Tuberculosis in Pakistan

Smoking is recognized as a significant risk factor for tuberculosis and a major public health concern in Pakistan. Surprisingly, this study did not find a statistical association between tuberculosis and diabetes comorbidity.

Diabetic patients face a substantial risk of developing tuberculosis compared to non-diabetic individuals.

The rising prevalence of diabetes mellitus (DM) poses a potential threat to tuberculosis control measures, particularly in middle- and low-income countries.

Khalid N, Ahmad F, Qureshi FM. Association amid the comorbidity of Diabetes Mellitus in patients of Active Tuberculosis in Pakistan: A matched case control study. Pak J Med Sci. 2021;37(3):816-820.

===

Another study:

Meghji, J., Kon, O.M. and Ainley, A., 2023. Clinical tuberculosis. Medicine, 51(11), pp.768-773.

Thirty high-TB-burden countries account for 87% of global cases, with incident rates exceeding 150/100,000. Two-thirds of these cases occur in just eight countries: India, Indonesia, China, the Philippines, Pakistan, Nigeria, Bangladesh, and the Democratic Republic of the Congo. England has been classified as a low-incidence area (<10/100,000) since 2017, with a rate of 7.3/100,000 in 2021. In 2020, England reported 4,125 TB cases, but national data obscure significant disparities, particularly among those born outside the UK, who accounted for 72.8% of cases (36.3/100,000).

See also: TB and dialysis

In 2020, 12.7% of TB patients had at least one social risk factor (alcohol or drug misuse, homelessness, imprisonment). Latent TB infection (LTBI) occurs when there is no clinical evidence of active disease despite a persistent immune response to TB antigens, with a 5-10% risk of progressing to active TB, typically within five years.

See also: Lin TB Lab

In 2020, 48.6% of TB cases in England were pulmonary. Symptoms of pulmonary TB (PTB) include cough, fever, night sweats, and weight loss, though haemoptysis is not always present. The WHO’s ‘End TB’ strategy promotes new diagnostic tools like Xpert MTB/RIF, a rapid assay detecting M. tuberculosis, despite its lower sensitivity in smear-negative or extrapulmonary cases. The Xpert MTB/RIF Ultra, recommended by WHO in 2017, has improved sensitivity, especially for CNS disease and HIV-positive patients.

See also: TB and Diabetes mellitus

The FluoroType MTBDR assay and the developing FluoroType XDR-TB offer advanced resistance profiling. Point-of-care strategies, such as urinary lipoarabinomannan detection, aim to decentralize TB diagnostics, particularly in low-resource settings. Cepheid's GeneXpert platforms are under review by WHO for use in these areas.

See also: TB Predictive Modelling

MDR/RR-TB patients are recommended all-oral regimens, avoiding injectables. NICE advises LTBI treatment in patients under 65 with close contact history to drug-sensitive pulmonary/laryngeal TB, with caution for those aged 35-65 due to hepatotoxicity risks. LTBI treatment typically involves rifampicin and isoniazid or monotherapy options, with newer WHO-endorsed regimens like weekly rifapentine. However, rifapentine access in Western Europe remains limited due to licensing issues. 

(Editor: Yoseph Leonardo Samodra)

Exploring Diagnostic Methods for Drug-Resistant Tuberculosis

Sanchini, A., Lanni, A., Giannoni, F. and Mustazzolu, A., 2024. Exploring Diagnostic Methods for Drug-Resistant Tuberculosis: A Comprehensive Overview. Tuberculosis, p.102522.

Laboratories are well-equipped to test resistance to established drugs like fluoroquinolones (FQs), linezolid (LZD), and second-line injectable drugs (SLIDs) such as amikacin (AMK) and kanamycin (KAN).

However, they are less prepared for novel drugs, like bedaquiline (BDQ), which are crucial for treating multidrug-resistant tuberculosis (MDR-TB).

Phenotypic drug susceptibility tests (pDSTs) classify bacteria as resistant if they grow in the presence of a drug, indicating the drug is ineffective.

Some pDSTs can determine the minimum inhibitory concentration (MIC) of a drug, while others only test for critical concentration (CC), determining whether a bacteria is susceptible or resistant.

pDSTs require a positive culture of Mycobacterium tuberculosis (MTB), which takes 2-3 weeks, and the tests need to be conducted in a biosafety level 3 laboratory.

Obtaining drug susceptibility results can take an additional 2-3 weeks, totaling 4-6 weeks from sample collection to pDST results.

The gold standards for pDST are the agar proportion method (on solid medium like Lowenstein-Jensen or 7H11 Middlebrook) or the Mycobacterium Growth Indicator Tube (MGIT) liquid medium, with MGIT being faster and more common.

The BACTEC MGIT 960 is a fully automated system that detects mycobacterial growth using fluorescence, testing only one drug concentration (CC) and categorizing isolates as susceptible or resistant.

