Leprosy in children in Cuba: Epidemiological and clinical description of 50 cases from 2012-2019.

INTRODUCTION: In 1993, Cuba achieved leprosy elimination according to the World Health Organization's (WHO) indicator of less than one case per 10,000 population. Despite this achievement, detection of new cases occurs every year among all age groups including children. Detection of new cases in children reveals persistent transmission of the infection. OBJECTIVE: To describe the clinical and epidemiological features of leprosy in individuals younger than 15 years (childhood leprosy) reported to the Cuban National Leprosy Control Program (NLCP) between 2012 and 2019. METHODS: We conducted a retrospective descriptive study between 2012 and 2019 to assess the clinical and epidemiologic features of individuals under the age of 15 years with a confirmed diagnosis of leprosy reported to the NLCP. We reviewed the NLCP database and collected data to better define the total number of cases of leprosy in adults, children (younger than 15 years). We assessed socio-demographic variables (age, gender, and province of residence) as well as variables of clinical interest including operational classification and staging at diagnosis, bacillary index, grade of disability by WHO staging. Additionally, we evaluated epidemiological variables including passive versus active surveillance of cases, contact investigation focusing specifically in household transmission, and the degree of kinship as well as standing of the child within the focus of transmission when there were additional cases. RESULTS: We identified fifty children during the study period corresponding to 3% of the overall cases of leprosy comprising all age groups in Cuba. In the age group younger than 15 years, the majorities of cases was from the Granma province and most were between the ages of 10 and 14 years. Clinically, multibacillary/lepromatous forms were the most common type identified with positive bacillary index. The majority of children diagnosed with leprosy during our study period had a history of a relative with a confirmed diagnosis of leprosy. CONCLUSIONS: Detection of cases of leprosy in individuals younger than 15 years of age in Cuba demonstrates ongoing transmission of M. leprae in specific geographic hotspots. Its frequency in the early adolescence, the predominant clinical forms, and the mode of detection associated with sources of suspected familiar infection demonstrated that there is a need for further efforts by the NLCP to conduct active surveillance activities among affected communities to identify cases of leprosy earlier with the goal of preventing further household and community transmission.

Leprosy in children: a Cuban experience on leprosy control.

BACKGROUND: Children are believed to be the most vulnerable group to leprosy. Childhood leprosy reflects disease transmission in the community as well as the efficiency of ongoing disease control programmes. In Cuba, leprosy is not a national health problem; however, new childhood leprosy cases are diagnosed every year. OBJECTIVE: We summarise the experience of Cuba on childhood leprosy control over the past two decades. RESULTS: Between 2000 and 2017, a total of 103 children in Cuba have been diagnosed with leprosy, showing that active transmission of cases remains in 13 of 15 provinces of Cuba. The majority of cases were multibacillary (66%), and 34% were paucibacillary cases. Clinically 60% of children have more than five lesions all over their body. Voluntary reporting was the principal method of case detection. The presence of familial and extrafamilial contact with leprosy cases may be a cause of concern, as it implies continuing transmission of the disease. Only four children had disabilities (one with grade 2 disabilities and three with grade 1 disabilities). A set of national investigations have been developed to intervene in a timely manner. Intervention strategies that combine clinical surveillance and laboratory test could be an option for early detection of childhood leprosy. CONCLUSIONS: Early detection of cases due to effective health education campaigns, regular and complete treatment with MDT, and contact tracing may be important in reducing the burden of leprosy in the community.

Diagnóstico de lepra en niños mediante seguimiento serológico de anticuerpos contra el glicolípido fenólico I / Leprosy diagnosis in children by antibodies serologic follow up against the phenolic glycolipid I

