Pharmacokinetic-Pharmacodynamic Determinants of Clinical Outcomes for Rifampin-Resistant Tuberculosis: A Multisite Prospective Cohort Study.

BACKGROUND: Rifampin-resistant and/or multidrug-resistant tuberculosis (RR/MDR-TB) treatment requires multiple drugs, and outcomes remain suboptimal. Some drugs are associated with improved outcome. It is unknown whether particular pharmacokinetic-pharmacodynamic relationships predict outcome. METHODS: Adults with pulmonary RR/MDR-TB in Tanzania, Bangladesh, and the Russian Federation receiving local regimens were enrolled from June 2016 to July 2018. Serum was collected after 2, 4, and 8 weeks for each drug's area under the concentration-time curve over 24 hours (AUC0-24). Quantitative susceptibility of the M. tuberculosis isolate was measured by minimum inhibitory concentrations (MICs). Individual drug AUC0-24/MIC targets were assessed by adjusted odds ratios (ORs) for favorable treatment outcome, and hazard ratios (HRs) for time to sputum culture conversion. K-means clustering algorithm separated the cohort of the most common multidrug regimen into 4 clusters by AUC0-24/MIC exposures. RESULTS: Among 290 patients, 62 (21%) experienced treatment failure, including 30 deaths. Moxifloxacin AUC0-24/MIC target of 58 was associated with favorable treatment outcome (OR, 3.75; 95% confidence interval, 1.21-11.56; P = .022); levofloxacin AUC0-24/MIC of 118.3, clofazimine AUC0-24/MIC of 50.5, and pyrazinamide AUC0-24 of 379 mg × h/L were associated with faster culture conversion (HR >1.0, P < .05). Other individual drug exposures were not predictive. Clustering by AUC0-24/MIC revealed that those with the lowest multidrug exposures had the slowest culture conversion. CONCLUSIONS: Amidst multidrug regimens for RR/MDR-TB, serum pharmacokinetics and M. tuberculosis MICs were variable, yet defined parameters to certain drugs-fluoroquinolones, pyrazinamide, clofazimine-were predictive and should be optimized to improve clinical outcome. CLINICAL TRIALS REGISTRATION: NCT03559582.

Insight into pathogenomics and phylogeography of hypervirulent and highly-lethal Mycobacterium tuberculosis strain cluster.

BACKGROUND: . The Mycobacterium tuberculosis Beijing genotype is globally spread lineage with important medical properties that however vary among its subtypes. M. tuberculosis Beijing 14717-15-cluster was recently discovered as both multidrug-resistant, hypervirulent, and highly-lethal strain circulating in the Far Eastern region of Russia. Here, we aimed to analyze its pathogenomic features and phylogeographic pattern. RESULTS: . The study collection included M. tuberculosis DNA collected between 1996 and 2020 in different world regions. The bacterial DNA was subjected to genotyping and whole genome sequencing followed by bioinformatics and phylogenetic analysis. The PCR-based assay to detect specific SNPs of the Beijing 14717-15-cluster was developed and used for its screening in the global collections. Phylogenomic and phylogeographic analysis confirmed endemic prevalence of the Beijing 14717-15-cluster in the Asian part of Russia, and distant common ancestor with isolates from Korea (> 115 SNPs). The Beijing 14717-15-cluster isolates had two common resistance mutations RpsL Lys88Arg and KatG Ser315Thr and belonged to spoligotype SIT269. The Russian isolates of this cluster were from the Asian Russia while 4 isolates were from the Netherlands and Spain. The cluster-specific SNPs that significantly affect the protein function were identified in silico in genes within different categories (lipid metabolism, regulatory proteins, intermediary metabolism and respiration, PE/PPE, cell wall and cell processes). CONCLUSIONS: . We developed a simple method based on real-time PCR to detect clinically significant MDR and hypervirulent Beijing 14717-15-cluster. Most of the identified cluster-specific mutations were previously unreported and could potentially be associated with increased pathogenic properties of this hypervirulent M. tuberculosis strain. Further experimental study to assess the pathobiological role of these mutations is warranted.

