Publications by authors named "Pitak Santanirand"

39 Publications

Performance of real-time PCR and immunofluorescence assay for diagnosis of Pneumocystis pneumonia in real-world clinical practice.

PLoS One 2020 21;15(12):e0244023. Epub 2020 Dec 21.

Division of Pulmonary and Critical Care, Department of Medicine, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand.

Background: PCR is more sensitive than immunofluorescence assay (IFA) for detection of Pneumocystis jirovecii. However, PCR cannot always distinguish infection from colonization. This study aimed to compare the performance of real-time PCR and IFA for diagnosis of P. jirovecii pneumonia (PJP) in a real-world clinical setting.

Methods: A retrospective cohort study was conducted at a 1,300-bed hospital between April 2017 and December 2018. Patients whose respiratory sample (bronchoalveolar lavage or sputum) were tested by both Pneumocystis PCR and IFA were included. Diagnosis of PJP was classified based on multicomponent criteria. Sensitivity, specificity, 95% confidence intervals (CI), and Cohen's kappa coefficient were calculated.

Results: There were 222 eligible patients. The sensitivity and specificity of PCR was 91.9% (95% CI, 84.0%-96.7%) and 89.7% (95% CI, 83.3%-94.3%), respectively. The sensitivity and specificity of IFA was 7.0% (95% CI, 2.6%-14.6%) and 99.2% (95% CI, 95.6%-100.0%), respectively. The percent agreement between PCR and IFA was 56.7% (Cohen's kappa -0.02). Among discordant PCR-positive and IFA-negative samples, 78% were collected after PJP treatment. Clinical management would have changed in 14% of patients using diagnostic information, mainly based on PCR results.

Conclusions: PCR is highly sensitive compared with IFA for detection of PJP. Combining clinical, and radiological features with PCR is useful for diagnosis of PJP, particularly when respiratory specimens cannot be promptly collected before initiation of PJP treatment.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0244023PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7751978PMC
February 2021

Mycobacterium haemophilum scleritis: two case reports and review of literature.

BMC Ophthalmol 2020 Sep 23;20(1):378. Epub 2020 Sep 23.

Department of Ophthalmology, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Rama VI Rd., Rajathevi, Bangkok, 10400, Thailand.

Background: Mycobacterium haemophilum is a rare and emerging nontuberculous mycobacteria (NTM). It normally causes localized or disseminated systemic diseases, particularly skin infections and arthritis in severely immunocompromised patients. There have been 5 cases of M. haemophilum ocular infections reported in the literature. Only 1 case presented with scleritis with keratitis. Here, we reported 2 cases of M. haemophilum scleritis. One of them was immunocompetent host and had keratitis with radial keratoneuritis as a presenting sign.

Case Presentation: Case 1: A 52-year-old Thai female with rheumatoid arthritis presented with scleritis. Conjunctival scraping was carried out and the culture result was positive for M. haemophilum. Despite receiving systemic and topical antibiotics, her clinical symptoms and signs worsened. Surgical debridement was performed. After surgery, the lesion was significantly improved and finally turned to conjunctival scarring. Case 2: A 32-year old healthy Thai male without underlying disease presented with nodular scleritis and keratouveitis with multiple radial keratoneuritis. Surgical debridement of the scleral nodule was performed. Initial microbiological investigations were negative. Herpes ocular infections was suspected. Topical antibiotics, oral acyclovir, low-dose topical steroids and systemic steroids were started. The scleral inflammation subsided but later the keratitis relapsed, requiring corneal biopsy. Histopathology of the specimen revealed acid-fast bacteria and M. haemophilum was identified by polymerase chain reaction (PCR) and sequencing. The diagnosis of Mycobacterial keratitis was made. Although using the combination of systemic and topical antibiotics, his clinical status progressively deteriorated. Multiple therapeutic penetrating keratoplasties were required to eradicate the infection. No recurrence was found during the 1-year follow-up in both cases.

Conclusions: M. haemophilum can cause scleritis and keratitis, even in immunocompenent host. Radial keraoneuritis is first described in M. haemophilum keratitis. NTM keratitis should be considered in the differential diagnosis of patients with radial keratoneuritis. Increased awareness and early diagnosis using appropriate culture conditions and molecular techniques are important for the proper treatment of this infection. Prompt surgical intervention appears to be vital for successful management of M. haemophilum scleritis and keratitis.
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http://dx.doi.org/10.1186/s12886-020-01649-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7513486PMC
September 2020

Microbial epidemiology and risk factors for relapse in gram-negative bacteria catheter-related bloodstream infection with a pilot prospective study in patients with catheter removal receiving short-duration of antibiotic therapy.

BMC Infect Dis 2020 Aug 17;20(1):604. Epub 2020 Aug 17.

Department of Pharmacy, Faculty of Pharmacy, Mahidol University, Bangkok, Thailand.

Background: Infectious Diseases Society of America (IDSA) guidelines suggest 7-14 days' duration of antibiotic treatment for uncomplicated Gram-negative bacteria (GNB) catheter-related bloodstream infection (CRBSI). The objectives of this study were to review microbial epidemiology, to determine rate and risk factors for relapse, and to compare clinical outcomes in patients receiving long- versus short-duration antibiotic therapy.

Methods: A retrospective phase 1 study was conducted between January 2010 and October 2016 to review microbial epidemiology and to determine the incidence of and risk factors for relapse in patients with GNB CRBSI, according to the IDSA guidelines diagnostic criteria. In phase 2 of the study, patients without risk factors for relapse between November 2016 and October 2017 were prospectively recruited to receive antibiotic therapy for 7 days after catheter removal. Matched patients from the retrospective phase 1 study who had received antibiotic therapy for ≥14 days were selected as a phase 2 control group to compare outcomes.

Results: In phase 1, three most common pathogens identified among 174 cases were Pseudomonas aeruginosa (22.0%), Klebsiella pneumoniae (16.7%), and Stenotrophomonas maltophilia (13.4%). Eighty-nine episodes of infection occurred while patients were receiving antibiotic therapy. Of 140 cases, the relapse rate was 6.4%. Catheter retention was the only risk factor strongly associated with relapse (odds ratio = 145.32; 95% confidence interval 12.66-1667.37, P < 0.001). In phase 2, 11 patients with catheter removal were prospectively recruited to receive short-duration therapy. The number of patients with relapse receiving long- or short-duration therapy was 1 (3%) and 0 (0%), respectively (P = 1.000).

Conclusions: For the management of patients with uncomplicated GNB CRBSI, empiric broad-spectrum antibiotic therapy with adequate coverage of P. aeruginosa should be chosen. Catheter removal should be performed to prevent relapse and shortening the duration of treatment could be considered.

Trial Registration: Thai Clinical Trial Registry: TCTR20190914001 . Retrospectively registered on 13 September 2019.
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http://dx.doi.org/10.1186/s12879-020-05312-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7430115PMC
August 2020

AcGI1, a novel genomic island carrying antibiotic resistance integron In687 in multidrug resistant Achromobacter xylosoxidans in a teaching hospital in Thailand.

