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1.
Sci Rep ; 11(1): 9859, 2021 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-33972644

RESUMO

Leprosy, a progressive, mutilating and highly stigmatized disease caused by Mycobacterium leprae (ML), continues to prevail in the developing world. This is due to the absence of rapid, specific and sensitive diagnostic tools for its early detection since the disease gets notified only with the advent of physical scarring in patients. This study reports the development of a Loop-mediated isothermal amplification (LAMP) technique for fast, sensitive and specific amplification of 16S rRNA gene of ML DNA for early detection of leprosy in resource-limited areas. Various parameters were optimized to obtain robust and reliable amplification of ML DNA. Blind clinical validation studies were performed which showed that this technique had complete concurrence with conventional techniques. Total absence of amplification of negative control DNA confirmed the specificity of this test. Various visual detection methods viz. colorimetric, turbidity differentiation and bridge flocculation were standardized to establish easy-to-read and rapid diagnosis. This technique eliminates the lack of accuracy and sensitivity in skin smear tests in patients and the requirement for expensive lab equipments and trained technicians. The technique holds promise for further expansion and has the potential to cater to the unmet needs of society for a cheap, highly-sensitive and robust rapid diagnosis of ML.


Assuntos
DNA Bacteriano/isolamento & purificação , Hanseníase/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Mycobacterium leprae/isolamento & purificação , Técnicas de Amplificação de Ácido Nucleico/métodos , Estudos de Viabilidade , Feminino , Humanos , Hanseníase/sangue , Hanseníase/microbiologia , Masculino , Mycobacterium leprae/genética , RNA Ribossômico 16S/genética , Sensibilidade e Especificidade , Estudos de Validação como Assunto
2.
Diagn Microbiol Infect Dis ; 100(2): 115337, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33610964

RESUMO

This systematic review (number register: CRD42018112736) was performed to compare the sensitivity and specificity of leprosy diagnostic methods. The search was conducted in 3 electronic databases in January 2021. Studies evaluating leprosy diagnostic tests were included according the eligibility criteria. Meta-analysis was performed to calculate the sensibility and specificity of the groups. We included 36 studies. The test sensitivity for paucibacillary patients was 0.31 (95%CI: 0.29-0.33) and the specificity was 0.92 (95%CI: 0.92-0.93). In multibacillary patients, the sensitivity was 0.78 (95%CI: 0.77-0.80) and specificity was 0.92 (95%CI: 0.92-0.93). Comparing the sensitivity and specificity of the different techniques included, it should be noted that polymerase chain reaction (PCR) test presented the highest sensitivity for paucibacillary patients, while the western blot technique showed the highest sensitivity for multibacillary patients. However, further studies are needed to optimise the diagnosis of leprosy, requiring research with a larger number of samples and more uniform protocols.


Assuntos
Hanseníase Multibacilar/diagnóstico , Hanseníase Paucibacilar/diagnóstico , Western Blotting/métodos , Reação em Cadeia da Polimerase/métodos , Sensibilidade e Especificidade
3.
Diagn Microbiol Infect Dis ; 100(1): 115325, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33556650

RESUMO

Although multidrug therapy is considered an effective treatment for leprosy, antimicrobial resistance is a serious concern. We performed a systematic review of studies on the diagnostic accuracy and screening of tests for antimicrobial resistance in leprosy. This review was registered in PROSPERO (CRD42020177958). In April 2020, we searched for studies in the PubMed, EMBASE, Web of Science, Scopus, Scielo, and LILACS databases. A random effects regression model was used for the meta-analysis. We included 129 studies. Molecular tests for dapsone resistance had a sensitivity of 78.8% (95% confidence interval [CI] = 65.6-87.9) and a specificity of 97.0% (95% CI = 94.0-98.6). Molecular tests for rifampicin resistance had a sensitivity and specificity of 88.7% (95% CI = 80.0-93.9) and 97.3% (95% CI = 94.3-98.8), respectively. Molecular tests for ofloxacin resistance had a sensitivity and specificity of 80.9% (95% CI = 60.1-92.3) and 96.1% (95% CI = 90.2-98.5), respectively. In recent decades, no increase in the resistance proportion was detected. However, the growing number of resistant cases is still a clinical concern.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana , Hanseníase , Testes de Sensibilidade Microbiana , Mycobacterium leprae/efeitos dos fármacos , DNA Bacteriano/genética , Humanos , Hanseníase/diagnóstico , Hanseníase/microbiologia , Testes de Sensibilidade Microbiana/métodos , Testes de Sensibilidade Microbiana/normas , Sensibilidade e Especificidade , Análise de Sequência de DNA
4.
Diagn Microbiol Infect Dis ; 99(2): 115232, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33130505

