The influence of ML Flow test in leprosy classification

Abstracts

Estudo descritivo e exploratório correlacionando o ML Flow, a baciloscopia e a classificação em paucibacilar (PB) e multibacilar (MB), envolveu 1.041 casos novos com hanseníase em 13 municípios de Minas Gerais, de outubro de 2002 a março de 2004. A concordância entre o ML Flow e a classificação pelo número de lesões cutâneas e a baciloscopia foi moderada (Kappa:0,51 e 0,48, respectivamente) e, substancial (Kappa:0,77) com a classificação final. De janeiro de 2000 a março de 2004, a proporção de casos novos MB no Estado, passou de 78,1 para 65,8%. A queda no percentual de MB foi maior nos serviços participantes da pesquisa ML Flow (73,1 para 53,3%). A diferença de PB e MB nos serviços participantes e não participantes, de janeiro a março de 2004, foi estatisticamente significativa, indicando implicação direta e benéfica no tratamento e no controle da endemia em Minas Gerais.

Hanseníase; Classificação; Sorologia; Baciloscopia

ARTICLE

The influence of ML Flow test in leprosy classification

Maria Aparecida de Faria Grossi^I,II; Maria Ana Araújo Leboeuf^II; Ana Regina Coelho de Andrade^I,II; Sandra Lyon^I,III; Carlos Maurício de Figueiredo Antunes^I; Samira Bührer-Sékula^IV

^IPost-graduate Program in Health Sciences: Infectious Diseases and Tropical Medicine, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil

^IIState Health Secretariat of Minas Gerais, Belo Horizonte, Brazil

^IIISanitary Dermatology Reference Center, Eduardo de Menezes Hospital, Belo Horizonte, MG, Brazil

^IVKIT-Biomedical Research, Royal Tropical Institute, Amsterdam, The Netherlands and Tropical Pathology and Public Health Institute, Federal University of Goiás, Goiânia, Goiás, Brazil

Address to

ABSTRACT

This is a descriptive, exploratory study correlating ML Flow, bacilloscopy and classification of paucibacillary (PB) and multibacillary (MB), involving 1,041 new leprosy cases in 13 municipalities of Minas Gerais State, from October 2002 to March 2004. Agreement between ML Flow and the classification of the number of skin lesions and bacilloscopy was moderate (K:0.51 and K:0.48, respectively); and substantial for final classification (K:0.77). From January 2000 to March 2004, the proportion of new MB cases in Minas Gerais decreased from 78.1 to 65.8%. The reduction in the percentage of MB cases was higher in health centers that participated in the ML Flow study (73.1% to 53.3%). The difference between PB and MB in the participating and non-participating health centers from January to March 2004 was statistically significant. Implementation of the ML Flow test influenced the classification of patients, suggesting a direct and beneficial impact on patient treatment and the control of the leprosy endemic in Minas Gerais, Brazil.

Key-words: Leprosy. Classification. Serology. Bacilloscopy.

Leprosy is a chronic, infectious disease that is of considerable importance for public health, given its magnitude and potential for causing disabilities and the fact that it mostly affects those of an economically active age³.

The interaction between Mycobacterium leprae and a human being can result in different clinical manifestations with varying signs and symptoms. This is the result of diverse physiopathological mechanisms, different levels of transmissibility and variations in evolution and prognosis, giving rise to innumerous classification systems throughout history¹.

The 1953 Madrid classification considered two stable and opposite ends of a spectrum: tuberculoid and Virchowian, and two unstable groups: indeterminate and dimorphous, which, in the natural evolution of the disease, move towards the ends of the spectrum. Indeterminate leprosy is considered the first clinical manifestation of the disease, which may lead to spontaneous cure or to another clinical form after a period that can vary from a few months to several years¹.

