Nicola Casali

Evolution and transmission of drug-resistant tuberculosis in a Russian population

The molecular mechanisms determining the transmissibility and prevalence of drug-resistant tuberculosis in a population were investigated through whole-genome sequencing of 1,000 prospectively obtained patient isolates from Russia. Two-thirds belonged to the Beijing lineage, which was dominated by two homogeneous clades. Multidrug-resistant (MDR) genotypes were found in 48% of isolates overall and in 87% of the major clades. The most common rpoB mutation was associated with fitness-compensatory mutations in rpoA or rpoC, and a new intragenic compensatory substitution was identified. The proportion of MDR cases with extensively drug-resistant (XDR) tuberculosis was 16% overall, with 65% of MDR isolates harboring eis mutations, selected by kanamycin therapy, which may drive the expansion of strains with enhanced virulence. The combination of drug resistance and compensatory mutations displayed by the major clades confers clinical resistance without compromising fitness and transmissibility, showing that, in addition to weaknesses in the tuberculosis control program, biological factors drive the persistence and spread of MDR and XDR tuberculosis in Russia and beyond.

Microevolution of extensively drug-resistant tuberculosis in Russia.

Extensively drug-resistant (XDR) tuberculosis (TB), which is resistant to both first- and second-line antibiotics, is an escalating problem, particularly in the Russian Federation. Molecular fingerprinting of 2348 Mycobacterium tuberculosis isolates collected in Samara Oblast, Russia, revealed that 72% belonged to the Beijing lineage, a genotype associated with enhanced acquisition of drug resistance and increased virulence. Whole-genome sequencing of 34 Samaran isolates, plus 25 isolates representing global M. tuberculosis complex diversity, revealed that Beijing isolates originating in Eastern Europe formed a monophyletic group. Homoplasic polymorphisms within this clade were almost invariably associated with antibiotic resistance, indicating that the evolution of this population is primarily driven by drug therapy. Resistance genotypes showed a strong correlation with drug susceptibility phenotypes. A novel homoplasic mutation in rpoC, found only in isolates carrying a common rpoB rifampicin-resistance mutation, may play a role in fitness compensation. Most multidrug-resistant (MDR) isolates also had mutations in the promoter of a virulence gene, eis, which increase its expression and confer kanamycin resistance. Kanamycin therapy may thus select for mutants with increased virulence, helping preserve bacterial fitness and promoting transmission of drug-resistant TB strains. The East European clade was dominated by two MDR clusters, each disseminated across Samara. Polymorphisms conferring fluoroquinolone resistance were independently acquired multiple times within each cluster, indicating that XDR TB is currently not widely transmitted.

Mycobacterium tuberculosis Pyrazinamide Resistance Determinants: a Multicenter Study

