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author:("thiel, T")
1.  Noninvasive assessment of mitochondrial organization in three-dimensional tissues reveals changes associated with cancer development 
International journal of cancer  2014;136(2):322-332.
Mitochondrial organization is often altered to accommodate cellular bioenergetic and biosynthetic demands. Changes in metabolism are a hallmark of a number of diseases, including cancer; however, the interdependence between mitochondrial metabolic function and organization is not well understood. Here, we present a noninvasive, automated and quantitative method to assess mitochondrial organization in three-dimensional (3D) tissues using exclusively endogenous two-photon excited fluorescence (TPEF) and show that mitochondrial organization reflects alterations in metabolic activities. Specifically, we examine the organization of mitochondria within live, engineered epithelial tissue equivalents that mimic normal and precancerous human squamous epithelial tissues. We identify unique patterns of mitochondrial organization in the different tissue models we examine, and we attribute these to differences in the metabolic profiles of these tissues. We find that mitochondria are clustered in tissues with high levels of glycolysis and are more highly networked in tissues where oxidative phosphorylation is more dominant. The most highly networked organization is observed within cells with high levels of glutamine consumption. Furthermore, we demonstrate that mitochondrial organization provides complementary information to traditional morphological hallmarks of cancer development, including variations in nuclear size. Finally, we present evidence that this automated quantitative analysis of endogenous TPEF images can identify differences in the mitochondrial organization of freshly excised normal and pre-cancerous human cervical tissue specimens. Thus, this method could be a promising new modality to assess the role of mitochondrial organization in the metabolic activity of 3D tissues and could be further developed to serve as an early cancer clinical diagnostic biomarker.
doi:10.1002/ijc.28992
PMCID: PMC4837461  PMID: 24862444
mitochondrial organization; cancer bioenergetics; auto-fluorescence; human papilloma virus; optical biomarkers
2.  The diagnostic accuracy of three rapid diagnostic tests for typhoid fever at Chittagong Medical College Hospital, Chittagong, Bangladesh 
Abstract
Objective
To determine the diagnostic accuracy of three rapid diagnostic tests (RDTs) for typhoid fever in febrile hospitalised patients in Bangladesh.
Methods
Febrile adults and children admitted to Chittagong Medical College Hospital, Bangladesh, were investigated with Bact/Alert® blood cultures and real‐time PCR to detect Salmonella enterica Typhi and Paratyphi A and assays for Rickettsia, leptospirosis and dengue fever. Acute serum samples were examined with the LifeAssay (LA) Test‐it™ Typhoid IgM lateral flow assay detecting IgM antibodies against S. Typhi O antigen, CTKBiotech Onsite Typhoid IgG/IgM Combo Rapid‐test cassette lateral flow assay detecting IgG and IgM antibodies against S. Typhi O and H antigens and SD Bioline line assay for IgG and IgM antibodies against S. Typhi proteins.
Results
In 300 malaria smear‐negative febrile patients [median (IQR) age of 13.5 (5–31) years], 34 (11.3%) had confirmed typhoid fever: 19 positive by blood culture for S. Typhi (three blood PCR positive) and 15 blood culture negative but PCR positive for S. Typhi in blood. The respective sensitivity and specificity of the three RDTs in patients using a composite reference standard of blood culture and/or PCR‐confirmed typhoid fever were 59% and 61% for LifeAssay, 59% and 74% for the CTK IgM and/or IgG, and 24% and 96% for the SD Bioline RDT IgM and/or IgG. The LifeAssay RDT had a sensitivity of 63% and a specificity of 91% when modified with a positive cut‐off of ≥2+ and analysed using a Bayesian latent class model.
Conclusions
These typhoid RDTs demonstrated moderate diagnostic accuracies, and better tests are needed.
doi:10.1111/tmi.12559
PMCID: PMC4832346  PMID: 26094960
typhoid fever; Salmonella enterica serovar Typhi; blood culture; real‐time PCR; rapid diagnostic tests; diagnostic accuracy; fièvre typhoïde; Salmonella enterica sérotype typhi; culture de sang; PCR en temps réel; tests de diagnostic rapide; précision diagnostique
3.  A novel ciprofloxacin-resistant subclade of H58 Salmonella Typhi is associated with fluoroquinolone treatment failure 
eLife  null;5:e14003.
The interplay between bacterial antimicrobial susceptibility, phylogenetics and patient outcome is poorly understood. During a typhoid clinical treatment trial in Nepal, we observed several treatment failures and isolated highly fluoroquinolone-resistant Salmonella Typhi (S. Typhi). Seventy-eight S. Typhi isolates were genome sequenced and clinical observations, treatment failures and fever clearance times (FCTs) were stratified by lineage. Most fluoroquinolone-resistant S. Typhi belonged to a specific H58 subclade. Treatment failure with S. Typhi-H58 was significantly less frequent with ceftriaxone (3/31; 9.7%) than gatifloxacin (15/34; 44.1%)(Hazard Ratio 0.19, p=0.002). Further, for gatifloxacin-treated patients, those infected with fluoroquinolone-resistant organisms had significantly higher median FCTs (8.2 days) than those infected with susceptible (2.96) or intermediately resistant organisms (4.01)(p<0.001). H58 is the dominant S. Typhi clade internationally, but there are no data regarding disease outcome with this organism. We report an emergent new subclade of S. Typhi-H58 that is associated with fluoroquinolone treatment failure.
Clinical trial registration: ISRCTN63006567.
DOI: http://dx.doi.org/10.7554/eLife.14003.001
eLife digest
People who ingest a type of bacteria called Salmonella Typhi can develop the symptoms of typhoid fever. This disease is common in low-income settings in Asia and Africa, and causes a high rate of death in people who are not treated with antimicrobial drugs.
