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 Table of Contents  
ORIGINAL ARTICLE
Year : 2021  |  Volume : 10  |  Issue : 4  |  Page : 393-397

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


1 Biomedical Sciences Center, Adolfo Lutz Institute, São Paulo, Brazil
2 Nursing Department, University of Medicine, São José do Rio Preto, São Paulo, Brazil

Date of Submission07-Oct-2021
Date of Decision26-Oct-2021
Date of Acceptance10-Nov-2021
Date of Web Publication13-Dec-2021

Correspondence Address:
Heloisa da Silveira Paro Pedro
São José of Rio Preto Laboratory, Adolfo Lutz Institute, São José of Rio Preto, São Paulo
Brazil
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmy.ijmy_206_21

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  Abstract 


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.

Keywords: Epidemiology, immunochromatography, leprosy


How to cite this article:
Ule Belotti NC, Tonelli Nardi SM, Arco Paschoal VD, Martins Montanha JO, Paro Pedro Hd, Gazetta CE. Laboratory diagnosis of leprosy: Two staining methods from bacilloscopy and rapid ml flow test. Int J Mycobacteriol 2021;10:393-7

How to cite this URL:
Ule Belotti NC, Tonelli Nardi SM, Arco Paschoal VD, Martins Montanha JO, Paro Pedro Hd, Gazetta CE. Laboratory diagnosis of leprosy: Two staining methods from bacilloscopy and rapid ml flow test. Int J Mycobacteriol [serial online] 2021 [cited 2022 Jan 21];10:393-7. Available from: https://www.ijmyco.org/text.asp?2021/10/4/393/332361




  Introduction Top


Leprosy is still considered a public health problem, especially in countries whose prevalence rates exceed 1 case/10 000 inhabitants.[1] It manifests itself mainly in agglomerated populations with precarious sanitary conditions and access to health systems; therefore, it is included within the group of Neglected Tropical Diseases.[2]

Diagnosis is based on the characteristic signs and symptoms of the disease, supported by laboratory tests. A case of Leprosy is defined by the World Health Organization by the presence of one or more of the following criteria: skin lesion with change in thermal or painful sensitivity; peripheral nerve involvement with neural thickening and the presence of Mycobacterium leprae bacilli confirmed by smear microscopy or skin biopsy.[3]

Bacillus is Gram positive, with a cell wall with a high content of structural lipids, responsible for causing great hydrophobicity, hindering the action of aqueous dyes differentiators. Thus, it stains with fuchsin and does not bleach with acids and alcohols when stained by the Ziehl–Neelsen method, giving it the characteristic of an acid-fast bacillus resistant (AFB).[3],[4]

Bacilloscopy is an easy-to-perform and low-cost complementary test for detecting AFB directly from intradermal smears of lesions or from other selected collection sites such as ear lobes, elbows, and knees in suspected leprosy.[4] The bacilloscopic exam, when positive, demonstrates the presence of the mycobacterium and indicates the most infective patients.[5] Its specificity is high; however, it has low sensitivity.[5],[6],[7] A negative bacilloscopy result does not rule out the diagnosis of leprosy, since bacilli are rarely detected in paucibacillary (PB) forms.[5]

Phenolic glycolipid-I, or PGL-I, is the bacillus cell wall molecule that confers the greatest immune response. It was one of the first Mycobacterium leprae specific antigens to be isolated and characterized.[8] The ML flow serological test, developed in 2003 by Bührer-Sékula, is a simple, low cost and easy to perform lateral flow immunochromatographic test for the detection of IgM anti-PGL-I antibodies in human blood or sérum.[8],[9]

The aim of this study was to assess the agreement between the results of the ML Flow test and bacilloscopy performed by two staining techniques: Ziehl–Neelsen and Auramine O, observing the epidemiological and clinical profile of patients, the positivity and contribution of the tests in the diagnosis of leprosy.


  Methods Top


This is a prospective and exploratory study, with data collection and biological materials carried out in patients with suspected leprosy. The study included 94 patients suspected of having the disease, treated on a spontaneous demand in health units in ten cities in the region of São José do Rio Preto, São Paulo, Brazil, belonging to the Epidemiological Surveillance Group (GVE-29), from August 2020 to May 2021.

After agreeing and signing the Informed Consent Form (ICF), all participants underwent clinical evaluation and collection of biological materials to undergo bacilloscopy and the ML Flow test. The exams were sent to be processed at the regional reference laboratory for Leprosy bacilloscopy.

For bacilloscopy, an intradermal scraping slide containing four smears from each patient was collected. The smears were first stained with Auramine O, and after reading under a fluorescence microscope, the smears were stained by Ziehl–Neelsen. The reading was performed qualitatively (positive or negative) for the fluorescence microscopy technique. For the Ziehl–Neelsen technique, the Bacilloscopic Index counting was used in a common optical microscope.

