Prospects for new leprosy diagnostic tools, a narrative review considering ELISA and PCR assays

Abstract Slit skin smear and histopathological examinations are currently the main laboratory tools used to aid the diagnosis of leprosy. However, their sensitivity is low, and many cases are not detected. New methodologies have been studied to develop more accurate tests. This narrative review aims to raise attention to the results of molecular (polymerase chain reaction) and serological (Enzyme-Linked Immunosorbent Assay) tests applied to the diagnosis of leprosy, and to summarize the available information about the former. Original scientific articles published in indexed international journals, whose study involved aspects of the diagnosis and classification of leprosy cases or home contacts, were selected. The data were extracted independently using a standardized method that dictated the inclusion of the following information: diagnosis in Paucibacillary and Multibacillary cases and in household contacts; sample number; sample type; study design; studied variables; statistical analysis employed; main results; and limitations identified. In clinical practice, the results from molecular and serological tests are assessed separately, with moderate sensitivity and specificity. However, an integrated study of these methodologies has been suggested for greater accuracy in diagnosis.

Enfermedad de Hansen. Una condición emergente en Chile / Hansen disease, an emergent condition in Chile

Resumen La enfermedad de Hansen (EH) es una infección secundaria a Mycobacterium leprae, de curso crónico y que afecta preferentemente la piel y los nervios periféricos. Es una condición emergente en Chile debido a la migración humana. Presentamos un caso que afectó a un trabajador inmigrante desde Haití, que presentó antes de su arribo múltiples lesiones cutáneas en placas, con hipoestesia y madarosis unilateral. El diagnóstico de la forma multibacilar se estableció por los hallazgos clínicos, la presencia de bacilos ácido-alcohol resistentes en una muestra cutánea y la detección de células inflamatorias alrededor de los terminales nerviosos y granulomas en una biopsia de piel, desde donde se amplificaron segmentos específicos de los genes rpoB y hsp65 de M. leprae. El paciente fue tratado con el esquema combinado estándar de la OMS para la forma multibacilar durante un año, con regresión parcial de las lesiones. Durante el seguimiento se detectó por RPC una disminución del número de copias del gen rpoB de M. leprae en muestras nasofaríngeas hasta el sexto mes. El registro de enfermedades de notificación obligatoria demuestra un incremento reciente de casos de EH en Chile, todos relacionados con población migrante. La enfermedad de Hansen es una nueva condición clínica y los clínicos deben estar atentos a esta posibilidad diagnóstica. Las pruebas moleculares pueden ayudar en el diagnóstico y seguimiento.

Hansen's disease (HD) is caused by Mycobacterium leprae. It has a chronic course and preferentially affects the skin and the peripheral nerves. It's an emergent infection in Chile due to migration waves. This case report affecting a migrant worker from Haiti that presented several compatible skin lesions, with hypoesthesia and unilateral madarosis that appeared before arrival. The diagnosis of a multibacillary form was established by clinical findings, presence of fast acid bacilli on a direct skin smear, and inflammatory cell surrounding nerve endings and granulomas on skin biopsy. Besides, specific rpoB and hsp65 gene segments from M. leprae were amplified from skin samples. Patient was treated with the WHO standard combined regimen for multibacillary forms during one year showing partial regression of skin lesions. Nasopharyngeal samples showed the presence of M. leprae rpoB copies detected by PCR decreasing until six months of therapy. Notifiable diseases databases showed a recent increment of cases, all related to migrant population. Hansen's disease is a new condition in Chile and clinicians should be aware of this possibility. Molecular tools may facilitate diagnosis and follow up.