RESUMO

Leprosy is an infectious disease caused by Mycobacterium leprae that affects the skin and nerves. The nerve damage in leprosy may be related to alterations in transcriptional factors, such as Krox-20, Oct-6, Sox-10. Thirty skin biopsies in leprosy patients and 15 non-leprosy skin biopsies were evaluated using RT-qPCR to assess Krox-20, Oct-6, and Sox-10 and these data was related with S-100 immunohistochemistry. Changes in gene expression were observed in the skin and dermal nerves of leprosy patients in Oct-6 and Sox-10. When comparing Oct-6 with S-100 IHC as diagnostic tests for leprosy, Oct-6 showed a sensitivity of 73.3%, and specificity of 100%, while S-100 IHC showed a sensitivity of 96.6% and specificity of 100%. Our data suggest Oct-6 could be an auxiliary biomarker specific to detecting changes in dermal nerves in leprosy and thus useful to health workers and pathologists with no expertise to observe nerve injuries in leprosy.


Assuntos
Hanseníase/diagnóstico , Fator 6 de Transcrição de Octâmero/genética , Adulto , Idoso , Anticorpos Antibacterianos/sangue , Carga Bacteriana , Biomarcadores/metabolismo , Biópsia , Estudos Transversais , Proteína 2 de Resposta de Crescimento Precoce/genética , Feminino , Humanos , Imunoglobulina G/sangue , Imuno-Histoquímica , Hanseníase/genética , Hanseníase/metabolismo , Hanseníase/patologia , Masculino , Pessoa de Meia-Idade , Mycobacterium leprae/imunologia , Proteínas S100/metabolismo , Fatores de Transcrição SOXE/genética , Sensibilidade e Especificidade , Pele/inervação , Pele/metabolismo , Pele/patologia , Transcrição Genética
5.
s.l; s.l; 2021. 10 p. ilus, tab.
Não convencional em Inglês | SES-SP, CONASS, HANSEN, SESSP-ILSLPROD, SES-SP, SESSP-ILSLACERVO, SES-SP | ID: biblio-1150427

RESUMO

Although multidrug therapy is considered an effective treatment for leprosy, antimicrobial resistance is a serious concern. We performed a systematic review of studies on the diagnostic accuracy and screening of tests for antimicrobial resistance in leprosy. This review was registered in PROSPERO (CRD42020177958). In April 2020, we searched for studies in the PubMed, EMBASE, Web of Science, Scopus, Scielo, and LILACS databases. A random effects regression model was used for the meta-analysis. We included 129 studies. Molecular tests for dapsone resistance had a sensitivity of 78.8% (95% confidence interval [CI] = 65.6−87.9) and a specificity of 97.0% (95% CI = 94.0−98.6). Molecular tests for rifampicin resistance had a sensitivity and specificity of 88.7% (95% CI = 80.0−93.9) and 97.3% (95% CI = 94.3−98.8), respectively. Molecular tests for ofloxacin resistance had a sensitivity and specificity of 80.9% (95% CI = 60.1−92.3) and 96.1% (95% CI = 90.2−98.5), respectively. In recent decades, no increase in the resistance proportion was detected. However, the growing number of resistant cases is still a clinical concern(AU).


Assuntos
Farmacorresistência Bacteriana , Hanseníase/terapia , Rifampina/uso terapêutico , Ofloxacino/uso terapêutico , Programas de Rastreamento , Sensibilidade e Especificidade , Análise de Sequência de DNA , Dapsona/uso terapêutico
6.
Rev Soc Bras Med Trop ; 53: e20200197, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33263683

RESUMO

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.


Assuntos
Hanseníase , Mycobacterium leprae , Ensaio de Imunoadsorção Enzimática , Humanos , Hanseníase/diagnóstico , Mycobacterium leprae/genética , Reação em Cadeia da Polimerase , Sensibilidade e Especificidade , Testes Sorológicos
7.
Indian J Med Res ; 152(4): 378-385, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-33380702

RESUMO

Background & objectives: : Early case detection is essential to interrupt transmission and to prevent further spread of tuberculosis (TB) in high endemic settings. Nucleic acid amplification tests (NAATs) with visual read-outs are ideal as point-of-care tests. Truenat™ MTB is an indigenous chip-based NAAT for detection of Mycobacterium tuberculosis, which involves extraction of DNA and real-time polymerase chain reaction (PCR) using portable, automated, battery-operated instruments. The current multicentric study was aimed to evaluate Truenat for detection of MTB in sputum samples obtained from patients with presumptive pulmonary TB with reference to culture as gold standard and Xpert as a comparator. Methods: : The study was conducted at four sites, namely ICMR-National Institute for Research in Tuberculosis, Chennai; All India Institute of Medical Sciences, New Delhi; ICMR-National JALMA Institute for Leprosy and Other Mycobacterial Diseases, Agra; and National Institute of TB and Respiratory Diseases, New Delhi. Patients suspected to have TB were screened for eligibility. Two sputum samples were collected from each patient. Tests included smear, Xpert and Truenat directly from the sputum sample and culture by Lowenstein-Jensen (L-J) medium and MGIT960 from decontaminated pellets. Sample used for Truenat assay was coded. Resolution of Truenat false positives was done using an in-house PCR with TRC4 primers. Results: : The study enrolled 2419 presumptive TB patients after screening 2465 patients, and 3541 sputum samples were collected from the enrolled patients. Results of 2623 samples were available for analysis. Truenat showed a positivity rate of 48.5 per cent as compared to 37.0 per cent by Xpert. The sensitivities of Truenat and Xpert were was 88.3 and 79.7 per cent, respectively in comparison with culture. Interpretation & conclusions: : Truenat MTB identified more positives among culture-confirmed samples than Xpert and had higher sensitivity. In addition, other advantageous operational features of Truenat MTB were identified which would be useful in field settings.


