Sami O. Simons

Validation of pncA Gene Sequencing in Combination with the Mycobacterial Growth Indicator Tube Method To Test Susceptibility of Mycobacterium tuberculosis to Pyrazinamide

ABSTRACT Pyrazinamide is important in the treatment of tuberculosis. Unfortunately, the diagnosis of pyrazinamide resistance is hampered by technical difficulties. We hypothesized that mutation analysis combined with the mycobacterial growth indicator tube (MGIT) phenotypic method would be a good predictor of pyrazinamide resistance. We prospectively analyzed 1,650 M. tuberculosis isolates referred to our tuberculosis reference laboratory in 2008 and 2009. In our laboratory, the MGIT 960 system was used for pyrazinamide resistance screening. If a pyrazinamide-resistant strain was detected, we performed a pncA gene mutation analysis. A second MGIT 960 susceptibility assay was performed afterwards to evaluate the accuracy of the pncA mutation analysis to detect true- or false-positive MGIT results. We observed pyrazinamide resistance in 69 samples using the first MGIT 960 analysis. In a second MGIT 960 analysis, 47 of the 69 samples proved susceptible (68% false positivity). Sensitivity of nonsynonymous pncA mutations for detecting resistant isolates was 73% (95% confidence interval [CI], 61% to 73%), and specificity was 100% (95% CI, 95% to 100%). A diagnostic algorithm incorporating phenotypic and molecular methods would have a 100% positive predictive value for detecting pyrazinamide-resistant isolates, indicating that such an algorithm, based on both methods, is a good predictor for pyrazinamide resistance in routine diagnostics.

Comparative Study on Genotypic and Phenotypic Second-Line Drug Resistance Testing of Mycobacterium tuberculosis Complex Isolates

ABSTRACT The mycobacterium growth indicator tube (MGIT960) automated liquid medium testing method is becoming the international gold standard for second-line drug susceptibility testing of multidrug- and extensively drug-resistant Mycobacterium tuberculosis complex isolates. We performed a comparative study of the current gold standard in the Netherlands, the Middlebrook 7H10 agar dilution method, the MGIT960 system, and the GenoType MTBDRsl genotypic method for rapid screening of aminoglycoside and fluoroquinolone resistance. We selected 28 clinical multidrug- and extensively drug-resistant M. tuberculosis complex strains and M. tuberculosis H37Rv. We included amikacin, capreomycin, moxifloxacin, prothionamide, clofazimine, linezolid, and rifabutin in the phenotypic test panels. For prothionamide and moxifloxacin, the various proposed breakpoint concentrations were tested by using the MGIT960 method. The MGIT960 method yielded results 10 days faster than the agar dilution method. For amikacin, capreomycin, linezolid, and rifabutin, results obtained by all methods were fully concordant. Applying a breakpoint of 0.5 μg/ml for moxifloxacin led to results concordant with those of both the agar dilution method and the genotypic method. For prothionamide, concordance was noted only at the lowest and highest MICs. The phenotypic methods yielded largely identical results, except for those for prothionamide. Our study supports the following breakpoints for the MGIT960 method: 1 μg/ml for amikacin, linezolid, and clofazimine, 0.5 μg/ml for moxifloxacin and rifabutin, and 2.5 μg/ml for capreomycin. No breakpoint was previously proposed for clofazimine. For prothionamide, a division into susceptible, intermediate, and resistant seems warranted, although the boundaries require additional study. The genotypic assay proved a reliable and rapid method for predicting aminoglycoside and fluoroquinolone resistance.

Prevalence and Localization of Pulmonary Embolism in Unexplained Acute Exacerbations of COPD: A Systematic Review and Meta-analysis

BACKGROUND: Patients with COPD experience episodes of increased inflammation, so‐called acute exacerbations of COPD (AE‐COPD). In 30% of AE‐COPD cases, no clear cause is found. Since there is well‐known cross talk between inflammation and thrombosis, the objectives of this study were to determine the prevalence, embolus localization, clinical relevance, and clinical markers of pulmonary embolism (PE) in unexplained AE‐COPD. METHODS: A systematic search was performed using MEDLINE and EMBASE platforms from 1974 to October 2015. Prospective and cross‐sectional studies that included patients with AE‐COPD and used pulmonary CT‐angiography for diagnosis of PE were included. RESULTS: The systematic search resulted in 1,650 records. The main reports of 22 articles were reviewed, and 7 studies were included. The pooled prevalence of PE in unexplained AE‐COPD was 16.1% (95% CI, 8.3%‐25.8%) in a total of 880 patients. Sixty‐eight percent of the emboli found were located in the main pulmonary arteries, lobar arteries, or interlobar arteries. Mortality and length of hospital admission seemed to be increased in patients with unexplained AE‐COPD and PE. Pleuritic chest pain and cardiac failure were more frequently reported in patients with unexplained AE‐COPD and PE. In contrast, signs of respiratory tract infection was less frequently related to PE. CONCLUSIONS: PE is frequently seen in unexplained AE‐COPD. Two‐thirds of emboli are found at locations that have a clear indication for anticoagulant treatment. These findings merit clinical attention. PE should receive increased awareness in patients with unexplained AE‐COPD, especially when pleuritic chest pain and signs of cardiac failure are present, and no clear infectious origin can be identified.

