoriasis (52.1%), followed by AK (17.4%) and SCC (17.4%).

Additionally, according to the hospital information system records, there were none who were diagnosed with HIV infection among those included in the study.

Demographic and clinical characteristics, as well as pathological diagnoses of Leishmania spp. positive participants are presented in table II.

Discussion

Some viruses and parasites exhibit tropism to the skin and present with characteristic clinical findings. Additionally, they may resemble certain skin tumours and proliferative skin diseases. In such cases, specific histopathological examination methods or molecular tests may be required for a definitive diagnosis. The role of certain parasites and viruses in the aetiology of chronic skin diseases is still under investigation. In this study, HHV-8 and Leishmania spp. were analysed in skin biopsy samples from patients diagnosed with six different skin diseases.

HHV-8 DNA was not detected in any of the samples, whereas Leishmania spp. DNA was identified in 8.4 per cent (23/275) of all cases. Leishmania spp. positivity was observed exclusively in samples from disease groups other than the control group (chronic dermatitis). In these cases, antileishmanial treatment may facilitate faster healing of the lesions. This would not only provide psychological and social relief to patients but also help prevent new cases by eliminating potential transmission routes.

The involvement of HHV-8 in the pathogenesis of KS, a malignant skin disease, has been well established⁷. However, data regarding the presence of HHV-8 in malignant and chronic proliferative skin diseases other than KS remain limited in the current literature. Although HHV-8 has been implicated in other malignancies, such as primary effusion lymphoma and Castleman’s disease, there is a notable lack of studies investigating its role in non-Kaposi cutaneous proliferative disorders. As noted by Becerril et al³, existing literature primarily focuses on the association between HHV-8 and KS, thereby highlighting a need for more systematic and comprehensive research on its potential involvement in other dermatological conditions³. Supporting this, KSHV sequences were identified in 82 per cent of 33 skin lesions including BCC, SCC, AK, verruca vulgaris, atypical squamous proliferation, and SK collected from four KSHV-positive transplant patients undergoing immunosuppressive therapy in the USA⁶. Inagi et al¹⁸ found HHV-8 positivity in 50 per cent of SCC cases and 33.3 per cent of AK cases. Nishimoto et al⁷ used a molecular method to detect HHV-8 DNA in both fresh and paraffin-embedded tissues from 118 benign and malignant skin diseases, reporting positivity in three out of 11 SCC samples, three out of 11 AK samples, two out of seven chronic dermatitis samples, and one out of 14 normal skin samples. However, in the present study, PCR analysis did not detect HHV-8 DNA in any of the FFPE samples from individuals diagnosed with malignant and chronic proliferative skin diseases. Nishimoto et al⁷ suggested that HHV-8 might persist at latent levels too low to be detected, particularly in immunosuppressed microenvironments, which could explain our findings. The absence of detectable HHV-8 DNA in our samples may be attributed to low viral copy numbers, differences in patient populations and lesion types, or methodological variations, such as the use of Southern hybridisation in Nishimoto et al⁷, which might account for differences in detection rates. Additionally, it has been reported that the sensitivity of PCR in detecting latent viruses in FFPE samples may be limited, especially in cases with low viral loads. Our results suggest that there is no relationship between these diseases and HHV-8 infection, which is consistent with studies that have not confirmed this association. Furthermore, some studies indicate that HHV-8 positivity reported in the literature may be due to laboratory contamination^19,20.

Trento et al²¹ investigated the prevalence of HHV-8 antibodies in a healthy control group and patients with inflammatory skin diseases. They did not detect a significant difference in positivity rates between inflammatory skin diseases (eczema, 9%; psoriasis, 18.2%) and the control group (13.9%), further supporting the lack of association. This study was conducted using FFPE samples; therefore, no data on HHV-8 serology in the study participants were available. Considering that serological analysis provides complementary information regarding past exposure and immune response, future studies incorporating both molecular and serological approaches may offer a more comprehensive understanding of the role of HHV-8 in chronic skin diseases.

