Efficacy of fluorescent microscopy versus modified Fite-Faraco stain in skin biopsy specimens of leprosy cases - a comparative study.

BACKGROUND: Histopathological examination of skin remains the cornerstone in the diagnosis of leprosy. At a few centers, fluorescent microscopy has been found to be useful in detecting more acid-fast bacilli (AFB) compared to modified Fite-Faraco staining but is sparsely documented. Hence, we studied the sensitivity of fluorescent microscopy and modified Fite-Faraco stain in the detection of Mycobacterium leprae in tissue sections. METHODS: Patients attending our outpatient department during January 2019 to June 2020 with the clinical features of leprosy were examined, and the diagnosis was confirmed by histopathology after informed consent. Tissue sections were stained by fluorescent stain and modified Fite-Faraco stain. Bacillary index was calculated for each case. RESULTS: Forty patients were recruited after following the inclusion and exclusion criteria. AFB were demonstrated in 20 patients by modified Fite-Faraco stain and in 27 patients with fluorescent stain. The sensitivity of fluorescent staining method (67.5%) was higher than modified Fite-Faraco stain (50%). Bacillary index was increased in 26 out of 40 cases by the fluorescent staining compared to the modified Fite-Faraco staining. Chi-square value was 69.3 and P value was 0.000, indicating a statistically significant correlation. LIMITATIONS: Fluorescent microscope is expensive, and trained people are needed to identify the bacilli. CONCLUSION: Fluorescent staining is more sensitive than modified Fite-Faraco staining in the detection of AFB in tissue sections. The bacilli detected per field were high with the fluorescent staining compared to the modified Fite-Faraco method.

Laboratory diagnosis of leprosy: Two staining methods from bacilloscopy and rapid ml flow test.

Background: The diagnosis of leprosy is based on the characteristic signs and symptoms of the disease, subsidized by laboratory tests. When positive, the bacilloscopy closes the diagnosis for leprosy. Phenolic glycolipid-I, or PGL-I, is a molecule in the bacillus cell wall that confers a greater immune response. The ML Flow test is an immunochromatographic test for the detection of anti-PGL-I IgM in human blood or serum. Methods: A prospective study with data collection and biological materials in patients with suspected leprosy from August 2020 to May 2021. For microscopy, intradermal smears were stained with Auramine O, and after reading under a fluorescence microscope, reviewed by Ziehl-Neelsen. The ML flow test was performed according to the Bührer-Sékula protocol. To assess the agreement between the methods, the Kappa index was estimated. Results: Of the 94 suspected leprosy patients, 31 (32.9%) were diagnosed with leprosy. There was moderate agreement between the results of the ML Flow and Auramine O tests (Kappa = 0.58) and substantial agreement between the ML Flow and Ziehl-Neelsen microscopy (Kappa = 0.72). In paucibacillary cases, serology was positive in 100% of patients. Conclusions: This study concluded that the Ziehl-Neelsen technique remains the best option for standard leprosy staining, and the ML flow test is more positive among the three techniques evaluated and can be an effective tool in the early diagnosis of leprosy cases.

Diagnostic Utility of Biplex/Multiplex Polymerase Chain Reaction in Infectious Granulomatous Dermatitis in North Indian Population.

BACKGROUND: A definite diagnosis of infectious granulomatous dermatitis (IGD) is difficult for both practicing dermatologists and dermatopathologists due to overlapping clinical and histomorphological features. We aimed to explore the role of multiplex polymerase chain reaction (PCR) for identifying a definite etiological agent for diagnosis and appropriate treatment in IGD in formalin-fixed paraffin-embedded tissue. MATERIALS AND METHODS: Sixty-two cases of IGD were included, excluding leprosy. The histochemical stains including Ziehl-Neelsen, periodic acid-Schiff, and Giemsa were performed in all cases. A multiplex PCR was designed for detection of tuberculosis (TB) (IS6110 and mpt64), fungal infections (ITS1, ITS2; ZM1, and ZM3), and leishmaniasis (kDNA). The results of histomorphology, histochemical stains, and multiplex PCR were compared. RESULTS: Among 62 cases, the sensitivity rate of PCR detection for organisms was 16.7%, 0%, 100%, 72%, 75%, and 66.7% in patients with TB, suggestive of TB, leishmaniasis, fungal infections, and granulomatous dermatitis not otherwise specified and granulomatous dermatitis suggestive of fungus, respectively. The TB PCR using IS6110 primers was negative in all cases; however, PCR using mpt64 primers was positive in 33.33% cases of scrofuloderma. The histochemical stains including Ziehl-Neelsen for acid-fast bacilli, periodic acid-Schiff for fungus, and Giemsa for Leishman-Donovan bodies showed positivity in 11.3%, 43.5%, and 3.2%, respectively. CONCLUSION: A multiplex PCR (Mycobacterium tuberculosis, Leishmania, and panfungal) is highly recommended in all cases of IGD where an etiological agent is difficult to establish by skin biopsy and histochemical stains along with a clinicopathological correlation. This will augment in appropriate treatment and will reduce empirical treatment and morbidity in such patients.

