Cindy W. S. Tse

Quasispecies of the D225G Substitution in the Hemagglutinin of Pandemic Influenza A(H1N1) 2009 Virus from Patients with Severe Disease in Hong Kong, China

The D225G (aspartic acid to glycine) substitution in the hemagglutinin of H1N1 influenza virus may alter its receptor-binding specificity. Direct analysis of polymorphisms in 126 amino acids spanning the receptor-binding site in the hemagglutinin of pandemic H1N1 2009 virus from 117 clinical specimens in Hong Kong found the D225G substitution for 7 (12.5%) of 57 patients with severe disease and for 0 (0%) of 60 patients with mild disease. D225G quasispecies were identified mainly in endotracheal aspirate samples and were identified less frequently in nasopharyngeal aspirate samples from patients with severe disease. Continuous monitoring of the prevalence and tissue tropism of this variant during its circulation among humans is important.

Novel Pan-Genomic Analysis Approach in Target Selection for Multiplex PCR Identification and Detection of Burkholderia pseudomallei, Burkholderia thailandensis, and Burkholderia cepacia Complex Species: a Proof-of-Concept Study

ABSTRACT Burkholderia pseudomallei, Burkholderia thailandensis, and the Burkholderia cepacia complex differ greatly in pathogenicity and epidemiology. Yet, they are occasionally misidentified by biochemical profiling, and even 16S rRNA gene sequencing may not offer adequate discrimination between certain species groups. Using the 23 B. pseudomallei, four B. thailandensis, and 16 B. cepacia complex genome sequences available, we identified gene targets specific to each of them (a Tat domain protein, a 70-kDa protein, and a 12-kDa protein for B. pseudomallei, B. thailandensis, and the B. cepacia complex, respectively), with an in-house developed algorithm. Using these targets, we designed a robust multiplex PCR assay useful for their identification and detection from soil and simulated sputum samples. For all 43 B. pseudomallei, seven B. thailandensis, and 20 B. cepacia complex (B. multivorans, n = 6; B. cenocepacia, n = 3; B. cepacia, n = 4; B. arboris, n = 2; B. contaminans, B. anthina, and B. pyrrocinia, n = 1 each; other unnamed members, n = 2) isolates, the assay produced specific products of predicted size without false positives or negatives. Of the 60 soil samples screened, 19 (31.6%) and 29 (48.3%) were positive for B. pseudomallei and the B. cepacia complex, respectively, and in four (6.7%) soil samples, the organisms were codetected. DNA sequencing confirmed that all PCR products originated from their targeted loci. This novel pan-genomic analysis approach in target selection is simple, computationally efficient, and potentially applicable to any species that harbors species-specific genes. A multiplex PCR assay for rapid and accurate identification and detection of B. pseudomallei, B. thailandensis, and the B. cepacia complex was developed and verified.

Elizabethkingia anophelis bacteremia is associated with clinically significant infections and high mortality

Unlike Elizabethkingia meningoseptica, the clinical importance of E. anophelis is poorly understood. We determined the clinical and molecular epidemiology of bacteremia caused by Elizabethkingia-like species from five regional hospitals in Hong Kong. Among 45 episodes of Elizabethkingia-like bacteremia, 21 were caused by Elizabethkingia, including 17 E. anophelis, three E. meningoseptica and one E. miricola; while 24 were caused by other diverse genera/species, as determined by 16S rRNA gene sequencing. Of the 17 cases of E. anophelis bacteremia, 15 (88%) were clinically significant. The most common diagnosis was pneumonia (n = 5), followed by catheter-related bacteremia (n = 4), neonatal meningitis (n = 3), nosocomial bacteremia (n = 2) and neutropenic fever (n = 1). E. anophelis bacteremia was commonly associated with complications and carried 23.5% mortality. In contrast, of the 24 episodes of bacteremia due to non-Elizabethkingia species, 16 (67%) were clinically insignificant. Compared to non-Elizabethkingia bacteremia, Elizabethkingia bacteremia was associated with more clinically significant infections (P < 0.01) and positive cultures from other sites (P < 0.01), less polymicrobial bacteremia (P < 0.01), and higher complication (P < 0.05) and mortality (P < 0.05) rates. Elizabethkingia bacteremia is predominantly caused by E. anophelis instead of E. meningoseptica. Elizabethkingia bacteremia, especially due to E. anophelis, carries significant morbidity and mortality, and should be considered clinically significant unless proven otherwise.

