Elena Shashkina

Molecular Epidemiology of Mycobacterium tuberculosis in a South African Community with High HIV Prevalence

To explore the relationship between human immunodeficiency virus (HIV) and Mycobacterium tuberculosis genotypes, we performed IS6110-based restriction fragment-length polymorphism analysis on M. tuberculosis culture specimens from patients with smear-positive tuberculosis in a periurban community in South Africa from 2001 through 2005. Among 151 isolates, 95 strains were identified within 26 families, with 54% clustering. HIV status was associated with W-Beijing strains (P = .009) but not with clustering per se. The high frequency of clustering suggests ongoing transmission in both HIV-negative and HIV-positive individuals in this community. The strong association between W-Beijing and HIV infection may have important implications for tuberculosis control.

Transmission of TB in a high HIV prevalent South African community

BACKGROUND In settings of high tuberculosis transmission, little is known of the interaction between human immunodeficiency virus (HIV) positive and HIV-negative tuberculosis disease and of the impact of antiretroviral treatment (ART) programs on tuberculosis transmission dynamics. METHODS Mycobacterium tuberculosis isolates were collected from patients with tuberculosis who resided in a South African township with a high burden of tuberculosis and HIV infection. Demographic and clinical data were extracted from clinic records. Isolates underwent IS6110-based restriction fragment length polymorphism analysis. Patients with unique (nonclustered) M. tuberculosis genotypes and cluster index cases (ie, the first tuberculosis case in a cluster) were defined as having tuberculosis due to reactivation of latent M. tuberculosis infection. Secondary cases in clusters were defined as having tuberculosis due to recent M. tuberculosis infection. RESULTS Overall, 311 M. tuberculosis genotypes were identified among 718 isolates from 710 patients; 224 (31%) isolates were unique strains, and 478 (67%) occurred in 87 clusters. Cluster index cases were significantly more likely than other tuberculosis cases to be HIV negative. HIV-positive patients were more likely to be secondary cases (P = .001), including patients receiving ART (P = .004). Only 8% of cases of adult-adult transmission of tuberculosis occurred on shared residential plots. CONCLUSIONS Recent infection accounted for the majority of tuberculosis cases, particularly among HIV-positive patients, including patients receiving ART. HIV-negative patients may be disproportionally responsible for ongoing transmission.

Survival and replication of clinical Mycobacterium tuberculosis isolates in the context of human innate immunity.

ABSTRACT The initial host response to Mycobacterium tuberculosis is driven by innate immunity. For this study, we examined the ability of 18 recent clinical isolates and 5 reference strains to survive and replicate in the context of host innate immunity by using whole blood culture. Six healthy tuberculin-negative volunteers served as subjects. H37Ra showed the least capacity to replicate of any of the strains tested, decreasing in viability 1.3 log CFU during 72 h of whole blood culture, whereas H37Rv increased 0.32 log. Clinical isolates varied greatly in their ability to replicate in blood cells, ranging from −0.4 to +0.8 log (P < 0.001). Four showed significantly more growth than H37Rv, and one showed significantly reduced growth. Host mechanisms for restricting intracellular mycobacterial growth were more effective during the first 24 h of whole blood culture than during the 24- to 72-h period. Certain mycobacterial isolates appeared preferentially able to withstand host defenses during each of these intervals. Although there was relatively more homogeneity among subjects than among strains, one of the six subjects showed a reduced capacity to restrict intracellular mycobacterial growth due to a defect expressed during the first 24 h of culture. Our findings indicate substantial variability in the capacity of clinical tuberculosis isolates to replicate in host cells in the face of innate host immunity.

MIC of Delamanid (OPC-67683) against Mycobacterium tuberculosis Clinical Isolates and a Proposed Critical Concentration.