FEMS Microbiol Lett 2020 Jul;367(14)

Institute of Integrative Biology, University of Liverpool, Liverpool L69 7ZB, UK.

This study investigated the genetic basis of multidrug resistance in two strains of Achromobacter xylosoxidans isolated from patients attending a hospital in Thailand in 2012. These isolates were highly resistant to cephalosporins, aminoglycosides, fluoroquinolones, co-trimoxazole and carbapenems. Whole genome sequencing revealed that the two isolates were not clonally related and identified a carbapenem resistance gene-habouring integron (In687), residing in a novel genomic island, AcGI1. This In687 shares 100% identical nucleotide sequence with ones found in Acinetobacter baumannii Aci 16, isolated from the same hospital in 2007. We report the first analysis of multidrug-resistant A. xylosoxidans isolated in Thailand, and the first example of this island in A. xylosoxidans. Our data support the idea that resistance has spread in Thailand via horizontal gene transfer between species and suggest the possibility of A. xylosoxidans may serve as a reservoir of antibiotic resistance, especially in hospital setting.
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http://dx.doi.org/10.1093/femsle/fnaa109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7610039PMC
July 2020

A megaplasmid family driving dissemination of multidrug resistance in Pseudomonas.

Nat Commun 2020 03 13;11(1):1370. Epub 2020 Mar 13.

Institute of Infection and Global Health, University of Liverpool, Liverpool, UK.

Multidrug resistance (MDR) represents a global threat to health. Here, we used whole genome sequencing to characterise Pseudomonas aeruginosa MDR clinical isolates from a hospital in Thailand. Using long-read sequence data we obtained complete sequences of two closely related megaplasmids (>420 kb) carrying large arrays of antibiotic resistance genes located in discrete, complex and dynamic resistance regions, and revealing evidence of extensive duplication and recombination events. A comprehensive pangenomic and phylogenomic analysis indicates that: 1) these large plasmids comprise an emerging family present in different members of the Pseudomonas genus, and associated with multiple sources (geographical, clinical or environmental); 2) the megaplasmids encode diverse niche-adaptive accessory traits, including multidrug resistance; 3) the accessory genome of the megaplasmid family is highly flexible and diverse. The history of the megaplasmid family, inferred from our analysis of the available database, suggests that members carrying multiple resistance genes date back to at least the 1970s.
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http://dx.doi.org/10.1038/s41467-020-15081-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7070040PMC
March 2020

Discovery of New and Potent InhA Inhibitors as Antituberculosis Agents: Structure-Based Virtual Screening Validated by Biological Assays and X-ray Crystallography.

J Chem Inf Model 2020 01 27;60(1):226-234. Epub 2019 Dec 27.

School of Cellular and Molecular Medicine , University of Bristol , Biomedical Sciences Building, University Walk , BS8 1TD Bristol , United Kingdom.

The enoyl-acyl carrier protein reductase InhA of is an attractive, validated target for antituberculosis drug development. Moreover, direct inhibitors of InhA remain effective against InhA variants with mutations associated with isoniazid resistance, offering the potential for activity against MDR isolates. Here, structure-based virtual screening supported by biological assays was applied to identify novel InhA inhibitors as potential antituberculosis agents. High-speed Glide SP docking was initially performed against two conformations of InhA differing in the orientation of the active site Tyr158. The resulting hits were filtered for drug-likeness based on Lipinski's rule and avoidance of PAINS-like properties and finally subjected to Glide XP docking to improve accuracy. Sixteen compounds were identified and selected for in vitro biological assays, of which two (compounds and ) showed MIC of 12.5 and 25 μg/mL against H37Rv, respectively. Inhibition assays against purified recombinant InhA determined IC values for these compounds of 0.38 and 0.22 μM, respectively. A crystal structure of the most potent compound, compound , bound to InhA revealed the inhibitor to occupy a hydrophobic pocket implicated in binding the aliphatic portions of InhA substrates but distant from the NADH cofactor, i.e., in a site distinct from those occupied by the great majority of known InhA inhibitors. This compound provides an attractive starting template for ligand optimization aimed at discovery of new and effective compounds against that act by targeting InhA.
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http://dx.doi.org/10.1021/acs.jcim.9b00918DOI Listing
January 2020

Outcomes of Empirical Antimicrobial Therapy for Pediatric Community-onset Febrile Urinary Tract Infection in the Era of Increasing Antimicrobial Resistance.

Pediatr Infect Dis J 2020 02;39(2):121-126

From the Department of Pediatrics.

Background: Urinary tract infection (UTI) is a common cause of fever in children. Despite the increasing numbers of extended-spectrum beta-lactamase-producing organisms in the community, the empirical therapy of choice is still third-generation cephalosporins. This study was performed to investigate whether inappropriate empirical therapy (IAT) of community-onset UTI results in adverse clinical outcomes.

Methods: We retrospectively studied a cohort of pediatric patients with first-episode community-onset UTI caused by Escherichia coli, Klebsiella pneumoniae and Proteus spp. at Ramathibodi Hospital from 2011 to 2017. The patients were classified into IAT and appropriate empirical therapy (AT) groups. Medical records were reviewed to assess clinical outcomes.

Results: One hundred fifty-one eligible patients were enrolled in this study. The most common causative organism was E. coli (88.8% and 96.2% in the AT and IAT groups, respectively). Among the causative organisms, 19.8% were extended-spectrum beta-lactamase-producing organisms. There was no significant difference in clinical failure, microbiologic failure, relapse or time to defervescence between the 2 groups. No patients in either group developed sepsis after receiving empirical therapy. However, the length of hospital stay was significantly longer in the IAT than AT group [4.00 (4.50-6.00) vs. 7.00 (5.00-11.25) days, respectively; P = 0.000].

Conclusions: No significant difference in treatment outcomes was found between pediatric patients receiving AT and IAT for the treatment of UTI. In the era of increasing antimicrobial resistance, third-generation cephalosporins may still be a good choice as an empirical antimicrobial for children diagnosed with community-onset UTI.
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http://dx.doi.org/10.1097/INF.0000000000002515DOI Listing
February 2020

Rapid screening and early precautions for carbapenem-resistant carriers decreased nosocomial transmission in hospital settings: a quasi-experimental study.

Antimicrob Resist Infect Control 2019 27;8:110. Epub 2019 Jun 27.

1Department of Infection Control and Prevention, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka, 565-0871 Japan.

Background: Active surveillance has the potential to prevent nosocomial transmission of carbapenem-resistant (CRAB). We assessed whether rapid diagnosis using clinical specimen-direct loop-mediated isothermal amplification (LAMP), a rapid molecular diagnostic assay, and subsequent intervention, could reduce CRAB nosocomial transmission in intensive care units (ICUs).

Methods: A before and after (quasi-experimental) study was conducted in two ICUs at the Mahidol University Faculty of Medicine Ramathibodi Hospital with 3 months of observational period followed by 9 months of interventional period. All patients were screened for CRAB using both the culture and LAMP method from rectal swab and/or bronchial aspirates (intubated patients only) upon admission, weekly thereafter, and upon discharge. During the pre-intervention period, we performed contact precautions based on culture results. In contrast, during the intervention period, we initiated contact precautions within a few hours after sample collection on the basis of LAMP results.