Insight into Population Structure and Drug Resistance of Pediatric Tuberculosis Strains from China and Russia Gained through Whole-Genome Sequencing.

This study aimed to determine phenotypic and genotypic drug resistance patterns of Mycobacterium tuberculosis strains from children with tuberculosis (TB) in China and Russia, two high-burden countries for multi/extensively-drug resistant (MDR/XDR) TB. Whole-genome sequencing data of M. tuberculosis isolates from China (n = 137) and Russia (n = 60) were analyzed for phylogenetic markers and drug-resistance mutations, followed by comparison with phenotypic susceptibility data. The Beijing genotype was detected in 126 Chinese and 50 Russian isolates. The Euro-American lineage was detected in 10 Russian and 11 Chinese isolates. In the Russian collection, the Beijing genotype and Beijing B0/W148-cluster were dominated by MDR strains (68% and 94%, respectively). Ninety percent of B0/W148 strains were phenotypically pre-XDR. In the Chinese collection, neither of the Beijing sublineages was associated with MDR/pre-XDR status. MDR was mostly caused by low fitness cost mutations (rpoB S450L, katG S315T, rpsL K43R). Chinese rifampicin-resistant strains demonstrated a higher diversity of resistance mutations than Russian isolates (p = 0.003). The rifampicin and isoniazid resistance compensatory mutations were detected in some MDR strains, but they were not widespread. The molecular mechanisms of M. tuberculosis adaptation to anti-TB treatment are not unique to the pediatric strains, but they reflect the general situation with TB in Russia and China.

Thermal tolerance of Cyclops kolensis lilljeborg, 1901 (Copepoda: Cyclopoida) and its relationships with pessimal to optimal performance.

Cyclops kolensis Lilljeborg, 1901 belongs to the Arctic complex of Palaeoarctic species, yet in the past 20 years, its occurrence has extended to the summer months in waterbodies with high water temperatures. This species is considered one of the most active migrants from the northern waterbodies in the Volga reservoir cascade to the Volga delta. Here, we explored the ranges of the preferred and avoidance temperatures of C. kolensis from two geographically isolated populations. Thermal tolerance was measured in a thermogradient installation and compared to the temperatures at which members of these populations occurred in their source waterbodies. Temperature preference was determined using the "chronic" method. Individuals of C. kolensis possessed a bimodal final temperature preferendum of 2-6 °C and 13-21 °C, which corresponds to the optimal thermal conditions of the species in a pond. These ranges were the same for individuals of both populations irrespective of the geographical location and water temperature of their source waterbodies. The temperature range of normal performance was 2-4 to 21-25 °C, and the pessimal temperature ranges were from 1 - 2 to 3-4 °C and from 22-25 °C to 26-30°Ð¡. These temperature ranges coincide with field observations over a recent 20-year period of temperature conditions under which the species develops in nature. Our results allow us to characterize C. kolensis as an ecologically plastic species, which, despite its strong association with the cold-water Complex species, is adapting to a wider temperature range as global warming occurs.

New epidemic cluster of pre-extensively drug resistant isolates of Mycobacterium tuberculosis Ural family emerging in Eastern Europe.

