Keith Waddell

Human Transcriptome Subtraction by Using Short Sequence Tags To Search for Tumor Viruses in Conjunctival Carcinoma

ABSTRACT Digital transcript subtraction (DTS) was developed to subtract in silico known human sequences from expression library data sets, leaving candidate nonhuman sequences for further analysis. This approach requires precise discrimination between human and nonhuman cDNA sequences. Database comparisons show high likelihood that small viral sequences can be successfully distinguished from human sequences. DTS analysis of 9,026 20-bp tags from an expression library of BCBL-1 cells infected with Kaposis sarcoma-associated herpesvirus (KSHV) resolved all but three candidate sequences. Two of these sequences belonged to KSHV transcripts, and the third belonged to an unannotated human expression sequence tag. Overall, 0.24% of transcripts from this cell line were of viral origin. DTS analysis of 241,122 expression tags from three squamous cell conjunctival carcinomas revealed that only 21 sequences did not align with sequences from human databases. All 21 candidates amplify human transcripts and have secondary evidence for being of human origin. This analysis shows that it is unlikely that distinguishable viral transcripts are present in conjunctival carcinomas at 20 transcripts per million or higher, which is the equivalent of approximately 4 transcripts per cell. DTS is a simple screening method to discover novel viral nucleic acids. It provides, for the first time, quantitative evidence against some classes of viral etiology when no viral transcripts are found, thereby reducing the uncertainty involved in new pathogen discovery.

Evaluating the role of human papillomaviruses in conjunctival neoplasia

Mucosal, cutaneous and Epidermodysplasia verruciformis (EV)-related human papillomaviruses (HPVs) were searched by broad-spectrum PCR in 86 conjunctival neoplasia biopsies and 63 conjunctival non-neoplastic control tissue from Ugandan subjects. Seven different EV-related HPV types, including a putative new HPV, and two mucosal HPVs were detected in 25% (14 out of 56) of HIV-positive, in 10% (three out of 30) of HIV-negative conjunctival neoplasia samples, and rarely (0–1.6%) in control subjects. The absence of high-risk HPVs and the low detection frequency of EV-related HPV types in more advanced tumour stages (10%) raise doubts about their role in conjunctival carcinomas.

Genital and cutaneous human papillomavirus (HPV) types in relation to conjunctival squamous cell neoplasia: A case-control study in Uganda

BackgroundWe investigated the role of infection with genital and cutaneous human papillomavirus types (HPV) in the aetiology of ocular surface squamous neoplasia (which includes both conjunctival intraepithelial neoplasia (CIN) and carcinoma) using data and biological material collected as part of a case-control study in Uganda.ResultsAmong 81 cases, the prevalence of genital and cutaneous HPV types in tumour tissue did not differ significantly by histological grade of the lesion. The prevalence of genital HPV types did not differ significantly between cases and controls (both 38%; Odds ratio [OR] 1.0, 95% confidence interval [CI] 0.4–2.7, p = 1.0). The prevalence of cutaneous HPV types was 22% (18/81) among cases and 3% (1/29) among controls (OR 8.0, 95% CI 1.0–169, p = 0.04).ConclusionWe find no evidence of an association between genital HPV types and ocular surface squamous neoplasia. The prevalence of cutaneous HPV was significantly higher among cases as compared to controls. Although consistent with results from two other case-control studies, the relatively low prevalence of cutaneous HPV types among cases (which does not differ by histological grade of tumour) indicates that there remains considerable uncertainty about a role for cutaneous HPV in the aetiology of this tumour.

A case-control study of ocular surface squamous neoplasia (OSSN) in Uganda.

HIV increases the risk of OSSN. Here we investigate other factors in a case‐control study from Uganda with 318 cases (48 CIN I, 66 CIN II, 81 CIN III and 123 with invasive disease) and 762 controls. Initial analyses were stratified by HIV serostatus (204 cases and 202 controls were HIV seropositive), but since findings were similar in infected and uninfected people, the combined results are presented here. The risk of OSSN increased with increasing time spent in direct sunlight (ptrend = 0.003, adjusted for age, sex, residential district and HIV serostatus): compared to those who reported spending up to 1 hr a day in direct sunlight, the risk was 1.7 (95% Confidence Interval [CI] 1.2–2.4) in those reporting 2–4‐hr exposure and 1.8 (95% CI 1.1–3.1) in those reporting 5+ hr. The risk was also increased among people reporting a previous injury to the affected eye (OR 2.4, 95% CI 1.2–4.5). Pinguecula in the nasal quadrant of the unaffected eye were evident on clinical examination for 98% of cases (293/300) and for 91% of the same quadrant in the right eye (246/271) of controls (OR = 6.4, 95% CI 2.5–16.1). We confirm associations with exposure to solar ultraviolet radiation and with the presence of pinguecula and report a role for previous ocular trauma in the aetiology of OSSN. We did not identify any additional factors that point to an underlying infectious cause, although this is an area of on‐going research.