ABSTRACT Pyrazinamide (PZA) is a prodrug that is converted to pyrazinoic acid by the enzyme pyrazinamidase, encoded by the pncA gene in Mycobacterium tuberculosis. Molecular identification of mutations in pncA offers the potential for rapid detection of pyrazinamide resistance (PZAr). However, the genetic variants are highly variable and scattered over the full length of pncA, complicating the development of a molecular test. We performed a large multicenter study assessing pncA sequence variations in 1,950 clinical isolates, including 1,142 multidrug-resistant (MDR) strains and 483 fully susceptible strains. The results of pncA sequencing were correlated with phenotype, enzymatic activity, and structural and phylogenetic data. We identified 280 genetic variants which were divided into four classes: (i) very high confidence resistance mutations that were found only in PZAr strains (85%), (ii) high-confidence resistance mutations found in more than 70% of PZAr strains, (iii) mutations with an unclear role found in less than 70% of PZAr strains, and (iv) mutations not associated with phenotypic resistance (10%). Any future molecular diagnostic assay should be able to target and identify at least the very high and high-confidence genetic variant markers of PZAr; the diagnostic accuracy of such an assay would be in the range of 89.5 to 98.8%. IMPORTANCE Conventional phenotypic testing for pyrazinamide resistance in Mycobacterium tuberculosis is technically challenging and often unreliable. The development of a molecular assay for detecting pyrazinamide resistance would be a breakthrough, directly overcoming both the limitations of conventional testing and its related biosafety issues. Although the main mechanism of pyrazinamide resistance involves mutations inactivating the pncA enzyme, the highly diverse genetic variants scattered over the full length of the pncA gene and the lack of a reliable phenotypic gold standard hamper the development of molecular diagnostic assays. By analyzing a large number of strains collected worldwide, we have classified the different genetic variants based on their predictive value for resistance which should lead to more rapid diagnostic tests. This would assist clinicians in improving treatment regimens for patients. Conventional phenotypic testing for pyrazinamide resistance in Mycobacterium tuberculosis is technically challenging and often unreliable. The development of a molecular assay for detecting pyrazinamide resistance would be a breakthrough, directly overcoming both the limitations of conventional testing and its related biosafety issues. Although the main mechanism of pyrazinamide resistance involves mutations inactivating the pncA enzyme, the highly diverse genetic variants scattered over the full length of the pncA gene and the lack of a reliable phenotypic gold standard hamper the development of molecular diagnostic assays. By analyzing a large number of strains collected worldwide, we have classified the different genetic variants based on their predictive value for resistance which should lead to more rapid diagnostic tests. This would assist clinicians in improving treatment regimens for patients.

Rapid diagnostics of tuberculosis and drug resistance in the industrialized world: clinical and public health benefits and barriers to implementation

In this article, we give an overview of new technologies for the diagnosis of tuberculosis (TB) and drug resistance, consider their advantages over existing methodologies, broad issues of cost, cost-effectiveness and programmatic implementation, and their clinical as well as public health impact, focusing on the industrialized world. Molecular nucleic-acid amplification diagnostic systems have high specificity for TB diagnosis (and rifampicin resistance) but sensitivity for TB detection is more variable. Nevertheless, it is possible to diagnose TB and rifampicin resistance within a day and commercial automated systems make this possible with minimal training. Although studies are limited, these systems appear to be cost-effective. Most of these tools are of value clinically and for public health use. For example, whole genome sequencing of Mycobacterium tuberculosis offers a powerful new approach to the identification of drug resistance and to map transmission at a community and population level.

Enhanced tuberculosis outbreak investigation using whole genome sequencing and IGRA

Whole genome sequencing (WGS) is a new powerful technology for characterisation of bacterial genomes and has been used successfully to investigate Mycobacterium tuberculosis isolates associated with tuberculosis (TB) outbreaks and to elucidate mutations conferring drug resistance [1–6]. Enhanced contact investigation and improved diagnosis and treatment of latent TB infection (LTBI) are an important strategy for TB control and elimination [7–10]. TB outbreak investigation can be enhanced by using whole genome sequencing, IGRA and social network analysis http://ow.ly/AzxfH

Heterogeneity of phenotypic characteristics of the modern and ancestral Beijing strains of Mycobacterium tuberculosis.

BACKGROUND The Beijing strain of Mycobacterium tuberculosis (MTB) is of great concern because this hypervirulent strain has caused numerous tuberculosis outbreaks. However, the mechanisms that allow the MTB Beijing strain to be highly pathogenic remain unclear and previous studies have revealed heterogeneity within this family. OBJECTIVE To determine the association between some phenotypic characteristics and phylogroups of the Beijing strain of MTB. METHODS Eight Beijing strains, 5 modern and 3 ancestral sublineages, were selected from the phylogroups of MTB. The selection was based on copy number of IS6110 at NTF, region of differences, and single nucleotide polymorphisms. The abilities of these strains to grow intracellularly in THP-1 macrophages, to induce apoptosis, necrosis, and cytokines production were examined using quantitative real-time PCR and commercially available ELISA kits, respectively. RESULTS There were some significant differences between the two sublineages of the Beijing strain of MTB. The ancestral Beijing sublineages showed higher intracellular growth rates (p < 0.05) and necrosis induction rates (p < 0.01) than the modern Beijing sublineages. By contrast, the modern Beijing sublineages induced lower apoptosis and protective cytokine responses, i.e., TNF-α (p < 0.05) and IL-6 (p < 0.01) and higher non-protective IL-10 response. The modern Beijing sublineages may have evolved so that they have greater ability to diminish host defense mechanisms. The slower growth rate and reduced necrosis induction in host cells might allow the bacteria to cause a persistent infection. CONCLUSION The results revealed a phylogroup-associated heterogeneity of phenotypes among MTB Beijing sublineages.

