Evaluation of the diagnostic value of immunocytochemistry and in situ hybridization in the pediatric leprosy.

Leprosy is characterized by a long and variable incubation period and a chronic clinical course. Diagnosis of leprosy is essentially based on clinical features. Although the majority of cases can be diagnosed clinically yet alternative methods for diagnosis are required especially for early cases. Immunocytochemistry and in situ hybridization can be a valuable tool for diagnosis for early cases. The present study is aimed to assess the diagnostic value of immunocytochemistry and in situ hybridization in cytological specimens and to compare these techniques with Z.N. staining. This prospective study was carried out in 26 patients below 18 years of age of leprosy. Clinical examination of each patient was done and categorized according to IAL. After taking consent, three skin smears was taken, one for Z.N. staining and remaining two for immunocytochemistry and in situ hybridization respectively. Routine skin smear examination by Z.N. staining method confirmed the diagnosis in 4/26 (15.83%) and these belonged to BB, BL category. Immunocytochemistry showed positivity in 10/15 (66.6%) in BT and 72.7% in BB/BL leprosy. Immunocytochemistry improved the diagnosis by 53.85%, and the results were statistically significant (p < 0.01). In situ hybridization showed the positive results in 80% cases of BT leprosy and 90.9% cases of BB/BL leprosy. In situ hybridization improved the diagnosis by 70% in comparison to ZN staining and the results were statistically significant (p < 0.01). This study supports that immunocytochemistry and in situ hybridization enhance the diagnosis of leprosy when compared to routine skin smears stained by Z.N staining. They are important diagnostictoolsfor definitive diagnosis in early as well as established cases of leprosy.

PCR and in-situ hybridization for diagnosis of leprosy.

OBJECTIVE: To assess the diagnostic value of Polymerase Chain Reaction (PCR) and in situ hybridization. METHODS: This prospective study was carried out in 22 patients RESULTS: The histopathological examination confirmed the clinical diagnosis in 27.2% cases only. In situ hybridization showed a positivity of 42.8% in early (I/BT) and 46.7% in BB/BL group. In situ hybridization thus enhanced the diagnosis by 18.1%. PCR targeting 36 kDa gene of M. leprae was performed on 15 cases. In these 15 cases, histopathology confirmed the diagnosis in 4 cases (26.6%) and PCR confirmed the diagnosis in 10 cases (66.6%), thus enhancing the diagnosis by 40%. CONCLUSION: 36 kDa PCR and in situ hybridization enhance the diagnosis of leprosy when compared to routine histopathology. They are important diagnostic tools for definitive diagnosis in early and doubtful cases of leprosy.

Evaluation of surgical tissue from patients with Crohn's disease for the presence of Mycobacterium avium subspecies paratuberculosis DNA by in situ hybridization and nested polymerase chain reaction.

Crohn's disease (CD) is a chronic inflammatory bowel disease (IBD) with tissue granuloma and histopathological alteration that resembles aspects in tuberculosis, leprosy, and paratuberculosis. Mycobacterium avium subsp paratuberculosis (MAP) is the causative agent of paratuberculosis, with a suspected role in the etiology of CD. We investigated the presence of MAP DNA in 31 surgical tissue samples from 20 subjects using fluorescence in situ hybridization (FISH) with the aid of confocal scanning laser microscopy and nested polymerase chain reaction (PCR) using the IS900 sequence unique to MAP. MAP DNA was detected by PCR in tissue from 10 of 12 (83%) patients with CD: 7/12 (58%) in inflamed, 6/11 (55%) in noninflamed and in 10 (83%) of either tissue and by FISH in 8 of 12 (67%) patients with CD: 7 of 12 (58%) in inflamed, 4 of 11 (36%) in noninflamed, and in 8(67%) of either tissue. In non-IBD subjects, MAP DNA was detected in the tissue of only 1 of 6 patients (17%) by PCR and 0 of 6 patients (0%) by FISH. MAP DNA was identified by PCR in inflamed tissue from 2 of 2 patients with ulcerative colitis. The detection of MAP DNA by either technique in tissue from subjects with CD is significant compared with non-IBD subjects (P < 0.005). Identification of MAP DNA in both inflamed and noninflamed tissue by both techniques suggests that MAP infection in patients with CD may be systemic. The data add more evidence toward a possible association of MAP in the pathogenesis of CD.

In situ hybridization in the histological diagnosis of early and clinically suspect leprosy.

