VIROLOGY RESEARCH DIVISION

During this period (January to December, 2010) the Virology Research Division was involved in three main research areas, namely, arbovirology, viral diarrhea and viruses causing acute respiratory infections. The research projects were mostly involved in disease surveillance of viral infections for timely prevention of disease outbreaks. Also, some of the studies were aimed to monitor the emergence of new viral strains or subtypes to provide base-line data for the formulation of effective candidate vaccines and for elucidating the contribution of viral genetics to the changing patterns of disease.

RESEARCH PROJECTS

COMMUNICABLE DISEASES

1.1. DIARRHOEA/ DYSENTERY

1.1.1. Surveillance of rotavirus diarrhoea in Yangon Children Hospital (WHO/SEARO)

The study involved collection of stool specimens from children less than 5 years of age admitted to the three medical wards of the Yangon Children’s Hospital, Yangon, Myanmar. Stool samples not less than 5 ml were collected from diarrheic children as soon as possible after hospital admission. The stool samples were transported daily to the laboratory in the Virology Research Division. A total of 673 stool samples were collected and clinical features were recorded among under 5 year old children admitted to the three medical wards for diarrhoea at the Yangon Children Hospital from January to December 2010. All the samples collected were tested for the presence of rotavirus antigen by a commercial enzyme immunoassay kit (ProSpect TTM Rotavirus from OXOID, UK). Rotavirus was detected in 356 (53 %) of 673 stool samples tested. In 2010, the Virology Research Division, of the Department of Medical Research (Lower Myanmar) participated in the EQAS (External Quality Assurance System) programme for rotavirus testing conducted by the WHO Reference Laboratory in Vellore, India with over 90% concordance on the EQAS results. Rotavirus diarrhoea was most prevalent in the 12 – 23 month age group and during the cooler, drier months of the year (January, November and December). A total of 120 Rotavirus positive samples from the year 2010 were sent to the Rotavirus Reference Laboratory in Vellore, India for genotyping. From 120 samples, 6 samples were G1P [8], 9 were G12P [6], 81were G12P [8], 11 were G2P [4], 4 were mixed type, 2 were partially typed and 7were untypable samples.

1.1.2. Hospital-based surveillance of intussusception among children in Yangon

Surveillance for intussusception among children less than 2 years of age admitted to the Yangon Children Hospital was conducted in the aim to provide base-line data for investigators and programmers in making decisions concerning future use of rotavirus vaccines. A total of 3 cases (all are males), ages ranging from 5 to 7 months were collected during this period. The types of intussusception were ileo-colic for all three cases. All patients presented with bloody mucus diarrhea, vomiting and fever. The stool samples were tested for the presence of rotavirus by a commercial ELISA kit (ProSpect TM Rotavirus, Oxoid, UK) and rotavirus was not detected in all three cases.

1.2. DENGUE HAEMORRHAGIC FEVER

1.2.1. Dengue serotypes among dengue haemorrhagic fever patients in Yangon Children Hospital

Paired sera samples were collected from clinically diagnosed dengue patients admitted to the medical wards of Yangon Children Hospital from January to October 2010. These were titrated for dengue haemagglutination inhibition antibody titres by the method of Clarke and Casals. Out of 121 pairs tested, 95 (78.5%) were serologically diagnosed as dengue infection of which 16 (16.8 %) had primary and 79 (83.2 %) had secondary infections. A new batch of dengue antigen was produced by intracerebral inoculation of suckling mice. A total of 509 suckling mice have been inoculated and dengue antigen produced by the sucrose acetone extraction method. The acute sero-negative samples from HAI testing were further processed by passaging in C6/36 mosquito cell line for 7days and the cell culture supernatant was harvested and typed by the immunofluorescent method using serotype-specific monoclonal antibodies to all four dengue serotypes. Out of 23 samples tested, 16 samples were typable. Of these 1 sample (4.3%) was dengue serotype 1, another 1 sample (4.3%) was dengue serotype 2, 12 samples (52.1%) were dengue serotype 4 and 2 samples (8.6%) showed dual infection of dengue serotype 2 and 4. The rest (7) samples, that is, (30.4%) were untypable.