Assuntos
Mycobacterium tuberculosis , Tuberculose Pulmonar , Humanos , Índia , Mycobacterium tuberculosis/genética , Padrões de Referência , Sensibilidade e Especificidade , Escarro , Tuberculose Pulmonar/diagnóstico
8.
Artigo em Inglês | MEDLINE | ID: mdl-33037158

RESUMO

Background: The biophysical and ultrasonographic properties of the skin change in papulosquamous diseases. Aims: : To identify biophysical and ultrasonographic properties for the differentiation of five main groups of papulosquamous skin diseases. Methods: Fifteen biophysical and ultrasonographic parameters were measured by multiprobe adapter system and high-frequency ultrasonography in active lesions and normal control skin in patients with chronic eczema, psoriasis, lichen planus, pityriasis rosea and parapsoriasis/mycosis fungoides. Using histological diagnosis as a gold standard, a decision tree analysis was performed based on the mean percentage changes of these parameters [(lesion-control/control) ×100] for differentiation of the diseases. Results: The accuracy of the decision tree model for differentiation of five diseases was 67% which developed based on changes in stratum corneum hydration, epidermal thickness, skin pH, melanin index, R0 (reciprocal of firmness) and erythema. Among the flowcharts for pairs of diseases, three models for differentiation had high accuracy (> 95%): those of psoriasis from lichen planus, pityriasis rosea, and parapsoriasis/mycosis fungoides. Limitations: Validation studies on a larger sample size in situations where the diagnosis is unclear are needed to confirm the accuracy and applicability of decision trees. Conclusion: Skin biophysical and ultrasonographic properties may help in the differentiation of papulosquamous diseases as simple and non-invasive tools.


Assuntos
Árvores de Decisões , Dermatopatias Papuloescamosas/diagnóstico por imagem , Dermatopatias Papuloescamosas/patologia , Adulto , Biometria , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Sensibilidade e Especificidade , Dermatopatias Papuloescamosas/fisiopatologia , Fenômenos Fisiológicos da Pele , Ultrassonografia , Adulto Jovem
9.
Int J Mycobacteriol ; 9(1): 18-23, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32474483

RESUMO

Background: Leprosy is a contagious disease and was eliminated globally in 2002. Since then, new cases were continuously detected from different parts of the world. Untreated leprosy cases shed millions of bacteria and are the main cause of dissemination of the disease. Currently, leprosy is detected by acid-fast bacilli (AFB) microscopy and has a low sensitivity ranging from 10% to 50%. The correlation between clinical findings and microscopy is unable to provide a conclusive case detection. Thus, in the present study, we compared to molecular methods, namely RLEP-polymerase chain reaction (RLEP-PCR) and inter-simple sequence repeat-PCR (ISSR-PCR) taking AFB microscopy as a gold standard for the detection of leprosy. Methods: A total of 168 clinically diagnosed leprosy patients were recruited in this study including 58 multibacillary and 110 paucibacillary patients. Slit-skin smear samples were taken for both microscopy and molecular study. Primers for RLEP-PCR were taken from the previous reports. The primers for ISSR-PCR were designed by screening the whole genome of Mycobacterium leprae TN strain (GenBank accession AL450380) for the presence of simple sequence repeats. One primer (TA)8CA3was synthesized and used for molecular amplification of ISSR-PCR. Results: We found that the efficacy of the AFB microscopy was 24.40%, whereas the efficacy of RLEP-PCR and ISSR-PCR was 63.09% and 73.21% (P = 0.000, 0.000, and 0.469), respectively. The area under the curve of receiver operating characteristic curve for the comparison of three diagnostic methods was 0.845. An enhancement of 48.81% in the case detection rate by ISSR-PCR over AFB microscopy and 10.12% over RLEP-PCR was also found. Our study clearly reveals that ISSR-PCR is a better tool for diagnosis of leprosy than AFB microscopy and RLEP-PCR. Interestingly, both the PCR techniques RLEP-PCR and ISSR-PCR are able to detect samples which were negative for AFB microscopy. Conclusion: Thus, the demonstration of ISSR-PCR in SSS samples can provide a better sensitive and confirmative tool for early diagnosis of leprosy.


