Isolation and full-genome sequences of Japanese encephalitis virus genotype I strains from Cambodian human patients, mosquitoes and pigs.

Japanese encephalitis remains the most important cause of viral encephalitis in humans in several southeast Asian countries, including Cambodia, causing at least 65 000 cases of encephalitis per year. This vector-borne viral zoonosis - caused by Japanese encephalitis virus (JEV) - is considered to be a rural disease and is transmitted by mosquitoes, with birds and pigs being the natural reservoirs, while humans are accidental hosts. In this study we report the first two JEV isolations in Cambodia from human encephalitis cases from two studies on the aetiology of central nervous system disease, conducted at the two major paediatric hospitals in the country. We also report JEV isolation from Culextritaeniorhynchus mosquitoes and from pig samples collected in two farms, located in peri-urban and rural areas. Out of 11 reverse-transcription polymerase chain reaction-positive original samples, we generated full-genome sequences from 5 JEV isolates. Five additional partial sequences of the JEV NS3 gene from viruses detected in five pigs and one complete coding sequence of the envelope gene of a strain identified in a pig were generated. Phylogenetic analyses revealed that JEV detected in Cambodia belonged to genotype I and clustered in two clades: genotype I-a, mainly comprising strains from Thailand, and genotype I-b, comprising strains from Vietnam that dispersed northwards to China. Finally, in this study, we provide proof that the sequenced JEV strains circulate between pigs, Culex tritaeniorhynchus and humans in the Phnom Penh vicinity.

Aetiology of acute meningoencephalitis in Cambodian children, 2010-2013.

Acute meningoencephalitis (AME) is associated with considerable morbidity and mortality in children in developing countries. Clinical specimens were collected from children presenting with AME at two Cambodian paediatric hospitals to determine the major aetiologies associated with AME in the country. Cerebrospinal fluid (CSF) and blood samples were screened by molecular and cell culture methods for a range of pathogens previously associated with AME in the region. CSF and serum (acute and convalescent) were screened for antibodies to arboviruses such as Japanese encephalitis virus (JEV), dengue virus (DENV), and chikungunya virus (CHIKV). From July 2010 through December 2013, 1160 children (one month to 15 years of age) presenting with AME to two major paediatric hospitals were enroled into the study. Pathogens associated with AME were identified using molecular diagnostics, cell culture and serology. According to a diagnostic algorithm, a confirmed or highly probable aetiologic agent was detected in 35.0% (n=406) of AME cases, with a further 9.2% (total: 44.2%, n=513) aetiologies defined as suspected. JEV (24.4%, n=283) was the most commonly identified pathogen followed by Orientia tsutsugamushi (4.7%, n=55), DENV (4.6%, n=53), enteroviruses (3.5%, n=41), CHIKV (2.0%, n=23) and Streptococcus pneumoniae (1.6%, n=19). The majority of aetiologies identified for paediatric AME in Cambodia were vaccine preventable and/or treatable with appropriate antimicrobials.

A prospective, comparative study of severe neurological and uncomplicated hand, foot and mouth forms of paediatric enterovirus 71 infections.

OBJECTIVES: In this study, we document the clinical characteristics and investigated risk factors for uncomplicated and severe forms of EV-A71 disease in Cambodian children. METHODS: From March to July 2014 inclusive, all patients with suspicion of EV-A71 infection presenting to Kantha Bopha Hospitals in Phnom Penh and Siem Reap and confirmed by the Virology Unit at the Institut Pasteur du Cambodge were prospectively enrolled in this study. Throat swabs, rectal swabs and serum samples were collected from all consecutive patients with suspected EV-A71 infection. In addition, CSF was also collected from patients with suspected EV-A71 associated encephalitis. A total of 122 patients (29 with uncomplicated disease and 93 with severe disease) with confirmed EV-A71 infection with all available demographic and clinical data for clinical classification and further analysis were included in the study. RESULTS: In this prospective EV-A71 study in Cambodia, we confirmed the previously reported association of male gender and absence of mouth or skin lesions with severe disease. We also highlighted the strong association of neutrophils in blood, but also in CSF in patients with pulmonary oedema. More importantly, we identified new putative nutrition-related risk factors for severe disease. CONCLUSIONS: EV-A71 is an important cause of encephalitis in the Asia-Pacific region. Further studies to determine the risk factors associated with severe EV-A71 disease are needed.