Natural environmental water sources in endemic regions of northeastern Brazil are potential reservoirs of viable Mycobacterium leprae

BACKGROUND The detection of live Mycobacterium leprae in soil and animals other than humans suggests that the environment plays a role in the transmission of leprosy. OBJECTIVE The objective of this study was to investigate the presence of viable M. leprae in natural water sources used by the local population in five municipalities in the state of Ceará, northeastern Brazil. METHODS Samples were collected from 30 different sources. Viable bacilli were identified by reverse transcriptase polymerase chain reaction (PCR) of the M. leprae gyrA gene and sequencing of the PCR products. Physicochemical properties of each water source were also assessed. FINDINGS M. leprae gyrA mRNA was found in 23 (76.7%) of the water sources. No association was found between depth of the water and sample positivity, nor was there any association between the type of water used by the population and sample positivity. An association between viable M. leprae and temperature and pH was found. Georeferencing showed a relation between the residences of leprosy cases and water source containing the bacterium. MAIN CONCLUSIONS The finding of viable M. leprae in natural water sources associated with human contact suggests that the environment plays an important role in maintaining endemic leprosy in the study region.

Presence of Mycobacterium leprae genotype 4 in environmental waters in Northeast Brazil

Abstract INTRODUCTION: This study quantified Mycobacterium leprae bacilli in environmental water samples from five municipalities in the State of Ceará by quantitative polymerase chain reaction (qPCR) and compared the identified genotypes with those obtained from leprosy patient biopsies. METHODS: We collected five replicas from each of the 30 selected reservoirs and skin lesion biopsies from 25 new leprosy cases treated at a reference center in Fortaleza, Ceará from 2010 to 2013. The 16S rRNA gene region of M. leprae was amplified by qPCR and a standard curve was created with the pIDTBlue 16SrRNAMlep plasmid. The Juazeiro do Norte water samples and the biopsies were genotyped (single nucleotide polymorphism [SNP] 1 to 4) and the SNP 4 genotypes were subtyped. RESULTS: Of the 149 water samples analyzed, 54.4% were positive for the M. leprae DNA. The M. leprae bacilli copy number ranged from 1.42 × 10 -1 to 1.44 × 10 + 2 . Most biopsies showed SNP type 4 (64%), while all samples from Juazeiro do Norte were SNP type 4, with subtype 4-N appearing at the highest frequency. CONCLUSIONS: We suggest that environmental waters containing M. leprae bacilli play an important role in disease transmission, justifying PGL-1 seropositivity in individuals living in areas where there is no reported case, and in leprosy cases individuals who report no previous contact with other case. Therefore, further investigation is needed to clarify disease transmission in this region and to explore the role of the environment. We also suggest that in this area surveillance for leprosy cases should be intensified.

qPCR detection of Mycobacterium leprae in biopsies and slit skin smear of different leprosy clinical forms

Abstract Leprosy, whose etiological agent is Mycobacterium leprae, is a chronic infectious disease that mainly affects the skin and peripheral nervous system. The diagnosis of leprosy is based on clinical evaluation, whereas histopathological analysis and bacilloscopy are complementary diagnostic tools. Quantitative PCR (qPCR), a current useful tool for diagnosis of infectious diseases, has been used to detect several pathogens including Mycobacterium leprae. The validation of this technique in a robust set of samples comprising the different clinical forms of leprosy is still necessary. Thus, in this study samples from 126 skin biopsies (collected from patients on all clinical forms and reactional states of leprosy) and 25 slit skin smear of leprosy patients were comparatively analyzed by qPCR (performed with primers for the RLEP region of M. leprae DNA) and routine bacilloscopy performed in histological sections or in slit skin smear. Considering clinical diagnostic as the gold standard, 84.9% of the leprosy patients were qPCR positive in skin biopsies, resulting in 84.92% sensitivity, with 84.92 and 61.22% positive (PPV) and negative (NPV) predictive values, respectively. Concerning bacilloscopy of histological sections (BI/H), the sensitivity was 80.15% and the PPV and NPV were 80.15 and 44.44%, respectively. The concordance between qPCR and BI/H was 87.30%. Regarding the slit skin smear, 84% of the samples tested positive in the qPCR. Additionally, qPCR showed 100% specificity, since all samples from different mycobacteria, from healthy individuals, and from other granulomatous diseases presented negative results. In conclusion, the qPCR technique for detection of M. leprae using RLEP primers proved to be specific and sensitive, and qPCR can be used as a complementary test to diagnose leprosy irrespective of the clinical form of disease.

