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Background To identify the clinical characteristics and risk factors for mortality of patients with cefepime-resistant Pseudomonas aeruginosa (FRPa) bacteremia. Methods This retrospective study analyzed adult patients with FRPa bacteremia hospitalized between January 2006 and December 2011. Results Seventy eight patients (46 male, 32 female; mean age: 72.2 ± 14.1 years) were included. Of them, 46 (59.0%) had ventilator use and 45 (57.7%) had intensive care unit stay. All the bacteremia episodes were health-care associated or hospital acquired, and 55.1% of FRPa blood isolates were multidrug resistant. The sources of bacteremia were identified in 42 patients (53.8%), with pneumonia being the most common one (28/42; 66.7%). The mean interval between admission and the sample date of the first FRPa-positive blood culture was 45.8 ± 52.6 days. The mean Pittsburgh bacteremia score was 5.0 ± 3.4. The 15-day and 30-day mortality rates were 50.0% and 65.4%, respectively. Patients (41; 52.6%) on appropriate antibiotic therapy within 72 hours of the first FRPa-positive blood culture had a higher 30-day survival rate than those without (48.8% vs. 18.9%, p = 0.011 by log-rank test). Multivariate analyses revealed that a higher Pittsburgh bacteremia score was an independent risk factor for either 15-day ( p = 0.002) or 30-day mortality ( p = 0.010), and appropriate antibiotic therapy within 72 hours was an independent protecting factor for either 15-day ( p = 0.049) or 30-day mortality ( p = 0.017). Conclusion FRPa bacteremia had a high mortality rate. The disease severity and appropriate antimicrobial therapy within 72 hours of positive blood culture were related to the patients' outcome.

Objectives Clostridium innocuum can cause extraintestinal infection in patients with underlying diseases. The role of C. innocuum in antibiotic-associated diarrhoea (AAD) remains unknown. Methods Clinical information of 103 patients from whom C. innocuum was isolated was reviewed. We carried out cellular and animal experiments to examine the pathogenic potential of C. innocuum in AAD. Results Eighty-eight per cent (91/103) of the 103 patients received antibiotics within 2 weeks of diarrhoea onset. Patients were further classified into two groups, severe colitis and diarrhoea, according to clinical severity level. The mortality rate was 13.6% (14/103) among the patients from whom C. innocuum was isolated. The lowest concentrations at which 90% of the isolates were inhibited for metronidazole and vancomycin were 0.5 and 16 mg/L, respectively. All isolates tested were susceptible to metronidazole but resistant to vancomycin. Nineteen randomly selected isolates (ten from severe colitis group, nine from diarrhoea group) were subjected to further in vitro cellular examinations. The level of cytotoxicity to Vero cells was significantly higher in isolates from the severe colitis group at both 24 and 48 hours after inoculation (24 and 48 hours, p 0.042 and 0.033, respectively). We observed apoptotic changes that subsequently led to cell death in C. innocuum –infected Vero cells. Tissue damages, necrotic changes and oedema were observed in the mouse ileal loop infected by C. innocuum. Conclusions Vancomycin-resistant C. innocuum may play a potential role as a causative agent of AAD. The clinical manifestations of AAD caused by C. innocuum were diarrhoea or severe colitis, including pseudomembranous colitis.

Although the mechanisms underlying the cytotoxic effect of NK cells on tumor cells and intracellular bacteria have been studied extensively, it remains unclear how these cells kill extracellular bacterial pathogens. In this study, we examine how human NK cells kill Mycobacterium kansasii and M.tb. The underlying mechanism is contact dependent and requires two cytolytic proteins: perforin and granulysin. Mycobacteria induce enhanced expression of the cytolytic proteins via activation of the NKG2D/NCR cell-surface receptors and intracellular signaling pathways involving ERK, JNK, and p38 MAPKs. These results suggest that NK cells use similar cellular mechanisms to kill both bacterial pathogens and target host cells. This report reveals a novel role for NK cells, perforin, and granulysin in killing mycobacteria and highlights a potential alternative defense mechanism that the immune system can use against mycobacterial infection.

Objectives Extra-intestinal clostridial infection (EICI) is rare but can be fatal. Traditional phenotypic methods can only assign many of the Clostridium species to the genus level. Methods A total of 376 non-repetitive Clostridium isolates from sterile sites were collected and subjected to matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) Biotyper analysis and 16S rRNA sequencing. Antimicrobial susceptibility was determined, and clinical characteristics of the patients were assessed. Clostridium innocuum isolates were characterized by genome sequencing and genotyping. We used molecular and cellular methods to explore the virulence and resistance mechanisms of C.innocuum. Results Clostridium innocuum was the second most common species to cause EICI, only next to Clostridium perfringens. All Clostridium isolates showed susceptibility to clindamycin, metronidazole, penicillin, piperacillin and ampicillin-sulbatam, while C. innocuum isolates were invariably resistant to vancomycin. Among 24 patients with EICI caused by C. innocuum, two (8.3%) had diarrhoea, three (12.5%) had soft-tissue infection, six (25%) had appendicitis and four (16.7%) each had shock and gastrointestinal perforation. The 30-day mortality was 16.7%. The C. innocuum isolated from different sites could not be separated from one another by genotyping. No known toxin genes were identified in the genome of C. innocuum but the species expressed cytotoxicity to epithelial cells. d-Alanine-d-alanine ligase, alanine racemase and d-alanyl-d-alanine carboxypeptidase are three main genes responsible for vancomycin resistance in C. innocuum. Conclusions Vancomycin-resistant C. innocuum is a previously unrecognized, yet prominent, cause for EICI. Genome analysis showed that the species could carry a lipopolysaccharide-like structure that is associated with cytotoxicity to cells in vitro.

Tularemia is a zoonotic infection seen primarily in the Northern Hemisphere. It is caused by the bacteria Francisella tularensis. Although the ulceroglandular form of the disease is the more common manifestation of infection, F tularensis is known to cause pneumonia. F tularensis has two predominant subspecies, namely subsp. tularensis (type A) and subsp. holarctica (type B). Type B tularemia is considered to be much less virulent than type A and barely caused lethal disease and pneumonia. We reported a case with a 68-year-old man immune-compromised patient diagnosed with bacteremic pneumonia engendered by type B tularemia with initial presentation of high fever, pneumonia with pleural effusion; the diagnosis was performed using 16S rRNA gene sequence analysis. The patient's fever, pneumonia, and pleural effusion were resolved with appropriate antibiotics for tularemia. This case involving severe bacteremic pneumonia in an immune-compromised patient is rare. This case suggests that low virulence F tularensis should be included in the differential diagnoses of bacteremic pneumonia for endemic tularemia.