ABSTRACT The increasing global burden of multidrug-resistant tuberculosis (MDR-TB) requires reliable drug susceptibility testing that accurately characterizes susceptibility and resistance of pathogenic bacteria to effectively treat patients with this deadly disease. Delamanid is an anti-TB agent first approved in the European Union in 2014 for the treatment of pulmonary MDR-TB in adults. Using the agar proportion method, delamanid MIC was determined for 460 isolates: 316 from patients enrolled in a phase 2 global clinical trial, 76 from two phase 2 early bactericidal activity trials conducted in South Africa, and 68 isolates obtained outside clinical trials (45 from Japanese patients and 23 from South African patients). With the exception of two isolates, MICs ranged from 0.001 to 0.05 μg/ml, resulting in an MIC50 of 0.004 μg/ml and an MIC90 of 0.012 μg/ml. Various degrees of resistance to other anti-TB drugs did not affect the distribution of MICs, nor did origin of isolates from regions/countries other than South Africa. A critical concentration/breakpoint of 0.2 μg/ml can be used to define susceptible and resistant isolates based on the distribution of MICs and available pharmacokinetic data. Thus, clinical isolates from delamanid-naive patients with tuberculosis have a very low MIC for delamanid and baseline resistance is rare, demonstrating the potential potency of delamanid and supporting its use in an optimized background treatment regimen for MDR-TB.

Molecular epidemiology of HIV-associated tuberculosis in Dar es Salaam, Tanzania: strain predominance, clustering and polyclonal disease

ABSTRACT Molecular typing of Mycobacterium tuberculosis can be used to elucidate the epidemiology of tuberculosis, including the rates of clustering, the frequency of polyclonal disease, and the distribution of genotypic families. We performed IS6110 typing and spoligotyping on M. tuberculosis strains isolated from HIV-infected subjects at baseline or during follow-up in the DarDar Trial in Tanzania and on selected community isolates. Clustering occurred in 203 (74%) of 275 subjects: 124 (80%) of 155 HIV-infected subjects with baseline isolates, 56 (69%) of 81 HIV-infected subjects with endpoint isolates, and 23 (59%) of 39 community controls. Overall, 113 (41%) subjects had an isolate representing the East Indian “GD” family. The rate of clustering was similar among vaccine and placebo recipients and among subjects with or without cellular immune responses to mycobacterial antigens. Polyclonal disease was detected in 6 (43%) of 14 patients with multiple specimens typed. Most cases of HIV-associated tuberculosis among subjects from this study in Dar es Salaam resulted from recently acquired infection. Polyclonal infection was detected and isolates representing the East Indian GD strain family were the most common.

pncA Gene Mutations Associated with Pyrazinamide Resistance in Drug-Resistant Tuberculosis, South Africa and Georgia

Although pyrazinamide is commonly used for tuberculosis treatment, drug-susceptibility testing is not routinely available. We found polymorphisms in the pncA gene for 70% of multidrug-resistant and 96% of extensively drug-resistant Mycobacterium tuberculosis isolates from South Africa and Georgia. Assessment of pyrazinamide susceptibility may be prudent before using it in regimens for drug-resistant tuberculosis.

Confirming Mycobacterium tuberculosis transmission from a cadaver to an embalmer using molecular epidemiology

Genotyping results and epidemiologic investigation were used to confirm tuberculosis transmission from a cadaver to an embalmer. This investigation highlights the utility of genotyping in identifying unsuspected epidemiologic links and unusual transmission settings. In addition, the investigation provides additional evidence for the occupational risk of tuberculosis among funeral service workers and indicates a need for education about tuberculosis risk and the importance of adhering to appropriate infection control measures among funeral service workers.

Colonization With Levofloxacin-resistant Extended-spectrum β-Lactamase-producing Enterobacteriaceae and Risk of Bacteremia in Hematopoietic Stem Cell Transplant Recipients

