This retrospective study examined the G/P type of rotavirus in RNA samples that have previously been e-typed by RNA-PAGE in 1996. The results were then compared to 2007 samples to ascertain the extent of changes that may have occurred in this 11-years time interval. The G and P genotypes were determined by hemi-nested PCR and further analysed by phylogenetic study. In 1996, the G/P combination G1P[8], G(UT)P[8] and G1P(UT) prevalence rate were 81%, 9% and 7%, respectively. As expected, the G9 genotype which has already emerged worldwide was identified in 42% of the 2007 samples with the remaining 33% G1P[8] and 25% G1P(UT) Analysis of the RNA pattern showed that majority of the isolates were long e-type in both series, nevertheless minor differences within electropherotypes were observed. Genetic diversity in some strains of the human group A rotaviruses was analysed by phylogenetic methods. These findings will help in the decision to introduce rotavirus vaccines within the next decade.
This study examined the temporal distribution of rotavirus genotypes in Malaysia. Rotaviruses from children with diarrhea admitted to hospitals in 1996 (n = 93) and 2007 (n = 12) in two different regions of Peninsular (West) Malaysia were analyzed for their G and P genotypes using a hemi-nested RT-PCR assay. In the 2007 samples, the dominant strain was G9P[8]. It was identified in 42% of the samples. Different strains all possessing the G1 genotype were identified in the rest of the samples. In contrast, 81% of the samples collected in 1996 were the G1P[8] strain. No strains with G9 genotype were detected in samples collected in 1996.
A total of 157 stool samples were examined for Group A rotaviruses in diarrheic children admitted to 8 different major hospitals in Malaysia. The overall incidence rate in this study was 19.7% (31 of 157) with a variation of 9.5% to 39.1% in different locations. Majority of the infections detected were in those under 2 years of age and there were fewer admissions in the older age group. The stool samples were initially screened for rotavirus Group A by latex agglutination method and followed by RNA electrophoresis. The size and the characteristics wheel-shaped morphology of the viral preparations when examined by electron-microscopy further confirmed the presence of rotaviruses in the positive stool samples. Analysis of the RNA pattern showed that majority of the isolates, 51.6% (16 of 31) were Type IIC ('long' with comigration of RNA segments 7 and 8), 35.5% (11 of 31) with Type IIG ('long' with comigration of segments 7, 8, 9), 9.7% (3 of 31) with Type IG ('short' with comigration of RNA segments 7, 8, 9) and 3.2% (1 of 31) of mixed or atypical pattern. It appeared that over a 12 year interval, only one new or unusual rotavirus electropherotype was found. This is the first comprehensive report on the electropherotypes of rotaviruses covering eight different geographical locations in Malaysia and the data obtained is useful for understanding the geographic distribution and types of rotaviruses transmitting in Malaysia.
Rotavirus and pathogenic free-living amoebae are causative agents of important health problems, especially for developing countries like Pakistan where the population has limited access to clean water supplies. Here, we evaluated the prevalence of rotavirus and free-living amoebae (Acanthamoeba spp., Balamuthia mandrillaris, Naegleria fowleri) in drinking water supplies of Karachi, Pakistan. Six water filtration plants that supply drinking water to the population of Karachi were investigated. Additionally, drinking water samples from households were analyzed for the presence of rotavirus and free-living amoebae. Rotavirus was present in 35% of the water samples collected from water filtration plants; however, domestic tap water samples had a prevalence of only 5%. Out of 20 water samples from filtration plants, 13 (65%) were positive for Acanthamoeba spp., and one (5%) was positive for B. mandrillaris. Out of 20 drinking water samples collected from different areas of Karachi, 35% were positive for Acanthamoeba spp. Rotavirus was detected in 5% of the drinking water samples tested. Overall, these findings showed for the first time the presence of rotavirus, in addition to pathogenic free-living amoebae in drinking water supplies of Karachi that could be an important public health risk for the affected population.