The Prevalence of Pulmonary Embolism in Acute Exacerbations of COPD

With great interest we read the systematic review by Rizkallah et al1 in CHEST reporting a high prevalence of pulmonary embolism (PE) during an acute exacerbation of COPD (AECOPD). The authors suggest that one of four COPD patients who require hospitalization for an acute exacerbation may have a PE. In our opinion however, the results of their study are skewed due to inadequate study sampling and a potential selection bias. For their review, Rizkallah et al1 selected five studies.2–6 Two studies2,3 did not estimate the prevalence of PE during an AECOPD, but rather calculated the coexistence of COPD in patients with a PE. Assuming that every patient with a PE presents with a AECOPD is in our opinion not justified. The three other studies selected4–6 display a high heterogeneity in PE prevalence. Rizkallah and colleagues1 argue that the higher prevalence is seen exclusively in hospitalized patients. However, all three studies also included patients from the emergency department. In our opinion, the discrepancy is due to a selection bias. In two studies,5,6 patients with an obvious infective cause of their AECOPD were excluded. Moreover, one of these studies6 only included patients with a d-dimer level 500 ng/mL. The only study4 not excluding these patient categories showed a low PE prevalence of 3%. We therefore argue that it may be both the exclusion of certain patient categories as well as the erroneous selection of studies that has artificially augmented the result presented by Rizkallah and colleagues.1 The prevalence of PE in AECOPD remains open to debate.

Drug susceptibility testing for optimizing tuberculosis treatment.

The principles of our current drug susceptibility testing (DST) for tuberculosis (TB) have already been laid out in 1963. Since then, DST has not gained much popularity owing to the long turn-around time and the introduction of potent antituberculosis drug regimens. These and other barriers have led to a critical gap in laboratory capacity in DST of Mycobacterium tuberculosis. However, owing to the emergence of multidrug resistant tuberculosis there is a pressing need for adequate and rapid DST. In recent years, methods for fastening the diagnosis of drug resistant tuberculosis have been developed. Semi-automated (non)- radiometric liquid culture systems reduced the turn-around-time significantly. With the introduction of molecular diagnostic methods, such as reverse line probes and the recently introduced semi-automated real-time PCR, the turn-around time of at least an indicative resistance testing has dropped from days to hours. However, much more can be gained in the development of fast phenotypic and molecular DST methodologies. Recently also pharmacodynamic studies have also added significantly to our understanding of resistance development in tuberculosis treatment. This article provides an overview of the most important DST techniques now available, with their characteristics, biosafety aspects, reproducibility and required quality control. Also the findings in pharmacodynamic studies and required future research are discussed. We will argue that drug susceptibility testing in TB treatment is an essential tool for adequate TB control and prevention of resistance and should be applied to all patients to guide TB treatment. Perhaps in the near future even individualized treatment doses could be an important help to prevent further emergence or further development of resistance.

Rapid diagnosis of pyrazinamide-resistant multidrug-resistant tuberculosis using a molecular-based diagnostic algorithm

There is an urgent need for rapid and accurate diagnosis of pyrazinamide-resistant multidrug-resistant tuberculosis (MDR-TB). No diagnostic algorithm has been validated in this population. We hypothesized that pncA sequencing added to rpoB mutation analysis can accurately identify patients with pyrazinamide-resistant MDR-TB. We identified from the Dutch national database (2007-11) patients with a positive Mycobacterium tuberculosis culture containing a mutation in the rpoB gene. In these cases, we prospectively sequenced the pncA gene. Results from the rpoB and pncA mutation analysis (pncA added to rpoB) were compared with phenotypic susceptibility testing results to rifampicin, isoniazid and pyrazinamide (reference standard) using the Mycobacterial Growth Indicator Tube 960 system. We included 83 clinical M. tuberculosis isolates containing rpoB mutations in the primary analysis. Rifampicin resistance was seen in 72 isolates (87%), isoniazid resistance in 73 isolates (88%) and MDR-TB in 65 isolates (78%). Phenotypic reference testing identified pyrazinamide-resistant MDR-TB in 31 isolates (48%). Sensitivity of pncA sequencing added to rpoB mutation analysis for detecting pyrazinamide-resistant MDR-TB was 96.8%, the specificity was 94.2%, the positive predictive value was 90.9%, the negative predictive value was 98.0%, the positive likelihood was 16.8 and the negative likelihood was 0.03. In conclusion, pyrazinamide-resistant MDR-TB can be accurately detected using pncA sequencing added to rpoB mutation analysis. We propose to include pncA sequencing in every isolate with an rpoB mutation, allowing for stratification of MDR-TB treatment according to pyrazinamide susceptibility.