Background Bacteremia caused by extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae (ESBL-E) is associated with inadequate empirical therapy and substantial mortality in neutropenic patients. Strategies are needed to identify neutropenic patients at high risk of these infections. Methods From April 2014 to September 2016, we collected perianal swabs, both at admission and weekly thereafter, from patients undergoing hematopoietic stem cell transplantation (HSCT). Patients received prophylactic levofloxacin while neutropenic. Swabs were plated onto selective agar, colonies were identified and underwent antimicrobial susceptibility testing, and phenotypic ESBL testing and polymerase chain reaction for β-lactamase genes were performed on ceftriaxone-resistant Enterobacteriaceae. We then determined the prevalence of pre-transplant ESBL-E colonization and risk of ESBL-E bacteremia. Colonizing and bloodstream isolates from patients with ESBL-E bacteremia underwent multilocus sequence typing and pulsed-field gel electrophoresis. Results We analyzed 312 patients, including 212 allogeneic and 100 autologous HSCT recipients. Ten percent (31/312) of patients had pre-transplant ESBL-E colonization. Susceptibility rates of colonizing ESBL-E were: levofloxacin, 25%; cefepime, 9%; piperacillin-tazobactam, 84%; and meropenem, 97%. Of 31 patients colonized with ESBL-E pre-transplant, 10 (32%) developed ESBL-E bacteremia during their transplant admission, compared to 1 (0.4%) of 281 patients not colonized with ESBL-E (P < .001). All bloodstream ESBL-E were levofloxacin-resistant and colonizing and bloodstream isolates from individual patients had identical genotypic profiles. Conclusions HSCT recipients who are colonized with levofloxacin-resistant ESBL-E pre-transplant and receive levofloxacin prophylaxis have high rates of bacteremia from their colonizing strain during neutropenia. Assessing for ESBL-E colonization in neutropenic patients could lead to optimization of empirical antibacterial therapy.

Exploring genotype concordance in epidemiologically linked cases of tuberculosis in New York City.

Comparing genotype results of tuberculosis (TB) isolates from individuals diagnosed with TB can support or refute transmission; however, these conclusions are based upon the criteria used to define a genotype match. We used a genotype-match definition which allowed for variation in IS6110 restriction fragment length polymorphism (RFLP) to support transmission between epidemiologically linked persons. Contacts of individuals with infectious TB (index cases) diagnosed in New York City from 1997 to 2003 who subsequently developed TB (contact cases) from 1997 to 2007 were identified. For each contact case and index case (case-pair), isolate genotypes (spoligotype and RFLP results) were evaluated. Isolates from case-pairs were classified as exact or non-exact genotype match. Genotypes from non-exact match case-pairs were reviewed at the genotyping laboratory to determine if the isolates met the near-genotype-match criteria (exactly matching spoligotype and similar RFLP banding patterns). Of 118 case-pairs identified, isolates from 83 (70%) had exactly matching genotypes and 14 (12%) had nearly matching genotypes (supporting transmission), while the remaining 21 (18%) case-pairs had discordant genotypes (refuting transmission). Using identical genotype-match criteria for isolates from case-pairs epidemiologically linked through contact investigation may lead to underestimation of transmission. TB programmes should consider the value of expanding genotype-match criteria to more accurately assess transmission between such cases.

Impact of gyrB and eis Mutations in Improving Detection of Second-Line-Drug Resistance among Mycobacterium tuberculosis Isolates from Georgia

ABSTRACT The country of Georgia has a high burden of multi- and extensively drug-resistant tuberculosis (XDR-TB). To evaluate whether mutations in gyrB and eis genes increased the sensitivity of detection of phenotypic resistance to ofloxacin and kanamycin or capreomycin compared to use of the first-generation MTBDRsl assay alone, which tests for mutations in gyrA and rrs genes, a retrospective study of stored Mycobacterium tuberculosis isolates was performed. All isolates underwent DNA sequencing of resistance-determining regions. Among 112 M. tuberculosis isolates with DNA extraction data, targeted sequencing was successfully performed for each gene as follows: for gyrA, 98% sensitivity; for gyrB, 96%; for rrs, 93%; for the eis gene and its promoter, 93%. The specificity and hence the positive predictive value of gyrA and gyrB mutations for detecting ofloxacin resistance were 100%. The addition of gyrB mutations increased the sensitivity of phenotypic ofloxacin resistance detection by 13% (75% to 88%). All rrs resistance-conferring mutations were A1401G, and this mutation had low sensitivity (40% and 18%) and high specificity (95% and 100%) in predicting phenotypic capreomycin and kanamycin resistance, respectively. The eis C-14T mutation increased the sensitivity of phenotypic kanamycin resistance detection by 9% (18% to 27%) and was found solely in kanamycin phenotypic resistance isolates. Our data showed that the inclusion of eis C-14T and gyrB mutations in addition to rrs and gyrA mutations improves the sensitivity of detection of phenotypic ofloxacin and kanamycin resistance, respectively.