Shubhada Shenai

Rapid Molecular Detection of Tuberculosis and Rifampin Resistance

BACKGROUND Global control of tuberculosis is hampered by slow, insensitive diagnostic methods, particularly for the detection of drug-resistant forms and in patients with human immunodeficiency virus infection. Early detection is essential to reduce the death rate and interrupt transmission, but the complexity and infrastructure needs of sensitive methods limit their accessibility and effect. METHODS We assessed the performance of Xpert MTB/RIF, an automated molecular test for Mycobacterium tuberculosis (MTB) and resistance to rifampin (RIF), with fully integrated sample processing in 1730 patients with suspected drug-sensitive or multidrug-resistant pulmonary tuberculosis. Eligible patients in Peru, Azerbaijan, South Africa, and India provided three sputum specimens each. Two specimens were processed with N-acetyl-L-cysteine and sodium hydroxide before microscopy, solid and liquid culture, and the MTB/RIF test, and one specimen was used for direct testing with microscopy and the MTB/RIF test. RESULTS Among culture-positive patients, a single, direct MTB/RIF test identified 551 of 561 patients with smear-positive tuberculosis (98.2%) and 124 of 171 with smear-negative tuberculosis (72.5%). The test was specific in 604 of 609 patients without tuberculosis (99.2%). Among patients with smear-negative, culture-positive tuberculosis, the addition of a second MTB/RIF test increased sensitivity by 12.6 percentage points and a third by 5.1 percentage points, to a total of 90.2%. As compared with phenotypic drug-susceptibility testing, MTB/RIF testing correctly identified 200 of 205 patients (97.6%) with rifampin-resistant bacteria and 504 of 514 (98.1%) with rifampin-sensitive bacteria. Sequencing resolved all but two cases in favor of the MTB/RIF assay. CONCLUSIONS The MTB/RIF test provided sensitive detection of tuberculosis and rifampin resistance directly from untreated sputum in less than 2 hours with minimal hands-on time. (Funded by the Foundation for Innovative New Diagnostics.)

Antituberculosis thiophenes define a requirement for Pks13 in mycolic acid biosynthesis

We report a new class of thiophene (TP) compounds that kill Mycobacterium tuberculosis (Mtb) by the novel mechanism of Pks13 inhibition. An F79S mutation near the catalytic Ser-55 site in Pks13 conferred TP-resistance in Mtb. Over-expression of wild-type pks13 resulted in TP-resistance and over-expression of the F79S pks13 mutant conferred high-level resistance. In vitro, TP inhibited fatty acyl-AMP loading onto Pks13. TP inhibited mycolic acid biosynthesis in wild-type Mtb, but to a much lesser extent in TP-resistant Mtb. TP treatment was bactericidal and equivalent to the first-line drug isoniazid, but it was less likely to permit emergent resistance. Combined isoniazid and TP treatment exhibited sterilizing activity. Computational-docking identified a possible TP-binding groove within the Pks13 ACP domain. This study confirms that Mtb Pks13 is required for mycolic acid biosynthesis, validates it as a druggable target and demonstrates the therapeutic potential of simultaneously inhibiting multiple targets in the same biosynthetic pathway.

Persisting positron emission tomography lesion activity and Mycobacterium tuberculosis mRNA after tuberculosis cure

The absence of a gold standard to determine when antibiotics induce a sterilizing cure has confounded the development of new approaches to treat pulmonary tuberculosis (PTB). We detected positron emission tomography and computerized tomography (PET–CT) imaging response patterns consistent with active disease, along with the presence of Mycobacterium tuberculosis (MTB) mRNA in sputum and bronchoalveolar lavage samples, in a substantial proportion of adult, HIV-negative patients with PTB after a standard 6-month treatment plus 1 year follow-up, including patients with a durable cure and others who later developed recurrent disease. The presence of MTB mRNA in the context of nonresolving and intensifying lesions on PET–CT images might indicate ongoing transcription, suggesting that even apparently curative treatment for PTB may not eradicate all of the MTB bacteria in most patients. This suggests an important complementary role for the immune response in maintaining a disease-free state. Sterilizing drugs or host-directed therapies, and better treatment response markers, are probably needed for the successful development of improved and shortened PTB-treatment strategies.The absence of a gold standard to determine when antibiotics have induced sterilizing cure confounds the development of new approaches to treat pulmonary tuberculosis (PTB). We detected PET-CT imaging response patterns consistent with active disease along with the presence of Mycobacterium tuberculosis mRNA in sputum and bronchoalveolar lavage samples in a substantial proportion of adult, HIV-negative PTB patients after standard 6-month treatment plus one year follow-up, including patients with a durable cure and others who later developed recurrent disease. The presence of MTB mRNA in the context of non-resolving and intensifying lesions on PET-CT might indicate ongoing transcription, suggesting that even apparently curative PTB treatment may not eradicate all organisms in most patients. This suggests an important complementary role for the immune response in maintaining a disease-free state. Sterilizing drugs or host-directed therapies and better treatment response markers are likely needed for the successful development of improved and shortened PTB treatment strategies.

