Tzong-Shi Chiueh

Experience of using convalescent plasma for severe acute respiratory syndrome among healthcare workers in a Taiwan hospital

Abstract Objectives: To describe the immunological responses and clinical outcome of coronavirus (SARS) infected healthcare workers (HCW) who had been administered with convalescent plasma as a treatment. Methods: Convalescent plasma (500 mL) was obtained from each of three SARS patients and transfused into the three infected HCW. Donors were blood type O and seronegative for hepatitis B and C, HIV, syphilis and human T-cell lymphotropic virus types I and II (HTLV-I and -II). Serum antibody (IgG) titre was >640. Apharesis was performed with a CS 3000 plus cell separator followed by the forming of the convalescent phase plasma. As part of the routine check with donated plasma, the convalescent plasma was confirmed free of residual SARS-CoV by RT–PCR. Serial serum samples obtained from the recipients of the convalescent plasma were collected to undertake real-time quantitative RT–PCR for SARS-CoV for direct measurement of viral concentration. Specific immunoglobulin IgM and IgG concentrations were titrated using an antigen microarray developed in-house. Results: Viral load dropped from 495 × 103, 76 × 103 or 650 × 103 copies/mL to zero or 1 copy/mL one day after transfusion. Anti-SARS-CoV IgM and IgG also increased in a time-dependent manner following transfusion. All three patients survived. One HCW became pregnant subsequently, delivering 13 months after discharge. Positive anti-SARS-CoV IgG was detected in the newborn. Passive transfer of anti-SARS-CoV antibody from the mother was considered as a possibility. Conclusions: All infected HCW whose condition had progressed severely and who had failed to respond to the available treatment, survived after transfusion with convalescent plasma.

Molecular typing and phenotype characterization of methicillin-resistant Staphylococcus aureus isolates from blood in Taiwan.

Background Staphylococcus aureus causes a variety of severe infections such as bacteremia and sepsis. At present, 60–80% of S. aureus isolates from Taiwan are methicillin resistant (MRSA). It has been shown that certain MRSA clones circulate worldwide. The goals of this study were to identify MRSA clones in Taiwan and to correlate the molecular types of isolates with their phenotypes. Methods A total of 157 MRSA isolates from bacteremic patients were collected from nine medical centers. They were typed based on polymorphisms in agr, SCCmec, MLST, spa, and dru. Phenotypes characterized included Panton-Valentine leucocidin (pvl), inducible macrolide-lincosamide-streptogramin B resistance (MLSBi), vancomycin (VA) and daptomycin (DAP) minimal inhibitory concentrations (MIC), and superantigenic toxin gene profiles. Difference between two consecutive samples was determined by Mann-Whitney-U test, and difference between two categorical variables was determined by Fishers exact test. Results Four major MRSA clone complexes CC1, CC5, CC8, and CC59 were found, including 4 CC1, 9 CC5, 111 CC8, and 28 CC59 isolates. These clones had the following molecular types: CC1: SCCmecIV and ST573; CC5: SCCmecII and ST5; CC8: SCCmecIII, ST239, and ST241, and CC59: SCCmecIV, SCCmecVT, ST59, and ST338. The toxin gene profiles of these clones were CC1: sec-seg-(sei)-sell-selm-(seln)-selo; CC5: sec-seg-sei-sell-selm-(seln)-selp-tst1; CC8: sea-selk-selq, and CC59: seb-selk-selq. Most isolates with SCCmecVT, ST59, spat437, and dru11 types were pvl + (13 isolates), while multidrug resistance (≥4 antimicrobials) were associated with SCCmecIII, ST239, spa t037, agrI, and dru14 (119 isolates) (p<0.001). One hundred and twenty four isolates with the following molecular types had higher VA MIC: SCCmecII and SCCmecIII; ST5, ST239, and ST241; spa t002, t037, and t421; dru4, dru10, dru12, dru13, and dru14 (p<0.05). No particular molecular types were found to be associated with MLSBi phenotype. Conclusions Four major MRSA clone complexes were found in Taiwan. Further studies are needed to delineate the evolution of MRSA isolates.

