Detection of drug resistant Mycobacterium tuberculosis by high-throughput sequencing of DNA isolated from acid fast bacilli smears.

BACKGROUND: Drug susceptibility testing for Mycobacterium tuberculosis (MTB) is difficult to perform in resource-limited settings where Acid Fast Bacilli (AFB) smears are commonly used for disease diagnosis and monitoring. We developed a simple method for extraction of MTB DNA from AFB smears for sequencing-based detection of mutations associated with resistance to all first and several second-line anti-tuberculosis drugs. METHODS: We isolated MTB DNA by boiling smear content in a Chelex solution, followed by column purification. We sequenced PCR-amplified segments of the rpoB, katG, embB, gyrA, gyrB, rpsL, and rrs genes, the inhA, eis, and pncA promoters and the entire pncA gene. RESULTS: We tested our assay on 1,208 clinically obtained AFB smears from Ghana (n = 379), Kenya (n = 517), Uganda (n = 262), and Zambia (n = 50). Coverage depth varied by target and slide smear grade, ranging from 300X to 12000X on average. Coverage of ≥20X was obtained for all targets in 870 (72%) slides overall. Mono-resistance (5.9%), multi-drug resistance (1.8%), and poly-resistance (2.4%) mutation profiles were detected in 10% of slides overall, and in over 32% of retreatment and follow-up cases. CONCLUSION: This rapid AFB smear DNA-based method for determining drug resistance may be useful for the diagnosis and surveillance of drug-resistant tuberculosis.

Optimising fluorescein diacetate sputum smear microscopy for assessing patients with pulmonary tuberculosis.

BACKGROUND: Assessing Mycobacterium tuberculosis (TB) viability by fluorescein diacetate (FDA) microscopy can predict TB culture results, treatment response and infectiousness. However, diverse methods have been published. We aimed to optimise FDA microscopy, minimising sputum processing, biohazard and complexity for use in resource-constrained settings. METHODS AND RESULTS: Optimization: Patients with smear-positive pulmonary TB before treatment and healthy control participants provided sputa. These were divided into equal aliquots that were tested directly or after NaOH centrifuge-decontamination. Each aliquot was cultured and used to prepare slides (n = 80). FDA microscopy used: 1 or 3 drops of sputum; with/out acid-alcohol wash; with/out phenol sterilization; with 0/30/60 seconds KMnO4 quenching. Control samples all had negative culture and microscopy results. FDA microscopy had higher sensitivity when performed directly (without centrifuge-decontamination) on 1 drop of sputum (P<0.001), because 3 drops obscured microscopy. Acid-alcohol wash and KMnO4 quenching made bacilli easier to identity (P = 0.005). Phenol sterilization did not impair microscopy (P>0.1). Validation: The 2 protocols that performed best in the optimization experiments were reassessed operationally by comparing duplicate slides (n = 412) stained with KMnO4 quenching for 30 versus 60 seconds. FDA microscopy results were similar (P = 0.4) and highly reproducible, with 97% of counts agreeing within +/-1 logarithm. Storage: Smear microscopy slides and aliquots of the sputum from which they were made were stored for 4 weeks. Twice-weekly, paired slides (n = 80) were stained with freshly prepared versus stored FDA and read quantitatively. Storing sputum, microscopy slides or FDA solution at 4°C or room temperature had no effect on FDA microscopy results (all P>0.2). Cost: Material costs for each slide tested by FDA microscopy using reagents purchased locally were USD $0.05 and required the same equipment, time and skills as auramine acid-fast microscopy. CONCLUSIONS: We recommend a simple, bio-secure protocol for FDA microscopy that provides sensitive and repeatable results without requiring centrifugation.

Evaluation of the GeneXpert MTB/RIF in patients with presumptive tuberculous meningitis.