The Sensititre system can determine the MIC of 12 anti-TB drugs using the microdilution method on a 96-well plate.

A colorimetric assay, such as the Crystal Violet Decolorization Assay, can detect resistance to rifampicin (RIF) and isoniazid (INH) with high sensitivity and specificity.

The microplate Alamar Blue Assay tests for BDQ resistance using resazurin dye, which turns pink and fluorescent when reduced by cellular metabolism. This assay found BDQ-resistant isolates without plausible resistance mutations that would have been misclassified by gene sequencing alone.

The Thin-Layer Agar (TLA) method for resistance detection involves inoculating a thin layer of agar with clinical samples, where growth in drug-containing sections indicates resistance. This method is promising for low-resource settings or field conditions.

Molecular drug susceptibility tests (DSTs) for detecting drug resistance in Mycobacterium tuberculosis (MTB) are based on identifying mutations in resistance-associated genes.

Molecular DSTs are simpler and faster than phenotypic DSTs (pDSTs) and can be applied directly to clinical samples without needing prior culture or a biosafety level 3 laboratory.

The main limitation of molecular DSTs is that they can only detect known mutations, potentially missing new, unknown, or rare mutations that confer drug resistance. Additionally, molecular DSTs cannot determine minimum inhibitory concentrations (MICs).

The gold standard molecular DSTs for detecting drug resistance in MTB are the PCR-based Xpert MTB/RIF and Xpert MTB/RIF Ultra (for detecting TB and rifampicin [RIF] resistance), endorsed by the World Health Organization (WHO).

Line probe assays (LPAs) such as GenoType MTBDRplus (Hain LifeScience) and the Nipro NTM + MDRTB detection kit (Nipro Corp.) are used for testing resistance to RIF and isoniazid (INH). The GenoType MTBDRsl (Hain LifeScience) is used for testing fluoroquinolones (FQs) and aminoglycosides.

The Xpert MTB/RIF system, developed by Cepheid, can identify MTB and its resistance to RIF from respiratory samples in approximately 90 minutes. This system uses automated nested real-time PCR, targeting mutations in the rpoB gene.

Xpert MTB/RIF Ultra improves sensitivity for RIF resistance detection by incorporating additional targets and lowering the detection limit compared to the original Xpert MTB/RIF.

The Xpert MTB/XDR system is approved for testing INH, FQs, amikacin (AMK), kanamycin (KAN), and ethionamide (ETH) after a positive result from Xpert MTB/RIF or Xpert MTB/RIF Ultra.

Xpert MTB/RIF used on bronchoalveolar lavage (BAL) samples shows a sensitivity of 87.0% and specificity of 92.0%. BAL samples are a useful alternative to sputum, especially in cases of sputum smear-negative results or when sputum production is insufficient.

The Xpert MTB/XDR assay can detect heteroresistance and serves as a useful follow-up test for MTB-positive samples to diagnose primary resistance in MDR-TB isolates.

The GeneXpert Omni system is a portable, point-of-care (POC) molecular diagnostic tool designed for field settings and remote areas. It is battery-powered, cloud-connected, and performs well under stressful field conditions.

The GenoType MTBDRplus system, developed by Hain Lifescience, identifies MTB complex and its resistance to RIF and INH directly from both pulmonary samples and isolated cultures using LPA, PCR amplification, and reverse hybridization. Turnaround time (TAT) is approximately five hours.

The GenoType MTBDRsl (Hain Lifescience) detects resistance to second-line drugs (SLDs) such as FQs (e.g., ofloxacin [OFL] and moxifloxacin [MOX]) and second-line injectable drugs (SLIDs) like KAN, AMK, and capreomycin (CAP).

The BD MAX™ MDR-TB test is an automated system that extracts DNA and performs real-time PCR to detect MTB and resistance to RIF and INH from respiratory samples.

BD MAX showed a sensitivity of 90.0% and specificity of 95.0% for RIF resistance, and sensitivity of 81.5% and specificity of 100% for INH resistance, compared to pDST.

The BD MAX system processes up to 24 specimens in one round, with a TAT of approximately four hours from start to results, making it a promising tool for drug-resistance testing. 

Whole-genome sequencing (WGS) analyzes all genes and compensatory mutations, and sequencing data can be analyzed retrospectively if new resistance genes or mutations are discovered.

Disadvantages of WGS include the need for a positive Mycobacterium tuberculosis (MTB) culture (typically a flagged positive MGIT culture), the requirement for complex bioinformatics expertise, high costs (around 100 EUR per sample), and labor-intensive laboratory work.

To reduce turnaround time (TAT), sequencing can be performed directly on sputum samples, but this may lower sensitivity due to contamination or low bacterial load.

Next-generation sequencing (NGS) approaches, such as targeted sequencing of specific DNA regions, provide higher depth of coverage.

Several platforms are available for WGS, including Illumina, Ion Torrent, PacBio SMRT RSII, and Oxford Nanopore MinION.