BACKGROUND: Ural genetic family is a part of the Euro-American lineage of Mycobacterium tuberculosis and is endemic in Northern Eurasia (former Soviet Union [FSU]). These strains were long described as drug susceptible and of low virulence, but recent studies reported an increasing circulation of the multidrug-resistant (MDR) and extensively drug-resistant (XDR) Ural strains. Here, we analyzed all publicly available whole genome sequence data of Ural genotype isolates, in order to elucidate their phylogenomic diversity with a special focus on MDR and potentially epidemic clones. RESULTS: A total of 149 M. tuberculosis genomes of Ural isolates from FSU countries were mined from the GMTV database and TB-ARC project. We identified 6002 variable amino acid positions that were assessed for functional significance and used to build ML, NJ trees and for Bayesian TMRCA estimation. Three robust monophyletic clades were identified: Clade A (31 isolates from Russia, Belarus, Moldova), Clade B (52 isolates from Russia), and Clade C (37 isolates from Moldova, 2 from Belarus). Clade C was significantly associated with XDR or pre-XDR status compared to the pooled Clades A and B (33/39 versus 5/83, P < 0.0001). Time of origin was estimated for Clade A at 77.7-137 years ago and for Clade B at 56.3-99.2 years ago compared to the significantly more recent origin for Clade C. in silico spoligotyping identified signatures specific of the Clade A (spoligotype SIT35), and Clades B and C (both SIT262). CONCLUSIONS: A genetically compact and evolutionarily young Ural Clade C, likely originated after collapse of the Soviet Union, and reached epidemic proportions in Moldova in the last 20 years. This epidemic pre-XDR clone (mostly rifampin, isoniazid and kanamycin resistant) is characterized by a specific combination of mutations: KatG Ser315Thr, fabG1 -15C > T, RpoB Ser450Leu, RpsL Lys88Arg, eis -12G > A and EmbB Ser297Ala/T > G. Its further dissemination may occur towards both Russia and European Union and should be taken into consideration by health authorities. The identified spoligotyping signatures can serve for rapid preliminary detection and surveillance of the more hazardous pre-XDR associated strains of the Ural family, both in populations from countries of their endemic circulation and migrant communities.

The preferred and avoidance temperatures of Thermocyclops crassus (Fischer, 1853) and their relation to the temperature of optimal, pessimal and normal performance of the species.

The ranges of the preferred and avoided temperatures in representatives of Thermocyclops crassus (Fischer, 1853) were determined by the results of experimental testing in a thermogradient aparatus and comparison of the obtained values with field observations of the optimal, pessimal and tolerated temperature conditions of development of these populations in nature. Copepods were sampled from pond located near Borok, Yaroslavl region, Russia (58o02'57'' N; 38o14'56'' E). The ambient water temperature was 15.0°Ð¡. Temperature preference was determined by the "chronic" method. The phenology of development of Th. crassus was observed in the field of water bodies in Central and Volga Federal Districts of Russia. The final thermal preferendum (FTP) achievement occurred with an increase in the preferred temperature: when animals were placed at a temperature in the thermal gradient (14.0-15.0°C) that approximated the temperature of the source pond, they moved to warmer water until they chose FTP (26.7°C). Obtained the values of FTP (25-30°C), temperature of normal performance (21-32°C) and pessimal temperatures of 9-20 and 33°Ð¡ well coincide with numerous field observations for temperature conditions of development of species in northern waterbodies of Holarctic and with the temperatures at which populations of Th. crassus thrive in southern waterbodies of Holarctic and in tropical lakes. It is concluded that, despite the historically long existence of the species in the reservoirs of the temperate climatic zone, the northern populations of Th. crassus retained the temperature responses characteristic of their southern sister populations. And although the species has adapted to life in northern reservoirs at lower temperatures, when it becomes possible to choose, cyclops prefer temperatures above 25°C, which are optimal for southern populations living in tropical waters. These data once again confirm that the horizontal thermal gradient method can be used to infer temperature tolerance of freshwater cyclopoid copepods in nature.

eis Promoter C14G and C15G Mutations Do Not Confer Kanamycin Resistance in Mycobacterium tuberculosis.

We studied the significance of particular eis mutations on Mycobacterium tuberculosis drug resistance using a specialized transduction strategy. Recombinant strains harboring eis promoter mutations C14T, C12T, and G10A exhibited kanamycin resistance with MICs of 40, 10, and 20 µg/ml, respectively, while recombinant strains harboring C14G and C15G mutations were kanamycin susceptible (MIC, 2.5 to 5 µg/ml). Each of the eis mutants tested remained amikacin susceptible (MIC, 0.5 to 4 µg/ml). The identification of specific eis mutations is needed for accurate genotypic susceptibility testing for kanamycin.