Assuntos
Hanseníase/diagnóstico , Técnicas de Diagnóstico Molecular/normas , Reação em Cadeia da Polimerase em Tempo Real/normas , Estudos Transversais , Primers do DNA , DNA Bacteriano/genética , Genoma Bacteriano , Humanos , Índia , Hanseníase/microbiologia , Técnicas de Diagnóstico Molecular/métodos , Mycobacterium leprae/genética , Estudos Prospectivos , Curva ROC , Reação em Cadeia da Polimerase em Tempo Real/métodos , Sensibilidade e Especificidade , Pele/microbiologia
10.
PLoS Negl Trop Dis ; 14(5): e0008325, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32453754

RESUMO

Leprosy urgently needs a precise and early diagnostic tool. The sensitivity of the direct (bacilli staining, Mycobacterium leprae DNA) and indirect (antibody levels, T cell assays) diagnostics methods vary based on the clinical form. Recently, PCR-based M. leprae DNA detection has been shown to differentially diagnose leprosy from other dermatological conditions. However, accuracy can still be improved, especially for use with less invasive clinical samples. We tested different commercial DNA extraction kits: DNeasy Blood & Tissue, QIAamp DNA Microbiome, Maxwell 16 DNA Purification, PowerSoil DNA Isolation; as well as in-house phenol-chloroform and Trizol/FastPrep methods. Extraction was performed on M. leprae-infected mouse footpads and different clinical samples of leprosy patients (skin biopsies and scrapings, lesion, oral and nasal swabs, body hair, blood on FTA cards, peripheral whole blood). We observed that the Microbiome kit was able to enrich for mycobacterial DNA, most likely due the enzymatic digestion cocktail along with mechanical disruption involved in this method. Consequently, we had a significant increase in sensitivity in skin biopsies from paucibacillary leprosy patients using a duplex qPCR targeting 16S rRNA (M. leprae) and 18S rRNA (mammal) in the StepOnePlus system. Our data showed that the presence of M. leprae DNA was best detected in skin biopsies and skin scrapings, independent of the extraction method or the clinical form. For multibacillary patients, detection of M. leprae DNA in nasal swabs indicates the possibility of having a much less invasive sample that can be used for the purposes of DNA sequencing for relapse analysis and drug resistance monitoring. Overall, DNA extracted with the Microbiome kit presented the best bacilli detection rate for paucibacillary cases, indicating that investments in extraction methods with mechanical and DNA digestion should be made.


Assuntos
DNA Bacteriano/isolamento & purificação , Mycobacterium leprae/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Animais , DNA Bacteriano/genética , Humanos , Camundongos , Mycobacterium leprae/genética , RNA Bacteriano/genética , RNA Ribossômico 16S/genética , Sensibilidade e Especificidade
11.
BMC Microbiol ; 20(1): 90, 2020 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-32293265

RESUMO

BACKGROUND: Visceral leishmaniasis in Ethiopia is a re-emerging threat to public health, with increased geographical distribution and number of cases. It is a fatal disease without early diagnosis and treatment; thus, the availability of affordable diagnostic tools is crucial. However, due to delays caused by import regulations, procurement and late delivery of imported test kits, accessibility remains a problem in the control program. Therefore, we aimed to produce and evaluate the performance of an in-house liquid (AQ) direct agglutination test (DAT) antigen. RESULT: The AQ-DAT was produced at the Armauer Hansen Research Institute, using Leishmania donovani strain (MHOM/ET/67/L82). Sera from 272 participants; 110 microscopically confirmed cases of VL, 76 apparently healthy and 86 patients who had infectious disease other than VL were tested with AQ-DAT, and standard kits: Freeze-dried DAT (FD-DAT) and rK39. Taking microscopy as a gold standard; the sensitivity and specificity of the AQ-DAT were 97.3 and 98.8%, respectively. It had high degrees of agreement (k > 0.8), with a significant (P < 0.05) correlation compared to microscopy, FD-DAT, and rK39. CONCLUSION: Although further standardization is required, the in-house AQ-DAT could improve diagnostic accessibility, minimize intermittent stock outs and strengthen the national VL control program.


