LP1 and LP2 primers were utilized for second-round PCR amplification as described above, except that this annealing temperature was lowered to 40C and 1 L first-round PCR was used as DNA template. MB were 97.96% and 89.8%, and these values for PB were 70% and 53.33%. However, the ND-O-BSA enzyme-linked immunosorbent assay (ELISA) experienced lower specificity, with relatively high false-positive results for TB patients (16.67%) and normal healthy controls (10%). Based on these encouraging findings, we propose the use of nested PCR of whole-blood samples along with ELISA test for early detection of leprosy cases. == Introduction == Leprosy is an infectious disease caused byMycobacterium leprae, which was identified by A. Hansen in 1873. It has a long incubation period and a wide spectrum of clinical manifestations, which make it hard to diagnose in the early stages of the disease. The leprosy bacillus has not been successfully culturedin vitroto date. Recently, numerous serological and cell-mediated immune methods have been developed, including a serological enzyme-linked immunosorbent assay (ELISA) test that detects antibodies against an epitope on phenolic glycolipid-1 (PGL-1) antigen, a natural disaccharide with octyl linkage conjugated Mequitazine to bovine serum albumin (ND-O-BSA), and a fusion protein of ML0405-ML2331 (LID-1) ofM. leprae.1These two antigens are specific to this bacillus, and the test can potentially lead to early diagnosis and predict Mequitazine individual outcome. The antibodies are found in the tissues and circulating blood of multibacillary (MB) patients. The test has been used worldwide,1,2and positivity is usually proportional to the bacillary weight, especially for MB patients. However, the potential drawback of the ELISA test is usually its cross-reactivity to other mycobacterial cell wall components and its low sensitivity for detection of paucibacillary disease. DNA amplification using polymerase chain reaction (PCR) seems to be more specific and sensitive for detecting bacilli in biopsies.3Woods and Cole4detected small figures ofM. lepraeby amplifying a specific repetitive sequence, whereas Plikaytis and others5detected as little as 3 fgM. lepraegenomic DNA using nested PCR targeting limited sequences of thegroELgene. Although PCR has already been applied to the detection ofM. lepraefor some years, it has been used mainly on biopsies and slit skin smears (SSSs) from suspected cases. Biopsies and SSSs are invasive procedures for the patient, and common lesions may not always be seen (for example, in indeterminate leprosy, where anesthesia is not obvious and early detection and proper multidrug therapy [MDT] is usually often missed). To improve the sensitivity and specificity of the current detection methods for diagnosing leprosy at an early stage, we applied a nested-PCR approach to Rabbit Polyclonal to GCF whole-blood specimens Mequitazine from different groups to detectM. lepraeby amplifying specific repetitive DNA sequences5,6and compared this method with the ELISA assays based on detection of antibodies to certainM. lepraeantigens for diagnosis of leprosy. == Methods == == Study populations. == This study was conducted at an endemic site in the Honghe Prefecture (detection rate at 5-12 months average, 20072011, of 1 1.08 per 100,000 populations) of Yunnan Province Mequitazine in southwest China. The newly diagnosed patients were assessed by clinical indicators of skin lesions, nerve involvement, and bacteriological (bacterial index by acid-fast staining) and histopathological methods, and they were classified according to the RidleyJopling level7carried out by qualified staff. Specimens included 49 MB (9 lepromatous leprosy [LL], 38 borderline lepromatous [BL], and 2 borderline borderline [BB]) and 30 paucibacillary (PB; 1 indeterminate [I], 3 paucibacillary tuberculoid [TT], and 26 borderline tuberculoid [BT]; 26 BT included 24 acid-fast bacilli [AFB] -positive and 2 AFB-negative). Household contacts (HHCs) were defined as individuals who lived in the same dwelling (i.e., sharing the same kitchen or interpersonal/recreational area). The HHC contacts were living with the leprosy patients during the treatment; 96 HHC were from MB patient families, 10 normal individuals were from the food handlers from Honghe Prefecture, Yunnan Province, and 25 normal individuals were from your medical staff, Beijing Friendship Hospital, during their annual physical checkup. The control including 18 treated tuberculosis patients. Whole-blood and plasma samples were collected after obtaining written informed consent as per the standards of the Ethical Committee of Beijing Companionship Hospital Institutional Committee, which approved this study. == Bacterial strains and antigens. == M. lepraestrain NHDP63,M..