Widespread nasal carriage of Mycobacterium lepraeamong a healthy population in a hyperendemic region of northeastern Brazil

A case-control study was conducted to determine the presence ofMycobacterium lepraeDNA in nasal secretions of leprosy cases and nonleprosy individuals in Fortaleza, Brazil. It included 185 cases identified by physicians at the Dona Libânia National Reference Centre for Sanitary Dermatology (CDERM). A control group (Co) (n = 136) was identified among individuals from CDERM not diagnosed as leprosy cases. To augment the spatial analysis of M. leprae specific repetitive element (RLEP) positive prevalence, an external group (EG) (n = 121), a convenience sample of healthy students, were included. Polymerase chain reaction for the RLEP sequence was conducted for all participants. Prevalence of RLEP positivity for cases and Co were 69.2% and 66.9%, respectively, significantly higher than for EG (28.1%), and reported elsewhere. Male sex, belonging to a lower socioeconomic status (D/E), history of a previous contact with a case and being older, were associated with being a leprosy case. Our geographical analysis demonstrated that the bacillus is widespread among the healthy population, with clusters of RLEP positive multibacillary cases concentrated in distinct areas of the city. Our results suggest that in endemic areas, as in Fortaleza, surveillance for both nonhousehold leprosy contacts and members of the general population living in cluster areas should be implemented.

Diagnostic challenges of single plaque-like lesion paucibacillary leprosy

The diagnosis of single-lesion paucibacillary leprosy remains a challenge. Reviews by expert dermatopathologists and quantitative polymerase chain reaction (qPCR) results obtained from 66 single-plaque biopsy samples were compared. Histological findings were graded as high (HP), medium (MP) or low (LP) probability of leprosy or other dermatopathy (OD). Mycobacterium leprae-specific genes were detected using qPCR. The biopsies of 47 out of 57 clinically diagnosed patients who received multidrug therapy were classified as HP/MP, eight of which were qPCR negative. In the LP/OD (n = 19), two out of eight untreated patients showed positive qPCR results. In the absence of typical histopathological features, qPCR may be utilised to aid in final patient diagnosis, thus reducing overtreatment and delay in diagnosis.

Is drug-resistant Mycobacterium leprae a real cause for concern?: First approach to molecular monitoring of multibacillary Colombian patients with and without previous leprosy treatment / ¿Es la resistencia de Mycobacterium leprae a los medicamentos un verdadero motivo de preocupación?: Primera aproximación a la vigilancia molecular de pacientes colombianos multibacilares con tratamiento previo para lepra y sin él

Introduction: There is no information in Colombia on Mycobacterium leprae primary and secondary drug resistance in regards to the WHO-multidrug therapy regime. On the other hand, public health authorities around the world have issued various recommendations, one of which prompts for the immediate organization of resistance surveillance through simple molecular methods. Objective: To determine the prevalence of Mycobacterium leprae drug resistance to rifampicin, ofloxacin and dapsone in untreated and previously treated patients at the Centro Dermatológico Federico Lleras Acosta during the 1985-2004 period. Materials and methods: We conducted a retrospective study which included multibacillary patient biopsies through elective sampling: 381 of them from new patients and 560 from previously treated patients. Using a microtome, we obtained six slides from each skin biopsy preserved in paraffin, and we extracted M. leprae DNA. We amplified three molecular targets through PCR and obtained the patterns of drug resistance to dapsone, rifampicin and ofloxacin by reverse hybridization. Finally, we collected epidemiological, clinical and demographical data for analyses. Results: From 941 samples under study, 4.14% of them were resistant to one or more drugs, and 5.77 and 3.04% had resistant genotypes in new and previously treated patients, respectively. Total resistance for each drug was 0.43% for dapsone, 3.19% for rifampicin and 1.17% for ofloxacin. We found statistically significant differences for rifampicin and for the total population when comparing the results from untreated versus previously treated patients. Two thirds of the resistant samples were resistant to rifampicin alone or combined. Conclusions: The standard multidrug therapy schemes continue being effective for leprosy cases; however, it is necessary to guarantee adherence and regularity. Surveillance to drug resistance in new and previously treated leprosy cases should be established.