During a study in Nepal, Thanh et al. tried to evaluate which of two antimicrobials was better for treating typhoid fever. One of the drugs – called gatifloxacin – did not work in some of the patients. To understand why this treatment failed, Thanh et al. decoded the entire DNA sequences of all the Salmonella Typhi bacteria isolated during the study. Comparing this genetic data to the clinical data of the patients identified a new variant of Salmonella Typhi. These bacteria have a specific combination of genetic mutations that render them resistant to the family of drugs that gatifloxacin belongs to – the fluoroquinolones.
Patients infected with the variant bacteria and treated with gatifloxacin were highly likely to completely fail treatment and have longer-lasting fevers. On further investigation Thanh et al. found these organisms were likely recently introduced into Nepal from India.
Fluoroquinolones are amongst the most effective and common antimicrobials used to treat typhoid fever and other bacterial infections. However, the presence of bacteria that are resistant to these compounds in South Asia means that they should no longer be the first choice of drug to treat typhoid fever in this location.
DOI: http://dx.doi.org/10.7554/eLife.14003.002
doi:10.7554/eLife.14003
PMCID: PMC4805543  PMID: 26974227
fluoroquinolones; randomised controlled trial; typhoid fever; nepal; treatment failure; H58; Human; S. enterica serovar Typhi
4.  A Clinical and Epidemiological Investigation of the First Reported Human Infection With the Zoonotic Parasite Trypanosoma evansi in Southeast Asia 
We show that the bovid-associated parasite Trypanosoma evansi is endemic in Vietnam and has zoonotic potential. Our study describes the first laboratory-confirmed human case of T. evansi in a previously healthy individual without apolipoprotein L1 deficiency.
Background. Trypanosoma is a genus of unicellular parasitic flagellate protozoa. Trypanosoma brucei species and Trypanosoma cruzi are the major agents of human trypanosomiasis; other Trypanosoma species can cause human disease, but are rare. In March 2015, a 38-year-old woman presented to a healthcare facility in southern Vietnam with fever, headache, and arthralgia. Microscopic examination of blood revealed infection with Trypanosoma.
Methods. Microscopic observation, polymerase chain reaction (PCR) amplification of blood samples, and serological testing were performed to identify the infecting species. The patient's blood was screened for the trypanocidal protein apolipoprotein L1 (APOL1), and a field investigation was performed to identify the zoonotic source.
Results. PCR amplification and serological testing identified the infecting species as Trypanosoma evansi. Despite relapsing 6 weeks after completing amphotericin B therapy, the patient made a complete recovery after 5 weeks of suramin. The patient was found to have 2 wild-type APOL1 alleles and a normal serum APOL1 concentration. After responsive animal sampling in the presumed location of exposure, cattle and/or buffalo were determined to be the most likely source of the infection, with 14 of 30 (47%) animal blood samples testing PCR positive for T. evansi.
Conclusions. We report the first laboratory-confirmed case of T. evansi in a previously healthy individual without APOL1 deficiency, potentially contracted via a wound while butchering raw beef, and successfully treated with suramin. A linked epidemiological investigation revealed widespread and previously unidentified burden of T. evansi in local cattle, highlighting the need for surveillance of this infection in animals and the possibility of further human cases.
doi:10.1093/cid/ciw052
PMCID: PMC4803109  PMID: 26908809
Vietnam; zoonosis; Trypanosoma evansi; case investigation; emerging infections
5.  The transfer and decay of maternal antibody against Shigella sonnei in a longitudinal cohort of Vietnamese infants 
Vaccine  2016;34(6):783-790.
Highlights
•Shigella sonnei is an emergent and highly drug resistant diarrheal pathogen.•The half-life of maternal S. sonnei IgG in infants is 43 days.•Maternal titer, antibody transfer ratio and gestational age influence birth titer.•Incidence of seroconversion in infants in southern Vietnam is 4/100 infant years.•Children should be vaccinated after 5 months of age if a candidate is licensed.
Background
Shigella sonnei is an emergent and major diarrheal pathogen for which there is currently no vaccine. We aimed to quantify duration of maternal antibody against S. sonnei and investigate transplacental IgG transfer in a birth cohort in southern Vietnam.
Methods and results
Over 500-paired maternal/infant plasma samples were evaluated for presence of anti-S. sonnei-O IgG and IgM. Longitudinal plasma samples allowed for the estimation of the median half-life of maternal anti-S. sonnei-O IgG, which was 43 days (95% confidence interval: 41–45 days). Additionally, half of infants lacked a detectable titer by 19 weeks of age. Lower cord titers were associated with greater increases in S. sonnei IgG over the first year of life, and the incidence of S. sonnei seroconversion was estimated to be 4/100 infant years. Maternal IgG titer, the ratio of antibody transfer, the season of birth and gestational age were significantly associated with cord titer.
Conclusions
Maternal anti-S. sonnei-O IgG is efficiently transferred across the placenta and anti-S. sonnei-O maternal IgG declines rapidly after birth and is undetectable after 5 months in the majority of children. Preterm neonates and children born to mothers with low IgG titers have lower cord titers and therefore may be at greater risk of seroconversion in infancy.
doi:10.1016/j.vaccine.2015.12.047
PMCID: PMC4742520  PMID: 26742945
Shigella; Maternal antibody; Placental transfer; Seroconversion
6.  The Ecological Dynamics of Fecal Contamination and Salmonella Typhi and Salmonella Paratyphi A in Municipal Kathmandu Drinking Water 
PLoS Neglected Tropical Diseases  2016;10(1):e0004346.
