Status of global virologic surveillance for rubella viruses.

The suspected measles case definition captures rubella cases. Therefore, measles surveillance will be improved in the course of the control and eventual elimination of rubella transmission. One aspect of rubella control, virologic surveillance, is reviewed here. A systematic nomenclature for rubella viruses (RVs) based on 13 genotypes has been established and is updated when warranted by increases in information about RVs. From 2005 through 2010, the genotypes of RVs most frequently reported were 1E, 1G, and 2B, and genotypes 1a, 1B, 1C, 1h, 1j, and 2C were less frequently reported. Virologic surveillance can support rubella control and elimination. Synopses of rubella virologic surveillance in various countries, regions, and globally are given, including characterization of viruses from imported cases in a country that has eliminated rubella and studies of endemic viruses circulating in countries without rubella control objectives. Current challenges are discussed.

Comparison of laboratory diagnostic methods for measles infection and identification of measles virus genotypes in Hong Kong.

The sensitivities of IgM detection, virus isolation, and RT-PCR for the diagnosis of measles infection were assessed using samples collected from confirmed measles cases from 2006 to 2009. The optimal timing of specimen collection and the preferred specimen type(s) for these tests were also determined. IgM detection showed highest sensitivity when serum samples were collected >or=5 days after rash onset. Virus isolation gave the highest sensitivity when samples were collected

Measles outbreak from Hong Kong International Airport to the hospital due to secondary vaccine failure in healthcare workers.

OBJECTIVE: To report an outbreak of measles with epidemiological link between Hong Kong International Airport (HKIA) and a hospital. METHODS: Epidemiological investigations, patients' measles serology, and phylogenetic analysis of the hemagglutinin (H) and nucleoprotein (N) genes of measles virus isolates were conducted. RESULTS: In total, 29 HKIA staff of diverse ranks and working locations were infected with measles within 1 month. Significantly fewer affected staff had history of travel than non-HKIA-related measles patients [10 of 29 (34.5%) vs 28 of 35 (80%); P < .01]. Of 9 airport staff who could recall detailed exposure history, 6 (66.7%) had visited self-service food premises at HKIA during the incubation period, where food trays, as observed during the epidemiological field investigation, were not washed after use. Furthermore, 1 airport baggage handler who was admitted to hospital A before rash onset infected 2 healthcare workers (HCWs) known to have 2 doses of MMR vaccination with positive measles IgG and lower viral loads in respiratory specimens. Infections in these 2 HCWs warranted contact tracing of another 168 persons (97 patients and 71 HCWs). Phylogenetic comparison of H and N gene sequences confirmed the clonality of outbreak strains. CONCLUSION: Despite good herd immunity with overall seroprevalence of >95% against measles, major outbreaks of measles occurred among HKIA staff having daily contact with many international pssengers. Lessons from severe acute respiratory syndrome (SARS) and measles outbreaks suggested that an airport can be a strategic epidemic center. Pre-exanthem transmission of measles from airport staff to HCWs with secondary vaccine failure poses a grave challenge to hospital infection control.

Alkanindiges hongkongensis sp. nov. A novel Alkanindiges species isolated from a patient with parotid abscess.

A bacterium was isolated from the abscess pus of a 72-year-old patient with Warthin's tumor and parotid abscess. The cells were aerobic, non-motile, Gram-negative but difficult to be destained, non-sporulating, coccobacillus. The bacterium grew poorly on sheep blood agar and MacConkey agar as non-hemolytic colonies of 0.5 mm in diameter after 24h of incubation at 37 degrees C in ambient air. Growth was enhanced by Tween 80. It produces catalase but not cytochrome oxidase. Sequencing of the cloned 16S rRNA PCR products of the bacterium revealed three different 16S rRNA gene sequences, with 12 - 31 bp differences among them. Phylogenetic analysis showed that the bacterium is closely related to Alkanindiges illinoisensis, with 5.0 - 5.9% differences between the 16S rRNA gene sequence of the bacterium and that of A. illinoisensis. Tryptophan auxotrophic strain of Acinetobacter trpE27 transformed with DNA extracted from the bacterium was unable to grow on tryptophan deficient medium, indicating that the bacterium was not a strain of Acinetobacter. The G+C content of the bacterium (mean +/-SD) was 46.9+4.3%. A new species, Alkanindiges hongkongensis sp. nov., is proposed, for which HKU9T is the type strain. Isolates with "small colonies" that are apparently Acinetobacter-like species should be carefully identified. Growth enhancement with aliphatic hydrocarbons should be looked for and 16S rRNA gene sequencing performed in order to find more potential cases of Alkanindiges infections, as well as to define the epidemiology, clinical spectrum, and outcome of infections associated with this genus.