Introducción. Colombia no dispone de información sobre farmacorresistencia primaria y secundaria de Mycobacterium leprae al esquema de terapia múltiple de la Organización Mundial de la Salud (OMS) y las autoridades de salud pública del mundo han emitido varias recomendaciones, entre las cuales está organizar de inmediato la vigilancia a la resistencia empleando métodos moleculares simples. Objetivo. Determinar la prevalencia de la resistencia de M. leprae a rifampicina, ofloxacina y dapsona en pacientes del Centro Dermatológico Federico Lleras Acosta con tratamiento previo y sin él durante el período de 1985 a 2004. Materiales y métodos. Se realizó un estudio retrospectivo. Mediante muestreo electivo se incluyeron biopsias de pacientes multibacilares: 381 de pacientes nuevos y 560 de pacientes previamente tratados. Se obtuvieron con micrótomo seis cortes de cada biopsia de piel incluida en parafina, y se realizó la extracción de ADN de M. leprae. Se llevó a cabo la amplificación de tres blancos moleculares mediante PCR y se obtuvieron los patrones de resistencia a los medicamentos dapsona, rifampicina y ofloxacina por hibridación inversa. Se recolectaron datos epidemiológicos, clínicos y demográficos para llevar a cabo los análisis. Resultados. De las 941 muestras estudiadas, 4,14 % era resistente a uno o más fármacos, y se detectaron 5,77 y 3,04 % con genotipos resistentes en pacientes nuevos y previamente tratados, respectivamente. La resistencia total para cada fármaco fue de 0,43 % a dapsona, 3,19 % a rifampicina y 1,17 % a ofloxacina. Se encontró una diferencia estadísticamente significativa para rifampicina y para la población total al comparar los resultados de los pacientes no tratados con los de los pacientes tratados previamente. Dos tercios de las muestras resistentes lo fueron a rifampicina sola o combinada. Conclusiones. Los esquemas de terapia múltiple estándar siguen siendo efectivos para los casos de lepra; sin embargo, es necesario garantizar el cumplimiento y la regularidad y establecer la vigilancia de la farmacorresistencia en pacientes nuevos y previamente tratados.

Leprosy: review of the epidemiological, clinical, and etiopathogenic aspects - Part 1

Leprosy is caused by Mycobacterium leprae and has been known since biblical times. It is still endemic in many regions of the world and a public health problem in Brazil. The prevalence rate in 2011 reached 1.54 cases per 10,000 inhabitants in Brazil. The mechanism of transmission of leprosy consists of prolonged close contact between susceptible and genetically predisposed individuals and untreated multibacillary patients. Transmission occurs through inhalation of bacilli present in upper airway secretion. The nasal mucosa is the main entry or exit route of M. leprae. The deeper understanding of the structural and biological characteristics of M. leprae, the sequencing of its genome, along with the advances in understanding the mechanisms of host immune response against the bacilli, dependent on genetic susceptibility, have contributed to the understanding of the pathogenesis, variations in the clinical characteristics, and progression of the disease. This article aims to update dermatologist on epidemiological, clinical, and etiopathogenic leprosy aspects.

Use of fine needle aspirate from peripheral nerves of pure-neural leprosy for cytology and PCR to confirm the diagnosis: A pilot study.