Evaluation of Auramine O staining and conventional PCR for leprosy diagnosis: A comparative cross-sectional study from Ethiopia.

BACKGROUND: Diagnosis of leprosy mainly relies on clinical examination due to the inconsistent sensitivity and poor reproducibility of the current laboratory tests. Utilisation of alternative methods to the standard Ziehl Neelsen (ZN), Fite-Faraco (FF) and Haematoxylin and Eosin (H&E) staining procedures may eventually improve leprosy diagnosis. METHODOLOGY/PRINCIPAL FINDINGS: In this comparative study, the performance of the fluorescent Auramine O (AO) staining and polymerase chain reaction (PCR) was assessed with different skin samples using a combination of ZN, FF and H&E staining as the gold standard. AO, ZN, FF, H&E and PCR tests were performed on slit skin smears (SSS) and/or punch biopsies collected from 141 clinically confirmed leprosy cases and 28 non-leprosy skin samples. DNA was extracted from punch biopsies using two different methods with or without mechanical lysis. Sensitivities were 87.6%, 59.3% and 77% for H&E, ZN and FF, respectively, whereas it reached 65.5% and 77.9% for AO in SSS and tissue sections and 91.1% for PCR in tissue samples. Morover, samples with low bacillary index, sensitivity of AO staining (61.8%) was similar to FF (60%, p>0.05) and lower than PCR (86.6%, p<0.05). Sensitivity of PCR also increased (96.8%, p<0.05) when mechanical lysis was used during DNA extraction compared to enzymatic treatment alone (84.6%). CONCLUSIONS/SIGNIFICANCE: Our results showed that for diagnostic purposes, analysis of skin section is more sensitive than SSS, especially for samples with low bacillary load. AO staining on SSS and tissue sections was not significantly better than other routine diagnostic tests but considerably more user friendly. The sensitivity of PCR was higher than current standard methods and increased when combined with more efficient DNA extraction using mechanical and chemical lysis. Therefore, we recommend AO staining for the diagnosis of leprosy in lower health facilities such as health centres and district hospitals and PCR diagnosis at referral level and research centres.

Role of S-100 Immunostain as An Auxiliary Diagnostic Aid in Leprosy.

INTRODUCTION: Histopathologic diagnosis of leprosy is difficult when Bacillary Index (BI) is zero and neural involvement are not easily identifiable on routine Hematoxylin and Eosin stain. This study was undertaken to study the role of S-100 immunostaining in demonstrating different patterns of nerve involvement in various types of leprosy. METHODS: Thirty one skin biopsies with clinico-histopathologic diagnoses of leprosy over a period of two years were included in the study. Ten cases of non-lepromatous granulomatous dermatoses (including eight cases of lupus vulgaris and two cases of erythema nodosum) were used as controls. Tissue sections from all cases and controls were stained with Hematoxylin and Eosin (H&E) stain, Fite stain and S-100 immunostain. The H&E stained slides were used to study the histopathological features, Fite stained slides for Bacillary Index and S-100 for nerve changes. RESULTS: Neural changes could be demonstrated in the entire spectrum of leprosy using S-100 immunostaining. The most common pattern of nerve destruction in the tuberculoid spectrum was fragmented and infiltrated whereas lepromatous spectrum showed mostly fragmented nerve twigs. Intact nerves were not detected in any of the leprosy cases. CONCLUSIONS: S-100 immunostain is a useful auxiliary aid to the routine H&E stain in the diagnosis of leprosy especially tuberculoid spectrum and intermediate leprosy.