Latin-American-Mediterranean lineage of Mycobacterium tuberculosis: Human traces across pathogen's phylogeography.

Currently, Mycobacterium tuberculosis isolates of Latin-American Mediterranean (LAM) family may be detected far beyond the geographic areas that coined its name 15years ago. Here, we established the framework phylogeny of this geographically intriguing and pathobiologically important mycobacterial lineage and hypothesized how human demographics and migration influenced its phylogeography. Phylogenetic analysis of LAM isolates from all continents based on 24 variable number of tandem repeats (VNTR) loci and other markers identified three global sublineages with certain geographic affinities and defined by large deletions RD115, RD174, and by spoligotype SIT33. One minor sublineage (spoligotype SIT388) appears endemic in Japan. One-locus VNTR signatures were established for sublineages and served for their search in published literature and geographic mapping. We suggest that the LAM family originated in the Western Mediterranean region. The most widespread RD115 sublineage seems the most ancient and encompasses genetically and geographically distant branches, including extremely drug resistant KZN in South Africa and LAM-RUS recently widespread across Northern Eurasia. The RD174 sublineage likely started its active spread in Brazil; its earlier branch is relatively dominated by isolates from South America and the derived one is dominated by Portuguese and South/Southeastern African isolates. The relatively most recent SIT33-sublineage is marked with enigmatic gaps and peaks across the Americas and includes South African clade F11/RD761, which likely emerged within the SIT33 subpopulation after its arrival to Africa. In addition to SIT388-sublineage, other deeply rooted, endemic LAM sublineages may exist that remain to be discovered. As a general conclusion, human mass migration appears to be the major factor that shaped the M. tuberculosis phylogeography over large time-spans.

Primary multidrug-resistant Mycobacterium tuberculosis in 2 regions, Eastern Siberia, Russian Federation.

Of 235 Mycobacterium tuberculosis isolates from patients who had not received tuberculosis treatment in the Irkutsk oblast and the Sakha Republic (Yakutia), eastern Siberia, 61 (26%) were multidrug resistant. A novel strain, S 256, clustered among these isolates and carried eis-related kanamycin resistance, indicating a need for locally informed diagnosis and treatment strategies.

Biodiversity of aquatic organisms in the Mekong Delta, Vietnam.

Background: The Mekong River is the 10th largest river in the world. It is recognised as the most productive river in Southeast Asia and economically essential to the region, with an estimated 60-65 million people living in the lower Mekong Basin. The Mekong Delta within Vietnam is considered a highly vulnerable ecosystem under threat from increasing anthropogenic pressure, such as dam construction and, as a consequence, the Delta is sinking and altering the natural hydrological cycle. Dams also lead to eutrophication and pollution of downstream water from regulated water flux and water stagnation. Another threat is climate change coupled with the lower rainfall, which could lead to an increased risk of drought in the Mekong Delta Basin. Thus, these project data represent an important baseline reference. The ecological health of the Mekong Delta's environment, as indicated by the quality and availability of its water and biological resources, largely determines the economic and social development of the region, which produces about half of the agriculture and aquaculture products of Vietnam. New information: This paper reports quantitative data on the biodiversity of six groups of aquatic organisms: bottom and pelagic fish, macrozoobenthos, microorganisms, phyto- and zooplankton in the Mekong Delta within Vietnam, as well as data on the physicochemical parameters of water and bottom sediments. The data were collected during 2018-2022 as part of the Ecolan E-3.4 programme within the framework of the research plan of the Joint Russian-Vietnamese Tropical Research and Technological Center. All presented datasets are published for the first time.

Transborder molecular analysis of drug-resistant tuberculosis in Mongolia and Eastern Siberia, Russia.

