Case report

The index case was born at term following an uncomplicated labour. Her mother had no clinical signs of human papillomavirus (HPV) infection. Psychomotor development was normal and the child had occasional mild upper respiratory tract infections. At the age of 2 years she gradually developed a hoarse voice and was referred to the local hospital. After ear-nose-throat (ENT) clinical examination and direct optical laryngoscopy, the patient was diagnosed with laryngeal papillomatosis and surgical laser ablation of papillomas was performed. Subsequent histological examination of tissue showed papillary structures covered by acanthotic squamous epithelium with mixed inflammatory cells in stroma. Final pathological diagnosis was squamous papilloma with mild dysplasia.

The patient was frequently seen by an ENT specialist during follow-up. Within 2 years of diagnosis there were six relapses with development of papillomas necessitating surgical treatment. These interventions were performed using laser ablation. Intervals between treatments shortened so that the shortest disease-free period was only 3 weeks. The last surgical excision was performed using surgical pliers under microscope control.

DNA from a biopsied specimen was isolated by means of a QIAamp DNA Mini Kit (tissue protocol) according to the manufacturer's handbook (QIAGEN, Hilden, Germany). Extracted DNA was amplified in a PTC 200 PCR thermocycler (MJ Research, Waltham, Massachusetts, USA). As an internal control, a 110 bp long fragment of the human β-globin gene was amplified using PC03/PC04 primers. Positive β-globin amplification showed that the sample contained enough DNA and that no PCR inhibitors were present. HPV DNA was detected by PCR using GP5+/GP6+ primers specific for the L1 region. Genotyping was performed by reverse line blot hybridisation (RLB), which permits the detection of 37 HPV types in a single assay.1 Genotyping in our case showed HPV type 11.

Immunological tests found no defects in humoral or cellular immunity. ELISA was used to test for anti-HPV antibodies using virus-like particles composed of capsid antigen L1 produced in a baculovirus expressing system in insect cells. No antibodies were found against HPV types 6, 11, 16, 18, 31 or 33. All HPV diagnostic procedures were undertaken in the National Reference Laboratory for Human Papillomavirus in The Institute of Hematology and Blood Transfusion, Prague.

The progressive course of the patient's disease led us to consider stimulating an immune response to HPV-11 as this might have a better chance of achieving long term remission compared to standard adjuvant treatments (such as cidofovir). We chose vaccination using a commercially approved vaccine with anti HPV-11 activity (GARDASIL, Merck, New Jersey, USA; regional brand name SILGARD in the Czech Republic). We used a three-dose schedule of vaccination (at 0, 2 and 6 months), with the first dose given 1 month after the last surgical excision.

The overall clinical picture and the course of disease changed markedly after vaccination. Since administration of the first vaccine dose, the patient has not experienced any episodes of voice hoarsening and repeated laryngoscopic examinations have found no recurrent papillomas. Two months after the third (and last) vaccination we checked anti-HPV antibodies and detected anti HPV-6 and anti HPV-11 antibodies in plasma.

Subsequent immune status study showed increased levels of IgM antibodies (1.88 g/l; reference range 0.4–1.6 g/l) and normal absolute CD3+ (3.04×10⁹/l; range 0.7–4.2), CD4+ (1.9×10⁹/l; range 0.3–2.0) and CD8+ (0.93×10⁹/l; range 0.3–1.8) T cell counts. At the time of writing, our patient has had no evidence of papillomatosis for 17 months. This is her longest disease-free period and her voice has regained normal physiological tone.