Whole Genome Sequence Analysis of a Large Isoniazid-Resistant Tuberculosis Outbreak in London: A Retrospective Observational Study

Background A large isoniazid-resistant tuberculosis outbreak centred on London, United Kingdom, has been ongoing since 1995. The aim of this study was to investigate the power and value of whole genome sequencing (WGS) to resolve the transmission network compared to current molecular strain typing approaches, including analysis of intra-host diversity within a specimen, across body sites, and over time, with identification of genetic factors underlying the epidemiological success of this cluster. Methods and Findings We sequenced 344 outbreak isolates from individual patients collected over 14 y (2 February 1998–22 June 2012). This demonstrated that 96 (27.9%) were indistinguishable, and only one differed from this major clone by more than five single nucleotide polymorphisms (SNPs). The maximum number of SNPs between any pair of isolates was nine SNPs, and the modal distance between isolates was two SNPs. WGS was able to reveal the direction of transmission of tuberculosis in 16 cases within the outbreak (4.7%), including within a multidrug-resistant cluster that carried a rare rpoB mutation associated with rifampicin resistance. Eleven longitudinal pairs of patient pulmonary isolates collected up to 48 mo apart differed from each other by between zero and four SNPs. Extrapulmonary dissemination resulted in acquisition of a SNP in two of five cases. WGS analysis of 27 individual colonies cultured from a single patient specimen revealed ten loci differed amongst them, with a maximum distance between any pair of six SNPs. A limitation of this study, as in previous studies, is that indels and SNPs in repetitive regions were not assessed due to the difficulty in reliably determining this variation. Conclusions Our study suggests that (1) certain paradigms need to be revised, such as the 12 SNP distance as the gold standard upper threshold to identify plausible transmissions; (2) WGS technology is helpful to rule out the possibility of direct transmission when isolates are separated by a substantial number of SNPs; (3) the concept of a transmission chain or network may not be useful in institutional or household settings; (4) the practice of isolating single colonies prior to sequencing is likely to lead to an overestimation of the number of SNPs between cases resulting from direct transmission; and (5) despite appreciable genomic diversity within a host, transmission of tuberculosis rarely results in minority variants becoming dominant. Thus, whilst WGS provided some increased resolution over variable number tandem repeat (VNTR)-based clustering, it was insufficient for inferring transmission in the majority of cases.

Combination of amikacin and doxycycline against multidrug-resistant and extensively drug-resistant tuberculosis

The objective of this study was to assess the activity of amikacin in combination with doxycycline against clinical strains of Mycobacterium tuberculosis in the search for new strategies against multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis. The study included 28 clinical M. tuberculosis strains, comprising 5 fully susceptible, 1 isoniazid-resistant, 17 MDR, 1 poly-resistant (streptomycin/isoniazid), 1 rifampicin-resistant and 3 XDR isolates, as well as the laboratory strain M. tuberculosis H37Rv. Minimum inhibitory concentrations (MICs) were determined using a modified chequerboard methodology in a BACTEC™ MGIT™ 960 System. Fractional inhibitory concentration indices (FICIs) were calculated, and synergy, indifference or antagonism was assessed. Whole-genome sequencing was performed to investigate the genetic basis of synergy, indifference or antagonism. The MIC50 and MIC90 values (MICs that inhibit 50% and 90% of the isolates, respectively) were, respectively, 0.5 mg/L and 1.0 mg/L for amikacin and 8 mg/L and 16 mg/L for doxycycline. The combination of amikacin and doxycycline showed a synergistic effect in 18 of the 29 strains tested and indifference in 11 strains. Antagonism was not observed. A streptomycin resistance mutation (K43R) was associated with indifference. In conclusion, the benefit of addition of doxycycline to an amikacin-containing regimen should be explored since in vitro results in this study indicate either synergy or indifference. Moreover, doxycycline also has immunomodulatory effects.