Eastern Siberia (Russia) and Mongolia are borderline regions in Asia with a high incidence of tuberculosis (TB). In this study, we investigated the transborder transmission of Mycobacterium tuberculosis with a focus on endemic and epidemic clones and drug resistance. M. tuberculosis isolates (287 from Mongolia and 754 from Russia) were collected using cross-sectional population-based surveys between 2010 and 2016. The isolates were genotyped using 24 variable number of tandem repeat loci and by testing of the key markers to discriminate within the Beijing genotype. All isolates were divided into 427 mycobacterial interspersed repetitive units types that were assigned to Lineage 2 (Beijing) and Lineage 4 (Ural, Haarlem, Latin American-Mediterranean [LAM], S, unclassified). The Beijing genotype was dominant in both countries (69% in Russia, 75% in Mongolia). However, the Beijing isolates differed significantly between the countries, in terms of the identified subtypes. LAM was the most common non-Beijing genotype (11.1% in Mongolia and 14.9% in Russia) and LAM isolates mostly belonged to the LAM-RUS branch in both countries. The multidrug-resistance (MDR) rate was higher in Russia than in Mongolia among newly diagnosed patients: 29.4% versus 5.6% (p < .001). In Mongolia, the MDR rate was similar in Beijing (29.7%) and non-Beijing (27.5%) genotypes. In Russia, a higher MDR rate was observed in (i) Beijing compared with non-Beijing (48.7% versus 38.3%, p = .03) and (ii) Beijing B0/W148 compared with Beijing Central Asian/Russian (63.4% versus 37.3%, p < .001). In conclusion, the M. tuberculosis population structure in Mongolia was shaped by mainly historical interaction with China (dominance of the Beijing genotype) and Northern Eurasia (presence of the LAM-RUS branch). In contrast, the transborder transmission of M. tuberculosis since the 1990s between Mongolia and its neighbours has been negligible, and the adverse trends of MDR-TB in Russia did not impact the current situation in Mongolia.

Influence of hypomagnetic field on the heartbeat in zebrafish embryos.

The magnetic environment may influence the functioning of the cardiovascular system. It was reported that low-frequency and static magnetic fields affect hemodynamics, heart rate, and heart rate variability in animals and humans. Moreover, recent data suggest that magnetic fields affect the circadian rhythms of physiological processes. The influence of the magnetic environment on heart functionating during early development has been studied insufficiently. We utilized transparent zebrafish embryos to evaluate the effect of the hypomagnetic field on the characteristics of cardiac function using a noninvasive optical approach based on photoplethysmographic microscopic imaging. The embryos were exposed to the geomagnetic and hypomagnetic fields from the second to the 116th hour post fertilization under a 16 h light/8 h dark cycle or constant illumination. The exposure of embryos to the hypomagnetic field in both lighting modes led to increased embryo mortality, the appearance of abnormal phenotypes, and a significant increase in the embryo's heartbeat rate. The difference between maximal and minimal heartbeat intervals, maximal to minimal heartbeat intervals ratio, and the coefficient of variation of heartbeat rate were increased in the embryos exposed to the hypomagnetic field under constant illumination from 96 to 116 h post fertilization. The dynamics of heartbeat rate changes followed a circadian pattern in all studied groups except zebrafish exposed to the hypomagnetic field under constant illumination. The results demonstrate the importance of natural magnetic background for the early development of zebrafish. The possible mechanisms of observed effects are discussed.

Process evaluation for the adaptation, testing and dissemination of a mobile health platform to support people with HIV and tuberculosis in Irkutsk, Siberia.