Introducción: Los niños contactos de pacientes con lepra se consideran las personas con mayores posibilidades de desarrollar la enfermedad. Objetivo: Valorar la utilidad del seguimiento serológico de anticuerpos contra el glicolípido fenólico I para el diagnóstico de lepra en niños. Métodos: Investigación prospectiva. Se incluyeron todos los niños contactos de pacientes diagnosticados con lepra en las provincias de La Habana, Santiago de Cuba y Guantánamo entre enero 2013-junio 2015. Los menores se evaluaron clínicamente mediante examen dermatoneurológico y se determinó la presencia de anticuerpos contra el glicolípido fenólico I de Mycobacterium leprae para el estudio serológico. Los niños con serología positiva se siguieron, con estos dos métodos, cada seis meses durante dos años. La confirmación de un caso nuevo de lepra se realizó mediante baciloscopía y biología molecular. Resultados: Se estudiaron 151 niños, de ellos 44 (29,13 por ciento) resultaron positivos al glicolípido fenólico I. Se diagnosticaron durante el período 12 casos, de los cuales 11 tuvieron serología positiva. Presentaron sospecha clínica 10 niños de los estudiados, solo se confirmó un caso nuevo, el cual tuvo serología negativa. En ocho de los niños diagnosticados se detectó presencia de bacilos ácido alcohol resistente en la lámina de baciloscopía. En los restantes cuatro niños el diagnóstico se confirmó por la reacción en cadena de la polimerasa. Conclusiones: Los resultados de esta investigación denotan la utilidad del seguimiento serológico de anticuerpos contra el glicolípido fenólico I en el diagnóstico de lepra en niños, en apoyo a la vigilancia clínica(AU)

Introduction: Children having contact with leprosy patients are considered the contacts with greater possibilities of developing the disease. Objective: To assess the usefulness of antibodies´ serologic follow up against the phenolic glycolipid I (PGL-1) for the diagnosis of leprosy in children. Methods: Prospective study in which were included all children contacts of patients diagnosed with leprosy in Havana, Santiago de Cuba and Guantanamo provinces between January 2013 and June 2015. They were evaluated clinically by the dermato-neurological examination and the presence of antibodies against the PGL-1 of M. leprae was determined. Children with positive serology were followed up using these same two methods every six months for two years. The confirmation of a new case of leprosy was made by smear microscopy and molecular biology / PCR-Rlep. Results: A total of 151 children were studied. Of these, 44 children (29.13 percent) were positive for phenolic glycolipid I. A total of 12 children were diagnosed during this period, of which 11 had positive serology. Only 10 children of the studied ones presented clinical suspicion and of these only one new case was confirmed, which had negative serology. In eight of the diagnosed children, the presence of acid-fast bacilli was detected in the smear microscopy. In the remaining four children, the diagnosis was confirmed by the PCR result. Conclusion: The results of this investigation show the usefulness of the antibodies´ serologic follow up against the phenolic glycolipid I in the diagnosis of leprosy in children as a support to clinical surveillance(AU)

Diagnóstico de lepra en niños mediante seguimiento serológico de anticuerpos contra el glicolípido fenólico I / Leprosy diagnosis in children by antibodies serologic follow up against the phenolic glycolipid I

Introducción: Los niños contactos de pacientes con lepra se consideran las personas con mayores posibilidades de desarrollar la enfermedad. Objetivo: Valorar la utilidad del seguimiento serológico de anticuerpos contra el glicolípido fenólico I para el diagnóstico de lepra en niños. Métodos: Investigación prospectiva. Se incluyeron todos los niños contactos de pacientes diagnosticados con lepra en las provincias de La Habana, Santiago de Cuba y Guantánamo entre enero 2013-junio 2015. Los menores se evaluaron clínicamente mediante examen dermatoneurológico y se determinó la presencia de anticuerpos contra el glicolípido fenólico I de Mycobacterium leprae para el estudio serológico. Los niños con serología positiva se siguieron, con estos dos métodos, cada seis meses durante dos años. La confirmación de un caso nuevo de lepra se realizó mediante baciloscopía y biología molecular. Resultados: Se estudiaron 151 niños, de ellos 44 (29,13 por ciento) resultaron positivos al glicolípido fenólico I. Se diagnosticaron durante el período 12 casos, de los cuales 11 tuvieron serología positiva. Presentaron sospecha clínica 10 niños de los estudiados, solo se confirmó un caso nuevo, el cual tuvo serología negativa. En ocho de los niños diagnosticados se detectó presencia de bacilos ácido alcohol resistente en la lámina de baciloscopía. En los restantes cuatro niños el diagnóstico se confirmó por la reacción en cadena de la polimerasa. Conclusiones: Los resultados de esta investigación denotan la utilidad del seguimiento serológico de anticuerpos contra el glicolípido fenólico I en el diagnóstico de lepra en niños, en apoyo a la vigilancia clínica(AU)

