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A comparison of the VP1, VP2, and VP4 regions for molecular typing of human enteroviruses

Perera D, Shimizu H, Yoshida H, Tu PV, Ishiko H, McMinn PC, et al.

J Med Virol, 2010 Apr;82(4):649-57.

PMID: 20166171 DOI: 10.1002/jmv.21652

The VP4, VP2, and VP1 gene regions were evaluated for their usefulness in typing human enteroviruses. Three published RT-PCR primers sets targeting separately these three gene regions were used. Initially, from a total of 86 field isolates (36 HEV-A, 40 HEV-B, and 10 HEV-C) tested, 100% concordance in HEV-A was identified from all three gene regions (VP4, VP2, and VP1). However, for HEV-B and HEV-C viruses, only the VP2 and VP1 regions, and not VP4, showed 100% concordance in typing these viruses. To evaluate further the usefulness of VP4 in typing HEV-A enteroviruses, 55 Japanese and 203 published paired VP4 and VP1 nucleotide sequences were also examined. In each case, typing by VP4 was 100% in concordance with typing using VP1. Given these results, it is proposed that for HEV-A enteroviruses, all three gene regions (VP4, VP2, and VP1), would be useful for typing these viruses. These options would enhance the capability of laboratories in identifying these viruses and would greatly help in outbreaks of hand, foot, and mouth disease.

Fulltext A decade of Japanese encephalitis surveillance in Sarawak, Malaysia: 1997-2006

Wong SC, Ooi MH, Abdullah AR, Wong SY, Krishnan S, Tio PH, et al.

Trop Med Int Health, 2008 Jan;13(1):52-5.

PMID: 18291002 DOI: 10.1111/j.1365-3156.2007.01967.x

Japanese encephalitis virus (JEV) is an important encephalitis virus in Asia, but there are few data on Malaysia. A hospital-based surveillance system for Japanese encephalitis (JE) has been in operation in Sarawak, Malaysia, for the last 10 years. JEV is endemic in Sarawak, with cases occurring throughout the year and a seasonal peak in the last quarter (one-way anova, P < 0.0001). Ninety-two per cent of 133 cases were children aged 12 years or younger; the introduction of JE vaccination in July 2001 reduced the number of JE cases (84 in the four seasons prior to vs. 49 in the six seasons after, McNemar's test, P = 0.0001). After implementation of the programme, the mean age of infected children increased from 6.3 to 8.0 years (Student's t-test, P = 0.0037), suggesting the need for a catch-up programme.

A dot enzyme immunoassay for dengue 3 virus: comparison with the haemagglutination inhibition test

Cardosa MJ, Noor Sham S, Tio PH, Lim SS

Southeast Asian J. Trop. Med. Public Health, 1988 Dec;19(4):591-4.

PMID: 3238470

A dot enzyme immunoassay (DEIA) was used to determine the levels of antibody to dengue 3 virus in the acute and convalescent sera of febrile patients with a clinical diagnosis of dengue fever or dengue haemorrhagic fever. The antibody titres were compared with titres determined by the haemagglutination inhibition (HI) test. The results of the study showed that, besides being more simple to perform, the DEIA is in order of magnitude more sensitive than the HI test. Furthermore, the data suggest that it is possible to use a single dilution as a cutoff point to predict with reasonable accuracy, if a patient has had a recent dengue infection. The DEIA test for antibodies to dengue virus is an appropriate technology highly suitable for rapid diagnosis and surveillance in developing countries.

A nitrocellulose membrane based IgM capture enzyme immunoassay for etiological diagnosis of dengue virus infections

Cardosa MJ, Baharudin F, Hamid S, Hooi TP, Nimmanitya S

Clin Diagn Virol, 1995 May;3(4):343-50.

PMID: 15566815

A nitrocellulose membrane based immunoassay for the detection of dengue virus specific IgM suitable for use in field situations or in peripheral laboratories would be useful for disease surveillance and control. This paper describes such an assay in an IgM capture format (MAC DOT) similar to the microplate based MAC ELISAs currently in use in several research and reference laboratories around the world. The MAC DOT was tested on several sample sets including a retrospective study of 119 patients from Children's Hospital, Bangkok, Thailand, with confirmed dengue infection. The sensitivity of the test was shown to be 94% taking only admission sera into consideration but rising to 99% when both an admission and a discharge specimen were considered. Other sample sets confirmed the high sensitivity and a study of 494 unselected febrile children showed that the specificity of the MAC DOT was 98%.

Fulltext A serological study of Japanese encephalitis virus infections in northern Peninsular Malaysia

Cardosa MJ, Choo BH, Zuraini I

Southeast Asian J. Trop. Med. Public Health, 1991 Sep;22(3):341-6.

