Invasive Non-typhoidal Salmonella Infections in Asia: Clinical Observations, Disease Outcome and Dominant Serovars from an Infectious Disease Hospital in Vietnam.

Invasive non-typhoidal Salmonella (iNTS) infections are now a well-described cause of morbidity and mortality in children and HIV-infected adults in sub-Saharan Africa. In contrast, the epidemiology and clinical manifestations of iNTS disease in Asia are not well documented. We retrospectively identified >100 cases of iNTS infections in an infectious disease hospital in Southern Vietnam between 2008 and 2013. Clinical records were accessed to evaluate demographic and clinical factors associated with iNTS infection and to identify risk factors associated with death. Multi-locus sequence typing and antimicrobial susceptibility testing was performed on all organisms. Of 102 iNTS patients, 71% were HIV-infected, >90% were adults, 71% were male and 33% reported intravenous drug use. Twenty-six/92 (28%) patients with a known outcome died; HIV infection was significantly associated with death (p = 0.039). S. Enteritidis (Sequence Types (ST)11) (48%, 43/89) and S. Typhimurium (ST19, 34 and 1544) (26%, 23/89) were the most commonly identified serovars; S. Typhimurium was significantly more common in HIV-infected individuals (p = 0.003). Isolates from HIV-infected patients were more likely to exhibit reduced susceptibility against trimethoprim-sulfamethoxazole than HIV-negative patients (p = 0.037). We conclude that iNTS disease is a severe infection in Vietnam with a high mortality rate. As in sub-Saharan Africa, HIV infection was a risk factor for death, with the majority of the burden in this population found in HIV-infected adult men

Non-typhoidal Salmonella serovars associated with invasive and non-invasive disease in the Lao People's Democratic Republic.

BACKGROUND: Invasive non-typhoidal Salmonella (iNTS) disease is a well-described cause of mortality in children and human immunodeficiency virus (HIV)-infected adults in sub-Saharan Africa. Additionally, there is an ill-defined burden of iNTS disease in Southeast Asia. METHODS: Aiming to investigate the causative serovars of non-invasive and iNTS disease and their associated antimicrobial susceptibility profiles in the Lao People's Democratic Republic, we performed multilocus sequence typing and antimicrobial susceptibility profiling on 168 NTS (63 blood and 105 faecal) organisms isolated in Lao between 2000 and 2012. RESULTS: Six different serovars were isolated from blood; Salmonella enterica serovar Enteritidis (n=28), S. enterica serovar Typhimurium (n=19) and S. enterica serovar Choleraesuis (n=11) accounted for >90% (58/63) of the iNTS disease cases. In contrast, the isolates from diarrhoeal faeces were comprised of 18 different serovars, the mostly commonly identified being S. enterica Typhimurium (n=28), S. enterica Weltevreden (n=14) and S. enterica Stanley (n=15). S. enterica Enteritidis and S. enterica Choleraesuis were significantly more associated with systemic disease than diarrhoeal disease in this patient group (p<0.001). CONCLUSIONS: We find a differing distribution of Salmonella sequence types/serovars between those causing iNTS disease and non-invasive disease in Lao. We conclude that there is a small but not insignificant burden of iNTS disease in Lao. Further clinical and epidemiological investigations are required to assess mortality and the role of comorbidities such as HIV

Multilocus sequence typing of Cryptococcus neoformans var. grubii from Laos in a regional and global context.

