Development of a rapid detection method for the macrolide resistance gene in Mycobacterium avium using the amplification refractory mutation system-loop-mediated isothermal amplification method.

Macrolide antibiotics such as clarithromycin (CLR) and azithromycin are the key drugs used in multidrug therapy for Mycobacterium avium complex (MAC) diseases. For these antibacterial drugs, drug susceptibility has been correlated with clinical response in MAC diseases. We have previously demonstrated the correlation between drug susceptibility and mutations in the 23S rRNA gene, which confers resistance to macrolides. Herein, we developed a rapid detection method using the amplification refractory mutation system (ARMS)-loop-mediated isothermal amplification (LAMP) technique to identify mutations in the 23S rRNA gene of M. avium. We examined the applicability of the ARMS-LAMP method to genomic DNA extracted from six genotypes of M. avium clinical isolates. The M. avium isolates were classified into 21 CLR-resistant and 9 CLR-susceptible strains based on the results of drug susceptibility tests; the 23S rRNA genes of these strains were sequenced and analyzed using the ARMS-LAMP method. Sequence analysis revealed that the 9 CLR-sensitive strains were wild-type strains, whereas the 21 CLR-resistant strains comprised 20 mutant-type strains and one wild-type strain. Using ARMS-LAMP, no amplification from genomic DNAs of the 10 wild-type strains was observed using the mutant-type mismatch primer sets (MTPSs); however, amplification from the 20 mutant-type strain DNAs was observed using the MTPSs. The rapid detection method developed by us integrates ARMS-LAMP with a real-time turbidimeter, which can help determine drug resistance in a few hours. In conclusion, ARMS-LAMP might be a new clinically beneficial technology for rapid detection of mutations.IMPORTANCEMultidrug therapy for pulmonary Mycobacterium avium complex disease is centered on the macrolide antibiotics clarithromycin and azithromycin, and resistance to macrolides is an important prognosticator for clinical aggravation. Therefore, it is important to develop a quick and easy method for detecting resistance to macrolides. Drug resistance is known to be correlated with mutations in macrolide resistance genes. We developed a rapid detection method using amplification refractory mutation system (ARMS)-loop-mediated isothermal amplification (LAMP) to identify a mutation in the 23S rRNA gene, which is a macrolide resistance gene. Furthermore, we examined the applicability of this method using M. avium clinical isolates. The rapid method developed by us for detection of the macrolide resistance gene by integrating ARMS-LAMP and a real-time turbidimeter can help in detection of drug resistance within a few hours. Since this method does not require expensive equipment or special techniques and shows high analytical speed, it would be very useful in clinical practice.

Active case detection and treatment of malaria in pregnancy using LAMP technology (LAMPREG): a pragmatic randomised diagnostic outcomes trial-study protocol.

INTRODUCTION: Malaria is one of the major public health problems in sub-Saharan Africa. It contributes significantly to maternal and fetal morbidity and mortality in affected countries. This study aims to evaluate the impact of enhanced case detection using molecular testing called loop-mediated isothermal amplification (LAMP) on birth outcomes in a prospective study design. METHODS AND ANALYSIS: A pragmatic randomised diagnostic outcomes trial will be conducted in several health institutes in different Ethiopian regions. Women (n=2583) in their first and second trimesters of pregnancy will be included in the study and individually randomised to the standard of care or enhanced case detection arms, and followed until delivery. Enrolment will encompass the malaria peak transmission seasons. In the standard of care arm, a venous blood sample will be collected for malaria diagnosis only in symptomatic patients. In contrast, in the intervention arm, mothers will be tested by a commercially available Conformité Européene (CE)-approved LAMP malaria test, microscopy and rapid diagnostic test for malaria regardless of their symptoms at each antenatal care visit. The primary outcome of the study is to measure birth weight. ETHICS AND DISSEMINATION: The study was approved by the following ethical research boards: Armauer Hansen Research Institute/ALERT Ethics Review Committee (FORM AF-10-015.1, Protocol number PO/05/20), the Ethiopia Ministry of Science and Higher Education National Research Ethics Review Committee (approval SRA/11.7/7115/20), the Ethiopia Food and Drug Administration (approval 02/25/33/I), UCalgary Conjoint Health Research Ethics Board (REB21-0234). The study results will be shared with the institutions and stakeholders such as the Ethiopia Ministry of Health, the Foundation for Innovative Diagnostics, WHO's Multilateral initiative on Malaria - Tropical Diseases Research (TDR-MIM), Roll Back Malaria and the Malaria in Pregnancy Consortium. The study results will also be published in peer-reviewed journals and presented at international conferences. TRIAL REGISTRATION NUMBER: NCT03754322.

