Genomics and zoonotic infections: Middle East respiratory syndrome.

The emergence of Middle East respiratory syndrome (MERS) and the discovery of MERS coronavirus (MERS-CoV) in 2012 suggests that another SARS-like epidemic is occurring. Unlike the severe acute respiratory syndrome (SARS) epidemic, which rapidly disappeared in less than one year, MERS has persisted for over three years. More than 1,600 cases of MERS have been reported worldwide, and the disease carries a worryingly high fatality rate of >30%. A total of 182 MERS-CoV genomes have been sequenced, including 94 from humans and 88 from dromedary camels. The 182 genomes all share >99% identity, indicating minimal variation among MERS-CoV genomes. MERS-CoV is a lineage C Betacoronavirus (ßCoV). MERS-CoV genomes can be roughly divided into two clades: clade A, which contains only a few strains, and clade B, to which most strains belong. In contrast to ORF1ab and structural proteins, the putative proteins encoded by ORF3, ORF4a, ORF4b, ORF5 and ORF8b in the MERS-CoV genome do not share homology with any known host or virus protein, other than those of its closely related lineage C ßCoVs. Human and dromedary viral genomes have intermingled, indicating that multiple camel-to-human transmission events have occurred. The multiple origins of MERS-CoV suggest that the virus has been resident in dromedaries for many years. This is consistent with the detection of anti-MERS-CoV antibodies in dromedary camels as early as the 1980s.

L'émergence du syndrome respiratoire du Moyen-Orient (SRMO, ou MERS d'après son sigle anglais) et l'identification en 2012 du coronavirus responsable de cette maladie (MERS-CoV) indiquent que nous sommes en présence d'une épidémie semblable à celle du syndrome respiratoire aigu sévère (SRAS). Toutefois, contrairement à l'épidémie du SRAS qui avait rapidement disparu en moins d'un an, le MERS persiste depuis plus de trois ans. Plus de 1 600 cas de MERS ont été notifiés dans le monde ; la maladie présente un taux de létalité particulièrement préoccupant, s'élevant à plus de 30 %. Au total, 182 génomes du MERS-CoV ont été séquencés jusqu'à présent, dont 94 provenaient de virus isolés chez l'homme et 88 chez des dromadaires. Ces 182 génomes ont en commun un pourcentage d'identité de 99 %, dénotant une très faible variabilité des génomes viraux. Le MERS-CoV appartient à la lignée C du genre Betacoronavirus (ßCoV). Les génomes du MERSCoV se répartissent, dans leurs grandes lignes, en deux clades : le clade A, qui ne contient que quelques souches, et le clade B regroupant l'immense majorité des souches. Contrairement à ce qui se produit avec la protéine ORF1ab et les protéines structurales, les protéines potentiellement codées par les gènes ORF3, ORF4a, ORF4b, ORF5 et ORF8b du génome du MERS-CoV ne présentent aucune homologie avec des protéines virales ou de l'hôte autres que celles d'autres bêtacoronavirus de la lignée C, qui lui sont étroitement apparentés. Les génomes des virus affectant l'homme et le dromadaire se sont entremêlés, ce qui montre que le virus a connu de multiples épisodes de transmission des camélidés à l'homme. Les origines multiples du MERS-CoV témoignent d'une présence prolongée du virus (plusieurs années) chez les dromadaires. Ce constat est corroboré par le fait que des anticorps anti-MERS-CoV ont été détectés chez des dromadaires dès le début des années 80.