The objective of this study was to ascertain the extent changes have occurred in the epidemiology of human rotavirus electropherotypes from the same location 7 to 8 years after an earlier study. Genomic RNA profiles of rotaviruses from diarrhoeic children admitted to the Kuala Lumpur Hospital from April to December 1996 were determined by polyacrylamide gel electrophoresis and silver staining. A total of 179 group A rotaviruses were detected from 870 children: 175 with legible staining of all RNA segments were classified into 14 distinct electropherotypes (10 and 4 with long and short migration patterns respectively). In addition, the results revealed: high predominance of long pattern electropherotypes (94% of the total electropherotypes); most long electropherotypes with RNA profiles which all 11 RNAs migrated separately (8 of 10 electropherotypes); all short electropherotypes had segments 2 and 3 that co-migrated; presence of a very numerically dominant electropherotype (75% of all electropherotypes); frequent co-circulation of the dominant electropherotype-present throughout the study period--with other electropherotypes present for limited periods; sequential temporal appearances by similar electropherotypes. These observations were similar to that of an earlier study conducted in 1988/89. Nevertheless, the dominant electropherotype in the present study was different and not among the electropherotypes detected in the earlier study.
The group A rotavirus staphylococcal co-agglutination test was evaluated and its sensitivity and specificity compared with an in-house enzyme-linked immunosorbent assay (ELISA) and a commercial latex agglutination test (Rotalex). In addition, the storage stability of the staphylococcal reagents was ascertained. Examination of 136 clarified suspensions of diarrhoeal faeces by the staphylococcal co-agglutination test revealed a high proportion of false positives (26%) and uninterpretable results (34%) due to non-specific agglutination. Non-specific agglutination could be removed effectively by prior absorption of the clarified faecal specimens with unsensitized staphylococci. The staphylococcal co-agglutination test was less sensitive and specific than the in-house enzyme-linked immunosorbent assay but was comparable to the Rotalex slide latex agglutination test. The staphylococcal reagents have a shelf life of at least 29 weeks.
The aim of this study was to optimize the conditions for the passive adsorption of polyclonal antibody onto plain surface polystyrene latex particles and its performance in a slide latex agglutination test for rotavirus antigen detection. Cleaning of latex particles by washing through repetitive centrifuging, decanting and resuspending in distilled water was adequate in removing surfactants from the particles' surfaces to enable coating. A study of antibody concentration, incubation temperature and buffer pH revealed that optimum coating was achieved with a 3-fold excess of antibody to the calculated total particle surface capacity for the antibody in a glycine-saline buffer of pH 9.2 at 40 degrees C for 4 hours. The ionic strength and pH of the latex suspending buffer and the sample buffer were critical factors determining the sensitivity of the test and the appearance of non-specific agglutination. Ultrasonication, addition of glycerol and Tween 20, either individually or in combination, were able to suppress non-specific agglutination in some batches of latex reagents. Polyethylene glycol 6000 enhanced the quality of agglutination as well as reduced the time of its appearance, especially in reagents that produced poor agglutination.
A 12-month study was carried out on the molecular epidemiology of rotavirus in urban and suburban Malaysian children. Analysis of faecal samples from 973 hospitalized diarrhoeic children by polyacrylamide gel electrophoresis detected 268 rotaviruses (28%). All isolates were group A rotaviruses, which produced 22 electropherotypes: 16 (91.5%) with long RNA migration patterns and 6 (8.5%) with short patterns. One of the long-pattern electropherotypes was the predominant strain (71.1% of the total electropherotypes) isolated during this study. Although 3 other strains were detected sporadically over the study period, 16 others were present only during the first 7 months and 2 others were confined to the last 5 months. Long- and short-pattern electropherotypes were found to co-circulate extensively. There was a significant association of short-pattern electropherotypes with infection in older children. In addition, the prevalence of vomiting and mean duration of diarrhoea were significantly associated with different electropherotypes.
A 1 year longitudinal study of 156 Malaysian children from urban and suburban areas in the Klang Valley revealed that the incidence rate of diarrhoea was 23.6 per 100 person-year with abnormal faeces reported on 0.26% of the total days of observation. Diarrhoea cases were detected in children from all socioeconomic classes. Rotavirus was isolated from 12% of the diarrheic children and asymptomatic rotavirus infection occurred in 3.2% of the children. All rotaviruses isolated were group A rotaviruses with long electrophoretypic pattern.