The ML Flow test was performed according to the protocol described by Bührer-Sékula.[8] To obtain the serum, a whole blood sample was collected to perform the serological test to verify the detection of anti-PGL-I IgM antibodies. The test is considered positive when the test area presents a red-colored line and negative due to the absence of this line. The results were registered in a qualitative way.

After the exams were performed, the cases confirmed by the laboratory or by the clinic were submitted to an interview with a questionnaire and Simplified Neurological Assessment.

The collected data were organized in spread sheets, and for analysis, the statistical program EPI INFO 7.1.2.0 Software (Atlanta, Georgia, USA) was used to obtain the distribution of frequency, mean, and standard deviation of the variables of interest. For the association of two variables, P < 0.05 was considered and Fisher's or Chi-square tests were used as appropriate. To assess the agreement between the methods, the Kappa index was estimated, and for the interpretation of this index, the Landis and Koch criteria were used.

This study was approved in June 2020 by the Research Ethics Committee according to CTC 62-L/2019.


  Results Top


Of the 94 suspected leprosy patients treated by spontaneous demand, 31 (32.9%) were diagnosed with leprosy (22 new cases and 9 cases that had already undergone treatment for more than 5 years and were considered suspected of relapse). Among the diagnosed patients, there was a predominance of males (58.0%), older than 60 years (45.2%) of age, and incomplete elementary education (45.1%). Regarding the history of comorbidities, hypertension and diabetes were prevalent. No participant was taking medication for leprosy.

Participants reported the onset of the clinical picture in 77.4% (n = 24) as anesthetic spots, 61.2% (n = 19) numbness, and 22.5% (n = 7) loss of strength. Direct contact with the most mentioned patients with the disease was, respectively, spouse (16.0%), children (13.0%), and siblings (13.0%). Four (12.9%) PB) cases and 27 (87.0%) multibacillary (MB) cases were diagnosed. Of the 22 newly diagnosed cases of leprosy, the most prevalent clinical form was borderline in 10 (45.4%) patients, and of the 9 suspected cases of recurrence, the most present clinical form was lepromatous in 7 (77.7%) patients. There was no diagnosis of the indeterminate form in the patients evaluated.

There was a 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 (Kappa = 0.72), according to the Landis and Koch criteria. It was observed that 27 patients had positive serology, while 21 and 15 had positive Ziehl–Neelsen and Auramine O results, respectively. It was observed that 8 (11.0%) patients with negative Ziehl–Neelsen bacilloscopy had positive serology, while 2 (9.5%) patients had positive Ziehl–Neelsen bacilloscopy (Smear Index below 1.0) with negative serology. These two patients suspected cases of relapsed and who had already treated the disease for more than 5 years. The ML flow test was more positive, considering that there were six more cases when compared to the bacilloscopy result [Table 1].
Table 1: Agreement between bacilloscopy and the ML flow serological test of the 94 patients evaluated

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[Table 2] presents the results obtained between the Ziehl–Neelsen and Auramine O techniques evaluated. The agreement observed between the two staining techniques was substantial (Kappa = 0.79), considered good according to the Kappa Index.
Table 2: Agreement of results between Ziehl–Neelsen and Auramine O bacilloscopy techniques of the 94 participants

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Of the 31 patients diagnosed with leprosy, two new cases were confirmed only by clinical evaluation, being negative in the three laboratory tests tested in this study, as shown in [Table 3].
Table 3: Frequency distribution of clinical data when related to positive results of the three laboratory tests evaluated

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  Discussion Top


Despite laboratory advances, even without an exam considered the gold standard in the diagnosis of Leprosy, laboratory tests have been very useful in helping to classify patients; however, some exams are not available in most basic health-care services. According to the Ministry of Health of Brazil,[4] dermatological and neurological examination and positive bacilloscopy are still considered sovereign in defining the diagnosis of leprosy.

The epidemiological data found in our study are in line with the literature regarding the male gender[10],[11] but differ in relation to the mean age, which was above the data reported by other authors who found involvement by leprosy in the more economically active adult stage.[11],[12] Low education and family income below two minimum wages are similar findings with data found in other studies[13] and reflect the socioeconomic precariousness that favors the spread of the disease.

The contacts that were most cited are considered consanguineous households, who present a higher risk of illness due to leprosy.[14] The clinical forms that most prevailed in the study were borderline and lepromatous, which attest to multibacillary forms of leprosy with greater ease in clinical and laboratory diagnosis, with higher number of reported cases,[14],[15],[16] suggesting the hypothesis of failure in the initial diagnosis by the health services.

