Frederick A. Sirgel

Long-term outcomes of patients with extensively drug-resistant tuberculosis in South Africa: a cohort study

BACKGROUND Long-term treatment-related outcomes in patients with extensively drug-resistant (XDR) tuberculosis are unknown. We followed up a cohort of patients to address knowledge gaps. METHODS Between March, 2008, and August, 2012, we prospectively followed up a cohort of 107 patients from three provinces in South Africa, who had been diagnosed with XDR tuberculosis between August 2002, and February, 2008. Available isolates from 56 patients were genotyped to establish strain type and used for extended susceptibility testing. FINDINGS All patients were treated empirically as inpatients with a median of eight drugs (IQR six to ten). 44 patients (41%) had HIV. 36 (64%) of 56 isolates were resistant to at least eight drugs, and resistance to an increasing number of drugs was associated with the Beijing genotype (p=0·01). After 24 months of follow-up, 17 patients (16%) had a favourable outcome (ie, treatment cure or completion), 49 (46%) had died, seven (7%) had defaulted (interruption of treatment for at least 2 consecutive months), and 25 (23%) had failed treatment. At 60 months, 12 patients (11%) had a favourable outcome, 78 (73%) had died, four (4%) had defaulted, and 11 (10%) had failed treatment. 45 patients were discharged from hospital, of whom 26 (58%) had achieved sputum culture conversion and 19 (42%) had failed treatment. Median survival of patients who had failed treatment from time of discharge was 19·84 months (IQR 4·16-26·04). Clustering of cases and transmission within families containing a patient who had failed treatment and been discharged were shown with genotypic methods. Net sputum culture conversion occurred in 22 patients (21%) and median time to net culture conversion was 8·7 months (IQR 5·6-26·4). Independent predictors of probability of net culture conversion were no history of multidrug-resistant tuberculosis (p=0·0007) and use of clofazamine (p=0·0069). Independent overall predictors of survival were net culture conversion (p<0·0001) and treatment with clofazamine (p=0·021). Antiretroviral therapy was also a predictor of survival in patients with HIV (p=0·003). INTERPRETATION In South Africa, long-term outcomes in patients with XDR tuberculosis are poor, irrespective of HIV status. Because appropriate long-stay or palliative care facilities are scarce, substantial numbers of patients with XDR tuberculosis who have failed treatment and have positive sputum cultures are being discharged from hospital and are likely to transmit disease into the wider community. Testing of new combined regimens is needed urgently and policy makers should implement interventions to minimise disease spread by patients who fail treatment. FUNDING European and Developing Countries Clinical Trials Partnership, South African National Research Foundation (SARChI), and the South African Medical Research Council.

Emergence and Spread of Extensively and Totally Drug-Resistant Tuberculosis, South Africa

Factors driving the increase in drug-resistant tuberculosis (TB) in the Eastern Cape Province, South Africa, are not understood. A convenience sample of 309 drug-susceptible and 342 multidrug-resistant (MDR) TB isolates, collected July 2008–July 2009, were characterized by spoligotyping, DNA fingerprinting, insertion site mapping, and targeted DNA sequencing. Analysis of molecular-based data showed diverse genetic backgrounds among drug-sensitive and MDR TB sensu stricto isolates in contrast to restricted genetic backgrounds among pre–extensively drug-resistant (pre-XDR) TB and XDR TB isolates. Second-line drug resistance was significantly associated with the atypical Beijing genotype. DNA fingerprinting and sequencing demonstrated that the pre-XDR and XDR atypical Beijing isolates evolved from a common progenitor; 85% and 92%, respectively, were clustered, indicating transmission. Ninety-three percent of atypical XDR Beijing isolates had mutations that confer resistance to 10 anti-TB drugs, and some isolates also were resistant to para-aminosalicylic acid. These findings suggest the emergence of totally drug-resistant TB.

gyrA mutations and phenotypic susceptibility levels to ofloxacin and moxifloxacin in clinical isolates of Mycobacterium tuberculosis

