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Abstract Background Commercial diagnostics are commonly used to identify gram-positive bacteria. Errors have been reported mostly at the species level. We have found certain phenotypic criteria used in API systems which significantly misidentify Leuconostoc, an emerging human pathogen, at the genus level. We also attempt to find practical, conventional phenotypic assays for accurate identification of this group of bacteria. Methods Clinical isolates of catalase-negative, gram-positive coccoid or coccobacillary bacteria with non-β hemolysis in our institute during 1997–2004 were subject to an identification aid by API 20 STREP, following the instruction manual, as an aid to conventional phenotypic tests. Those identified as Leuconostoc by API 20 STREP were re-examined by the same kit and also by API 50 CHL according to the instruction manuals, by our Leuconostoc conventional phenotypic assays, by Leuconostoc- and Lactobacillus-specific PCR's, and, where possible, by 16S rDNA sequence analysis. In addition, catalase-negative gram-positive isolates during 2005–2006 which were resistant to vancomycin at high levels were also evaluated by the same phenotypic and genotypic assays. Results Out of several thousands of clinical gram-positive isolates, 26 catalase negative gram-positive isolates initially identified as Leuconostoc by API 20 STREP and 7 vancomycin-resistant gram-positive catalase-negative bacteria entered the study. 11 out of the 26 isolates and all the 7 isolates were identified as Leuconostoc by API 20 STREP. Only 5 isolates, however, were confirmed by both genotypic and all defined conventional phenotypic criteria. API 50 CHL also failed to reliably provide accurate identification of Leuconostoc. We have identified key problem tests in API 20 STREP leading to misidentification of the bacteria. A simple, conventional set of phenotypic tests for Leuconostoc identification is proposed. Conclusion The current API systems cannot accurately identify Leuconostoc. Identification of vancomycin-resistant, catalase-negative gram-positive bacteria should be performed by a few practical phenotypic assays, with assistance of genotypic assays where available.

The increasing occurrence of hospital-associated infections, particularly bacteremia, caused by extensively drug-resistant (XDR) carbapenemase-producing colistin-resistant Klebsiella pneumoniae highlights a critical requirement to discover new therapeutic alternatives. Bacteriophages having host-specific bacteriolytic effects are promising alternatives for combating these pathogens. Among 12 phages isolated from public wastewater in Thailand, two phages-vB_kpnM_05 (myovirus) and vB_kpnP_08 (podovirus) showed broad-host range, producing bacteriolytic activities against 81.3% (n = 26) and 78.1% (n = 25) of 32 XDR carbapenemase-producing colistin-resistant K. pneumoniae, with capsular types-K15, K17, K50, K51, K52/wzi-50 and K2/wzi-2. Both phages showed short replication times, large burst sizes with rapid adsorptions. They exhibited significant stability under various environmental conditions. Genomic analysis revealed that both phages are genetically distinct phages from Myoviridae and Podoviridae family, with the lack of toxin, virulence, lysogeny and antibiotic resistance genes. These characteristics highlighted their promising potential for utilizing in phage therapy for combating XDR K. pneumoniae. Although phage cocktail combining vB_kpnM_05 and vB_kpnP_08 provided significant bacteriolysis for longer duration (8 h) than its monophage (6 h), bacterial regrowth was observed which suggested an evitable development of phage resistance under phages' selection pressures. Future study will be undertaken to elucidate the precise mechanisms by which these XDR K. pneumoniae developed phage resistance and their associated fitness cost. Remarkably, combining phage cocktail with amikacin at their sub-inhibitory concentrations produced potent synergy by completely suppressing bacterial regrowth in vitro. Our study demonstrated the significant therapeutic and prophylactic effectiveness of a phage cocktail-amikacin combination as a promising alternative strategy for overcoming bacteremia associated with XDR K. pneumoniae having carbapenemase and colistin resistance in vivo., Competing Interests: Declaration. Competing interests: The authors declare no competing interests. Informed consent: For this retrospective study of anonymous clinical isolates, the requirement for informed consent from patients was waived by the Institutional Review Board (IRB) of the Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand (IRB No.2865/65)., (© 2024. The Author(s).)