The present study tests the utility of the in situ hybridization procedure for M. leprae rRNA in the histological diagnosis of early leprosy and clinically suspect leprosy, both diagnostically demanding situations. The histological confirmation obtained with routine histopathology (Haematoxylin-Eosin staining for studying morphologic alterations and Fite-Faraco staining for demonstration of acid-fast bacilli) were 32% for early leprosy and 25% for clinically suspect leprosy. With performance of the in situ hybridization on the histologically unconfirmed cases, the positivity rates obtained were 58.8% and 55%, respectively. The results of the study confirm the utility of the procedure in the diagnostically difficult situations of early and suspect leprosy, and it is proposed that the procedure be employed in situations of clinical doubt.

Expression of CC and CXC chemokines and chemokine receptors in human leprosy skin lesions.

We have investigated the expression of chemokines and their receptors in leprosy skin lesions using immunohistochemistry. Skin biopsies from 25 leprosy patients across the leprosy spectrum, 11 patients undergoing type I reversal reactions and four normal donors were immunostained by ABC peroxidase method using antibodies against CC and CXC chemokines and their receptors. Using an in situ hybridization technique we have also studied the expression of monocyte chemoattractant protein 1 (MCP-1), RANTES and interleukin (IL)-8 chemokines mRNA in leprosy skin lesions. Chemokines and receptor expression was detected in all leprosy skin biopsies. Expression of CC chemokines MCP-1 (P < 0.01) and RANTES (P < 0.01) were elevated significantly in borderline tuberculoid leprosy in reversal reaction compared to non-reactional borderline tuberculoid leprosy, but there was no difference in the expression of IL-8 chemokine. Surprisingly, there was no significant difference in the expression of CC (CCR2 and CCR5) and CXC (CXCR2) chemokine receptors across the leprosy spectrum. Similarly, there was no significant difference in the expression of mRNA for MCP-1, regulated upon activation normal T cell expressed and secreted (RANTES) and IL-8 chemokines. Here, the presence of a neutrophil chemoattractant IL-8 in leprosy lesions, which do not contain neutrophils, suggests strongly a role of IL-8 as a monocyte and lymphocyte recruiter in leprosy lesions. These results suggest that the chemokines and their receptors, which are known to chemoattract T lymphocytes and macrophages, are involved in assembling the cellular infiltrate found in lesions across the leprosy spectrum.

Expression of transforming growth factor-beta isoforms and their receptors in lepromatous and tuberculoid leprosy.

Leprosy is an infectious disease with two polar forms, tuberculoid leprosy (TT) and lepromatous leprosy (LL), that are characterized by strong cell-mediated immunity (CMI) and CMI anergy, respectively. Transforming growth factor-beta (TGF-beta) belongs to a family of pleiotropic cytokines (TGF-beta1, TGF-beta2 and TGF-beta3) that participate in the control of cell differentiation and proliferation, as well as tissue repair. This cytokine family is unique because it suppresses CMI. In this study, we compared the expression of the three TGF-beta isoforms and their receptors in skin biopsies from LL and TT patients (LL = 20; TT = 20) using immunohistochemistry and automated morphometry. The percentage of cells immunostained for the three TGF-beta isoforms and cells positive for the three TGF-beta receptors in the inflammatory infiltrate located in the papillary dermis, reticular dermis and periadnexal tissue were significantly higher in LL than that in TT, with macrophages being the most common and strongest immunoreactive cells. Some lymphocytes, fibroblasts, keratinocytes and epithelial cells from sweat glands and hair roots were also positive. In situ reverse-transcription polymerase chain reaction corroborated the capacity of these cells to synthesize TGF-beta1 and TGF-beta receptor 2. This high expression of TGF-beta isoforms and their receptors could contribute to CMI anergy and other clinical characteristic features of leprosy, like skin atrophy.

Expression of transforming growth factor-beta isoforms and their receptors in lepromatous and tuberculoid leprosy