Cryptococcosis causes approximately 180 000 deaths each year in patients with human immunodeficiency virus (HIV). Patients with other forms of immunosuppression are also at risk, and disease is increasingly recognized in apparently immunocompetent individuals. Cryptococcus neoformans var. grubii, responsible for the majority of cases, is distributed globally. We used the consensus ISHAM Multilocus sequence typing (MLST) scheme to define the population structure of clinical C. neoformans var. grubii isolates from Laos (n = 81), which we placed into the global context using published MLST data from other countries (total N = 1047), including a reanalysis of 136 Vietnamese isolates previously reported. We observed a phylogeographical relationship in which the Laotian population was similar to its neighbor Thailand, being dominated (83%) by Sequence Types (ST) 4 and 6. This phylogeographical structure changed moving eastwards, with Vietnam's population consisting of an admixture of isolates dominated by the ST4/ST6 (35%) and ST5 (48%) lineages. The ST5 lineage is the predominant ST reported from China and East Asia, where it accounts for >90% of isolates. Analysis of genetic distance (Fst) between different populations of C. neoformans var. grubii supports this intermediate structure of the Vietnamese population. The pathogen and host diversity reported from Vietnam provide the strongest epidemiological evidence of the association between ST5 and HIV-uninfected patients. Regional anthropological genetic distances suggest diversity in the C. neoformans var. grubii population across Southeast Asia is driven by ecological rather than human host factors. Where the ST5 lineage is present, disease in HIV-uninfected patients is to be expected

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

Evaluation of Luminex xTAG Gastrointestinal Pathogen Panel Assay for Detection of Multiple Diarrheal Pathogens in Fecal Samples in Vietnam.

Diarrheal disease is a complex syndrome that remains a leading cause of global childhood morbidity and mortality. The diagnosis of enteric pathogens in a timely and precise manner is important for making treatment decisions and informing public health policy, but accurate diagnosis is a major challenge in industrializing countries. Multiplex molecular diagnostic techniques may represent a significant improvement over classical approaches. We evaluated the Luminex xTAG gastrointestinal pathogen panel (GPP) assay for the detection of common enteric bacterial and viral pathogens in Vietnam. Microbiological culture and real-time PCR were used as gold standards. The tests were performed on 479 stool samples collected from people admitted to the hospital for diarrheal disease throughout Vietnam. Sensitivity and specificity were calculated for the xTAG GPP for the seven principal diarrheal etiologies. The sensitivity and specificity for the xTAG GPP were >88% for Shigellaspp.,Campylobacterspp., rotavirus, norovirus genotype 1/2 (GI/GII), and adenovirus compared to those of microbiological culture and/or real-time PCR. However, the specificity was low (∼60%) for Salmonella species. Additionally, a number of important pathogens that are not identified in routine hospital procedures in this setting, such as Cryptosporidiumspp. and Clostridium difficile, were detected with the GPP. The use of the Luminex xTAG GPP for the detection of enteric pathogens in settings, like Vietnam, would dramatically improve the diagnostic accuracy and capacity of hospital laboratories, allowing for timely and appropriate therapy decisions and a wider understanding of the epidemiology of pathogens associated with severe diarrheal disease in low-resource settings

South Asia as a Reservoir for the Global Spread of Ciprofloxacin-Resistant Shigella sonnei: A Cross-Sectional Study.

BACKGROUND: Antimicrobial resistance is a major issue in the Shigellae, particularly as a specific multidrug-resistant (MDR) lineage of Shigella sonnei (lineage III) is becoming globally dominant. Ciprofloxacin is a recommended treatment for Shigella infections. However, ciprofloxacin-resistant S. sonnei are being increasingly isolated in Asia and sporadically reported on other continents. We hypothesized that Asia is a primary hub for the recent international spread of ciprofloxacin-resistant S. sonnei. METHODS AND FINDINGS: We performed whole-genome sequencing on a collection of 60 contemporaneous ciprofloxacin-resistant S. sonnei isolated in four countries within Asia (Vietnam, n = 11; Bhutan, n = 12; Thailand, n = 1; Cambodia, n = 1) and two outside of Asia (Australia, n = 19; Ireland, n = 16). We reconstructed the recent evolutionary history of these organisms and combined these data with their geographical location of isolation. Placing these sequences into a global phylogeny, we found that all ciprofloxacin-resistant S. sonnei formed a single clade within a Central Asian expansion of lineage III. Furthermore, our data show that resistance to ciprofloxacin within S. sonnei may be globally attributed to a single clonal emergence event, encompassing sequential gyrA-S83L, parC-S80I, and gyrA-D87G mutations. Geographical data predict that South Asia is the likely primary source of these organisms, which are being regularly exported across Asia and intercontinentally into Australia, the United States and Europe. Our analysis was limited by the number of S. sonnei sequences available from diverse geographical areas and time periods, and we cannot discount the potential existence of other unsampled reservoir populations of antimicrobial-resistant S. sonnei. CONCLUSIONS: This study suggests that a single clone, which is widespread in South Asia, is likely driving the current intercontinental surge of ciprofloxacin-resistant S. sonnei and is capable of establishing endemic transmission in new locations. Despite being limited in geographical scope, our work has major implications for understanding the international transfer of antimicrobial-resistant pathogens, with S. sonnei acting as a tractable model for studying how antimicrobial-resistant Gram-negative bacteria spread globally