The classification system proposed by Ridley and Jopling in 1966 is the one most used in research and takes immunity into consideration within a spectrum of host resistance and histopathology. This, however, makes it more difficult to use in the field in smaller health centers. The forms are described as: tuberculoid, borderline (subdivided as borderline-tuberculoid, borderline-borderline and borderline-lepromatous), subpolar lepromatous and lepromatous^{1 19}.

In 2000, the World Health Organization (WHO) recommended a simplified method of leprosy classification for treatment purposes, based on the counting of skin lesions. This system was adopted by the Brazilian Ministry of Health (MoH) in 2002. Patients with five cutaneous lesions or less are classified as PB, whereas those with more than five lesions are MB^{24 25}. The clinical classification of leprosy cases, as proposed by the WHO, simply for treatment, involves the risk of overestimating the number of MB cases¹².

In Brazil, skin smear bacilloscopy is used as an auxiliary exam, when available, to classify patients as PB or MB. A positive bacilloscopy classifies the patient as MB, regardless of the number of skin lesions², but a negative result does not exclude the possibility of leprosy diagnosis³.

Due to the limited availability of bacilloscopy in many leprosy control programs, other clinical methods were developed for patient classification^{10 12 18 20 23}. Sensitivity and specificity were established for clinical criteria, based on bacilloscopic references²⁰. The use of the three cardinal signs of leprosy: skin lesions with altered sensation, thickened peripheral nerves and a positive bacilloscopy, resulted in a sensitivity of 97%, with a positive predictive value of 98% to establish leprosy diagnosis⁵.

This classification is necessary for the allocation of patients within the two treatment regimens currently in existence and would have no importance if a single course of chemotherapy existed for all patients. However, it is important to remember that PB and MB present very different bacterial loads and distinct risks of developing relapse, disabilities and deformities. Therefore, correct classification is an important tool to ensure quality treatment for patients²⁰.

The introduction of multi-drug therapy (MDT) for leprosy occurred in Brazil in 1986. The detection rate of MB forms increased from 5.6 to 11.7/10,000 population in the period from 1986 to 1993. That increase took place in nearly all Brazilian states, with a greater tendency to administer 24 doses of MDT/MB, in contrast to the six doses given to PB patients. The percentage of new MB cases in Brazil as whole, increased from 57% to 71.4% over this period and in the State of Minas Gerais, the shift was from 67.7% MB to 94.7%. The introduction of MDT, led to a massive change on a national scale. The alteration in the frequency of MB case treatment with MDT seems to more closely reflect the conservative clinical attitudes regarding the treatment regimen to be administered rather than any real change in epidemiological profile¹⁵.

Beginning in 1980, PGL-I was described as the immunogenic antigen specific to Mycobacterium leprae⁴ and the first serological tests appeared. The most commonly applied was the enzyme linked immunosorbent assay (ELISA), used to detect anti-PGL-I antibodies, specifically IgM⁹.

ML Flow, developed in 2003, is an immunochromatographic test that reveals the presence of IgM antibodies against Mycobacterium leprae-specific PGL-I. It is a rapid test that uses either serum or whole blood and can be used directly by health professionals without the need for laboratories or special equipment⁶. The reagents are highly stable and can be stored at room temperature⁶.

The test can be used as a tool for the correct classification of new leprosy cases as PB or MB and for use in the identification of contacts with a higher risk of developing the disease in the future⁶. More recent reports indicate that if serology was used to classify leprosy, the number of patients treated as MB would drop.

In a multicentric study of 2,632 new leprosy cases, ML Flow seropositivity was 50.8% in Brazil, 62.9% in Nigeria and 31.9% in Nepal. The proportion of MB cases, according to the counting of anesthetic skin lesions was 39.5% in Brazil, 19.4% in Nigeria and 35.6% in Nepal. Bacilloscopy was positive in 27.1% in Brazil and 11.6% in Nepal⁷. In Nigeria, bacilloscopy is not part of the clinical routine and a strong tendency to classify patients as MB was observed as a result, such that 95.7% of patients received MDT/MB.