One of the UN sustainable development goals is to achieve universal access to safe and affordable drinking water by 2030. It is locations like Kathmandu, Nepal, a densely populated city in South Asia with endemic typhoid fever, where this goal is most pertinent. Aiming to understand the public health implications of water quality in Kathmandu we subjected weekly water samples from 10 sources for one year to a range of chemical and bacteriological analyses. We additionally aimed to detect the etiological agents of typhoid fever and longitudinally assess microbial diversity by 16S rRNA gene surveying. We found that the majority of water sources exhibited chemical and bacterial contamination exceeding WHO guidelines. Further analysis of the chemical and bacterial data indicated site-specific pollution, symptomatic of highly localized fecal contamination. Rainfall was found to be a key driver of this fecal contamination, correlating with nitrates and evidence of S. Typhi and S. Paratyphi A, for which DNA was detectable in 333 (77%) and 303 (70%) of 432 water samples, respectively. 16S rRNA gene surveying outlined a spectrum of fecal bacteria in the contaminated water, forming complex communities again displaying location-specific temporal signatures. Our data signify that the municipal water in Kathmandu is a predominant vehicle for the transmission of S. Typhi and S. Paratyphi A. This study represents the first extensive spatiotemporal investigation of water pollution in an endemic typhoid fever setting and implicates highly localized human waste as the major contributor to poor water quality in the Kathmandu Valley.
Author Summary
Aiming to understand the ecology of municipal drinking water and measure the potential exposure to pathogens that cause typhoid fever (Salmonella Typhi and Salmonella Paratyphi A) in Kathmandu, Nepal, we collected water samples from 10 water sources weekly for one year and subjected them to comprehensive chemical, bacteriological and molecular analyses. We found that Kathmandu drinking water exhibits longitudinal fecal contamination in excess of WHO guidelines. The chemical composition of water indicated site-specific pollution profiles, which were likely driven by localized contamination with human fecal material. We additionally found that Salmonella Typhi and Salmonella Paratyphi A could be detected throughout the year in every water sampling location, but specifically peaked after the monsoons. A microbiota analysis (a method for studying bacterial diversity in biological samples) revealed the water to be contaminated by complex populations of fecal bacteria, which again exhibited a unique profile by both location and time. This study shows that Salmonella Typhi and Salmonella Paratyphi A can be longitudinally detected in drinking water in Kathmandu and represents the first major investigation of the spatiotemporal dynamics of drinking water pollution in an endemic typhoid setting.
doi:10.1371/journal.pntd.0004346
PMCID: PMC4703202  PMID: 26735696
9.  Clinical implications of reduced susceptibility to fluoroquinolones in paediatric Shigella sonnei and Shigella flexneri infections 
Objectives
We aimed to quantify the impact of fluoroquinolone resistance on the clinical outcome of paediatric shigellosis patients treated with fluoroquinolones in southern Vietnam. Such information is important to inform therapeutic management for infections caused by this increasingly drug-resistant pathogen, responsible for high morbidity and mortality in young children globally.
Methods
Clinical information and bacterial isolates were derived from a randomized controlled trial comparing gatifloxacin with ciprofloxacin for the treatment of paediatric shigellosis. Time–kill experiments were performed to evaluate the impact of MIC on the in vitro growth of Shigella and Cox regression modelling was used to compare clinical outcome between treatments and Shigella species.
Results
Shigella flexneri patients treated with gatifloxacin had significantly worse outcomes than those treated with ciprofloxacin. However, the MICs of fluoroquinolones were not significantly associated with poorer outcome. The presence of S83L and A87T mutations in the gyrA gene significantly increased MICs of fluoroquinolones. Finally, elevated MICs and the presence of the qnrS gene allowed Shigella to replicate efficiently in vitro in high concentrations of ciprofloxacin.
Conclusions
We found that below the CLSI breakpoint, there was no association between MIC and clinical outcome in paediatric shigellosis infections. However, S. flexneri patients had worse clinical outcomes when treated with gatifloxacin in this study regardless of MIC. Additionally, Shigella harbouring the qnrS gene are able to replicate efficiently in high concentrations of ciprofloxacin and we hypothesize that such strains possess a competitive advantage against fluoroquinolone-susceptible strains due to enhanced shedding and transmission.
doi:10.1093/jac/dkv400
PMCID: PMC4743702  PMID: 26679253
10.  In vitro activity of colistin in antimicrobial combination against carbapenem-resistant Acinetobacter baumannii isolated from patients with ventilator-associated pneumonia in Vietnam 
Journal of Medical Microbiology  2015;64(Pt 10):1162-1169.
Acinetobacter baumannii has become one of the major infection threats in intensive care units (ICUs) globally. Since 2008, A. baumannii has been the leading cause of ventilator-associated pneumonia (VAP) in our ICU at an infectious disease hospital in southern Vietnam. The emergence of this pathogen in our setting is consistent with the persistence of a specific clone exhibiting resistance to carbapenems. Antimicrobial combinations may be a strategy to treat infections caused by these carbapenem-resistant A. baumannii. Therefore, we assessed potential antimicrobial combinations against local carbapenem-resistant A. baumannii by measuring in vitro interactions of colistin with four antimicrobials that are locally certified for treating VAP. We first performed antimicrobial susceptibility testing and multilocus variable number tandem repeat analysis (MLVA) genotyping on 74 A. baumannii isolated from quantitative tracheal aspirates from patients with VAP over an 18-month period. These 74 isolates could be subdivided into 21 main clusters by MLVA and >80 % were resistant to carbapenems. We selected 56 representative isolates for in vitro combination synergy testing. Synergy was observed in four (7 %), seven (13 %), 20 (36 %) and 38 (68 %) isolates with combinations of colistin with ceftazidime, ceftriaxone, imipenem and meropenem, respectively. Notably, more carbapenem-resistant A. baumannii isolates (36/43; 84 %) exhibited synergistic activity with a combination of colistin and meropenem than carbapenem-susceptible A. baumannii isolates (2/13; 15 %) (P = 0.023; Fisher's exact test). Our findings suggest that combinations of colistin and meropenem should be considered when treating carbapenem-resistant A. baumannii infections in Vietnam, and we advocate clinical trials investigating combination therapy for VAP.
doi:10.1099/jmm.0.000137
PMCID: PMC4755130  PMID: 26297024
11.  The role of MicroRNA in castration resistant prostate cancer 
Urologic oncology  2014;32(5):517-523.