Genomic surveillance for hypervirulence and multi-drug resistance in invasive Klebsiella pneumoniae from South and Southeast Asia

Abstract: Background: Klebsiella pneumoniae is a leading cause of bloodstream infection (BSI). Strains producing extended-spectrum beta-lactamases (ESBLs) or carbapenemases are considered global priority pathogens for which new treatment and prevention strategies are urgently required, due to severely limited therapeutic options. South and Southeast Asia are major hubs for antimicrobial-resistant (AMR) K. pneumoniae and also for the characteristically antimicrobial-sensitive, community-acquired “hypervirulent” strains. The emergence of hypervirulent AMR strains and lack of data on exopolysaccharide diversity pose a challenge for K. pneumoniae BSI control strategies worldwide. Methods: We conducted a retrospective genomic epidemiology study of 365 BSI K. pneumoniae from seven major healthcare facilities across South and Southeast Asia, extracting clinically relevant information (AMR, virulence, K and O antigen loci) using Kleborate, a K. pneumoniae-specific genomic typing tool. Results: K. pneumoniae BSI isolates were highly diverse, comprising 120 multi-locus sequence types (STs) and 63 K-loci. ESBL and carbapenemase gene frequencies were 47% and 17%, respectively. The aerobactin synthesis locus (iuc), associated with hypervirulence, was detected in 28% of isolates. Importantly, 7% of isolates harboured iuc plus ESBL and/or carbapenemase genes. The latter represent genotypic AMR-virulence convergence, which is generally considered a rare phenomenon but was particularly common among South Asian BSI (17%). Of greatest concern, we identified seven novel plasmids carrying both iuc and AMR genes, raising the prospect of co-transfer of these phenotypes among K. pneumoniae. Conclusions: K. pneumoniae BSI in South and Southeast Asia are caused by different STs from those predominating in other regions, and with higher frequency of acquired virulence determinants. K. pneumoniae carrying both iuc and AMR genes were also detected at higher rates than have been reported elsewhere. The study demonstrates how genomics-based surveillance—reporting full molecular profiles including STs, AMR, virulence and serotype locus information—can help standardise comparisons between sites and identify regional differences in pathogen populations

An open label randomized controlled trial of tamoxifen combined with amphotericin B and fluconazole for cryptococcal meningitis

Background: Cryptococcal meningitis has high mortality. Flucytosine is a key treatment but is expensive and rarely available. The anti-cancer agent tamoxifen has synergistic anti-cryptococcal activity with amphotericin in vitro. It is off-patent, cheap, and widely available. We performed a trial to determine its therapeutic potential. Methods:Open label randomized controlled trial. Participants received standard care - amphotericin combined with fluconazole for the first two weeks - or standard care plus tamoxifen 300mg/day. The primary end point was Early Fungicidal Activity (EFA) - the rate of yeast clearance from cerebrospinal fluid (CSF). Trial registration https://clinicaltrials.gov/ct2/show/NCT03112031 . Results: 50 patients were enrolled, (median age 34 years, 35 male). Tamoxifen had no effect on EFA (- 0.48log10 colony-forming units/mL/CSF control arm versus -0.49 tamoxifen arm, difference - 0.005log10CFU/ml/day, 95%CI: -0.16, 0.15, P=0.95). Tamoxifen caused QTc prolongation. Conclusion: High dose tamoxifen does not increase the clearance rate of Cryptococcus from CSF. Novel, affordable therapies are needed. Funding:The trial was funded through the Wellcome Trust Asia Programme Vietnam Core Grant 106680 and a Wellcome Trust Intermediate Fellowship to JND grant number WT097147MA

New Variant of Multidrug-Resistant Salmonella enterica Serovar Typhimurium Associated with Invasive Disease in Immunocompromised Patients in Vietnam.