Background: The diagnosis of pure neural leprosy (PNL) remained subjective because of over-dependence of clinical expertise and a lack of simple yet reliable diagnostic tool. The criteria for diagnosis, proposed by Jardim et al., are not routinely done by clinicians in developing country as it involves invasive nerve biopsy and sophisticated anti-PGL-1 detection. We conducted a study using fi ne needle aspiration cytology (FNAC) coupled with Ziehl Neelsen staining (ZN staining) and Multiplex- Polymerase Chain Reaction (PCR) specifi c for M. leprae for an objective diagnosis of pure neural leprosy (PNL), which may be simpler and yet reliable. Aim: The aim of the study is to couple FNAC with ZN staining and multiplex PCR to diagnose pure neural leprosy patients rapidly, in simpler and yet reliable way. Methods: Thirteen patients of PNL as diagnosed by two independent consultants were included as case, and 5 patients other than PNL were taken as control in the study. Fine needle aspiration was done on the affected nerve, and aspirates were evaluated for cytology, ZN staining and multiplex- PCR. Results: Out of the 13 cases where fi ne needle aspiration was done, M. leprae could be elicited in the nerve tissue aspirates in 5 cases (38.4%) with the help of conventional acid-fast staining and 11 cases (84.6%) with the help of multiplex PCR. On cytological examination of the aspirates, only 3 (23%) cases showed specifi c epithelioid cells, whereas 8 (61.5%) cases showed non-specifi c infl ammation, and 2 (15.3%) cases had no infl ammatory cells. Conclusion: Our study demonstrates that in the fi eld of laboratory diagnosis of PNL cases, FNAC in combination with ZN staining for acid-fast bacilli (AFB) and Multiplex-PCR can provide a rapid and defi nitive diagnosis for the majority of PNL cases. FNAC is a less-invasive, outdoor-based and simpler technique than invasive nerve biopsy procedure. Thus, this study may enlighten the future path for easy and reliable diagnosis of PNL.

New insights in the pathogenesis of type 1 and type 2 lepra reaction.

In the current scenario of leprosy elimination, lepra reactions (LRs) remain a major persistent problem. Type 1 LR (T1LR) and type 2 LR (T2LR) are the major causes of nerve damage and permanent disabilities. The immunopathogenesis of LR have recently become an important fi eld of research, since it may provide the relevant targets for the early detection and control of these episodes. Presently, there are no uniformly acceptable laboratory markers for LR. Genetic and serum markers in human host may predict susceptibility to reactions as well as progression of nerve damage in leprosy. Therefore, a deeper understanding of the molecular mechanisms involved in LR may provide a rational strategy for early diagnosis and prevention of the catastrophic consequences of LR.

Detection of Mycobacterium leprae in saliva and the evaluation of oral sensitivity in patients with leprosy

The aim of this study was to investigate sensitivity disorders in the oral cavity related to the presence of Mycobacterium leprae in the saliva of treatment-naïve patients with leprosy in the state of Amazonas, Brazil. A cross-sectional study was conducted involving 45 subjects with leprosy. The subjects were interviewed to evaluate the sensitivity of the oral cavity. For the detection of M. leprae, saliva and slit-skin smear samples were collected. The samples were analysed using a bacteriological index (BI) protocol and the real-time quantitative polymerase chain reaction (qPCR). The results indicated that 15 of the 45 (33.3%) subjects with leprosy showed decreased oral sensitivity, which confirmed the importance of the oral cavity sensitivity evaluation. There was not a direct relationship between the presence of M. leprae in saliva and changes in oral sensitivity. Positive saliva qPCR results from six (31.6%) of 19 paucibacillary (PB) patients suggested the possibility of a new site for sample collection. Positive results using these diagnostic techniques (BI, slit-skin smear and saliva qPCR) increased to 55.5%, thus opening the possibility of combining these different techniques to increase the rate of positive diagnoses, especially in PB patients.

Genotyping of Mycobacterium leprae present on Ziehl-Neelsen-stained microscopic slides and in skin biopsy samples from leprosy patients in different geographic regions of Brazil