Since Brazil has a high burden of leprosy, the introduction of new laboratory techniques that contribute to the diagnosis of the disease, such as the ML Flow serological test, should be evaluated and compared to existing techniques for its routine application.[17]

In this study, ML Flow test positivity occurred in 87.0% (n = 27) of patients diagnosed with the disease, a finding consistent with studies carried out in Brazil that described seropositivity between 50.8% and 94.2%.[9],[10] The test showed strong agreement with the two bacilloscopy techniques evaluated, with a greater association with Ziehl–Neelsen, a result superior to those described in the literature.[10],[15] This finding shows that serological testing can be a great ally to help in the clinical classification of patients and to define the treatment.[3]

Previously, the superiority of fluorescence microscopy over the Ziehl–Neelsen method was shown in histological sections of skin fragments[18],[19] or in intradermal scraping.[20] In this study, positivity by the Ziehl–Neelsen technique was superior to the fluorescence technique, with results of 22.3% and 15.9%, respectively. According to previous researches, the introduction of the fluorescent method in the laboratory routine should initially occur as a screening of positive or negative smears, and later, after familiarization with the technique, the Bacilloscopic Index can be performed.[20] There were no reports in the literature comparing staining using the Auramine O technique with serological tests, perhaps because serological tests such as ML Flow were developed at the research level and are not yet established as a routine laboratory.

Unlike other studies citing sensitivity of only 40% of the ML Flow test in paucibacillary cases,[8],[9] our results detected positive serology in 100% of patients classified as PB. Despite the limited that may have been an obstacle to this research, it is noteworthy that the ML Flow test is not recommended as the only diagnostic test, as most PB cases may not develop detectable antibodies in serology.[8],[21] These four (4) PB patients also had up to five skin lesions with anesthetic areas and reported having direct contact with patients with the disease in the questionnaires.

Most patients diagnosed with leprosy had one or more compromised nerves, and this finding is strongly related to the positive results of the ML Flow test (89.4%) and of bacilloscopy using the Ziehl–Neelsen technique (78.9%). Although the region studied is considered a nonendemic region, our findings showed that the diagnosed cases present some physical disability already installed (Degree of Physical Disability 1 and 2), which suggests a late diagnosis of the disease.

As for the number of skin lesions, half (51.7%) of the patients had five or more lesions, mainly associated with the positive result of the ML Flow test (86.6%) followed by the positive result of the Ziehl–Neelsen bacilloscopy (80.0%). Of the 11 patients who reported absence of a Bacillus Calmette-Guérin (BCG) vaccine scar, 100% had positive serology. There are reports that, in unvaccinated individuals, there is a tendency toward an increase in antibody titers to PGL-I, reaffirming vaccination as an important measure of disease control.[22],[23]

The multivariate analysis of the variables of interest in [Table 3] did not show statistical significance, however, as this was a small number of patients diagnosed in a region considered nonendemic, this was a limiting factor for this research.

Molecular methods have shown effective results for detecting new cases of the disease. A comparative study between the molecular methods, repetitive element (RLEP) polymerase chain reaction (RLEP-PCR), and inter-simple sequence repetition (ISSR-PCR), with AFB microscopy, identified superior efficacy of the ISSR-PCR technique in the early diagnosis of cases of leprosy using skin smear samples.[24] However, another recently published study detected a greater sensitivity by the multiplex PCR technique in paucibacillary cases of the disease compared to the RLEP-PCR.[25]

To achieve global elimination of the disease, it is necessary to monitor newly detected cases, regular and complete treatment of patients, and monitor cases with early detection of Degree of Physical Disabilities. According to Naaz et al.,[26] a social marketing approach also has a lot of potential to help improve leprosy health education programs, by motivating people to seek appropriate treatment early and community acceptance of leprosy patients.

Thus, as there is no gold standard test for the disease, our results show how important it is to complement the standard diagnosis with serology to add or direct to a more reliable laboratory result in the classification of cases and in the appropriate choice of treatment for the patient.


  Conclusions Top


There was greater agreement between the results of the ML Flow serological test and the bacilloscopy by Ziehl–Neelsen, when compared to the results of the bacilloscopy by Auramine O. Among the proposed microscopy techniques, it is concluded that the Ziehl–Neelsen technique remains the best option for standard leprosy staining. Related to the positive results of the ML Flow test and bacilloscopy using the Ziehl–Neelsen technique, most patients diagnosed with leprosy in our study had one or more compromised nerves, Degree of Physical Disability 1, five or more skin lesions, and no scar BCG vaccine.

The ML Flow test showed the highest positivity among the three techniques evaluated. As it is a quick and easy-to-perform test, it can be a useful and effective auxiliary tool in the early diagnosis of leprosy cases, to be used routinely at any level of the health service, as there is no gold standard laboratory test for leprosy.

