Discovery of 4 exonic and 1 intergenic novel susceptibility loci for leprosy.

Seven new risk coding variants have been identified through an exome-wide association study (EWAS), which studied the contributions of protein-coding variants to leprosy susceptibility. But some potential susceptibility loci were not studied in the previous EWAS study because of the project consideration. Seventeen unstudied potential susceptibility loci of the previous EWAS were validated in 3169 cases and 9814 controls in this study. Four disease-associated exonic loci were identified: rs671 in ALDH2 (P = 2.0 × 10-20 , odds ratio [OR] = 1.35), rs13259978 in SLC7A2 (P = 1.74 × 10-8 , OR = 1.28), rs925368 in GIT2 (P = 9.18 × 10-17 , OR = 1.44), and rs75680863 in TCN2 (P = 8.37 × 10-21 , OR = 0.74). Potentially implicating ZFP36L1 as a new susceptibility gene, 1 intergenic single nucleotide polymorphism (SNP), rs1465788 (P = 7.81 × 10-6 , OR = 0.88), was also suggested to be associated with leprosy. A luciferase reporter assay showed that the rs1465788 risk allele notably decreased the transcription activity of the flanking sequence. These findings suggest the possible involvement of lipid metabolism, NF-κB homeostasis and macrophage antimicrobial pathways in leprosy pathogenesis.

HLA-B*13:01 and the dapsone hypersensitivity syndrome.

BACKGROUND: Dapsone is used in the treatment of infections and inflammatory diseases. The dapsone hypersensitivity syndrome, which is associated with a reported mortality of 9.9%, develops in about 0.5 to 3.6% of persons treated with the drug. Currently, no tests are available to predict the risk of the dapsone hypersensitivity syndrome. METHODS: We performed a genomewide association study involving 872 participants who had received dapsone as part of multidrug therapy for leprosy (39 participants with the dapsone hypersensitivity syndrome and 833 controls), using log-additive tests of single-nucleotide polymorphisms (SNPs) and imputed HLA molecules. For a replication analysis, we genotyped 24 SNPs in an additional 31 participants with the dapsone hypersensitivity syndrome and 1089 controls and performed next-generation sequencing for HLA-B and HLA-C typing at four-digit resolution in an independent series of 37 participants with the dapsone hypersensitivity syndrome and 201 controls. RESULTS: Genomewide association analysis showed that SNP rs2844573, located between the HLA-B and MICA loci, was significantly associated with the dapsone hypersensitivity syndrome among patients with leprosy (odds ratio, 6.18; P=3.84×10(-13)). HLA-B*13:01 was confirmed to be a risk factor for the dapsone hypersensitivity syndrome (odds ratio, 20.53; P=6.84×10(-25)). The presence of HLA-B*13:01 had a sensitivity of 85.5% and a specificity of 85.7% as a predictor of the dapsone hypersensitivity syndrome, and its absence was associated with a reduction in risk by a factor of 7 (from 1.4% to 0.2%). HLA-B*13:01 is present in about 2 to 20% of Chinese persons, 1.5% of Japanese persons, 1 to 12% of Indians, and 2 to 4% of Southeast Asians but is largely absent in Europeans and Africans. CONCLUSIONS: HLA-B*13:01 was associated with the development of the dapsone hypersensitivity syndrome among patients with leprosy. (Funded by the National Natural Science Foundation of China and others.).

First Report of Root Rot Caused by Phytophthora sansomeana on Soybean in China.