Introduction
Castration resistant prostate cancer (CRPC) has a historically low median survival rate but recent advances and discoveries in micro RNAs (miRNAs) have opened the potential for new prognostication modalities to enhance therapeutic success. As new chemotherapies and immunotherapies are developed there is an increasing need for precision and stratification of CRPC to allow for optimization and personalization of therapy.
Methods
A systematic literature review was conducted via electronic database resulting in the selection of forty-two articles based on title, abstract, study format, and content by a consensus of all participating authors. The majority of selected articles were published between 2002 and 2013. In this review, we will discuss the robustness of miRNAs as a biomarker platform, miRNAs associated with prostate cancer, and recent discoveries of miRNA associations with CRPC.
Results
The associations discovered have been of interest due to the ability to differentiate between CRPC and localized prostate cancer. With evaluation of multiple miRNAs, it is possible to provide a profile in regards to tumor characteristics. Furthermore, actions of miRNAs on CRPC tumor cells have the ability to suppress metastatic phenotypes.
Conclusion
miRNAs may have a growing role in CRPC prognostication and potentially transform into a therapeutic potential.
doi:10.1016/j.urolonc.2013.11.004
PMCID: PMC4570565  PMID: 24935732
Castration Resistant Prostate Cancer; micro RNA; Review
12.  AB026. SCN1A mutational analysis in 20 Vietnamese children with Dravet syndrome 
Annals of Translational Medicine  2015;3(Suppl 2):AB026.
Background
Dravet syndrome is one of the most catastrophic types of epilepsy in infants. It is found that 70-80% of cases of Dravet syndrome are caused by mutations in SCN1A, the gene encoding alpha-1 subunit of the sodium channel. Mutations of the SCN1A gene have an autosomal dominant inheritance pattern. To date, over 1,000 SCN1A mutations have been reported all over the world, however, no SCN1A mutation studies have been performed in the Vietnamese population, and genetic characteristics of Vietnamese Dravet patients are not yet clear. In this study, we analyzed SCN1A gene in 20 Vietnamese patients with clinical features of Dravet syndrome at Children’s Hospital 2, Ho Chi Minh City, Vietnam.
Methods
Direct sequencing and multiple ligation-dependent probe amplification (MLPA) were performed to screen the entire coding regions as well as exon-intron boundaries of the gene.
Results
Fourteen mutations (14/20; 70%) were identified including 13 point mutations detected by PCR-Sequencing and 1 large deletion mutation spanning nearly whole exon 7 detected by MLPA. Five mutations were classified as truncations (2 frameshift and 3 nonsense mutations) and 9 were classified as missense mutations. There were 7 mutations were localized at pore-forming loop (connecting S5-S6); 5 mutations were localized at cytoplasmic loops (connecting 2 nearby homologous domains), 1 mutations were localized at transmembrane segments, and 1 mutation in a intronic region. Nine of these 14 SCN1A mutations were novel and parental DNA analysis for the identified mutations in 11 available cases show that all of the mutations were de nono. Besides well-known genotype–phenotype correlations, our study results strongly suggests the existence of modifying factors.
Conclusions
The proportion of SCN1A mutations among Vietnamese Dravet patients in this study appeared to be consistent with other populations (70%). Our study also expands the spectrum of SCN1A mutations and confirms the current understanding of genotype–phenotype correlations.
doi:10.3978/j.issn.2305-5839.2015.AB026
PMCID: PMC4563513
Dravet syndrome; SCN1A; Vietnamese
13.  White Matter Changes of Neurite Density and Fiber Orientation Dispersion during Human Brain Maturation 
PLoS ONE  2015;10(6):e0123656.
Diffusion tensor imaging (DTI) studies of human brain development have consistently shown widespread, but nonlinear increases in white matter anisotropy through childhood, adolescence, and into adulthood. However, despite its sensitivity to changes in tissue microstructure, DTI lacks the specificity to disentangle distinct microstructural features of white and gray matter. Neurite orientation dispersion and density imaging (NODDI) is a recently proposed multi-compartment biophysical model of brain microstructure that can estimate non-collinear properties of white matter, such as neurite orientation dispersion index (ODI) and neurite density index (NDI). In this study, we apply NODDI to 66 healthy controls aged 7–63 years to investigate changes of ODI and NDI with brain maturation, with comparison to standard DTI metrics. Using both region-of-interest and voxel-wise analyses, we find that NDI exhibits striking increases over the studied age range following a logarithmic growth pattern, while ODI rises following an exponential growth pattern. This novel finding is consistent with well-established age-related changes of FA over the lifespan that show growth during childhood and adolescence, plateau during early adulthood, and accelerating decay after the fourth decade of life. Our results suggest that the rise of FA during the first two decades of life is dominated by increasing NDI, while the fall in FA after the fourth decade is driven by the exponential rise of ODI that overcomes the slower increases of NDI. Using partial least squares regression, we further demonstrate that NODDI better predicts chronological age than DTI. Finally, we show excellent test—retest reliability of NODDI metrics, with coefficients of variation below 5% in all measured regions of interest. Our results support the conclusion that NODDI reveals biologically specific characteristics of brain development that are more closely linked to the microstructural features of white matter than are the empirical metrics provided by DTI.