Seroprevalence and Transmission of Human Influenza A(H5N1) Virus before and after Virus Reassortment, Cambodia, 2006-2014.

Thirty-five human influenza A(H5N1) cases were reported in Cambodia during 2013-2014 after emergence of a clade 1.1.2 reassortant virus. We tested 881 villagers and found 2 cases of pauci- or asymptomatic infection. Seroprevalence after emergence of the reassortant strain (0.2%) was lower than the aggregate seroprevalence of 1.3% reported in earlier studies.

Molecular epidemiology of human enterovirus 71 at the origin of an epidemic of fatal hand, foot and mouth disease cases in Cambodia.

Human enterovirus 71 (EV-A71) causes hand, foot and mouth disease (HFMD). EV-A71 circulates in many countries and has caused large epidemics, especially in the Asia-Pacific region, since 1997. In April 2012, an undiagnosed fatal disease with neurological involvement and respiratory distress occurred in young children admitted to the Kantha Bopha Children's Hospital in Phnom Penh, Cambodia. Most died within a day of hospital admission, causing public panic and international concern. In this study, we describe the enterovirus (EV) genotypes that were isolated during the outbreak in 2012 and the following year. From June 2012 to November 2013, 312 specimens were collected from hospitalized and ambulatory patients and tested by generic EV and specific EV-A71 reverse transcription PCR. EV-A71 was detected in 208 clinical specimens while other EVs were found in 32 patients. The VP1 gene and/or the complete genome were generated. Our phylogenetic sequencing analysis demonstrated that 80 EV-A71 strains belonged to the C4a subgenotype and 3 EV-A71 strains belonged to the B5 genotype. Furthermore, some lineages of EV-A71 were found to have appeared in Cambodia following separate introductions from neighboring countries. Nineteen EV A (CV-A6 and CV-A16), 9 EV B (EV-B83, CV-B3, CV-B2, CV-A9, E-31, E-2 and EV-B80) and 4 EV C (EV-C116, EV-C96, CV-A20 and Vaccine-related PV-3) strains were also detected. We found no molecular markers of disease severity. We report here that EV-A71 genotype C4 was the main etiological agent of a large outbreak of HFMD and particularly of severe forms associated with central nervous system infections. The role played by other EVs in the epidemic could not be clearly established.

Identification of molecular markers associated with alteration of receptor-binding specificity in a novel genotype of highly pathogenic avian influenza A(H5N1) viruses detected in Cambodia in 2013.

Human infections with influenza A(H5N1) virus in Cambodia increased sharply during 2013. Molecular characterization of viruses detected in clinical specimens from human cases revealed the presence of mutations associated with the alteration of receptor-binding specificity (K189R, Q222L) and respiratory droplet transmission in ferrets (N220K with Q222L). Discovery of quasispecies at position 222 (Q/L), in addition to the absence of the mutations in poultry/environmental samples, suggested that the mutations occurred during human infection and did not transmit further.

The sensitivity of an interferon-γ release assay in microbiologically confirmed pediatric tuberculosis.

UNLABELLED: This study aimed at determining the sensitivity of a whole blood interferon-γ release assay (IGRA) among children with microbiologically confirmed tuberculosis in a high-burden country. Children with a diagnosis of tuberculosis based on clinical and radiographic assessment were tested with an IGRA in addition to microbiologic examination of appropriate specimens for acid-fast bacilli, mycobacterial rRNA, and observation for growth of Mycobacterium tuberculosis on appropriate culture media. Of the 405 children with a clinical diagnosis of tuberculosis, 91 (22.5 %) had microbiologically confirmed tuberculosis, of whom 81 were tested with an IGRA. A positive result was obtained in 43 (sensitivity 53.1 %, 95 % confidence interval 42.3 to 63.6 %), uninfluenced by age, sex, or disease manifestation. CONCLUSIONS: The sensitivity of a whole blood interferon-γ release assay in microbiologically confirmed pediatric tuberculosis was low. An IGRA cannot, thus, be used as rule-in test, but it might be useful to rule in tuberculosis among children in whom tuberculosis is notoriously difficult to confirm microbiologically.