OBJECTIVES To compare mutations in the quinolone resistance-determining region of the gyrA gene and flanking sequences with the MICs of ofloxacin and moxifloxacin for Mycobacterium tuberculosis. METHODS The presence of mutations in 177 drug-resistant M. tuberculosis isolates was determined by DNA sequencing and the MICs quantified by MGIT 960. RESULTS Single nucleotide polymorphisms were detected at codons 94 (n = 30), 90 (n = 12), 91 (n = 3), 89 (n = 1), 88 (n = 1) and 80 (n = 1). Four isolates with double mutations D94G plus A90V (n = 2) and D94G plus D94N (n = 2) reflect mixed populations. Agreement between genotypic and phenotypic susceptibility was high (≥97%) for both drugs. Mutant isolates had an MIC(50) of 8.0 mg/L and an MIC(90) of >10 mg/L for ofloxacin compared with an MIC(50) and MIC(90) of 2.0 mg/L for moxifloxacin. Codons 94 and 88 were linked to higher levels of fluoroquinolone resistance compared with codons 90, 91 and 89. The MIC distributions for the wild-type isolates ranged from ≤0.5 to 2.0 mg/L for ofloxacin and from ≤0.125 to 0.25 mg/L for moxifloxacin. However, 96% of the isolates with genetic alterations had MICs ≤2.0 mg/L for moxifloxacin, which is within its achievable serum levels. CONCLUSIONS This study provides quantitative evidence that the addition of moxifloxacin to extensively drug-resistant tuberculosis (XDR-TB) regimens based on a clinical breakpoint of 2.0 mg/L has merit. The use of moxifloxacin in the treatment of multidrug-resistant tuberculosis may prevent the acquisition of additional mutations and development of XDR-TB.

Impact of Nonlinear Interactions of Pharmacokinetics and MICs on Sputum Bacillary Kill Rates as a Marker of Sterilizing Effect in Tuberculosis

ABSTRACT The relationships between antituberculosis drug exposure and treatment effects on humans receiving multidrug therapy are complex and nonlinear. In patients on treatment, an analysis of the rate of decline in the sputum bacillary burden reveals two slopes. The first is the α-slope, which is thought to reflect bactericidal effect, followed by a β-slope, which is thought to reflect sterilizing activity. We sought to characterize the effects of standard first-line treatment on sterilizing activity. Fifty-four patients receiving combination therapy for pulmonary tuberculosis in a clinical trial had drug concentrations measured and Mycobacterium tuberculosis isolates available for MIC identification. Sputum sample cultures were performed at baseline and weekly for 8 weeks. A time-to-event model based on the days to positivity in the liquid cultures was used to estimate the β-slope. The pharmacokinetic parameters of rifampin, isoniazid, ethambutol, and pyrazinamide were determined for each patient. Multivariate adaptive regression splines analyses, which simultaneously perform linear and nonlinear analyses, were used to identify the relationships between the predictors and the β-slope. The potential predictors examined included HIV status, lung cavitation, 24-h area under the concentration-time curve (AUC), peak drug concentration (Cmax), AUC/MIC ratio, Cmax/MIC ratio, and the time that that concentration persisted above MIC. A rifampin Cmax of >8.2 mg/liter and a pyrazinamide AUC/MIC of >11.3 were key predictors of the β-slope and interacted positively to increase the β-slope. In patients with a rifampin AUC of <35.4 mg · h/liter, an increase in the pyrazinamide AUC/MIC and/or ethambutol Cmax/MIC increased the β-slope, while increasing isoniazid Cmax decreased it, suggesting isoniazid antagonism. Antibiotic concentrations and MICs interact in a nonlinear fashion as the main drivers of a sterilizing effect. The results suggest that faster speeds of sterilizing effect might be achieved by omitting isoniazid and by increasing rifampin, pyrazinamide, and ethambutol exposures. However, isoniazid and ethambutol exposures may only be of importance when rifampin exposure is low. These findings need confirmation in larger studies. (This study has been registered at controlled-trials.com under registration no. ISRCTN80852505.)

Early bactericidal activity of amoxicillin in combination with clavulanic acid in patients with sputum smear-positive pulmonary tuberculosis