Antibodies against human papillomavirus type 16 (HPV-16) and conjunctival squamous cell neoplasia in Uganda

Antibodies against human papillomavirus type 16 (HPV-16) and conjunctival squamous cell neoplasia in Uganda

The aetiology and associations of conjunctival intraepithelial neoplasia: further evidence

Kiire and Dhillon draw attention to the surge in cases of conjunctival neoplasia,1 but do not make clear that this has occurred mostly in sub-Saharan Africa—in Uganda, for example, the reported incidence has more than tripled over the past decade.2,3 HIV infection leads to a roughly 10-fold increase in the risk of conjunctival neoplasia, and in Africa, most people with conjunctival neoplasia are HIV positive.4–8 In a recent study of 414 cases in Uganda, 64% of people with conjunctival neoplasia were HIV positive; this applied to intraepithelial and invasive cases.9 Ophthalmologists in Western countries may see …

Clinical features and survival among children with retinoblastoma in Uganda

Aims To characterise the clinical features, treatment and outcome of children diagnosed with retinoblastoma in Uganda. Methods The study comprised a 6-year nationwide enrolment with follow-up. Results In total, 282 cases were enrolled, 26% (72) were bilateral; 6% were lost to follow-up. Almost all diagnoses in the first affected eye were International Classification of Retinoblastoma group E or worse. Histology was available for 92%; of those, 45%, had extraocular tumour at diagnosis. Enucleation of the first eye was done for 271; 94 received radiotherapy to the socket and in the last 2 years, 70 children received chemotherapy. At close of study, 139 children had died. Survival, as determined in a proportional hazards model adjusted for age, sex, laterality and treatment era (pre or post introduction of chemotherapy), varied by extent of the tumour (p<0.001); children with only intraocular involvement were 80% less likely to die (HR=0.21, 95% CI 0.12 to 0.35) compared with children with extraocular involvement. Conclusions Diagnostic delay results in relatively high mortality among children with retinoblastoma in Uganda. There is an urgent need for more effective treatment modalities, particularly chemotherapy, and nationwide efforts to encourage earlier access to medical care.

Identification of Human Herpesvirus 8 Sequences in Conjunctiva Intraepithelial Neoplasia and Squamous Cell Carcinoma of Ugandan Patients.

The incidence of squamous cell carcinoma of the conjunctiva is particularly high in sub-Saharan Africa with temporal trends similar to those of Kaposi sarcoma (KS). Human herpesvirus type 8 (HHV8), has not yet been investigated in conjunctiva tumors. In this study biopsies and PBMCs of conjunctiva neoplasia patients along with nonneoplastic conjunctiva tissues have been analyzed for HHV8 sequences by PCR targeting ORF26. All amplimers were subjected to nucleotide sequencing followed by phylogenetic analysis. HHV8 DNA has been identified in 12 out of 48 (25%) HIV-positive, and in 2 out of 24 (8.3%) HIV-negative conjunctiva neoplastic tissues and in 4 out of 33 (12.1%) PBMC samples from conjunctiva neoplasia diseased patients as well as in 4 out of 60 (6.7%) nontumor conjunctiva tissues. The viral load ranged from 1 to 400 copies/105 cells. Phylogenetic analysis showed that the majority of HHV8 ORF26 amplimers clustered with subtypes R (n = 11) and B2 (n = 6). This variant distribution is in agreement with that of HHV8 variants previously identified in Ugandan KS cases. The presence of HHV8 in conjunctiva tumors from HIV-positive patients warrants further studies to test whether HHV8 products released by infected cells may have paracrine effects on the growth of conjunctiva lesions.

Improving survival of retinoblastoma in Uganda

Background Diagnostic delay results in relatively high mortality among children with retinoblastoma in Uganda, where treatment was limited to surgery and, for some, radiotherapy. In order to improve outcomes, a simple programme of neoadjuvant and adjuvant chemotherapy was introduced. Here we report survival before and after this change to medical practice. Methods Affordable standard off-patent chemotherapy agents were administered by trained paramedical staff to groups of patients at the same time. Survival before and after the introduction of chemotherapy was monitored. Between 2006 and 2013 a total of 270 patients with retinoblastoma were included, 181 treated prior to chemotherapy and 89 after (beginning in 2009). We had 94% follow-up and 249 had histological verification of diagnosis. Results Using a proportional hazards model adjusted for age, sex and laterality, children treated after chemotherapy was introduced had a 37% lower risk of dying (HR 0.63, 95% CI 0.41 to 0.99) compared with children treated before. Prior to the introduction of chemotherapy only 15% of children who survived bilateral disease retained vision after treatment compared with 71% after chemotherapy. Conclusions The introduction of chemotherapy proved safe and cost-effective in non-specialist hands and was associated with significant improvements in survival and, among bilateral cases, in preserving vision.