Background Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been widely used in microbial identification. This study evaluated the performance of MALDI-TOF MS and investigated the economic and medical impact of MALDI-TOF MS implementation. Methods A total of 12,202 clinical isolates collected from April to September 2013 were identified using MALDI-TOF MS, and the success rates in identifying isolates were analyzed. The differences in the processing time, cost of consumables, weight of waste, and clinical impact between MALDI-TOF MS and biochemical reaction were compared. Results MALDI-TOF MS successfully identified 96% of 12,202 isolates, including 96.8% of 10,502 aerobes, 90.5% of 1481 anaerobes, 93.8% of 81 yeasts, and 90.6% of 138 nontuberculous mycobacteria at the genus level. By using MALDI-TOF MS, the processing time for aerobes decreased from 32.5 hours to 4.1 hours, and that for anaerobes decreased from 71.5 hours to 46 hours. For detection of aerobes and anaerobes, the cost of consumables was estimated to decrease by US$0.9 per isolate, thus saving US$94,500 in total annual isolation. Furthermore, the weight of waste decreased six-fold, resulting in a reduction of 350 kg/month or 4.2 tons/year. MALDI-TOF MS also increased the percentage of correct antibiotics treatment for Escherichia coli and Klebsiella pneumonia from 56.1% to 75% and shortened the initiation time of the correct antibiotic action from 3.3 hours to 2.5 hours. Conclusions MALDI-TOF MS is a rapid, reliable, economical, and environmentally friendly method for routine microbial identification and may contribute to early appropriate antibiotic treatment in clinical settings.

Higher vancomycin MIC values (≥1.5 mg/L via Etest) may be associated with vancomycin treatment failure among patients with serious methicillin-resistant Staphylococcus aureus (MRSA) infections. As there were limited similar data for teicoplanin, this retrospective cohort study intended to determine the predictive value of teicoplanin MICs for treatment failure among patients with MRSA bacteraemia.All patients with at least one blood culture positive for MRSA admitted to the hospital between January 2010 and January 2011 were reviewed. Patients with an age ≥18 years and receipt of teicoplanin therapy throughout the course or receipt of72 h of vancomycin therapy and then teicoplanin for3 days were enrolled. Teicoplanin Etest(®) MICs and treatment outcomes for MRSA bacteraemia were reviewed to identify the breakpoint of teicoplanin MICs influencing treatment outcomes.Of the 101 patients enrolled, 56 had a lower teicoplanin MIC (≤1.5 mg/L) for MRSA and 45 had a higher MIC (1.5 mg/L) for MRSA. A lower teicoplanin MIC was associated with a favourable outcome [37 (66.1%) versus 13 (28.9%); P0.001] and a lower rate of bloodstream infection-related mortality [15 (26.8%) versus 22 (48.9%); P=0.022]. Patients with chronic obstructive pulmonary disease, bacteraemic pneumonia or higher Pittsburgh bacteraemia score had an unfavourable outcome (P=0.028, 0.022 and0.001, respectively). Multivariate analysis showed that teicoplanin MIC1.5 mg/L, higher Pittsburgh bacteraemia score and bacteraemic pneumonia were independent risk factors for unfavourable outcome.A higher teicoplanin MIC value (1.5 mg/L) may predict an unfavourable outcome and higher mortality rate among teicoplanin-treated MRSA bacteraemic patients.

An increasing incidence of carbapenem-resistant Klebsiella pneumoniae (CRKP) infections has been reported worldwide. The aim of this study was to investigate the mechanism underlying carbapenem resistance and its relationship to antibiotic exposure. Sixteen isolates with various carbapenem susceptibilities recovered from five patients between 2003 and 2006 were subjected to molecular study. The medical records of the patients were also reviewed. All of the patients were admitted for complicated respiratory illness, had a prolonged hospital stay, and were exposed to antibiotics. Carbapenems were prescribed before the emergence of the CRKP. Various combinations of extended-spectrum cephalosporinase genes belonging to the SHV, CTX-M, and AmpC groups were found among the isolates. Other carbapenem resistance-associated genes, such as bla(IMP), bla(VIM), bla(OXA), and bla(KPC), were not found. OmpK35 was not expressed in any of the isolates, and additional loss of OmpK36 was observed in all CRKP isolates. Two insertion elements, ISPa13 or IS5, were found inserted into OmpK36 in the isolates derived from three patients. These IS elements were also identified in their parental carbapenem-susceptible isolates, suggesting that an internal transposition into OmpK36 resulted in resistance. OmpK36 loss represents the major mechanism for the development of CRKP in extended-spectrum cephalosporinase-producing isolates. A prolonged hospital stay and recent carbapenem exposure may predispose patients to CRKP, impacting the clinical outcome.

Nosocomial infections caused by multidrug-resistant (MDR) Acinetobacter baumannii have been increasing in recent years, posing a threat to public health worldwide. The susceptibility to eight antimicrobial agents of 35 clinical A. baumannii isolates from Taiwan was tested. Isolates were examined by polymerase chain reaction (PCR) and sequencing for β-lactamase genes and mutations in the gyrA and parC genes. Expression of AdeB, an efflux pump protein, was evaluated by real-time quantitative PCR. The level of adeB expression correlated with resistance to ciprofloxacin and ampicillin/sulbactam in A. baumannii isolates. Almost all isolates with full resistance to ciprofloxacin had both high adeB expression and point mutations in parC and gyrA , but 4 intermediate-resistant isolates had only high adeB expression without point mutations in gyrA or parC , in contrast to 18 susceptible isolates with low adeB expression and without mutations in gyrA or parC . Sixteen isolates (45.7%) carrying a type 1 integron were MDR as well as being more resistant to imipenem, amikacin, gentamicin, ceftazidime or cefepime than those without the integron. The class 1 integron in A. baumannii carried different resistance gene cassettes, including 5′CS- bla IMP-1 – aadA4 –3′CS, 5′CS– aacA4 – aadA1 –3′CS and 5′CS– aacC1 – aadA1 –3′CS. In conclusion, expression of the adeB gene was associated with resistance to ciprofloxacin and ampicillin/sulbactam in A. baumannii . Multiple mutations in gyrA and parC also played a role in ciprofloxacin resistance. The major metallo-β-lactamase contributing to imipenem resistance in A. baumannii in Taiwan was bla IMP-1 , which was carried by the class 1 integron. The class 1 integron was associated with the MDR phenotype in A. baumannii .