PMID: 1667957

This study describes the status of viral encephalitis in Perak, Malaysia during the year 1990. In addition, 14 cases selected from Penang and Perak during the years 1989 and 1990 are presented, with data showing titers of neutralizing antibodies against Japanese encephalitis virus (JEV) and dengue 2 virus, titers of antibodies against JEV and dengue virus antigens as determined by DEIA, and a comparison of these with the presence of IgM to JEV and dengue virus. These data show that there probably is far more viral encephalitis due to JEV in Malaysia than the national figures reflect.

Adenovirus type 21-associated acute flaccid paralysis during an outbreak of hand-foot-and-mouth disease in Sarawak, Malaysia

Ooi MH, Wong SC, Clear D, Perera D, Krishnan S, Preston T, et al.

Clin Infect Dis, 2003 Mar 1;36(5):550-9.

PMID: 12594634

We report the virological and clinical features of 8 children who presented with adenovirus-associated acute flaccid paralysis (AFP) during an epidemic of enterovirus type 71 (EV71)-associated hand-foot-and-mouth disease (HFMD) in Sarawak, Malaysia, in 1997. Neutralization tests and phylogenetic analysis revealed adenovirus type 21 (Ad21), although DNA restriction digests suggested that this virus was different from the prototype Ad21. Four children had upper-limb monoparesis, 2 had lower-limb monoparesis (one of whom had changes in the anterior spinal cord noted on magnetic resonance imaging), and 2 had flaccid paraparesis. At follow-up, 4 children were noted to have made full recoveries and 3 had residual flaccid weakness and wasting. Neurophysiological investigation revealed a mixture of axonal and demyelinating features in motor and sensory nerves, with denervation. These findings suggest that Ad21 might cause AFP by anterior horn cell damage or neuropathy of the brachial or lumbosacral plexus. The occurrence of these unusual adenovirus infections during an outbreak of EV71-associated HFMD suggests that an interaction between the 2 viruses may have occurred.

Antibodies against prM protein distinguish between previous infection with dengue and Japanese encephalitis viruses

Cardosa MJ, Wang SM, Sum MS, Tio PH

BMC Microbiol, 2002 May 5;2:9.

PMID: 12019028

In Southeast Asia, dengue viruses often co-circulate with other flaviviruses such as Japanese encephalitis virus, and due to the presence of shared antigenic epitopes it is often difficult to use serological methods to distinguish between previous infections by these flaviviruses.

Fulltext Comparison of an IgM capture ELISA with a dot enzyme immunoassay for laboratory diagnosis of dengue virus infections

Cardosa MJ, Zuraini I

Southeast Asian J. Trop. Med. Public Health, 1991 Sep;22(3):337-40.

PMID: 1818383

This study describes the use of an IgM capture ELISA using cell culture derived antigens and a polyclonal rabbit antiflavivirus antisera for the detection of dengue positive cases. The IgM capture ELISA is compared with the dot enzyme immunoassay and the results are discussed in the context of dengue endemicity.

Convenient and versatile DNA extraction using agarose plugs for ribotyping of problematic bacterial species

Nair S, Karim R, Cardosa MJ, Ismail G, Pang T

J Microbiol Methods, 1999 Oct;38(1-2):63-7.

PMID: 10520586

We describe a convenient, versatile and safe method for preparing bacterial DNA for ribotyping analysis. In this method, extraction of bacterial DNA from Salmnonella typhi and Burkholderia pseudomallei. and subsequent restriction endonuclease digestion, was performed in agarose blocks/plugs thus minimizing shearing and loss of DNA, problems commonly associated with liquid phase phenol extraction. Digested DNA in the plugs was then electrophoresed directly, transferred to nylon membranes and hybridized with labeled rDNA probes in the usual manner to provide reproducible restriction patterns. This method is particularly useful for bacterial species where standard DNA extraction in the liquid phase using phenol has been problematic (e.g. B. pseudomallei) but can be used for any bacterial species. The DNA extracted within the agarose plugs can be stored for long periods and can be used in other, widely-used typing methods such as pulsed-field gel electrophoresis (PFGE) and PCR-based techniques. Embedding live cells directly in agarose plugs also minimizes the risk of exposure to these virulent human pathogens among laboratory workers.

Dengue fever and dengue haemorrhagic fever in Malaysia

Cardosa MJ

Southeast Asian J. Trop. Med. Public Health, 1988 Sep;19(3):483-6.

PMID: 3217825

Dengue haemorrhagic fever: questions of pathogenesis

Cardosa MJ

Curr. Opin. Infect. Dis., 2000 Oct;13(5):471-475.