Results: A total of 1335 patients were admitted to the ICUs, of which 866 patients (pre-intervention period: 187; intervention period: 679) were eligible for this study. Incidence rate of CRAB infection decreased to 20.9 per 1000 patient-days in the intervention period from 35.2 in the pre-intervention period ( < 0.02). The calculated hazard ratio of CRAB transmission was 0.65 (95% confidence interval [CI], 0.44-0.97). Risk factors for CRAB acquisition included exposure to carbapenem (hazard ratio, 2.54 [95% CI: 1.61-5.57]).

Conclusions: LAMP screening for CRAB upon ICU admission proved feasible for routine clinical practice. Rapid screening using LAMP followed by early intervention may reduce CRAB transmission rates in ICUs when compared to conventional intervention.
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http://dx.doi.org/10.1186/s13756-019-0564-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6598269PMC
June 2020

Epidemiology of carbapenem-resistant Enterobacteriaceae: a 5-year experience at a tertiary care hospital.

Infect Drug Resist 2019 20;12:461-468. Epub 2019 Feb 20.

Clinical Microbiology Laboratory, Department of Pathology, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand.

Purpose: The incidence of carbapenem-resistant Enterobacteriaceae (CRE) has been increasing worldwide. Ertapenem resistance is mediated by non-carbapenemase mechanisms, and has less of an effect on susceptibility to imipenem and meropenem. This study aimed to study the epidemiology of CRE, and to compare risk factors and related mortality between non-susceptibility to ertapenem alone Enterobacteriaceae (NSEE), with non-susceptibility to other carbapenems (imipenem, meropenem, or doripenem) Enterobacteriaceae (NSOCE) at a tertiary care hospital in Thailand.

Methods: All CRE isolated were identified between December 2011 and December 2016. Quarterly incidence rate was estimated. Hospital-wide carbapenem consumption was calculated as defined daily doses (DDD). Relationships between hospital-wide carbapenem consumption and incidence of CRE were tested. Factors associated with NSEE and NSOCE, and risk factors associated with 14- and 30-day mortality in patients with CRE infection were determined.

Results: The quarterly CRE incidence increased significantly from 3.37 per 100,000 patient-days in the last quarter of 2011 to 32.49 per 100,000 patient-days in the last quarter of 2016. ( for trend <0.001). Quarterly hospital-wide carbapenem consumption increased 1.58 DDD per 1,000 patient-days ( for trend=0.004). The Poisson regression showed the expected increase of CRE incidence was 1.02 per 100,000 patient-days for a 1 DDD per 1,000 patient-days increase in carbapenem consumption (<0.001). There were 40 patients with NSEE and 134 patients with NSOCE in the 5-year study period. The NSEE group had significantly lower carbapenem exposure compared with the NSOCE group (adjusted odds ratio: 0.25; =0.001). No difference in 14-day and 30-day all-cause mortality between the two groups was observed.

Conclusion: The incidence of CRE has risen significantly at our institution. Previous carbapenem use was associated with NSOCE. This hospital-wide carbapenem use was significantly associated with the increasing incidence of CRE.
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http://dx.doi.org/10.2147/IDR.S192540DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6390851PMC
February 2019

PCR-Dipstick-Oriented Surveillance and Characterization of - and Carbapenemase-Carrying in a Thai Hospital.

Front Microbiol 2019 8;10:149. Epub 2019 Feb 8.

Research Institute for Microbial Diseases, Osaka University, Suita, Japan.

Colistin is used as an alternative therapeutic for carbapenemase-producing (CPE) infections which are spreading at a very high rate due to the transfer of carbapenemase genes through mobile genetic elements. Due to the emergence of , the plasmid-mediated colistin resistance gene, -positive (MCRPEn) pose a high risk for the transfer of -carrying plasmid to CPE, leading to a situation with no treatment alternatives for infections caused by possessing both and carbapenemase genes. Here, we report the application of PCR-dipstick-oriented surveillance strategy to control MCRPEn and CPE by conducting the PCR-dipstick technique for the detection of MCRPEn and CPE in a tertiary care hospital in Thailand and comparing its efficacy with conventional surveillance method. Our surveillance results showed a high MCRPEn (5.9%) and CPE (8.7%) carriage rate among the 219 rectal swab specimens examined. Three different CPE clones were determined by pulsed-field gel electrophoresis (PFGE) whereas only two MCRPEn isolates were found to be closely related as shown by single nucleotide polymorphism-based phylogenetic analysis. Whole genome sequencing (WGS) and plasmid analysis showed that MCRPEn carried in two plasmids types-IncX4 and IncI2 with ~99% identity to the previously reported -carrying plasmids. The identification of both MCRPEn and CPE in the same specimen indicates the plausibility of plasmid-mediated transfer of genes leading to the emergence of colistin- and carbapenem-resistant . The rapidity (<2 h) and robust sensitivity (100%)/specificity (~99%) of PCR-dipstick show that this specimen-direct screening method could aid in implementing infection control measures at the earliest to control the dissemination of MCRPEn and CPE.
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http://dx.doi.org/10.3389/fmicb.2019.00149DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6375898PMC
February 2019

Correlation between antimicrobial consumption and the prevalence of carbapenem-resistant Escherichia coli and carbapenem-resistant Klebsiella pneumoniae at a university hospital in Thailand.

J Clin Pharm Ther 2019 Apr 21;44(2):292-299. Epub 2018 Dec 21.

Department of Pharmacy, Faculty of Pharmacy, Mahidol University, Bangkok, Thailand.

What Is Known And Objective: Carbapenem-resistant Enterobacteriaceae (CRE) are virulent gram-negative bacilli and cause urgent healthcare problems worldwide. One of the main factors leading to the emergence of CRE is antimicrobial consumption. The objective of this study was to assess how closely the rate of antimicrobial consumption and the prevalences of carbapenem-resistant Escherichia coli (CR-EC) and carbapenem-resistant Klebsiella pneumoniae (CR-KP) are correlated.

Methods: A retrospective study was performed at a university hospital in Thailand from January 2013 to September 2016. The prevalence of E coli and K pneumoniae was represented as percentages per species per quarter. The antimicrobial consumption rate per quarter was expressed as the defined daily dose (DDD)/1000 patient-days. Evaluation of the relationships between the rate of antimicrobial consumption and the prevalences of CR-EC and CR-KP was conducted via Pearson's or Spearman's correlation analyses.

Results And Discussion: During the study period, the prevalence of CR-EC and CR-KP was less than 6%; however, significantly increasing prevalences were reported for both CR-EC (r = 0.55, P = 0.03) and CR-KP (r = 0.87, P < 0.01). There was a significant increasing trend in the consumption of meropenem (r = 0.65, P = 0.01), levofloxacin (r = 0.63, P = 0.01), ceftriaxone (r = 0.55, P = 0.03), ertapenem (r = 0.52, P = 0.05) and the carbapenem group (r = 0.64, P = 0.01). A significant correlation was observed between CR-KP prevalence and total carbapenem consumption (r = 0.55, P = 0.04). Moreover, levofloxacin consumption had a significant positive relationship with the prevalence of CR-KP (r = 0.65, P = 0.01). No positive correlation was found with the prevalence of CR-EC.