Eight pairs of Escherichia coli isolates with various carbapenem susceptibilities from 8 patients were prospectively collected to study the development of resistance. All carbapenem-resistant E. coli isolates were resistant to all tested ss-lactams antibiotics except tigecycline. Identical pulsed-field gel electrophoresis (PFGE) patterns were found in carbapenem-susceptible and -resistant isolates but different PFGE patterns occurred among patients. A CMY-2 ss-lactamase was found in all E. coli isolates. No previously reported carbapenemase genes were detected. Examination of outer membrane protein (OMP) profiles revealed that OmpA was not found in all isolates, while OmpC and OmpF were lost in carbapenem-resistant isolates. Loss of both OmpC and OmpF represents the major mechanism of the development of carbapenem resistance in those patients with CMY-2-producing E. coli infections.

Objectives: In 2008, a new set of penicillin breakpoints was published in the CLSI revised guideline, M100-S18, to define the susceptibility of non-meningeal isolates of Streptococcus pneumoniae. The impact of the change is studied and discussed. Methods: Laboratory data on pneumococcal isolates collected from Chang Gung Memorial Hospital during 2000―07 were analysed using the original and modified penicillin CLSI breakpoints. Results: A total of 3729 non-duplicate isolates were identifed, including 43 (1.2%) meningeal isolates showing high rates of penicillin (79.1%) and ceftriaxone (34.9%) resistance. For non-meningeal isolates, penicillin non-susceptibility was reduced significantly from 75.1% (72.4% in 2000―03 increasing to 77.4% in 2004―07) to 16% (28.6% in 2000 decreasing to 2.4% in 2007) if the modified breakpoints were applied. However, isolates for which penicillin MICs were 1―2 mg/L increased significantly from 34.2% in 2000 to 59.8% in 2007. Ceftriaxone non-susceptibility also increased significantly from 2.8% before 2005 to 18.4% thereafter. A quarter (25.7%) of the pneumococcal isolates were recovered from patients

The aim of this study was to evaluate immunity in HIV-uninfected children with bacille Calmette-Guerin-induced disease (BCG-ID) over an 8-year period, with particular emphasis on underlying diseases.Patient afflicted with BCG-ID proven by clinical courses, dermatologic features, pathology, specific polymerase chain reaction, and/or spoligotyping were enrolled between 2000 and 2007. Lymphocyte proliferation, polymorphonuclear function, interleukin (IL)-12/23-interferons (IFN)-gamma circuit, and Toll-like receptor 2-associated signaling were investigated.Of the 271,618 total live births who received the BCG vaccine, eight patients (seven males) with BCG-ID were enrolled during an 8-year period and presented as three disseminated, two distant, and three regional BCG-ID. Their age at onset ranged from 1 to 28 months. All had a vaccine-injection scar except for one with lower CD3 and natural killer cells, compatible with severe combined immunodeficiency (SCID) identified by IL-2 receptor common gamma chain (IL2RG) mutation (Arg226Lys). The other SCID patient with de novo IL2RG mutation (Trp74Gly) had more recurrent infections. The third patient with primary autoimmune neutropenia had disseminated BCG-ID extending to abdominal wall. The fourth patient with chronic mucocutaneous candidiasis had regional BCG-ID and impaired lymphocyte proliferation to Candida and BCG antigens. No defective evidence of polymorphonuclear functions, IL-12/23-IFN-gamma circuit, and Toll-like receptor 2-associated signaling was detected in the remaining four patients.Immunologic analysis in HIV-uninfected patients with BCG-ID reveals primary immunodeficiency diseases, especially in those with deficiencies in T-cell and neutrophil functions observed in our cohort, including primary autoimmune neutropenia and chronic mucocutaneous candidiasis.

The rapid identification of mycobacteria from smear-positive sputum samples is an important clinical issue. Furthermore, the availability of a cheap, technically simple, and accurate method also would benefit mycobacterial laboratories in developing countries. In the present study, we aimed to develop an assay allowing the identification of the Mycobacterium tuberculosis complex (MTBC) and other frequently isolated nontuberculous mycobacteria (NTM) directly from smear-positive sputum samples. A nested PCR-restriction fragment length polymorphism analysis (nested-PRA) assay that focuses on the analysis of the hsp65 gene was developed and evaluated for its efficiency compared to that of traditional culture methods and 16S rRNA gene sequencing identification. A total of 204 smear-positive and culture-positive sputum specimens were prospectively collected for analysis between November 2005 and May 2006. The samples were classified according to an acid-fast bacillus (AFB) staining scale as rare/1+, 2+, or 3+. The results of the nested-PRA showed that the identification rate for AFB 3+, AFB 2+, and AFB rare/1+ samples was 100, 95, and 53%, respectively, and that the overall identification rate was 89%. All positive results by the nested-PRA method agreed with the results by culture and 16S rRNA gene sequence analysis. The nested-PRA appears to have clinical applicability when used for the direct identification of mycobacterial organisms (both MTBC and NTM) that are present in smear-positive sputum samples, especially for countries in which MTBC is endemic.

Chronic sclerosing osteomyelitis caused by Chryseobacterium meningosepticum was observed in a woman with steroid use. Her chronic sclerosing osteomyelitis was cured with ciprofloxacin. English literature review found 4 cases of bone-joint infection due to C. meningosepticum. The implications of treatment with a fluoroquinolone were discussed.

Forty-six cephamycin-resistant Escherichia coli isolates from patients diagnosed with community-acquired urinary tract infection were selected in order to study their resistance mechanism. With the exception of one isolate producing CMY-4, all isolates produced a CMY-2 β-lactamase. Molecular typing showed that the CMY-2-producing isolates were not related. Cephamycin resistance was plasmid encoded and conjugatively transferred. Plasmid digest profiles suggested that the plasmids were different. Thirty-nine of the 45 CMY-2-producing isolates harboured a plasmid containing a specific DNA fragment, IS Ecp1 – bla CMY-2 – blc – sugE , which was identical to those previously published in CMY-2-producing Salmonella enterica serotype Choleraesuis (SCB67) and Salmonella enterica serotype Typhimurium (pNF1358) from Taiwan and the USA, respectively. Among the remaining six isolates, insertion of IS 1294 and IS 1 at different positions was observed in one and five isolates, respectively. The regions surrounding bla CMY-2 of the six isolates were identical to the other 39 isolates as well as to SCB67 and pNF1358. Since the present identical transmissible bla CMY-2 -carrying element was observed in food animal sources both in the USA and Taiwan, its possible transmission to humans, as revealed in this study, is of great concern. Awareness of this mobile resistance element is required to prevent introduction into hospitals and to reduce the spread of this emerging resistance within the community.