Assuntos
Testes de Aglutinação/métodos , Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/imunologia , Leishmania donovani/imunologia , Leishmaniose Visceral/diagnóstico , Adolescente , Adulto , Testes Diagnósticos de Rotina , Doenças Endêmicas , Etiópia/epidemiologia , Feminino , Humanos , Leishmaniose Visceral/imunologia , Masculino , Pessoa de Meia-Idade , Sensibilidade e Especificidade , Adulto Jovem
12.
J Appl Microbiol ; 128(6): 1814-1819, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31981442

RESUMO

AIMS: Diagnosis of leprosy, a chronic infection caused by Mycobacterium leprae, predominantly depends on clinical manifestations and histopathological analysis, hampering rapid and accurate diagnostics. Our aim was to increase accuracy of leprosy diagnosis by improving M. leprae's DNA detection based on polymerase chain reaction (PCR) technique using new specific primers for the RLEP repetitive sequence. METHODS AND RESULTS: The specific target region, RLEP, of M. leprae's genome was selected based on comparative genomics. After confirming the specificity of this region, using blastn analysis, primers were designed and tested for their in silico specificity. To evaluate the specificity and sensitivity of these primers in vitro, 184 blood samples from patients were used in qPCR. The new primer pair LYON1/LYON2 produced 91% positive samples, whereas the current primer pair LP1/LP2 produced 46%. Specificity and DNA detection limit test were carried out to compare the efficiency of the developed primer pair. The LYON1/LYON2 primer showed 100% specificity, whereas LP1/LP2 showed 64%. The DNA detection limit of LYON1/LYON2 was 10 copies of bacterial genomes per millilitre, whereas LP1/LP2 was 1000 copies of bacterial genomes per millilitre. CONCLUSIONS: In conclusion, the developed LYON1/LYON2 primer pair presented to be a specific and sensitive new molecular marker for the diagnosis of leprosy. SIGNIFICANCE AND IMPACT OF THE STUDY: The development of a specific primer pair for the detection of the M. leprae genome through qPCR technique contributes to a fast, sensitive and specific diagnosis, which is essential to prevent spreading and progression of this disease.


Assuntos
Hanseníase/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Mycobacterium leprae/isolamento & purificação , DNA Bacteriano/genética , Feminino , Genoma Bacteriano/genética , Humanos , Sequências Repetitivas Dispersas/genética , Hanseníase/sangue , Hanseníase/microbiologia , Mycobacterium leprae/genética , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e Especificidade
13.
Rev. Soc. Bras. Med. Trop ; 53: e20200197, 2020.
Artigo em Inglês | LILACS, ColecionaSUS, SES-SP | ID: biblio-1143857

RESUMO

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.


Assuntos
Humanos , Hanseníase/diagnóstico , Mycobacterium leprae/genética , Ensaio de Imunoadsorção Enzimática , Testes Sorológicos , Reação em Cadeia da Polimerase , Sensibilidade e Especificidade
14.
s.l; s.n; 2020. 15 p. ilus, graf, tab.
Não convencional em Inglês | SES-SP, CONASS, HANSEN, SESSP-ILSLPROD, SES-SP, SESSP-ILSLACERVO, SES-SP | ID: biblio-1146399

RESUMO

Leprosy is difficult to diagnose since it is caused by a bacterium that does not grow in vitro. Bacilli direct detection or the presence of specific antibodies can vary greatly depending on the clinical form. M. leprae direct DNA detection can aid clinical diagnosis, although invasive skin biopsies are still necessary to detect the pathogen or histological features consistent with leprosy. Here we show that a kit combining mechanical and chemical lysis efficiently removes host DNA and enriches for M. leprae DNA, allowing better detection of paucibacillary cases. We believe our findings can contribute to improving disease diagnosis, as well as early detection and that could help monitoring strategies(AU).


Assuntos
Humanos , Animais , Camundongos , DNA Bacteriano/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Mycobacterium leprae/isolamento & purificação , DNA Bacteriano/genética , RNA Bacteriano/genética , RNA Ribossômico 16S/genética , Sensibilidade e Especificidade , Mycobacterium leprae/genética
15.
Indian J Med Res ; 152(5): 482-489, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33707390

RESUMO

Background & objectives: There is a need for an affordable, easy, high-sensitivity test usable at the peripheral health facility for diagnosis of drug-resistant (DR) tuberculosis (TB) to interrupt disease transmission. Nucleic acid amplification tests (NAATs) for early detection of DR-TB are ideal to bring testing near to the patient. TruenatTM MTB (Mycobacterium tuberculosis) and TruenatTM MTB-RIF (rifampicin) is an indigenous chip-based real-time polymerase chain reaction (PCR) based test for detection of multidrug-resistant (MDR) TB. The test involves extraction of DNA using automated, battery operated Trueprep instrument and real-time PCR performed on the Truelab analyzer. We report here multicentric validation of Truenat MTB-RIF for detection of DR-TB in suspected DR-TB patients. Methods: Consecutive patients aged 18-65 yr, with symptoms suggestive of TB and with a history of previous treatment, reporting to the National TB Elimination Programme (NTEP) clinics under four national institutes, namely AIIMS (All India Institute of Medical Sciences, New Delhi), NITRD (National Institute of Tuberculosis and Respiratory Diseases, New Delhi), NIRT (National Institute for Research in Tuberculosis, Chennai) and ICMR-National JALMA Institute for Leprosy and other Mycobacterial Diseases, Agra, were included in the study. Two sputum samples (one spot and one morning) were collected from each patient, after obtaining informed written consent. The samples were subjected to smear, GeneXpert and MGIT 960 culture (and drug susceptibility testing to RIF) (surrogate for MDR-TB) to serve as reference tests. The samples were coded to ensure blinding and subjected to Truenat MTB-RIF. Truenat MTB-RIF Version 1.5 was used for testing 1084 samples for RIF resistance, while Version 2.0 was used to test another 1201 samples. Results: Truenat MTB-RIF Version 1.5 in comparison with comprehensive laboratory reference standards yielded sensitivity and specificity of 76.2 and 94.7 per cent, respectively for the detection of RIF resistance in 1084 samples, collected across four sites. Based on the analysis of discordant samples, Version 2.0 of Truenat was developed by the manufacturer and this was further tested on additional 1201 samples, yielding a sensitivity of 87.5 per cent and specificity of 99.5 per cent. Interpretation & conclusions: Multicentric trial of TruenatTM MTB-RIF demonstrated a great potential of this point of care NAAT for detection of MDR-TB. The test would be useful in limited resource settings and inaccessible areas without need for any additional infrastructure.