[Importance of quality control of baciloscopy in laboratories that perform diagnosis of tuberculosis]. / Importancia del control de la calidad de la baciloscopia en los laboratorios de diagnóstico de tuberculosis.

BACKGROUND: Baciloscopy is the primary tool for pulmonary tuberculosis diagnosis, being this technique the most used internationally in the search for infectious cases. Quality control is the process of the rechecking smears by a highly qualified observer. AIM: To evaluate and highlight the importance of quality control of smear microscopy in the Provincial Laboratories diagnosticians of Tuberculosis in Cuba. METHODS: This study was conducted at the National Reference Laboratory and Research in Tuberculosis, Leprosy and Mycobacteria in the Institute of Tropical Medicine "Pedro Kouri", Havana, Cuba, Were evaluated 2676 smears received from January 2013 to December 2014, from Provincial Centers of Hygiene, Epidemiology and Microbiology of Cuba, including the special municipality Isla de la Juventud. RESULTS: 2,664 (99.5%) were concordant smears, the correlation obtained for the positive smears were 96.5% and 99.8% for negative. Were identified12 reading errors: 7 (3.5%) false positive and 5 (0.2%) false negatives. Slides were classified with adequate quality of smears in 2039 (76.2%), showed difficulties in realizing the extension in 1464 (54.7%) and staining were adequate in 2343 (87.6%). The kappa index was 0.9674. CONCLUSION: Although there was good agreement between observations it is recommended to improve the quality of extended, maintain staff training program that performs this activity, like regular supervision by specialists, to further improve the quality of diagnosis.

Importancia del control de la calidad de la baciloscopia en los laboratorios de diagnóstico de tuberculosis / Importance of quality control of baciloscopy in laboratories that perform diagnosis of tuberculosis

Introducción: La baciloscopia es la herramienta primaria en el diagnóstico de la tuberculosis (TBC) pulmonar activa, siendo esta la técnica más utilizada internacionalmente en la búsqueda de casos infecciosos. El control de calidad consiste en la relectura de las láminas por un observador altamente calificado. Objetivo: Evaluar y destacar la importancia del control de la calidad de la baciloscopia en los laboratorios provinciales encargados del diagnóstico de TBC en Cuba. Material y Métodos: Este estudio fue realizado en el Laboratorio Nacional de Referencia e Investigaciones de Tuberculosis, Lepra y Micobacterias del Instituto de Medicina Tropical "Pedro Kourí", La Habana, Cuba. Fueron evaluadas 2.676 láminas recibidas en el período de enero de 2013-diciembre de 2014, procedentes de los diferentes Centros Provinciales de Higiene, Epidemiología y Microbiología de Cuba, incluido el Municipio Especial Isla de la Juventud. Resultados: Hubo 2.664 (99,5%) láminas concordantes, la concordancia obtenida para las láminas positivas fue 96,5% y las negativas 99,8%. Se identificaron 12 errores de lectura: 7 (3,5%) falsos positivos, 5 (0,2%) falsos negativos. Se calificaron láminas con calidad de la muestra adecuada en 2.039 (76,2%), presentaron deficiencias en la realización de la extensión 1.464 (54,7%), y la tinción fue adecuada en 2.343 (87,6%). El índice de kappa fue de 0.9674. Conclusión: Aunque hubo una adecuada concordancia entre las observaciones realizadas, se recomienda mejorar la calidad del extendido, mantener programa de entrenamiento al personal que realiza esta actividad, al igual que las supervisiones periódicas por parte de especialistas, para continuar mejorando la calidad del diagnóstico.