Single gene target bacterial identification. groEL gene sequencing for discriminating clinical isolates of Burkholderia pseudomallei and Burkholderia thailandensis.

Proper identification of Burkholderia pseudomallei and Burkholderia thailandensis is crucial in guiding clinical management of patients with suspected melioidosis, as more than 99% of cases of melioidosis are caused by B. pseudomallei, whereas B. thailandensis is only responsible for causing less than 1% of the cases. However, the difference between the 16S ribosomal RNA gene sequences of B. pseudomallei and that of B. thailandensis is only 1%, and is therefore not discriminative enough for distinguishing the 2 species confidently. In this study, we amplified and sequenced the groEL genes of 7 strains of B. thailandensis and 6 strains of B. pseudomallei, and compared the sequences with 7 other groEL gene sequences of Burkholderia species. BLAST analysis revealed that the putative protein encoded by the groEL gene of B. thailandensis has 99.6%, 99.5%, 98.4%, 98.5%, and 96.5% amino acid identity with the groEL of B. pseudomallei, B. mallei, B. cepacia, B. vietnamiensis, and B. fungorum respectively. The amino acid sequences of GroEL of the strains of B. thailandensis and B. pseudomallei all showed >99.5% amino acid identity with each other. The nucleotide sequence of the groEL gene of any of the strains of B. thailandensis showed >99.8% nucleotide identity with that of any of the other strains of B. thailandensis, and the nucleotide sequence of the groEL gene of any of the strains of B. pseudomallei showed >99.5% nucleotide identity with that of any of the other strains of B. pseudomallei. However, the nucleotide sequence of any of the strains of B. thailandensis showed <97.6% nucleotide identity with any of the strains of B. pseudomallei. The amino acid sequences of GroEL of the 20 strains of Burkholderia species all showed >96% amino acid identity with each other. Furthermore, the nucleotide sequence of the groEL genes of the 2 strains of B. cepacia showed >99.5% nucleotide identity with each other, and the nucleotide sequence of the groEL gene of B. mallei showed >99.5% nucleotide identity with any of the strains of B. pseudomallei. The groEL gene sequence is therefore good for distinguishing between B. thailandensis and B. pseudomallei, and the GroEL amino acid and groEL nucleotide sequences of this single gene locus may potentially be useful for a 2-tier hierarchical identification of medically important Burkholderia at the genus and species levels respectively.

Eggerthella hongkongensis sp. nov. and eggerthella sinensis sp. nov., two novel Eggerthella species, account for half of the cases of Eggerthella bacteremia.

Eggerthella, one of the human gut flora, was rarely reported to cause bacteremia in the literature. We describe the application of 16S ribosomal RNA gene sequencing in defining the epidemiology and clinical significance of Eggerthella bacteremia during a 4-year period. Among 55 clinically significant blood culture isolates of anaerobic Gram-positive bacilli, 5 were identified as E. lenta and 5 belonged to 2 novel Eggerthella species, proposed as E. hongkongensis and E. sinensis, respectively. The 10 patients with Eggerthella bacteremia were adults, and 9 had underlying diseases. In all cases, the source of the bacteremia was likely from endogenous flora. Septic shock was a complication in 4 patients, and 3 patients died. The present study suggests that Eggerthella bacteremia is much more common than expected and is associated with significant morbidity and mortality. Moreover, the 2 novel species account for half of the cases of Eggerthella bacteremia.