Objectives: To evaluate the accuracy of beta-lactamase gene detection directly from urine samples by Nanopore sequencing., Methods: DNA was extracted from bacterial pellets in spun urine. The purified DNA was then sequenced in native form by a Nanopore sequencer (MinION) to identify the organisms and beta-lactamase genes. Results were compared to routine urine cultures and standard antimicrobial susceptibility tests (AST)., Results: We processed 60 urine samples of which routine cultures grew Enterobacteriaceae, including 28 carbapenem-resistant (CRE), 17 extended-spectrum beta-lactamase (ESBL) or AmpC producing, and 15 non-ESBL/AmpC phenotypes. We excluded 7 samples with extremely low DNA amounts (<1 ng/μl) for a final case of 53 in total. The sensitivity of antimicrobial resistance gene detection within 6 h, the optimal duration from real-time simulation, of Nanopore sequencing for the diagnosis of carbapenem-resistant and ceftriaxone-resistant phenotypes was 73.9 % (95%CI 56.0-91.9 %) and 81.1 % (95%CI 68.5-93.7 %), while the specificity was 96.7 % (95%CI 90.2-100.0 %) and 56.3 % (95%CI 31.9-80.6 %), respectively. The median times for MinION to generate DNA reads containing carbapenemase and ESBL/AmpC genes were 93 min (IQR 17-245.5) and 99 min (IQR 31.25-269.75) after sequencing commencement, respectively., Conclusions: Nanopore sequencing can identify bacterial genotypic resistance in urine and may enable clinicians to adjust antimicrobial therapy earlier than routine AST., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Given the rise of multidrug-resistant (MDR) Pseudomonas aeruginosa infections, alternative treatments are needed. Anti-pseudomonal phage therapy shows promise, but its clinical application is limited due to the development of resistance and a lack of biofilm penetration. Recently, adjuvants like CaEDTA have shown the ability to enhance the effectiveness of combined antimicrobial agents. Here, we tested a phage-adjuvant combination and demonstrated the effectiveness of intranasally inhaled phage (KKP10) + CaEDTA in addition to ceftazidime/avibactam (CZA) for chronic P. aeruginosa lung infections. The results emphasize that intranasal inhalation of phage along with CaEDTA can successfully re-sensitize MDR P. aeruginosa to CZA in a triple combination treatment. This promising approach shows potential as a therapy for chronic respiratory tract infections., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Developing an effective therapy to overcome carbapenemase-positive Klebsiella pneumoniae (CPKp) is an important therapeutic challenge that must be addressed urgently. Here, we explored a Ca-EDTA combination with aztreonam or ceftazidime-avibactam in vitro and in vivo against diverse CPKp clinical isolates. The synergy testing of this study demonstrated that novel aztreonam-Ca-EDTA or ceftazidime-avibactam-Ca-EDTA combination was significantly effective in eliminating planktonic and mature biofilms in vitro , as well as eradicating CPKp infections in vivo . Both combinations revealed significant therapeutic efficacies in reducing bacterial load in internal organs and protecting treated mice from mortality. Conclusively, this is the first in vitro and in vivo study to demonstrate that novel aztreonam-Ca-EDTA or ceftazidime-avibactam-Ca-EDTA combinations provide favorable efficacy and safety for successful eradication of carbapenemase-producing Klebsiella pneumoniae planktonic and biofilm infections., Competing Interests: The authors declare no competing interests., (© 2023 The Author(s).)