In Taiwan, beginning in 2013, the 13-valent pneumococcal conjugate vaccine (PCV13) was provided free of charge to children 2–5 years of age. In 2014, this was extended to children 1–5 years old. During 2012–2014, 953 cases of culture-confirmed pneumococcal disease (CCPD), including 104 invasive pneumococcal disease (IPD), were prospectively identified and analyzed at a 3,700-bed hospital in Taiwan. From 2012 to 2014, the incidence per 10,000 admissions decreased from 26.7 to 20.4 for CCPD (P P P 15A, ST8315B and ST33823A. From 2012 to 2014, isolates with penicillin minimum inhibitory concentrations >2 mg/L decreased from 27.8% to 8.1% (P

PURPOSE This study aimed to report the surgical outcome of patch grafts using glycerol-preserved corneas in perforated or near-perforated rheumatoid arthritis-related peripheral ulcerative keratitis (PUK). DESIGN This is a retrospective chart review of a case series. METHODS The medical records at a single institution were reviewed between July 2004 and July 2011. Of the 19 patients with PUK, 7 (36.8%) underwent glycerol-preserved cornea patch grafts for rheumatoid arthritis-related PUK. The clinical features, precipitating factors, adjuvant therapy, and therapeutic outcomes were analyzed. RESULTS The age of patients ranged from 49 to 82 years (mean [SD], 64.43 [13.53] years) for the 4 women and 3 men with perforated or near-perforated PUK who were managed with systemic and local immunosuppressive therapy. A wound culture revealed 1 Staphylococcus aureus and 1 filamentary fungal infection. The mean (SD) area of the graft was 13.28 (6.11) mm; the mean (SD) time to reepithelializaiton was 7.0 (1.60) days. One patient had high intraocular pressure managed by topical medication; another had a complicated cataract formation. One graft melting was successfully managed by topical and systemic medication. CONCLUSIONS Glycerol-preserved cornea patch grafts were successful in all patients and may be a viable option in perforated or near-perforated PUK, particularly when fresh donor corneas are neither available nor indicted.

The prevalence of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae in a tertiary hospital in Taiwan was assessed over a 16-month period. A total of 125 nonrepetitive ESBL-producing isolates of Enterobacter cloacae, Escherichia coli, and Klebsiella pneumoniae were available for investigation using molecular methods. Four predominant intensive care units (ICUs) were identified, and SHV-12 (59%), CTX-M- 3 (36%), and CTX-M-14 (14%) were the three most frequent ESBLs. SHV-12 was predominant among E. cloacae in the burn unit and K. pneumoniae in the other three chest medicine-related ICUs. CTX-M-3 was predominant among E. coli and K. pneumoniae in three other ICUs. The dissemination of ESBL-producing Enterobacteriaceae in four ICUs of a medical center in Taiwan is a consequence of the clonal dissemination of a few epidemic strains along with the horizontal transmission of resistance genes-carrying plasmids among bacterial organisms.

Objectives: The aim of the study was to characterize the genetic basis of b-lactam resistance developed in clinical isolates of Klebsiella pneumoniae after exposure to cefuroxime. Methods: Clinical features of two episodes of liver abscess caused by K. pneumoniae in a diabetic patient were reported. Four isolates (KP1/KP2 and KP3/KP4 )o fK. pneumoniae were recovered from cultures of blood/pus in the first and second episodes, respectively. Laboratory investigation of the K. pneumoniae isolates included genotyping by PFGE, resistance gene analysis by PCR amplification and DNA sequencing, and outer membrane protein analysis by SDS–PAGE. Results :K P3 and KP4 were recovered after a 21 day cefuroxime therapy and demonstrated identical genotypes to that of KP1 and KP2. However, compared with KP1 and KP2, emerging resistance to piperacillin, cefalotin, cefuroxime and cefoxitin was observed. The other antibiotics tested, except ampicillin, retained the same effectiveness against the four isolates, although increases (4- to 8-fold) in the MICs of cefotaxime, ceftriaxone, ceftazidime, cefepime, flomoxef and aztreonam were observed in KP3 and KP4. None of the isolates produced extended-spectrum b-lactamases or plasmid-mediated AmpC b-lactamases. Deficiency in the expression of an outer membrane protein (OmpK35) was observed in the cefuroxime-resistant isolates, KP3 and KP4. Conclusions: The increased resistance to cephalosporins in these clinical isolates of K. pneumoniae after exposure to cefuroxime might be related to the loss of OmpK35.

Infection by extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae has been increasing in Taiwan. Accurate identification of the ESBL genes is necessary for surveillance and for epidemiological studies of the mode of transmission in the hospital setting. We describe herein the development of a novel system, which consists of a multiplex PCR to identify bla SHV , bla CTX-M-3 -like, and bla CTX-M-14 -like genes and a modified SHV melting-curve mutation detection method to rapidly distinguish six prevalent bla SHV genes ( bla SHV-1 , bla SHV-2 , bla SHV-2a , bla SHV-5 , bla SHV-11 , and bla SHV-12 ) in Taiwan. Sixty-five clinical isolates, which had been characterized by nucleotide sequencing of the bla SHV and bla CTX-M genes, were identified by the system. The system was then used to genotype the ESBLs from 199 clinical isolates, including 40 Enterobacter cloacae , 68 Escherichia coli , and 91 Klebsiella pneumoniae , collected between August 2002 and March 2003. SHV-12 (80 isolates) was the most prevalent type of ESBL identified, followed in order of frequency by CTX-M-3 (65 isolates) and CTX-M-14 (36 isolates). Seventeen (9%) of the 199 clinical isolates harbored both SHV- and CTX-M-type ESBLs. In contrast to Enterobacter cloacae , the majority of which produced SHV-type ESBLs, E. coli and K. pneumoniae were more likely to possess CTX-M-type ESBLs. Three rare CTX-M types were identified through sequencing of the bla CTX-M-3 -like (CTX-M-15) and bla CTX-M-14 -like (CTX-M-9 and CTX-M-13) genes. The system appears to provide an efficient differentiation of ESBLs among E. coli , K. pneumoniae , and Enterobacter cloacae in Taiwan. Moreover, the design of the system can be easily adapted for similar purposes in areas where different ESBLs are prevalent.