Improving molecular tools for global surveillance of measles virus.

BACKGROUND: The genetic characterization of wild-type measles viruses plays an important role in the description of viral transmission pathways and the verification of measles elimination. The 450 nucleotides that encode the carboxyl-terminus of the nucleoprotein (N-450) are routinely sequenced for genotype analysis. OBJECTIVES: The objectives of this study were to develop improved primers and controls for RT-PCR reactions used for genotyping of measles samples and to develop a method to provide a convenient, safe, and inexpensive means to distribute measles RNA for RT-PCR assays and practice panels. STUDY DESIGN: A newly designed, genetically defined synthetic RNA and RNA isolated from cells infected with currently circulating genotypes were used to compare the sensitivity of primer pairs in RT-PCR and nested PCR. FTA® cards loaded with lysates of measles infected cells were tested for their ability to preserve viral RNA and destroy virus infectivity. RESULTS: A new primer pair, MeV216/MeV214, was able to amplify N-450 from viruses representing 10 currently circulating genotypes and a genotype A vaccine strain and demonstrated 100-fold increased sensitivity compared to the previously used primer set. A nested PCR assay further increased the sensitivity of detection from patient samples. A synthetic positive control RNA was developed that produced PCR products that are distinguishable by size from PCR products amplified from clinical samples. FTA® cards completely inactivated measles virus and stabilized RNA for at least six months. CONCLUSIONS: These improved molecular tools will advance molecular characterization of circulating measles viruses globally and provide enhanced quality control measures.

Catabacter hongkongensis gen. nov., sp. nov., isolated from blood cultures of patients from Hong Kong and Canada.

Four bacterial isolates were recovered from the blood cultures of four patients, two of whom were from Hong Kong and two of whom were from Canada. The two Hong Kong strains were isolated from a 48-year-old man with intestinal obstruction and secondary sepsis (strain HKU16T) and from a 39-year-old man with acute appendicitis (strain HKU17), while the two Canadian strains were isolated from a 74-year-old man with biliary sepsis (strain CA1) and from a 66-year-old woman with metastatic carcinoma and sepsis (strain CA2). While the first three patients survived, the last patient died 2 weeks after the episode of bacteremia. All four isolates are strictly anaerobic, nonsporulating, gram-positive coccobacilli that were unidentified by conventional phenotypic tests and commercial identification systems. They grow on sheep blood agar as nonhemolytic pinpoint colonies after 48 h of incubation at 37 degrees C in an anaerobic environment. All are catalase positive and motile, with flagella. They produce acid from arabinose, glucose, mannose, and xylose. They do not produce indole or reduce nitrate. They are sensitive to penicillin, vancomycin, and metronidazole but resistant to cefotaxime. 16S rRNA gene sequence analysis showed 16.0%, 16.8%, and 21.0% base differences from Clostridium propionicum, Clostridium neopropionicum, and Atopobium minutum, respectively. The G+C content of strain HKU16T is 40.2% +/- 2.2%. Based on their phylogenetic affiliation, unique G+C content, and phenotypic characteristics, we propose a new genus and species, Catabacter hongkongensis gen. nov., sp. nov., to describe the bacterium, for which HKU16 is the type strain, and suggest that it be assigned to a new family, Catabacteriaceae. The gastrointestinal tract was probably the source of the bacterium for at least three of the four patients. The isolation of a catalase-positive, motile, nonsporulating, anaerobic gram-positive bacillus in clinical laboratories should raise the possibility of C. hongkongensis. Further studies should be performed to ascertain the epidemiology and other disease associations of this bacterium.

Typhoid fever associated with acute appendicitis caused by an H1-j strain of Salmonella enterica serotype Typhi.

