Whole genome enrichment approach for rapid detection of Mycobacterium tuberculosis and drug resistance-associated mutations from direct sputum sequencing.

Tuberculosis is the leading cause of death among infectious diseases worldwide. Detection of Mycobacterium tuberculosis (Mtb), using routine culture-based methods is time consuming resulting in delayed diagnosis and poor treatment outcomes. Currently available molecular tests provide faster diagnosis but are able to screen only limited hot-spot mutations. Whole genome sequencing from direct sputum offers a potential solution, however, due to the presence of other microbes and host DNA its use in diagnostic testing remains challenging. In this study, we present a targeted Mtb-enrichment assay for lineage-4 coupled with an improved analysis pipeline that uses 1657 bacterial taxa as background for reducing non-Mtb genome from sputum DNA. This method drastically improved the recovery of Mtb DNA from sputum (Mtb alignment increased from 3% to >65%) as compared to non-enrichment-based sequencing. We obtained >99% Mtb genome coverage as compared to 49% in non-enriched sputum sequencing. We were able to identify Mtb positive samples from controls with 100% accuracy using Mpt64 gene coverage. Our method not only achieved 100% sensitivity to resistance variants profiled by line probe assay (LPA), but also outperformed LPA in determining drug resistance based on phenotypic drug susceptibility tests for 6 anti-tuberculosis drugs (accuracy of 97.7% and 92.8% by enriched WGS and LPA, respectively).

Identifying and mapping TB hot spots in an urban slum by integratingGeographic positioning system and the local postman - A pilot study.

SETTING: Mahavir DOT Centre, Hyderabad, Telangana, India INTRODUCTION: Urban slums are characterized by crowding, poverty. In such setting due to lack of infection control the transmission of tuberculosis is known to rise, thereby creating a "Hot" spot. Distribution of residences in such areas does not necessarily follow postal codes, making it difficult for health workers to locate TB patients unless accompanied by the STLS. OBJECTIVE: To investigate the utility of integrating the help of local postman and geographic positioning system (GPS) to identify and create map of hot spots in an area under a regional DOT centre. MATERIALS & METHODS: Retrospective and prospective demographic data of TB patients enrolled during 12 years (1999-2011) was analysed from the TB register at a ward where number of cases continued to increase despite active implementation of DOTS strategy. Non-Spatial data was generated with the local postman identifying individual house addresses. The corresponding co-ordinates were recorded with GPS and uploaded in Google Earth to identify the locations. Area map was created by software (AutoCAD, Map R3, MapInfo Pro 7.5 Trial Version and MS office Tools). Residences of Index patients were marked in different colours year wise on the map. RESULTS: Maps displayed in the DOT centre area helped in identifying HOT SPOT and visualization of the clustering of TB cases in the area. Time interval between subsequent infections (3 months-5 years) could be calculated in the locality, within household, neighbourhood and random contacts. Average distances (<1 m) between houses indicated the probable source of infection. Risk factors included crowding, poor ventilation and sanitation contributed to TB transmission in HOT spot area. CONCLUSION: Integrating local postman and information technology to identify HOT SPOT in RNTCP, will help in early intervention by health personnel to arrest TB transmission.

Modern and ancestral genotypes of Mycobacterium tuberculosis from Andhra Pradesh, India.

Traditionally, the distribution of the Mycobacterium tuberculosis genotypes in India has been characterized by widespread prevalence of ancestral lineages (TbD1+ strains and variants) in the south and the modern forms (TbD1(-) CAS and variants) predominating in the north of India. The pattern was, however, not clearly known in the south-central region such as Hyderabad and the rest of the state of Andhra Pradesh where the prevalence of both tuberculosis (TB) and human immunodeficiency virus (HIV) infection is one of the highest in the country; this area has been the hotspot of TB vaccine trials. Spoligotyping of 101 clinical isolates obtained from Hyderabad and rural Andhra Pradesh confirmed the occurrence of major genogroups such as the ancestral (or the TbD1+ type or the East African Indian (EAI) type), the Central Asian (CAS) or Delhi type and the Beijing lineage in Andhra Pradesh. Sixty five different spoligotype patterns were observed for the isolates included in this study; these were further analyzed based on specific genetic signatures/mutations. It was found that the major genogroups, CAS and "ancestral," were almost equally prevalent in our collection but followed a north-south compartmentalization as was also reported previously. However, we observed a significant presence of MANU lineage in south Andhra Pradesh, which was earlier reported to be overwhelmingly present in Mumbai. This study portrays genotypic diversity of M. tuberculosis from the Indian state of Andhra Pradesh and provides a much needed snapshot of the strain diversity that will be helpful in devising effective TB control programs in this part of the world.