The global prevalence of colistin-resistant Klebsiella pneumoniae (ColRkp) facilitated by chromosomal and plasmid-mediated Ara4N or PEtN-remodeled LPS alterations has steadily increased with increased colistin usage for treating carbapenem-resistant K. pneumoniae (CRkp). Our study demonstrated the rising trend of ColRkp showing extensively and pandrug-resistant characteristics among CRkp, with a prevalence of 28.5%, which was mediated by chromosomal mgrB, pmrB, or phoQ mutations (91.5%), and plasmid-mediated mcr-1.1, mcr-8.1, mcr-8.2 alone or in conjunction with R256G PmrB (8.5%). Several genetic alterations in mgrB (85.1%) with increased expressions of Ara4N-related phoPQ and pmrK were critical for establishing colistin resistance in our isolates. In this study, we discovered the significant associations between extensively drug-resistant bacteria (XDR) and pandrug-resistant bacteria (PDR) ColRkp in terms of moderate, weak or no biofilm-producing abilities, and altered expressions of virulence factors. These ColRkp would therefore be very challenging to treat, emphasizing for innovative therapy to combat these infections. Regardless of the underlying colistin-resistant mechanisms, colistin-EDTA combination therapy in this study produced potent synergistic effects in both in vitro and in vivo murine bacteremia, with no ColRkp regrowth and improved animal survival, implying the significance of colistin-EDTA combination therapy as systemic therapy for unlocking colistin resistance in ColRkp-associated bacteremia., (© 2022. The Author(s).)

Development of an effective therapy to overcome colistin resistance in Klebsiella pneumoniae, a common pathogen causing catheter-related biofilm infections in vascular catheters, has become a serious therapeutic challenge that must be addressed urgently. Although colistin and EDTA have successful roles for eradicating biofilms, no in vitro and in vivo studies have investigated their efficacy in catheter-related biofilm infections of colistin-resistant K. pneumoniae. In this study, colistin resistance was significantly reversed in both planktonic and mature biofilms of colistin-resistant K. pneumoniae by a combination of colistin (0.25-1 µg/ml) with EDTA (12 mg/ml). This novel colistin-EDTA combination was also demonstrated to have potent efficacy in eradicating colistin-resistant K. pneumoniae catheter-related biofilm infections, and eliminating the risk of recurrence in vivo. Furthermore, this study revealed significant therapeutic efficacy of colistin-EDTA combination in reducing bacterial load in internal organs, lowering serum creatinine, and protecting treated mice from mortality. Altered in vivo expression of different virulence genes indicate bacterial adaptive responses to survive in hostile environments under different treatments. According to these data discovered in this study, a novel colistin-EDTA combination provides favorable efficacy and safety for successful eradication of colistin-resistant K. pneumonia catheter-related biofilm infections., (© 2021. The Author(s).)

Candida Albicans: is abundant in the human gut mycobiota but this species does not colonize the mouse gastrointestinal tract. C. albicans administration in dextran-sulfate solution (DSS) induced-colitis mouse model (DSS+ Candida ) might resemble more to human condition, therefore, a DSS colitis model with Candida administration was studied; first, to test the influence of fungi in DSS model and second, to test the efficacy of Lactobacillus rhamnosus L34. We demonstrated serum (1→3)-β-D-glucan (BG) elevation in patients with IBD and endoscopic moderate colitis in clinical remission, supporting the possible influence of gut fungi toward IBD in human. Then, in mouse model, Candida gavage was found to worsen the DSS model indicated by higher mortality rate, more severe colon histology and enhanced gut-leakage (FITC-dextran assay, endotoxemia, serum BG and blood bacterial burdens) but did not affect weight loss and diarrhea. DSS+ Candida induced higher pro-inflammatory cytokines both in blood and in intestinal tissue. Worsened systemic pro-inflammatory cytokine responses in DSS+ Candida compared with DSS alone was possibly due to the more severe translocation of LPS, BG and bacteria (not fungemia) from gut into systemic circulation. Interestingly, bacteremia from Pseudomonas aeruginosa was more frequently isolated from DSS+ Candida than DSS alone. In parallel, P. aeruginosa was also isolated from fecal culture in most of the mice in DSS+ Candida group supported by prominent Gammaproteobacteria in fecal microbioata analysis. However, L. rhamnosus L34 attenuated both DSS+ Candida and DSS model through the attenuation of gut local inflammation (cytokines and histology), gut-leakage severity, fecal dysbiosis (culture method and microbiome analysis) and systemic inflammation (serum cytokines). In conclusion, gut Candida in DSS model induced fecal bacterial dysbiosis and enhanced leaky-gut induced bacteremia. Probiotic treatment strategy aiming to reduce gut-fungi and fecal dysbiosis could attenuate disease severity. Investigation on gut fungi in patients with IBD is highly interesting.