La aparición del síndrome respiratorio de Oriente Medio (MERS, por sus siglas en inglés) y el descubrimiento del coronavirus que lo causa (MERS-CoV) en 2012 parecen apuntar al advenimiento de una nueva epidemia análoga a la del síndrome respiratorio agudo severo (SRAS). Pero a diferencia de lo ocurrido con la epidemia de SRAS, que en menos de un año había desaparecido, el MERS lleva más de tres años presente. En el mundo se han notificado más de 1.600 casos de MERS, y la enfermedad presenta una tasa de letalidad muy alta y preocupante, superior al 30%. Hasta ahora se han secuenciado un total de 182 genomas del MERS-CoV, 94 de ellos obtenidos a partir de personas y 88 a partir de dromedarios. Estos 182 genomas comparten identidad en más de un 99%, lo que pone de manifiesto un nivel mínimo de variación entre los genomas coronavíricos. El coronavirus del MERS pertenece al linaje C del género Betacoronavirus (ßCoV). Los genomas de este virus pueden ser divididos, a grandes rasgos, en dos clados: el clado A, que agrupa unas pocas cepas; y el clado B, al que pertenecen la gran mayoría de las cepas. A diferencia de lo que ocurre con la proteína ORF1ab y las proteínas estructurales, las proteínas que supuestamente codifican los genes ORF3, ORF4a, ORF4b, ORF5 y ORF8b del genoma del MERS-CoV no comparten homología con ninguna proteína conocida de otros virus o anfitriones, salvo con proteínas de otros betacoronavirus del linaje C estrechamente emparentados con él. Los genomas de los virus que afectan a personas y dromedarios se han entremezclado, lo que indica que se han producido numerosos episodios de transmisión de camélidos a humanos. De los múltiples orígenes del MERS-CoV se deduce que el virus lleva muchos años siendo residente en dromedarios, lo que concuerda con el hecho de que ya en los años ochenta se detectaran anticuerpos anti-MERS-CoV en dromedarios.

Development of monoclonal antibody-based galactomannoprotein antigen-capture ELISAs to detect Aspergillus fumigatus infection in the invasive aspergillosis rabbit models.

Aspergillus fumigatus is one of the most prominent opportunistic fungal pathogens in immunocompromised hosts. Early recognition of this infection along with prompt antifungal therapy may increase the survival rate. We expressed two potential bio-markers of A. fumigatus infection-galactomannoprotein Afmp1p and Afmp4p in Pichia pastoris. We generated 33 monoclonal antibodies (MAbs), 20 against recombinant Afmp1p (rAfmp1p) and the other 13 against recombinant Afmp4p (rAfmp4p). Subsequently, we developed two antigen-capture enzyme-linked immunosorbent assays (ELISAs) which employed MAbs as both the capture and the detection antibodies for rAfmp1p and rAfmp4p. The two antigen-capture ELISAs specifically detected Afmp1p/Afmp4p in cultures of A. fumigatus and had no cross-reaction with other tested pathogenic fungi, including Penicillium marneffei and other pathogenic Aspergillus species. The Afmp1p-captured ELISA would be positive even when the culture supernatant of A. fumigatus had been diluted to 128-fold of its original concentration. The two antigen ELISAs could capture circulating or excreted antigens during the acute phase of invasive aspergillosis (IA) in the animal model, and had no cross-reactivity to other Aspergillus-challenged animal models. We developed two antigen-capture ELISAs for the laboratory diagnosis of A. fumigatus infection. These two antigen-capture ELISAs may be useful in the clinical diagnosis of aspergillosis.

First report of chronic implant-related septic arthritis and osteomyelitis due to Kytococcus schroeteri and a review of human K. schroeteri infections.

We report the first case of Kytococcus schroeteri implant-related septic arthritis and osteomyelitis, identified by phenotypic tests and 16S rRNA sequencing, which responded to implant removal and doxycycline. 16S rRNA sequencing was useful for the accurate and rapid identification of the organism as it exhibited three different colonial morphologies in vitro.

Epidemiology of coronavirus-associated respiratory tract infections and the role of rapid diagnostic tests: a prospective study.

1. Coronaviruses accounted for 1.6% (98/6272) of respiratory tract infections based on nasopharyngeal aspirate samples. 2. HCoV-OC43 was the most common coronavirus detected,followed by HCoV-NL63, CoVHKU1,and HCoV-229E. 3. Although CoV-HKU1 infections were most often associated with the upper respiratory tract, more severe illness (pneumonia,acute bronchiolitis, and asthmatic exacerbation) may occur, especially in those with underlying disease. In young children, CoV-HKU1 infection is associated with a high rate of febrile seizures (50%). 4. CoV-HKU1 and HCoV-OC43 infections peaked in winter, in contrast to HCoV-NL63, which mainly occurred in early summer and autumn, but was absent in winter. 5. Reverse transcriptase polymerase chain reaction is useful for the rapid diagnosis of coronavirus infections.

Wild animal surveillance for coronavirus HKU1 and potential variants of other coronaviruses.