Genetic diversity of the Mycobacterium tuberculosis Beijing family based on SNP and VNTR typing profiles in Asian countries.

The Mycobacterium tuberculosis (MTB) Beijing strain is highly virulent, drug resistant, and endemic over Asia. To explore the genetic diversity of this family in several different regions of eastern Asia, 338 Beijing strains collected in Taiwan (Republic of China) were analyzed by mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing and compared with published MIRU-VNTR profiles and by the Hunter-Gaston diversity index (HGDI) of Beijing strains from Japan and South Korea. The results revealed that VNTR2163b (HGDI>0.6) and five other loci (VNTR424, VNTR4052, VNTR1955, VNTR4156 and VNTR 2996; HGDI>0.3) could be used to discriminate the Beijing strains in a given geographic region. Analysis based on the number of VNTR repeats showed three VNTRs (VNTR424, 3192, and 1955) to be phylogenetically informative loci. In addition, to determine the geographic variation of sequence types in MTB populations, we also compared sequence type (ST) data of our strains with published ST profiles of Beijing strains from Japan and Thailand. ST10, ST22, and ST19 were found to be prevalent in Taiwan (82%) and Thailand (92%). Furthermore, classification of Beijing sublineages as ancient or modern in Taiwan was found to depend on the repeat number of VNTR424. Finally, phylogenetic relationships of MTB isolates in Taiwan, South Korea, and Japan were revealed by a minimum spanning tree based on MIRU-VNTR genotyping. In this topology, the MIRU-VNTR genotypes of the respective clusters were tightly correlated to other genotypic characters. These results are consistent with the hypothesis that clonal evolution of these MTB lineages has occurred.

A Truncated AdeS Kinase Protein Generated by ISAba1 Insertion Correlates with Tigecycline Resistance in Acinetobacter baumannii

Over-expression of AdeABC efflux pump stimulated continuously by the mutated AdeRS two component system has been found to result in antimicrobial resistance, even tigecycline (TGC) resistance, in multidrug-resistant Acinetobacter baumannii (MRAB). Although the insertion sequence, ISAba1, contributes to one of the AdeRS mutations, the detail mechanism remains unclear. In the present study we collected 130 TGC-resistant isolates from 317 carbapenem resistant MRAB (MRAB-C) isolates, and 38 of them were characterized with ISAba1 insertion in the adeS gene. The relationship between the expression of AdeABC efflux pump and TGC resistant was verified indirectly by successfully reducing TGC resistance with NMP, an efflux pump inhibitor. Further analysis showed that the remaining gene following the ISAba1 insertion was still transcribed to generate a truncated AdeS protein by the Pout promoter on ISAba1 instead of frame shift or pre-termination. Through introducing a series of recombinant adeRS constructs into a adeRS knockout strain, we demonstrated the truncated AdeS protein was constitutively produced and stimulating the expression of AdeABC efflux pump via interaction with AdeR. Our findings suggest a mechanism of antimicrobial resistance induced by an aberrant cytoplasmic sensor derived from an insertion element.

Overexpression of the adeB Gene in Clinical Isolates of Tigecycline-Nonsusceptible Acinetobacter baumannii without Insertion Mutations in adeRS

ABSTRACT Thirteen clinical isolates of multidrug-resistant Acinetobacterbaumanniiresistant to carbapenems (MRAB-C) with tigecycline nonsusceptibility were collected from individual patients in this study. None of the 13 isolates shared the same strain characteristics in molecular typing. All of them showed increased adeB transcription, as predicted. However, none of these tigecycline-nonsusceptible MRAB-C isolates were found to possess previously known adeRS mutations. Upregulation of adeB transcription may result from cross stimulation by other mechanisms.

