Serial measurement of the circulating levels of tumour necrosis factor and its soluble receptors 1 and 2 for monitoring leprosy patients during multidrug treatment

Leprosy is an infectious and contagious spectral disease accompanied by a series of immunological events triggered by the host response to the aetiologic agent, Mycobacterium leprae . The induction and maintenance of the immune/inflammatory response in leprosy are linked to multiple cell interactions and soluble factors, primarily through the action of cytokines. The purpose of the present study was to evaluate the serum levels of tumour necrosis factor (TNF)-α and its soluble receptors (sTNF-R1 and sTNF-R2) in leprosy patients at different stages of multidrug treatment (MDT) in comparison with non-infected individuals and to determine their role as putative biomarkers of the severity of leprosy or the treatment response. ELISA was used to measure the levels of these molecules in 30 healthy controls and 37 leprosy patients at the time of diagnosis and during and after MDT. Our results showed increases in the serum levels of TNF-α and sTNF-R2 in infected individuals in comparison with controls. The levels of TNF-α, but not sTNF-R2, decreased with treatment. The current results corroborate previous reports of elevated serum levels of TNF-α in leprosy and suggest a role for sTNF-R2 in the control of this cytokine during MDT.     

Identification of clinical,epidemiological and laboratory risk factors for leprosy reactions during and after multidrug therapy

This cross-sectional retrospective study evaluated 440 leprosy patients; 57% (251/440) had leprosy reactions during and/or after multidrug therapy, 80.5% (202/251) of whom presented with multibacillary leprosy. At diagnosis, positive bacterial index (BI) [odds ratio (OR) = 6.39; 95% confidence interval (CI): 4.1-10.1)] or polymerase chain reaction (PCR) (OR = 9.15; 95% CI: 5.4-15.5) in skin smears, anti-phenolic glycolipid-1 (anti-PGL-1) ELISA (OR = 4.77; 95% CI: 2.9-7.9), leucocytosis (OR = 9.97; 95% CI: 3.9-25.7), thrombocytopenia (OR = 5.72; 95% CI: 2.3-14.0) and elevated lactate dehydrogenase (OR = 2.38; 95% CI: 1.4-4.0) were potential markers for the development of reactions during treatment. After treatment, positive BI (OR = 8.47; 95% CI: 4.7-15.3) and PCR (OR = 6.46; 95% CI: 3.4-12.3) in skin smears, anti-PGL-1 ELISA (OR = 2.25; 95% CI: 1.3-3.9), anaemia (OR = 2.36; 95% CI: 1.2-4.5), leucocytosis (OR = 4.14; 95% CI: 1.5-11.6) and thrombocytopenia (OR = 3.70; 95% CI: 1.3-2.2) were risk factors for the occurrence of reactions during the study period. The identification of groups with an increased risk for developing reactions will allow for the timely development of a treatment plan to prevent nerve damage and, therefore, the appearance of the disabling sequelae associated with the stigma of leprosy.     

DNA fingerprinting of Mycobacterium leprae strains using variable number tandem repeat (VNTR) - fragment length analysis (FLA)

The study of the transmission of leprosy is particularly difficult since the causative agent, Mycobacterium leprae, cannot be cultured in the laboratory. The only sources of the bacteria are leprosy patients, and experimentally infected armadillos and nude mice. Thus, many of the methods used in modern epidemiology are not available for the study of leprosy. Despite an extensive global drug treatment program for leprosy implemented by the WHO, leprosy remains endemic in many countries with approximately 250,000 new cases each year. The entire M. leprae genome has been mapped and many loci have been identified that have repeated segments of 2 or more base pairs (called micro- and minisatellites). Clinical strains of M. leprae may vary in the number of tandem repeated segments (short tandem repeats, STR) at many of these loci. Variable number tandem repeat (VNTR) analysis has been used to distinguish different strains of the leprosy bacilli. Some of the loci appear to be more stable than others, showing less variation in repeat numbers, while others seem to change more rapidly, sometimes in the same patient. While the variability of certain VNTRs has brought up questions regarding their suitability for strain typing, the emerging data suggest that analyzing multiple loci, which are diverse in their stability, can be used as a valuable epidemiological tool. Multiple locus VNTR analysis (MLVA) has been used to study leprosy evolution and transmission in several countries including China, Malawi, the Philippines, and Brazil. MLVA involves multiple steps. First, bacterial DNA is extracted along with host tissue DNA from clinical biopsies or slit skin smears (SSS). The desired loci are then amplified from the extracted DNA via polymerase chain reaction (PCR). Fluorescently-labeled primers for 4-5 different loci are used per reaction, with 18 loci being amplified in a total of four reactions. The PCR products may be subjected to agarose gel electrophoresis to verify the presence of the desired DNA segments, and then submitted for fluorescent fragment length analysis (FLA) using capillary electrophoresis. DNA from armadillo passaged bacteria with a known number of repeat copies for each locus is used as a positive control. The FLA chromatograms are then examined using Peak Scanner software and fragment length is converted to number of VNTR copies (allele). Finally, the VNTR haplotypes are analyzed for patterns, and when combined with patient clinical data can be used to track distribution of strain types.     