Fatal Nosocomial MDR TB Identified through Routine Genetic Analysis and Whole-Genome Sequencing

To the Editor: In November 2012, a 44-year-old HIV-negative white man (patient 1) with fever, fatigue, and breathlessness sought care at a hospital in the United Kingdom. He had never traveled abroad but had biopsy-proven alcoholic cirrhosis. No acid-fast bacilli were seen on multiple samples, including ascitic fluid, and he received treatment for presumptive abdominal tuberculosis (TB). Mycobacterium tuberculosis was subsequently cultured after 12 days. His clinical condition deteriorated, and he died of multiorgan failure 44 days after admission. The cultured M. tuberculosis was subsequently confirmed as multidrug resistant (Technical Appendix Table). Routine mycobacterial interspersed repetitive unit–variable-number tandem-repeat (MIRU-VNTR) testing was performed (1) (Technical Appendix Table). A matching MIRU-VNTR profile was identified from a 42-year-old South African–born, HIV-positive health care worker (patient 2) who had died in 2008 after admission to the same hospital. She has been described previously in detail because she had worked at Tugela Ferry hospital in KwaZulu-Natal, South Africa, which was associated with a 2005 outbreak of multidrug-resistant TB (MDR TB) and extensively drug-resistant TB (2,3) (Technical Appendix Figure 1). To ascertain whether these isolates could have matching MIRU-VNTR patterns by chance alone, we compared the MIRU-VNTR results with a national database of ≈11,745 isolates typed since the UK typing service began in 2010. Only 2 other isolates matched (from patients 3 and 4), originating from a UK hospital ≈100 miles away. Although both patients were HIV-positive health care workers from sub-Saharan Africa, no history of contact could be established with patients 1 or 2. A review of admission records established that patients 1 and 2 were admitted to the same medical ward in 2008 for 8 days, suggesting a high probability of nosocomial transmission. The ward had a traditional “Nightingale” configuration with beds for male and female patients arranged dormitory-style. In 2009, patient 1 had been identified as a contact of patient 2 and was offered screening for latent infection but had failed to attend appointments and was not under regular medical follow-up. No other common contact was identified. The estimated time from known contact between patients 1 and 2 until the clinical presentation of patient 1 was 49 months. Sequencing libraries from genomic DNA extracted from the 4 UK M. tuberculosis isolates that had matching MIRU-VNTR profiles were paired-end sequenced by using Illumina MiSeq (Illumina, San Diego, CA, USA). To investigate the origins of the infections, they were compared with 36 South Africa strains (including 1 from the Tugela Ferry outbreak [4]) sequenced by using Illumina HiSeq 2000 platforms. For each sequenced strain, a random subset of reads was aligned at ≈100× coverage to the M. tuberculosis H37Rv reference genome by using BWA version 0.5.9-r16 (5). Pilon v1.5 (http://www.broadinstitute.org/software/pilon/) was run in variant discovery to generate a list of single-nucleotide polymorphisms (SNPs) and insertions and deletions. We estimated a phylogeny using RAxML v7.7.8 (6) using a general time reversible + gamma substitution model with 1,000 bootstrap replicates. Pairwise comparison of whole-genome sequences from M. tuberculosis isolated from patients 1 and 2 found that the 2 sequences differed at only 4 SNPs (Table). Based on previous estimates of background mutations rates of 0.5 SNP/year (7), the pairwise distance between isolates from patient 1 and 2 increases confidence in the epidemiologic data implicating transmission >4 years earlier, although uncertainties exist around such estimates. Comparison between samples from patient pairs (1+2 vs. 3+4) found differences of 69–72 SNPs, which strongly argues against transmission between them. Table Pairwise distances between 2 pairs of Mycobacterium tuberculosis isolates from patients in the United Kingdom, an isolate from the 2005 Tugela Ferry outbreak in KwaZulu-Natal, South Africa (KZN605), and reference strain H37Rv In comparison with isolates sampled from KwaZulu-Natal (Technical Appendix Figure 1), isolates from patients 1 and 2 were closely related to a strain associated with the Tugela Ferry outbreak (KZN605; Technical Appendix Figure 2). Isolates from patients 3 and 4 were less closely related to isolates from the Tugela Ferry outbreak but were closely related to other isolates circulating within the region, consistent with the hypothesis that both infections originally occurred within South Africa. This investigation illustrates the power of current technology to inform our understanding of the links in MDR TB transmission between low- and high-incidence areas. Whole-genome sequencing of pathogens is becoming part of routine practice for establishing transmission and resistance patterns (8). The greater certainty it brings to transmission data can provide evidence to justify more active policies of screening and isolation as part of infection control. The nosocomial transmission described here is consistent with the fact that a person with pulmonary TB (patient 2) was managed on an open ward before being put into respiratory isolation and had not been previously screened by occupational health services. Recent data reviewing MDR TB transmission in the United Kingdom before 2007 did not identify cases of nosocomial transmission during that period (9). However, the emergence of MDR TB in regions of high HIV prevalence is relatively recent (10), and the cases described here suggest that increased vigilance for TB and MDR TB among migrating health care workers might be required. Technical Appendix: Characteristics of 4 clinical isolates of multidrug-resistant Mycobacterium tuberculosis; isolates sequenced from KwaZulu-Natal, South Africa; and phylogenetic representation of isolates collected from the United Kingdom and KwaZulu-Natal. Click here to view.(179K, pdf)