Introduction: Children having contact with leprosy patients are considered the contacts with greater possibilities of developing the disease. Objective: To assess the usefulness of antibodies´ serologic follow up against the phenolic glycolipid I (PGL-1) for the diagnosis of leprosy in children. Methods: Prospective study in which were included all children contacts of patients diagnosed with leprosy in Havana, Santiago de Cuba and Guantanamo provinces between January 2013 and June 2015. They were evaluated clinically by the dermato-neurological examination and the presence of antibodies against the PGL-1 of M. leprae was determined. Children with positive serology were followed up using these same two methods every six months for two years. The confirmation of a new case of leprosy was made by smear microscopy and molecular biology / PCR-Rlep. Results: A total of 151 children were studied. Of these, 44 children (29.13 percent) were positive for phenolic glycolipid I. A total of 12 children were diagnosed during this period, of which 11 had positive serology. Only 10 children of the studied ones presented clinical suspicion and of these only one new case was confirmed, which had negative serology. In eight of the diagnosed children, the presence of acid-fast bacilli was detected in the smear microscopy. In the remaining four children, the diagnosis was confirmed by the PCR result. Conclusion: The results of this investigation show the usefulness of the antibodies´ serologic follow up against the phenolic glycolipid I in the diagnosis of leprosy in children as a support to clinical surveillance(AU)

Rhopalurus junceus scorpion venom induces antitumor effect in vitro and in vivo against a murine mammary adenocarcinoma model.

OBJECTIVES: In Cuba the endemic scorpion species Rhopalurus junceus has been used in traditional medicine for cancer treatment and related diseases. However there is no scientific evidence about its therapeutic potential for cancer treatment. The aim of the study was to determine the antitumor effect of scorpion venom against a murine mammary adenocarcinoma F3II. MATERIALS AND METHODS: The cytotoxic activity was determined by MTT assay with venom concentrations ranging from 0.1-1 mg/ml. Apoptosis was determined by RT-PCR and flow cytometry. Toxic effect in healthy animals and tumor growth kinetics in F3II bearing-mice were evaluated by using scorpion venom doses (0.2; 0.8; 3.2 mg/kg) after one and ten injections respectively by the intraperitoneal route . RESULTS: Scorpion venom induced a significant cytotoxic effect (P<0.05) in F3II cells in a concentration-dependent manner. The cell death event involves the apoptotic pathway due to up-regulation of pro-apoptotic genes (p53, bax), down-regulation of antiapoptotic gene (bcl-2), and 33% of Annexin V+/PI- cells at early apoptosis and 10.21% of Annexin V+/PI+ cells at late apoptosis. Scorpion venom induced significant inhibition of tumor progression (P<0.05) in F3II bearing-mice in a dose-dependent manner. The antitumor effect was confirmed due to dose-dependent reduction of Ki-67 and CD31 proteins present in tumor tissue. CONCLUSION: Evidence indicates that scorpion venom can be an attractive natural product for deep investigation and developing a novel therapeutic agent for breast cancer treatment.

Rhopalurus junceus scorpion venom induces apoptosis in the triple negative human breast cancer cell line MDA-MB-231.

Rhopalurus junceus scorpion venom has demonstrated high cytotoxic activity in epithelial cancer cells. In the present study, the effect of scorpion venom on cell viability and apoptosis was evaluated in the MDA-MB-231 human breast carcinoma cell line. Cell viability was analyzed using MTT assay. The cell death event was examined trough end-point RT-PCR to identify the expression of apoptosis-related genes, fluorescent microscopy and mitochondrial membrane potential (ΔΨm) alteration. The results demonstrated that scorpion venom induced apoptosis in MDA-MB-231 cells in a time-dependent manner. Besides, scorpion venom treatment also resulted in p53, bax, noxa, puma, caspase 3 and p21 over-expression, while the expression of bcl-2 and bcl-xl was down-regulated. Apoptosis was associated with depolarization of ΔΨm. The overall effect indicates that the selective cytotoxic effect of the scorpion venom is associated with its apoptosis-inducing effect through the mitochondrial pathway. Therefore, R. junceus scorpion venom may be an interesting natural extract for further investigation in breast cancer treatment strategies.

