Preliminary analysis of the value of Fite staining in the diagnosis of leprosy / 中华皮肤科杂志

Objective To evaluate the diagnostic value of Fite staining in leprosy histopathology.Methods Between 2013 and 2017,13 patients diagnosed with leprosy or suspected leprosy (high suspicion of leprosy based on clinical manifestations and hematoxylin-eosin staining,but negative acid-fast staining) in our department,were enrolled into this study.The histopathological sections were subjected to Fite staining,and the results were compared with those of acid-fast staining,so as to assess the value of Fite staining in the diagnosis of leprosy.Results Six patients with positive acid-fast staining still showed positive Fite staining.Among 7 patients with suspected leprosy and negative acid-fast staining,6 patients showed positive Fite staining with varying numbers of Mycobacterium leprae,and 1 showed negative Fite staining.Conclusion Fite staining can increase the detection rate of Mycobacterium leprae.

Diseño del kit de tinción Ziehl Neelsen del Instituto nacional de Higiene "Rafael Rangel" / Design Kit Ziehl Neelsen the Instituto Nacional de Higiene "Rafael Rangel"

La tinción de Ziehl Neelsen (ZN), es una técnica de coloración de microorganismos para la identificación de patógenos, como Mycobacterium tuberculosis causante de la tuberculosis, que requiere de tres (03) soluciones: Carbol Fucsina Fenicada (Fucsina Básica), Azul de Metileno al 1% y Solución Decolorante, que se elaboran en la Sección de Reactivos y Colorantes del Instituto Nacional de Higiene "Rafael Rangel" y se emplean en el diagnóstico de tuberculosis. Esta investigación surgió con el propósito de comprobar el tiempo de caducidad y condiciones de almacenamiento de dichos productos y presentarlos en un estuche tipo kit para su distribución, en apoyo a la Red Nacional de Laboratorios de Salud Pública y comercialización con otros entes. Se realizó el ensayo de tres (3) lotes del kit de Ziehl Neelsen; con su respectiva contramuestra que fue evaluada en el análisis final. Se registraron los parámetros físicos de temperatura y humedad relativa bajo condiciones normales de almacenamiento en el laboratorio, con las muestras protegidas de la luz. Se evaluó la funcionalidad por medio de la tinción ZN observada bajo microscopio, de tres (03) muestras con ATCC 700686: M. peregrinum y ATCC 29213: S. aureus por lote; tomando en cuenta el exceso de colorante, y la definición de las coloraciones. Estas evaluaciones se realizaron durante dos (02) años encontrándose como resultado que física y funcionalmente los productos contentivos en el kit se mantenían estables, fijándose un tiempo de caducidad de dos (02) años.

Ziehl Neelsen (ZN) is a staining technique of microorganisms for the identification of pathogens as Mycobacterium tuberculosis, causative of tuberculosis, which requires three (03) solutions: Carbol Fuchsin combined with Phenol (Basic Fuchsin), Methylene Blue 1% and Bleaching solution, which are prepared in Section of Reagents and Coloring of the Instituto Nacional de Higiene "Rafael Rangel" and are used in the diagnosis of tuberculosis. This investigation was made with the purpose of checking the shelf life and storage conditions of these products and present them in a kit type container for distribution in support of the National Network of Public Health Laboratories and marketing with other entities. The analysis was performed in three (3) batches of the Ziehl Neelsen kit; with their respective counter sample that was evaluated in the final analysis. The physical parameters of temperature and relative humidity were recorded in the laboratory under normal storage conditions with samples protected from light. The functionality was evaluated through ZN staining being observed under a microscope three (03) samples with ATCC 700686: M. peregrinum and ATCC 29213: S. aureus by Batch; taking into consideration the excess dye, and the definition of the colors. These evaluations were conducted for two (02) years found as main result that physically and functionally the products in the kit were stable, and can set an expiration time of two years.

Role of fine needle aspiration cytology in diagnosing leprosy: in a tertiary care hospital.