Development and validation of a multiplex real-time qPCR assay using GMP-grade reagents for leprosy diagnosis.

Leprosy is a chronic dermato-neurological disease caused by Mycobacterium leprae, an obligate intracellular bacterium. Timely detection is a challenge in leprosy diagnosis, relying on clinical examination and trained health professionals. Furthermore, adequate care and transmission control depend on early and reliable pathogen detection. Here, we describe a qPCR test for routine diagnosis of leprosy-suspected patients. The reaction simultaneously amplifies two specific Mycobacterium leprae targets (16S rRNA and RLEP), and the human 18S rRNA gene as internal control. The limit of detection was estimated to be 2.29 copies of the M. leprae genome. Analytical specificity was evaluated using a panel of 20 other skin pathogenic microorganisms and Mycobacteria, showing no cross-reactivity. Intra- and inter-operator Cp variation was evaluated using dilution curves of M. leprae DNA or a synthetic gene, and no significant difference was observed between three operators in two different laboratories. The multiplex assay was evaluated using 97 patient samples with clinical and histopathological leprosy confirmation, displaying high diagnostic sensitivity (91%) and specificity (100%). Validation tests in an independent panel of 50 samples confirmed sensitivity and specificity of 97% and 98%, respectively. Importantly, assay performance remained stable for at least five months. Our results show that the newly developed multiplex qPCR effectively and specifically detects M. leprae DNA in skin samples, contributing to an efficient diagnosis that expedites the appropriate treatment.

A simple assay to quantify mycobacterial lipid antigen-specific T cell receptors in human tissues and blood.

T cell receptors (TCRs) encode the history of antigenic challenge within an individual and have the potential to serve as molecular markers of infection. In addition to peptide antigens bound to highly polymorphic MHC molecules, T cells have also evolved to recognize bacterial lipids when bound to non-polymorphic CD1 molecules. One such subset, germline-encoded, mycolyl lipid-reactive (GEM) T cells, recognizes mycobacterial cell wall lipids and expresses a conserved TCR-ɑ chain that is shared among genetically unrelated individuals. We developed a quantitative PCR assay to determine expression of the GEM TCR-ɑ nucleotide sequence in human tissues and blood. This assay was validated on plasmids and T cell lines. We tested blood samples from South African subjects with or without tuberculin reactivity or with active tuberculosis disease. We were able to detect GEM TCR-ɑ above the limit of detection in 92% of donors but found no difference in GEM TCR-ɑ expression among the three groups after normalizing for total TCR-ɑ expression. In a cohort of leprosy patients from Nepal, we successfully detected GEM TCR-ɑ in 100% of skin biopsies with histologically confirmed tuberculoid and lepromatous leprosy. Thus, GEM T cells constitute part of the T cell repertoire in the skin. However, GEM TCR-ɑ expression was not different between leprosy patients and control subjects after normalization. Further, these results reveal the feasibility of developing a simple, field deployable molecular diagnostic based on mycobacterial lipid antigen-specific TCR sequences that are readily detectable in human tissues and blood independent of genetic background.

RLEP LAMP for the laboratory confirmation of leprosy: towards a point-of-care test.