What Is New And Conclusion: The rate of consumption of levofloxacin and carbapenems was the important key factor correlated with the rate of emergence of CR-KP. This is the first report demonstrating the correlation between levofloxacin consumption and CR-KP prevalence.
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http://dx.doi.org/10.1111/jcpt.12791DOI Listing
April 2019

Phenotypic and genotypic characterizations of extended-spectrum beta-lactamase-producing in Thailand.

Infect Drug Resist 2018 5;11:2151-2157. Epub 2018 Nov 5.

Department of Microbiology, Faculty of Pharmacy, Mahidol University, Bangkok, Thailand,

Purpose: Extended-spectrum β-lactamases (ESBLs) have become an issue in community worldwide due to an increase in antibiotic resistance over the past decade. This study was aimed to investigate the phenotypic and genotypic characteristics of ESBL-producing in Thailand.

Materials And Methods: In this study, all clinical isolates collected from tertiary hospitals in Thailand were identified as by biochemical tests and MALDI-TOF mass spectrometry. ESBL-producing was preliminary screened with disk diffusion method by cephalosporin disks and confirmed by the method of combination disk diffusion. Antimicrobial susceptibility test was used to determine MIC values of all ESBL-producing . For genotypic detection, a variety of ESBL genes were determined by PCR. Moreover, multilocus sequence typing (MLST) analysis was performed on internal portions of seven housekeeping genes for the diversity and phylogenetic relatedness of clonal group.

Results: Of the 285 ESBL-producing , most were susceptible to carbapenems. These strains showed a high resistance rate to ciprofloxacin (85.26%). The most frequently detected gene was group at about 71.23% followed by group (38.95%). The , , , , and genes were identified in 31.93%, 5.96%, 4.56%, 3.51%, and 0.70% of ESBL-producing isolates, respectively. The OXA-10 gene was detected in only one strain. ESBL-producing isolates with high antimicrobial resistance were further investigated. Among those, sequence type ST38 was mostly found, followed by ST405, ST410, and ST131. It is noteworthy that the gene was mainly detected in all four ST-type clones (ST38, ST405, ST410, and ST131).

Conclusion: This study provided a recent evidence of the genetic diversity of ESBL-producing in Thailand. In addition, the profile related to antimicrobial resistance pattern in this region was also demonstrated.
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http://dx.doi.org/10.2147/IDR.S174506DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6223337PMC
November 2018

Genomic Characterization of Carbapenemase-Producing Klebsiella pneumoniae with Chromosomally Carried .

Antimicrob Agents Chemother 2018 12 26;62(12). Epub 2018 Nov 26.

Japan-Thailand Research Collaboration Center for Infectious Diseases, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan.

We report here strains carrying chromosomal in Thailand. The genomes of these two isolates include a 160-kbp insertion containing , which is almost identical to that in the IncHI1B-like plasmid. Further analysis indicated that IS-mediated intermolecular transposition and Tn transposase-mediated homologous recombination resulted in the integration of into the chromosome from an IncHI1B-like plasmid. The spread of this type of carbapenem-resistant may threaten public health and warrants further monitoring.
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http://dx.doi.org/10.1128/AAC.01520-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6256778PMC
December 2018

Naked eye detection of the Mycobacterium tuberculosis complex by recombinase polymerase amplification-SYBR green I assays.

J Clin Lab Anal 2019 Feb 20;33(2):e22655. Epub 2018 Aug 20.

Research Group of Innovative Diagnosis of Antimicrobial Resistance, Department of Transfusion Medicine and Clinical Microbiology, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok, Thailand.

Background: Rapid diagnosis of Mycobacterium tuberculosis (Mtb) is key to controlling the spread of tuberculosis, which is a global health concern. In this study, isothermal recombinase polymerase amplification (RPA) was developed to detect specific targets of Mtb, IS6110 and IS1081. Additionally, SYBR Green I was used for endpoint detection of the RPA products by the naked eye.

Method: A total of 146 genomic Mtb DNA samples and 24 genomic nontuberculous mycobacteria (NTM) DNA samples were amplified at IS6110 and IS1081 by RPA. After a complete amplification, the RPA amplicons were examined by agarose gel electrophoresis (RPA-AGE) and SYBR Green I (RPA-S) assays. The performance of the RPA assays was evaluated by comparing them to a conventional PCR.

Results: The RPA assay demonstrated to have a good capability to differentiate Mtb from NTM with a very short turnaround time at a constant temperature. Compared to conventional PCR, the sensitivities and specificities of RPA-AGE for IS6110 and IS1081 were 100%. The specificity of RPA-S was 100% for both targets; however, its sensitivities for IS6110 and IS1081 were 97.95% and 99.32%, respectively. The limits of detection of IS6110 RPA-AGE and RPA-S were 0.05 and 0.5 ng, respectively, while the LODs of IS1081 RPA-AGE and RPA-S were 0.00005 and 0.05 ng, respectively. Both RPA assays showed a satisfying diagnostic specificity, with no cross-reaction with other bacteria.

Conclusion: A rapid, sensitive, naked eye RPA assay can be integrated into point-of-care diagnosis for Mtb detection, especially in remote areas where laboratory instrument resources are limited.
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http://dx.doi.org/10.1002/jcla.22655DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6818612PMC
February 2019

Biochemical and genetic analyses of the oomycete provide new insights into clinical identification and urease-based evolution of metabolism-related traits.

PeerJ 2018 5;6:e4821. Epub 2018 Jun 5.

Systems Biology and Bioinformatics Research Group, Pilot Plant Development and Training Institute, King Mongkut's University of Technology Thonburi, Bangkok, Thailand.

The oomycete microorganism, , causes the life-threatening infectious condition, pythiosis, in humans and animals worldwide. Affected individuals typically endure surgical removal of the infected organ(s). Detection of by the established microbiological, immunological, or molecular methods is not feasible in non-reference laboratories, resulting in delayed diagnosis. Biochemical assays have been used to characterize , some of which could aid in the clinical identification of this organism. Although hydrolysis of maltose and sucrose has been proposed as the key biochemical feature useful in discriminating from other oomycetes and fungi, this technique requires a more rigorous evaluation involving a wider selection of strains. Here, we evaluated 10 routinely available biochemical assays for characterization of 26 strains, isolated from different hosts and geographic origins. Initial assessment revealed diverse biochemical characteristics across the strains tested. Failure to hydrolyze sugars is observed, especially in slow-growing strains. Because hydrolysis of maltose and sucrose varied among different strains, use of the biochemical assays for identification of should be cautioned. The ability of to hydrolyze urea is our focus, because this metabolic process relies on the enzyme urease, an important virulence factor of other pathogens. The ability to hydrolyze urea varied among strains and was not associated with growth rates. Genome analyses demonstrated that urease- and urease accessory protein-encoding genes are present in both urea-hydrolyzing and non-urea-hydrolyzing strains of . Urease genes are phylogenetically conserved in and related oomycetes, while the presence of urease accessory protein-encoding genes is markedly diverse in these organisms. In summary, we dissected biochemical characteristics and drew new insights into clinical identification and urease-related evolution of .
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http://dx.doi.org/10.7717/peerj.4821DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5993020PMC
June 2018

Carbapenemase-Producing Carbapenem-Resistant Enterobacteriaceae from Bangkok, Thailand, and Their Detection by the Carba NP and Modified Carbapenem Inactivation Method Tests.