doi:10.1371/journal.pone.0123656
PMCID: PMC4482659  PMID: 26115451
14.  A Prospective Multi-Center Observational Study of Children Hospitalized with Diarrhea in Ho Chi Minh City, Vietnam 
We performed a prospective multicenter study to address the lack of data on the etiology, clinical and demographic features of hospitalized pediatric diarrhea in Ho Chi Minh City (HCMC), Vietnam. Over 2,000 (1,419 symptomatic and 609 non-diarrheal control) children were enrolled in three hospitals over a 1-year period in 2009–2010. Aiming to detect a panel of pathogens, we identified a known diarrheal pathogen in stool samples from 1,067/1,419 (75.2%) children with diarrhea and from 81/609 (13.3%) children without diarrhea. Rotavirus predominated in the symptomatic children (664/1,419; 46.8%), followed by norovirus (293/1,419; 20.6%). The bacterial pathogens Salmonella, Campylobacter, and Shigella were cumulatively isolated from 204/1,419 (14.4%) diarrheal children and exhibited extensive antimicrobial resistance, most notably to fluoroquinolones and third-generation cephalosporins. We suggest renewed efforts in generation and implementation of policies to control the sale and prescription of antimicrobials to curb bacterial resistance and advise consideration of a subsidized rotavirus vaccination policy to limit the morbidity due to diarrheal disease in Vietnam.
doi:10.4269/ajtmh.14-0655
PMCID: PMC4426562  PMID: 25802437
15.  Clinically and Microbiologically Derived Azithromycin Susceptibility Breakpoints for Salmonella enterica Serovars Typhi and Paratyphi A 
Azithromycin is an effective treatment for uncomplicated infections with Salmonella enterica serovar Typhi and serovar Paratyphi A (enteric fever), but there are no clinically validated MIC and disk zone size interpretative guidelines. We studied individual patient data from three randomized controlled trials (RCTs) of antimicrobial treatment in enteric fever in Vietnam, with azithromycin used in one treatment arm, to determine the relationship between azithromycin treatment response and the azithromycin MIC of the infecting isolate. We additionally compared the azithromycin MIC and the disk susceptibility zone sizes of 1,640 S. Typhi and S. Paratyphi A clinical isolates collected from seven Asian countries. In the RCTs, 214 patients who were treated with azithromycin at a dose of 10 to 20 mg/ml for 5 to 7 days were analyzed. Treatment was successful in 195 of 214 (91%) patients, with no significant difference in response (cure rate, fever clearance time) with MICs ranging from 4 to 16 μg/ml. The proportion of Asian enteric fever isolates with an MIC of ≤16 μg/ml was 1,452/1,460 (99.5%; 95% confidence interval [CI], 98.9 to 99.7) for S. Typhi and 207/240 (86.3%; 95% CI, 81.2 to 90.3) (P < 0.001) for S. Paratyphi A. A zone size of ≥13 mm to a 5-μg azithromycin disk identified S. Typhi isolates with an MIC of ≤16 μg/ml with a sensitivity of 99.7%. An azithromycin MIC of ≤16 μg/ml or disk inhibition zone size of ≥13 mm enabled the detection of susceptible S. Typhi isolates that respond to azithromycin treatment. Further work is needed to define the response to treatment in S. Typhi isolates with an azithromycin MIC of >16 μg/ml and to determine MIC and disk breakpoints for S. Paratyphi A.
doi:10.1128/AAC.04729-14
PMCID: PMC4394775  PMID: 25733500
16.  Immunological and Biochemical Correlates of Adjunctive Dexamethasone in Vietnamese Adults with Bacterial Meningitis 
Adjunctive treatment to improve outcome from bacterial meningitis has centered on dexamethasone. Among Vietnamese patients with bacterial meningitis, cerebrospinal fluid (CSF) opening pressure and CSF:plasma glucose ratios were significantly improved and levels of CSF cytokines interleukin (IL)–6, IL-8, and IL-10 and were all statistically significantly lower after treatment in patients who were randomized to dexamethasone, compared with levels in patients who received placebo.
doi:10.1086/630207
PMCID: PMC4340500  PMID: 19814625
17.  Emergence of carbapenem-resistant Acinetobacter baumannii as the major cause of ventilator-associated pneumonia in intensive care unit patients at an infectious disease hospital in southern Vietnam 
Journal of Medical Microbiology  2014;63(Pt 10):1386-1394.
Ventilator-associated pneumonia (VAP) is a serious healthcare-associated infection that affects up to 30 % of intubated and mechanically ventilated patients in intensive care units (ICUs) worldwide. The bacterial aetiology and corresponding antimicrobial susceptibility of VAP is highly variable, and can differ between countries, national provinces and even between different wards in the same hospital. We aimed to understand and document changes in the causative agents of VAP and their antimicrobial susceptibility profiles retrospectively over an 11 year period in a major infectious disease hospital in southern Vietnam. Our analysis outlined a significant shift from Pseudomonas aeruginosa to Acinetobacter spp. as the most prevalent bacteria isolated from quantitative tracheal aspirates in patients with VAP in this setting. Antimicrobial resistance was common across all bacterial species and we found a marked proportional annual increase in carbapenem-resistant Acinetobacter spp. over a 3 year period from 2008 (annual trend; odds ratio 1.656, P = 0.010). We further investigated the possible emergence of a carbapenem-resistant Acinetobacter baumannii clone by multiple-locus variable number tandem repeat analysis, finding a blaOXA-23-positive strain that was associated with an upsurge in the isolation of this pathogen. We additionally identified a single blaNDM-1-positive A. baumannii isolate. This work highlights the emergence of a carbapenem-resistant clone of A. baumannii and a worrying trend of antimicrobial resistance in the ICU of the Hospital for Tropical Diseases in Ho Chi Minh City, Vietnam.