Dynamic of H5N1 virus in Cambodia and emergence of a novel endemic sub-clade.

In Cambodia, the first detection of HPAI H5N1 virus in birds occurred in January 2004 and since then there have been 33 outbreaks in poultry while 21 human cases were reported. The origin and dynamics of these epizootics in Cambodia remain unclear. In this work we used a range of bioinformatics methods to analyze the Cambodian virus sequences together with those from neighboring countries. Six HA lineages belonging to clades 1 and 1.1 were identified since 2004. Lineage 1 shares an ancestor with viruses from Thailand and disappeared after 2005, to be replaced by lineage 2 originating from Vietnam and then by lineage 3. The highly adapted lineage 4 was seen only in Cambodia. Lineage 5 is circulating both in Vietnam and Cambodia since 2008 and was probably introduced in Cambodia through unregistered transboundary poultry trade. Lineage 6 is endemic to Cambodia since 2010 and could be classified as a new clade according to WHO/OIE/FAO criteria for H5N1 virus nomenclature. We propose to name it clade 1.1A. There is a direct filiation of lineages 2 to 6 with a temporal evolution and geographic differentiation for lineages 4 and 6. By the end of 2011, two lineages, i.e. lineages 5 and 6, with different transmission paths cocirculate in Cambodia. The presence of lineage 6 only in Cambodia suggests the existence of a transmission specific to this country whereas the presence of lineage 5 in both Cambodia and Vietnam indicates a distinct way of circulation of infected poultry.

Molecular epidemiology of Orientia tsutsugamushi in Cambodia and Central Vietnam reveals a broad region-wide genetic diversity.

Scrub typhus is an acute infectious disease caused by an obligate intracellular bacterium Orientia tsutsugamushi following the bite of infected trombiculid mites of the genus Leptotrombidium. This zoonotic disease is a major cause of febrile illness in the Asia-Pacific region, with a large spectrum of clinical manifestations from unapparent or mild disease to fatal disease. O. tsutsugamushi is characterized by a very high genomic plasticity and a large number of antigenic variants amongst strains. The 56-kDa type specific antigen (TSA) gene, encoding the major antigenic protein, was used as reference to investigate the genetic relationships between the strains and to genotype O. tsutsugamushi isolates. The open reading frame of the 56-kDa TSA gene of 41 sequences (28 Cambodian and 13 Vietnamese strains) from patient samples were sequenced and used for genotyping. The 28 Cambodian isolates clustered into 5 major groups, including Karp (43.5%), JG-v (25%), Kato/TA716 (21.5%), TA763 (3.5%) and Gilliam (3.5%). Karp (77%), TA763 (15.5%) and JG-v (7.5%) strains were identified amongst the 13 Vietnamese isolates. This is the first countrywide genotyping description in Cambodia and in Central Vietnam. These results demonstrate the considerable diversity of genotypes in co-circulation in both countries. The genotyping result might raise awareness amongst Cambodian and Vietnamese clinicians of the high genetic diversity of circulating O. tsutsugamushi strains and provides unique and beneficial data for serological and molecular diagnosis of scrub typhus infections as well as raw materials for future studies and vaccine development.

Reemergence of Chikungunya virus in Cambodia.

Chikungunya virus (CHIKV), probably Asian genotype, was first detected in Cambodia in 1961. Despite no evidence of acute or recent CHIKV infections since 2000, real-time reverse transcription PCR of serum collected in 2011 detected CHIKV, East Central South African genotype. Spatiotemporal patterns and phylogenetic clustering indicate that the virus probably originated in Thailand.

Non-invasive diagnosis of lung tuberculosis in children by single voxel ¹H-magnetic resonance spectroscopy.