While most strains of Salmonella enterica serotype Typhi, the etiologic agent of typhoid fever, have only a phase 1 flagellar antigen, H1-d, variations of the flagellar antigen have been observed. Although H1-j strains (one of the flagellar antigen variants) account for 10 to 50% of S. enterica serotype Typhi strains found in Indonesia, there have been no published data to suggest its existence in other parts of the world. We describe a case of typhoid fever associated with acute appendicitis caused by an S. enterica serotype Typhi H1-j strain in a Chinese woman in Hong Kong. A gram-negative, motile rod was recovered from her blood and stool cultures. Conventional biochemical tests and the Vitek system (GNI+) showed that the bacterium was S. enterica serotype Typhi. The isolate agglutinated with poly(O), 9O, Vi and H1-j Salmonella antisera but not with poly(H) antisera. The patient developed antibodies against only S. enterica serotype Typhi O antigens but not against H1-d antigen by the Widal test. Flagellin C gene (fliC) sequencing showed a 261-bp deletion in the fliC gene of the isolate, confirming that the isolate possessed the H1-j antigen. The patient had no past history of travel to Indonesia or personal contact with any Indonesian. She recovered with appendectomy and antibiotic treatment. Further studies should be performed to determine the prevalence of this unusual S. enterica serotype Typhi strain in our locality.

Anaerospora hongkongensis gen. nov. sp. nov., a novel genus and species with ribosomal DNA operon heterogeneity isolated from an intravenous drug abuser with pseudobacteremia.

A bacterium was isolated from the blood culture of an intravenous drug abuser with pseudobacteremia. The cells were strictly anaerobic, straight or slightly curved, sporulating, Gram-negative rods. It grew on sheep blood agar as non-hemolytic, pinpoint colonies after 48 hr of incubation at 37 C in an anaerobic environment. It was motile but did not produce catalase or cytochrome oxidase. 16S ribosomal DNA (rDNA) sequencing revealed three different copies of 16S rDNA sequences. More than 90% of the differences among them were due to differences in the lengths of the sequences. Phylogenetically, the bacterium is clustered with Dendrosporobacter, Sporomusa, and Propionispora, the other three genera of anaerobic, sporulating, Gram-negative rods. There were 8.6-11.1% differences between the 16S rDNA sequences of the bacterium and that of D. quercicolus, 4.7-15.1% differences between the 16S rDNA sequences of it and those of S. acidovorans, S. aerivorans, S. malonica, S. ovata, S. paucivorans, S. silvacetica, S. spaeroides, and S. termitida, and 7.6-13.1% differences between the 16S rDNA sequences of it and those of P. hippei and P. vibrioides. The G+C content of the bacterium (mean +/- SD) was 46.8 +/- 3.2%. For these reasons, a new genus and species, Anaerospora hongkongensis gen. nov. sp. nov., is proposed, for which HKU15T is the type strain.

SARS coronavirus detection methods.

Using clinical samples from patients with severe acute respiratory syndrome, we showed that the sensitivities of a quantitative reverse transcription-polymerase chain reaction (80% for fecal samples and 25% for urine samples) were higher than those of the polyclonal (50% and 5%) and monoclonal (35% and 8%) antibody-based nucleocapsid antigen capture enzyme-linked immunosorbent assays.

Catheter-related Microbacterium bacteremia identified by 16S rRNA gene sequencing.

We describe the application of 16S rRNA gene sequencing in defining two cases of catheter-related Microbacterium bacteremia. In the first case, a gram-positive bacillus was isolated from both the blood culture and central catheter tip of a 39-year-old woman with chronic myeloid leukemia. The API Coryne system identified the isolate as 98.9% Aureobacterium or Corynebacterium aquaticum. In the second case, a gram-positive bacillus was recovered from five sets of blood cultures from both central catheter and percutaneous venipuncture of a 5-year-old girl with acute myeloid leukemia. The isolate was identified by the API Coryne system as 99.7% Cellulomonas or Microbacterium species. Further phenotypic tests failed to identify the two isolates. 16S rRNA gene sequencing showed 99.4% similarity between the first isolate and Microbacterium oxydans and 98.7% similarity between the second isolate and Microbacterium trichotecenolyticum, indicating that both isolates were Microbacterium species. Microbacterium infections are rarely reported in the literature. Although the central venous catheter was previously proposed to be a source of bacteremia, the first case in this report represents the first culture-documented case of catheter-related Microbacterium bacteremia.