Microb Drug Resist 2018 Sep 21;24(7):1006-1011. Epub 2018 May 21.

1 Microbiology Laboratory, Department of Pathology, Faculty of Medicine Ramathibodi Hospital, Mahidol University , Bangkok, Thailand .

Aim: The purpose of the study was to determine the epidemiology of carbapenemase genes among carbapenem-resistant Enterobacteriaceae and evaluate the Carba NP and modified carbapenem inactivation method (mCIM) tests in their detection.

Materials And Methods: A total of 287 nonduplicated Enterobacteriaceae isolates, which were at least resistant to one of the carbapenems, were identified and detected for carbapenemase genes by multiplex PCR covering bla, bla, bla, bla, and bla. All positive genes were then sequenced. These isolates were phenotypically tested for the production of carbapenemases by mCIM and Carba NP tests to evaluate the efficacy of these methods.

Results: Seven species of carbapenem-resistant isolates mainly Klebsiella pneumoniae, Escherichia coli, and Enterobacter cloacae were detected. Of these isolates, three families of carbapenemase genes, including bla (bla, , , ), bla (bla, , ), and bla, were found. Of these, 223 (77.70%) carried at least one of the carbapenemase genes. The bla was detected in 160/223 (71.75%) isolates, of which 153/160 (95.63%) were the bla. Three types of the bla group, bla, bla, and bla, were found, 91/104 (87.5%) harbored the bla. In addition, 25.11% (56/223) of the carbapenemase-producing isolates harbored a combination of bla and bla. Phenotypic detection methods, mCIM and Carba NP, showed 100% sensitivity and specificity to bla, bla, and bla, while the mCIM was positive in all bla and bla isolates, only 12.5% (1/8) and 28.95% (11/38), respectively, were detected by the Carba NP test.

Conclusions: This study revealed a unique prevalence of carbapenemase genes in Bangkok, Thailand, as well as demonstrated the efficacy and limitation of phenotypic detection methods of carbapenemase in the area where bla and bla were predominant.
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http://dx.doi.org/10.1089/mdr.2018.0080DOI Listing
September 2018

Prevalence and risk factors of drug-resistant extrapulmonary tuberculosis.

Clin Respir J 2018 Jun 6;12(6):2101-2109. Epub 2018 Mar 6.

Microbiology Laboratory, Department of Pathology, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok 10400, Thailand.

Background: Physicians are usually aware of the occurrence of drug-resistant (DR) pulmonary tuberculosis (PTB), but lack concern about DR-extrapulmonary TB (EPTB). Data regarding the prevalence and risk factors of DR-EPTB remain limited.

Objectives: To determine the prevalence and risk factors of DR-EPTB.

Methods: A retrospective study was performed in patients who had culture-proven Mycobacterium tuberculosis (MTB) from various specimens between January 2013 and December 2015. Patients were classified into three groups: PTB, EPTB and concomitant PTB and EPTB (PTB + EPTB). Clinical data, chest radiographic extent of disease and patterns of DR were collected.

Results: There were 1014 culture-proven MTB specimens (716 pulmonary specimens and 298 extrapulmonary specimens) from 986 patients (648 PTB, 218 EPTB and 120 PTB + EPTB). The prevalences of isoniazid-, rifampicin- and multidrug-resistant EPTB were 7.8%, .5% and .5%, respectively, which were lower than those of PTB. When PTB and EPTB coexisted, a higher rate of DR-TB was observed than for PTB alone. Of 338 EPTB patients, the extent of radiographic disease was associated with isoniazid-, rifampicin- and multidrug-resistant TB. Previous history of TB and use of steroids/immunosuppressive drugs were also associated with rifampicin- and multidrug-resistant TB in multivariate analysis.

Conclusions: The prevalence of DR-EPTB was high in patients who had concomitant PTB. Although the prevalences of rifampicin- and multidrug-resistant TB were low in isolated EPTB, the prevalence of isoniazid-resistant TB remained high. Therefore, drug susceptibility testing should be performed in EPTB patients, especially those who carry the aforementioned risk factors.
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http://dx.doi.org/10.1111/crj.12779DOI Listing
June 2018

A randomized controlled trial of sitafloxacin vs. ertapenem as a switch therapy after treatment for acute pyelonephritis caused by extended-spectrum β-lactamase-producing Escherichia coli: A pilot study.

J Infect Chemother 2017 Aug 3;23(8):556-562. Epub 2017 Jun 3.

Department of Medicine, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand. Electronic address:

Background: The overuse and misuse of carbapenems have contributed to the antibiotic resistance crisis. The role of oral fluoroquinolones as a switch therapy for the treatment of urinary tract infection from Escherichia coli (ESBL-EC) is limited.

Objective: To compare the clinical and bacteriological efficacy of sitafloxacin and ertapenem for non-bacteremic acute pyelonephritis caused by ESBL-EC.

Methods: A prospective randomized controlled trial of patients with acute pyelonephritis caused by ESBL-EC was performed as a pilot study. One of the carbapenems was initially given to the patients. After day 3, patients were randomized to receive either sitafloxacin or ertapenem.

Results: Thirty-six patients were enrolled: 19 (52.8%) in the sitafloxacin group and 17 (47.2%) in the ertapenem group. There was no statistically significant difference in baseline characteristics between the two groups except a lower proportion of previous urinary catheter insertion in the sitafloxacin group (15.8% vs. 52.9%, p = 0.018). Signs and symptoms at presentation were similar between the two groups except a higher proportion of patients with chills in the sitafloxacin group (68.4% vs. 29.4%, p = 0.019). At day 10, all but one patient in the ertapenem group had clinical cure. Microbiological eradication was comparable between the sitafloxacin and ertapenem groups (84.2% vs. 75%, p = 0.677). There were no significant adverse effects.

Conclusions: Treatment of non-bacteremic acute pyelonephritis caused by ESBL-EC with carbapenem followed by oral sitafloxacin is effective and well-tolerated. Sitafloxacin may be considered as an alternative choice of switch therapy in this clinical setting.
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http://dx.doi.org/10.1016/j.jiac.2017.05.005DOI Listing
August 2017

PCR-Dipstick Chromatography for Differential Detection of Carbapenemase Genes Directly in Stool Specimens.

Antimicrob Agents Chemother 2017 06 24;61(6). Epub 2017 May 24.