BACKGROUND: Meningitis caused by Mycobacterium tuberculosis is a major cause of morbidity and mortality worldwide. We evaluated the performance of cerebrospinal fluid (CSF) testing with the GeneXpert MTB/RIF assay versus traditional approaches for diagnosing tuberculosis meningitis (TBM). METHODS: Patients were adults (n = 37) presenting with suspected TBM to the Hospital Nacional Dos de Mayo, Lima, Peru, during 12 months until 1st January 2015. Each participant had a single CSF specimen that was divided into aliquots that were concurrently tested for M. tuberculosis using GeneXpert, Ziehl-Neelsen smear and culture on solid and liquid media. Drug susceptibility testing used Mycobacteria Growth Indicator Tube (MGIT 960) and the proportions method. RESULTS: 81% (30/37) of patients received a final clinical diagnosis of TBM, of whom 63% (19/30, 95% confidence intervals, CI: 44-80%) were HIV-positive. 22% (8/37, 95%CI: 9.8-38%), of patients had definite TBM. Because definite TBM was defined by positivity in any laboratory test, all laboratory tests had 100% specificity. Considering the 30 patients who had a clinical diagnosis of TBM: diagnostic sensitivity was 23% (7/30, 95%CI: 9.9-42%) for GeneXpert and was the same for all culture results combined; considerably greater than 7% (2/30, 95%CI: 0.82-22%) for microscopy; whereas all laboratory tests had poor negative predictive values (20-23%). Considering only the 8 patients with definite TBM: diagnostic sensitivity was 88% (7/8, 95%CI: 47-100%) for GeneXpert; 75% (6/8, 95%CI: 35-97%) for MGIT culture or LJ culture; 50% (4/8, 95%CI 16-84) for Ogawa culture and 25% (2/8, 95%CI: 3.2-65%) for microscopy. GeneXpert and microscopy provided same-day results, whereas culture took 20-56 days. GeneXpert provided same-day rifampicin-susceptibility results, whereas culture-based testing took 32-71 days. 38% (3/8, 95%CI: 8.5-76%) of patients with definite TBM with data had evidence of drug-resistant TB, but 73% (22/30) of all clinically diagnosed TBM (definite, probable, and possible TBM) had no drug-susceptibility results available. CONCLUSIONS: Compared with traditional culture-based methods of CSF testing, GeneXpert had similar yield and faster results for both the detection of M. tuberculosis and drug-susceptibility testing. Including use of the GeneXpert has the capacity to improve the diagnosis of TBM cases.

Sputum Microscopy With Fluorescein Diacetate Predicts Tuberculosis Infectiousness.

Background: Sputum from patients with tuberculosis contains subpopulations of metabolically active and inactive Mycobacterium tuberculosis with unknown implications for infectiousness. Methods: We assessed sputum microscopy with fluorescein diacetate (FDA, evaluating M. tuberculosis metabolic activity) for predicting infectiousness. Mycobacterium tuberculosis was quantified in pretreatment sputum of patients with pulmonary tuberculosis using FDA microscopy, culture, and acid-fast microscopy. These 35 patients' 209 household contacts were followed with prevalence surveys for tuberculosis disease for 6 years. Results: FDA microscopy was positive for a median of 119 (interquartile range [IQR], 47-386) bacteria/µL sputum, which was 5.1% (IQR, 2.4%-11%) the concentration of acid-fast microscopy-positive bacteria (2069 [IQR, 1358-3734] bacteria/µL). Tuberculosis was diagnosed during follow-up in 6.4% (13/209) of contacts. For patients with lower than median concentration of FDA microscopy-positive M. tuberculosis, 10% of their contacts developed tuberculosis. This was significantly more than 2.7% of the contacts of patients with higher than median FDA microscopy results (crude hazard ratio [HR], 3.8; P = .03). This association maintained statistical significance after adjusting for disease severity, chemoprophylaxis, drug resistance, and social determinants (adjusted HR, 3.9; P = .02). Conclusions: Mycobacterium tuberculosis that was FDA microscopy negative was paradoxically associated with greater infectiousness. FDA microscopy-negative bacteria in these pretreatment samples may be a nonstaining, slowly metabolizing phenotype better adapted to airborne transmission.

The seasonality of tuberculosis, sunlight, vitamin D, and household crowding.

BACKGROUND: Unlike other respiratory infections, tuberculosis diagnoses increase in summer. We performed an ecological analysis of this paradoxical seasonality in a Peruvian shantytown over 4 years. METHODS: Tuberculosis symptom-onset and diagnosis dates were recorded for 852 patients. Their tuberculosis-exposed cohabitants were tested for tuberculosis infection with the tuberculin skin test (n = 1389) and QuantiFERON assay (n = 576) and vitamin D concentrations (n = 195) quantified from randomly selected cohabitants. Crowding was calculated for all tuberculosis-affected households and daily sunlight records obtained. RESULTS: Fifty-seven percent of vitamin D measurements revealed deficiency (<50 nmol/L). Risk of deficiency was increased 2.0-fold by female sex (P < .001) and 1.4-fold by winter (P < .05). During the weeks following peak crowding and trough sunlight, there was a midwinter peak in vitamin D deficiency (P < .02). Peak vitamin D deficiency was followed 6 weeks later by a late-winter peak in tuberculin skin test positivity and 12 weeks after that by an early-summer peak in QuantiFERON positivity (both P < .04). Twelve weeks after peak QuantiFERON positivity, there was a midsummer peak in tuberculosis symptom onset (P < .05) followed after 3 weeks by a late-summer peak in tuberculosis diagnoses (P < .001). CONCLUSIONS: The intervals from midwinter peak crowding and trough sunlight to sequential peaks in vitamin D deficiency, tuberculosis infection, symptom onset, and diagnosis may explain the enigmatic late-summer peak in tuberculosis.