Phosphorylation of KasB Regulates Virulence and Acid-Fastness in Mycobacterium tuberculosis

Mycobacterium tuberculosis bacilli display two signature features: acid-fast staining and the capacity to induce long-term latent infections in humans. However, the mechanisms governing these two important processes remain largely unknown. Ser/Thr phosphorylation has recently emerged as an important regulatory mechanism allowing mycobacteria to adapt their cell wall structure/composition in response to their environment. Herein, we evaluated whether phosphorylation of KasB, a crucial mycolic acid biosynthetic enzyme, could modulate acid-fast staining and virulence. Tandem mass spectrometry and site-directed mutagenesis revealed that phosphorylation of KasB occurred at Thr334 and Thr336 both in vitro and in mycobacteria. Isogenic strains of M. tuberculosis with either a deletion of the kasB gene or a kasB_T334D/T336D allele, mimicking constitutive phosphorylation of KasB, were constructed by specialized linkage transduction. Biochemical and structural analyses comparing these mutants to the parental strain revealed that both mutant strains had mycolic acids that were shortened by 4–6 carbon atoms and lacked trans-cyclopropanation. Together, these results suggested that in M. tuberculosis, phosphorylation profoundly decreases the condensing activity of KasB. Structural/modeling analyses reveal that Thr334 and Thr336 are located in the vicinity of the catalytic triad, which indicates that phosphorylation of these amino acids would result in loss of enzyme activity. Importantly, the kasB_T334D/T336D phosphomimetic and deletion alleles, in contrast to the kasB_T334A/T336A phosphoablative allele, completely lost acid-fast staining. Moreover, assessing the virulence of these strains indicated that the KasB phosphomimetic mutant was attenuated in both immunodeficient and immunocompetent mice following aerosol infection. This attenuation was characterized by the absence of lung pathology. Overall, these results highlight for the first time the role of Ser/Thr kinase-dependent KasB phosphorylation in regulating the later stages of mycolic acid elongation, with important consequences in terms of acid-fast staining and pathogenicity.

Mutation Detection and Accurate Diagnosis of Extensively Drug-Resistant Tuberculosis: Report from a Tertiary Care Center in India

ABSTRACT We screened and spoligotyped 150 consecutive phenotypically confirmed extensively drug-resistant Mycobacterium tuberculosis (XDR-TB) isolates (January 2008 to March 2009) for rifampin, isoniazid, fluoroquinolone, and aminoglycoside resistance targeting rpoB, inhA, katG, gyrA, gyrB, and rrs. Mutations predominant among XDR-TB were S315T (katG) (100% of isolates), S531L (rpoB) (97% of isolates), D94G (gyrA) (53% of isolates), and A1401G (rrs) (71% of isolates). Spoligotyping revealed 62% of the isolates to be Beijing.

Rapid speciation of 15 clinically relevant mycobacteria with simultaneous detection of resistance to rifampin, isoniazid, and streptomycin in Mycobacterium tuberculosis complex.

OBJECTIVE To design and standardize an in-house reverse line blot hybridization (RLBH) assay for the accurate identification of 15 clinically relevant species of mycobacteria and for the detection of drug resistance to rifampin (RIF), isoniazid (INH), and streptomycin (STR) in Mycobacterium tuberculosis complex (MTB). MATERIAL AND METHODS Oligonucleotides specific for 15 different species of mycobacteria and wild type and mutant alleles of selected codons in the rpobeta, inhA, katG, rpsL, and rrs genes were designed and immobilized on a membrane. A multiplex PCR was standardized to amplify all target genes. The assay was optimized using ATCC and known mutant strains. Three hundred MTB isolates, 85 non-tuberculous mycobacteria (NTM) isolates, and 48 smear-positive specimens were analyzed. Results were confirmed by PCR restriction enzyme assay and sequencing. RESULTS Upon RLBH analysis, among the NTM, 14% were identified as Mycobacterium fortuitum, 16% were identified as Mycobacterium abscessus, 20% showed 99% homology with Mycobacterium intracellulare, and 31% showed 98% homology with Mycobacterium simiae. Of the 300 MTB isolates analyzed, 75% RIF-resistant isolates had Ser531Leu mutation in the rpobeta gene. Of the INH-resistant isolates, 89% showed Ser315Thr mutation in the katG gene, whereas 16% showed -15 C-->T mutation in the promoter region of the inhA gene. Among STR-resistant isolates, 75% had A-->G mutation in the rpsL gene at codon 43. RLBH results showed 96-99% concordance with phenotypic culture results. CONCLUSION This is a first attempt at combining speciation with detection of drug resistance to RIF, INH, and STR in MTB for accurate and rapid management of mycobacterial infections as well as for compiling genotypic epidemiological data.