The study assessed and compared bacterial vaginosis (BV) prevalence in Thai women in reproductive age in four study groups - group 1, HIV-positive with copper intrauterine device (Cu-IUD); group 2, HIV-positive without Cu-IUD; group 3, HIV-negative with Cu-IUD; and group 4, HIV-negative without Cu-IUD. We conducted a cross-sectional study. BV prevalence was assessed by Nugent score and Amsel criteria. Descriptive statistics was used to present baseline characteristics; kwallis rank test - to compare variables between the four groups; logistic regression - to assess factors, related to BV prevalence. The analysis included 137 women in the four study groups with a median age of 39 years. Median BV prevalence by Nugent score was 45%, intermediate vaginal flora - 7% and normal vaginal flora - 48%. There was no statistically significant difference in the BV prevalence between the four study groups, p = 0.711. Threefold lower BV prevalence was found, assessed by Amsel criteria compared to Nugent score. Women with body mass index (BMI) < 20 had higher probability to have BV or intermediate vaginal flora, OR = 3.11, 95% CI (1.2-8.6), p = 0.025. The study found a high BV prevalence in the four study groups, related neither to HIV status, nor to Cu-IUD use. BV prevalence was associated only with low BMI. Thus, Cu-IUD could be a good contraceptive choice for HIV-positive women. Research in defining normal vaginal microbiota and improve diagnostic methods for BV should continue.

Gastrointestinal (GI) bacterial translocation in sepsis is well known, but the role of Lactobacillus species probiotics is still controversial. We evaluated the therapeutic effects of Lactobacillus rhamnosus L34 in a new sepsis model of oral administration of pathogenic bacteria with GI leakage induced by either an antibiotic cocktail (ATB) and/or dextran sulfate sodium (DSS). GI leakage with ATB, DSS, and DSS plus ATB (DSS+ATB) was demonstrated by fluorescein isothiocyanate (FITC)-dextran translocation to the circulation. The administration of pathogenic bacteria, either Klebsiella pneumoniae or Salmonella enterica serovar Typhimurium, enhanced translocation. Bacteremia was demonstrated within 24 h in 50 to 88% of mice with GI leakage plus the administration of pathogenic bacteria but not with GI leakage induction alone or bacterial gavage alone. Salmonella bacteremia was found in only 16 to 29% and 0% of mice with Salmonella and Klebsiella administrations, respectively. Klebsiella bacteremia was demonstrated in 25 to 33% and 10 to 16% of mice with Klebsiella and Salmonella administrations, respectively. Lactobacillus rhamnosus L34 attenuated GI leakage in these models, as shown by the reductions of FITC-dextran gut translocation, serum interleukin-6 (IL-6) levels, bacteremia, and sepsis mortality. The reduction in the amount of fecal Salmonella bacteria with Lactobacillus treatment was demonstrated. In addition, an anti-inflammatory effect of the conditioned medium from Lactobacillus rhamnosus L34 was also demonstrated by the attenuation of cytokine production in colonic epithelial cells in vitro In conclusion, Lactobacillus rhamnosus L34 attenuated the severity of symptoms in a murine sepsis model induced by GI leakage and the administration of pathogenic bacteria., (Copyright © 2017 American Society for Microbiology.)