Assuntos
Mycobacterium tuberculosis , Tuberculose Resistente a Múltiplos Medicamentos , Tuberculose Pulmonar , Adolescente , Adulto , Idoso , Humanos , Índia , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Mycobacterium tuberculosis/genética , Rifampina/farmacologia , Rifampina/uso terapêutico , Sensibilidade e Especificidade , Escarro , Tuberculose Resistente a Múltiplos Medicamentos/diagnóstico , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Tuberculose Resistente a Múltiplos Medicamentos/genética , Tuberculose Pulmonar/diagnóstico , Tuberculose Pulmonar/tratamento farmacológico , Adulto Jovem
16.
PLoS Negl Trop Dis ; 13(12): e0007946, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31881061

RESUMO

BACKGROUND: Although leprosy is efficiently treated by multidrug therapy, resistance to first-line (dapsone, rifampin) and second-line (fluoroquinolones) drugs has been described worldwide. However, the characteristics of drug resistance in Southwest China remain unknown. Furthermore, the sensitivity of polymerase chain reaction (PCR)/sequencing for resistance detection is limited, especially for paucibacillary (PB) leprosy patients. The current study aimed to develop a nested PCR/sequencing and TaqMan SNP Genotyping Assay to increase the sensitivity of the method used to detect drug resistance in Mycobacterium leprae and to reveal the nature of M. leprae drug resistance in Southwest China. METHODOLOGY/PRINCIPAL FINDINGS: Seventy-six specimens, including skin biopsy (n = 64), formalin-fixed paraffin-embedded (FFPE) (n = 11) and skin-slit smear (SSS) (n = 1) samples from multibacillary (MB, n = 70) and PB (n = 6) leprosy patients from Southwest China, were included in this study. The presence of mutations in drug resistance-determining regions (DRDRs) of the rpoB, folP1, and gyrA genes, which are associated with rifampicin, dapsone, and quinolone resistance, respectively, was detected by PCR/sequencing, as recommended by the WHO, and the nested PCR and TaqMan SNP Genotyping Assay developed in this study. Mutations in the folP gene were detected in 19 (25.00%) samples, indicating dapsone-resistant M. leprae, with one (1.31%) sample showing mutations in two genes, folP and gyrA, reflecting multidrug-resistant strains to dapsone and ofloxacin. However, no rpoB mutation was detected. Compared with PCR/sequencing, nested PCR increased the sensitivity of detecting rpoB (from 51.39% to 78.94% for leprosy patients and from 0.00% to 50.00% for PB), gyrA (from 75.00% to 80.26% for leprosy patients and from 50.00% to 66.67% for PB), and folP1 (from 5.26% to 84.21% for leprosy patients and from 0.00% to 66.67% for PB). Moreover, the TaqMan SNP Genotyping Assay showed greater sensitivity for folP1 detection (from 5.26% to 78.94-86.84% for leprosy patients and from 0.00% to 33.33%-83.33% for PB patients) than the PCR/sequencing method. In addition, the latter method was able to more easily distinguish heterozygous genotypes and mutant homozygous genotypes from homozygous genotypes. CONCLUSIONS/SIGNIFICANCE: Nested PCR/sequencing and the TaqMan SNP Genotyping Assay are rapid and highly sensitive methods for detecting drug resistance in leprosy cases. The current study revealed that diamino-diphenylsulfone (DDS; also known as dapsone) resistance in M. leprae, as indicated by folP1 gene detection, is still the most concerning form of drug resistance in leprosy patients from Southwest China.