Exploring Alternative Biomaterials for Diagnosis of Pulmonary Tuberculosis in HIV-Negative Patients by Use of the GeneXpert MTB/RIF Assay

ABSTRACT The utility of the GeneXpert MTB/RIF (Xpert) assay for detection of Mycobacterium tuberculosis in sputum samples has been extensively studied. However, the performance of the Xpert assay as applied to other readily accessible body fluids such as exhaled breath condensate (EBC), saliva, urine, and blood has not been established. We used the Xpert assay to test EBC, saliva, urine, and blood samples from HIV-negative, smear- and culture-positive pulmonary tuberculosis (TB) patients for the presence of M. tuberculosis. To compare the ability of the assay to perform bacterial load measurements on sputum samples with versus without sample processing, the assay was also performed on paired direct and processed sputum samples from each patient. The Xpert assay detected M. tuberculosis in none of the 26 EBC samples (sensitivity, 0.0%; 95% confidence interval [95% CI], 0.0%, 12.9%), 10 of the 26 saliva samples (sensitivity, 38.5%; 95% CI, 22.4%, 57.5%), 1 of 26 urine samples (sensitivity, 3.8%; 95% CI, 0.7%, 18.9%), and 2 of 24 blood samples (sensitivity, 8.3%; 95% CI, 2.3%, 25.8%). For bacterial load measurements in the different types of sputum samples, the cycle thresholds of the two M. tuberculosis-positive sputum types were well correlated (Spearman correlation of 0.834). This study demonstrates that the Xpert assay should not be routinely used to detect M. tuberculosis in EBC, saliva, urine, or blood samples from HIV-negative patients suspected of having pulmonary tuberculosis. As a test of bacterial load, the assay produced similar results when used to test direct versus processed sputum samples. Sputum remains the optimal sample type for diagnosing pulmonary tuberculosis in HIV-negative patients with the Xpert assay.

Host blood RNA signatures predict the outcome of tuberculosis treatment

Biomarkers for tuberculosis treatment outcome will assist in guiding individualized treatment and evaluation of new therapies. To identify candidate biomarkers, RNA sequencing of whole blood from a well-characterized TB treatment cohort was performed. Application of a validated transcriptional correlate of risk for TB revealed symmetry in host gene expression during progression from latent TB infection to active TB disease and resolution of disease during treatment, including return to control levels after drug therapy. The symmetry was also seen in a TB disease signature, constructed from the TB treatment cohort, that also functioned as a strong correlate of risk. Both signatures identified patients at risk of treatment failure 1–4 weeks after start of therapy. Further mining of the transcriptomes revealed an association between treatment failure and suppressed expression of mitochondrial genes before treatment initiation, leading to development of a novel baseline (pre-treatment) signature of treatment failure. These novel host responses to TB treatment were integrated into a five-gene real-time PCR-based signature that captures the clinically relevant responses to TB treatment and provides a convenient platform for stratifying patients according to their risk of treatment failure. Furthermore, this 5-gene signature is shown to correlate with the pulmonary inflammatory state (as measured by PET-CT) and can complement sputum-based Gene Xpert for patient stratification, providing a rapid and accurate alternative to current methods.

Bacterial Loads Measured by the Xpert MTB/RIF Assay as Markers of Culture Conversion and Bacteriological Cure in Pulmonary TB.

Introduction Biomarkers are needed to monitor tuberculosis (TB) treatment and predict treatment outcomes. We evaluated the Xpert MTB/RIF (Xpert) assay as a biomarker for TB treatment during and at the end of the 24 weeks therapy. Methods Sputum from 108 HIV-negative, culture-positive pulmonary TB patients was analyzed using Xpert at time points before and during anti-TB therapy. Results were compared against culture. Direct Xpert cycle-threshold (Ct), a change in the Ct (delta Ct), or a novel “percent closing of baseline Ct deficit” (percent closing) were evaluated as classifiers of same-day and end-of-treatment culture and therapeutic outcomes. Results Xpert was positive in 29/95 (30.5%) of subjects at week 24; and positive one year after treatment in 8/64 (12.5%) successfully-treated patients who remained free of tuberculosis. We identified a relationship between initial bacterial load measured by baseline Xpert Ct and time to culture conversion (hazard ratio 1.06, p = 0.0023), and to the likelihood of being among the 8 treatment failures at week 24 (AUC = 72.8%). Xpert Ct was even more strongly associated with culture conversion on the day the test was performed with AUCs 96.7%, 99.2%, 86.0% and 90.2%, at Day 7, Week 4, 8 and 24, respectively. Compared to baseline Ct measures alone, a combined measure of baseline Ct plus either Delta Ct or percent closing improved the classification of treatment failure status to a 75% sensitivity and 88.9% specificity. Conclusions Genome loads measured by Xpert provide a potentially-useful biomarker for classifying same day culture status and predicting response to therapy.

Fading of Auramine-Stained Mycobacterial Smears and Implications for External Quality Assurance

ABSTRACT Light-emitting diode fluorescence microscopy is being scaled up for tuberculosis control, but fading of auramine-stained slides could compromise external quality assurance. We stored auramine-stained slides and reexamined them over time. Slides stored in all environments faded quickly, with significant changes in the proportion of positive slides in as little as 1 week.