Lepra Tardía. Comportamiento clínico epidemiológico. La Habana. Período 2008-2016 / No disponible

La lepra es una enfermedad incluida en el grupo de enfermedades desatendidas según la Organización Mundial de la Salud, en Cuba la enfermedad dejó de ser un problema de salud desde 1993. En La Habana se notifican cada año casos de lepra de forma tardía con diferentes grados de discapacidad. El objetivo de este estudio fue caracterizar a los enfermos notificados de lepra tardíamente en La Habana. Se realizó un estudio longitudinal, descriptivo y retrospectivo de los casos de lepra diagnosticados tardíamente en la provincia entre 2008 y 2016, se incluyeron 96 casos a los cuales se revisó la Encuesta Epidemiológica para enfermos de lepra para evaluar variables como edad, sexo, tiempo transcurrido entre los primeros síntomas y el diagnóstico, grado de discapacidad al diagnóstico, formas clínicas y especialidades involucrados en el diagnóstico tardío. El grupo de edad más afectado fue el de 25 a 59 años, los municipios más afectados fueron Lisa y Arroyo Naranjo, predominó la forma clínica multibacilar, y la discapacidad grado ll se presentó en el 18% de los casos. Se evidenció la importancia de un diagnóstico precoz para evitar la aparición de discapacidades y que los municipios periféricos tienen la mayor posibilidad de diagnosticar casos tardíos

Leprosy is a disease included in the group of neglected diseases by The World Health Organization. In Cuba, the disease has ceased to be a health problem since 1993. In Havana, each year cases of late leprosy are reported with various grades of disability. The aim of this study was to characterize the behavior of patient whom reported with late leprosy in Havana. We conducted a longitudinal, descriptive and retrospective study of the leprosy cases diagnosed late in the province between 2008 and 2016. We included 96 cases in which the epidemiological survey for leprosy patients was carried out, variables such as age, sex, time elapsed between the first symptoms and the diagnosis, degree of disability at diagnosis, clinical forms, and specialties involved in the late diagnosis were evaluated. The most affected age group was between 25-59 years old, the most affected municipalities were Lisa and Arroyo Naranjo, the multibacillary clinical form predominated, and the grade II disability was present in 18% of the cases. The importance of an early diagnosis to avoid the appearance of disability in the patients was put in evidence by the series studied and the most peripheral municipalities are the one with the greatest possibility of diagnosing late cases

Transient glutathione depletion in the substantia nigra compacta is associated with neuroinflammation in rats.

Glutathione (GSH) deficiency has been identified as an early event in the progression of Parkinson's disease. However, the role of GSH in the etiology and pathogenesis of this neurodegenerative disorder is not well established. The aim of this study is to assess the effect of transient GSH depletion in the substantia nigra pars compacta (SNpc) on neuroinflammation after the injection of a single dose of l-buthionine sulfoximine (BSO) into the SNpc of male Sprague-Dawley rats. The results showed that BSO treatment stimulates microglia (p<0.01) and astroglial response (p<0.01), c-Jun N-terminal kinase and inducible nitric oxide synthase (iNOS) (p<0.001) in the SNpc, accompanied by dopaminergic dysfunction. In addition, high levels of tumor necrosis factor α (p<0.01), interleukins IL-1ß p<0.01), IL-6 p<0.001) and nitric oxide p<0.01) were found in the treated animals compared to control groups, while no significant differences were found in IL-10 levels. These results suggest that transient GSH depletion can increase the susceptibility of SNpc to degeneration by promoting an inflammatory response and nitrosative stress, reinforcing the possible role of GSH unbalance, oxygen/nitrogen reactive species and neuroinflammation as causal factors on the degeneration of the SNpc.

Enzymatic analysis of venom from Cuban scorpion Rhopalurus junceus.

