Fite-Faraco staining in combination with multiplex polymerase chain reaction: a new approach to leprosy diagnosis.

BACKGROUND: Leprosy is not always an easy disease to diagnose, and patients can remain undiagnosed for longtime, not only at the peripheral clinics but also even at places with higher medical facilities, so, there is an urgent need for rapid and definitive modalities for leprosy diagnosis. This prospective study evaluates the ability of Fite-Faraco staining (FF staining) and multiplex polymerase chain reaction (PCR) over hematoxylin and eosin staining (H and E staining) and Ziehl-Neelsen staining (ZN staining). AIMS: The aim of this perspective study is to evaluate the effectiveness of FF staining in combination with multiplex PCR for the early and rapid diagnosis of leprosy than any other coexisting diagnosis tool. METHODS: Patients with new skin patches or nodules with or without evidence of nerve damage were selected for the study. Punch biopsy was collected according to standard procedures. Each biopsy sample was divided into two equal parts, one half was fixed in 4% (v/v) buffered neutral formalin and then accordingly embedded in paraffin. Sections were stained by three different methods: H and E staining for histopathological examination, ZN staining, and FF staining for detection of acid-fast bacilli (AFB). And the other part was subjected for DNA extraction and PCR was carried out by the obtained DNA sample. RESULTS: H and E staining, ZN staining, FF staining, and PCR yield 58.2%, 50.9%, 60%, and 67.7% successful diagnosis of leprosy. The true diagnostic performances for these techniques were as follows: H and E staining - sensitivity 70.6%, positive predictive value (PPV) 81.9%, negative predictive value (NPV) 53.6%. For ZN staining - sensitivity 59.9%, PPV 69%, NPV 45.7%. For FF staining - sensitivity 74.6%, PPV 85.9%, NPV 56.7%, and for PCR - sensitivity 87.8%, PPV 95.6%, NPV 71.2%. CONCLUSION: The combination of FF staining and PCR was shown to provide a rapid and definitive diagnosis in the majority of leprosy suspected cases with a higher positive likelihood ratio (+LR) of 7.76 and 2.716, respectively, than H and E staining of 2.244 and ZN staining of 1.378.

Cyclooxygenase 2 expression in vessels and nerves in reversal reaction leprosy.

Tissue expression of cyclooxygenase (COX)2, an inducible enzyme synthesizing eicosanoids in inflammation, was studied in reversal reaction (RR) leprosy in comparison with nonreactionary leprosy. COX2 was consistently expressed in cells of the mononuclear-macrophage lineage across the leprosy spectrum. Only in RR, the following two additional sites showed COX2 expression in the dermis and subcutis: 1) microvessels and 2) nerve bundles and isolated nerve fibers. The same sites also express vascular endothelial growth factor (VEGF). This is in keeping with experimental models relating VEGF to COX2 expression, with VEGF enhancing prostaglandin production through COX2 stimulation and prostaglandin synthase expression. We postulate that selective COX2 inhibitors, which are currently used in several inflammatory conditions, could be considered for RR treatment to reduce acute symptoms caused by tissue edema and possibly prevent long-term nerve damage, the main complication of RR.

Short report: Cyclooxygenase 2 expression in vessels and nerves in reversal reaction leprosy

Tissue expression of cyclooxygenase (COX)2, an inducible enzyme synthesizing eicosanoids in inflammation, was studied in reversal reaction (RR) leprosy in comparison with nonreactionary leprosy. COX2 was consistently expressed in cells of the mononuclear-macrophage lineage across the leprosy spectrum. Only in RR, the following two additional sites showed COX2 expression in the dermis and subcutis: 1) microvessels and 2) nerve bundles and isolated nerve fibers. The same sites also express vascular endothelial growth factor (VEGF). This is in keeping with experimental models relating VEGF to COX2 expression, with VEGF enhancing prostaglandin production through COX2 stimulation and prostaglandin synthase expression. We postulate that selective COX2 inhibitors, which are currently used in several inflammatory conditions, could be considered for RR treatment to reduce acute symptoms caused by tissue edema and possibly prevent long-term nerve damage, the main complication of RR.