The incorrect classification of a patient can lead to insufficient treatment for those MB cases misclassified as PB and excessive treatment for PB cases classified as MB²². Correct classification will make leprosy control more effective, avoiding excessive treatment and preventing future relapse due to insufficient treatment⁶.

Early diagnosis, correct classification of leprosy patients and adequate treatment are challenges to the elimination of the disease, principally where the management of patients is being integrated into basic healthcare services²². This study showed that the implementation of this test influenced the classification of patients in Minas Gerais, Brazil.

PATIENTS AND METHODS

As part of a multicentric study in Brazil, Nepal and Nigeria, a descriptive, exploratory study to compare the results of the ML Flow serological test, bacilloscopy and classification of paucibacillary (PB) and multibacillary (MB) leprosy was conducted by the Minas Gerais State Health Secretariat, coordinated by the Biomedical Research Department, Royal Tropical Institute, Amsterdam and financed by Netherlands Leprosy Relief, Amsterdam, Holland ¹¹. It was approved by the Research Ethics Committee of the Santa Casa de Misericórdia of Belo Horizonte on 22/11/2001, under protocol no. 39/01 and that of the Federal University of Minas Gerais, on 16/02/2004, nº 312/04. All research subjects agreed to participate in the study and signed a free informed consent form.

Study population. Field research was conducted between October 2002 and March 2004, in 14 healthcare institutions in 13 municipalities of the State of Minas Gerais, in 8 local health centers, 4 regional referral centers and 2 state referral centers, involving 1,041 new leprosy cases. Cases with positive bacilloscopy and/or serology were classified as MB for treatment purposes, regardless of the number of cutaneous lesions or affected nerves.

Laboratory exams. The ML Flow test was conducted as described by Bührer-Sékula et al⁶ and the results recorded qualitatively as either positive or negative.

The collection of skin smears for bacilloscopy was performed at four sites: skin lesion; earlobes; and elbow on the opposite side of the body from the lesion, or in the absence of lesions, from both elbows. The slides were stained using the Ziehl-Neelsen method and the smears examined in immersion at 100x. The bacteriological index (BI) was calculated according to Ridley's logarithmic scale from 0 to 6¹⁹.

Data analysis. The data were collected by health service professionals who agreed to participate in the study and were trained to conduct tests. The results were noted on the patient's medical record and a research form was sent to the State Health Secretariat monthly, together with the dipsticks of the serological tests, duly conserved for a second, confirmatory reading.

The data were entered using Epi-info software, version 6.0, with double entry, corrected, validated, checked for consistency and, when necessary, returned to the health center to clarify doubts. The identity of the patients was kept in strict confidence throughout the study.

The agreement between the first reading of the ML Flow test, as recorded by the local health professionals involved in the study, and the second reading taken by a single, independent examiner in Belo Horizonte was nearly perfect (kappa: 0.81) with 91% agreement in the results.

Agreement between the ML Flow results and a) classification using the number of skin lesions, b) bacilloscopy and c) final classification used by health center to treat the patient was analyzed, taking into consideration clinical signs, number of affected nerves, bacilloscopy and the ML Flow outcome.

Cohen's kappa coefficient was used to evaluate any agreement according to Landis and Koch's criteria for interpretation¹³: no agreement for an index below 0.00; slight agreement between 0.00 and 0.20; fair agreement from 0.21 to 0.40; moderate agreement from 0.41 to 0.60; substantial agreement from 0.61 to 0.80 and almost perfect agreement from 0.81 to 1.00.

RESULTS

Seropositivity in the ML Flow test was verified in 50.8% of new cases, while bacilloscopy was positive in 27.1% and 39.5% of patients tested presented six or more cutaneous lesions (Table 1).

Agreement between ML Flow and classification by skin lesion counts and bacilloscopy was moderate (Kappa: 0.51 and Kappa: 0.48, respectively), but substantial for final classification given by the health center for treatment purposes (Kappa: 0.77) (Table 1).