doi:10.1099/jmm.0.076646-0
PMCID: PMC4170484  PMID: 25038137
18.  Two Cases of Bacteriemia Caused by Nontoxigenic, Non-O1, Non-O139 Vibrio cholerae Isolates in Ho Chi Minh City, Vietnam 
Journal of Clinical Microbiology  2014;52(10):3819-3821.
The toxigenic bacterium Vibrio cholerae belonging to the O1 and O139 serogroups is commonly associated with epidemic diarrhea in tropical settings; other diseases caused by this environmental pathogen are seldom identified. Here we report two unassociated cases of nonfatal, nontoxigenic V. cholerae non-O1, non-O139 bacteremia in patients with comorbidities in Ho Chi Minh City, Vietnam, that occurred within a 4-week period.
doi:10.1128/JCM.01915-14
PMCID: PMC4187776  PMID: 25122858
19.  Cells of origin and tumor-initiating cells for nonmelanoma skin cancers 
Cancer letters  2012;338(1):82-88.
The epidermis of the skin is a multilayered stratified epithelium whose primary function is to provide a barrier against our external environment. As a result, cells in the epidermis are subject to constant assault from environmental pathogens, many of which can cause deleterious mutations. However, most of these mutations do not lead to skin cancer. One explanation is that most genetic hits are sustained by mature or transit cells with limited proliferative capacity and only stem cells that acquire genetic alterations have the potential to propagate a frank tumor. In this mini-review we will discuss recent studies that provide some of the first genetic evidence to support a stem cell origin for a number of skin cancer types.
doi:10.1016/j.canlet.2012.05.008
PMCID: PMC3422447  PMID: 22579650
epidermis; hair follicle; keratinocyte; stem cell; cancer stem cell
20.  Salmonella Typhi and Salmonella Paratyphi A elaborate distinct systemic metabolite signatures during enteric fever 
eLife  2014;3:e03100.
The host–pathogen interactions induced by Salmonella Typhi and Salmonella Paratyphi A during enteric fever are poorly understood. This knowledge gap, and the human restricted nature of these bacteria, limit our understanding of the disease and impede the development of new diagnostic approaches. To investigate metabolite signals associated with enteric fever we performed two dimensional gas chromatography with time-of-flight mass spectrometry (GCxGC/TOFMS) on plasma from patients with S. Typhi and S. Paratyphi A infections and asymptomatic controls, identifying 695 individual metabolite peaks. Applying supervised pattern recognition, we found highly significant and reproducible metabolite profiles separating S. Typhi cases, S. Paratyphi A cases, and controls, calculating that a combination of six metabolites could accurately define the etiological agent. For the first time we show that reproducible and serovar specific systemic biomarkers can be detected during enteric fever. Our work defines several biologically plausible metabolites that can be used to detect enteric fever, and unlocks the potential of this method in diagnosing other systemic bacterial infections.
DOI: http://dx.doi.org/10.7554/eLife.03100.001
eLife digest
Enteric fever is estimated to affect over 37 million people every year. Although treatable with antimicrobial drugs, a slow and/or incorrect diagnosis can result in serious and often life-threatening complications.
Enteric fever is the combined name for typhoid fever and paratyphoid fever. While the symptoms of these diseases are indistinguishable, the strains of Salmonella bacteria that cause them are genetically distinct. Moreover, the two organisms that cause the disease exhibit different propensities to develop resistance to antimicrobials. It is important, therefore, to be able to distinguish between typhoid fever and paratyphoid fever so that the correct treatment can be prescribed. However, the diagnostic tools available today struggle to discriminate between Salmonella Typhi (which causes typhoid fever) and Salmonella Paratyphi A (which causes paratyphoid fever).
Now, Näsström et al. have developed a methodology that can determine if an individual is infected by Salmonella Typhi or Salmonella Paratyphi A, or neither. Rather than trying to detect the bacteria themselves, the test relies on measuring the levels of various metabolites—molecules produced during metabolism—in the blood. Näsström et al. discovered a set of six metabolites that are affected in different ways by typhoid and paratyphoid fever. The next challenge is to develop this approach so it can be used in endemic settings.
DOI: http://dx.doi.org/10.7554/eLife.03100.002
doi:10.7554/eLife.03100
PMCID: PMC4077204  PMID: 24902583
metabolites; mass spectrometry; enteric fever; typhoid; Salmonella Typhi; Salmonella Paratyphi A; human
21.  Evaluation of the Diagnostic Accuracy of a Typhoid IgM Flow Assay for the Diagnosis of Typhoid Fever in Cambodian Children Using a Bayesian Latent Class Model Assuming an Imperfect Gold Standard 
Rapid diagnostic tests are needed for typhoid fever (TF) diagnosis in febrile children in endemic areas. Five hundred children admitted to the hospital in Cambodia between 2009 and 2010 with documented fever (≥ 38°C) were investigated using blood cultures (BCs), Salmonella Typhi/Paratyphi A real-time polymerase chain reactions (PCRs), and a Typhoid immunoglobulin M flow assay (IgMFA). Test performance was determined by conventional methods and Bayesian latent class modeling. There were 32 cases of TF (10 BC- and PCR-positive cases, 14 BC-positive and PCR-negative cases, and 8 BC-negative and PCR-positive cases). IgMFA sensitivity was 59.4% (95% confidence interval = 41–76), and specificity was 97.8% (95% confidence interval = 96–99). The model estimate sensitivity for BC was 81.0% (95% credible interval = 54–99). The model estimate sensitivity for PCR was 37.8% (95% credible interval = 26–55), with a specificity of 98.2% (95% credible interval = 97–99). The model estimate sensitivity for IgMFA (≥ 2+) was 77.9% (95% credible interval = 58–90), with a specificity of 97.5% (95% credible interval = 95–100). The model estimates of IgMFA sensitivity and specificity were comparable with BCs and better than estimates using conventional analysis.