Bacteremia due to Clostridium hathewayi in a patient with acute appendicitis.

Clostridium hathewayi is a newly described Clostridium species isolated from the feces of healthy human individuals, but its clinical significance is not known. We describe a case of human infection associated with C. hathewayi. The bacterium (strain HKU18) was isolated from the blood culture of a 39-year-old patient with acute gangrenous appendicitis complicated by septic shock. The cells were strictly anaerobic, nonmotile rods that stained gram negative. Conventional phenotypic tests and commercial identification systems failed to identify HKU18 to the species level. 16S rRNA gene analysis showed 1.4% nucleotide difference between the sequence of HKU18 and that of C. hathewayi, indicating that HKU18 was a strain of C. hathewayi. The patient responded to appendectomy and antibiotic treatment. 16S rRNA gene sequencing would be useful in further characterizing the clinical disease spectrum of C. hathewayi.

Detection of severe acute respiratory syndrome (SARS) coronavirus nucleocapsid protein in sars patients by enzyme-linked immunosorbent assay.

We report the development of an enzyme-linked immunosorbent assay (ELISA) for the detection of severe acute respiratory syndrome (SARS) coronavirus (CoV) nucleocapsid protein. The assay was carried out with hyperimmune polyclonal nucleocapsid-specific antibodies from guinea pigs and rabbits immunized with recombinant His(6)-tagged SARS CoV nucleocapsid protein. The assay was used for the detection of SARS CoV nucleocapsid protein in nasopharyngeal aspirate, urine, and fecal samples collected from patients with confirmed SARS between days 2 and 33 after the onset of illness. The ELISA was capable of detecting this protein in SARS CoV cell culture lysates at 15 50% tissue culture infective doses/ml but did not produce positive signals when tested with cell culture lysates of human coronaviruses OC43 and 229E. When tested with 120 nasopharyngeal aspirate, 100 urine, and 100 fecal specimens from hospitalized patients without SARS, the assay was shown to have high specificities-96.7, 99, and 96%, respectively. In an evaluation of clinical specimens from SARS patients, 34 (52%) of 66 nasopharyngeal aspirate samples from 50 patients, 5 (5%) of 94 urine samples from 94 patients, and 36 (55%) of 65 fecal samples from 65 patients tested positive for SARS CoV nucleocapsid protein. Nucleocapsid protein could be detected from days 6 to 24 in nasopharyngeal aspirate specimens, from days 11 to 31 in urine specimens, and from days 8 to 32 in fecal specimens after the onset of illness. Moreover, the protein could be detected in 25 (83%) of 30 nasopharyngeal aspirate specimens obtained from days 11 to 15 and in all 7 fecal specimens obtained from days 21 to 32. Since the present ELISA is more convenient and economical than reverse transcription-PCR, it may serve as an alternative tool for the early diagnosis of SARS CoV infection in laboratories with limited resources and expertise and for mass screening for the reservoir of SARS CoV. Further studies on serial clinical specimens should reveal the duration of nucleocapsid protein shedding and may reveal a higher detection rate in SARS patients.

Seronegative bacteremic melioidosis caused by Burkholderia pseudomallei with ambiguous biochemical profile: clinical importance of accurate identification by 16S rRNA gene and groEL gene sequencing.

An aerobic gram-negative bacterium was isolated from the blood and sputum of an 84-year-old, chair-bound nursing home resident with acute bacteremic pneumonia. Although the phenotypic characteristics suggested that the bacterium could be Burkholderia pseudomallei, the Vitek 1 system (GNI+), which can successfully identify 99% of B. pseudomallei strains, showed that the bacterium was "unidentified." Immunoglobulin G against the lipopolysaccharide (LPS) of B. pseudomallei, as detected by an LPS-based enzyme-linked immunosorbent assay with 95% sensitivity, was negative in both the acute-phase and convalescent-phase sera. Sequencing of the groEL gene showed that the isolate was B. pseudomallei. Proper identification of the bacterium in this study is crucial, since there would be a radical difference in the duration of antimicrobial therapy.