Molecular drug susceptibility testing in the Netherlands: performance of the MTBDRplus and MTBDRsl assays.

BACKGROUND The performance of molecular drug susceptibility testing in countries with a low prevalence of drug resistance, such as the Netherlands, has not been adequately studied. OBJECTIVE To evaluate the diagnostic accuracy of the GenoType(®) MTBDRplus and MTBDRsl assays to detect resistance to first- and second-line anti-tuberculosis drugs in the context of a nationwide screening programme in the Netherlands. RESULTS The MTBDRplus assay had a sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of 100%, 99%, 80% and 100% for detecting rifampicin resistance. The sensitivity, specificity, PPV and NPV of either a katG or inhA mutation for detecting isoniazid resistance were 88%, 100%, 100% and 99%. The MTBDRsl assay had a sensitivity, specificity, PPV and NPV of 100%, 99%, 83%, and 100% for detecting moxifloxacin resistance; 62%, 71%, 58% and 74%, respectively, for detecting ethambutol resistance; 86%, 99%, 86% and 99% for detecting amikacin resistance; and 50%, 96%, 71% and 91% for detecting capreomycin resistance. CONCLUSION The MTBDRplus and MTBDRsl assays may aid in decision making in tuberculosis treatment in low-level drug resistance settings and should preferably be used to exclude resistance.

Montelukast for bronchiolitis obliterans syndrome after lung transplantation.

www.chestpubs.org association. Further, nearly all published studies uniformly recommend that the inclusion of atelectasis as a cause of postoperative fever be discarded as historical conjecture and unsupported dogma. Mavros and colleagues 1 conclude that there is a need for additional large studies to precisely evaluate whether there is an association that the prior studies have not detected. We respectfully disagree, given the fi ndings of our most recent study. From our perspective, based on our large cohort of patients and the outcome of our study, rather than performing additional studies, it would seem more prudent and appropriate to simply discard old dogma.

Tissue plasminogen activator and DNase in empyema.

To the Editor: Rahman and colleagues (Aug. 11 issue)1 found improved fluid drainage in patients with pleural infections and a reduced frequency of surgical referral and duration of hospital stay after combined therapy with intrapleural tissue plasminogen activator (t-PA) and DNase. In pleural effusions, the viscosity correlates with the concentrations of albumin, protein, and lactate dehydrogenase.2,3 In an experimental model, the peak in lactate dehydrogenase levels occurs about 12 hours after the peak in numbers of inflammatory cells in the pleural fluid.4 Hence, antibiotic treatment before randomization might have had an influence on the pleural inflammatory response and consequently on the results. Can the authors provide information about antibiotic treatment before randomization and chest-tube drainage?

The effects of signal transducer and activator of transcription three mutations on human platelets

Abstract Involvement of signal transducer and activator of transcription 3 (STAT3) in inflammation is well known. Recently, a role for STAT3 in platelet activation and platelet production has been suggested. Platelets exhibit important immune functions and engagement of STAT3 in platelet physiology may link inflammation and hemostasis. This study investigated the effects of STAT3 loss-of-function mutations and single nucleotide polymorphisms (SNPs) in STAT3 on glycoprotein VI (GPVI)-mediated platelet activation and platelet numbers in humans. Two cohorts were studied. The first cohort concerned patients with STAT3 loss-of-function mutations. Platelet numbers were investigated in eight patients and GPVI-mediated platelet activation was functionally tested in four patients. Additional experiments were performed to investigate underlying mechanisms. The second cohort concerned 334 healthy volunteers and investigated the consequences of SNPs in STAT3 on GPVI-mediated platelet activation and platelet numbers. Platelet activation was lower in STAT3 loss-of-function patients at baseline and after stimulation of the GPVI receptor, reflected by decreased P-selectin expression. This was independent of gene transcription. Blockade of the adenosine di-phosphate (ADP) pathway resulted in a further decrease of P-selectin expression, particularly in STAT3 loss-of-function patients. In contrast, the SNPs in STAT3 did not influence GPVI-mediated platelet activation. Also, platelet numbers were not affected by STAT3 loss-of-function mutations, nor was there an association with the SNPs. In conclusion, STAT3 signaling does not seem to play a major role in thrombopoiesis. We confirm that STAT3 is involved in GPVI-mediated platelet activation in humans, independent of gene transcription. GPVI-mediated platelet activation is highly dependent on secondary ADP release. Our findings suggest that STAT3 modulation may affect inflammation, hemostasis, and their interaction.