Self-mortification and the stigma of leprosy in northern India

This article examines the biocultural dynamics of social discrimination and physical disfigurement among people with leprosy, or Hansen's disease (HD), in Banaras, northern India. Based on the narratives and observations ofpeople living in colony and street settings, I trace three destructive processes by which the social stigmata of leprosy become physically expressed. First, strategies of concealment further the progression and spread of HD through late detection and undertreatment. Second, the internalization of stigma can lead to bodily dissociation and injury through self-neglect. Finally, some people intentionally seek injuries under conditions of desperate poverty. As a result of such mortification processes, these people came to embody, quite literally, the prejudices that exacerbated their condition in the first place.     

Immunostimulatory activity of major membrane protein-II from Mycobacterium leprae

We examined the antigenicity of an immunomodulatory protein, major membrane protein (MMP)-II, from Mycobacterium leprae, since host defense against M. leprae largely depends on adaptive immunity. Both unprimed and memory T cells from healthy individuals were stimulated by autologous MMP-II-pulsed monocyte-derived dendritic cells (DCs) to produce IFN-gamma. The DC-mediated IFN-gamma production was dependent on the expression of MHC, CD86, and MMP-II antigens. Memory T cells from paucibacillary (PB) leprosy more extensively responded to MMP-II-pulsed DCs than T cells from healthy individuals, while comparable IFN-gamma was produced by unprimed T cells. Memory T cells from multibacillary leprosy, which are normally believed to be anergic, were activated similarly to those from healthy individuals by MMP-II-pulsed DCs. These results suggest that memory T cells from PB leprosy are primed with MMP-II prior to the manifestation of the disease, and MMP-II is highly antigenic in terms of activation of adaptive immunity.     

Testing conditional independence in diagnostic palaeoepidemiology

Leprosy was a well-known and dreaded disease in the Middle Ages. A substantial fraction of the adult population carried leprosy-related lesions. Previous research analyzed the occurrence and implications of seven such lesions in samples of medieval skeletons. These analyses were carried out under the assumption of conditional independence among lesion scores. The present paper examines this assumption by developing a test based on the odds ratios and applying the test to three rural medieval samples from Europe: Tirup from the 12th-14th century AD in Jutland, Denmark; Refshale from the 12th-14th century AD on the island of Lolland, Denmark; and Lauchheim from AD 460-680 in southern Germany. Signs of nonzero prevalence of leprosy at death were found in all three samples: Tirup, 25.5% (95% CI, 17.2-34.6%); Refshale, 39.1% (95% CI, 25.5-54.7%); and Lauchheim, 16.2% (95% CI, 10.0-22.9%). It is shown that when leprosy is the prime factor creating variation in the lesion scores in and between samples, the assumption of conditional independence cannot be rejected.     

Comparative cytomorphology of skin, lymph node, liver and bone marrow in patients with lepromatous leprosy1

OBJECTIVE: The aim of this study was to compare the cytological changes in skin, lymph nodes, liver and bone marrow in patients with lepromatous leprosy. METHODS: Skin lesion, lymph node, liver and bone marrow aspirates were analysed. May-Grunwald-Giemsa (MGG) and Ziehl-Neelsen (Z-N) stains were employed. Comparative cytomorphology was studied. RESULTS: Twenty patients with lepromatous leprosy were studied. Lepra cells (LC) predominated in the skin aspirates of 12 patients with lepromatous leprosy (LL), lymphocytes accompanied LC in eight patients with borderline-lepromatous (BL) leprosy. Three patients of LL leprosy and two of BL leprosy in type 2 reaction additionally had numerous neutrophils. Two patterns of lymph node aspirates were seen: partial replacement with few LC in a reactive lymphoid background (10), complete replacement with either only LC or LC in a background of degenerating neutrophils (10), the latter a feature of type 2 reaction. Liver aspiration was performed in seven patients and of bone marrow in eight patients. Occasional LC were present in five liver-aspirated patients, steatosis and Kupffer cell hyperplasia in four patients, and myelopoiesis in two patients. Bone marrow smears invariably had occasional LC and a relative increase in mature plasma cells; sea-blue histiocytes were seen in six patients. CONCLUSION: Lepra cells predominated in skin and lymph node aspirates with complete replacement. In comparison, liver, bone marrow and lymph node aspirates with partial replacement were dominated by a preponderance of cells native to these organs with only few or occasional LC.     

The measurement of adenylate energy charge (AEC) in mycobacteria, including Mycobacterium leprae

Abstract The adenylate energy charge (AEC) of Mycobacterium leprae, Mycobacterium lepraemurium and the cultivable Mycobacterium smegmatis were determined following incubation in a variety of culture conditions. The AEC values for M. smegmatis were similar to those reported for other cultivable bacteria. The AEC values for M. leprae and M. lepraemurium purified from host tissue were lower than those of in vitro-grown organisms. The possible use of the AEC in in vitro studies with M. leprae is discussed.     

Genotypic analysis of Mycobacterium leprae isolates from Japan and other Asian countries reveals a global transmission pattern of leprosy

The genotype of single-nucleotide polymorphism type 3, CTC, at positions 14676, 164275, and 2935685, along with four copies of 6 bp repeats in the rpoT gene, was predominant for isolates originating in the Japanese mainland. Type 1, CGA, type 2, CTA, and type 3 were detected from Korea, Indonesia, and Myanmar. No isolates with four copies of 6 bp were detected from Myanmar, Okinawa, and Japanese Brazilian patients. Type 4, TTC, with three copies of 6 bp, was detected only from Japanese Brazilians. The results indicate that infection occurred in Brazil and the disease developed later in Japan.