Gastrointestinal (GI) leakage in Clostridium difficile-associated diarrhea (CDAD) is well known but is not routinely assessed in clinical practice. Serum (1→3)-β-D-glucan (BG), a fungal cell wall component used as a biomarker for invasive fungal disease, was tested in a CDAD mouse model with and without probiotics. Higher serum fluorescein isothiocyanate-dextran (FITC-dextran) and spontaneous gram-negative bacteremia, GI leakage indicators, were frequently found in CDAD mice, which died compared with those which survived. BG, serum macrophage inflammatory protein-2 and FITC-dextran but not quantitative blood bacterial count differentiated the clinical severity. Interestingly, a specific dose of Lactobacillus rhamnosus L34 attenuated CDAD and decreased serum BG and FITC-dextran, but not other parameters. BG also showed a higher area under the receiver operating characteristic curve for 7-day mortality than FITC-dextran. Fifty-five percent of CDAD mice with BG ≥ 60 pg/ml (the human negative cut-off value for invasive fungal disease) at 1 day after C. difficile gavage died within 7 days. In conclusion, S: erum BG was elevated in mice with severe CDAD, an established model of GI leakage with a strong association with mortality rate. BG monitoring in patients with CDAD is of interest as both a potential prognostic tool and a therapeutic efficacy indicator., (© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Objective: To compare the effectiveness of ceftriaxone versus cefazolin for the prevention of febrile morbidity and postoperative infections among patients after abdominal hysterectomy., Methods: In a double-blind, randomized, controlled trial in Bangkok, Thailand, 320 patients undergoing abdominal hysterectomy between July 2008 and July 2009 were randomly assigned to receive 1g of either ceftriaxone or cefazolin intravenously in a single dose before surgery. The participants were evaluated for postoperative fever and infection for up to 4 weeks. χ(2) or Fisher exact tests were used for statistical analysis., Results: There was no significant difference between the ceftriaxone and cefazolin groups in incidence of febrile morbidity (9.4% versus 11.2%), wound infection (3.8% versus 1.9%), vaginal cuff infection (3.8% versus 1.9%), or urinary tract infection (1.9% versus 1.9%)., Conclusion: There was no difference between the use of single-dose preoperative ceftriaxone and cefazolin in preventing infectious morbidity among patients undergoing hysterectomy., (Copyright © 2012 International Federation of Gynecology and Obstetrics. Published by Elsevier Ireland Ltd. All rights reserved.)

Objective: There has been a development of automated and continuous-monitoring blood culture systems that are more sensitive than conventional systems for the detection of microorganisms. Whether two or three blood culture specimens obtained during a 24-hour period using these automated systems achieving a higher recovery rate of microorganism remains to be determined. The present study was aimed to compare the recovery rates of microorganism of blood-stream infections (BSIs) using two and three blood culture specimens., Material and Method: A prospective investigator-blinded study was carried out in patients who needed to have blood cultures in medicine wards and intensive care units as well as an emergency room of King Chulalongkorn Memorial Hospital, Bangkok, Thailand, between October 1, 2010 and March 31, 2011. Three blood culture specimens were obtained from each patient during a 24-hour period. Each specimen was inoculated into an aerobic bottle of blood culture broth (TREK Diagnostics, Cleveland, OH, US), and then incubated at 37 degrees C for seven days., Results: Of 568 patients, there were 116 (20.4%) unimicrobial episodes with three blood cultures obtained during a 24-hour period. There were 70 (12.3%) and 46 (8.1%) episodes of true pathogen and contaminant, respectively. The recovery rates of true pathogen were 75.7% (53 isolates), 87.1% (61 isolates), and 100% (70 isolates) with the first, second, and third blood culture specimens, respectively (p < 0.05 between the recovery rate with the first two and the third blood culture specimens). There were 25 (35.7%), 38 (58.6%) isolates, and four (5.7%) of Gram-positive, Gram-negative bacteria, and fungi, respectively. Among 25 Gram-positive bacteria, Staphylococcus aureus was the most common isolate (10, 14.3%), followed by Streptococcus pneumoniae (5, 7.1%) and Enterococcus faecalis, Enterococcus faecium, coagulase-negative Staphylococcus (3, 10% each). Among 38 Gram-negative bacteria, Escherichia coli was the most common isolate (13, 18. 6%), followed by Pseudomonas aeruginosa (8, 11.4%), and Klebsiella pneumoniae (6, 8.6%). The sensitivity and specificity of the recovery rate of microorganisms using two blood culture specimens were 85.7% and 92.3%, respectively. The sensitivity and specificity of the recovery rate of microorganisms using three blood culture specimens were 100% and 90.8%, respectively., Conclusion: To the best of the authors'knowledge, the present study is the first prospective study to compare the recovery rate of microorganisms of BSIs between the two and three blood culture specimens using the VersaTREK blood culture system. Three blood culture specimens are required to achieve the recovery rate of more than 99%.