Salmonella infection is a distressing health problem worldwide. This study reports the changing epidemiology of Salmonella infections in Taiwan during 1999-2003, with emphasis on increasing ceftriaxone resistance.Records of Salmonella clinical isolates in Chang Gung Memorial Hospital during 1999-2003 were reviewed. All isolates were identified and antimicrobial susceptibility determined by standard methods. A total of 22 ceftriaxone-resistant isolates were investigated by PCR sequencing of the bla(TEM), bla(SHV), bla(CTX-M) and ampC genes. Southern-blot hybridization was used to localize the ampC gene. Infrequent-restriction-site PCR was used to genotype these isolates.A total of 3635 Salmonella isolates, including 3592 (98.8%) non-typhoid Salmonella, were identified. Serogroup B (55.6%) remained the most predominant, but the prevalence has been decreasing. In contrast, serogroup D infections have increased significantly from 13.6 to 22.8%. Overall resistance to ampicillin and chloramphenicol remained high, with the highest rate (91% to both drugs) observed in Salmonella enterica serotype Choleraesuis in 2003. A sudden upsurge of ciprofloxacin resistance from zero to 69% was found in S. Choleraesuis. Ceftriaxone resistance increased in several serogroups (0.8-2.1%; average, 1.5%). The resistance was associated with plasmid-mediated bla(CMY-2) in 14 cases and extended-spectrum beta-lactamases (ESBLs), including CTX-M-3 (n=6), SHV-2a (n=1) and SHV-12 (n=1), in others. Diverse serotypes and genotypes were found among the ceftriaxone-resistant isolates.Increasing ceftriaxone resistance in non-typhoid Salmonella appears to link to the spread of plasmid-mediated ampC or ESBL genes. Effective measures should be taken to prevent the problem worsening.

The emergence of resistance to antimicrobial agents within the salmonellas is a worldwide and severe problem. A case of treatment failure due to the emergence of resistance to ceftriaxone in Salmonella enterica serotype Anatum was studied. S. enterica serotype Anatum and Escherichia coli , both of which are susceptible to ceftriaxone, were initially isolated from a diabetic patient hospitalized for the treatment of wound and urinary tract infections. Resistant S. enterica serotype Anatum and E. coli strains were isolated concomitantly 2 weeks after the initiation of ceftriaxone therapy. The patient eventually died of a sepsis caused by the ceftriaxone-resistant salmonella. PCR, nucleotide sequence analysis, and DNA-DNA hybridization identified a bla CTX-M-3 gene located on a 95.1-kb plasmid from the ceftriaxone-resistant isolates of S. enterica serotype Anatum and E. coli . The plasmid was proved to be conjugative. Molecular fingerprinting showed that the susceptible and resistant strains were genetically indistinguishable. The emergence of resistance to ceftriaxone in S. enterica serotype Anatum was due to the in vivo acquisition of a plasmid containing the bla CTX-M-3 gene and was the cause for treatment failure in this patient.

Background: Cell-free DNA is detectable in circulating blood. Numerous reports in the literature have pointed out that cell-free DNA in plasma or serum has the clinical potential to be a more specific tumor marker for the diagnosis and prognosis, as well as the early detection, of cancer. Methods: In order to adapt cell-free DNA to a routine clinical laboratory test, we used commercial kits such as the QIAamp blood kit for DNA extraction and the PicoGreen DNA kit for DNA quantification. This was done so our results and the normal reference value established would allow to be compared by other laboratories. We have established the normal reference level of cell-free DNA for females and males from age 20–70 years. We also detected elevated cell-free DNA in all cancers that were tested in this study, including carcinomas, leukemia and lymphoma. Results: Our study indicates that the elevation of serum cell-free DNA was usually detected in specimens containing elevated tumor markers and is most likely associated with tumor metastases. The electrophoretic pattern of cell-free DNA showed that cell-free DNA from cancer patient is fragmented, containing smaller DNA (100 bp) not found in normal cell-free DNA. Conclusions: Measuring cell-free DNA may complement currently used tumor markers for the management of cancer patients.

Cases of bacteremia caused by vancomycin-resistant E. faecium (VRE-fm) increased significantly in Taiwan. The present multicenter surveillance study was performed to reveal the associated epidemiological characteristics. In 2012, 134 non-repetitive VRE-fm isolates were prospectively collected from 12 hospitals in Taiwan. Antimicrobial susceptibility, pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and analysis of van genes and Tn1546 structures were investigated. Two isolates carried vanB genes, while all the remaining isolates carried vanA genes. Three isolates demonstrated a specific vanA genotype - vanB phenotype. Nine (6.7%) isolates demonstrated tigecycline resistance, and all were susceptible to daptomycin and linezolid. Molecular typing revealed 58 pulsotypes and 13 sequence types (STs), all belonged to three major lineages 17, 18, and 78. The most frequent STs were ST17 (n = 48, 35.8%), ST414 (n = 22, 16.4%), and ST78 (n = 16, 11.9%). Among the vanA harboring isolates, eight structure types of the Tn1546-like element were demonstrated. Type I (a partial deletion in the orf1 and insertion of IS1251-like between the vanS - vanH genes) and Type II (Type I with an additional insertion of IS1678 between orf2 - vanS genes) were the most predominant, consisted of 60 (45.5%) and 62 (47.0%) isolates, respectively. The increase of VRE-fm bacteremia in Taiwan may be associated with the inter- and intra-hospital spread of some major STs and horizontal transfer of vanA genes mostly carried on two efficient Tn1546-like elements. The prevailing ST414 and widespread of the Type II Tn1546-like elements are an emerging problem that requires continuous monitoring.

Background/purposeCarbapenem-resistant Pseudomonas aeruginosa infections have been a challenge and issue in hospital settings. However, the clinical impact of P. aeruginosa blood isolates resistant only to carbapenems has never been discussed previously.MethodsTo assess the risk factors and clinical significance of bacteremia caused by carbapenem resistance only P. aeruginosa (CROPA), a 6-year retrospective case–control study was conducted. The CROPA strains were defined as isolates susceptible to ciprofloxacin, antipseudomonal penicillins and cephalosporins, and aminoglycosides but resistant to one antipseudomonal carbapenem (imipenem or meropenem) or both. The controls were selected among patients with bacteremia due to P. aeruginosa susceptible to all above classes of antipseudomonal antibiotics, which was defined as all-susceptible P. aeruginosa.ResultsTwenty-five patients had at least one blood culture positive for CROPA, and 50 controls had all-susceptible P. aeruginosa bacteremia. CROPA bacteremia had a high 30-day mortality rate (72.0%), as compared to 26.0% for the controls (p

Streptococcus mitis (a common and usually harmless bacterium found in the nose, mouth and throat) can have an unusually high level of resistance to β-lactam antibiotics. We report a patient who developed fatal Streptococcus mitis septicaemia following unrelated cord blood transplantation. Administration of vancomycin to patients with recurrent fever during allogeneic stem cell transplantation might be indicated.