CL is frequently observed in tropical and subtropical regions²². However, studies have also been conducted in non-endemic countries^23,24. In Turkey, the majority of CL cases occur in the Southern provinces, including Şanlıurfa, Adana, Gaziantep, Hatay, Osmaniye, Kahramanmaraş, and Mersin. A significant increase in cases has been reported in the Southeastern provinces following the arrival of refugees from endemic regions²⁵. Additionally, CL cases have been reported in Burdur, Ankara, and Istanbul, which are non-endemic provinces, with some patients having a history of travel to other regions in the country^16,26,27. Similarly, global studies have identified cases without documented travel history^22,23. Although Ankara, where our study was conducted, is not an endemic area for CL, it receives immigration from both within and outside the country. Patients also visited our hospital from rural settlements. Among the Leishmania spp. positive cases, two patients resided in the Black Sea Region; however, their travel history to endemic areas could not be confirmed due to limited information in their medical records.

CL lesions typically present as single or multiple lesions on visible areas of the body, such as the extremities and the head and neck region²⁸. Rarely, cases and case series were reported in less common locations, including the lips, eyelids, nasal vestibule, nose, thighs, armpits, back, hips, and groin^9,13,22-24. In this study, consistent with the literature, most Leishmania spp. positive patients had lesions on the face and extremities, which are more exposed to the phlebotomes responsible for CL transmission. However, three cases exhibited atypical lesion locations (scalp, palm, and infraorbital region).

While some Leishmania spp. exhibit tropism to the skin, others show tropism to internal organs. Classically, L. infantum and L. donovani are recognised as the causative agents of visceral leishmaniasis (VL), whereas L. tropica and L. major are associated with CL. However, VL cases caused by L. tropica and CL cases caused by L. donovani and L. infantum have also been reported¹². This variation influences the location and visual characteristics of skin lesions, as well as the clinical progression of the disease^23,24. However, since retrospectively archived samples were used in the present study, factors such as the clinical course of the disease and variations in treatment response could not be assessed. Additionally, species discrimination was not possible due to the design of the PCR kit used. Studies conducted in our country have reported cases with atypical clinical presentations, in addition to lesions specific to CL, such as noduloulcerative lesions, multiple nodular lesions, superficial ulcerations, erythematous plaques, crusted ulcers, and erythematous scaly papuloplaques. In the differential diagnosis of these cases, diseases such as granuloma annulare, zosteriform lichen planus, linear inflammatory verrucous epidermal nevus, and epidermoid carcinoma were considered^12,16,27. Because CL can be confused with many granulomatous diseases, a differential diagnosis is crucial, particularly in chronic cases and malignancies that may develop from scarring. This may be attributed to the atrophy of adnexal structures in wound areas, making the affected tissues more susceptible to ultraviolet radiation and other exogenous carcinogens. Additionally, radiation exposure in these areas may further contribute to malignant transformation¹⁰. Besides granulomatous diseases, CL can also mimic conditions such as leprosy, deep fungal infections, mycobacterial infections, necrobiosis lipoidica, sporotrichosis, and pyoderma gangrenosum. This diagnostic overlap increases the risk of misdiagnosis and treatment delays, highlighting the importance of thorough clinical and histopathological evaluation^14,29.

Histopathological examination results in the diagnosis of CL may vary depending on the location and quantity of the biopsy material, the presence of necrosis in the lesion, parasite load, staining process, and the experience of the evaluating specialists²⁸.We believe that negative histopathological examination results may be attributed to the following reasons. In chronic diseases with a low parasite load in lesions, RT-PCR, which has higher sensitivity, may be preferred. In addition to fresh biopsy samples, FFPE biopsy samples can also be utilised for molecular methods³⁰. Another advantage of molecular methods is their ability to obtain epidemiological data by identifying the causative agent at the species level. This also provides valuable guidance in determining the appropriate treatment approach⁸.

A limitation of this study was that species discrimination could not be performed because the primers used in the molecular analysis targeted Leishmania species. Another limitation was that positive samples were not confirmed by sequencing.

In conclusion, this study demonstrated that HHV-8 DNA was not detected in biopsy samples from malignant and chronic proliferative skin diseases, suggesting no association between HHV-8 and these conditions. On the other hand, Leishmania spp. DNA was identified in 8.4 per cent of all samples, indicating that factors such as climate change, environmental conditions, and migration may contribute to an increased incidence in non-endemic regions. Considering its potential to mimic other dermatological conditions, CL may be included in the differential diagnosis of chronic, non-healing, and treatment-refractory skin lesions, especially in cases with atypical clinical presentations or in patients from endemic areas, to facilitate early detection and minimise scarring. Additionally, the use of FFPE biopsy samples in molecular analyses may aid in the diagnosis of CL cases with atypical clinical presentations.