BACKGROUND: Nucleic acid-based amplification tests (NAAT), above all (q)PCR, have been applied for the detection of Mycobacterium leprae in leprosy cases and household contacts with subclinical infection. However, their application in the field poses a range of technical challenges. Loop-mediated isothermal amplification (LAMP), as a promising point-of-care NAAT does not require sophisticated laboratory equipment, is easy to perform, and is applicable for decentralized diagnosis at the primary health care level. Among a range of gene targets, the M. leprae specific repetitive element RLEP is regarded as highly sensitive and specific for diagnostic applications.  METHODS: Our group developed and validated a dry-reagent-based (DRB) RLEP LAMP, provided product specifications for customization of a ready-to-use kit (intended for commercial production) and compared it against the in-house prototype. The assays were optimized for application on a Genie® III portable fluorometer. For technical validation, 40 "must not detect RLEP" samples derived from RLEP qPCR negative exposed and non-exposed individuals, as well as from patients with other conditions and a set of closely related mycobacterial cultures, were tested together with 25 "must detect RLEP" samples derived from qPCR confirmed leprosy patients. For clinical validation, 150 RLEP qPCR tested samples were analyzed, consisting of the following categories: high-positive samples of multibacillary (MB) leprosy patients (> 10.000 bacilli/extract), medium-positive samples of MB leprosy patients (1.001-10.000 bacilli/extract), low-positive samples of MB leprosy patients (1-1.000 bacilli/extract), endemic controls and healthy non-exposed controls; each n = 30.  RESULTS: Technical validation: both LAMP formats had a limit of detection of 1.000 RLEP copies, i.e. 43-27 bacilli, a sensitivity of 92% (in-house protocol)/100% (ready-to-use protocol) and a specificity of 100%. Reagents were stable for at least 1 year at 22 °C. Clinical validation: Both formats showed a negativity rate of 100% and a positivity rate of 100% for high-positive samples and 93-100% for medium positive samples, together with a positive predictive value of 100% and semi-quantitative results. The positivity rate for low-positive samples was 77% (in-house protocol)/43% (ready-to-use protocol) and differed significantly between both formats.  CONCLUSIONS: The ready-to-use RLEP DRB LAMP assay constitutes an ASSURED test ready for field-based evaluation trials aiming for routine diagnosis of leprosy at the primary health care level.

Rapid diagnosis of Leishmania infection with a portable loopmediated isothermal amplification device.

L. donovani is an intracellular protozoan parasite, that causes visceral leishmaniasis (VL), and consequently, post-kala azar dermal leishmaniasis (PKDL). Diagnosis and treatment of leishmaniasis is crucial for decreasing its transmission. Various diagnostic techniques like microscopy, enzyme-linked immunosorbent assays (ELISA) and PCR-based methods are used to detect leishmaniasis infection. More recently, loop-mediated isothermal amplification (LAMP) assay has emerged as an ideal diagnostic measure for leishmaniasis, primarily due to its accuracy, speed and simplicity. However, point-of-care diagnosis is still not been tested with the LAMP assay. We have developed a portable LAMP device for the monitoring of Leishmania infection. The LAMP assay performed using our device can detect and amplify as little as 100 femtograms of L. donovani DNA. In a preliminary study, we have shown that the device can also amplify L. donovani DNA present in VL and PKDL patient samples with high sensitivity (100%), specificity (98%) and accuracy (99%), and can be used both for diagnostic and prognostic analysis. To our knowledge, this is the first report to describe the development and application of a portable LAMP device which has the potential to evolve as a point-of-care diagnostic and prognostic tool for Leishmania infections in future.

Fiocruz cria teste molecular para hanseníase inédito no Brasil

Um novo teste de diagnóstico desenvolvido por pesquisadores da Fundação Oswaldo Cruz (Fiocruz) pode contribuir para o enfrentamento da hanseníase

Development of a novel loop-mediated isothermal amplification assay for rapid detection of Mycobacterium leprae in clinical samples.