Beijing clades of Mycobacterium tuberculosis are associated with differential survival in HIV-negative Russian patients

We conducted a prospective study to establish factors associated with survival in tuberculosis patients in Russia including social, clinical and pathogen-related genetic parameters. Specifically we wished to determine whether different strains/clades of the Beijing lineage exerted a differential effect of survival. HIV-negative culture-confirmed cases were recruited during 2008-2010 across Samara Oblast and censored in December 2011. Molecular characterization was performed by a combination of spoligotyping, multilocus VNTR typing and whole genome sequencing (WGS). We analyzed 2602 strains and detected a high prevalence of Beijing family (n=1933; 74%) represented largely by two highly homogenous dominant clades A (n=794) and B (n=402) and non-A/non-B (n=737). Multivariable analysis of 1366 patients with full clinical and genotyping data showed that multi- and extensive drug resistance (HR=1.86; 95%CI: 1.52, 2.28 and HR=2.19; 95%CI: 1.55, 3.11) had the largest impact on survival. In addition older age, extensive lung damage, shortness of breath, treatment in the past and alcohol abuse reduced survival time. After adjustment for clinical and demographic predictors there was evidence that clades A and B combined were associated with poorer survival than other Beijing strains (HR=0.48; 95%CI 0.34, 0.67). All other pathogen-related factors (polymorphisms in genes plcA, plcB, plcC, lipR, dosT and pks15/1) had no effect on survival. In conclusion, drug resistance exerted the greatest effect on survival of TB patients. Nevertheless we provide evidence for the independent biological effect on survival of different Beijing family strains even within the same defined geographical population. Better understanding of the role of different strain factors in active disease and their influence on outcome is essential.