Background: Leprosy, a chronic inflammatory granulomatous disease chiefly involving skin and peripheral nerves and occasionally other organ systems, caused by Mycobacterium leprae. It has tormented the human civilization through time immemorial. Leprosy remains a significant public health problem worldwide, especially in developing countries like India. The diagnosis of leprosy is not always easy because of long incubation period, over dependence of clinical expertise and a lack of rapid and simple diagnostic tool, patients remain undiagnosed for longer time. Fine needle aspiration (FNAC) technique is an inexpensive, rapid and accurate procedure for diagnosis of leprosy. We conducted a prospective study evaluating the ability of fine needle aspiration cytology in diagnosing and classifying leprosy lesions on Ridley-Jopling scale (R-J scale). The aim of this prospective study was to assess the usefulness of fine needle aspiration cytology in early diagnosis of leprosy, to identify specific cytological characteristics of diagnosis and to correlate the cytological smear findings with histopathology and to evaluate merits of relatively non-invasive procedure of FNAC over more invasive procedure - biopsy. Methods: The study is a hospital based prospective study carried out in the Department of Pathology and Department of Skin, Venereal Diseases, Leprosy, N.S.C.B. Medical College & Hospital, Jabalpur (M.P.) September 2010 to September 2013. Patients with new skin lesions were selected for the study. FNAC was performed and aspirates were evaluated for cytology using Hematoxylin and Eosin staining (H&E staining), Ziehl-Neelsen staining (ZN staining) and punch biopsy was collected. Results: Out of 50 cases, clinical and cytological correlation was seen in 88% tuberculoid leprosy, 93.7% of borderline tuberculoid, 33% of borderline lepromatous leprosy and 66% of lepromatous leprosy. While clinical with histopathological correlation revealed 100% specificity in tuberculoid leprosy, 100% in borderline tuberculoid, 66.6% in borderline lepromatous, 83.3% in lepromatous leprosy and 80% in indeterminate leprosy and 100% in histoid leprosy in our study. The overall cytodiagnostic accuracy has been 92% in present study. Conclusion: Our study demonstrates that the combination of FNAC and ZN staining for Acid Fast Bacilli (AFB) can provide a rapid diagnosis in majority of leprosy suspected cases. FNAC is a safe, simple, rapid, less-invasive, OPD procedure for early diagnosis and classification of leprosy cases.

Is universal sample processing methodology better than conventional techniques for detection of tuberculosis.

Background: The identification of infectious cases is a crucial first step for tuberculosis control programmes worldwide. It relies exclusively on the detection of acid‑fast bacilli in sputum by smear microscopy. Therefore, there is an urgent and definite need to improve the sensitivity of smear microscopy. Objective: The USP method was compared with the two most commonly used conventional methods of smear microscopy namely; direct smear microscopy and the microscopy by modified Petroff’s method. Materials and Methods: Two samples from each patient were taken from 197 patients of presumptive tuberculosis. One smear was made for direct Ziehl‑Neelsen staining and two smears were made after processing by two concentration methods i.e., modified Petroff’s and USP solution. LJ media were inoculated for culture after processing by both concentration methods. Results: Among 197 cases 93 were culture positive by either method. Out of 93 culture‑positive sample, 78.5% were direct smear positive, 89% were 4%NaOH smear positive and 96% were USP smear‑positive samples but difference in diagnostic accuracy of USP (96%) and modified Petroff method (93%) is not statistically significant (P > 0.01). Conclusion: The present study evaluated the smear microscopy by USP method with two conventional methods, direct microscopy and microscopy by modified Petroff’s method. The study concludes that although USP method is more sensitive than conventional methods, it is not feasible to include it in diagnosis of early tuberculosis within RNTCP.

Same day sputum smear microscopy approach with modified ZN staining for the diagnosis of pulmonary tuberculosis in a microscopy centre at Rajahmundry.

Background: Sputum smear microscopy is the main‑stay in the diagnosis of pulmonary tuberculosis in many developing countries. To overcome the drop outs, same day diagnosis is ideal. Materials and Methods: In the current study, two spot sputum samples (SS2 approach) are collected within a gap of one hour (same day sputum smear microscopy) in addition to the standard spot morning (SM) approach. The smears were stained with standard Ziehl Neelsen (ZN) and modified ZN staining techniques. Results: Out of 1537 patients, sputum smear positivity (SSP) was 9.43% (146 patients) in SM approach with standard ZN staining. Smear positivity was increased to 9.8% (151 patients) with modified ZN staining. For SS2 approach, SSP was 9.37% (144 patients) and 9.8% (151 patients) with standard and modified ZN staining procedures, respectively. Conclusions: Diagnosis of lung tuberculosis is possible with two spot sputum samples with modified ZN staining.

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 st aining - 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.