Enhancement in the histological diagnosis of leprosy in patients with only sensory loss by demonstration of mycobacterial antigens using anti-BCG polyclonal antibodies.

This study was undertaken to assess whether the immunoperoxidase technique using anti-BCG serum is able to confirm the diagnosis of early leprosy among patients whose unique clinical manifestation is a localized area of sensory loss, in a higher proportion than the routine mycobacterial staining methods, namely hematoxylin-eosin and Wade. The study was held in the north of a hyper-endemic area of leprosy, Manaus, Amazonas (Brazil). Fifty-one paraffin-embedded skin biopsy blocks were retrieved and processed for the immunohistochemical study, by means of anti-BCG polyclonal antibodies for the detection of mycobacterial antigens. The routine stains confirmed the leprosy diagnosis in 17% of the cases, while the immunostaining method confirmed it in 47%. The McNemar test showed that the observed difference between these two techniques was statistically significant (p = < 0.05). In the same way, 50 blocks of skin conditions considered in the differential histopathological diagnosis of early leprosy were processed for the immunohistochemical test to analyze the possibility of false-positive results which occurred in 8 (16%) patients. The study suggests that immunostaining may increase the proportion of the routine histological diagnosis of leprosy in patients who have sensory loss only, even while using biopsies obtained in fieldwork conditions. This is very advantageous in hyper-endemic areas and in areas that are in the post-elimination period of leprosy control where sensory loss may be a sentinel sign of the disease.

[Histopathologic findings for the research of acid-fast bacilli in erythema nodosum]. / Características histopatológicas necessárias para a pesquisa de bacilos álcool-ácido resistentes no eritema nodoso.

It was possible to prove, by means of retrospective histopathological studies of 51 glass slides of common erythema nodosum (EN) and 39 glass slides of erythema nodosum leprosum (ENL), that the histopathological features of the two groups differed in several ways. It was found that the most prominent divergent findings between these two diseases was the presence of a lepromatous granulomatous infiltrate in the ENL; the predominant location of the inflammatory infiltrate was derma-hypodermal in ENL and hypodermal in common EN; there was a preferential septa attack in common EN and lobular in ENL; foreign giant cells occurred only in common EN; the lymphocyte cell infiltration in common EN and of neutrophils in ENL and the presence of inflammatory infiltrate around nerves in ENL. Our study has proved that the search for M. leprae does not have to be made in all cases of EN as there are histological differences in the routine histopathological exam between common EN and ENL that can alert to the real need for this procedure. This search must be performed when during the routine histopathological exam of sections stained by hematoxilin-eosin the ENL histopathological characteristics delineated herein are observed.

Histological methods for assessing myelin sheaths and axons in human nerve trunks.

Although there are many histological techniques for assessing myelin sheaths and axons in paraffin embedded or frozen sections of the peripheral nervous system, modern approaches usually use plastic embedded material. Although plastic embedding is superior for small cutaneous branches, this method has limited value for histological assessment of nerve trunks. We report three methods which together yield a comprehensive approach for thorough and detailed investigation of human nerve trunks. The rapid osmication method permitted assessment of myelinated nerve fibers from frozen sections at operation, thus providing the surgeon with guidance on the extent of nerve resection. The modification presented here resulted in permanent slides, allowing comparison of results with those of the other two procedures. The new osmium-hematoxylin technique could be performed on paraffin embedded nerves. Paraffin, unlike plastic, permitted the study of the whole cross sectional area of the nerve in single sections. Moreover, the sharp image of the myelin permitted computerized morphometry. The significantly modified axonal silver impregnation technique was performed on frozen sections mounted on glass slides, as opposed to the time-consuming impregnation of free-floating sections. The latter technique had a high success rate and permitted semiquantitative assessment of axons in nerve trunks. These methods can be performed in any routine histology laboratory and resulted in greater accuracy compared to conventional methods.