Comparing classification by skin lesion counts and the result of the ML Flow test, 188 (29.8%) of patients classified as PB were seropositive, while 70 (17%) of those classified as MB were seronegative (Table 1).

When the final classification given by the health centers is analyzed with the ML Flow results, 107 (17.1%) patients treated as MB had a negative ML Flow, while 11 (2.6%) of those treated as PB had a positive ML Flow (Table 1).

A reduction in the percentage of MB patients among the total of new cases diagnosed in Minas Gerais was observed, falling from 78.1% in 2000, to 65.8%, in March 2004. The reduction in MB cases was greater in health services that participated in the ML Flow study, decreasing from 73.1 to 53.3%, than in the non-participating services, which showed a decrease from 80.6 to 72.2% (Figure 1). The reduction between the percentages of MB cases was greater in the participating institutions and the difference among PB and MB patients diagnosed from January to March 2004 in participating and non-participating centers was statistically significant, with a χ² = 23.8and P-value < 0.001 (Figure 1).

DISCUSSION

The difference in seropositivity of the ML Flow test in this study (50.8%), compared to that obtained by other authors in the same State, Lyon et al (57%)¹⁴ and Castorina-Silva⁸ (70%) and in other countries, such as Nigeria (62.9%) and Nepal (35.6%)⁷, may be explained by the epidemiological and clinical characteristics of the patients, as well as the type of healthcare institution where treatment was administered.

Bacilloscopy positivity varied from 10.6 to 46.2% in the 14 centers involved in the study (data not presented) and the average of 27.1% was shown to be lower than that described in articles from referral centers in Minas Gerais, Lyon et al (35.9%)¹⁴ and Castorina-Silva (40%)⁸; although it was much higher that results from Nepal (11.6%)⁷ and others frequently cited in the literature^{1 5 12 16 20 21}. The high percentage found in the study of Gallo et al (77.9%)¹⁰, can probably be explained by the fact that it was conducted within a national leprosy reference center. Smear bacilloscopy, while a relatively simple and easy test to perform, has a number of operational limitations and the results are often not reliable^{5 20}, in contrast with the high reproducibility and reliability observed with ML Flow; not to mention the fact that the serological test reflects the body's general immunological response to the presence of bacilli. This fact is confirmed by the variation in the ML Flow positivity that accompanied the variation in bacilloscopy. It was positive in 95.7% of patients with positive bacilloscopy, detecting Mycobacterium Leprae-specific antibodies in more than a third of new leprosy cases with negative bacilloscopy.

In the cases studied, the percentage of seropositivity was almost twice that of bacilloscopic positivity, corroborating the lower sensitivity of bacilloscopy that has been reported by several authors^{1 5 12 16 20 21}. The use of serology by health centers may assist in the correct classification and appropriate treatment of true multibacillary patients, thus eliminating many sources of transmission and possibly preventing relapse in those cases where a negative skin smear result leads to the administration of insufficient treatment.

Agreement between ML Flow and the final classification made by the health Centers was substantial, witnessed by the fact that the ML Flow results altered the final classification. Agreement was only moderate compared with the clinical classification standardized by the WHO.

The proportion of MB cases (39.5%) using the criteria of skin lesion counts, as proposed by the WHO and adopted in Brazil, was higher than that observed in Nepal (35.6%)⁷, and cited by Castorina-Silva (35%)⁸. It was considerably higher than the percentage observed in Nigeria (19.4%)⁷ and in Ethiopia (20%)¹⁶, yet lower than in studies conducted in state and national reference centers by Lyon et al (43.7%)¹⁴ and Gallo et al (73.4%)¹⁰, reinforcing the probable differences in the clinical characteristics of patients attended in referral centers.

