W06　リーシュマニア

リーシュマニア症は、吸血昆虫のサシチョウバエによって媒介される。近年サシチョウバエが吸血時に注入する唾液に対する免疫応答を宿主に付与することにより、リーシュマニア感染を防御できるという報告がなされ、サシチョウバエ唾液成分が新しいリーシュマニア感染防御ワクチンとして期待されている。本研究では、リーシュマニアワクチン候補となるサシチョウバエ唾液タンパクの同種内での抗原性について比較検討した。[材料と方法] 地理的に隔離された地域（アフリカのマリおよびケニア）から採取した、Leishmania majorを媒介するサシチョウバエP. duboscqiの唾液腺からcDNAライブラリーを作製、それぞれランダムに約1,000クローンずつのcDNAの塩基配列を決定し、遺伝子転写産物を網羅的に解析をした。また、それぞれの唾液タンパクについてT細胞エピトープの解析を行い、抗原性について比較検討した。[結果と考察] P. duboscqi唾液の主要タンパクは、近縁種であるP. papatasiのSP12, SP14, SP15, SP28, SP30, SP32, apyrase, yellow-related proteinと相同性をもつタンパク、旧大陸サシチョウバエ種 (Phlebotomus sp.) では報告がないadenosine deaminaseなどから構成されていた。これらのタンパクをマリおよびケニアのP. duboscqi間で比較したところ、非常に高い相同性がみとめられ、T細胞エピトープがよく保存されていることが明らかになった。これらのことから、サシチョウバエ唾液成分を利用したリーシュマニアワクチンは、地理的分布が隔離されている場合でも、同種のサシチョウバエが媒介するリーシュマニア感染に対しては効果的であることが示唆された。

We previously reported a highly sensitive (94.0%) and specific (99.6%) urine-based ELISA using a recombinant kinesin-related protein with a molecular weight of 42-kDa (rKRP42 urine ELISA) for the diagnosis of visceral leishmaniasis (VL). We also applied this newly developed rKRP42 urine ELISA, in a endemic village named ‘Nobai Bot Tala’ to detect rKRP42 antigen specific antibodies in urine samples, and found to be very useful in early detection of clinical VL cases. This time, we analysed anti-rKRP42 IgG levels in urine and serum, and also compared the results with commercially available (serum-based) rK39 dipstick test, with paired urine and serum samples collected from 70 confirmed VL cases and 439 volunteers of the endemic village. Among 70 confirmed VL cases 65 were positive and 4 were negative by all three tests. One serum ELISA and dipstick positive gave negative result with urine ELISA. Among 439 endemic volunteers 47 were positive and 351 were negative by all three tests. Forty-one gave variable results with 3 different tests. More detailed analysis will be presented with these 41 samples. Positive correlations were observed between anti-rKRP42 IgG in urine and serum both in confirmed VL and endemic volunteers. We previously reported that measurement of rKRP42 antigen specific IgG in urine samples will help to diagnose VL in its early stage, and high IgM titer could be utilized to predict possible clinical development. The present study confirmed the usefulness of urine for serum for the diagnosis of VL.

Protozoan parasite Leishmania donovani is the causative agent of Kala-azar or visceral leishmaniasis (VL). In Nepal, VL is endemic in 12 districts of central and eastern regions which border with endemic areas Bihar state of India, where VL endemic has been known. The main purposes of this study are to identify species and genotype of Leishmania isolates from Nepal and to characterize their infectivity to experimental animals. We applied PCR-restriction fragment-length polymorphism (RFLP) analysis of the cysteine proteinase b (cpb) gene and mini-exon gene to analyze Leishmania isolates from Nepal. One isolate from India and Kenya were used as control. These isolates were also studied by single-strand conformation polymorphism (SSCP) analysis of the ribosomal internal transcribed spacer (ITS). Isolates were obtained from bone-marrow aspirates from suspected patients. Out of one hundred sixty-six aspirates, seventy-two were microscopically positive and twenty-nine were successfully isolated in culture. Twenty-nine isolates were identified as L. donovani by RFLP and SSCP. Result obtained from RFLP and SSCP revealed that all isolates collected from Nepal have the same pattern with the standard Indian isolate but different from a Kenyain isolate. This is due to no genetic heterogeneity within L. donovani species collected from Nepal. Twenty-nine isolates were inoculated in nude and BALB/c mice. Twenty isolates were considered to have infectivity in nude mice, while ten isolates can infect to BALB/c mice.

Determinants of the clinical presentation of the leishmaniases are poorly understood but Leishmania species and strain differences are important. To examine the relationship between clinical presentation, species and isoenzyme polymorphisms, 56 Leishmania isolates from distinct presentations of American tegumentary leishmaniasis (ATL) from Ecuador were analyzed. Isolates were characterized by MLEE for 11 isoenzymes. Six Leishmania species constituting 21 zymodemes were identified: L. (Viannia) panamensis (21 isolates, 7 zym), L. (V.) guyanensis (7 isolates, 4 zym), L. (V.) braziliensis (5 isolates, 3 zym), L. (Leishmania) mexicana (11 isolates, 4 zym), L. (L.) amazonensis (10 isolates, 2 zym) and L. (L.) major (2 isolates, 1 zym). L. panamensis the species most frequently identified in the Pacific region was associated with several clinical variants of cutaneous disease (CL); eight cases of recidiva cutis (LRC) found in the Pacific highlands were associated with 3 zymodemes. Mucocutaneous leishmaniasis found only in the Amazon was associated with 3 zymodemes of L. braziliensis. The Uta lesions, found in the Andean highlands were related predominantly with a single zymodeme of L. mexicana. Our data show a high degree of phenotypic variation within species, and some evidence for associations between specific variants of ATL (i.e. Uta and LRC) and specific Leishmania zymodemes. This study further defines the geographic distribution of Leishmania sp. and clinical variants of ATL in Ecuador.

内臓リーシュマニア症（VL症）は全世界で毎年５０万人が新たに感染しており、その２０％がバングラデシュ北東部、インド東部，ネパール南東部一帯に集中している。この地方のVL症はヒト以外に宿主はなく、サシチョウバエ（Phlebotomus argentipes）によって媒介される。バングラデシュのVL症は５０-６０年代に行われたマラリア対策のためのDDT残留噴霧により激減したが、７０年代の散発的な発生の後、８０年代にはPabna Districtで大流行した。現在、６４のDistrictのうち、３４DistrictsでVL症の発生が報告されているが、その多くはOld Brahmaputra川とPadma川にはさまれる北西部の１０ Districtsに集中しており、１９９４-２００４年の発生数の９０％を占めている。９０年代後半は、Padma川北側のPabna Districtで多くのVL症の発生が見られたが最近では減少傾向にあり，かわってOld Brahmaputra川流域のMymensingh Districtでの発生が増加しており、バングラデシュのVL症の発生の５０％を占めるようになってきている。
VL症の治療は従来通りアンチモニー製剤の投与（４週間の筋注）であるが、副作用が多く薬剤耐性の出現も報告されている。現在、経口投与ができるMiltefosineが期待されており治験が始まっている。VL症controlには、簡便な診断法の開発普及、それを用いた広範囲な疫学調査による流行地の特定、患者の早期発見、早期治療、サシチョウバエ対策が不可欠である。また、VL症が貧困層の疾患であることも考慮すべき重要な要因である。