We analysed 16 variable number tandem repeats (VNTR) and three single-nucleotide polymorphisms (SNP) in Mycobacterium leprae present on 115 Ziehl-Neelsen (Z-N)-stained slides and in 51 skin biopsy samples derived from leprosy patients from Ceará (n = 23), Pernambuco (n = 41), Rio de Janeiro (n = 22) and Rondônia (RO) (n = 78). All skin biopsies yielded SNP-based genotypes, while 48 of the samples (94.1%) yielded complete VNTR genotypes. We evaluated two procedures for extracting M. leprae DNA from Z-N-stained slides: the first including Chelex and the other combining proteinase and sodium dodecyl sulfate. Of the 76 samples processed using the first procedure, 30.2% were positive for 16 or 15 VNTRs, whereas of the 39 samples processed using the second procedure, 28.2% yielded genotypes defined by at least 10 VNTRs. Combined VNTR and SNP analysis revealed large variability in genotypes, but a high prevalence of SNP genotype 4 in the Northeast Region of Brazil. Our observation of two samples from RO with an identical genotype and seven groups with similar genotypes, including four derived from residents of the same state or region, suggest a tendency to form groups according to the origin of the isolates. This study demonstrates the existence of geographically related M. leprae genotypes and that Z-N-stained slides are an alternative source for M. leprae genotyping.

Unveiling healthy carriers and subclinical infections among household contacts of leprosy patients who play potential roles in the disease chain of transmission

Leprosy transmission still occurs despite the availability of highly effective treatment. The next step towards successfully eliminating leprosy is interrupting the chain of transmission of the aetiological agent, Mycobacterium leprae. In this investigation, we provide evidence that household contacts (HHCs) of leprosy patients might not only have subclinical infections, but may also be actively involved in bacilli transmission. We studied 444 patients and 1,352 contacts using anti-phenolic glycolipid-I (PGL-I) serology and quantitative polymerase chain reaction (qPCR) to test for M. leprae DNA in nasal swabs. We classified the patients according to the clinical form of their disease and the contacts according to the characteristics of their index case. Overall, 63.3% and 34.2% of patients tested positive by ELISA and PCR, respectively. For HHCs, 13.3% had a positive ELISA test result and 4.7% had a positive PCR test result. The presence of circulating anti-PGL-I among healthy contacts (with or without a positive PCR test result from nasal swabs) was considered to indicate a subclinical infection. DNA detected in nasal swabs also indicates the presence of bacilli at the site of transmission and bacterial entrance. We suggest that the concomitant use of both assays may allow us to detect subclinical infection in HHCs and to identify possible bacilli carriers who may transmit and disseminate disease in endemic regions. Chemoprophylaxis of these contacts is suggested.

Insights from animal models on the immunogenetics of leprosy: a review

A variety of host immunogenetic factors appear to influence both an individual's susceptibility to infection with Mycobacterium leprae and the pathologic course of the disease. Animal models can contribute to a better understanding of the role of immunogenetics in leprosy through comparative studies helping to confirm the significance of various identified traits and in deciphering the underlying mechanisms that may be involved in expression of different disease related phenotypes. Genetically engineered mice, with specific immune or biochemical pathway defects, are particularly useful for investigating granuloma formation and resistance to infection and are shedding new light on borderline areas of the leprosy spectrum which are clinically unstable and have a tendency toward immunological complications. Though armadillos are less developed in this regard, these animals are the only other natural hosts of M. leprae and they present a unique opportunity for comparative study of genetic markers and mechanisms associable with disease susceptibility or resistance, especially the neurological aspects of leprosy. In this paper, we review the recent contributions of genetically engineered mice and armadillos toward our understanding of the immunogenetics of leprosy.

Mycobacterium leprae in six-banded (Euphractus sexcinctus) and nine-banded armadillos (Dasypus novemcinctus) in Northeast Brazil

Human beings are the main reservoir of the causative agent of leprosy, Mycobacterium leprae. In the Americas, nine-banded armadillos (Dasypus novemcinctus) also act as a reservoir for the bacillus. In the state of Ceará (CE), which is located in Northeast Brazil and is an endemic area of leprosy, there are several species of armadillos, including D. novemcinctus and Euphractus sexcinctus (six-banded armadillo). Contact between humans and armadillos occur mainly through hunting, cleaning, preparing, cooking and eating. This study identified M. leprae DNA in the two main species of armadillos found in Northeast Brazil. A total of 29 wild armadillos (27 D. novemcinctus and 2 E. sexcinctus) were captured in different environments of CE countryside. Samples from the ear, nose, liver and spleen from each of these animals were tested by a nested M. leprae-specific repetitive element polymerase chain reaction assay. The samples that tested positive were confirmed by DNA sequencing. M. leprae was detected in 21% (6/29) of the animals, including five D. novemcinctus and one E. sexcinctus. This is the first Brazilian study to identify the presence of a biomarker of M. leprae in wild armadillos (D. novemcinctus and E. sexcinctus) in a leprosy hyperendemic area where there is continuous contact between humans and armadillos.