PMID: 11964816 DOI: 10.1097/00001432-200010000-00007

The year under review has seen a remarkable proliferation of papers on dengue. Four prospective studies have been carried out across the dengue belt, many groups have been pushing at the question of pathogenesis of dengue haemorrhagic fever, and a breakthrough has been achieved in the development of a mouse model for human dengue haemorrhagic fever.

Fulltext Dengue vaccine design: issues and challenges

Cardosa MJ

Br Med Bull, 1998;54(2):395-405.

PMID: 9830205 DOI: 10.1093/oxfordjournals.bmb.a011696

Dengue virus infection is now a global problem affecting tens of millions of people. The spread of the four dengue virus serotypes had led to increased incidence of dengue haemorrhagic fever (DHF) reported and with 2.5 billion people at risk, efforts towards the development of safe and effective vaccines against dengue must be accelerated. This chapter reviews some of the important lessons of pathogenesis which may be learnt from classical studies in the field and place these in the context of current knowledge about the molecular biology of the virus. The issues which have to be addressed in designing a safe vaccine against dengue are raised and the problems of designing subunit as well as whole virus vaccines are pointed out, particularly with regard to the phenomenon of antibody dependent enhancement and, more generally, the problem of immune potentiation of disease. More efforts must be made to understand the basis of pathogenesis in DHF and in finding out what nature has to teach about protection against and recovery from dengue virus infection.

Dengue virus isolation by antibody-dependent enhancement of infectivity in macrophages

Cardosa MJ

Lancet, 1987 Jan 24;1(8526):193-4.

PMID: 2880019

Acute-phase serum samples collected during an outbreak of dengue fever and dengue haemorrhagic fever in Penang, Malaysia, were tested by a method involving antibody-dependent enhancement of infectivity in the mouse macrophage-like cell line, P388D1. 58 of 71 (81.7%) serologically positive cases yielded virus.

Development of a dot enzyme immunoassay for dengue 3: a sensitive method for the detection of antidengue antibodies

Cardosa MJ, Hooi TP, Shaari NS

J Virol Methods, 1988 Oct;22(1):81-8.

PMID: 3058737

Partially purified DEN3 virus was used as antigen in a sensitive dot enzyme immunoassay (DEIA) for the detection of antibodies to flavivirus antigens. We describe here the method used to prepare and optimise the antigen-bearing nitrocellulose membranes and present the results obtained from screening 20 acute phase sera from patients shown to have had recent dengue infections by the haemagglutination inhibition (HI) test. Sixteen pairs of acute and convalescent sera from dengue-negative patients had no detectable antibody to dengue virus by HI. These were shown to have no antibody detectable by DEIA. Sera positive for dengue antibodies by HI had DEIA titers ranging from 10 to several thousand times greater than the titers detected by HI.

Development of a sandwich ELISA to detect virus-like-particles in enterovirus A71 vaccines

Lim PY, Cardosa MJ

J Virol Methods, 2019 08;270:113-119.

PMID: 31100287 DOI: 10.1016/j.jviromet.2019.05.005

The goal of this paper was to develop a sandwich ELISA that can detect intact human enterovirus A71 (EV-A71) virus-like particles (VLPs) in vaccines. This assay specifically detected EV-A71 viruses from different sub-genogroups as well as EV-A71 VLPs, and treatment of VLPs with high heat and low pH reduced or completely abolished detection of the VLPs suggesting that the ELISA detected assembled particles. Using a purified VLP as a reference standard, a quantitative sandwich ELISA (Q-ELISA) was established which was used to monitor the yield and purity of the VLPs during manufacturing. Coupled with immunogenicity studies, the Q-ELISA was used to evaluate the performance of the VLPs and formalin-inactivated EV-A71 vaccine. This assay has the potential to play an important role in the development of an efficient process to produce and purify the VLPs and in examining the quality of EV-A71 vaccines.

Diagnosis and surveillance of dengue virus infections: gold standards and new directions

Cardosa MJ

Malays J Pathol, 1989 Aug;11:7-10.

PMID: 2698983

Differential Diagnosis of Flavivirus Infections in Horses Using Viral Envelope Protein Domain III Antigens in Enzyme-Linked Immunosorbent Assay

Piyasena TBH, Setoh YX, Hobson-Peters J, Prow NA, Bielefeldt-Ohmann H, Khromykh AA, et al.

Vector Borne Zoonotic Dis, 2017 12;17(12):825-835.