BACKGROUND: Sensitive and definitive diagnostic tests are required for timely treatment of leprosy and to control its transmission. AIM: In the present study, we report the development of loop-mediated isothermal amplification assay using six primers targeting the RLEP gene sequence uniquely present in Mycobacterium leprae. METHODS: Tissue punch samples (n = 50) and slit aspirates (n = 50) from confirmed cases of leprosy (M. leprae positive by quantitative polymerase chain reaction), reporting at the Department of Dermatology, Safdarjung Hospital, New Delhi, were analyzed using newly developed closed tube loop-mediated isothermal amplification assay. The sensitivity and specificity; positive predictive value, negative predictive value and accuracy were calculated using MedCalc statistical software. RESULTS: The loop-mediated isothermal amplification assay specifically amplified M. leprae genomic DNA with an analytical sensitivity of 100 fg. About 47 Out of the 50 quantitative polymerase chain reactions confirmed M. leprae positive tissue samples, 47 were positive by loop-mediated isothermal amplification assay (sensitivity 94%; 95% confidence interval 83.5%-98.8%) while only 31/50 were positive by histopathology (sensitivity 62%; 95% confidence interval 47.2%-75.4%) . Using slit aspirate samples of these 50 patients, 42 were positive by both quantitative polymerase chain reaction and loop-mediated isothermal amplification assay (sensitivity 84%; 95% confidence interval 70.9%-92.8%) while only 23/50 (sensitivity 46%; 95% confidence interval 31.8%-60.7%) were positive by microscopy. LIMITATIONS: In the present study, the leprosy patient cohort was not uniform, as it comprised a lower number of paucibacillary cases (22%) compared to multibacillary (78%) cases. CONCLUSION: Loop-mediated isothermal amplification assay established here provides a rapid and accurate diagnostic test for leprosy in terms of sensitivity and specificity. The assay is simple to perform in comparison with other molecular techniques (polymerase chain reaction/quantitative polymerase chain reaction) and has potential for field applicability.

Development of a Loop-mediated isothermal amplification (LAMP) technique for specific and early detection of Mycobacterium leprae in clinical samples.

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.

Loop-mediated isothermal amplification (LAMP) assay targeting RLEP for detection of Mycobacterium leprae in leprosy patients.

OBJECTIVE: Leprosy is a chronic infectious disease caused by Mycobacterium leprae and it remains a significant health problem in several parts of the world. Early and accurate diagnosis of this disease is therefore essential. Previously published loop-mediated isothermal amplification (LAMP) protocols for detecting mycobacterial species used conventional primers targeting the 16S rRNA, gyrB and insertion sequence genes. METHODS: In this study, we conducted a LAMP assay for leprosy and compared it with quantitative polymerase chain reaction (q-PCR) and conventional PCR assays to determine the efficiency, sensitivity and specificity of each technique. We chose conserved sequence RLEP as a suitable molecular target for assays. RESULTS: The LAMP assay provided rapid and accurate results, confirming leprosy in 91/110 clinical skin tissue samples from leprosy patients and amplifying the target pathogen in <60 min at 65 °C. The assay was more sensitive than conventional PCR and more straightforward and faster than the q-PCR assay. CONCLUSIONS: The LAMP assay has the potential for developing quicker, more accessible visual methods for the detection of M. leprae, which will enable early diagnosis and treatment and prevent further infection in endemic areas.

Rapid Multiplex Loop-Mediated Isothermal Amplification (m-LAMP) Assay for Differential Diagnosis of Leprosy and Post-Kala-Azar Dermal Leishmaniasis.