Use of the polymerase chain reaction to detect Mycobacterium leprae in urine

Leprosy is an infectious disease caused by Mycobacterium leprae. The polymerase chain reaction (PCR) has been applied to detect M. leprae in different clinical samples and urine seems to be attractive for this purpose. PCR was used to improve the sensitivity for diagnosing leprosy by amplifying a 151-bp PCR fragment of the M. leprae pra gene (PCR-Pra) in urine samples. Seventy-three leprosy patients (39 males and 34 females, 14 to 78 years old) were selected for leprosy diagnosis at a reference laboratory in Maringá, PR, Brazil. Of these, 36 were under anti-leprosy multidrug therapy with dapsone and rifampicin for tuberculoid (TT) and dapsone, rifampicin and clofazimine for borderline (BB) and lepromatous (LL) forms. The control group contained 50 healthy individuals without any clinical history of leprosy. DNA isolated from leprosy patients’ urine samples was successfully amplified by PCR-Pra in 46.6 percent (34/73) of the cases. The positivity of PCR-Pra for patients with the TT form was 75 percent for both patients under treatment and non-treated patients (P = 0.1306). In patients with the LL form, PCR-Pra positivity was 52 and 30 percent for patients under treatment and non-treated patients, respectively (P = 0.2386). PCR-Pra showed a statistically significant difference in detecting M. leprae between the TT and LL forms of leprosy in patients under treatment (P = 0.0033). Although the current study showed that the proposed PCR-Pra has some limitations in the detection of M. leprae, this method has the potential to be a useful tool for leprosy diagnosis mainly in TT leprosy where the AFB slit-skin smear is always negative.

Comparação entre microssatélites e o gene Ml MntH como alvos para a identificação do Mycobacterium leprae por PCR na hanseníase / Comparison between microsatellites and Ml MntH gene as targets to identify Mycobacterium leprae by PCR in leprosy

FUNDAMENTOS: PCR tem sido frequentemente utilizada no diagnóstico molecular da hanseníase. OBJETIVOS: comparar os resultados da PCR com 4 pares de primers específicos para Mycobacterium leprae, bem como os resultados da PCR à classificação operacional, segundo a OMS, de multibacilar (MB) e paucibacilar (PB) da hanseníase. MÉTODO: Vinte e oito amostras de DNA, extraído de biópsias congeladas de pele e de imprint de biópsias em papel de filtro de 23 pacientes (14 MB e 9 PB), foram utilizadas na PCR com primers que amplificam 131pb, 151pb e 168pb de regiões de microssatélites, e um fragmento de 336pb do gene Ml MntH (ML2098) do bacilo. RESULTADOS: O bacilo pôde ser detectado em 22 (78,6 por cento) das 28 amostras. Nove (45 por cento) das 20 amostras de biópsia e 6 (75 por cento) das 8 amostras de imprints foram positivas para TTC. Sete (35,5 por cento) amostras de biópsias e 5 (62,5 por cento) imprints foram positivos para AGT, e 11 (55 por cento) biópsias e 4 (50 por cento) imprints foram positivos para AT. Oito (38 por cento) amostras de biópsias e 5 (62,5 por cento) imprints foram positivos para o gene Ml MntH. Dentre o grupo MB, os microssatélites detectaram o bacilo em 78,5 por cento das amostras, e o gene Ml MntH, em 57,1 por cento das amostras, independentemente do material clínico. No grupo PB, 55,5 por cento das amostras foram positivas para os microssatélites, enquanto que 22,2 por cento o foram para o gene Ml MntH. CONCLUSÕES: Estes resultados mostram que, tanto as regiões específicas de microssatélites quanto o gene Ml MntH, podem representar ferramentas úteis na detecção do Ml MntH por PCR em amostras de biópsias e imprint de biópsias.