Assuntos
Farmacorresistência Bacteriana , Técnicas de Genotipagem/métodos , Hanseníase/microbiologia , Testes de Sensibilidade Microbiana/métodos , Mycobacterium leprae/efeitos dos fármacos , Mycobacterium leprae/genética , Reação em Cadeia da Polimerase/métodos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , China , Feminino , Genes Bacterianos , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Mycobacterium leprae/isolamento & purificação , Sensibilidade e Especificidade , Análise de Sequência de DNA/métodos , Adulto Jovem
17.
PLoS Negl Trop Dis ; 13(10): e0007731, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31577795

RESUMO

BACKGROUND: Detection and pathology analysis of Mycobacterium leprae using skin biopsy tissues are essential for leprosy diagnosis and monitoring response to treatment. Although formalin fixation of patient tissues may not be ideal for molecular studies, biopsy samples are the most accessible material from suspected cases. Therefore, clinical molecular laboratories must be able to utilize formalin-fixed, paraffin-embedded (FFPE) material. OBJECTIVE: To determine the best molecular method for diagnosing and monitoring leprosy in FFPE specimens, we developed a single-tube nested PCR (STNPCR) (131 bp) and SYBRGreen PCR (101 bp) assay using primers for the M. leprae-specific repetitive element (RLEP) gene and evaluated the results compared to those using previously established RLEP primers (372 bp). METHODS: FFPE biopsy samples obtained from 145 leprosy patients (during or after multidrug therapy (MDT)) and patients with 29 other confounding dermatoses were examined by the bacteria index (BI) and by simple PCR, STNPCR, and SYBRGreen PCR using primers amplifying a 372-bp, 131-bp or 101-bp fragment of RLEP, respectively. RESULTS: In leprosy patients receiving MDT, STNPCR showed a highest specificity of 100% and a positive predictive value (PPV) of 100%. For multibacillary (MB), paucibacillary (PB) and all leprosy patients, the highest sensitivities were 91.42%, 39.13%, and 67.92%, negative predictive values (NPVs) were 8.57%, 60.36%, and 32.07%, and the highest accuracies were 93.93%, 62.67%, and 74.81%, respectively, higher than the results of SYBRGreen PCR and simple PCR. For post-MDT leprosy patients, SYBRGreen PCR showed the highest sensitivity of 50.0%, highest specificity of 100%, a PPV of 100%, an NPV of 100% and the highest accuracy of 83.72% for MB patients, which were higher than those of STNPCR and simple PCR. STNPCR showed the highest sensitivity of 26.66% and 34.48%, highest specificity of 100% and 100%, a PPV of 100% and 100%, NPV of 72.50% and 60.21%, and highest accuracy of 75.00% and 67.24% for PB and leprosy patients, respectively, higher than those of SYBRGreen PCR and simple PCR. CONCLUSIONS: These findings suggest that STNPCR or SYBRGreen PCR (131-bp and 101-bp fragment amplification, respectively) for RLEP using FFPE specimens performs better as a diagnostic test and for monitoring response to MDT than does simple PCR based on 372-bp fragment amplification. Additionally, STNPCR showed increased sensitivity for PB diagnosis using FFPE specimens, which can be transferred remotely or retrieved from previous leprosy patients.


Assuntos
Formaldeído , Hanseníase/diagnóstico , Mycobacterium leprae/isolamento & purificação , Inclusão em Parafina/métodos , Reação em Cadeia da Polimerase/métodos , Biópsia/métodos , China , Primers do DNA , DNA Bacteriano/genética , Quimioterapia Combinada , Humanos , Hansenostáticos/uso terapêutico , Hanseníase/tratamento farmacológico , Mycobacterium leprae/genética , Sequências Repetitivas de Ácido Nucleico/genética , Sensibilidade e Especificidade , Pele/microbiologia
18.
BMC Infect Dis ; 19(1): 753, 2019 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-31462296

RESUMO

BACKGROUND: Leprosy continues to be a health problem in endemic areas. More than 200,000 new cases of leprosy per year suggest that transmission of the disease is still ongoing, presumably as airborne infection through nasal droplets. Late diagnosis supports continued transmission and increases the individual risk for functional disabilities. Laboratory tools are considered beneficial to facilitate early detection and clinical assessment of cases. The aim of this study was to validate molecular tools allowing detection, quantification and assessment of viability of M. leprae from nasal swab samples which are easy to obtain without the need of any invasive procedures. METHODS: Validation of two real-time PCRs detecting M. leprae DNA (RLEP qPCR) and RNA (16S rRNA RT qPCR) was conducted on "must not detect"/"must detect" samples and 160 pre-treatment nasal swab samples from 20 clinically diagnosed multibacillary (MB) leprosy patients from Togo. RESULTS: Both assays were 100% M. leprae specific and showed analytical sensitivities of three templates each. Out of 20 clinically diagnosed MB leprosy patients, 15 (75.0%) had a positive RLEP qPCR result from nasal swab samples. The 16S rRNA RT qPCR detected viable bacilli in nasal swab samples of ten out of these 15 RLEP positive patients (66.7%). CONCLUSION: The combined RLEP/16S rRNA (RT) qPCR assay provides a sensitive and specific tool to determine the bacterial load and viability of M. leprae from nasal swab samples and is applicable for early diagnosis, monitoring treatment response and investigating the role of nasal carriage of M. leprae in human-to-human transmission through aerosol infection.