Leprosy and post-kala-azar dermal leishmaniasis (PKDL) are co-endemic neglected tropical diseases often misdiagnosed because of close resemblance in their clinical manifestations. The test that aids in differential diagnosis of leprosy and PKDL would be useful in endemic areas. Here, we report development of a multiplex loop-mediated isothermal amplification (m-LAMP) assay for differential detection of Mycobacterium leprae and Leishmania donovani using a real-time fluorometer. The m-LAMP assay was rapid with a mean amplification time of 15 minutes, and analytical sensitivity of 1 fg for L. donovani and 100 fg for M. leprae. The distinct mean Tm values for M. leprae and L. donovani allowed differentiation of the two organisms in the m-LAMP assay. Diagnostic sensitivity of the assay was evaluated by using confirmed cases of leprosy (n = 40) and PKDL (n = 40) (tissue and slit aspirate samples). All the leprosy and PKDL samples used in this study were positive by organism-specific QPCR and loop-mediated isothermal amplification assays. The diagnostic sensitivity of the m-LAMP assay was 100% (95% CI: 91.2-100.0%) for detecting PKDL and 95% for leprosy (95% CI: 83.1-99.4%). Our m-LAMP assay was successfully used to detect both M. leprae and L. donovani in a patient coinfected with leprosy and macular PKDL. The m-LAMP assay is rapid, accurate, and applicable for differential diagnosis of leprosy versus PKDL, especially in endemic areas.

Novel Diagnostics for Kaposi Sarcoma and Other Skin Diseases in Resource-Limited Settings.

In resource-limited settings, point-of-care diagnostic devices have the potential to reduce diagnostic delays and improve epidemiologic surveillance of dermatologic conditions. We outline novel-point-of care diagnostics that have recently been developed for dermatologic conditions that primarily affect patients living in resource-limited settings, namely, Kaposi sarcoma, cutaneous leishmaniasis, leprosy, Buruli ulcer, yaws, onchocerciasis, and lymphatic filariasis. All of the technologies described in this article are prototypes, and some have undergone field testing. These devices still require validation in real-world settings and effective pricing to have a major impact on dermatologic care in resource-limited settings.

Dominant marker (inter-simple sequence repeat-polymerase chain reaction) versus codominant marker (RLEP-polymerase chain reaction) for laboratory diagnosis of leprosy: A comparative evaluation.

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.

Novel PCR primers for improved detection of Mycobacterium leprae and diagnosis of leprosy.

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.

Quantitative PCR for leprosy diagnosis and monitoring in household contacts: A follow-up study, 2011-2018.

Household contacts (HHC) of leprosy patients exhibit high-risk of developing leprosy and contact tracing is helpful for early diagnosis. From 2011 to 2018,2,437 HHC were examined in a clinic in Rio de Janeiro, Brazil and 16S qPCR was used for diagnosis and monitoring of contacts. Fifty-four HHCs were clinically diagnosed with leprosy at intake. Another 25 exhibited leprosy-like skin lesions at intake, 8 of which were confirmed as having leprosy (50% of which were qPCR positive) and 17 of which were diagnosed with other skin diseases (6% qPCR positive). In skin biopsies, qPCR presented a sensitivity of 0.50 and specificity of 0.94. Furthermore, 955 healthy HHCs were followed-up for at least 3 years and skin scrapings were collected from earlobes for qPCR detection. Positive qPCR indicated a non-significant relative risk of 2.52 of developing the disease. During follow-up, those who progressed towards leprosy exhibited 20% qPCR positivity, compared to 9% of those who remained healthy. Disease-free survival rates indicated that age had a significant impact on disease progression, where patients over 60 had a greater chance of developing leprosy [HR = 32.4 (3.6-290.3)]. Contact tracing combined with qPCR may assist in early diagnosis and age is a risk factor for leprosy progression.

A novel integrated molecular and serological analysis method to predict new cases of leprosy amongst household contacts.