1. Although CoV-HKU1 was not identified in any of the studied animals, a coronavirus closely related to SARS-CoV (bat-SARS-CoV) was identified in 23 (19%) of 118 wild Chinese horseshoe bats by reverse transcriptase polymerase chain reaction (RT-PCR). 2. Complete genome sequencing and phylogenetic analysis showed that bat-SARS-CoV formed a distinct cluster with SARS-CoV as group 2b coronaviruses, distantly related to known group 2 coronaviruses. 3. Most differences between the bat-SARS-CoV and SARS-CoV genomes were observed in the spike gene. The presence of a29-bp insertion in ORF 8 of bat-SARS-CoV genome, not in most human SARS-CoV genomes, suggests that it has a common ancestor with civet SARS-CoV. 4. Antibody against recombinant bat-SARS-CoV nucleocapsid protein was detected in 84% of Chinese horseshoe bats using an enzyme immunoassay.Neutralising antibody to human SARS-CoV was also detected in those with lower viral loads.5. This study also revealed a previously unknown diversity of coronaviruses in bats, which are important natural reservoir for coronaviruses including SARS-CoV-like viruses.

Primary infective spondylodiscitis caused by Lactococcus garvieae and a review of human L. garvieae infections.

We report the first case of primary infective spondylodiscitis due to Lactococcus garvieae, confirmed by 16S rRNA gene sequencing, in the absence of concomitant endocarditis in a patient with long-standing gastritis on famotidine. He responded to a 6-week course of ampicillin. The gastrointestinal tract is probably the source of infection.

Human enterovirus 71 and hand, foot and mouth disease.

Hand, foot and mouth disease (HFMD) is generally a benign febrile exanthematous childhood disease caused by human enteroviruses. The route of transmission is postulated to be faeco-oral in developing areas but attributed more to respiratory droplet in developed areas. Transmission is facilitated by the prolonged environmental survival of these viruses and their greater resistance to biocides. Serious outbreaks with neurological and cardiopulmonary complications caused by human enterovirus 71 (HEV-71) seem to be commoner in the Asian Pacific region than elsewhere in the world. This geographical predilection is unexplained but could be related to the frequency of intra- and inter-typic genetic recombinations of the virus, the host populations' genetic predisposition, environmental hygiene, and standard of healthcare. Vaccine development could be hampered by the general mildness of the illness and rapid genetic evolution of the virus. Antivirals are not readily available; the role of intravenous immunoglobulin in the treatment of serious complications should be investigated. Monitoring of this disease and its epidemiology in the densely populated Asia Pacific epicentre is important for the detection of emerging epidemics due to enteroviruses.

Environmental surveillance for Laribacter hongkongensis, a diarrhoeal pathogen discovered in Hong Kong.

1. Laribacter hongkongensis was isolated from the midguts and hindguts of 86 (24%) of 360 freshwater fish from retail markets, including grass carp (60%), bighead carp (53%), mud carp (25%), and large-mouth bass (5%). 2. This study is the first to demonstrate the presence of L hongkongensis in natural water environments, with the bacterium being isolated from the waters of six reservoirs, with higher recovery rates in summer and during days of higher water and ambient temperatures. 3. Molecular typing using pulsed field gel electrophoresis revealed a heterogeneous population of L hongkongensis in both the freshwater fish and drinking water reservoir isolates, suggesting that the bacterium is endemic in our freshwater environments. 4. Since freshwater fish are common food items for our population, the general public should be educated on the proper preparation and thorough cooking of freshwater fish before consumption to avoid L hongkongensis-associated gastroenteritis. 5. Although it is unlikely that treated drinking water is a significant source of L hongkongensis-associated gastroenteritis, it is important to be aware of the possibility of other contaminated water as a source of human infection.

An oral mucosal DNA vaccine for SARS coronavirus infections.

1. When different forms of SARS coronavirus (SARS-CoV) spike protein-based vaccines for generation of a neutralising antibody response to SARS-CoV were injected into a mouse model, all the mice immunised with intramuscular tPA-optimised 800 DNA vaccine boosted with intraperitoneal recombinant spike polypeptide generated by Escherichia coli and intramuscular CTLA4Hinge SARS800 DNA vaccine boosted with intraperitoneal S-peptide had neutralising antibody titres of>1:1280.2. This observation may have major practical value for field studies, such as the immunisation of civet cats, as the cost of recombinant proteins produced by E coli is much lower than those produced by eukaryotic systems.3. This study indicates that the type of vaccine used for priming is crucial for determining the type of immune response developed.Subsequent doses will boost the immune response generated by the first dose of vaccine.

Clinical features and molecular epidemiology of coronavirus-HKU1-associated community-acquired pneumonia.