OBJECTIVES: We developed and tested a mobile health-based programme to enhance integration of HIV and tuberculosis (TB) care and to promote a patient-centred approach in a region of high coinfection burden. Phases of programme development included planning, stakeholder interviews and platform re-build, testing and iteration. SETTING: In Irkutsk, Siberia, HIV/TB coinfection prevalence is high relative to the rest of the Russian Federation. PARTICIPANTS: Pilot testing occurred for a cohort of 60 people with HIV and TB. RESULTS: Key steps emerged to ensure the mobile health-based programme could be operational and adequately adapted for the context, including platform language adaptation, optimisation of server management, iteration of platform features, and organisational practice integration. Pilot testing of the platform rebuild yielded favourable patient perceptions of usability and acceptability at 6 months (n=47 surveyed), with 18 of 20 items showing scores above 4 (on a scale from 1 to 5) on average. Development of this mobile health-based programme for integrated care of infections highlighted the importance of several considerations for tailoring these interventions contextually, including language adaptation and technological capacity, but also, importantly, contextualised patient preferences related to privacy and communication with peers and/or providers, existing regional capacity for care coordination of different comorbidities, and infection severity and treatment requirements. CONCLUSIONS: Our experience demonstrated that integration of care for TB and HIV can be well served by using multimodal mobile health-based programmes, which can enhance communication and streamline workflow between providers across multiple collaborating institutions and improve continuity between inpatient and outpatient care settings. Further study of programme impact on contextual disease-related stigma and social isolation as well as evaluation of implementation on a broader scale for HIV care is currently under way. TRIAL REGISTRATION NUMBER: NCT03819374.

Implementation of a Mobile Health Strategy to Improve Linkage to and Engagement with HIV Care for People Living with HIV, Tuberculosis, and Substance Use in Irkutsk, Siberia.

In Irkutsk, Siberia, there is a high prevalence of HIV and tuberculosis (TB) coinfection. Mobile health (mHealth) strategies have shown promise for increasing linkage to and engagement in care for people living with HIV (PLWH) in other contexts. We evaluated outcomes for a cohort of PLWH, TB, and substance use in Irkutsk after participation in a multi-feature mHealth intervention called MOCT. Sixty patients were enrolled during hospitalization for TB. We evaluated participant app usage, linkage to HIV care postdischarge, perception of self-efficacy related to HIV care, and HIV-related clinical outcomes at 6 months. We also performed an exploratory analysis to compare a subset of 49 patients with a pre-intervention cohort matched for age and gender. Participants demonstrated engagement with app features examined at 6 months. The majority linked to HIV care by 6 months (83%). Self-scoring of confidence in ability to communicate with HIV providers improved from baseline (median score 8, scale 1-10) to 6 months (10, p = 0.004). A higher proportion of the MOCT subset refilled antiretroviral therapy (69% vs. 43% in pre-intervention cohort, p = 0.01), with fewer deaths in the MOCT subset at 6 months (1 death vs. 10 deaths in pre-intervention cohort, p = 0.02) and a decreased likelihood of developing the composite outcome of death/failure to achieve viral suppression at 6 months (adjusted odds ratio = 0.33, p = 0.029). This study demonstrates preliminary intervention uptake and improvement in short-term outcomes for an urban cohort of PLWH, TB, and substance use enrolled in a multi-feature mHealth intervention, a novel strategy for the context. Clinical Trial Registration Number: NCT03819374.

Extremely lethal and hypervirulent Mycobacterium tuberculosis strain cluster emerging in Far East, Russia.