Leprosy is an infectious disease with two polar forms, tuberculoid leprosy (TT) and lepromatous leprosy (LL), that are characterized by strong cell-mediated immunity (CMI) and CMI anergy, respectively. Transforming growth factor-beta (TGF-beta) belongs to a family of pleiotropic cytokines (TGF-beta1, TGF-beta2 and TGF-beta3) that participate in the control of cell differentiation and proliferation, as well as tissue repair. This cytokine family is unique because it suppresses CMI. In this study, we compared the expression of the three TGF-beta isoforms and their receptors in skin biopsies from LL and TT patients (LL = 20; TT = 20) using immunohistochemistry and automated morphometry. The percentage of cells immunostained for the three TGF-beta isoforms and cells positive for the three TGF-beta receptors in the inflammatory infiltrate located in the papillary dermis, reticular dermis and periadnexal tissue were significantly higher in LL than that in TT, with macrophages being the most common and strongest immunoreactive cells. Some lymphocytes, fibroblasts, keratinocytes and epithelial cells from sweat glands and hair roots were also positive. In situ reverse-transcription polymerase chain reaction corroborated the capacity of these cells to synthesize TGF-beta1 and TGF-beta receptor 2. This high expression of TGF-beta isoforms and their receptors could contribute to CMI anergy and other clinical characteristic features of leprosy, like skin atrophy.

Expression of CC and CXC chemokines and chemokine receptors in human in human leprosy skin lesions

We have investigated the expression of chemokines and their receptors in leprosy skin lesions using immunohistochemistry. Skin biopsies from 25 leprosy patients across the leprosy spectrum, 11 patients undergoing type I reversal reactions and four normal donors were immunostained by ABC peroxidase method using antibodies against CC and CXC chemokines and their receptors. Using an in situ hybridization technique we have also studied the expression of monocyte chemoattractant protein 1 (MCP-1), RANTES and interleukin (IL)-8 chemokines mRNA in leprosy skin lesions. Chemokines and receptor expression was detected in all leprosy skin biopsies. Expression of CC chemokines MCP-1 (P < 0.01) and RANTES (P < 0.01) were elevated significantly in borderline tuberculoid leprosy in reversal reaction compared to non-reactional borderline tuberculoid leprosy, but there was no difference in the expression of IL-8 chemokine. Surprisingly, there was no significant difference in the expression of CC (CCR2 and CCR5) and CXC (CXCR2) chemokine receptors across the leprosy spectrum. Similarly, there was no significant difference in the expression of mRNA for MCP-1, regulated upon activation normal T cell expressed and secreted (RANTES) and IL-8 chemokines. Here, the presence of a neutrophil chemoattractant IL-8 in leprosy lesions, which do not contain neutrophils, suggests strongly a role of IL-8 as a monocyte and lymphocyte recruiter in leprosy lesions. These results suggest that the chemokines and their receptors, which are known to chemoattract T lymphocytes and macrophages, are involved in assembling the cellular infiltrate found in lesions across the leprosy spectrum.

Overexpression of vascular endothelial growth factor and its endothelial cell receptor KDR in type 1 leprosy reaction.

The sites of expression of vascular endothelial growth factor (VEGF) and of KDR, its endothelial cell receptor, were investigated in leprosy reaction Type 1, or reversal reaction (RR), by immunohistochemistry and in situ hybridization. In comparison with nonreactional leprosy, overexpression of both VEGF and KDR was seen in granuloma cells, especially epithelioid and foreign body-type giant cells, the epithelium and the vascular endothelium of RR specimens. In granuloma cells, hybridization for VEGF was stronger than immunostaining, a finding that may reflect the rapid turnover of VEGF in an immunologically dynamic situation such as RR. In the epidermis, double immunohistochemistry revealed VEGF overexpression in CDla-positive dendritic cells. The VEGF may not only be relevant for hyperpermeability and mononuclear cell differentiation (the key morphologic features in the acute, clinically evident phase of RR), but it could also be implicated in RR onset, when dendritic cells are activated in response to antigen stimulation.

Do nerve growth factor-related mechanisms contribute to loss of cutaneous nociception in leprosy?

While sensory loss in leprosy skin is the consequence of invasion by M. leprae of Schwann cells related to unmyelinated fibres, early loss of cutaneous pain sensation, even in the presence of nerve fibres and inflammation, is a hallmark of leprosy, and requires explanation. In normal skin, nerve growth factor (NGF) is produced by basal keratinocytes, and acts via its high affinity receptor (trk A) on nociceptor nerve fibres to increase their sensitivity, particularly in inflammation. We have therefore studied NGF- and trk A-like immunoreactivity in affected skin and mirror-site clinically-unaffected skin from patients with leprosy, and compared these with non-leprosy, control skin, following quantitative sensory testing at each site. Sensory tests were within normal limits in clinically-unaffected leprosy skin, but markedly abnormal in affected skin. Sub-epidermal PGP 9.5- and trk A- positive nerve fibres were reduced only in affected leprosy skin, with fewer fibres contacting keratinocytes. However, NGF-immunoreactivity in basal keratinocytes, and intra-epidermal PGP 9.5-positive nerve fibres, were reduced in both sites compared to non-leprosy controls, as were nerve fibres positive for the sensory neurone specific sodium channel SNS/PN3, which is regulated by NGF, and may mediate inflammation-induced hypersensitivity. Keratinocyte trk A expression (which mediates an autocrine role for NGF) was increased in clinically affected and unaffected skin, suggesting a compensatory mechanism secondary to reduced NGF secretion at both sites. We conclude that decreased NGF- and SNS/PN3-immunoreactivity, and loss of intra-epidermal innervation, may be found without sensory loss on quantitative testing in clinically-unaffected skin in leprosy; this appears to be a sub-clinical change, and may explain the lack of cutaneous pain with inflammation. Sensory loss occurred with reduced sub-epidermal nerve fibres in affected skin, but these still showed trk A-staining, suggesting NGF treatment may restore pain sensation.