1.2.2. Sentinel surveillance of dengue in endemic regions of Myanmar This project is implemented in collaboration with the WHO collaborating centre for Arbovirus Reference and Research, Brisbane, Australia. Material for sera collections and dengue rapid test kits were distributed to all sentinel sites (Mandalay, Sittwe, Mawlamyaing, Lashio and Pathein). These samples were tested with PANBIO Dengue Duo IgM and IgG rapid test kit from Australia. The sera which were sent from Mawlamyaing hospital were from 41 patients, of which 17 patients (42.5%) were positive for dengue infection. Of these patients, 3 ( 17.6%) were positive for anti-DENV IgM which denotes primary dengue infection. The remaining 14 patients ( 82.4% ) had either anti-DENV IgG alone or anti- DENV IgG together with IgM, thus denotimg secondary dengue infections. A total of 28 blood samples were sent from Pathein hospital taken from patients who were clinically suspected of dengue infection. Of these 28 patients 22 patients (79.6%) were confirmed to have dengue infection of which 1 patient (4.5%) had primary infection and the rest (21 patients) had secondary infections, that is ( 95.5%). From Sittwe General Hospital, 62 sera samples were sent of which 37 samples (46%) were positive for dengue infections. Of these 37 samples 7 ( 19% ) had primary infection and 30 ( 81% ) had secondary dengue infections. From Mandalay General Hospital, a total of 99 sera samples taken from patients with clinically suspected dengue infection were sent. Of these 99 patients, 57 patients (58%)were confirmed to have dengue infection of which 11 patients (18.3%) had primary infection and the rest 46 patients (80.7%) had secondary infection. Some positive samples were further processed by doing RNA extraction with the QIAGEN RNA mini extraction kit and further serotyped using multiplex dengue PCR primers (Veredus Laboratories, Singapore) specific for all 4 serotypes of dengue using Reverse Transcriptase Polymerase Chain Reaction (RTPCR). From the Pathein samples, out of 11 samples tested, one sample (9%) was dengue serotype 2, three samples (27.2%) was dengue serotype 4 and another one sample (9%) showed dual infection of dengue serotype 2 and 4, six samples (54.5%) was untypable. A total of 8 samples from Sittwe were tested for serotyping and 1 sample (12.5%) showed dengue serotype 1, 3 samples (37.5%) showed dengue serotype 2 and 4 samples (50%) was untypable. From the Mawlamyaing samples, out of 6 samples tested, one sample (16.66%) was dengue serotype 2, two samples (33.33%) was dengue serotype 4 and the rest three samples (50%) were untypable. From the Mandalay samples, out of 20 samples tested, one sample (5%) was dengue serotypes 1, nine samples (45%) was dengue serotype 4, three samples ((15%) showed dual infection of dengue serotype 1 and 4, and seven samples (35%) were untypable.

1.3. JAPANESE ENCEPHALITIS

1.3.1 Hospital-based surveillance of Japanese encephalitis virus infections in Yangon Serum and Cerebro-spinal Fluid samples were collected from the patients with sign and symptoms of encephalitis who were admitted to Yangon General Hospital, New Yangon General Hospital, Yangon Children Hospital, Insein General Hospital and Thingangyun Sanpya General Hospital. Thirty-seven sera and one CSF sample were obtained from January 2010 to October 2010. These samples were tested with JE IgM/IgG antibody rapid test kits (Standard Diagnostics, Korea). Among the sera and CSF tested three samples were JE IgG positive. These samples were again confirmed by JE DENGUE IgM Combo ELISA, Panbio Australia. Three out of 37 human sera were JE antibody positive and five were Dengue antibody positive. These three positive cases were from Bogalay, South Dagon and Thanlyin with the ages of 11, 12 and 23 years, and they were admitted to hospitals during July 2010, presenting with signs and symptoms of fever, neck stiffness, impaired consciousness and meningo-encephalitis.

1.3.2 Isolation of Japanese encephalitis virus from piglets in Thaketa Township, Yangon. Japanese encephalitis virus exists in an enzootic cycle between mosquitoes and pigs and /or water birds. It is transmitted by Culex mosquitoes which has flight range of 2- 12km. Two human cases of confirmed Japanese encephalitis were reported in 2010 September who were residing in Thaketa town-ship. At the same time a small pig-farm, which is about 2km distance from the residing place of these two human JE cases was selected to isolate JE virus from piglets. Blood samples were collected from 5 piglets weekly for 14 weeks starting from the age of 8 weeks. A total of 70 blood samples were collected, sera were separated and kept at -80°C. Five sera samples taken at the 14th week of sample collection were tested with JE Dengue IgM Combo ELISA, PanBio, Australia and two sera were JE IgM positive.