Rhopalurus junceus scorpion venom has been identified as a natural extract with anticancer potential. Interestingly, this scorpion venom does not cause adverse symptoms in humans. However, there is scarce information about its composition and enzymatic activity. In this work, we determined the electrophoretic profile of the venom, the gelatinase and caseinolytic activity, and the phospholipase A2 (PLA2) and hemolytic activity. The effect of different venom doses (6.25, 12.5 and 25 mg/kg) on gastrocnemius muscle was also measured as CK and LDH activity in serum. The presence of hyaluronidase was determined by turbidimetric assay. The effect of different fractions obtained by gel filtration chromatography were evaluated at different concentrations (0.05, 0.1, 0.2, 0.4, 0.6mg/ml) against lung cancer cell A549 and lung normal cell MRC-5 using MTT assay. The electrophoretic profile demonstrated the presence of proteins bands around 67kDa, 43kDa, 18.4kDa and a majority band below 14.3kDa. The venom did not showed caseinolytic, gelatinase, PLA2 and hemolytic activity even at highest venom concentration used in the study. Scorpion venom only showed a significant toxic effect on gastrocnemius muscles identified by CK and LDH release after subcutaneous injection of 12.5 and 25mg/kg. Low molecular weight fractions (<4kDa) induced a significant cytotoxicity in A549 cells while high molecular weight proteins (45-60kDa) were responsible for hyaluronidase activity and toxic effect against MRC-5. Experiments indicate that Rhopalurus junceus scorpion venom has low enzymatic activity, which could contribute to the low toxic potential of this scorpion venom.

Evaluación de diferentes muestras para la detección molecular de Mycobacterium leprae en Cuba / Evaluation of different samples for Mycobacterium leprae molecular detection in Cuba

Introducción: en la actualidad, la principal estrategia para controlar la infección por Mycobacterium leprae es la detección precoz y la multiterapia. El objetivo del presente estudio fue evaluar la utilidad del empleo de diferentes muestras clínicas en el diagnóstico molecular de la infección por M. leprae. Métodos: se evaluaron 22 pacientes con clínica sugestiva de lepra. Se tomaron muestras de linfa de cuatro puntos (ambos lóbulos auriculares y ambos codos) recogidas en una lámina porta objetos (lámina de baciloscopía) y en un hisopo, además muestra de hisopado nasal, muestra de sangre total y de tejido de la lesión. Las muestras se analizaron mediante baciloscopía, histología y PCR según correspondió en cada caso. Por otro lado, para determinar la sensibilidad y especificidad del método, se realizó un estudio de casos y controles en el que se emplearon 40 láminas de baciloscopías negativas de pacientes con lepra paucibacilar y 40 láminas negativas de personas sin lepra. Resultados: el 54,5 por ciento de los pacientes resultó positivo por baciloscopía. Solo en el 41 por ciento de los pacientes la histología tuvo resultados concluyentes de lepra. En el 100 por ciento de los pacientes se detectó la presencia de ADN de M. leprae a partir de la lámina de baciloscopía y el hisopado de linfa. En el 95,45 por ciento de los pacientes se pudo amplificar la secuencia diana a partir de la sangre total y solo en el 31,8 por ciento de los pacientes el hisopado nasal resultó positivo. El estudio de casos y controles mostró que la sensibilidad y especificidad de la PCR respecto al diagnóstico convencional fue de 100 por ciento. Conclusión: el diagnóstico de M. leprae mediante PCR, es de gran utilidad cuando las técnicas convencionales no son concluyentes. La lámina de baciloscopía y el hisopado de linfa constituyen las muestras clínicas más útiles para la confirmación molecular de la infección por M. leprae(AU)