UNLABELLED: Our previous study showed that (1)H-magnetic resonance spectroscopy ((1)H-MRS) can detect lipid peaks characteristic for Mycobacterium tuberculosis infection in cerebral lesions of young children; therefore, we aimed to extend and validate the application of (1)H-MRS for the diagnosis of active pulmonary tuberculosis lesions in three adolescent patients. Here, we document lipid peaks characteristic for M. tuberculosis infection by (1)H-MRS from lung tissue surrounding lung cavities of two patients whose sputum samples were positive for acid-fast bacilli by microscopy and positive for M. tuberculosis by genetic testing, indicating active tuberculosis. A similar lipid peak was found also in the pleural effusion of a third patient with concurrent lung cavity compatible with active tuberculosis. However, in a patient with a pyogenic pulmonary abscess, (1)H-MRS of the drained pus displayed different characteristic peaks but no lipid peak at all. CONCLUSION: Our findings further validate (1)H-MRS as a rapid, non-invasive, and specific diagnostic tool for active tuberculosis in children with microbiologically documented infection outside the central nervous system, specifically in the lungs.

Neuroimaging of pediatric intracranial infection--part 2: TORCH, viral, fungal, and parasitic infections.

In the second half of this 2-part review, the neuroimaging features of the most common viral, fungal, and parasitic infections of the pediatric central nervous system are discussed. Brief discussions of epidemiology and pathophysiology will be followed by a review of the imaging findings and potential differential considerations.

Neuroimaging of pediatric intracranial infection--part 1: techniques and bacterial infections.

Conventional and advanced neuroimaging have become central to the diagnosis of infectious diseases of the pediatric central nervous system. Imaging modalities used by (pediatric) neuroradiologists include cranial ultrasound, computed tomography, and magnetic resonance imaging, including advanced techniques such as diffusion weighted or tensor imaging, perfusion weighted imaging, susceptibility weighted imaging, and (1) H magnetic resonance spectroscopy. In this first of a two part review, imaging techniques in general and the imaging findings of bacterial infections of the intracranial compartment including epidural empyema, subdural empyema, meningitis, cerebritis, cerebral abscess, and pyogenic intraventricular empyema (ventriculitis) are discussed.

The diagnosis of brain tuberculoma by (1)H-magnetic resonance spectroscopy.

UNLABELLED: Toddlers are more prone to develop severe and extrapulmonary tuberculosis (TB) than older children. This is partially explained by differences in the immune response. Early and specific diagnosis is essential to start adequate treatment, especially if the central nervous system (CNS) is involved. The lack of sputum production and inherent dangers or impossibility of sampling CNS lesions may delay diagnosis. In addition, the magnetic resonance imaging (MRI) features of TB abscesses are non-specific and may mimic abscesses of other infectious etiology. (1)H-magnetic resonance spectroscopy ((1)H-MRS) may increase specificity of diagnosis by identifying lipids within the lesions that are considered characteristic for TB. Therefore, we studied four children with presumable CNS-TB with MRI and (1)H-MRS. In vivo and in vitro (1)H-MRS showed elevated lipid peaks within the TB lesions. CONCLUSION: (1)H-MRS allows to non-invasively identifying TB with high specificity and may allow early installment of targeted antimicrobial treatment.

Influenza A/H5N1 virus infection in humans in Cambodia.

BACKGROUND: Between January 2005 and April 2006, six patients of influenza A/H5N1 virus infection were reported in Cambodia, all with fatal outcome. OBJECTIVES: We describe the virological findings of these six H5N1 patients in association with clinical and epidemiologic findings. STUDY DESIGN: Broncho-alveolar lavage, nasopharyngeal, throat and rectal swabs and sera were cultured for virus isolation and viral load quantified in clinical specimens by real-time RT-PCR. We compared sequences obtained from different body sites within the same patient to detect viral quasi-species. RESULTS: H5N1 virus strains isolated in Cambodia belong to genotype Z, clade 1 viruses. H5N1 viruses were isolated from serum and rectal swab specimens in two patients. The haemagglutinin gene sequences of the virus in different body sites did not differ. Amino acid substitutions known to be associated with a change in virus binding were not observed. CONCLUSION: The high frequency of virus isolation from serum and faecal swabs highlights that H5N1 is likely to be a disseminated infection in humans and this has implications for antiviral treatment, biosafety in clinical laboratories and on risks for nosocomial and human-to-human transmission. There were no tissue-specific adaptive mutations in the HA gene from viruses isolated from different organs.