1.4. CHIKUNGUNYA VIRUSES

1.4.1 Clinical profile and prevalence of Chikungunya infection Sera samples were collected from adult patients from the medical wards of Yangon General Hospital and New Yangon General Hospital who were clinically suspected of having Chikungunya infection. A total of 102 sera samples were tested by the SD Chikungunya IgM rapid test and 35 patients (33 %) were confirmed serologically to have Chikungunya infection. Regarding the clinical profile of these patients, 100% (35/35) presented with fever, 71% presented with rash (25/35), 74% (26/35) presented with joint pains, headache was seen in 49% (17/35) of patients, lymph node enlargement in 7 patients, that is 20%. Bleeding manifestations such as epistaxis and gastrointestinal bleeding were also seen in 20% (7/35) of patients, abdominal pain in 9% (3/35) of patients and lastly vomiting in 11% (4/35) of patients. Sera samples were also collected from children under 12 years of age from the medical wards of Yangon Children Hospital who were clinically suspected of having Chikungunya infection. A total of 13 sera samples were tested by the SD Chikungunya IgM rapid test and 5 patients (33%) were confirmed serologically to have Chikungunya infection. In these children, 100% (5/5) presented with joint pain and fever, 80% (4/5) presented with rash, 40% (2/5) presented with bleeding manifestation like epistaxis and gastrointestinal bleeding, lymph node enlargement in 1 patient, that is 20%, abdominal pain was seen in 40% (2/5).

1.5. ACUTE RESPIRATORY INFECTION

1.5.1. Surveillance of Influenza and Parainfluenza viruses in children in Yangon

Surveillance for Influenza (Influenza A and B viruses) and Parainfluenza viruses (Parainfluenza 1 , 2 and 3) in children attending the Out Patient Department of Yangon Children Hospital presenting with signs and symptoms of acute respiratory infection (ARI) was conducted in the aim to detect the prevalence of these viruses. Throat swabs were taken from a total of 127 cases in 2010. One hundred and thirty nine samples from September 2009 to August 2010 were subjected to virus isolation in MDCK (Madin Darby Canine Kidney) cell lines. After second passage, respiratory viruses were screened by Indirect Immunofluorescent assay (IFA). One hundred and nine samples (81%) showed respiratory virus positive. These respiratory positive samples proceeded to further passages. After fourth passage, influenza and parainfluenza viruses were identified by IFA using specific monoclonal antibodies. Influenza virus-A was detected in 10 cases (7.4%), influenza virus-B in 10 cases (7.4%), PIV-1 in 13 cases (9.6%), PIV-2 in 9 cases (6.7%) and PIV-3 in 8 cases (5.9%). Dual infection of influenza virus-A and PIV-3 was detected in one case, influenza virus-B and PIV-1 in one case, influenza virus-B and PIV-3 in one case and PIV-1 and PIV-3 in one case. Majority of the cases were between 2 to 36 months of age presenting mainly with cough (100%), fever (60-90%) and rhinorrhoea (60-85%).

1.5.2. Aetiology, modifiable risk factors, clinical features and immunological status of children with acute respiratory infections attending general practitioner’s clinic in periurban setting, Yangon
(Please refer to annual report of Bacteriology Research Division )

2. ACADEMIC AND TECHNOLOGY DEVELOPMENT

2.1. QUALITY CONTROL TESTS

2.1.1. Diagnostic Evaluation of SD BIOLINE Dengue Duo Rapid Cassette Test in detection of Dengue samples from Yangon Children Hospital

Sera samples were collected from clinically diagnosed dengue patients admitted to the medical wards of Yangon Children Hospital. Evaluation of the SD BIOLINE Dengue Duo Rapid Cassette Test was done by comparing the results of this test with the haemagglutination inhibition test and dengue specific ELISA test. One hundred and seventy (170) samples have been tested with SD BIOLINE Dengue Duo Rapid Cassette Test and compared with both the ELISA and HAI test.The agreement between the SD BIOLINE Dengue Duo Rapid Cassette Test and the dengue specific ELISA test for secondary dengue infections is 94.2% which is much higher when compared with the agreement between these two tests for primary dengue infections which is only 16.7% (Kappa value 0.14, P= 0.06) This is the same when comparing the SD BIOLINE Dengue Duo Rapid Cassette Test and the haemagglutination inhibition test (HAI). The agreement between these two tests for secondary dengue infections is 98.9% whereas for primary dengue infections is 14.8% (Kappa value 0.19, P= 0.002). With the ROC (Receiver Operator Curve) on detection of secondary dengue infections by the SD BIOLINE Dengue Duo Rapid Cassette Test, the area under the curve value is 0.8 (95% CI 0.6-1.0, P= 0.03). So the SD BIOLINE Dengue Duo Rapid Cassette Test is useful for detection of secondary dengue infections.

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