Introduction: currently, the early detection and treatment with multitherapy are the main strategy to control the infection by Mycobacterium leprae. The objective of the present study was to evaluate the usefulness of different clinical samples for molecular diagnosis of M. leprae infection. Methods: twenty two patients with suggestive clinical leprosy were analyzed. Different clinical samples were taken by slit skin smear, histopathology and PCR. To determine the sensitivity and specificity of the PCR method, a case-control study was also performed using 40 slides from negative smears of patients with leprosy paucibacillary and 40 from individuals without leprosy. Results: from all patient studied fifty-four percent were positive by slit skin smear and 41 percent were conclusive of leprosy by histopathology. M. leprae DNA was detected in slit skin smears and lymph swabs in 100 percent of patients. In 95,45 percent of patients were detected M. leprae DNA in whole blood and in 31,8 percent of them in nasal swab. The sensitivity of PCR respect to conventional diagnostic was 100 percent, the specificity was 97.5 percent, and the positive predictive value was 97.56 and the negative predictive value was100 percent. Conclusion: the diagnosis of M. leprae by PCR is very useful when conventional techniques are inconclusive. The slit skin smears and lymph swabs are the most useful clinical samples for molecular confirmation of infection with M. leprae(AU)

Evaluación de diferentes muestras para la detección molecular de Mycobacterium leprae en Cuba / Evaluation of different samples for Mycobacterium leprae molecular detection in Cuba

Introducción: en la actualidad, la principal estrategia para controlar la infección por Mycobacterium leprae es la detección precoz y la multiterapia. El objetivo del presente estudio fue evaluar la utilidad del empleo de diferentes muestras clínicas en el diagnóstico molecular de la infección por M. leprae. Métodos: se evaluaron 22 pacientes con clínica sugestiva de lepra. Se tomaron muestras de linfa de cuatro puntos (ambos lóbulos auriculares y ambos codos) recogidas en una lámina porta objetos (lámina de baciloscopía) y en un hisopo, además muestra de hisopado nasal, muestra de sangre total y de tejido de la lesión. Las muestras se analizaron mediante baciloscopía, histología y PCR según correspondió en cada caso. Por otro lado, para determinar la sensibilidad y especificidad del método, se realizó un estudio de casos y controles en el que se emplearon 40 láminas de baciloscopías negativas de pacientes con lepra paucibacilar y 40 láminas negativas de personas sin lepra. Resultados: el 54,5 por ciento de los pacientes resultó positivo por baciloscopía. Solo en el 41 por ciento de los pacientes la histología tuvo resultados concluyentes de lepra. En el 100 por ciento de los pacientes se detectó la presencia de ADN de M. leprae a partir de la lámina de baciloscopía y el hisopado de linfa. En el 95,45 por ciento de los pacientes se pudo amplificar la secuencia diana a partir de la sangre total y solo en el 31,8 por ciento de los pacientes el hisopado nasal resultó positivo. El estudio de casos y controles mostró que la sensibilidad y especificidad de la PCR respecto al diagnóstico convencional fue de 100 por ciento. Conclusión: el diagnóstico de M. leprae mediante PCR, es de gran utilidad cuando las técnicas convencionales no son concluyentes. La lámina de baciloscopía y el hisopado de linfa constituyen las muestras clínicas más útiles para la confirmación molecular de la infección por M. leprae(AU)

Introduction: Currently, the early detection and treatment with multitherapy are the main strategy to control the infection by Mycobacterium leprae. The objective of the present study was to evaluate the usefulness of different clinical samples for molecular diagnosis of M. leprae infection. Methods: Twenty two patients with suggestive clinical leprosy were analyzed. Different clinical samples were taken by slit skin smear, histopathology and PCR. To determine the sensitivity and specificity of the PCR method, a case-control study was also performed using 40 slides from negative smears of patients with leprosy paucibacillary and 40 from individuals without leprosy. Results: From all patient studied fifty-four percent were positive by slit skin smear and 41 percent were conclusive of leprosy by histopathology. M. leprae DNA was detected in slit skin smears and lymph swabs in 100 percent of patients. In 95,45 percent of patients were detected M. leprae DNA in whole blood and in 31,8 percent of them in nasal swab. The sensitivity of PCR respect to conventional diagnostic was 100 percent, the specificity was 97.5 percent, and the positive predictive value was 97.56 and the negative predictive value was100 percent. Conclusion: The diagnosis of M. leprae by PCR is very useful when conventional techniques are inconclusive. The slit skin smears and lymph swabs are the most useful clinical samples for molecular confirmation of infection with M. leprae(AU)

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.