Superinfection by hepatitis D virus (HDV) leads to acute hepatitis and causes progression to liver cirrhosis in a significant proportion of hepatitis B surface antigen (HBsAg) carriers. Current regimens (interferon) to treat hepatitis D patients has only transient but no lasting effects. New approaches are, therefore, warranted. Recently, several laboratory studies have discovered interesting properties of HDV that may become targets for antiviral chemicals. Viral replication requires the small hepatitis delta antigen (s-HDAg). The s-HDAg is a nuclear phosphoprotein. There is evidence indicating that phosphorylation is important for HDV replication. A second step of replication requires HDV-RNA self-cleavage and self-ligation. Interestingly, one group of antibiotics, the aminoglycosides, exerts strong suppression effects on HDV ribozyme activities. In the following stage of viral assembly, two post-translational modifications, namely isoprenylation of large HDAg and glycosylation of HBsAg are involved. Agents capable of blocking the two modifications should reduce viral production. These four possible targets are reviewed. For prevention, effective vaccines are not yet available. Two novel approaches are discussed. The first demonstrates the immunogenicity of a nucleic acid vaccine in mice. The second approach assembled an empty HDV particle in yeast. Advances on such laboratory investigations may provide new methods for the control of hepatitis D in the future.

SUMMARYA review of the annual prevalence of Pseudomonas aeruginosa at a regional hospital in Taiwan revealed a significant increase in the incidence of extensive drug-resistant P. aeruginosa (XDRPA) from 2·1% in 2003 to 5·8% in 2007. The first XDRPA isolate was recovered in 2001 from the emergency ward. The widespread dissemination of XDRPA isolates to more than 10 other wards was discovered the following year. Six pulsotypes of 67 XDRPA isolates from 2006 onwards were identified and 91% were a single strain, suggesting the existence of a hidden outbreak. Prior to the recognition of the outbreak, the majority of cases were not considered to be healthcare-associated infections until molecular evidence was provided. A cohort measure was launched by the infection control practitioners that effectively controlled the outbreak. Patients with XDRPA were mostly referred from neighbouring long-term care facilities, which may have been the reservoir of the XDRPA clone.

Background/PurposeThe objective of this study was to determine the predictive value of teicoplanin minimal inhibitory concentrations (MICs) for treatment failure among patients with methicillin-resistant Staphylococcus aureus (MRSA) pneumonia.MethodsIn this study, all patients with ≥1 tracheal aspirates or sputum cultures positive for MRSA admitted to the hospital between April 2011 and September 2011 were reviewed. We enrolled patients who are ≥18 years of age, with a diagnosis of pneumonia, and with a receipt of teicoplanin therapy throughout the course. The relationship between teicoplanin Etest MICs and treatment outcomes of MRSA pneumonia was analyzed to identify the breakpoint of teicoplanin MICs influencing treatment outcomes.ResultsOf the 80 patients enrolled, 31 had a lower teicoplanin MIC level (

Introduction Mycobacterium marinum causes skin and soft tissue, bone and joint, and rare disseminated infections. In this study, we aimed to investigate the relationship between treatment outcome and antimicrobial susceptibility patterns. A total of 27 patients with M. marinum infections were enrolled. Methods Data on clinical characteristics and therapeutic methods were collected and analyzed. We also determined the minimum inhibitory concentrations of 7 antibiotics against 30 isolates from these patients. Results Twenty-seven patients received antimycobacterial agents with or without surgical debridement. Eighteen patients were cured, 8 failed to respond to treatment, and one was lost to follow-up. The duration of clarithromycin (147 vs. 28; p = 0.0297), and rifampicin (201 vs. 91; p = 0.0266) treatment in the cured patients was longer than that in the others. Surgical debridement was performed in 10 out of the 18 cured patients, and in 1 of another group (p = 0.0417). All the 30 isolates were susceptible to clarithromycin, amikacin, and linezolid; 29 (96.7%) were susceptible to ethambutol; 28 (93.3%) were susceptible to sulfamethoxazole; and 26 (86.7%) were susceptible to rifampicin. However, only 1 (3.3%) isolate was susceptible to doxycycline. Discussion Early diagnosis of the infection and appropriate antimicrobial therapy with surgical debridement are the mainstays of successful treatment. Clarithromycin and rifampin are supposed to be more effective agents.

The rapid identification of mycobacteria from smear-positive sputum samples is very important. To identify the Mycobacterium tuberculosis complex (MTBC) and frequently isolated nontuberculous mycobacteria strains directly from smear-positive sputum samples, an improved multiplex polymerase chain reaction (PCR) assay was developed. Nine pairs of primers targeting the 16S–23S rDNA internal transcribed spacer-1, hsp65, and the early secretory antigen (ESAT-6) gene sequences were developed, and their efficacy was evaluated in comparison to traditional culturing and 16S rRNA gene sequencing methods. A total of 200 smear- and culture-positive sputum specimens collected between November 2005 and May 2006 were used for the analysis. The results of the assay showed an accurate identification rate for acid-fast bacilli (AFB) 3+, AFB 2+, and AFB rare/1+ samples of 98%, 95%, and 53%, respectively. The improved multiplex PCR method saves time and has advantages for identifying mycobacteria from AFB 2+ and 3+ sputum samples. The method is suitable for use in countries with a high MTBC prevalence rate.

Between 2003 and 2009, the prevalence of extensively drug-resistant Pseudomonas aeruginosa (XDR-PA) increased significantly in northern Taiwan from 1.0% to 2.1%. Molecular methods were used to investigate the genetic relatedness and carbapenem resistance mechanisms of a collection of 203 non-repetitive XDR-PA isolates available for study. Using pulsed-field gel electrophoresis (PFGE), 52 genotypes were observed; one predominant genotype (pulsotype 1) was found in 57.6% of the isolates. Polymerase chain reaction (PCR), sequencing and quantitative reverse-transcriptase PCR analyses demonstrated that one horizontally acquired mechanism [metallo-β-lactamase (MBL) genes] and two mutational mechanisms (efflux and porins) accounted for the carbapenem resistance. The most predominant horizontally acquired mechanism was carriage of bla(VIM-3), which was found in 61.1% of isolates. Decreased expression of oprD was the most prevalent mutational mechanism and was found in 70.0% of the XDR-PA isolates, whereas overexpression of mexA was found in 27.6% of the isolates. The highlight of this study was the discovery of statistically significant relationships between certain horizontally acquired and mutational resistance mechanisms and their contribution to carbapenem susceptibility. MBL-producers expressed significantly lower MexAB and higher OprD than non-MBL-producers. Amongst isolates without an acquired β-lactamase gene, oprD expression was significantly reduced, whilst expression of efflux pumps was increased. Reduced OprD expression alone or the production of VIM-type MBLs showed similar contributions to a low to intermediate MIC(50) (minimum inhibitory concentration for 50% of the organisms) for carbapenems. Isolates with reduced OprD expression that simultaneously harboured bla(VIM) exhibited high levels of resistance to carbapenems, which implied that these two mechanisms had a synergistic effect on the MICs.