Detection and typing of vancomycin-resistance genes of enterococci from clinical and nosocomial surveillance specimens by multiplex PCR

Ninety-three clinical isolates of vancomycin-resistant enterococci (VRE) collected from nine hospitals in Taiwan were examined for the presence of vanA, vanB, vanC1, or vanC2/vanC3 genes by a multiplex PCR. Forty-seven of these VRE isolates were vanA positive, 1 contained both vanC1 and vanA, 40 harboured vanB, 2 were vanC1, and 3 were identified to be vanC2/vanC3. Twenty-four vanA isolates were sensitive to teicoplanin and thus did not have a typical VanA phenotype. Five isolates with the VanC phenotype harboured vanB. None of the 40 clinically isolated vancomycin-susceptible E. faecium or E. faecalis and the vancomycin-resistant Leuconostoc and Pediococcus isolates were positive for any of the van genes. While performing nosocomial surveillance, VRE were isolated from 47 of 467 rectal swabs by culture. Compared with the conventional culture method, the sensitivity and specificity of the multiplex PCR for detecting and identifying vancomycin-resistance genes in enterococci directly from culture-positive broth were 97.9% and 100%, respectively. The results suggest that genotypic characterization of vancomycin-resistance is necessary for all clinical VRE isolates and that the multiplex PCR assay can be an alternative method for this purpose.

A 10-year analysis of thyrotoxic periodic paralysis in 135 patients: focus on symptomatology and precipitants

Background A comprehensive analysis has not been performed on patients with thyrotoxic periodic paralysis (TPP) characterized by acute hypokalemia and paralysis in the setting of thyrotoxicosis. Purpose The aim of this study was to analyze the detailed symptomatology of thyrotoxicosis and precipitating factors for the attack in a large cohort of TPP patients. Patients and methods A prospective observational study enrolled patients with TPP consecutively over 10 years at an academic medical center. Clinical features, including signs/symptoms of thyrotoxicosis and precipitating factors, were analyzed. The Waynes index was used to assess the severity of thyrotoxicosis at presentation. Patients who agreed to receive an oral glucose-loading test after recovery were evaluated. Results Among the 135 TPP patients (male:female, 130:5), 70% of paralytic attacks occurred in the morning, especially during the seasons of summer and fall. Two-thirds of patients did not have a known family or personal history of hyperthyroidism. Only 17% of TPP patients manifested overt signs/symptoms of thyrotoxicosis (Waynes index >19). A clear precipitating factor, such as high carbohydrate load, acute upper respiratory tract infection, strenuous exercise, high-salt diet, or the use of steroids or bronchodilators, was identified in only 34% of TPP patients. A glucose load to stimulate insulin secretion induced acute hypokalemia (K+2.47±0.6 mmol/l) with reparalysis in only 18% (10/55) of TPP patients. Conclusions Most TPP patients have only subtle clinical signs/symptoms of thyrotoxicosis and only a small fraction has clear precipitating factors. In addition to the effects of hyperinsulinemia, other insulin-independent mechanisms may participate in the pathogenesis of TPP.