Phytophthora sansomeana E.M. Hansen & Reeser is a newly described species and infects Douglas-fir, alfalfa, and soybean (1). Soybean production is an important part of the local economy in Yili State in Xinjiang Uygur Autonomous Region, northwest China. Unfortunately since 2005, root and stem rot disease has emerged on a number of farms. To identify the causal agent, plant samples with symptoms, including whole plant wilting or yellowing and stunting, were collected from fields during 2005 and 2008. Tissue from the edges of stem lesions was placed on selective lima bean agar (LBA) at 20°C for 3 to 4 days (2,3). Four single zoospore isolates of Phytophthora were obtained and maintained on LBA or 10% V8 juice liquid medium for examination of morphological and physiological characteristics. The colonies on LBA were aerial and rosaceous. The isolates were homothallic, and oogonia and oospores were readily produced in culture after 7 days on LBA plates. Oogonia averaged 38 µm and oospore width ranged from 23 to 48 µm and averaged 31 µm. Antheridia were approximately 15 × 12 µm and predominantly amphigynous in V8 juice. Sporangia were terminal or paragynous on persistent sporangiophores, nonpapillate, ovoid to obpyriform, and measured 52 × 35 µm with an average length/breadth ratio of 1.5. Hyphal swellings were produced in V8 juice 2 days after inoculation. The optimum temperature for growth was approximately 25°C and none occurred at 0 or 35°C. The internal transcribed spacer (ITS) sequence of this Phytophthora species (GenBank FJ966880) agreed 100% with sequences of P. sansomeana isolates deposited in GenBank (GQ853880 and EU925375). Pathogenicity tests were performed by hypocotyl inoculation method (2) using isolate Yili71 and potted soybean cv. Williams. Plants were grown in a growth chamber for 10 days before inoculation in 16-cm-diameter pots (2). Plants were inoculated with 2- × 2-mm plugs of mycelium grown for 4 days on LBA at 25°C, the plugs were adhered to the sides of wounded lower hypocotyls. As controls, plants were inoculated with LBA agar plugs without mycelium (2). Inoculated plants were maintained in a growth chamber at approximately 25°C with a 10-h dark/14-h light cycle and 50% relative humidity and symptom development was monitored daily for 1 week. Wounded stems inoculated with mycelium developed water-soaked lesions, which were similar to those seen on naturally infected plants. A Phytophthora sp. was reisolated from the margins of expanding lesions on wounded stems. To our knowledge, this is the first report of P. sansomeana infection of soybean in China and the threat it may pose to soybean production is unclear. References: (1) E. M. Hansen et al. Mycologia 101:129. 2009. (2) Z. Y. Wang et al. Fungal Genet. Biol. 43:826, 2006. (3) X. B. Zheng. Methods in Phytophthora. Chinese Agriculture Press. Beijing, China, 1995.

An investigation of attitudes, beliefs and behaviour of leprosy patients, family members and PHC workers towards multidrug therapy in Yangzhou and Dongtai Districts of China.

To improve the operational efficiency of multidrug therapy (MDT) implementation in rural areas, an investigation into the attitudes, beliefs and behaviour of leprosy patients and their family members as well as primary health care (PHC) workers towards MDT was carried out in Yangzhou and Dongtai Districts of China. A sample of 370 leprosy patients, 594 family members and 730 PHC workers was interviewed or investigated individually using questionnaires. The results showed that: 1, the presently used MDT is acceptable to a wide range of patients although a small number of patients have various problems in their treatment; 2, the patients' habit in daily drug administration, their awareness of the risk of default and confidence in MDT have a positive influence in increasing drug compliance; and 3, the supervision and encouragement of family members to patients' treatment which is associated with their knowledge on MDT is also beneficial to patients' drug compliance. However, only half of the PHC workers had a basic knowledge of MDT and a desire to participate in MDT implementation, a finding which clearly calls for urgent attention and improvement. In order to ensure the effective implementation of MDT, there is a need to educate leprosy patients and their family members as well as PHC workers to establish the patients' correct awareness of MDT, obtain the family support and motivate the PHC workers.

Study on nude mice inoculated with Mycobacterium leprae by multiple routes.