Mycobacterium tuberculosis strains of the early ancient sublineage of the Beijing genotype are mostly drug susceptible and mainly circulate in East Asia. We have recently discovered two clusters of this sublineage emerging in the Asian part of Russia (VNTR-defined 1071-32 and 14717-15 types) and, to our surprise, both were strongly MDR/XDR-associated. Here, we evaluated their pathogenic features. The clinical isolates and reference laboratory strain H37Rv were investigated in the C57BL/6 mouse model to assess their virulence and lethality properties. The BACTEC MGIT 960 system was used to study the in vitro growth characteristics. In the murine model, strains 396 (14717-15-cluster, from Buryatia, Far East) and 6691 (1071-32-cluster, from Omsk, Siberia) demonstrated contrasting properties. The 396-infected group had significantly higher mortality, more weight loss, higher bacterial burden, and more severe lung pathology. Furthermore, compared to the previously published data on other Russian epidemic Beijing strains (B0/W148, CAO, Central Asian Russian), strain 396 demonstrated the highest mortality. Under the in vitro growth experiment, cluster 14717-15 isolates had significantly shorter lag-phase. To conclude, low-virulent MDR strain 6691 belongs to the Beijing 1071-32-cluster widespread across FSU countries but at low prevalence. This corresponds to common expectation that multiple drug resistance mutations reduce fitness and virulence. In contrast, highly lethal and hypervirulent MDR strain 396 represents an intriguing Beijing 14717-15 cluster predominant only in Buryatia, Far East (16%), sporadically found beyond it, but not forming clusters of transmission. Further in-depth study of this most virulent Russian Beijing cluster is warranted.

Practical approach to detection and surveillance of emerging highly resistant Mycobacterium tuberculosis Beijing 1071-32-cluster.

Ancient sublineage of the Mycobacterium tuberculosis Beijing genotype is endemic and prevalent in East Asia and rare in other world regions. While these strains are mainly drug susceptible, we recently identified a novel clonal group Beijing 1071-32 within this sublineage emerging in Siberia, Russia and present in other Russian regions. This cluster included only multi/extensive drug resistant (MDR/XDR) isolates. Based on the phylogenetic analysis of the available WGS data, we identified three synonymous SNPs in the genes Rv0144, Rv0373c, and Rv0334 that were specific for the Beijing 1071-32-cluster and developed a real-time PCR assay for their detection. Analysis of the 2375 genetically diverse M. tuberculosis isolates collected between 1996 and 2020 in different locations (European and Asian parts of Russia, former Soviet Union countries, Albania, Greece, China, Vietnam, Japan and Brazil), confirmed 100% specificity and sensitivity of this real-time PCR assay. Moreover, the epidemiological importance of this strain and the newly developed screening assay is further stressed by the fact that all identified Beijing 1071-32 isolates were found to exhibit MDR genotypic profiles with concomitant resistance to additional first-line drugs due to a characteristic signature of six mutations in rpoB450, rpoC485, katG315, katG335, rpsL43 and embB497. In conclusion, this study provides a set of three concordant SNPs for the detection and screening of Beijing 1071-32 isolates along with a validated real-time PCR assay easily deployable across multiple settings for the epidemiological tracking of this significant MDR cluster.

Epidemiology of Viliuisk encephalomyelitis in Eastern Siberia.

BACKGROUND: Viliuisk encephalomyelitis is a disorder that starts, in most cases, as an acute meningoencephalitis. Survivors of the acute phase develop a slowly progressing neurologic syndrome characterized by dementia, dysarthria, and spasticity. An epidemic of this disease has been spreading throughout the Yakut Republic of the Russian Federation. Although clinical, neuropathologic, and epidemiologic data suggest infectious etiology, multiple attempts at pathogen isolation have been unsuccessful. METHODS: Detailed clinical, pathologic, laboratory, and epidemiologic studies have identified 414 patients with definite Viliuisk encephalomyelitis in 15 of 33 administrative regions of the Yakut Republic between 1940 and 1999. All data are documented in a Registry. RESULTS: The average annual Viliuisk encephalomyelitis incidence rate at the height of the epidemic reached 8.8 per 100,000 population and affected predominantly young adults. The initial outbreak occurred in a remote isolated area of the middle reaches of Viliui River; the disease spread to adjacent areas and further in the direction of more densely populated regions. The results suggest that intensified human migration from endemic villages led to the emergence of this disease in new communities. Recent social and demographic changes have presumably contributed to a subsequent decline in disease incidence. CONCLUSIONS: Based on the largest known set of diagnostically verified Viliuisk encephalomyelitis cases, we demonstrate how a previously little-known disease that was endemic in a small indigenous population subsequently reached densely populated areas and produced an epidemic involving hundreds of persons.

