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.

Diagnosing leprosy: revisiting the role of the slit-skin smear with critical analysis of the applicability of polymerase chain reaction in diagnosis.

BACKGROUND: Diagnosing leprosy is challenging, especially in early-stage cases, and the need for a sensitive diagnostic tool is urgent. Polymerase chain reaction (PCR) holds promise as a simple and sensitive diagnostic tool, but its usefulness in the Indian context requires further evaluation. Slit-skin smear (SSS) remains the conventional method of leprosy detection. Hence, this study was undertaken to evaluate and compare the diagnostic efficacy of PCR versus that of SSS. METHODS: Punch biopsy of skin and SSS were obtained from the active margins of lesions. Cases were clinically grouped according to whether they were multibacillary (MB) or paucibacillary (PB) and classified into tuberculoid (TT), borderline tuberculoid (BT), borderline lepromatous (BL), lepromatous (LL), histoid, and indeterminate groups after clinicopathological correlation. DNA was extracted from biopsy specimens, and multiplex PCR was carried out incorporating primers intended for the amplification of a specific 372-bp fragment of a repetitive sequence of Mycobacterium leprae DNA. RESULTS: Among 164 patients, PCR was positive in 82.3%. The sensitivity of PCR was significantly greater (P < 0.0001) than that of SSS in both the MB (85.9% vs. 59.8%) and PB (75.4% vs. 1.8%) subgroups; the difference in sensitivity in the PB subgroup is remarkable. Positivity by PCR and SSS was found in 100% of LL and histoid leprosy, but PCR had significantly greater (P < 0.0001) positivity in BT leprosy and was of definite increased value in indeterminate and TT leprosy. CONCLUSIONS: Polymerase chain reaction had higher sensitivity compared with SSS, especially in diagnostically challenging and PB cases. Thus, the use of this costly but sensitive tool should be restricted to this subgroup, because SSS is sufficiently sensitive in the diagnosis of LL and histoid leprosy.