1. Coronavirus (CoV)-HKU1 accounts for 2.4% of community-acquired pneumonia. 2. Clinical features alone cannot differentiate this entity from other community-acquired pneumonia. 3. Further studies are needed to understand the significance of CoV-HKU1 in upper respiratory tract infection and its potential to cause outbreaks of acute viral respiratory illnesses.

Viral loads in clinical specimens and SARS manifestations.

1. A high viral load in nasopharyngeal aspirate (with or without a high viral load in serum) is a useful prognostic indicator of respiratory failure or mortality. The presence of viral RNA in multiple body sites is also indicative of poor prognosis. 2. Early treatment with an effective antiviral agent before day 10 may decrease the peak viral load, and thus ameliorate the clinical symptoms and mortality, and reduce viral shedding and the risk of transmission

Outbreak of human metapneumovirus infection in psychiatric inpatients: implications for directly observed use of alcohol hand rub in prevention of nosocomial outbreaks.

Nosocomial outbreaks of infectious diseases in psychiatric facilities are not uncommon but the implementation of infection control measures is often difficult. Here, we report an outbreak of an acute respiratory illness in a psychiatric ward between 29 July and 20 August 2005 involving 31 patients. Human metapneumovirus was detected in seven (23%) patients by reverse transcription-polymerase chain reaction and nucleotide sequencing. A review of outbreak surveillance records showed that six nosocomial outbreaks occurred in the year 2005, of which four (67%) were confirmed or presumably related to a respiratory viral infection. Directly observed deliveries of alcohol hand rub 4-hourly during daytime to all psychiatric patients was instituted in December 2005. Only one nosocomial respiratory viral outbreak occurred in the following year. The total number of patients and staff involved in nosocomial outbreaks due to presumed or proven respiratory virus infections decreased significantly from 60 to six (P<0.001), whereas those due to all types of nosocomial outbreaks also decreased from 70 to 24 (P=0.004). Alcohol hand rub has been shown to have potent bactericidal and virucidal activity against a wide range of nosocomial pathogens. Regular use of directly observed alcohol hand rub may decrease the incidence and scale of nosocomial outbreaks due to enveloped respiratory viruses especially in mentally incapacitated patients.

Clinical predictors and outcomes of Klebsiella pneumoniae bacteraemia in a regional hospital in Hong Kong.

BACKGROUND: Klebsiella pneumoniae (KP) infection is associated with high morbidity and mortality. Multidrug resistance, especially extended-spectrum ß-lactamase (ESBL) production, in KP is endemic worldwide. AIM: To evaluate the clinical characteristics and outcomes of patients with KP bacteraemia in critical care and general ward settings. METHODS: Adult patients admitted to a regional hospital in Hong Kong from January 1st, 2009 to June 30th, 2016 (7.5 years) with KP bacteraemia were included. Demographics, clinical features, microbiological characteristics, and outcomes were analysed. FINDINGS: Among 853 patients, 178 (20.9%) required critical care and 176 (20.6%) died within 30 days of hospital admission. Thirty-day survivors were younger (P<0.001), had milder disease (defined by Sequential Organ Failure Assessment score) (P<0.001), presented with hepatobiliary sepsis (P<0.001) or urosepsis (P<0.001), less septic shock (P=0.013), fewer invasive organ supports (P<0.001), and had appropriate empirical antibiotics (P<0.001). Cox regression analysis showed that respiratory tract infection (hazard ratio: 2.99; 95% confidence interval: 2.061-4.337; P≤0.001), gastrointestinal tract infection (excluding hepatobiliary system) (2.763; 1.761-4.337; P≤0.001), mechanical ventilation (2.202; 1.506-3.221; P≤0.001), medical case (1.830; 1.253-2.672; P=0.002), inappropriate empirical antibiotics (1.716; 1.267-2.324; P≤0.001), female (1.699; 1.251-2.307; P<0.001), age >65 years (1.692; 1.160-2.467; P=0.006), and presence of solid tumour (1.457; 1.056-2.009; P=0.022) were independent risk factors for 30-day mortality. Unexpectedly, diabetes mellitus was associated with better 30-day survival (P=0.002). A total of 102 patients (12.0%) had infections with ESBL-producing strains, which were not associated with higher 30-day mortality. CONCLUSION: KP bacteraemia is associated with high 30-day mortality. Site of infection, patients' comorbidities and appropriate use of empirical antibiotic are important predictors of patients' outcomes.

