Kaposi’s sarcoma-associated herpesvirus protein ORF75 among HIV-1 patients in Kenya

Background Histology is used to identify Kaposi’s sarcoma (KS) in countries with low resources to fund healthcare costs. Approximately 95% of KS cases can be detected using a polymerase chain reaction. Objective To determine the presence of the open reading frame 75 (ORF75) gene associated with Kaposi’s sarcoma herpes virus among HIV-1/AIDS patients and to describe morphological presentations of KS. Methods This was a retrospective, descriptive study of archived tissue blocks collected from 2013 to 2016. Haematoxylin and eosin staining was used to identify KS. Deoxyribonucleic acid from archived tissue blocks was extracted and a nested polymerase chain reaction was used to detect the ORF75 gene. Results All 81 cases in this study had been diagnosed as HIV-1 positive, of which 68 had hallmark features of KS in the histology report and 13 had features suggestive of KS (‘KS-like’). Microscopic identification of KS by haematoxylin and eosin staining was considered a significant indicator of KS herpes virus ORF75 gene positivity (p = 0.002). The ORF75 gene was detected in 60.5% (49/81) of tissue blocks; 27.2% were men (22/81) and 33.3% were women (27/81). The ORF75 gene was observed to be present in up to 15.4% (2/13) of the cases reported to have KS-like features. Conclusion Following the initial diagnosis of KS by histology, the ORF75 gene was fur-ther detected from both cases that had hallmark features of KS as well as among cases with KS-like fea-tures.


Introduction
Kaposi sarcoma (KS) is a tumor formed from blood vessels; it later shows lesions on the skin or organs of HIV-positive people. 1 All forms of KS are caused by Kaposi's sarcoma herpesvirus (KSHV), also known as human herpesvirus 8 (HHV-8). 2,3 The genome of HHV-8 contains a minimum of 100 open reading frames (ORF), of which 4 to 75 are known to be unique to herpesvirus. 4 The KSHV genome encodes more than 84 proteins that play a role in viral replication and host-virus interaction. 5 The replication cycle of KSHV entails latent and lytic phases. During the lytic cycle, the ORF75 genes are expressed resulting in the manifestation of KS. 6,7 The ORF75 gene product has been proven to aid in lytic replication and enhancement of virus pathogenesis in host cells. 8 Kaposi's sarcoma is listed among the defining malignancies of HIV/AIDS. 9,10,11 The distinct feature of HIV-associated KS is that it might affect the lymph nodes, gastrointestinal tract, lungs or liver. 12,13 Despite the fact that saliva is the main route by which KSHV is transmitted, 14 HHV-8 has been isolated from other body fluids. 14,15,16 The main route of HHV-8 transmission to the opposite sex is through sexual relations. 17 The pathogenesis of KS presents as an abnormal neoangiogenesis, proliferation of cancer cells and inflammation of endothelial cells. 18 A classic KS lesion manifests various features ranging from maculopapular to nodular or plaque-like and, in most cases, is painless. 12,19,20,21 http://www.ajlmonline.org Open Access biopsies by use of histological staining techniques should not be underestimated. 24 In tissue biopsies, microscopic examination involves identification of proliferated spindle cells and oedema. 25 Clinical diagnoses of KS have been shown to have limited predictive value. 26 The use of molecular techniques such as polymerase chain reaction (PCR) permits the detection of the HHV-8 gene even for patients who present with early vascular lesions that histological techniques might miss. 27 The use of PCR in the diagnosis of KS can detect approximately 95% of cases. 28 The HHV-8 DNA has been successfully amplified using nested PCR previously. 29,30 This study was aimed at determining the presence of the ORF75 gene linked to KSHV among HIV-1/AIDS patients. In addition, the objective of this current study was to describe the morphological presentations of KS among the studied cases.

Methods
The present study only included patients aged 18 years and older. Data on clinical information that was useful for this study were extracted from the registry records with the help of the data clerk. The following data were obtained from the registry records: sex, age, HIV-1 status, if patient was on antiretroviral or Highly Active Antiretroviral Therapy treatment, anatomic location of KS lesions, number of KS lesions, distribution of KS lesions, cluster of differentiation 4 cell count and histology diagnosis.

Ethical considerations
Study approval number P682/11/2014 was assigned by Kenyatta National Hospital/University of Nairobi Ethics and Research Committee.

Study design
A cross-sectional, descriptive, hospital-based study was used. Formalin-fixed, paraffin-embedded tissue blocks were retrieved from archives following histological reports of the patients who were diagnosed with KS or KS-like disease between 2013 and 2016. A consecutive sampling technique was used to select the archived tissue blocks from Thematic Unit of Anatomic Pathology, Department of Human Pathology, College of Health Sciences, University of Nairobi, and Department of Laboratory Medicine, Cytology Section, Kenyatta National Hospital.
For this study, a total of 81 tissue blocks were selected and analysed. A rotary microtome was used to section the formalin-fixed, paraffin-embedded blocks. A different blade was used for every formalin-fixed, paraffin-embedded block so as to avoid carry-over of genetic material. Once a block was cut, the microtomes surface was decontaminated using DNAZap TM PCR DNA degradation solution (catalog number: AM9890; Thermo Fisher Scientific, Waltham, Massachusetts, United States). Each tissue section was cut to 10 µm thick. The tissue sections were processed for haematoxylin and eosin staining and a qualified pathologist reported on the results.