Tumour necrosis factor-alpha (TNF-alpha) synthesis is associated with the skin and peripheral nerve pathology of leprosy reversal reactions.

Leprosy may be complicated by episodes of increased cell-mediated immunity towards Mycobacterium leprae (reversal reactions) which result in severe local immunopathology in skin lesions and peripheral nerves. Using in situ hybridization and MoAb techniques we have demonstrated TNF-alpha mRNA and TNF-alpha protein in macrophages infiltrating leprosy skin and peripheral nerve. Levels of TNF-alpha mRNA are significantly increased in reactional skin and nerve, particularly in borderline tuberculoid patients. TNF-alpha mRNA and TNF-alpha protein levels are higher in reactional nerves then reactional skin. In both reactional skin and nerve TNF-alpha mRNA is more abundant than TNF-alpha protein; this may reflect the rapid turnover of TNF-alpha protein in an immunologically dynamic situation, such as is seen in reversal reaction. Our findings emphasize the importance of documenting both mRNA and protein production when assessing the role of cytokines in pathology. The leprosy reversal reaction may be regarded as a useful model of tissue immunopathology in which TNF-alpha is generated as part of the host response to infection, but also produces local tissue damage.

The molecular diagnosis of cutaneous infection.

Molecular biologic techniques are having an impact on many different aspects of medicine. In the realm of infectious agents, they are enhancing our diagnostic capabilities, allowing earlier detection of infection, avoiding the need of culturing infectious agents for the purpose of diagnosis, and broadening our concepts to include the presence of infection in the absence of a culturable agent or serologic evidence of infection. As one might expect, the applicability of these techniques varies with the type of infectious agent being considered. For example, in most bacterial infections the infectious agent can be cultured and accurately identified within 48 hours. It is therefore unlikely that molecular diagnostic techniques will replace the "gold standard" of culture in instances of bacterial infection. Mycobacterial and spirochetal agents, by contrast, do not fall into this category. Although the rapid growers (Mycobacterium fortuitum and Mycobacterium chelonae) pose little problem because they can be cultured within 1 week, M. tuberculosis, M. leprae and the slow growing mycobacteriae require long intervals to culture and/or identify. These organisms may in the future be identified with molecular diagnostic techniques. In the group of spirochetal pathogens, Treponema pallidum infection is, for the most part, easily diagnosed with serologic studies. In early seronegative cases, spirochetes can be detected in primary inoculation lesions by darkfield microscopy or in tissue sections with appropriate stains. Conversely, detection of Borrelia burgdorferi is currently fraught with difficulties. Tissue sections stained for these spirochetes are difficult to interpret, and serologic studies have shown widely variable results. The polymerase chain reaction has already been applied to the study of Borrelia infection with encouraging early results.(ABSTRACT TRUNCATED AT 250 WORDS)

Use of an ordered cosmid library to deduce the genomic organization of Mycobacterium leprae.

In an attempt to unify the genetic and biological research on Mycobacterium leprae, the aetiological agent of leprosy, a cosmid library was constructed and then ordered by a combination of fingerprinting and hybridization techniques. The genome of M. leprae is represented by four contigs of overlapping clones which, together, account for nearly 2.8Mb of DNA. Several arguments suggest that the gaps between the contigs are small in size and that virtually complete coverage of the chromosome has been obtained. All of the cloned M. leprae genes have been positioned on the contig maps together with the 29 copies of the dispersed repetitive element, RLEP. These have been classified into four groups on the basis of differences in their organization. Several key housekeeping genes were identified and mapped by hybridization with heterologous probes, and the current genome map of this uncultivable pathogen comprises 72 loci.