Assuntos
Hanseníase/microbiologia , Mycobacterium leprae/genética , Cavidade Nasal/microbiologia , RNA Ribossômico 16S , Reação em Cadeia da Polimerase em Tempo Real/métodos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , DNA Bacteriano/genética , Humanos , Hanseníase/diagnóstico , Hanseníase Multibacilar/diagnóstico , Hanseníase Multibacilar/microbiologia , Pessoa de Meia-Idade , Mycobacterium leprae/isolamento & purificação , Mycobacterium leprae/patogenicidade , RNA Ribossômico 16S/genética , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Togo , Adulto Jovem
19.
EBioMedicine ; 47: 301-308, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31422044

RESUMO

BACKGROUND: Transmission of Mycobacterium leprae, the pathogen causing leprosy, is still persistent. To facilitate timely (prophylactic) treatment and reduce transmission it is vital to both early diagnose leprosy, and identify infected individuals lacking clinical symptoms. However, leprosy-specific biomarkers are limited, particularly for paucibacillary disease. Therefore, our objective was to identify new biomarkers for leprosy and assess their applicability in point-of-care (POC) tests. METHODS: Using multiplex-bead-arrays, 60 host-proteins were measured in a cross-sectional approach in 24-h whole blood assays (WBAs) collected in Bangladesh (79 patients; 54 contacts; 51 endemic controls (EC)). Next, 17 promising biomarkers were validated in WBAs of a separate cohort (55 patients; 27 EC). Finally, in a third cohort (36 patients; 20 EC), five candidate markers detectable in plasma were assessed for application in POC tests. FINDINGS: This study identified three new biomarkers for leprosy (ApoA1, IL-1Ra, S100A12), and confirmed five previously described biomarkers (CCL4, CRP, IL-10, IP-10, αPGL-I IgM). Overnight stimulation in WBAs provided increased specificity for leprosy and was required for IL-10, IL-1Ra and CCL4. The remaining five biomarkers were directly detectable in plasma, hence suitable for rapid POC tests. Indeed, lateral flow assays (LFAs) utilizing this five-marker profile detected both multi- and paucibacillary leprosy patients with variable immune responses. INTERPRETATION: Application of novel host-biomarker profiles to rapid, quantitative LFAs improves leprosy diagnosis and allows POC testing in low-resource settings. This platform can thus aid diagnosis and classification of leprosy and also provides a tool to detect M.leprae infection in large-scale contact screening in the field.


Assuntos
Biomarcadores , Interações Hospedeiro-Patógeno , Hanseníase/sangue , Hanseníase/diagnóstico , Testes Imediatos , Adolescente , Adulto , Biomarcadores/sangue , Criança , Pré-Escolar , Estudos Transversais , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Lactente , Recém-Nascido , Hanseníase/microbiologia , Hanseníase/transmissão , Masculino , Testes Imediatos/normas , Curva ROC , Sensibilidade e Especificidade , Fluxo de Trabalho , Adulto Jovem
20.
Emerg Microbes Infect ; 8(1): 1178-1185, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31381478

RESUMO

ABSTRACT Visceral Leishmaniasis (VL) causes high morbidity and mortality in low-to-middle-income countries worldwide. In this study, we used Laser Direct-Write (LDW) technology to develop a new Lateral Flow Device (LFD) with double-channel geometry on a low-cost paper platform as a rapid and accurate serodiagnostic assay for human VL. This Duplex VL-LFD was based on a laser-patterned microfluidic device using two recombinant Leishmania proteins, ß-tubulin and LiHyp1, as novel diagnostic antigens. The VL-LFD assay was tested with blood/serum samples from patients diagnosed with VL, Tegumentary Leishmaniasis, Leishmaniasis of unknown identity, other parasitic diseases with similar clinical symptoms, i.e. Leprosy Disease and Chagas Disease, and blood from healthy donors, and compared in parallel with commercial rK39 IT-LEISH® Kit. Clinical diagnosis and real-time Polymerase Chain Reaction assay were used as reference standards. VL-LFD Sensitivity (S ± 95% Confidence Intervals (CI)) of 90.9 (78.9-100) and Specificity (Sp ± 95% CI) of 98.7 (96.1-100) outperformed the IT-LEISH® Kit [S = 77.3 (59.8-94.8), Sp = 94.7 (89.6-99.8)]. This is the first study reporting successful development of an LFD assay using the LDW technology and the VL-LFD warrants comparative testing in larger patient cohorts and in areas with endemic VL in order to improve diagnosis and disease management.


Assuntos
Imunoensaio/métodos , Leishmaniose Visceral/diagnóstico , Testes Sorológicos/métodos , Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/imunologia , Humanos , Sensibilidade e Especificidade , Fatores de Tempo
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