doi:10.4269/ajtmh.13-0384
PMCID: PMC3886406  PMID: 24218407
22.  Fitness benefits in fluoroquinolone-resistant Salmonella Typhi in the absence of antimicrobial pressure 
eLife  2013;2:e01229.
Fluoroquinolones (FQ) are the recommended antimicrobial treatment for typhoid, a severe systemic infection caused by the bacterium Salmonella enterica serovar Typhi. FQ-resistance mutations in S. Typhi have become common, hindering treatment and control efforts. Using in vitro competition experiments, we assayed the fitness of eleven isogenic S. Typhi strains with resistance mutations in the FQ target genes, gyrA and parC. In the absence of antimicrobial pressure, 6 out of 11 mutants carried a selective advantage over the antimicrobial-sensitive parent strain, indicating that FQ resistance in S. Typhi is not typically associated with fitness costs. Double-mutants exhibited higher than expected fitness as a result of synergistic epistasis, signifying that epistasis may be a critical factor in the evolution and molecular epidemiology of S. Typhi. Our findings have important implications for the management of drug-resistant S. Typhi, suggesting that FQ-resistant strains would be naturally maintained even if fluoroquinolone use were reduced.
DOI: http://dx.doi.org/10.7554/eLife.01229.001
eLife digest
The fluoroquinolones are a group of antimicrobials that are used to treat a variety of life-threatening bacterial infections, including typhoid fever. Before the introduction of antimicrobials, the mortality rate from typhoid fever was 10–20%. Prompt treatment with fluoroquinolones has reduced this to less than 1%, and has also decreased the severity of symptoms suffered by people with the disease.
Now, however, the usefulness of many antimicrobials, including the fluoroquinolones, is threatened by the evolution of antimicrobial resistance within the bacterial populations being treated. Drug resistance in bacteria typically arises through specific mutations, or following the acquisition of antimicrobial resistance genes from other bacteria. It is thought that the frequent use of antimicrobials in human and animal health puts selective pressure on bacterial populations, allowing bacterial strains with mutations or genes that confer antimicrobial resistance to survive, while bacterial strains that are sensitive to the antimicrobials die out.
At first it was thought that specific mutations conferring antimicrobial resistance came at a fitness cost, which would mean that such mutations would be rare in the absence of antimicrobials. Now, based on research into typhoid fever, Baker et al. describe a system in which the majority of evolutionary routes to drug resistance are marked by significant fitness benefits, even in the absence of antimicrobial exposure.
Typhoid is caused by a bacterial pathogen known as Salmonella Typhi, and mutations in two genes—gyrA and parC—result in resistance to fluoroquinolones. Baker et al. show that mutations in these genes confer a measurable fitness advantage over strains without these mutations, even in the absence of exposure to fluoroquinolones. Moreover, strains with two mutations in one of these genes exhibited a higher than predicted fitness, suggesting that there is a synergistic interaction between the two mutations. This work challenges the dogma that antimicrobial resistant organisms have a fitness disadvantage in the absence of antimicrobials, and suggests that increasing resistance to the fluoroquinolones is not solely driven by excessive use of this important group of drugs.
DOI: http://dx.doi.org/10.7554/eLife.01229.002
doi:10.7554/eLife.01229
PMCID: PMC3857714  PMID: 24327559
Salmonella; typhoid; fitness cost; epistasis; fluoroquinolone; Other
23.  Identification of Salmonella enterica Serovar Typhi Genotypes by Use of Rapid Multiplex Ligation-Dependent Probe Amplification 
Journal of Clinical Microbiology  2013;51(9):2950-2958.
Salmonella enterica serovar Typhi, the causative agent of typhoid fever, is highly clonal and genetically conserved, making isolate subtyping difficult. We describe a standardized multiplex ligation-dependent probe amplification (MLPA) genotyping scheme targeting 11 key phylogenetic markers of the S. Typhi genome. The MLPA method demonstrated 90% concordance with single nucleotide polymorphism (SNP) typing, the gold standard for S. Typhi genotyping, and had the ability to identify isolates of the H58 haplotype, which is associated with resistance to multiple antimicrobials. Additionally, the assay permitted the detection of fluoroquinolone resistance-associated mutations in the DNA gyrase-encoding gene gyrA and the topoisomerase gene parC with a sensitivity of 100%. The MLPA methodology is simple and reliable, providing phylogenetically and phenotypically relevant genotyping information. This MLPA scheme offers a more-sensitive and interpretable alternative to the nonphylogenetic subgrouping methodologies that are currently used in reference and research laboratories in areas where typhoid is endemic.
doi:10.1128/JCM.01010-13
PMCID: PMC3754622  PMID: 23824765
24.  Differential Epidemiology of Salmonella Typhi and Paratyphi A in Kathmandu, Nepal: A Matched Case Control Investigation in a Highly Endemic Enteric Fever Setting 
Background
Enteric fever, a systemic infection caused by the bacteria Salmonella Typhi and Salmonella Paratyphi A, is endemic in Kathmandu, Nepal. Previous work identified proximity to poor quality water sources as a community-level risk for infection. Here, we sought to examine individual-level risk factors related to hygiene and sanitation to improve our understanding of the epidemiology of enteric fever in this setting.