Urine colorimetry for levofloxacin pharmacokinetics and personalized dosing in people with drug-resistant tuberculosis.

Background: Levofloxacin is a preferred drug for multidrug-resistant (MDR)-tuberculosis (TB) with bactericidal activity that correlates with the pharmacokinetic exposures of serum peak concentration (Cmax) and total area under the concentration time curve (AUC0-24). Pharmacokinetic exposures can be measured to personalize dosing to reach targets, but this practice requires venepuncture, chromatographic or mass spectrometry equipment, and technical expertise. We sought to demonstrate the accuracy of using urine colorimetry as a more feasible estimation of levofloxacin exposure. Method: A colorimetric method using bromocresol green was tested on spiked urine samples with levofloxacin measured using a spectrophotometer. This method was tested in urine samples of healthy volunteers given one 750 mg dose of levofloxacin with urine collected at 0-4 h, 4-8 h, and 8-24 h intervals, and concomitant serum samples were collected and analyzed by high-performance liquid chromatography. Validation of this assay was done in a cohort of people living with human immunodeficiency virus (PLWH), initiating a levofloxacin containing MDR-TB regimen. Results: Urine colorimetry was reproducible in spiked samples and the calibration was curve linear for levofloxacin concentrations ranging from 7.8 µg/ml to 250 µg/ml, with r = 0.98. In healthy volunteers, correlation between urine absorbance values and serum AUC0-24 was highest in urine collected between 4 and 8 h (r = 0.91, P = 0.01), yet in PLWH, urine collected between 0 and 4 h had highest correlation (r = 0.66, P = 0.05). The area under the receiver operating characteristics curve was >0.8 in the derivation, as well as the validation cohort for the urine absorbance values identifying people with total serum exposure below target. Conclusion: Urine colorimetry was highly sensitive in predicting target serum concentrations. Colorimetric methods to determine levofloxacin in urine may improve the feasibility of therapeutic drug monitoring and personalized dose adjustment in TB endemic settings.

Metagenomic analysis of mycobacterial transrenal DNA in patients with HIV and tuberculosis coinfection.

The existence of "transrenal" DNA (tr-DNA), i.e. cell-free DNA that has distributed through the renal barrier to the urine, was first shown from a pathogen in 2000 (Botezatu et al., 2000). However, a targeted search for tr-DNA from Mycobacterium tuberculosis (MBT) started relatively recently (Cannas et al., 2008; Green et al., 2009). While other MBT cellular components found in the urine, e.g. lipoarabinomannan, have been used as an enhanced diagnostic tool, tr-DNA has the potential for strain specific identification or a more persistent biomarker during treatment of active disease. We therefore sought to identify by high-throughput next generation sequencing (NGS) MBT genome fragments in the urine of people with human immunodeficiency virus and tuberculosis (HIV-TB) co-infection living in a co-epidemic setting, and to evaluate whether these DNA targets are suitable for the development a quantitative TaqMan polymerase chain reaction with real-time detection (rt-PCR). Selection and mapping to the reference MBT genome of strain H37Rv (NC_000962) revealed 158 fragments of mycobacterial DNA with length from 19 to 44 base pairs (bp) repeated in different DNA samples. Five targets were chosen for design of rt-PCR primers and probes. Comparative analysis of the newly developed tests that were based on the results of NGS did not reveal a significant increase in sensitivity and specificity relative to the previous empirically designed targets. Howver, highly reproducible NGS reads of mycobacterial tr-DNA were obtained. rt-PCR test development suitable for more practical clinical use was likely limited by the small size of the secreted DNA fragments. It is necessary to develop further molecular approaches for the detection of mycobacterial tr-DNA or rely on NGS techniques with inherent bioinformatics requirements.