Background: Baciloscopy is the primary tool for pulmonary tuberculosis diagnosis, being this technique the most used internationally in the search for infectious cases. Quality control is the process of the rechecking smears by a highly qualified observer. Aim: To evaluate and highlight the importance of quality control of smear microscopy in the Provincial Laboratories diagnosticians of Tuberculosis in Cuba. Methods: This study was conducted at the National Reference Laboratory and Research in Tuberculosis, Leprosy and Mycobacteria in the Institute of Tropical Medicine "Pedro Kouri", Havana, Cuba, Were evaluated 2676 smears received from January 2013 to December 2014, from Provincial Centers of Hygiene, Epidemiology and Microbiology of Cuba, including the special municipality Isla de la Juventud. Results: 2,664 (99.5%) were concordant smears, the correlation obtained for the positive smears were 96.5% and 99.8% for negative. Were identified12 reading errors: 7 (3.5%) false positive and 5 (0.2%) false negatives. Slides were classified with adequate quality of smears in 2039 (76.2%), showed difficulties in realizing the extension in 1464 (54.7%) and staining were adequate in 2343 (87.6%). The kappa index was 0.9674. Conclusion: Although there was good agreement between observations it is recommended to improve the quality of extended, maintain staff training program that performs this activity, like regular supervision by specialists, to further improve the quality of diagnosis.

Jorge Lobo's disease: immunohistochemical characterization of dendritic cells in cutaneous lesions.

BACKGROUND: Jorge Lobo's disease (JLD) is a cutaneous chronic mycosis caused by Lacazia loboi. We studied Factor XIIIa + dermal dendrocytes (FXIIIa + DD), Langerhans cells (LC) through the expression of langerin and the expression of S100 protein. METHODS: A total of 41 biopsies and 10 normal skins (control) were developed with a polymer-based immunohistochemical method. RESULTS: Lesions presented infiltrate comprising macrophages, some asteroid corpuscles, lymphocytes, multinucleated giant cells and a large number of fungi. LCs presented short dendrites and were scarcely distributed. Dermal langerin + cells were detected in nine JLD lesions. FXIIIa + DD were hypertrophic, visualized in the inflammatory infiltrate of JLD lesions. Cells S100+ were present in JLD and control group with a similar number of cells. A total of 14 specimens did not express FXIIIa, and this considerable number probably contributed to the statistical similarity with the control group. CONCLUSIONS: The results indicate that LCs are present in the immune response against Lacazia loboi. Some dermal langerin + cells could be another subset of dendritic cells. Our data indicate changes of LCs in JLD cutaneous lesions and present, for the first time, results that show langerin + cells in the dermis and corroborate previous observations on the participation of FXIIIa + DD in the in situ immune response in JLD.

Comparison of four DNA extraction methods for the detection of Mycobacterium leprae from Ziehl-Neelsen-stained microscopic slides.

OBJECTIVE/BACKGROUND: The diagnosis of leprosy has been a challenge due to the low sensibility of the conventional methods and the impossibility of culturing the causative organism. In this study, four methods for Mycobacterium leprae nucleic-acid extraction from Ziehl-Neelsen-stained slides (ZNS slides) were compared: Phenol/chloroform, Chelex 100 resin, and two commercial kits (Wizard Genomic DNA Purification Kit and QIAamp DNA Mini Kit). METHODS: DNA was extracted from four groups of slides: a high-codification-slide group (bacteriological index [BI]⩾4), a low-codification-slide group (BI=1), a negative-slide group (BI=0), and a negative-control-slide group (BI=0). Quality DNA was evidenced by the amplification of specific repetitive element present in M. leprae genomic DNA (RLEP) using a nested polymerase chain reaction. RESULTS: This is the first report comparing four different extraction methods for obtaining M. leprae DNA from ZNS slides in Cuban patients, and applied in molecular diagnosis. Good-quality DNA and positive amplification were detected in the high-codification-slide group with the four methods, while from the low-codification-slide group only the QIAGEN and phenol-chloroform methods obtained amplification of M. leprae. In the negative-slide group, only the QIAGEN method was able to obtain DNA with sufficient quality for positive amplification of the RLEP region. No amplification was observed in the negative-control-slide group by any method. Patients with ZNS negative slides can still transmit the infection, and molecular methods can help identify and treat them, interrupting the chain of transmission and preventing the onset of disabilities. CONCLUSION: The ZNS slides can be sent easily to reference laboratories for later molecular analysis that can be useful not only to improve the diagnosis, but also for the application of other molecular techniques.

Relative value of immunohistochemistry in detection of mycobacterial antigen in suspected cases of tuberculosis in tissue sections.