SUMMARYTo monitor the changing trend of extended-spectrum β-lactamase (ESBL)-producing bacteria, a 7-year continuous study was launched in 2001 at the largest tertiary hospital in Taiwan. A significant increase over the study period was evident for ESBL-producing isolates ofEscherichia coli(4·8–10·0%) andKlebsiella pneumoniae(15·0–23·4%). Molecular investigation conducted in three separate periods revealed the prevalent ESBL types and their genetic relatedness. CTX-M-producing isolates (73·8%) were more prevalent than SHV-type ESBLs (37·0%), the most frequent being CTX-M-14 (34·3%), CTX-M-3 (25·9%), and SHV-12 (25·7%). However, a marked increase of CTX-M-15-producing isolates from 2·1% in 2002 to 29·6% in 2007 was also noted. The increase of ESBL-producing isolates in both species may be mainly due to the horizontal transmission of resistance plasmids, while clonal expansion of some epidemic strains further added to the dispersion of ESBL-producingK. pneumoniae.

Objectives: Mycobacterium kansasii causes a variety of infections. Although previous reports on the prognosis of antimicrobial therapy have been mostly satisfactory, problems involving treatment failure or relapse have been encountered. The purpose of this study was to establish a relationship between the clinical treatment outcomes of M. kansasii infections and bacterial drug susceptibility, and their clonality. Methods: A total of 37 M. kansasii clinical isolates and clinical information on 34 patients were retrospectively collected in a tertiary medical centre in Taiwan. Bacterial drug susceptibility was determined by the microdilution method. The phylogenetic relationship was analysed by PFGE analysis. Results: Results of PFGE typing revealed a major cluster (cluster I) and eight other divergent patterns. Two/three strains leading to treatment failure were also multidrug resistant and belonged to cluster I. Conclusions: A relationship between high drug resistance and genetic relatedness of some M. kansasii strains was established. This was associated with clinical treatment failure.

An extended-spectrum beta-lactamase (CTX-M-15 type)-producing Escherichia coli persisted in the intestinal tract for5 months in a pediatric patient after cord blood stem-cell transplantation and caused 2 episodes of sepsis. The bla(CTX-M-15) is carried by a large plasmid of approximately 130 kb in size. The prolonged shedding of the highly resistant E. coli posed a great challenge to infection control and public health.

Enterococcal infections at the Chang Gung Memorial Hospital, Taiwan, have increased significantly in recent years, accompanied by a significant growth of vancomycin resistance from1% to 3.8%. However, the significant increase in vancomycin resistance was only found in Enterococcus faecium (from 0.5% to 17.4%).A total of 172 patients infected with vancomycin-resistant enterococci (85 E. faecium and 87 E. faecalis) during 1998-2004 were retrospectively studied. Clinical and laboratory features were analyzed using Stata for Windows (version 8.2). Genotypes of the isolates were determined by infrequent-restriction-site polymerase chain reaction.Multivariate analysis revealed that prior use of imipenem (odds ratio [OR], 30.1; 95% confidence interval [CI], 4.2-215.9) or clindamycin (OR, 6.5; 95% CI, 1.5-28.1), positive urine cultures (OR, 6.1; 95% CI, 2.1-17.8) and penicillin resistance (OR, 55.9; 95% CI, 18.5-168.3) were significantly associated with the infections caused by vancomycin-resistant E. faecium. Genotyping analysis demonstrated a predominant genotype in 71 (83.5%) of the E. faecium isolates, while diverse genotypes were found among the E. faecalis isolates. No apparent correlation between genotype and any specific ward was found. Up to the end of 2005, primary efforts to restrict imipenem usage and reinforce infection control measures have reduced by half the infections caused by vancomycin-resistant E. faecium.Multiple factors were associated with the unusual increase of vancomycin-resistant E. faecium infections in this hospital. Continuous monitoring of appropriate antimicrobial usage and stringent compliance to infection control measures are required to control the increase of such infections.

Objectives : To investigate the possibility of identifying DNA hypermethylation in the circulation of prostate cancer patients. Methods : Plasma DNA samples were extracted from 36 prostate cancer patients and 27 benign prostate hyperplasia (BPH) cases. After extensive methylation-sensitive restriction enzyme digestion, the DNA samples were subjected to the real-time quantitative PCR amplification. Dissociation curve analysis was applied to determine if hypermethylation occurred in the promoter region flanking the GSTP1 gene, a well-documented epigenetic event among prostate cancer cells, in these plasma DNA samples. Results : 11 of 36 prostate cancer patients showed positive peak pattern, indicating methylation changes occurred. Concordant data were obtained from the corresponding paraffin-embedded tissue samples available from the Tumor Bank. Twenty-five of the 27 BPH cases showed negative results, suggesting no methylation changes happened in the CpG islands in these cases. Conclusions : We have successfully identified prostate cancer genome hypermethylation in the peripheral circulation in prostate cancer patients with this protocol. This method can effectively distinguish BPH from prostate neoplasm. Although a larger number of samples are necessary to validate the capability of the protocol in practice, using plasma DNA sample is an ideal non-invasive approach for prostate neoplasm detection.

Received 29 August 2005; returned 25 September 2005; revised 16 November 2005; accepted 12 December 2005 Objectives: The dissemination of cephamycin resistance in Enterobacteriaceae and its correlation with a transposon-like DNA element consisting of a specific tnpA-blaCMY-2-blc-sugE structure were investigated. Methods: A total of 140 enterobacterial isolates belonging to 17 species (10 genera) of Enterobacteriaceae phenotypically characterized as putative AmpC-producers were evaluated. The isolates were examined by PCR analysis, DNA–DNA hybridization and nucleotide sequencing. Results: The blaCMY-2-carrying element was detected in 34 isolates from 10 species (9 genera), including all 14 Salmonella and 4 Shigella isolates as well as 7 of the 10 Escherichia coli isolates tested. The remaining 9 isolates were from 112 isolates of the other 14 species tested. The genetic structure of the blaCMY-2-carrying element was identical in 29 isolates, while in 3 E. coli and 2 Citrobacter isolates an additional insertion sequence IS1 was found inserted at various nucleotide positions close to the 3 0 end, either within or downstream, of tnpA. In 12 of the 14 representative isolates examined, the blaCMY-2-carrying element was found inserted in the finQ gene of various-sized plasmids with highly conserved 8 bp direct repeats flanking the junction regions. Among the other 106 non-CMY-2-producing isolates, plasmid-mediated ampC genes were found only in one isolate of Enterobacter aerogenes which carried a blaDHA-1-like gene. Conclusions: blaCMY-2 is the most prevalent plasmid-mediated ampC gene among Enterobacteriaceae. All the blaCMY-2 genes identified in the present study were associated with a specific transposon-like element that may be responsible for the spread of blaCMY-2 among Enterobacteriaceae.