Immune-deficient nude mice were inoculated with nude mouse derived Mycobacterium leprae by multiple routes (intravenously, subcutaneously at the foot pads and ears). The results showed that these inoculated animals were capable of producing a great number of Mycobacterium leprae to a level 10 per gram of tissue and were detected histopathologically to have heavy lepromatous lesions. The dissemination of the infection was found particularly in sites with lower body temperature. The organisms have a partiality to striated muscles and peripheral nerves. The authors suggest that experimental leprosy in nude mice is a very useful tool for leprosy research, especially in countries without armadillos. Compared with the single-route inoculation reported previously, multiple-route inoculation is more available.

The susceptibility testing of 13 strains of Mycobacterium leprae to rifampicin and the determination of minimal effective dosage.

By use of the mouse footpad technique, the susceptibility testing of 13 strains of Mycobacterium leprae to rifampicin (RFP) and the determination of minimal effective dosage (MED) were carried out. Among these strains of M. leprae, 8 were obtained from previously untreated multibacillary leprosy patients and 5 from relapsed leprosy patients without using RFP previously. The results showed that the MED of all strains to RFP were less than or equal to 0.001% FRP in the diet, 5 strains being equal to 0.001%, 5 less than or equal to 0.0001%, 2 greater than or equal to 0.0003% and 1 less than or equal to 0.0003%. The results indicated that the MED value of RFP could be lower than that of other reports. Because the critical concentration of RFP for assessment of RFP-resistant strains is not well established a further study would be worthwhile. The results of the determination of sera RFP concentrations in mice administered the RFP diet were identical with that of Holmes' report. Five of the 13 strains also showed that the growth of bacilli were suppressed by 10 mg/kg RFP using the gavage method.

Preliminary observations on experimental leprosy in tupaias (Tupaia belangeri yunalis).

The Tupaia belangeri yunalis (tree shrew) is one of the primitive primates. They were inoculated subcutaneously in the footpad or intravenously with Mycobacterium leprae from a patient with multibacillary leprosy. As controls, the footpads of CFW mice were inoculated with the same suspension of M. leprae. The results showed growth of acid-fast bacilli (AFB) in the footpads of locally inoculated CFW mice and in the footpads of both locally and intravenously inoculated tupaias. Whereas the numbers of AFB declined in the footpads of CFW mice after 12 months, they increased in the tupaia footpads, up to 2.44 x 19(9) AFB/g of tissue. The footpads of one tupaia were swollen, which on section revealed a granulomatous infiltration, including foamy and heavily infected macrophages. M. leprae were also seen in the branches of cutaneous nerves. Also AFB occurred in some viscera. Preliminary studies indicate that the AFB multiplying in tupaias are M. leprae.

[Multiplication of mycobacterium leprae in nude mice].

This article reported the multiplication of mycobacterium leprae in immune-deficient animals-nude mice (NIH/nu/nu). Mycobacterium leprae taken from multibacillary leprosy patient were inoculated with 1.0 x 10(4)/foot pad into right hind foot pad(FP) of nude mice and CFW mice. The results of bacterial enumeration showed that acid-fast bacillus (AFB) in the nude mice continuously multiplied. At 21 months after inoculation, the numbers of AFB increased by over 108,000-folds and reached greater than 1.08 x 10(9)/FP (greater than 5.4 x 10(10)/g tissues), while the CFW mice developed only 3.68 x 10(6)/FP and exhibited their characteristic limited growth pattern. It is indicated that the phase of logarithmic growth was prolonged in nude mice. The AFB multiplied were identified as mycobacterium leprae by following identification test: (1) failure to growth on artificial media; (2) loss of acid-fastness by pyridine extraction; (3) typical growth curve of mycobacterium leprae in foot-pad and sensitivity to antileprosy drugs. The histopathological examination of nude mice foot-pad inoculated were found lepromatous lesions, in which enormous numbers of AFB were seen on 476 days postinoculation, AFB disseminated into the fear, nose, sciatic nerve, testicle, lymphnodes, liver, spleen, lung, and noninoculated foot-pad. As mentioned above, it is considered that experimental leprosy was established in nude mice.