Various bioinformatic tools are available to predict drug resistance profiles from raw WGS data, such as KvarQ, TBProfiler, CASTB, Mykrobe Predictor, PhyResSE, Resistance Sniffer, and Treesist-TB.

Benefits of replacing routine phenotypic DST (pDST) with WGS include reduced TAT, shorter time on inappropriate treatment, reduced treatment duration, fewer patients in treatment, and cost savings (up to £7.2 million in 10 years).

Both the MiSeq and MinION platforms have similar TAT (approximately three days), but MinION is more flexible as sequencing can be terminated once sufficient data is collected. MinION has a lower cost, at 57 USD per sample compared to 130 USD with MiSeq.

MinION's portability, flexibility, and cost-effectiveness make it valuable for clinical settings.

The QuantaMatrix Multiplexed Assay Platform (QMAP) is an assay based on reverse hybridization, using magnetic microparticles to identify resistance to RIF, INH, EMB, FQs, and SLIDs within six hours from a positive MTB culture.

MALDI-TOF MS (Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry) uses a laser to ionize molecules and analyze them by mass, useful for detecting resistance mutations. The MassARRAY system identifies resistance mutations for streptomycin (SM), RIF, INH, EMB, and FQs.

Key factors for choosing a drug-resistance test for MTB include rapid TAT, simplicity of procedure, cost, equipment requirements, and test sensitivity and specificity.

BACTEC MGIT 960 remains a widely used pDST method, offering susceptibility testing for a full panel of drugs, though it requires significant investment in installation, maintenance, and ongoing costs, which can be a barrier in low-resource settings.

MGIT supports WGS by helping interpret results and identifying new resistance mutations, though there is contamination risk during sample handling in culture-based methods.

Common molecular tests include the Hain MTBDRplus, MTBDRsl, and GenXpert systems, but WGS offers the most comprehensive and reliable resistance profiling, along with molecular typing for identifying clones and clusters.

Regional centers for WGS could support peripheral hospitals and labs by allowing them to send strains or extracted DNA for sequencing.

MALDI-TOF is a valid competitor to WGS, offering similar information but with limitations related to instrument costs and the need for skilled personnel to interpret results.

=-=

Menzies, N.A., Cohen, T., Lin, H.H., Murray, M. and Salomon, J.A., 2012. Population health impact and cost-effectiveness of tuberculosis diagnosis with Xpert MTB/RIF: a dynamic simulation and economic evaluation. PLoS medicine, 9(11), p.e1001347.

·       TB diagnosis in high-burden settings mainly relies on sputum smear microscopy, which has limited sensitivity, especially among HIV-infected patients.

·       Traditional culture-based diagnosis and drug sensitivity testing are costly and slow, with many resource-limited settings lacking the capacity for high-volume testing.

·       The Xpert MTB/RIF automated DNA test provides rapid and sensitive detection of TB and rifampicin resistance, using a cartridge-based system that integrates sample processing and real-time PCR.

·       Xpert can be used by relatively unskilled healthcare workers and delivers results in less than 2 hours.

·       WHO recommended Xpert for initial diagnosis in suspected cases of multidrug-resistant TB (MDR-TB) or HIV-associated TB disease in December 2010.

·       Xpert has the potential to significantly reduce TB morbidity and mortality, especially in southern Africa, by diagnosing and enabling early treatment for smear-negative TB cases.

·       Over time, Xpert could reduce TB transmission and the reservoir of latent TB infection in the population.

·       Despite these benefits, TB incidence is projected to remain substantial after three decades of Xpert use without other changes to TB control strategies.

·       This persistence is due to the large pool of latently infected individuals and the lower transmission likelihood of smear-negative cases diagnosed by Xpert.

·       The adoption of Xpert increases demands on health systems, including the need for additional first-line TB treatment, HIV treatment for coinfected individuals, and second-line TB treatment for drug-resistant cases. 

[Yoseph Leonardo Samodra]

A social network analysis model approach to understand TB transmission in Madagascar

Pando, C., Hazel, A., Tsang, L.Y., Razafindrina, K., Andriamiadanarivo, A., Rabetombosoa, R.M., Ambinintsoa, I., Sadananda, G., Small, P.M., Knoblauch, A.M. and Rakotosamimanana, N., 2023. A social network analysis model approach to understand tuberculosis transmission in remote rural Madagascar. BMC Public Health, 23(1), p.1511.

Social network analysis (SNA), informed by ethnographic data on the nature and structure of human interactions, can provide valuable insights and enhance the realism of compartmental models. 

Despite 15 years of intervention, the prevalence of latent TB infection only slightly decreased, indicating the persistence of TB reservoirs even after systematic treatment of active cases. The intensity of social contacts may increase TB exposure, and these inter-community differences are often overlooked by conventional transmission models. 

(Editor: Yoseph Samodra)