BACKGROUND: The Polymerase Chain Reaction (PCR) technique has been frequently used in the molecular diagnosis of leprosy. OBJECTIVES: To compare the results of PCR with four pairs of Mycobacterium leprae specific primers as well as to compare these results to multibacillary (MB) and paucibacillary (PB) leprosy according to the WHO operational classification. METHOD: 28 DNA samples, collected from the frozen skin biopsies and biopsy imprints on filter paper of 23 patients (14 MB and PB 9), were examined for PCR using primers which amplify 131, 151 and 168bp of specific microsatellite regions and a 336 fragment of the Ml MntH (ML2098) gene. RESULTS: M.leprae bacillus could be detected in 22 (78.6 percent) of the 28 samples. 9 (45 percent) of the 20 biopsy samples and 6 (75 percent) of the 8 imprints were positive to TTC. 7 (35.5 percent) skin biopsy specimens and 5 (62.5 percent) imprints were positive to AGT, and 11 (55 percent) biopsies and 4 (50 percent) were positive to AGT. 11 (55 percent) skin biopsies and 4 (50 percent) imprints were positive to AT. 8(38 percent) skin biopsies and 5 (62.5 percent) imprints were positive to the Ml MntH gene. In the MB group, the microsatellites detected the bacillus in 78.5 percent of the samples, and the Ml MntH gene in 57.1 percent of the samples, independent of the clinical material. In the PB group 55.5 percent of samples were positive to the microsatellite primers, while 22.2 percent were positive to the Ml MntH gene. CONCLUSIONS: These results show that both the specific regions of microsatellites, as well as the Ml MntH gene fragment can be useful tools for detecting the M. leprae DNA by PCR in frozen skin biopsy samples and filter paper biopsy imprints.

Genetic risk factors for human susceptibility to infections of relevance in dermatology / Fatores de risco genético para a suscetibilidade humana à infecções de relevância em dermatologia

BACKGROUND: In the pre-microbiological era, it was widely accepted that diseases, today known to be infectious, were hereditary. With the discovery of microorganisms and their role in the pathogenesis of several diseases, it was suggested that exposure to the pathogen was enough to explain infection. Nowadays, it is clear that infection is the result of a complex interplay between pathogen and host, therefore dependant on the genetic make-up of the two organisms. Dermatology offers several examples of infectious diseases in different stages of understanding of their molecular basis. In this review, we summarize the main advances towards dissecting the genetic component controlling human susceptibility to infectious diseases of interest in dermatology. Widely investigated diseases such as leprosy and leishmaniasis are discussed from the genetic perspective of both host and pathogen. Others, such as rare mycobacterioses, fungal infections and syphilis, are presented as good opportunities for research in the field of genetics of infection.

INTRODUÇÃO: Durante a era pré-microbiológica, era comum a visão de que doenças, hoje sabidamente infecciosas, eram hereditárias. Com a descoberta dos microorganismos e seu papel na patogênese de diversas patologias, chegou-se a propor que a exposição ao patógeno era condição suficiente para explicar infecção. Hoje, está claro que infecção é o resultado de uma complexa interação entre patógeno e hospedeiro, dependendo portanto, em última análise, do make-up genético de ambos os organismos. A dermatologia oferece diversos exemplos de doenças infecciosas em diferentes graus de entendimento de suas bases moleculares. Nesta revisão, resumimos os principais avanços na direção da dissecção do componente genético controlando suscetibilidade do ser humano a doenças infecciosas de importância na dermatologia. Doenças amplamente estudadas, como a hanseníase e a leishmaniose, são discutidas sob o ponto de vista da genética tanto do hospedeiro quanto do patógeno. Outras, como micobacterioses raras, micoses e sífilis, são apresentadas como boas oportunidades para pesquisa na área de genética de infecção.