Methodology and principal findings
A matched case-control analysis was performed through enrollment of 103 blood culture positive enteric fever patients and 294 afebrile community-based age and gender-matched controls. A detailed questionnaire was administered to both cases and controls and the association between enteric fever infection and potential exposures were examined through conditional logistic regression. Several behavioral practices were identified as protective against infection with enteric fever, including water storage and hygienic habits. Additionally, we found that exposures related to poor water and socioeconomic status are more influential in the risk of infection with S. Typhi, whereas food consumption habits and migration play more of a role in risk of S. Paratyphi A infection.
Conclusions and significance
Our work suggests that S. Typhi and S. Paratyphi A follow different routes of infection in this highly endemic setting and that sustained exposure to both serovars probably leads to the development of passive immunity. In the absence of a polyvalent vaccine against S. Typhi and S. Paratyphi A, we advocate better systems for water treatment and storage, improvements in the quality of street food, and vaccination with currently available S. Typhi vaccines.
Author Summary
Enteric fever, caused by ingestion of bacteria Salmonella Typhi or Salmonella Paratyphi A, is common in regions with poor water quality and sanitation. We sought to identify individual-level risks for infection in Kathmandu, Nepal, a region endemic for enteric fever. In this study, we enrolled patients presenting to hospital who were blood-culture positive for enteric fever and a series of community controls matched for age, gender and residential ward. Our findings suggest that while some risks for infection with S. Typhi and S. Paratyphi A overlap, these organisms also have distinctive routes of infection in this setting; poor water and socioeconomic status seemed more influential in infection with S. Typhi, whereas food consumption habits and migratory status were shown to play a larger role in infection with S. Paratyphi A. Additionally, serological evaluation of IgG levels against the Vi (Salmonella Typhi) and the O:2 (Salmonella Paratyphi A) antigens demonstrated high titers against both antigens throughout life, suggesting frequent and constant exposure to these organisms in Kathmandu. As major improvements in sanitation infrastructure are unlikely in this setting, we recommend water treatment and storage-based prevention strategies, as well as street food quality regulation, and the promotion of vaccination with existing typhoid vaccines.
doi:10.1371/journal.pntd.0002391
PMCID: PMC3749961  PMID: 23991240
25.  Identification of Immunogenic Salmonella enterica Serotype Typhi Antigens Expressed in Chronic Biliary Carriers of S. Typhi in Kathmandu, Nepal 
Background
Salmonella enterica serotype Typhi can colonize and persist in the biliary tract of infected individuals, resulting in a state of asymptomatic chronic carriage. Chronic carriers may act as persistent reservoirs of infection within a community and may introduce infection to susceptible individuals and new communities. Little is known about the interaction between the host and pathogen in the biliary tract of chronic carriers, and there is currently no reliable diagnostic assay to identify asymptomatic S. Typhi carriage.
Methodology/Principal Findings
To study host-pathogen interactions in the biliary tract during S. Typhi carriage, we applied an immunoscreening technique called in vivo-induced antigen technology (IVIAT), to identify potential biomarkers unique to carriers. IVIAT identifies humorally immunogenic bacterial antigens expressed uniquely in the in vivo environment, and we hypothesized that S. Typhi surviving in the biliary tract of humans may express a distinct antigenic profile. Thirteen S. Typhi antigens that were immunoreactive in carriers, but not in healthy individuals from a typhoid endemic area, were identified. The identified antigens included a number of putative membrane proteins, lipoproteins, and hemolysin-related proteins. YncE (STY1479), an uncharacterized protein with an ATP-binding motif, gave prominent responses in our screen. The response to YncE in patients whose biliary tract contained S. Typhi was compared to responses in patients whose biliary tract did not contain S. Typhi, patients with acute typhoid fever, and healthy controls residing in a typhoid endemic area. Seven of 10 (70%) chronic carriers, 0 of 8 bile culture-negative controls (0%), 0 of 8 healthy Bangladeshis (0%), and 1 of 8 (12.5%) Bangladeshis with acute typhoid fever had detectable anti-YncE IgG in blood. IgA responses were also present.
Conclusions/Significance
Further evaluation of YncE and other antigens identified by IVIAT could lead to the development of improved diagnostic assays to identify asymptomatic S. Typhi carriers.
Author Summary
Salmonella enterica serotype Typhi is the cause of typhoid fever and infects over 21 million individuals and causes 200,000 deaths each year. With adequate treatment, most patients recover from their acute stage of illness and clear infection. However, a small percentage of S. Typhi infected individuals develop a chronic but asymptomatic infection in the biliary tract that can persist for decades. Since S. Typhi is a human-restricted pathogen, chronic carriers may act as reservoirs of infection. Correctly identifying and treating asymptomatic chronic carriers could be critical for ultimate control of typhoid fever. Using an immunoscreening technique called in vivo-induced antigen technology (IVIAT), we have identified potential biomarkers unique to S. Typhi chronic carriers. Further evaluation of these antigens could lead to the development of improved diagnostic assays to detect asymptomatic S. Typhi carriers in typhoid endemic zones, and to an improved understanding of the pathogenesis of S. Typhi in the chronic carrier state.
doi:10.1371/journal.pntd.0002335
PMCID: PMC3731212  PMID: 23936575

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