Department of Infection Control and Prevention, Graduate School of Medicine, Osaka University, Osaka, Japan.

A PCR-dipstick chromatography technique was designed and evaluated for differential identification of , , , and carbapenemase genes directly in stool specimens within 2 h. It is a DNA-DNA hybridization-based detection system where PCR products can be easily interpreted by visual observation without electrophoresis. The PCR-dipstick showed high sensitivity (93.3%) and specificity (99.1%) in directly detecting carbapenemase genes in stool specimens compared with multiplex PCR for genomic DNA of the isolates from those stool specimens.
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http://dx.doi.org/10.1128/AAC.00067-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5444138PMC
June 2017

Endobronchial ultrasound-guided transbronchial needle aspiration rinse fluid polymerase chain reaction in the diagnosis of intrathoracic tuberculous lymphadenitis.

Infect Dis (Lond) 2017 Mar 21;49(3):193-199. Epub 2016 Oct 21.

b Microbiology Laboratory, Department of Pathology, Faculty of Medicine , Ramathibodi Hospital, Mahidol University , Bangkok , Thailand.

Background: Intrathoracic tuberculous (TB) lymphadenitis is a diagnostic challenge to the clinician. Although endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) can obtain a sample from the affected lymph node, the diagnosis of TB lymphadenitis by cytopathology remains inaccurate.

Objective: To evaluate the efficacy of EBUS-TBNA rinse fluid TB polymerase chain reaction (PCR) assay for the diagnosis of intrathoracic TB lymphadenitis.

Methods: A retrospective study was conducted on 102 patients who underwent EBUS-TBNA for diagnostic evaluation of intrathoracic lymphadenopathy. EBUS-TBNA specimens were evaluated by cytopathological examination. Rinse fluid of the needle was routinely submitted for acid-fast bacillus (AFB) staining, mycobacterial culture, and TB-PCR using the Anyplex MTB/NTM real-time detection kit.

Results: Of 102 patients, 16 were diagnosed with intrathoracic TB lymphadenitis by either microbiology, cytopathology, or on clinical grounds. The sensitivity, specificity, positive predictive value, and negative predictive value of rinse fluid TB PCR assay were 56.2%, 100.0%, 100.0%, and 92.5%, respectively. Using the area under the ROC curve (AUC) as a measure of a diagnostic performance, TB-PCR had the highest AUC, compared with mycobacterial culture, AFB smear, and finding of necrotizing granulomatous inflammation (0.78, 0.75, 0.56, and 0.72, respectively). A combination of TB PCR, mycobacterial culture, and finding of necrotizing granulomatous inflammation provided the best diagnostic performance (sensitivity, specificity, positive predictive value, negative predictive value, and AUC of 75.0%, 100.0%, 100.0%, 95.6%, and 0.88, respectively).

Conclusions: EBUS-TBNA rinse fluid TB-PCR is useful in the diagnosis of intrathoracic TB lymphadenitis. Combining TB-PCR with mycobacterial culture and cytopathological findings improved the diagnosis performance.
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http://dx.doi.org/10.1080/23744235.2016.1244613DOI Listing
March 2017

EPIDEMIOLOGY AND CONTROL OF THE FIRST REPORTED VANCOMYCIN-RESISTANT ENTEROCOCCUS OUTBREAK AT A TERTIARY-CARE HOSPITAL IN BANGKOK, THAILAND.

Southeast Asian J Trop Med Public Health 2016 May;47(3):494-502

This retrospective study described the first reported vancomycin-resistant enterococci (VRE) outbreak from June 2013 through January 2014 at a tertiary-care hospital in Bangkok, Thailand. After the index case was detected in an 18-bed medical intermediate care unit, a number of interventions was implemented, including targeted active surveillance for VRE, strict contact precautions, enhanced standard precautions, dedicated units for VRE cases, extensive cleaning of the environment and the restricted use of antibiotics. VRE isolates were evaluated by polymerase chain reaction and random amplified polymorphic DNA (RAPD) testing. A prevalence case-control study was conducted. Among 3,699 culture samples from 2,671 patients screened, 74 patients (2.8%) had VRE. The positivity rate declined from 15.1% during week 1 to 8.2% during week 2 and then 1.4% during week 3. By weeks 4-9, the prevalences were 0-2.7%. However, the prevalence rose to 9.4% during week 10 and then subsequently declined. All VRE isolates were Enterococcus faecium and had the vanA gene. RAPD analysis revealed a single predominant clone. Multivariate analysis showed mechanical ventilation for ≥ 7 days was a predictive factor for VRE colonization [odds ratio (OR) 11.47; 95% confidence interval (CI): 1.75-75.35; p = 0.011]. This experience demonstrates VRE can easily spread and result in an outbreak in multiple-bed units. Active surveillance, early infection control interventions and rapid patient cohorting were important tools for control of this outbreak. Patients requiring mechanical ventilator for ≥ 7 days were at higher risk for VRE acquisition.
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May 2016

Incidence of Fluoroquinolone Resistant Aerobic Organisms and Efficacy of Rectal Cleaning in Men Undergoing Transrectal Ultrasound Guided Prostate Biopsy.

J Med Assoc Thai 2016 Jun;99(6):691-6

Objective: To evaluate the incidence of fluoroquinolone resistant organisms in rectum and efficacy of rectal cleansing in men undergoing transrectal ultrasound guided prostate biopsy (TRUS-Bx) in Ramathibodi Hospital.

Material And Method: Between December 2012 and March 2013, 105 male patients who had prostate specific antigen (PSA) more than 4 ng/ml or abnormal digital rectal examination (DRE) underwent TRUS-Bx were enrolled. Two specimens of rectal swab for bacterial culture were taken from each patient. The first rectal swab was obtained at the beginning of the procedure (BC), another after cleaning the rectum with betadine solution (AC). All gram-negative enteric bacteria were isolated. The results of both specimens were analyzed by Chi-square test and McNemar test.

Results: One hundred five men that underwent TRUS-Bx were included in the present study. Of the 105 patients, 15 men were found to have no bacterial growth while 90 men showed bacterial growth at the BC procedure. After the AC procedure, 53 men (59%) remained having positive culture for bacterial strains (p<0.001), and 37 (41%) showed no bacterial growth. There was no change in the bacterial strains in 36 men while another four men demonstrated an increasing number of bacterial strains at the AC stage. Of 90 patients, 81 (90%) men carried ciprofloxacin resistant organisms including Escherichia coli (E. coli) (55.56%), extended-spectrum β-lactamase (ESBL)-producing E. coli (35.80%), Klebsiella pneumoniae (6.17%), and Enterobacter cloacae (2.47%).

Conclusion: Incidence of fluoroquinolone resistant organisms in rectum of men undergoing TRUS-Bx at Ramathibodi Hospital was approximately 90%. E. coli was the most common organism. The results indicated that rectal cleaning significantly decreases the incidence of overall bacterial colonization in rectum before TRUS-Bx.
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June 2016

Hemoculture and Direct Sputum Detection of mecA-Mediated Methicillin-Resistant Staphylococcus aureus by Loop-Mediated Isothermal Amplification in Combination With a Lateral-Flow Dipstick.