Comparing the classification by skin lesions and the result of the ML Flow test, 188 (29.8%) of patients classified as PB were seropositive, indicating the treatment received could be insufficient. Meanwhile, 70 (17%) of those classified as MB were seronegative and may have been administered excessive treatment if this was the only criterion used for classification (Table 1).

In relation to the final classification given by the health centers, observation showed that 60% of patients were treated as MB. When the final classification is analyzed in conjunction with the ML Flow results, 107 (17.1%) patients treated as MB presented a negative ML Flow, while 11 (2.6%) of those treated as PB presented a positive ML Flow (Table 1). These data seem to suggest that health professionals place greater value on clinical observations.

A strong tendency exists among health professionals to classify leprosy patients as MB, as per the example from Nigeria, where 95.7% were treated as MB, even though only 19.4% had 6 or more cutaneous lesions⁷. This appears to be related to the absence of laboratory exams, such as bacilloscopy or biopsy (histopathology), which provide security to the professional when making the decision. All this data indicates the likely benefit of including the ML Flow test as an auxiliary tool in the classification of leprosy cases, above all for Family Health Strategy teams, which often have less experience with the disease. This fact suggests that the use of ML Flow could have a significant impact on the prevention of excessive or insufficient treatment regimens and promote a reduction in MB diagnosed cases.

A reduction was seen in the MB proportion of all new leprosy cases detected in the health centers that participated in the study, which subsequently had a strong repercussion on the MB percentage in the State of Minas Gerais as a whole (Figure 1). The use of the ML Flow test to clarify leprosy classification effectively modified the classification of new cases in the State by reducing the number of patients treated as MB during the research period, thereby reducing the use of MDT/MB blisters and the number of consultations, a direct and beneficial impact on treatment and the control of the leprosy endemic.

The results of this study indicate that the use of ML Flow as an auxiliary tool in the classification of leprosy patients and the definition of the most appropriate course of treatment may facilitate the work of basic healthcare professionals, especially those who have minimal experience with the disease and who do not have laboratory access, thereby helping to increase the coverage and impact of leprosy control measures.

ACKNOWLEDGEMENTS

The authors are grateful to the patients, healthcare professionals and institutions involved in the study; the Minas Gerais State Health Secretariat; Professor Dr. Manoel Otávio da Costa Rocha, Coordinator of the Postgraduate Program in Health Sciences: Infectious Diseases Tropical Medicine of the Federal University of Minas Gerais; the Biomedical Research Department of the Royal Tropical Institute for its confidence in our efforts to undertake this research in Minas Gerais and to Netherlands Leprosy Relief (NLR) in Amsterdam for its financial support.