The pattern of rotavirus infection in babies of the neonatal special care nursery (SCN) of the Kuala Lumpur Maternity Hospital was studied. The presence of rotavirus in the neonates' stools was ascertained using the method of polyacrylamide gel electrophoresis and silver staining. No rotavirus was detected in the 511 stools and rectal swabs collected from the 164 neonates over a 8-week period. Thus the babies admitted to the SCN from the labour rooms and the postnatal wards of the hospital were unlikely to be carriers of rotavirus or infected by rotavirus during their stay. It was concluded that rotavirus was not endemic in the nursery or the postnatal wards of this maternity hospital.
The prevalence of human rotavirus enteritis in children admitted to the gastroenteritis ward of the Kuala Lumpur General Hospital was studied in 1982. Human rotavirus in the stool of the patients was detected by enzyme-linked immunosorbent assay. The survey showed that rotavirus enteritis in children were observed throughout the year, with two broad peaks of rotavirus infection occurring around March and September. The lowest incidence was recorded in July, however, no prolonged period of low prevalence of rotavirus enteritis was observed. The average prevalence for the whole of 1982 was 40% of the total diarrhoeal cases. No significant relationship was noted between the prevalence of the disease and rainfall.
A human-porcine reassortant rotavirus, strain R1207, was identified from 74 group A rotaviruses detected in 197 (37.6%) stool samples collected from patients who attended a tertiary care hospital in Ragama, Sri Lanka. This is the first report of a human-porcine reassortant rotavirus in Sri Lanka. The patient was a 12-month-old boy who had been hospitalized with fever and acute diarrhea with a duration of 6 days. The family had pigs at home before the birth of this boy. However, the neighbors still practice pig farming. The genotype constellation of R1207 was G4-P[6]-I1-R1-C1-M1-A1-N1-T1-E1-H1. This is based on the assignment of all the eleven gene segments a full genome-based genotyping system. R1207 showed a 4-2-3-2 genomic electrophoretic migration pattern, which is characteristic of group A rotaviruses. Our analyses revealed that five (NSP2, NSP4, VP1, VP2, and VP7) of the 11 genes were closely related to the respective genes of porcine strains. Although the remaining six genes (NSP1, NSP3, NSP5, VP3, VP4, and VP6) were related to human strains, with the exception of the gene sequence of NSP1, all of these human strains were human-porcine reassortants. With a genogroup 1 genetic backbone, this strain was possibly formed via multiple genetic reassortments. We do not know whether this strain is circulating in pigs, as no data are available on porcine rotaviruses in Sri Lanka. Surveillance should be strengthened to determine the epidemiology of this genotype of rotavirus in Sri Lanka and to assess whether the infection was limited or sustained by ongoing human-to-human transmission.
Factors affecting the mechanical transmission of rotavirus by the legs and wings of the housefly, Musca domestica L., were examined in a laboratory study. Rotavirus was picked up when houseflies walked on thin smears of clarified rotavirus suspensions. The addition of glycerol, which increased viscosity of the virus suspension, and particulate human feces slightly increased the proportion of flies contaminated with virus. However, the addition of glycerol greatly reduced the average number of virus particles picked up per fly, whereas feces greatly increased the number of particles. The proportion of flies with virus-contaminated legs, which transferred virus to > 1 contact surface, was increased by longer contact time with the surface and when the contact surface was agar instead of glass. Most virus particles were deposited on 1st contact with the surface. Most flies dislodged virus particles inoculated on the underside of their wings soon after the start of simulated flight. Our data indicated that the nature of the virus-suspending medium has a greater effect on the level of virus contamination than on the ability to become contaminated. The importance of walking as a mode of virus transport depends on the nature of the contact surface, the risk of the contaminated fly settling first on a surface likely to come into contact with humans, and fly numbers.
