BackgroundThe analysis of transmission of tuberculosis (TB) is challenging in areas with a large migrant population. Standard genotyping may fail to differentiate transmission within the host country from new importations, which is key from an epidemiological perspective.AimTo propose a new strategy to simplify and optimise cross-border surveillance of tuberculosis and to distinguish between recent transmission in the host country and new importationsMethodsWe selected 10 clusters, defined by 24-locus mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR), from a population in Spain rich in migrants from eastern Europe, north Africa and west Africa and reanalysed 66 isolates by whole-genome sequencing (WGS). A multiplex-allele-specific PCR was designed to target strain-specific marker single nucleotide polymorphisms (SNPs), identified from WGS data, to optimise the surveillance of the most complex cluster.ResultsIn five of 10 clusters not all isolates showed the short genetic distances expected for recent transmission and revealed a higher number of SNPs, thus suggesting independent importations of prevalent strains in the country of origin. In the most complex cluster, rich in Moroccan cases, a multiplex allele-specific oligonucleotide-PCR (ASO-PCR) targeting the marker SNPs for the transmission subcluster enabled us to prospectively identify new secondary cases. The ASO-PCR-based strategy was transferred and applied in Morocco, demonstrating that the strain was prevalent in the country.ConclusionWe provide a new model for optimising the analysis of cross-border surveillance of TB transmission in the scenario of global migration.

BackgroundTuberculosis (TB) is a major global health problem and remains the leading cause of morbidity and mortality in developing countries. Routinely used TB diagnostic methods, in most endemic areas, are time-consuming, often less-sensitive, expensive and inaccessible to most patients. Therefore, there is an urgent need for the development of early, easy to use and effective diagnosis tools of TB, which can be effectively integrated into resource limited settings, to anticipate the early treatment and limit further spread of the disease.Over the last decade, Loop-mediated isothermal amplification (LAMP) assays have become a powerful tool for rapid diagnosis of infectious diseases because of the simplicity of device requirements. Indeed, LAMP is a simple, quick and cost effective Isothermal Nucleic Acid Amplification diagnostic test (INAAT) that has the potential to be used in TB endemic settings of resource-poor countries.MethodsIn the present study, we have developed a simple and rapid TB molecular diagnostic test using a Single-Step Loop-mediated isothermal DNA amplification (SS-LAMP) method for the detection of Mycobacterium tuberculosis complex (MTBC) strains, with a simplified sample preparation procedure, eliminating DNA extraction prior to LAMP amplification, DNA initial denaturation and enzymatic inactivation steps during the amplification process.To perform our in-house SS-LAMP assay, a set of six specific primers was specifically designed to recognize eight distinct regions on the MTBC species-specific repetitive insertion sequence 6110 (IS6110). The amplification of the targeted DNA was carried out under isothermal conditions at 65 °C within 1 h. Our protocol was firstly optimized using 60 of confirmed MTBC isolates and a recombinant pGEMeasy-IS6110 vector for sensitivity testing. Thereafter, the assay was evaluated on liquefied sputum specimens collected from 157 Moroccan patients suspected of having TB.ResultsOur SS-LAMP developed assay was able to detect MTBC DNA directly from liquefied sputum samples without any prior DNA extraction, denaturation nor the final enzymatic inactivation step. When compared to routinely used Löwenstein Jensen (LJ) Culture method, our SS-LAMP assay is rapid and showed specificity and sensitivity of 99.14 % and 82.93 % respectively which are within the international standards. In addition, the limit of detection of our assay was found to be as little as 10 copies of bacterial DNA.ConclusionTo our knowledge, this is the first study using a single step LAMP (SS-LAMP) procedure as a rapid, easy to perform and cost effective testing for TB early detection. This innovative assay could be suitable for low-income countries with restricted health equipment facilities.

BackgroundThe frequency of occurrence of extrapulmonary tuberculosis (EPTB) has been increasing globally over the last two decades. In Morocco, EPTB cases account for 46% of the patients reported with a new episode of tuberculosis (TB). Lymph node TB (LNTB) is the most common form of EPTB. In line with the guidelines of the National TB Program, the diagnosis is mainly based on clinical evidence, including histopathology.ObjectiveThis study aimed to evaluate the yield of histopathology testing in the diagnosis of LNTB.MethodsThis cross-sectional, prospective study was conducted among patients with cervical lymph node who were enrolled in the study from November 2016 to May 2017 in three regions of Morocco. We compared the outcomes of histopathological testing with those of bacteriology. Sensitivity (Se), specificity (Sp), positive predictive value (PPV), and negative predictive value (NPV) of histopathology testing were calculated. Culture and Xpert tests were used as the gold standard Laboratoty Testing.ResultsA total of 262 patients were enrolled in this study. The Se, Sp, PPV, and NPV of histopathology testing were 95.6% (129/135), 64.6% (82/127), 74.1% (129/174), and 93.2% (82/88), respectively, in the presence of granuloma with or without caseous necrosis and were 84.4% (114/135), 74.8% (95/127), 78.1% (114/146), and 81.9% (95/116), respectively, in the presence of granuloma with caseous necrosis. The granuloma with caseous necrosis was associated with increased PPV and Sp of histopathology testing (P<.05).ConclusionsThe presence of the granuloma with caseous necrosis in the histopathological examination had significantly improved the yield of histopathology testing for the diagnosis of LNTB. The findings recommend to maintain histopathology testing in establishing the LNTB diagnosis and to explore other techniques to improve it.

BackgroundRapid diagnosis of drug resistance in tuberculosis (TB) is pivotal for the timely initiation of effective antibiotic treatment to prevent the spread of drug-resistant strains. The development of low-cost, rapid and robust methods for drug-resistant TB detection is highly desirable for resource-limited settings.MethodsWe report the use of an in house plasmid-based quantitative polymerase chain reaction-high-resolution melting (qPCR-HRM) analysis for the detection of mutations related to rifampicin-resistant Mycobacterium tuberculosis (MTB) in clinical isolates from Moroccan patients. Five recombinant plasmids containing predominant mutations (S531L, S531W, H526Y and D516V) and the wild-type sequence of the Rifampicin Resistance-Determining Region (RRDR) have been used as controls to screen 45 rifampicin-resistant and 22 rifampicin-susceptible MTB isolates.ResultsThe sensitivity and the specificity of the qPCR-HRM analysis were 88.8% and 100% respectively as compared to rifampicin Drug Susceptibility Testing (DST). The results of qPCR-HRM and DNA sequencing had a concordance of 100%.ConclusionOur qPCR-HRM assay is a sensitive, accurate and cost-effective assay for the high-throughput screening of mutation-based drug resistance in TB reference laboratories.

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