BACKGROUND: Early detection of Mycobacterium leprae is a key strategy for disrupting the transmission chain of leprosy and preventing the potential onset of physical disabilities. Clinical diagnosis is essential, but some of the presented symptoms may go unnoticed, even by specialists. In areas of greater endemicity, serological and molecular tests have been performed and analyzed separately for the follow-up of household contacts, who are at high risk of developing the disease. The accuracy of these tests is still debated, and it is necessary to make them more reliable, especially for the identification of cases of leprosy between contacts. We proposed an integrated analysis of molecular and serological methods using artificial intelligence by the random forest (RF) algorithm to better diagnose and predict new cases of leprosy. METHODS: The study was developed in Governador Valadares, Brazil, a hyperendemic region for leprosy. A longitudinal study was performed, including new cases diagnosed in 2011 and their respective household contacts, who were followed in 2011, 2012, and 2016. All contacts were diligently evaluated by clinicians from Reference Center for Endemic Diseases (CREDEN-PES) before being classified as asymptomatic. Samples of slit skin smears (SSS) from the earlobe of the patients and household contacts were collected for quantitative polymerase chain reaction (qPCR) of 16S rRNA, and peripheral blood samples were collected for ELISA assays to detect LID-1 and ND-O-LID. RESULTS: The statistical analysis of the tests revealed sensitivity for anti-LID-1 (63.2%), anti-ND-O-LID (57.9%), qPCR SSS (36.8%), and smear microscopy (30.2%). However, the use of RF allowed for an expressive increase in sensitivity in the diagnosis of multibacillary leprosy (90.5%) and especially paucibacillary leprosy (70.6%). It is important to report that the specificity was 92.5%. CONCLUSION: The proposed model using RF allows for the diagnosis of leprosy with high sensitivity and specificity and the early identification of new cases among household contacts.

Diagnostic accuracy of tests for leprosy: a systematic review and meta-analysis.

OBJECTIVES: Owing to difficulties in the clinical diagnosis of leprosy, several complementary tests have been developed and used. The aim was to systematically summarize the accuracy of diagnostic tests for leprosy. METHODS: We searched for relevant articles in Embase, Medline, and Global Health databases, until June 2017. Studies evaluating the accuracy of any diagnostic techniques for differentiating between people with and without leprosy were included. Studies solely focusing on differentiating between the separate forms of leprosy were excluded. Our protocol was registered on PROSPERO (CRD42017071803). We assessed study quality using the QUADAS-2 checklist. A bivariate random effects regression model was used for the meta-analyses. RESULTS: We included 78 studies, most of those evaluating the detection of IgM antibodies against phenolic glycolipid I using ELISA. Sensitivity of the 39 studies evaluating ELISA was 63.8% (95% CI 55.0-71.8); specificity 91.0% (95% CI 86.9-93.9). The lateral flow test (nine studies) and the agglutination test (five studies) had a slightly higher sensitivity and a slightly lower specificity. Sensitivity of qPCR was (five studies) 78.5% (95% CI 61.9-89.2) and specificity 89.3% (95% CI 61.4-97.8). Sensitivity of conventional PCR was (17 studies) 75.3% (95% CI 67.9-81.5) and specificity 94.5% (95% CI 91.4-96.5). CONCLUSIONS: Although the test accuracy looks reasonable, the studies suffered from heterogeneity and low methodological quality.

Performance of diagnostic tests for pulmonary tuberculosis in indigenous populations in Brazil: the contribution of Rapid Molecular Testing.

OBJECTIVE: To evaluate the accuracy of rapid molecular testing as a diagnostic tool and estimate the incidence of smear-positive pulmonary tuberculosis among the indigenous population. METHODS: This is an epidemiological study based on secondary data. We calculated the incidence of smear-positive pulmonary tuberculosis between January 1st, 2011 and December 31, 2016, and the performance of bacilloscopy and rapid molecular testing in diagnosing pulmonary tuberculosis compared to sputum culture (standard test). RESULTS: We included 4,048 cases of indigenous people with respiratory symptoms who provided sputum samples for analysis. Among them, 3.7%, 6.7%, and 3.7% had positive results for bacilloscopy, sputum culture, and rapid molecular testing, respectively. The mean incidence of pulmonary tuberculosis was 269.3/100 thousand inhabitants. Rapid molecular testing had 93.1% sensitivity and 98.2% specificity, compared to sputum culture. Bacilloscopy showed 55.1% sensitivity and 99.6% specificity. CONCLUSIONS: Rapid molecular testing can be useful in remote areas with limited resources and a high incidence of tuberculosis, such as indigenous villages in rural regions of Brazil. In addition, the main advantages of rapid molecular testing are its easy handling, fast results, and the possibility of detecting rifampicin resistance. Together, these attributes enable the early start of treatment, contributing to reduce the transmission in communities recognized as vulnerable to infection and disease.