BACKGROUND: The diagnosis of tuberculosis (TB) depends on identification of the infecting organism. The diagnosis presents as a challenge due to its diverse clinical presentation and low yield of acid-fast bacilli (AFB) in tissue sections. AIM: The aim of the present study is immunohistochemical localization of tubercle bacilli or their components that persist in the granulomas, but have lost the property of staining with acid-fast stain, assess the advantage of immunostaining over conventional Ziehl-Neelsen (ZN) staining and further to study the staining pattern on immunohistochemistry (IHC). MATERIALS AND METHODS: The study population comprised 100 suspected cases of TB. Tissue sections from these were subjected to hematoxylin and eosin, ZN and IHC staining using polyclonal antibody to Mycobacterium tuberculosis followed by a comparative analysis of the results. Cases of lepromatous leprosy were used as a positive control. RESULTS: Acid-fast bacilli were identified by ZN stain in 23% of cases. IHC identified 72% cases. In the present study, IHC had higher sensitivity (95.56%) and negative predictive value (96.43%), but lower specificity (35.06%) and positive predictive value (30.56%) than ZN stain which had the sensitivity, specificity, positive predictive value and negative predictive values of 30.56%, 96.43%, 95.65% and 41.56% respectively. CONCLUSION: Immunohistochemistry is a simple and sensitive technique for localization of tubercle bacilli and their components on tissue sections. It can be easily incorporated in routine histopathology laboratory and serve as an efficient diagnostic adjunct to conventional ZN staining. This will help reduce the practice of prescribing empirical antitubercular treatment based on clinical suspicion alone.

Clinicopathologic assessment of Candida colonization of oral leukoplakia.

BACKGROUND: Leukoplakia is the most common premalignant lesion of the oral mucosa. We studied the colonization of Candida in oral leukoplakia using direct microscopy, culture and histopathology to determine if there is a statistical correlation between Candida invasion and the clinical appearance and presence of epithelial dysplasia in leukoplakia. METHODS: Samples were collected from 40 patients with oral leukoplakia and 21 controls. The swabs collected were used to inoculate Sabouraud's dextrose agar slant and for direct microscopy with Gram's stain. Culture growths were subjected to germ tube and corn meal agar tests to differentiate between Candida albicans and non-albicans groups. Biopsies were also done in all patients for histopathological confirmation; Gomori's methanamine silver stain was used to identify fungal invasion of lesional epithelium. RESULTS AND CONCLUSIONS: Nineteen cases of leukoplakia showed Candida on direct smears, compared to 3 controls. Eighteen cases and one control showed growth of Candida on culture. Non-homogenous leukoplakia showed a higher positivity rate on microscopy and culture than homogenous lesions. All these correlations were statistically significant. Forty percent of leukoplakia cases were simultaneously positive for Candida on direct microscopy, culture and histopathologic evaluation. No significant difference was found between non-dysplastic and distinctly dysplastic lesions with respect to Candida detection on microscopy or culture.

Immunocytochemical detection of mycobacterial antigen in extrapulmonary tuberculosis.

The aim of the study is to determine whether immunostaining for mycobacterial antigen can contribute to the cytological diagnosis of extrapulmonary tuberculosis (EPTB). The study was carried out on aspirated material of lymph nodes, and other accessible sites, from 65 patients with clinical diagnosis of tuberculosis (TB). Twenty patients, diagnosed by fine-needle aspiration, with non-tuberculous granulomas served as controls. The diagnosis of TB was based on the demonstration of acid-fast bacilli (AFB), culture positivity for Mycobacterium tuberculosis (M. tuberculosis), or response to treatment with standard anti-tubercular therapy. Immunostaining was done using polyclonal antibody to mycobacteria. AFB positivity by Ziehl Neelsen (ZN) staining was 21%, 65.38%, and 68% respectively in Pattern 1 (granulomas alone), in Pattern 2 (granulomas with necrosis), and in Pattern 3 (necrosis alone). Overall AFB positivity was 56.92%. Twenty-eight of 65 cases were negative for AFB on direct smear. Culture was positive in 46% (13/28). Sensitivity and specificity of immunostaining were 96.92% (63/65) and 95%, respectively. Immunoreactivity was seen in 26 (92.8%) of 28 cases which were negative by ZN staining. Except in the case of leprosy, in which cross reactivity was seen, there was no immunoreactivity in the control group. Immunocytochemistry (ICC) had high sensitivity (96.2%) and specificity (95%) in the diagnosis of EPTB. ICC may be a useful adjunct to evaluation of cytomorphology and ZN staining.