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Absence of Chlamydia psittaci in ocular adnexal lymphoma from Japanese patients

Low-grade malignant lymphomas arising from mucosa-associated lymphoid tissue (MALT) represent a distinct clinicopathological entity of B-cell non-Hodgkin lymphoma (Isaacson & Wright, 1984). MALT lymphoma tends to remain localised and the extranodal sites of involvement include the gastrointestinal tract, salivary gland, thyroid and ocular region. MALT lymphoma often occurs in relation to autoimmune disorders or chronic antigen stimulation, but the pathogenetic mechanisms of this type of lymphoma have not been well defined. Recently, Ferreri et al (2004) reported that 32 (80%) of 40 Italian patients with ocular adnexal lymphoma harboured Chlamydia psittaci DNA. The findings suggested that C. psittaci infection might contribute to the development of these lymphomas. Soon after this, two other groups independently investigated ocular adnexal lymphoma for the presence of C. psittaci DNA in 57 patients from south Florida (Rosado et al, 2005) and 11 patients from the north-eastern USA (Vargas et al, 2005) and found that none of them carried C. psittaci DNA. One explanation for this discrepancy concerns geographic differences in the incidence of C. psittaci infection. However, all of these reports are based on findings from patients in the USA and Europe, and therefore additional surveys evaluating the association between C. psittaci and ocular adnexal lymphomas including MALT lymphoma in other geographical regions of the world would be warranted. There has been no report from Asia thus far. In this study, we looked for C. psittaci DNA in tumour specimens from Japanese patients with primary ocular adnexal lymphoma. Tissues obtained by an incisional biopsy from 24 Japanese patients (12 males and 12 females, mean age 56 years; range 34–73 years) with localised lymphoproliferative conditions of the ocular adnexa, including 18 MALT lymphomas, three nonMALT lymphomas and three reactive ocular lymphoid hyperplasias, were studied. The localities of the tumours were the orbit in 14 cases, lacrimal glands in four cases, and conjunctiva in six cases. DNA was obtained from frozen or paraffinembedded tissues using the phenol/chroroform extraction technique or DNA extraction system according to the manufacturer’s instructions (Takara, Tokyo, Japan). All samples were successfully amplified by polymerase chain reaction (PCR) for human b-globin sequences, yielding a 123-bp amplicon, which indicated that amplifiable DNAs were present. Three independent PCRs were used to detect C. psittaci DNA in this study. First, we used the same method employed in the previous studies (Ferreri et al, 2004; Rosado et al, 2005; Vargas et al, 2005). The touchdown enzyme time release PCR with CPS100 and CPS101 primers produces a 111-bp amplicon (Madico et al, 2000). The primers amplify a highly conserved region encoding the 16S ribosomal RNA gene. The second method was a nested PCR to detect the same DNA region with the first-step primers (5¢-ACGGAATAATGACTTCGG-3¢ and 5¢-TACCTGGTACGCTCAATT-3¢) and the second-step primers (5¢-ATAATGACTTCGGTTGTTATT-3¢ and 5¢-TGTTTTA GATGCCTAAACAT-3¢), generating a 127-bp amplicon. The PCR primers for the third C. psittaci PCR were derived from another DNA region encoding the ompA gene, and the primer sequences were 5¢-GCCTTAAACATCTGGGATCG-3¢ and 5¢-GCACAACCACATTCCCATAAAG-3¢, giving a 248-bp fragment. All three PCRs failed to detect C. psittaci DNA in any of our samples, while positive control reactions using DNA prepared from C. psittaci strain Cal-10 yielded amplicons of expected size. In addition, our samples were negative for Chlamydia pneumoniae and Chlamydia trachomatis DNA sequences. Our results showed no correlation between C. psittaci infection and ocular adnexal lymphoma in Japan, supporting the observation from the USA (Rosado et al, 2005; Vargas et al, 2005). All these studies were based on testing C. psittaci DNA sequences. Given the serological evidence of an association between chronic chlamydia infections (C. pneumoniae and C. trachomatis) and lymphomas (Anttila et al, 1998), serological surveys for C. psittaci may also help to better understand the relationship between C. psittaci and ocular adnexal lymphoma. Although the ‘hit-and-run’ mechanism cannot be fully excluded, the present findings indicate that C. psittaci is unlikely to have played a pathogenetic role in ocular adnexal lymphoma in the Japanese population.