The early bactericidal activity (EBA) of an antituberculosis agent is the rate of decrease in viable colony-forming units (CFU) per milliliter of sputum during the first 2 d of treatment of patients with previously untreated smear-positive pulmonary tuberculosis. The objective of this open randomized study was to evaluate the EBA of the combination of amoxicillin 3 g and clavulanic acid 750 mg. Ten patients with a mean age of 34 y and a mean weight of 56 kg received amoxicillin/clavulanic acid and 5 patients with a mean age of 34 y and a mean weight of 57 kg received no drug. In the patients receiving 1 dose of amoxicillin/clavulanic acid daily for 2 d the mean log10CFU/ml of sputum before treatment was 6.7402 (SD 0.539) and after 2 d of treatment 6.7046 (SD 0.609); the corresponding values in patients receiving no drug were 6.7823 (SD 0.563) and 6.7502 (SD 0.673), respectively. The EBA of 0.018 (SD 0.130) in patients receiving amoxicillin/clavulanic acid did not differ significantly from that of 0.016 (SD 0.069) in patients receiving no drug. It is unlikely that the combination of amoxicillin/clavulanic acid has an important place in the treatment of tuberculosis with the exception of those patients with multidrug-resistant tuberculosis who are otherwise therapeutically destitute.The early bactericidal activity (EBA) of an antituberculosis agent is the rate of decrease in viable colony-forming units (CFU) per milliliter of sputum during the first 2 d of treatment of patients with previously untreated smear-positive pulmonary tuberculosis. The objective of this open randomized study was to evaluate the EBA of the combination of amoxicillin 3 g and clavulanic acid 750 mg. Ten patients with a mean age of 34 y and a mean weight of 56 kg received amoxicillin/clavulanic acid and 5 patients with a mean age of 34 y and a mean weight of 57 kg received no drug. In the patients receiving 1 dose of amoxicillin/clavulanic acid daily for 2 d the mean log10CFU/ml of sputum before treatment was 6.7402 (SD 0.539) and after 2 d of treatment 6.7046 (SD 0.609); the corresponding values in patients receiving no drug were 6.7823 (SD 0.563) and 6.7502 (SD 0.673), respectively. The EBA of 0.018 (SD 0.130) in patients receiving amoxicillin/clavulanic acid did not differ significantly from that of 0.016 (SD 0.069) in patients receiving no drug. It is unlikely that the combination of amoxicillin/clavulanic acid has an important place in the treatment of tuberculosis with the exception of those patients with multidrug-resistant tuberculosis who are otherwise therapeutically destitute.

Mutations in the rrs A1401G gene and phenotypic resistance to amikacin and capreomycin in mycobacterium tuberculosis

The aminoglycosides amikacin (AMK)/kanamycin (KAN) and the cyclic polypeptide capreomycin (CAP) are important injectable drugs in the treatment of multidrug-resistant tuberculosis. Cross-resistance among these drug classes occurs and information on the minimum inhibitory concentrations (MICs), above the normal wild-type distribution, may be useful in identifying isolates that are still accessible to drug treatment. Isolates from the Eastern Cape Province of South Africa were subjected to DNA sequencing of the rrs (1400-1500 region) and tlyA genes. Sequencing data were compared with (i) conventional susceptibility testing at standard critical concentrations (CCs) on Middlebrook 7H11 agar and (ii) MGIT 960-based MIC determinations to assess the presence of AMK- and CAP-resistant mutants. Isolates with an rrs A1401G mutation showed high-level resistance to AMK (>20 mg/L) and decreased phenotypic susceptibility to CAP (MICs 10-15 mg/L). The MICs of CAP were below the bioavailability of the drug, which suggests that it may still be effective against multi- or extensively drug resistant tuberculosis [M(X)DR-TB]. Agar-based CC testing was found to be unreliable for resistance recognition of CAP in particular.

Emergence and treatment of multidrug resistant (MDR) and extensively drug-resistant (XDR) tuberculosis in South Africa

Drug resistant tuberculosis (TB) has reached alarming proportions in South Africa, draining valuable resources that are needed to fight drug susceptible TB. It is currently estimated that 9.6% of all TB cases have multi-drug resistant (MDR)-TB, thereby ranking South Africa as one of the highest MDR-TB burden countries in the world. Molecular epidemiological studies have demonstrated the complexity of the epidemic and have clearly shown that the epidemic is driven by transmission as a consequence of low cases detection and diagnostic delay. The latter has in turn fueled the amplification of drug resistance, ultimately leading to the emergence of extensively drug resistant (XDR)-TB. Despite the introduction of new drugs to combat this scourge, culture conversion rates for XDR-TB remain below 20%. Failure to achieve cure may be explained from DNA sequencing results which have demonstrated mutations in 7 genes encoding resistance to at least 8 anti-TB drugs. This review shows how molecular epidemiology has provided novel insights into the MDR-TB epidemic in South Africa and thereby has highlighted the challenges that need to be addressed regarding the diagnosis and treatment of MDR-TB. An important step towards for curbing this epidemic will be collaboration between clinicians, laboratories and researchers to establish scientific knowledge and medical expertise to more efficiently guide public health policy.

The rationale for using Rifabutin in the treatment of MDR and XDR tuberculosis outbreaks.