Nontyphoidal Salmonella (NTS), particularly Salmonella enterica serovar Typhimurium, is among the leading etiologic agents of bacterial enterocolitis globally and a well-characterized cause of invasive disease (iNTS) in sub-Saharan Africa. In contrast, S Typhimurium is poorly defined in Southeast Asia, a known hot spot for zoonotic disease with a recently described burden of iNTS disease. Here, we aimed to add insight into the epidemiology and potential impact of zoonotic transfer and antimicrobial resistance (AMR) in S Typhimurium associated with iNTS and enterocolitis in Vietnam. We performed whole-genome sequencing and phylogenetic reconstruction on 85 human (enterocolitis, carriage, and iNTS) and 113 animal S Typhimurium isolates isolated in Vietnam. We found limited evidence for the zoonotic transmission of S Typhimurium. However, we describe a chain of events where a pandemic monophasic variant of S Typhimurium (serovar I:4,[5],12:i:- sequence type 34 [ST34]) has been introduced into Vietnam, reacquired a phase 2 flagellum, and acquired an IncHI2 multidrug-resistant plasmid. Notably, these novel biphasic ST34 S Typhimurium variants were significantly associated with iNTS in Vietnamese HIV-infected patients. Our study represents the first characterization of novel iNTS organisms isolated outside sub-Saharan Africa and outlines a new pathway for the emergence of alternative Salmonella variants into susceptible human populations.IMPORTANCESalmonella Typhimurium is a major diarrheal pathogen and associated with invasive nontyphoid Salmonella (iNTS) disease in vulnerable populations. We present the first characterization of iNTS organisms in Southeast Asia and describe a different evolutionary trajectory from that of organisms causing iNTS in sub-Saharan Africa. In Vietnam, the globally distributed monophasic variant of Salmonella Typhimurium, the serovar I:4,[5],12:i:- ST34 clone, has reacquired a phase 2 flagellum and gained a multidrug-resistant plasmid to become associated with iNTS disease in HIV-infected patients. We document distinct communities of S Typhimurium and I:4,[5],12:i:- in animals and humans in Vietnam, despite the greater mixing of these host populations here. These data highlight the importance of whole-genome sequencing surveillance in a One Health context in understanding the evolution and spread of resistant bacterial infections

A Multi-Center Randomized Trial to Assess the Efficacy of Gatifloxacin versus Ciprofloxacin for the Treatment of Shigellosis in Vietnamese Children

The bacterial genus Shigella is the most common cause of dysentery (diarrhea containing blood and/or mucus) and the disease is common in developing countries with limitations in sanitation. Children are most at risk of infection and frequently require hospitalization and antimicrobial therapy. The WHO currently recommends the fluoroquinolone, ciprofloxacin, for the treatment of childhood Shigella infections. In recent years there has been a sharp increase in the number of organisms that exhibit resistance to nalidixic acid (an antimicrobial related to ciprofloxacin), corresponding with reduced susceptibility to ciprofloxacin. We hypothesized that infections with Shigella strains that demonstrate resistance to nalidixic acid may prevent effective treatment with ciprofloxacin. We performed a randomized controlled trial to compare 3 day ciprofloxacin therapy with 3 days of gatifloxacin, a newer generation fluoroquinolone with greater activity than ciprofloxacin. We measured treatment failure and time to the cessation of individual disease symptoms in 249 children with dysentery treated with gatifloxacin and 245 treated with ciprofloxacin. We could identify no significant differences in treatment failure between the two groups or in time to the cessation of individual symptoms. We conclude that, in Vietnam, ciprofloxacin and gatifloxacin are similarly effective for the treatment of acute dysentery