REFERENCES

• 1. Araújo MG. Hanseníase no Brasil. Artigo de atualização. Revista da Sociedade Brasileira de Medicina Tropical 36: 373-82, 2003.
• ²
  Brasil. Ministério da Saúde. Área Técnica de Dermatologia Sanitária. Hanseníase- Atividades de Controle e Manual de Procedimentos. Brasília, 2001.
• ³
  Brasil. Ministério da Saúde. Hanseníase in Vigilância em Saúde. Brasília, 2007. Cadernos de Atenção Básica, no 21, p. 70-104
• 4. Breman PJ, Barrow WW. Evidence for species lipid antigens in Mycobacterium leprae International Journal of Leprosy and Other Mycobacterial Diseases 48: 382-387, 1980.
• 5. Britton WJ, Lockwood DNJ. Seminar leprosy. The Lancet 363: 1209-1219, 2004.
• 6. Bührer-Sékula S, Smits HL, Gussenhoven GC, Leeuwen J, Amador S, Fujiwara T, Klatser PR, Oskam L. Simple and fast lateral flow test for classification of leprosy patients and identification of contacts with high risk of developing leprosy. Journal of Clinical Microbiology 41: 1991-1995, 2003.
• 7. Bührer-sékula S, Visschedijk J, Grossi MAF, Dhakal KP, Namadi AU, Klatser PR, Oskam L. The ML Flow test as a point of care test for leprosy control programmes: potential effects on classification of leprosy patients. Leprosy Review 78: 70-79, 2007.
• 8. Castorina-Silva R. Estudo do comportamento dos testes sorológicos ML Flow e ELISA (PGL-I) em áreas endêmica e não endêmica de hanseníase. Tese (Doutorado em Ciências da Saúde). Universidade Federal de Minas Gerais, Belo Horizonte 2008.
• 9. Cho SN, Yanagibara DL, Hunter SW, Gelber RH, Brennan PJ. Serological specificity of phenolic glicolipid I from Mycobacterium leprae and use in serodiagnosis of leprosy . Infection and Immunity 41: 1077-1083, 1983.
• 10. Gallo Men, Nery AC, Junior Lanr Sales AM, Albuquerque ECA. Alocação do paciente hanseniano na poliquimioterapia: correlação da classificação baseada no número de lesões cutâneas com os exames baciloscópicos. Anais Brasileiros de Dermatologia, 78: 415-424, 2003.
• ¹¹
  Introdução do teste ML Flow no Controle da hanseníase. Manual geral do projeto. Belo Horizonte, abr. 2002.
• 12. Keita S, Faye O, Konare HD, Sow SO, Ndiaye HT, Traore I. Evaluation de la classification clinique des nouveaux cas de lèpre. Etude réalisée à l'Institut Marchoux (Bamako, Mali). Annales de Dermatologie et de Venerologie 130: 184-186, 2003.
• 13. Landis JR; Koch GG. The measurement of observer agreement for categorical data. Biometrics 33: 159-74, 1977.
• 14. Lyon S, Lyon AC, Silva RC, Grossi MAF, Lyon SH, Bührer-Sékula S, Rocha MOC. A comparison of ML Flow serology and slit skin smears to assess the bacterial load in newly diagnosed leprosy patients in Brazil. Leprosy Review 79: 1-9, 2008.
• 15. Martelli CMT, Andrade ALSS, Grossi MAF, Leboeuf MAA, Lombardi C, Zicker F. Changes in leprosy pattern after multidrug therapy implementation. International Journal of Leprosy 63: 95-97, 1995.
• 16. Norman G, Joseph G, Richard J. Validity of the WHO Operational classification and value of other clinical signs in the classification of leprosy. International Journal of Leprosy and Other Mycobacterial Diseases 72: 278-283, 2004.
• 17. Oskam L, Slim E, Bührer-SÉkula S. Serology: recent developments, strengths, limitations and prospects: A state of the art overview. Leprosy Review 74: 196-205, 2003.
• 18. Porichha D. A Plea to revive skin smear examination. International Journal of Leprosy and Other Mycobacterial Diseases 69: 116-119, 2001.
• 19. Ridley DS, Jopling WH. Classification of leprosy according to immunity: a five group system. International Journal of Leprosy and Other Mycobacterial Diseases 34: 255-273, 1966.
• 20. Summary of the Report of The International Leprosy Association Technical Forum.International Journal of Leprosy and Other Mycobacterial Diseases 70:(suppl 1), 2002.
• 21. Ustianowski AP, Lockwood DNJ. Leprosy: current diagnostic and treatment approaches. Current Opinion Infectious Diseases 16: 421-427, 2003.
• 22. Visschedijk J, Engelhard A, Lever P, Grossi MAF, Feenstra P. Leprosy control strategies and the integration of health services: an international perspective. Cadernos de Saúde Pública 19: 1567-1581, 2003.
• 23. Waters MFR. To smear or not to smear? Leprosy Review 73: 211-214, 2002.
• 24. World Health Organization. PQT: perguntas e respostas. Revisão 1997. WHO/LEP/97.8. Geneva, 1997.
• 25. World Health Organization. A guide to leprosy control. Geneve, 1997.

Publication Dates

• Publication in this collection
  17 July 2009
• Date of issue
  2008