Rotavirus is the leading causative viral agent of pediatric acute gastroenteritis globally, infecting mostly children 5 years old and below. Data on rotavirus prevalence in Malaysia is scarce, despite the WHO's recommendation for continuous rotavirus surveillance, and has underestimated the need for national rotavirus vaccination. Characteristics of the current rotavirus strains in Malaysia have to be determined to understand the rotavirus epidemiology and vaccine compatibility. This study sought to determine the genetic relatedness of Sarawak rotavirus strains with global strains and to determine the antigenic coverage and epitope compatibility of Rotarix and RotaTeq vaccines with the Sarawak rotavirus strains via in silico analysis. A total of 89 stool samples were collected from pediatric patients (<5 years old) with acute gastroenteritis at private hospitals in Kuching, Sarawak. Rotavirus was detected using reverse transcription-polymerase chain reaction. Positive amplicons were analyzed using nucleotide sequencing before phylogenetic analyses and assessment of epitope compatibility. Genotyping revealed G1P[8] (1/13; 7.7%), G3P[8] (3/13; 23%), G9P[4] (1/13; 7.7%), and G9P[8] (3/13; 23%), G9P[X] (1/13; 7.7%), GXP[4] (1/13; 7.7%), and GXP[8] (3/13; 23%) in samples. All wild-type Sarawak rotavirus strains, with the exception of G1, showed variations in their phylogenetic and antigenic epitope characteristics.
A diarrhoea outbreak had occurred among neonates delivered in a private hospital in Kedah from 15 August to 8 September 2002 involving 27 (55.1%) cases out of a total of 49 deliveries. Thirteen of them (48.1%) were admitted to either government or private hospitals for treatnzent while fourteen of them (51.9%) were managed at home. The main presenting feature was frequent yellowish to greenish watery stool not associated with vomitting. Investigations include active case finding, environmental inspection, sampling of stool specimens, identifying causative agents and identuying human carriers. All the diarrhoea eases (100%) were noted to have received infant formula feeding while in the private hospital. Staphylococcus aureus was isolated hom the milk scoop which was used for milk preparation. Nasal swabs of four (50%) nursing personnel were also positive for Staphylococcus aureus. One of them was positive for methycilline resistant Staphylococcus aureus (MRSA). The milk and water samples showed no signuicant bacterial contamination. Stool samples of these cases were negative for Rotavirus, Vibrio sp., Salmonella sp., Shigella sp. and Entamoeba coli. This outbreak of diarrhoea was noted to have a strong association with infant formula feeding in the hospital. Breastfeeding should be continuously promoted. Baby friendly hospital initiatives in private hospital settings need to be initiated.
Amongst 107 diarrheal cases studied a bacterial agent was isolated from 71 (66%) cases of which 60 (85%) were due to a single agent and the remaining 11 (15%) were of mixed infections. Enterotoxigenic Escherichia coli (ETEC) was isolated from 65 cases. Other pathogens isolated included Salmonella spp, Shigella spp and rotavirus. There was a higher isolation rate of ETEC from females and rotavirus from males. The infection rate was found to higher for the 0-2 year age group as compared to the 3-5 year age group. Amongst the ETEC isolated the STa 2 toxotype was the predominant type.
The VP4 genetic groups of 151 field strains of human rotaviruses obtained from infants and young children with diarrhea from four locations in Malaysia were analyzed. The strains were adapted to growth in tissue culture and studied further by molecular hybridization of northern blotted RNA to PCR-generated cDNA probes representing amino acids 84-180 of the KU strain VP4, 83-181 of the DS-1 strain VP4, and 83-180 of either the 1076 or K8 strain VP4, representing VP4 genetic groups 1-4 (P1A, P1B, P2, and P3), respectively. The majority (79% of the field strains hybridized with the KU VP4 genetic group 1 probe and were associated with G1, G3, G4, untypable, or mixed G serotypes. VP4 genetic group 1 (P1A) strains were the most common in all locations in Malaysia between 1978-1988. Three strains which exhibited G3 and subgroup I specificity hybridized with the K8 VP4 genetic group 4 probe. These three VP4 genetic group 4 (P3) strains were detected in two different years and locations, extending the initial detection of this VP4 genetic group (the K8 strain) in Japan to a larger geographical area of Asia.