J Clin Lab Anal 2016 Sep 17;30(5):760-7. Epub 2016 Mar 17.

Bioengineering and Sensing Technology Laboratory, BIOTEC, National Science and Technology Development Agency (NSTDA), Pathum Thani, Thailand.

This study reports loop-mediated isothermal amplification (LAMP) for rapid detection of methicillin-resistant Staphylococcus aureus from direct clinical specimens. Four primers including outer and inner primers were specifically designed on the two target sequences-femB to identify S. aureus and mecA to identify antibiotic-resistant gene. Reference strains including various species of gram-positive/gram-negative isolates were used to evaluate and optimize LAMP assays. The optimum LAMP condition was found at 63°C within 70 min assay time (include hybridization with FITC probe for 5 min and further 5 min for reading the results on the lateral flow dipstick). The detection limits of LAMP for mecA was 10 pg of total DNA or 100 CFU/ml. The LAMP assays were applied to a total of 155 samples of direct DNA extraction from sputum and hemoculture bottles. The sensitivity of LAMP for mecA detection in sputum and hemoculture bottles was 93.3% (28/30) and 100% (52/52), respectively. In conclusion, LAMP assay is an alternative technique for rapid detection of MRSA infection with a technical simplicity and cost-effective method in a routine diagnostic laboratory.
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http://dx.doi.org/10.1002/jcla.21935DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6807216PMC
September 2016

Development of an Anti-Elicitin Antibody-Based Immunohistochemical Assay for Diagnosis of Pythiosis.

J Clin Microbiol 2016 Jan;54(1):43-8

Department of Pathology, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand

Pythiosis is an emerging and life-threatening infectious disease of humans and animals living in tropical and subtropical countries and is caused by the fungus-like organism Pythium insidiosum. Antifungals are ineffective against this pathogen. Most patients undergo surgical removal of the infected organ, and many die from advanced infections. Early and accurate diagnosis leads to prompt management and promotes better prognosis for affected patients. Immunohistochemical assays (IHCs) have been developed using rabbit antibodies raised against P. insidiosum crude extract, i.e., culture filtrate antigen (CFA), for the histodiagnosis of pythiosis, but cross-reactivity with pathogenic fungi compromises the diagnostic performance of the IHC. Therefore, there is a need to improve detection specificity. Recently, the elicitin protein, ELI025, was identified in P. insidiosum, but it was not identified in other human pathogens, including true fungi. The ELI025-encoding gene was successfully cloned and expressed as a recombinant protein in Escherichia coli. This study aims to develop a new IHC using the rabbit anti-ELI025 antibody (anti-ELI) and to compare its performance with the previously reported anti-CFA-based IHC. Thirty-eight P. insidiosum histological sections stained positive by anti-ELI-based and anti-CFA-based IHCs indicating 100% detection sensitivity for the two assays. The anti-ELI antibody stained negative for all 49 negative-control sections indicating 100% detection specificity. In contrast, the anti-CFA antibody stained positive for one of the 49 negative controls (a slide prepared from Fusarium-infected tissue) indicating 98% detection specificity. In conclusion, the anti-ELI based IHC is sensitive and specific for the histodiagnosis of pythiosis and is an improvement over the anti-CFA-based assay.
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http://dx.doi.org/10.1128/JCM.02113-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4702728PMC
January 2016

Pediatric extended spectrum β-lactamase infection: Community-acquired infection and treatment options.

Pediatr Int 2016 May 13;58(5):338-46. Epub 2016 Apr 13.

Department of Pediatrics, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand.

Background: Infection caused by extended spectrum β-lactamase (ESBL)-producing Enterobacteriaceae in pediatric patients has been increasing and spreading to the community, compromising the options for effective antibiotics. This retrospective study was conducted to identify which antibiotics ESBL-producing Enterobacteriaceae remain susceptible to. In addition, the prevalence of community-acquired infection caused by these organisms, and the possibility of association between these organisms and septic shock, were explored.

Methods: Antibiotic susceptibility of ESBL-producing and non-ESBL-producing Escherichia coli and Klebsiella pneumoniae strains isolated from pediatric patients were reviewed to determine the rates of susceptibility to various antibiotics. A chart review was performed to clarify the prevalence of community-acquired infection and the severity.

Results: Of 849 strains analyzed, 40% were ESBL positive. Apart from cephalosporins, ESBL-producing strains were also less likely to be susceptible to other antibiotics, such as quinolones, gentamicin, netilmicin, and cotrimoxazole, more than 90% of which were still susceptible to amikacin, carbapenems, colistin, and tigecycline. Around 20% of community-acquired infections in the present study were caused by ESBL-producing strains. ESBL-producing strains found in the community were more likely to be susceptible to gentamicin, netilmicin, and cefepime than those found in hospital. Infection caused by ESBL-producing strains was not significantly associated with septic shock.

Conclusion: The increase in infection caused by ESBL-producing Enterobacteriaceae limits the availability of effective antibiotics. Given that carbapenems are necessary for treating serious infections, amikacin, cefepime, and piperacillin/tazobactam are possible options for consolidative therapy or for non-serious infection.
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http://dx.doi.org/10.1111/ped.12845DOI Listing
May 2016

Clinical Specimen-Direct LAMP: A Useful Tool for the Surveillance of blaOXA-23-Positive Carbapenem-Resistant Acinetobacter baumannii.

PLoS One 2015 28;10(7):e0133204. Epub 2015 Jul 28.

Division of Infection Control and Prevention, Osaka University Graduate School of Medicine, Osaka, Japan.

Healthcare-associated infections are a leading cause of morbidity and mortality worldwide. Treatment is increasingly complicated by the escalating incidence of antimicrobial resistance. Among drug-resistant pathogens, carbapenem-resistant Acinetobacter baumannii (CRAb) is of increasing concern because of the limited applicable therapies and its expanding global distribution in developed countries and newly industrialized countries. Therefore, a rapid detection method that can be used even in resource-poor countries is urgently required to control this global public health threat. Conventional techniques, such as bacterial culture and polymerase chain reaction (PCR), are insufficient to combat this threat because they are time-consuming and laborious. In this study, we developed a loop-mediated isothermal amplification (LAMP) method for detecting blaOXA-23-positive CRAb, the most prevalent form of CRAb in Asia, especially in Thailand, and confirmed its efficacy as a surveillance tool in a clinical setting. Clinical samples of sputum and rectal swabs were collected from patients in a hospital in Bangkok and used for LAMP assays. After boiling and centrifugation, the supernatants were used directly in the assay. In parallel, a culture method was used for comparison purposes to evaluate the specificity and sensitivity of LAMP. As a first step, a total of 120 sputum samples were collected. The sensitivity of LAMP was 88.6% (39/44), and its specificity was 92.1% (70/76) using the culture method as the "gold standard". When surveillance samples including sputum and rectal swabs were analyzed with the LAMP assay, its sensitivity was 100.0%. This method enables the direct analysis of clinical specimens and provides results within 40 minutes of sample collection, making it a useful tool for surveillance even in resource-poor countries.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0133204PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4517775PMC
May 2016

SPR-DNA array for detection of methicillin-resistant Staphylococcus aureus (MRSA) in combination with loop-mediated isothermal amplification.