Infection by extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae has been increasing in Taiwan. Accurate identification of the ESBL genes is necessary for surveillance and for epidemiological studies of the mode of transmission in the hospital setting. We describe herein the development of a novel system, which consists of a multiplex PCR to identify bla(SHV), bla(CTX-M-3)-like, and bla(CTX-M-14)-like genes and a modified SHV melting-curve mutation detection method to rapidly distinguish six prevalent bla(SHV) genes (bla(SHV-1), bla(SHV-2), bla(SHV-2a), bla(SHV-5), bla(SHV-11), and bla(SHV-12)) in Taiwan. Sixty-five clinical isolates, which had been characterized by nucleotide sequencing of the bla(SHV) and bla(CTX-M) genes, were identified by the system. The system was then used to genotype the ESBLs from 199 clinical isolates, including 40 Enterobacter cloacae, 68 Escherichia coli, and 91 Klebsiella pneumoniae, collected between August 2002 and March 2003. SHV-12 (80 isolates) was the most prevalent type of ESBL identified, followed in order of frequency by CTX-M-3 (65 isolates) and CTX-M-14 (36 isolates). Seventeen (9%) of the 199 clinical isolates harbored both SHV- and CTX-M-type ESBLs. In contrast to Enterobacter cloacae, the majority of which produced SHV-type ESBLs, E. coli and K. pneumoniae were more likely to possess CTX-M-type ESBLs. Three rare CTX-M types were identified through sequencing of the bla(CTX-M-3)-like (CTX-M-15) and bla(CTX-M-14)-like (CTX-M-9 and CTX-M-13) genes. The system appears to provide an efficient differentiation of ESBLs among E. coli, K. pneumoniae, and Enterobacter cloacae in Taiwan. Moreover, the design of the system can be easily adapted for similar purposes in areas where different ESBLs are prevalent.

A 68-year-old diabetic woman suffered from mycotic aneurysm due to Klebsiella pneumoniae over her abdominal aorta; she received surgical intervention, followed by treatment with first-generation cephalosporins for 6 months. She was hospitalized again 11 months later because of another episode of mycotic aneurysm caused by K. pneumoniae on her thoracic aorta. Fingerprinting generated by pulsed-field gel electrophoresis and infrequent-restriction-site polymerase indicated K. pneumoniae isolates of the identical clonal strain were responsible for these two mycotic-aneurysm episodes. Unfortunately, nosocomial pneumonia developed at the second hospitalization; blood and purposefully sampled feces specimen cultures both grew CTX-M-24-producing K. pneumoniae, which were of the same strain and genetically nonrelated to the K. pneumoniae strain causing mycotic aneurysms earlier. This is the first report on infection due to CTX-M-24-producing K. pneumoniae. It is unclear whether the prolonged use of first-generation cephalosporins in this case selected a strain of enteric organism possessing the ESBL in question, which was capable of passing this ESBL plasmid to the K. pneumoniae strain causing the nosocomial infection. This report suggests that further observation is needed before one can draw a conclusion on the possibility of the selection of ESBL enteric organism by extensive exposure to first-generation cephalosporins.

The clinical presentations of laboratory-confirmed Bordetella pertussis infection in Chang Gung Children's Hospital during 1997 and 2001 were analyzed. Of the 46 cases, 25 (54.3%) were male. The patients ages ranged from 24 days to 37 years, with a mean and median of 4.3 years and 10.5 years, respectively. Forty four patients had vaccination records, among them 23 patients (52.2%) had receivedor = 3 doses of pertussis vaccine. Of the patients who were partially vaccinated (received 1 or 2 doses vaccine) or unvaccinated, 16 (69.6%) presented with whooping cough, 5 (22.2%) with post-tussive vomiting, and 13 (59.1%) with cyanosis. Leukocytosis (white blood cellsor = 15,000 cells/microL) and lymphocytosis (lymphocytesor = 10,000 cells/microL) were observed in 17 (47.2%) and 16 (44.4%) of the patients, respectively. Fourteen patients (30.4%) developed complications, among which pneumonia was the most common (92.3%). Among infantsor = 1 year of age, 95.2% were partially vaccinated (20/21), compared with 5% (1/20) of the patients1 year of age (p0.05). The overall complication rate was 37.5%, compared with 18.2% for patients1 year of age (p0.05). One 2-month-old patient required ventilatory support after the development of cardiopulmonary failure. There was no mortality in this study. In summary, pertussis most commonly occurred in infants who were unvaccinated or partially vaccinated. These patients usually presented with atypical symptoms such as cyanosis or apnea. The importance of vaccination still cannot be overemphasized because immunized patients usually present with milder disease than those who are not immunized.

Accurate diagnosis of Bordetella pertussis infection is difficult. Polymerase chain reaction (PCR) tests are more sensitive than culture, but the reported sensitivity is variable. We prospectively compared the performance of culture, serology, and a multiplex PCR for the detection of B. pertussis.A total of 193 paired nasopharyngeal (NP) swab specimens were examined by both culture and a multiplex PCR. Serology results were available in 103 patients. Medical charts of the patients with discrepant laboratory findings were reviewed and compared with the United States Centers for Disease Control and Prevention (CDC) clinical case definition.Of the 193 specimens, 11 were positive on both culture and PCR, and 14 were positive on PCR only. Of the 103 specimens with serology results, 3 were positive with all three methods, and 69 were negative with all methods. Eleven of the 14 PCR-positive only cases and 6 of the 19 serology-positive only cases were defined as true pertussis cases according to an expanded standard which includes either (1) culture positive or (2) PCR or serology positive with clinical features fulfilling the CDC clinical case definition and the patients having received macrolides treatment for more than 10 days. The sensitivity and specificity of the multiplex PCR were 79% and 98%, respectively, while those for serology were 47% and 85%, and for culture 39% and 100%.Our data confirm the superior sensitivity of the multiplex PCR in detection of B. pertussis, compared with conventional culture and serology. Clinical validation indicates that the multiplex PCR offers specific detection of B. pertussis from NP specimens.