Development and evaluation of a droplet digital PCR assay for the diagnosis of paucibacillary leprosy in skin biopsy specimens.

BACKGROUND: The reduced amounts of Mycobacterium leprae (M. leprae) among paucibacillary (PB) patients reflect the need to further optimize methods for leprosy diagnosis. An increasing number of reports have shown that droplet digital polymerase chain reaction (ddPCR) is a promising tool for diagnosis of infectious disease among samples with low copy number. To date, no publications have investigated the utility of ddPCR in the detection of M. leprae. The aim of this study was to develop and evaluate a ddPCR assay for the diagnosis of PB leprosy. METHODOLOGY: The two most sensitive DNA targets for detection of M. leprae were selected from electronic databases for assessment of sensitivity and specificity by quantitative polymerase chain reaction (qPCR) and ddPCR. Control patients (n = 59) suffering from other dermatological diseases were used to define the cut-off of the duplex ddPCR assay. For comparative evaluation, qPCR and ddPCR assays were performed in 44 PB patients and 68 multibacillary (MB) patients. PRINCIPAL FINDINGS: M. leprae-specific repetitive element (RLEP) and groEL (encoding the 65 kDa molecular chaperone GroEL) were used to develop the ddPCR assay by systematically analyzing specificity and sensitivity. Based on the defined cut-off value, the ddPCR assay showed greater sensitivity in detecting M. leprae DNA in PB patients compared with qPCR (79.5% vs 36.4%), while both assays have a 100% sensitivity in MB patients. CONCLUSIONS/SIGNIFICANCE: We developed and evaluated a duplex ddPCR assay for leprosy diagnosis in skin biopsy samples from leprosy patients. While still costly, ddPCR might be a promising diagnostic tool for detection of PB leprosy.

Quantitative polymerase chain reaction in paucibacillary leprosy diagnosis: A follow-up study.

OBJECTIVE: The diagnosis of paucibacillary (PB) leprosy cases remains a challenge because of the absence of a confirmatory laboratory method. While quantitative polymerase chain reaction (qPCR) has been shown to provide reliable sensitivity and specificity in PB diagnoses, a thorough investigation of its efficacy in clinical practice has not yet been published. The present study evaluated patients with suspected leprosy skin lesions by using qPCR to identify PB individuals in the Leprosy Outpatient clinic at the Oswaldo Cruz Foundation in Rio de Janeiro, Brazil. METHODS: One hundred seventy-two suspected PB cases were included in the study. The patients were evaluated by a dermatologist at three different times. The clinical dermato-neurological examination and collected samples were performed on the first visit. On the second visit, the results of the histopathological analysis and PCR assay (DNA-based Mycobacterium leprae qPCR-targeting 16S gene) results were analyzed, and a decision regarding multi-drug therapy was made. A year later, the patients were re-examined, and the consensus diagnosis was established. RESULTS: In 58% (100/172) of cases, a conclusive diagnosis via histopathological analysis was not possible; however, 30% (30/100) of these cases had a positive PCR. One hundred ten patients (110/172) attended the third visit. The analysis showed that while the sensitivity of the histopathological test was very low (35%), a qPCR alone was more effective for identifying leprosy, with 57% sensitivity. CONCLUSION: The use of qPCR in suspected PB cases with an inconclusive histology improved the sensitivity of leprosy diagnoses.