PMID: 29083957 DOI: 10.1089/vbz.2017.2172

In Australia, infection of horses with the West Nile virus (WNV) or Murray Valley encephalitis virus (MVEV) occasionally results in severe neurological disease that cannot be clinically differentiated. Confirmatory serological tests to detect antibody specific for MVEV or WNV in horses are often hampered by cross-reactive antibodies induced to conserved epitopes on the envelope (E) protein. This study utilized bacterially expressed recombinant antigens derived from domain III of the E protein (rE-DIII) of MVEV and WNV, respectively, to determine whether these subunit antigens provided specific diagnostic markers of infection with these two viruses. When a panel of 130 serum samples, from horses with known flavivirus infection status, was tested in enzyme-linked immunosorbent assay (ELISA) using rE-DIII antigens, a differential diagnosis of MVEV or WNV was achieved for most samples. Time-point samples from horses exposed to flavivirus infection during the 2011 outbreak of equine encephalitis in south-eastern Australia also indicated that the rE-DIII antigens were capable of detecting and differentiating MVEV and WNV infection in convalescent sera with similar sensitivity and specificity to virus neutralization tests and blocking ELISAs. Overall, these results indicate that the rE-DIII is a suitable antigen for use in rapid immunoassays for confirming MVEV and WNV infections in horses in the Australian context and warrant further assessment on sensitive, high-throughput serological platforms such as multiplex immune assays.

Fulltext Disability after encephalitis: development and validation of a new outcome score

Lewthwaite P, Begum A, Ooi MH, Faragher B, Lai BF, Sandaradura I, et al.

Bull World Health Organ, 2010 Aug 01;88(8):584-92.

PMID: 20680123 DOI: 10.2471/BLT.09.071357

OBJECTIVE: To develop a simple tool for assessing the severity of disability resulting from Japanese encephalitis and whether, as a result, a child is likely to be dependent.

METHODS: A new outcome score based on a 15-item questionnaire was developed after a literature review, examination of current assessment tools, discussion with experts and a pilot study. The score was used to evaluate 100 children in Malaysia (56 Japanese encephalitis patients, 2 patients with encephalitis of unknown etiology and 42 controls) and 95 in India (36 Japanese encephalitis patients, 41 patients with encephalitis of unknown etiology and 18 controls). Inter- and intra-observer variability in the outcome score was determined and the score was compared with full clinical assessment.

FINDINGS: There was good inter-observer agreement on using the new score to identify likely dependency (Kappa = 0.942 for Malaysian children; Kappa = 0.786 for Indian children) and good intra-observer agreement (Kappa = 1.000 and 0.902, respectively). In addition, agreement between the new score and clinical assessment was also good (Kappa = 0.906 and 0.762, respectively). The sensitivity and specificity of the new score for identifying children likely to be dependent were 100% and 98.4% in Malaysia and 100% and 93.8% in India. Positive and negative predictive values were 84.2% and 100% in Malaysia and 65.6% and 100% in India.

CONCLUSION: The new tool for assessing disability in children after Japanese encephalitis was simple to use and scores correlated well with clinical assessment.

Fulltext Dot enzyme immunoassay: an alternative diagnostic aid for dengue fever and dengue haemorrhagic fever

Cardosa MJ, Tio PH

Bull World Health Organ, 1991;69(6):741-5.

PMID: 1786623

A dot enzyme immunoassay (DEIA) for the detection of antibodies to dengue virus was tested for use as a tool in the presumptive diagnosis of dengue fever and dengue haemorrhagic fever. Paired sera from the following groups of patients were tested using the DEIA and the haemagglutination inhibition (HI) test: those with primary dengue fever; those experiencing a second dengue infection; and febrile patients who did not have dengue. The data obtained show that the DEIA can be effectively used at a serum dilution of 1:1000 to confirm presumptive recent dengue in patients with a second dengue infection. However, demonstration of seroconversion proved necessary for patients with primary dengue. At a serum dilution of 1:1000 the DEIA has a specificity of 97.3%. The role of this simple and rapid test in improving the effectivity of programmes for the control of dengue virus infection is discussed.

Enterovirus 71 encephalomyelitis and Japanese encephalitis can be distinguished by topographic distribution of inflammation and specific intraneuronal detection of viral antigen and RNA

Wong KT, Ng KY, Ong KC, Ng WF, Shankar SK, Mahadevan A, et al.

Neuropathol. Appl. Neurobiol., 2012 Aug;38(5):443-53.

PMID: 22236252 DOI: 10.1111/j.1365-2990.2011.01247.x

To investigate if two important epidemic viral encephalitis in children, Enterovirus 71 (EV71) encephalomyelitis and Japanese encephalitis (JE) whose clinical and pathological features may be nonspecific and overlapping, could be distinguished.

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