Usefulness of the MicroSeq 500 16S rDNA bacterial identification system for identification of anaerobic Gram positive bacilli isolated from blood cultures.

Using full 16S ribosomal RNA (rRNA) gene sequencing as the gold standard, 20 non-duplicating anaerobic Gram positive bacilli isolated from blood cultures were analysed by the MicroSeq 500 16S rDNA bacterial identification system. The MicroSeq system successfully identified 13 of the 20 isolates. Four and three isolates were misidentified at the genus and species level, respectively. Although the MicroSeq 500 16S rDNA bacterial identification system is better than three commercially available identification systems also evaluated, its database needs to be expanded for accurate identification of anaerobic Gram positive bacilli.

Globicatella bacteraemia identified by 16S ribosomal RNA gene sequencing.

BACKGROUND: Globicatella are streptococcus-like organisms that have been rarely isolated from clinical specimens. Their epidemiology and clinical significance remain largely unknown. AIMS: To describe two cases of Globicatella bacteraemia identified by 16S ribosomal RNA (rRNA) gene sequencing. METHODS: Two unidentified streptococcus-like bacteria isolated from blood cultures of patients were subject to 16S rRNA gene sequencing. RESULTS: Two cases of Globicatella bacteraemia were identified by 16S rRNA gene sequencing. In the first case, a gram positive coccus was isolated from the blood culture of an 80 year old woman with diabetes mellitus and nosocomial sepsis, who died the day after developing the bacteraemia. The bacterium was unidentified by conventional phenotypic tests, the Vitek (gram positive identification) and the ATB expression (ID32 Strep) systems. In the second case, a similar bacterium was isolated from the blood culture of a 92 year old woman with polymicrobial acute pyelonephritis complicated by septic shock, who subsequently recovered after antibiotic treatment. 16S rRNA gene sequencing of the two isolates showed 0.5% nucleotide difference from that of G. sulfidifaciens and 0.7% nucleotide difference from that of G. sanguinis, indicating that they were Globicatella species. CONCLUSIONS: Because Globicatella is rarely encountered in clinical microbiology laboratories, it may have been overlooked or misidentified in these cases. 16S rRNA gene sequencing is a useful tool to better characterise the epidemiology and clinical significance of Globicatella.

Bacteraemia caused by Anaerotruncus colihominis and emended description of the species.

BACKGROUND: Anaerotruncus colihomonis is a newly described bacterial genus and species isolated from the stool specimens of children. Its clinical significance, however, is unknown. AIMS: To describe a case of A colihominis bacteraemia identified by 16S ribosomal RNA (rRNA) gene sequencing and provide an emended description of the species. METHODS: An unidentified anaerobic bacillus (strain HKU19) that stains Gram negative was subjected to characterisation by 16S rRNA gene sequencing, G+C content determination and electron microscopy. RESULTS: Strain HKU19 was isolated from the blood culture of a 78-year-old woman with nosocomial bacteraemia. It was found to be an anaerobic, non-motile, pleomorphic, thin bacillus that stains Gram negative. It produces Indole and utilises glucose and mannose. Identifying the strain to the species level was not possible by conventional phenotypic tests and commercial identification systems. The G+C content of strain HKU19 was found to be 53.43 mol%. A similarity of 99.3% nucleotide identities was found between the 16S rRNA gene sequence of strain HKU19 and that of A colihominis WAL 14 565(T), which was isolated from a human faecal specimen. In contrast with the original description of A colihominis, HKU19 was found to produce occasional oval, terminal spores, although the other phenotypic characteristics matched. Spores were also occasionally observed when the two previously reported strains were re-examined. CONCLUSIONS: Although the source of the bacteraemia in the patient cannot be determined, this report suggests that A colihominis is of clinical significance. Spore formation is proposed as an emended description of A colihominis.

First genome sequences of buffalo coronavirus from water buffaloes in Bangladesh.

We report the complete genome sequences of a buffalo coronavirus (BufCoV HKU26) detected from the faecal samples of two domestic water buffaloes (Bubalus bubalis) in Bangladesh. They possessed 98-99% nucleotide identities to bovine coronavirus (BCoV) genomes, supporting BufCoV HKU26 as a member of Betacoronavirus 1. Nevertheless, BufCoV HKU26 possessed distinct accessory proteins between spike and envelope compared to BCoV. Sugar-binding residues in the N-terminal domain of S protein in BCoV are conserved in BufCoV HKU26.