Life-threatening invasive Helcococcus kunzii infections in intravenous-drug users and ermA-mediated erythromycin resistance

ABSTRACT We report the first two cases of life-threatening invasive Helcococcus kunzii infection, with primary bacteremia and empyema thoracis, respectively. Gram smears of both H. kunzii isolates showed a mixture of gram-positive and gram-negative cocci. The isolate from the first patient, resistant to erythromycin and clindamycin, possessed an ermA gene.

MP1 Homologue-Based Multilocus Sequence System for Typing the Pathogenic Fungus Penicillium marneffei: a Novel Approach Using Lineage-Specific Genes

ABSTRACT A highly reproducible and discriminative typing system is essential for better understanding of the epidemiology of Penicillium marneffei, the most important thermal dimorphic fungus causing respiratory, skin, and systemic mycosis in Southeast Asia. The sequences of 11 housekeeping genes were identical among 10 strains of P. marneffei, but those of MP1 and its 13 homologues, a novel superfamily of mannoproteins in the subdivision Pezizomycotina of Ascomycetes, mostly species of Penicillium and Aspergillus, showed significant variations. Therefore, a multilocus sequence typing (MLST) system for P. marneffei was constructed using MP1 (549 bp) and the four of its homologues (MPLP4 [337 bp], MPLP7 [347 bp], MPLP10 [546 bp], and MPLP13 [422 bp]) that showed the greatest variations. Among the 2,201 bp of the five loci, 183 polymorphic sites were observed in 44 strains of P. marneffei. The median number of alleles at each locus was five (range, 5 [MPLP4, MPLP7, and MPLP13] to 15 [MPLP10]). Four of the five genes had nonsynonymous substitution/synonymous substitution (dn/ds) ratios of >1. A total of 35 different sequence types (STs) were assigned to the 44 P. marneffei isolates, with 28 of the 35 STs identified only once. The discriminatory power was 0.9884. MP1 and its homologues were better than housekeeping genes for MLST in P. marneffei. Due to their more rapid evolutionary rates, lineage-specific genes may be better candidates than housekeeping genes for sequence-based typing, especially in microbes that evolve slowly or have evolved recently.

Reduced Levofloxacin Susceptibility and Tetracycline Resistance in a Clinical Isolate of Haemophilus quentini Identified by 16S rRNA Sequencing

ABSTRACT This paper reports the first case of Haemophilus quentini bacteremia with reduced susceptibility to levofloxacin and resistance to nalidixic acid identified by 16S rRNA sequencing. There was an S84L substitution in gyrA and an S84I substitution in parC. The isolate had coresistance to ampicillin (β-lactamase positive) and tetracycline mediated by the tet(B) gene.

A novel MLST sequence type discovered in the first fatal case of Laribacter hongkongensis bacteremia clusters with the sequence types of other human isolates.

Laribacter hongkongensis is a gram-negative, facultative anaerobic, motile, S-shaped, asaccharolytic, urease-positive bacillus in the Neisseriaceae family of β-proteobacteria. To date, all patients with L. hongkongensis infection have survived, including the two patients with L. hongkongensis bacteremia and patients with L. hongkongensis gastroenteritis. In this study, we describe the clinical, microbiological and molecular characterization of the first fatal case associated with L. hongkongensis bacteremia in a patient with colonic carcinoma that metastasized to the liver. The identity of the isolate was confirmed via phenotypic tests and 16S rRNA gene sequencing. Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI–TOF MS), using the Bruker database extended with L. hongkongensis reference strains, also identified the isolate as L. hongkongensis, with a top match score of 2.473. Multilocus sequence typing revealed a new sequence type (ST), and phylogenetic analysis and eBURST demonstrated unambiguously that the ST of the isolate was clustered with two other STs found exclusively in human patients, consistent with the theory that some clones of L. hongkongensis could be more virulent than others. Underlying liver diseases and ascites potentially represent distinct risk factors for invasive L. hongkongensis infection. More widespread use of MALDI–TOF MS for identification and improvements of selective media should facilitate the identification of more cases of L. hongkongensis infection.