High Prevalence of VanB2 Vancomycin-Resistant Enterococcus faecium in Taiwan

ABSTRACT Thirty-six VanB glycopeptide-resistant Enterococcus faecium isolates were collected from patients in five different hospitals in Taiwan. The vancomycin resistance genes were amplified by the long vanB PCR, which amplifies the 6,373-bpvanB gene cluster including thevanRB2, vanSB2, vanYB2, vanWB2, vanHB2, vanB2, andvanXB2 genes. The deduced amino acid sequences were found to be 95 to 98% homologous to those of thevanB1 gene cluster: VanRB1, 97%; VanSB1, 97%; VanYB1, 96%; VanHB1, 95%; VanB1, 96%; and VanXB1, 98%. Restriction enzyme analysis of the long vanB PCR products revealed that all 36 isolates had the same vanB2-specific pattern. DNA sequence analysis of the vanB2 gene, which is ad-Ala–d-Lac ligase gene, revealed that none of the 36 sequences were identical to the previously publishedvanB2 sequence. Thirty-one isolates had 1 nucleotide different from the published vanB2 sequence. The sequences of the other five isolates differed from the publishedvanB2 sequence by 2 or 3 nucleotides. Four isolates with a low or moderate resistance to vancomycin (MIC = 4 to 32 μg/ml) were found to have the same leucine-to-methionine change at amino acid position 308 of the vanB2 gene. The genomic DNAs of all 36 isolates were digested with SmaI and then typed by pulsed-field gel electrophoresis (PFGE). Eight different PFGE types (I to VIII) were observed, and type I was found to be prevalent in all hospitals examined in this study. This result suggests that intra- and interhospital dissemination of this E. faecium strain has occurred in Taiwan.

Changing trends in antimicrobial resistance of major bacterial pathogens, 1985–2005: A study from a medical center in northern Taiwan

BACKGROUND Antimicrobial resistance is a major health problem worldwide. We evaluated the antimicrobial resistance trends of 16 major bacterial pathogens at a tertiary medical center in northern Taiwan. METHODS We conducted a retrospective review of annual summary documents for antimicrobial susceptibility of clinically isolated gram-positive and gram-negative bacteria from 1985 to 2005. The numbers of isolates and susceptibilities were calculated for three 7-year periods: first period, 1985-1991; second period, 1992-1998; and the third period, 1999-2005. RESULTS During the 21-year period, 219,715 bacterial pathogens were identified. A significant increase in incidence over time was found for methicillin-resistant Staphylococcus aureus, methicillin-resistant S epidermidis, penicillin-nonsusceptible Streptococcus pneumoniae, erythromycin-resistant S pneumoniae, vancomycin-resistant enterococci, cefotaxime/ceftriaxone-resistant Escherichia coli and Klebsiella pneumoniae, and imipenem-resistant Acinetobacter baumannii. Additionally, a significant increase in ciprofloxacin resistance rates over time from 1996 to 2005 was noted for E coli, Enterobacter cloacae, and A baumannii (through 1997 to 2005). However, a significant decrease in erythromycin resistance rate with time from 1999 to 2005 was found for Groups A and B streptococci, non-A, B, D streptococci, and S pneumoniae. CONCLUSION Resistance to antimicrobial agents increased rapidly in the past two decades in Taiwan and has become very common in major bacterial pathogens. Continuous enforcement of policies to limit use of antimicrobial agents and active surveillance of antimicrobial resistance through a nationwide system are both warranted.

Molecular and Phenotypic Characteristics of Methicillin-Resistant and Vancomycin-Intermediate Staphylococcus aureus Isolates from Patients with Septic Arthritis

ABSTRACT Staphylococcus aureus is one of the most common pathogens in community- and hospital-associated infections and frequently causes severe and intractable infections in osteoarticular tissues. S. aureus isolates that are resistant to methicillin (meticillin) (MRSA isolates) and intermediately resistant to vancomycin (VISA isolates) have emerged. In this report, we described two patients, one female and one male, diagnosed with septic arthritis due to S. aureus infections. A total of 13 MRSA isolates were obtained from these two patients. All but one isolate belonged to the VISA group. All seven isolates from the female patient were determined to be community associated, multilocus sequence type (MLST) 59, and staphylococcal cassette chromosome mecA (SCCmec) type IV; had direct repeat units (DRUs) of nine repeats; were spa type t437; and were susceptible to sulfa and quinolone antibiotics. The other six isolates, from the male patient, were determined to be hospital associated, MLST 239, and SCCmec type III; had DRUs of 14 repeats; were spa type t037; and were resistant to sulfa and quinolone antibiotics. All 13 MRSA isolates were in agr group I, were pvl negative, and showed no evidence of any association between vancomycin resistance and autolysis.