Genetically related Mycobacterium tuberculosis strains with alterations at codon 516 in the rpoB gene were observed amongst a substantial number of patients with drug resistant tuberculosis in the Eastern Cape Province (ECP) of South Africa. Mutations at codon 516 are usually associated with lower level rifampicin (RIF) resistance, while susceptibility to rifabutin (RFB) remains intact. This study was conducted to assess the rationale for using RFB as a substitution for RIF in the treatment of MDR and XDR tuberculosis outbreaks. Minimum inhibitory concentrations (MICs) of 34 drug resistant clinical isolates of M tuberculosis were determined by MGIT 960 and correlated with rpoB mutations. RFB MICs ranged from 0.125 to 0.25 µg/ml in the 34 test isolates thereby confirming phenotypic susceptibility as per critical concentration (CC) of 0.5 µg/ml. The corresponding RIF MICs ranged between 5 and 15 µg/ml, which is well above the CC of 1.0 µg/ml. Molecular-based drug susceptibility testing provides important pharmacogenetic insight by demonstrating a direct correlation between defined rpoB mutation and the level of RFB susceptibility. We suggest that isolates with marginally reduced susceptibility as compared to the epidemiological cut-off for wild-type strains (0.064 µg/ml), but lower than the current CC (≤0.5 µg/ml), are categorised as intermediate. Two breakpoints (0.064 µg/ml and 0.5 µg/ml) are recommended to distinguish between susceptible, intermediate and RFB resistant strains. This concept may assist clinicians and policy makers to make objective therapeutic decisions, especially in situations where therapeutic options are limited. The use of RFB in the ECP may improve therapeutic success and consequently minimise the risk of ongoing transmission of drug resistant M. tuberculosis strains.

The pyrazinamide susceptibility breakpoint above which combination therapy fails

Objectives To identify the pyrazinamide MIC above which standard combination therapy fails. Methods MICs of pyrazinamide were determined for Mycobacterium tuberculosis isolates, cultured from 58 patients in a previous randomized clinical trial in Cape Town, South Africa. The MICs were determined using BACTEC MGIT 960 for isolates that were collected before standard treatment with isoniazid, rifampicin, pyrazinamide and ethambutol commenced. Weekly sputum collections were subsequently made for 8 weeks in order to culture M. tuberculosis in Middlebrook broth medium. Classification and regression tree (CART) analysis was utilized to identify the pyrazinamide MIC predictive of sputum culture results at the end of pyrazinamide therapy. The machine learning-derived susceptibility breakpoints were then confirmed using standard association statistics that took into account confounders of 2 month sputum conversion. Results The pyrazinamide MIC range was 12.5 to >100 mg/L for the isolates prior to therapy. The epidemiological 95% cut-off value was >100 mg/L. The 2 month sputum conversion rate in liquid cultures was 26% by stringent criteria and 48% by less stringent criteria. CART analysis identified an MIC breakpoint of 50 mg/L, above which patients had poor sputum conversion rates. The relative risk of poor sputum conversion was 1.5 (95% CI: 1.2–1.8) for an MIC >50 mg/L compared with an MIC ≤50 mg/L. Conclusions We propose a pyrazinamide susceptibility breakpoint of 50 mg/L for clinical decision making and for development of rapid susceptibility assays. This breakpoint is identical to that identified using computer-aided simulations of hollow fibre system output.

Novel katG mutations causing isoniazid resistance in clinical M. tuberculosis isolates.

We report the discovery and confirmation of 23 novel mutations with previously undocumented role in isoniazid (INH) drug resistance, in catalase-peroxidase (katG) gene of Mycobacterium tuberculosis (Mtb) isolates. With these mutations, a synonymous mutation in fabG1g609a, and two canonical mutations, we were able to explain 98% of the phenotypic resistance observed in 366 clinical Mtb isolates collected from four high tuberculosis (TB)-burden countries: India, Moldova, Philippines, and South Africa. We conducted overlapping targeted and whole-genome sequencing for variant discovery in all clinical isolates with a variety of INH-resistant phenotypes. Our analysis showed that just two canonical mutations (katG 315AGC-ACC and inhA promoter-15C-T) identified 89.5% of resistance phenotypes in our collection. Inclusion of the 23 novel mutations reported here, and the previously documented point mutation in fabG1, increased the sensitivity of these mutations as markers of INH resistance to 98%. Only six (2%) of the 332 resistant isolates in our collection did not harbor one or more of these mutations. The third most prevalent substitution, at inhA promoter position -8, present in 39 resistant isolates, was of no diagnostic significance since it always co-occurred with katG 315. 79% of our isolates harboring novel mutations belong to genetic group 1 indicating a higher tendency for this group to go down an uncommon evolutionary path and evade molecular diagnostics. The results of this study contribute to our understanding of the mechanisms of INH resistance in Mtb isolates that lack the canonical mutations and could improve the sensitivity of next generation molecular diagnostics.