We describe the first case of typhoid spondylodiscitis in Southeast Asia, and the literature were also reviewed. A 57-year-old diabetic Thai man who presented with a one-month course of progressive low back pain associated with paraparesis and bowel-bladder dysfunction. Examination revealed local tenderness over T12 area, spastic paraparesis, impaired pinprick sensation up to T12 level, and loose anal sphincter tone. Magnetic resonance imaging showed spondylodiscitis of T11 and T12 and epidural abscess causing spinal cord compression. T11 and T12 laminectomy, T11/12 discectomy, and debridement of epidural abscess were performed, and the cultures of the pus grew Salmonella Typhi. He was treated with intravenous ciprofloxacin for three weeks and was discharged from the hospital with oral ciprofloxacin and trimethoprim-sulfamethoxazole for another five months of treatment. The patient was doing well when last seen two months after discontinuation of antimicrobial treatment. In addition, a total of ten cases of typhoid spondylitis/spondylodiscitis were reviewed.

Background: Commercial diagnostics are commonly used to identify gram-positive bacteria. Errors have been reported mostly at the species level. We have found certain phenotypic criteria used in API systems which significantly misidentify Leuconostoc, an emerging human pathogen, at the genus level. We also attempt to find practical, conventional phenotypic assays for accurate identification of this group of bacteria., Methods: Clinical isolates of catalase-negative, gram-positive coccoid or coccobacillary bacteria with non-beta hemolysis in our institute during 1997-2004 were subject to an identification aid by API 20 STREP, following the instruction manual, as an aid to conventional phenotypic tests. Those identified as Leuconostoc by API 20 STREP were re-examined by the same kit and also by API 50 CHL according to the instruction manuals, by our Leuconostoc conventional phenotypic assays, by Leuconostoc- and Lactobacillus-specific PCR's, and, where possible, by 16S rDNA sequence analysis. In addition, catalase-negative gram-positive isolates during 2005-2006 which were resistant to vancomycin at high levels were also evaluated by the same phenotypic and genotypic assays., Results: Out of several thousands of clinical gram-positive isolates, 26 catalase negative gram-positive isolates initially identified as Leuconostoc by API 20 STREP and 7 vancomycin-resistant gram-positive catalase-negative bacteria entered the study. 11 out of the 26 isolates and all the 7 isolates were identified as Leuconostoc by API 20 STREP. Only 5 isolates, however, were confirmed by both genotypic and all defined conventional phenotypic criteria. API 50 CHL also failed to reliably provide accurate identification of Leuconostoc. We have identified key problem tests in API 20 STREP leading to misidentification of the bacteria. A simple, conventional set of phenotypic tests for Leuconostoc identification is proposed., Conclusion: The current API systems cannot accurately identify Leuconostoc. Identification of vancomycin-resistant, catalase-negative gram-positive bacteria should be performed by a few practical phenotypic assays, with assistance of genotypic assays where available.