Biosens Bioelectron 2015 Dec 25;74:335-40. Epub 2015 Jun 25.

Materials Science and Engineering Programme, Faculty of Science, Mahidol University, Rama VI Rd., Phayathai, Bangkok 10400, Thailand; Physics Department, Faculty of Science, Mahidol University, Rama VI Rd., Phayathai, Bangkok 10400, Thailand. Electronic address:

In this study, we evaluated surface plasmon resonance imaging (SPR imaging) as a DNA biosensor for the detection of methicillin-resistant Staphylococcus aureus (MRSA) which is one of the most common causes of nosocomial infections. The DNA sample were collected from clinical specimens, including sputum and blood hemoculture were undergone LAMP amplification for 0.18 kbp and 0.23 kbp DNA fragments of femB and mecA genes, respectively. The self-assembled monolayer surface (SAMs) was used for immobilized streptavidin-biotinylated probes on the sensor surface for the detection of LAMP amplicons from MRSA. Both LAMP amplicons were simultaneously hybridized with ssDNA probes immobilized onto a bio-functionalized surface to detect specific targets in the multiplex DNA array platform. In addition, the sensor surface could be regenerated allowing at least five cycles of use with a shortened assay time. The detection limit of LAMP-SPR sensing was 10 copies/µl and LAMP-SPR sensing system showed a good selectivity toward the MRSA.
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http://dx.doi.org/10.1016/j.bios.2015.06.038DOI Listing
December 2015

Treatment with intrastromal and intracameral voriconazole in 2 eyes with Lasiodiplodia theobromae keratitis: case reports.

Medicine (Baltimore) 2015 Feb;94(6):e541

From the Department of Ophthalmology (KL, MN, NN); and Department of Pathology (PS), Ramathibodi Hospital, Mahidol University, Bangkok, Thailand.

To report the clinical presentation and the role of intrastromal and intracameral voriconazole injection in the management of rare cases of fungal keratitis caused by Lasiodiplodia theobromae.Two eyes of 2 patients with Lasiodiplodia keratitis unresponsive to topical and oral antifungal medications were included in this study. Diagnosis of Lasiodiplodia keratitis was confirmed by microbiological analysis, including culture-based (case 1 and 2) and DNA sequencing techniques (case 2 only).The first patient presented with multiple satellite lesions and one of these infiltrates spread deeply into the cornea, forming a stromal abscess. Another patient had a large full-thickness corneal infiltrates with several fungal balls in the anterior chamber, requiring a limbus-to-limbus therapeutic penetrating keratoplasty. Despite aggressive topical therapy, the stromal abscess continued to worsen in the first case and recurrent keratitis was observed postoperatively in the second case. Voriconazole 50 μg/0.1 mL was administered intracamerally and intrastromally around the fungal abscess as adjuncts to topical antimycotics in the first case. The second patient who needed therapeutic keratoplasty was treated with an intracameral injection of 50 μg/0.1 mL voriconazole at the end of surgery. Postoperatively, 100 μg/0.1 mL voriconazole was also injected intracamerally after the recurrence of infection was noted in the graft. Reinjections were given 48 hours apart in both cases. After the injections, all corneal and anterior chamber lesions were reduced in size and density and completely resolved within 4 weeks.Intrastromal and intracameral voriconazole injections may offer safe and effective treatment options for L theobromae keratitis.
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http://dx.doi.org/10.1097/MD.0000000000000541DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4602755PMC
February 2015

Arcanobacterium pyogenes endocarditis: a case report and literature review.

Southeast Asian J Trop Med Public Health 2014 Jan;45(1):142-8

We report the case of a 64-year-old man with Arcanobacterium pyogenes endocarditis. The patient presented with dyspnea and asymmetrical progressive quadriparesis. A transthoracic echocardiogram revealed mobile vegetations on both leaflets of his mitral valve measuring 0.5 x 3 cm, thickening of the mitral valve with severe mitral regurgitation due to dehiscence of the papillary muscle to the posterior mitral leaflet. He also had aortic sclerosis with a vegetation measuring 0.5 x 1 cm causing aortic valve dehiscence and free flow aortic regurgitation. An initial hemoculture grew out pleomorphic, gram-positive, non-motile, anaerobic to microaerophilic bacilli. A diagnosis of infective endocarditis was made using modified Duke criteria. He was treated with intravenous ampicillin and gentamicin. Four days after admission, he developed acute respiratory failure and succumbed to the disease. A pre-mortem hemoculture and post-mortem heart valve culture grew Arcanobacterium pyogenes. Septic thromboemboli involving the brain, kidneys, lungs and spleen were documented. The patient also had ischemic vasculopathy with focal spinal arteriolitis and bilateral demyelination of the cervical corticospinal tracts. There are three published reports of human A. pyogenes endocarditis in the literature. Neurological involvement with ischemic spinal vasculopathy and demyelination has not been reported. We report the first autopsy proven case of A. pyogenes infective endocarditis with ischemic spinal vasculopathy. We review the clinicopathologic features of systemic A. pyogenes infection.
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January 2014

Clinical characteristics and mortality risk factors of cryptococcal infection among HIV-negative patients.

J Med Assoc Thai 2014 Jan;97(1):36-43

Objective: Describe the clinical characteristics, treatment, outcomes, complications, and factors associated with mortality of cryptococcosis in HIV-negative patients.

Material And Method: A retrospective cohort study was conducted among HIV-negative adult patients with positive culture for Cryptococcus neoformans between 2005 and 2010.

Results: Forty-nine HIV-negative patients were identified with median (IQR) age of 62.5 (45.5-71.9) years of which 40.8% were male. The common underlying medical conditions were cardiovascular diseases (36.7%). The common sites of positive culture were cerebrospinal fluid/intracerebral abscess (46.9%), blood (36%), and sputum/bronchoalveolar larvage fluid (28.6%). Twenty-nine (59.2%) patients had co-infections with another organism, such as Gram-negative bacteria (24.4%), M. tuberculosis (17.8%), and Gram-positive bacteria (13.3%). The common clinical presentations were fever (67.3%), alteration of consciousness (34.7%), and headache (26.5%). Complication was detected in 61.2% such as acute kidney injury (47.0%), coma (38.8%), and shock (22.4%). The overall mortality was 51%. By multivariate logistic regression, factors associated with mortality were alteration of consciousness (adjusted OR = 6.85; 95% CI: 1.41-33.28, p = 0.017) and co-infections (adjusted OR = 5.32; 95% CI: 1.25-22.69, p = 0.024).

Conclusion: The mortality rate of HIV-negative patients with cryptococcosis is very high. Early recognition and treatment of cryptococcosis in HIV-negative patients are crucial and may improve the outcome.
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January 2014