Comparative analysis for the detection of tuberculosis by conventional and flurosence microscopy.

Total of 300 TB patients sample were collected from NIDCH (Indoor and Outdoor) for six months period where three different specimens (sputum, pus, BAL) were collected among different age group (male and female). In sputum sample, percentage of identified samples in Auramine-O stain was 41% whereas in Z-N stain 32%; in pus sample, Auramine stain is 22% and Z-N stain 15%; In BAL sample, Auramine stain is 12% and Z-N stain is 10%. Therefore, out of 300 samples, 25% of positive samples were identified by Auramine-O staining whereas 19% of positive samples were identified by Z-N stain. In all the cases Auramine-O is found better for identification of TB than Z-N. In sputum, male positive 10%, female positive 3.66%; in pus sample, male positive 5% and female positive 2.33%; in BAL sample, male positive 3% and female positive 1%. Age group of 26 to 45 years were found high rate of TB in male patients.

FNAC in Tuberculous Lymphadenitis Experience from a teritiary level referral centre.

Background: In developing countries like India, tuberculous lymphadenitis is one of the most common causes of lymphadenopathy. However, anti-tubercular treatment cannot be given only on clinical suspicion. Cytomorphology with acid fast staining proves to be a valuable tool in diagnosing these cases. Aims: To study the utility, limitations of fine needle aspiration cytology and various cytomorphological presentations in reference to Ziehl-Neelsen staining in tuberculous lymphadenitis. Material and Methods: In a study period of July to October 2010, three hundred and eighteen consecutive superficial lymph nodes, clinically suspected to be tuberculous were subjected to cytological evaluation with Hematoxylin & Eosin, Giemsa and Ziehl-Neelsen stained smears. In addition, demographic profile of these patients with clinical presentation was also studied. Results: Incidence of tuberculous lymphadenitis was 55%. Overall AFB positivity was 71.0%. Only Necrosis without epithelioid cell granulomas was the most common cytological picture and that showed highest AFB positivity also. Threefourth of the patients presented in second to fourth decade of life. Cervical region was the most common site of involvement with solitary lymphadenopathy as the most common presentation in contrast to matted lymph nodes as reported by others. Conclusions: Fine needle aspiration cytology is a safe, cheap procedure requiring minimal instrumentation and is highly sensitive to diagnose tuberculous lymphadenitis. The sensitivity can be further increased by complementing cytomorphology with acid fast staining. In acid fast staining negative cases, yield of acid fast bacilli positivity can be increased by doing Ziehl-Neelsen staining on second smear or decolourized smear revealing necrosis or by repeat aspiration. Microbiological assessment should also be done in such cases.

Comparison of Ziehl Neelsen and Auramine O staining methods on direct and concentrated smears in clinical specimens.

Background: In developing countries like ours with a large number of tuberculosis (TB) cases and limited resources, the diagnosis of TB relies primarily on smear microscopy for Acid Fast Bacilli (AFB) but its sensitivity is limited in paucibacillary cases. Aim: To evaluate the increase in efficacy of smear microscopy when smears are prepared from clinical samples after concentration by Petroff’s method and stained by Auramine O (AO) fluorescent dye as against Ziehl Neelsen (ZN) staining of similar taking culture as the gold standard. Methods: Smears were prepared from 393 clinical samples both by direct and after Petroff’s concentration and examined by fluorescent microscopy and Ziehl Neelsen method .The concentrated material was also cultured on Lowenstein Jensen media and the results of the two microscopy methods were compared with the culture results taken as the gold standard. Results: Mycobacterial growth was detected in 137(35.77%) specimens, out of which three were non-tubercular mycobacteria. Using culture as the reference method, the sensitivity of direct staining was 55.55% for ZN and 71.85% for AO. Direct fluorescent microscopy detected 9.29% paucibacillary sputum samples that were missed on ZN staining. On concentration, the sensitivity increased by 6.67% for ZN and 11.11% for AO. The sensitivity of AFB smear microscopy increased by 27.41% and was statistically significant (p=<.001) when both methods were combined. The specificity was 99.19% for both ZN and AO. Conclusion: Fluorescent microscopy has higher sensitivity and comparable specificity which is further enhanced by concentration. Now with the advent of newer inexpensive Light Emitting Diode (LED) based fluorescent microscopes (FM), which are easier to use, fluorescent microscopy can be widely used even in peripheral laboratories where culture facilities are not available.