The emergence of hospital acquired infections with bacteria resistant to antimicrobials such as vancomycin resistant enterococci (VRE) has become a worldwide concern. The authors studied the prevalence and surveillance of 5 years study of VRE in King Chulalongkon Memorial Hospital and phenotype of these resistance strains. A total of enterococci 1854 isolates were collected from clinical specimens from 1995 to 1999. Screening vancomycin resistance was identified by the agar plated method and minimal inhibitory concentration (MIC) of vancomycin was determined for vancomycin-resistance strains by E-test. The results demonstated that 15 (0.81%) VRE were isolated from 1,854 specimens. Fourteen VRE were identified as Enterococcus faecium and one strain was Enterococcus faecalis. All of these strains, carrying the VanB phenotype, were susceptible to teicoplanin. Similar to other studies, most VRE strains are E. faecium. To the authors' knowledge, this is the first VRE study carried out in King Chulalongkorn Memorial Hospital. The results showed a low prevalence of VRE and surveillance of 5 years study demonstated a gradual increase of VRE. Therefore, it is important to continue periodic surveys of VRE to prevent the spread of VRE in hospitals.

A large outbreak of group A Streptococcus (GAS) pharyngitis occurred among 285 cadets at the Chulachomklao Royal Military Academy, Nakhon Nayok between September 22 and 27, 1996. An epidemiologic investigation was conducted to determine the source of infection. It seemed that respiratory droplets were the mechanism of spread, although foodborne transmission could not be excluded. To assess the presence of the outbreak strain in the community, conventional serotyping and molecular typing by emm sequencing were performed. The results demonstrated the advantage of molecular typing compared to serotyping and suggested that the outbreak was caused by the same strain. Interestingly, the involvement of the causative emm79 strain, which is an uncommon type of GAS disease, in a large number of patients may imply the invasive potential of this organism.

Mycoplasma pneumoniae is a causative agent of human respiratory tract infection of which the clinical features are not significantly different from those of infections caused by other respiratory pathogens. The diagnosis is based principally on laboratory tests. Since conventional methods such as culture and serological tests are time-consuming, insensitive, and non-specific, polymerase chain reaction (PCR) was employed for laboratory diagnostics. This study was aimed to develop PCR method to detect M. pneumoniae by designing primers to amplify fragment of the P1 adhesin gene. Two protocols, PCR-probe hybridization and nested PCR, were carried out. False-positive result due to amplicon carry over was prevented by using dUTP instead of dTTP and the addition of enzyme uracil DNA glycosylase (UDG). For nested PCR, UDG was added only in the first round reaction mixture. The sensitivity of PCR was 10 fg of M, pneumoniae DNA as detected by agarose gel electrophoresis and increased to be 1 fg as detected by either probe hybridization or nested PCR. The specificity of PCR was tested with DNAs from Mycoplasma spp, a variety of different bacterial genera and human leukocyte. All gave negative results. Considering of the speed, sensitivity, specificity and the prevention of amplicon carryover, the developed PCR-based protocols were suitable and reliable for the detection of M. pneumoniae in routine laboratory.

Emm (M protein gene) typing was used to analyze group A streptococcal (GAS) clinical isolates in Thailand from in-patients at Chulalongkorn University Hospital (CUH) between January 1, 1995 and December 31, 1999. Forty GAS isolates were recovered from blood and other sterile sites from 40 patients presenting with different types of infections. A variety of emm sequences (24 types) have been reported in this study including the identification of 2 novel emm variants demonstrating the diverse population of invasive GAS isolates in Thailand. The common emm types include emm1 (5 isolates, 12.5%), emm22 (4 isolates, 10%), emm25 (3 isolates, 7.5%), emm61 (3 isolates, 7.5%), and STNS1033 (3 isolates, 7.5%). No particular emm types of GAS tend to be frequently associated with specific clinical presentation, complication, or anatomic site of infection. This report provides epidemiological information from Thailand where streptococcal infections and their sequelae are important public health problems. In addition, the results are useful for the development of a suitable M protein based vaccine in the future.