Deoxyribonucleic acid extraction and polymerase chain reaction
Isolation of DNA from tissue sections was done using a GeneRead DNA FFPE kit (Qiagen, Hilden, Germany). The extraction kit removes paraffin and reverses formalin cross-links from tissue before DNA is bound to the QIAampMinElute column (Qiagen, Hilden, Germany). The eluted DNA is then ready to be used for nested PCR to detect the ORF75 gene in HHV-8. A Taq

Statistical analysis
The data were analysed using Statistical Package for Social Sciences version 21 (SPSS Inc Binghamton, New York, United States); the relationship between the ORF75 gene and clinical characteristics were tested by using chi-square and t-tests. A p-value of less than 0.05 was considered to be statistically significant. Odds ratios in a cross-sectional study are known as prevalence odds ratios and were used as a measure of association. 33

Results
Of the 81 tissue samples included in the study, 43.2% (35/81) were from women and 56.8% (46/81) were from men (

Kaposi sarcoma morphology and distribution of lesions
In the histology report, 68 cases had hallmark features of KS, whereas 13 cases had features suggestive of KS (KS-like). The types of KS morphology identified included patchy, nodular, plaque and KS-like ( Figure 1). The amplified ORF75 genes of HHV-8 were identified by 1% agarose gel electrophoresis ( Figure 2).

Discussion
Retrieved clinical data revealed that all of the tissue blocks retrieved in the present study were collected from patients who had been diagnosed with HIV-1. These patients might have developed KS lesions due to immunosuppression or because they were immunocomprised due to increased viral load that impaired their immune system. Other studies have also associated KS as an HIV/AIDS-defining illness. 9,13,24,34,35,36 The findings of this study revealed that men were more prone to development of KS: 56.8% (46/81) compared with women 43.2% (35/81). This observation is concordant with others who also noted more frequent development of KS among men. 37,38,39,40 There is a lack of consensus as to why all forms of KS are more common among men than women. 41  http://www.ajlmonline.org Open Access diagnosis of KS in HIV-positive patients, despite the availability of highly active antiretroviral therapy. 49 Other studies have stated that patients infected with AIDSassociated KS respond to combined antiretroviral therapy by 50% depending on geographical location and severity of the presentation, thereby resulting in immune reconstitution and HIV suppression. 50,51,52 In the current study, these manifestations of KS could be attributed to the weakening of the immune system by HIV-1.
This study found patchy, plaque and nodular morphological presentations of KS. The morphological appearance of KS shows progression from plaques to nodular form and fungiform. 1 Kaposi sarcoma lesions are known to progress from asymptomatic to macule, papule, plaque and nodule forms. 53 The findings of this study revealed that the KS lesions were disseminated in different body regions, including the lower limbs, upper limbs, genitalia, eyelids, palate, oral cavity and trunk (chest and back). In another study, fatality was witnessed in HIV-positive patients who had KS lesions manifested in the gastrointestinal tract, lungs and lymph nodes. 54 The decision in this study to use tissue biopsy for detection of the ORF75 gene of HHV-8 is in agreement with another study that supported the use of tumor biopsies as suitable for viral DNA identification due to high viral load as opposed to the use of blood. 30 Further to that, nested PCR has been used successfully to assess the prevalence of HHV-8 among HIV-positive patients in Brazil. 27  KSHV DNA. 55 The detection of the ORF75 gene implies that this gene was present in 49 (60.5%) of the studied cases.

Strength and limitations
Our study used the haematoxylin and eosin staining technique and the nested PCR method for detection of the ORF75 genes of the KSHV. However, HHV-8 immunohistochemical biopsy has been demonstrated to be the 'gold standard' for KS diagnosis. 56 Cases in the present study had a dark skin pigmentation. In another study of dark-skinned patients, KS had been confirmed to mimic a number of non-KS-like dermatological conditions. 56 In another study, it was observed that it is difficult to identify KS in dark-skinned individuals, who presented with violaceous skin lesions. 56 The use of the PCR technique in the detection of KSHV has been shown to give the utmost specificity compared to the use of tests that determine exposure to infection. 28 In addition, the PCR technique can detect approximately 95% of all KS cases. 28 However, the cost associated with the use of PCR is quite high, which would limit the clinical application of HHV-8 DNA detection in resource-limited facilities. 28

Implications and recommendations
The present study considered microscopic detection of KS by haematoxylin and eosin as a significant indicator of KSHV ORF75 gene positivity. It therefore recommends the use of both clinical diagnosis and routine microscopy in the diagnosis of KS in resource-limited facilities. However, among individuals with dark skin pigmentation, there is the need to employ the use of a robust diagnostic technique to ascertain the true causative agent.

Conclusion
The presence of the ORF75 gene of KSHV among immunosuppressed patients due to HIV-1 was successfully detected. Following the initial diagnosis of KS by histology,