Ethical clearance

This work was approved at the Adolfo Lutz Institute by Scientific Committee and Ethical Committee, under number CTC 62-L/2019.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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WHO. World Health Organization. Global Leprosy Strategy 2016-2020: Accelerating Towards a Leprosy-Free World; 2016. Available from: https://apps.who.int/iris/bitstream/handle/10665/208824/9789290225096_en.pdf?sequence=14 & isAllowed=y. [Last accessed on 2020 Dec 10].  Back to cited text no. 1
    
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Moschella SL. An update on the diagnosis and treatment of leprosy. J Am Acad Dermatol 2004;51:417-26.  Back to cited text no. 7
    
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Bührer-Sékula S, Smits HL, Gussenhoven GC, van Leeuwen J, Amador S, Fujiwara T, et al. Simple and fast lateral flow test for classification of leprosy patients and identification of contacts with high risk of developing leprosy. J Clin Microbiol 2003;41:1991-5.  Back to cited text no. 8
    
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Contin LA, Alves CJM, Fogagnolo L, Nassif PW, Barreto JA, Lauris JRP, et al. Use of the ML-Flow test as a tool in classifying and treating leprosy. Braz Ann Dermatol 2011;86:91-5.  Back to cited text no. 9
    
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Lyon S, Castorina da Silva R, Lyon AC, Grossi MA, Lyon SH, Azevedo Mde L, et al. Association of the ML Flow serologic test to slit skin smear. Rev Soc Bras Med Trop 2008;41 Suppl 2:23-6.  Back to cited text no. 10
    
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Sarubi JC. Comparative Study of Ziehl-Neelsen and Auramine O Techniques in Smear Microscopy of Four-and Six-Site Dermal Scrapings in New Cases of Leprosy, in a Reference Service in Belo Horizonte. [Master's Dissertation] Federal University of Minas Gerais – Faculty of Medicine; 2008.  Back to cited text no. 11
    
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Limeira OM. A Study on the Agreement Between the ML Flow and Bacilloscopy Tests in the Control of Leprosy in New and Reactive Multibacillary Cases and their Contacts. [Master's Dissertation] University of Brasília; 2006.  Back to cited text no. 12
    
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Frade MA, de Paula NA, Gomes CM, Vernal S, Bernardes Filho F, Lugão HB, et al. Unexpectedly high leprosy seroprevalence detected using a random surveillance strategy in midwestern Brazil: A comparison of ELISA and a rapid diagnostic test. PLoS Negl Trop Dis 2017;11:e0005375.  Back to cited text no. 16
    
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Deepa SA, Hippargi SB, Rao G, Saha D, Yelikar BR, Karigoudar M. Evaluation of Fluorescent Staining for Diagnosis of Leprosy and its Impact on Grading of the Disease: Comparison with Conventional Staining. J Clin Diagnostic Res 2016;10:23-6.  Back to cited text no. 19
    
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Gonzales Prendes MA, Fors Carbonell A, Pardo-Castello V, Curbelo Hernandez A. Fluorescent microscopy in leprology; comparative evaluation of the fluorescent method and the Ziehl-Neelsen technic in the bacteriological diagnosis. Int J Lepr 1953;21:35-40.  Back to cited text no. 20
    
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Niitsuma EN, Bueno IC, Arantes EO, Carvalho AP, Xavier Junior GF, Fernandes GD, et al. Factors associated with the development of leprosy in contacts: A systematic review and meta-analysis. Rev Bras Epidemiol 2021;24:e210039.  Back to cited text no. 22
    
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Lopes LQ, Nascimento GP, Ahmed AM, Sultan MAI, Barraza RAS, Moura RS, et al. Seropositivity to PGL-I in a reintegration center for people affected by leprosy and different regions in the municipality of Anápolis-Goiás. Braz J Health Rev Curitiba 2021;4:6834-45.  Back to cited text no. 23
    
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Mohanty PS, Bansal AK, Naaz F, Patil SA, Arora M, Singh M. Dominant marker (inter-simple sequence repeat-polymerase chain reaction) versus codominant marker (RLEP-polymerase chain reaction) for laboratory diagnosis of leprosy: A comparative evaluation. Int J Mycobacteriol 2020;9:18-23.  Back to cited text no. 24
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Chaitanya VS, Cuello L, Das M, Sudharsan A, Ganesan P, Kanmani K, et al. Analysis of a novel multiplex polymerase chain reaction assay as a sensitive tool for the diagnosis of indeterminate and tuberculoid forms of leprosy. Int J Mycobacteriol 2017;6:1-8.  Back to cited text no. 25
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Naaz F, Mohanty PS, Bansal AK, Kumar D, Gupta UD. Challenges beyond elimination in leprosy. Int J Mycobacteriol 2017;6:222-8.  Back to cited text no. 26
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