Leera Kittigul

Molecular characterization of rotaviruses, noroviruses, sapovirus, and adenoviruses in patients with acute gastroenteritis in Thailand

Outbreaks of viral gastroenteritis occur worldwide including Thailand. Unfortunately, there is limited information since etiologic agents have not been identified in several outbreaks of nonbacterial gastroenteritis. The genotype of enteric viruses causing acute gastroenteritis in Thailand was determined using reverse transcription‐multiplex polymerase chain reaction and DNA sequencing. From January 2006 to February 2007, stool samples were collected from patients with acute gastroenteritis of all age groups attending a hospital in Thailand, and patients with nonbacterial acute gastroenteritis (262 patients) were tested for enteric viruses. The overall positive detection rate of enteric viruses was 14.9%; group A rotaviruses (6.1%), noroviruses (6.5%): GI (0.8%) and GII (5.7%), adenoviruses (1.5%), and sapoviruses (0.8%) were found. Group B and C rotaviruses, and astroviruses were not detected in the enrolled patients. Viral acute gastroenteritis occurred in children less than 15 years of age (25.2%, 33/131) with higher frequency than in adults (4.6%, 6/131), P‐value <0.001. Rotavirus G1 was the most predominant genotype, followed by G3, and G9. Among noroviruses, GI‐2 was identified; whereas, GII was predominant with a high frequency of GII‐4 observed, followed by GII‐16, GII‐2, GII‐3, and GII‐12. Sapovirus GII‐3 and human adenoviruses were identified. This study suggests that enteric viruses play an essential role in patients with acute gastroenteritis attending hospital and mainly in children who have a higher prevalence of group A rotaviruses and noroviruses. The genetic analyses provide molecular epidemiological data for viruses important to public health. J. Med. Virol. 81:345–353, 2009.

Norovirus GII-4 2006b Variant Circulating in Patients With Acute Gastroenteritis in Thailand During a 2006-2007 Study

Noroviruses (NoVs) are recognized as a significant cause of acute gastroenteritis in children and adults. A 14‐month study, from January 2006 to February 2007, was undertaken in a hospital in Thailand to determine the prevalence and genetic characterization of NoVs in patients of all ages with acute gastroenteritis. Based on reverse transcription‐nested polymerase chain reaction (RT‐nested PCR), NoVs were detected in 122 of 273 (44.7%) collected stool samples. Of the 122 NoV‐positive samples, 28 (23%) belonged to GI, 79 (64.8%) belonged to GII, and 15 (12.2%) were mixed infections of GI and GII strains. Three NoV GI‐positive and 42 NoV GII‐positive samples were characterized successfully by DNA sequencing of the RT‐nested PCR products and phylogenetic analysis. For NoV GI, two genotypes were identified: GI‐2 (one sample) and GI‐6 (two samples). NoV GII could be classified further into five distinct genotypes: GII‐2 (1 sample), GII‐3 (3 samples), GII‐4 (14 samples), GII‐6 (3 samples), and GII‐17 (2 samples), and one unclassified genotype (19 samples). All NoV GII‐4 strains showed 88–98% nucleotide identity with NoV GII‐4 2006b variants reported worldwide. Among genotypes of NoV characterized, one co‐infected stool sample exhibited NoVs GI‐6 and GII‐4 2006b. This study suggests that there is an important role of NoVs as etiologic agents in patients with acute gastroenteritis. The predominant circulating genotype of NoV infections is GII‐4 2006b variant. J. Med. Virol. 82: 854–860, 2010.

An improved method for concentrating rotavirus from water samples

A modified adsorption-elution method for the concentration of seeded rotavirus from water samples was used to determine various factors which affected the virus recovery. An enzyme-linked immunosorbent assay was used to detect the rotavirus antigen after concentration. Of the various eluents compared, 0.05M glycine, pH 11.5 gave the highest rotavirus antigen recovery using negatively charged membrane filtration whereas 2.9% tryptose phosphate broth containing 6% glycine; pH 9.0 was found to give the greatest elution efficiency when a positively charged membrane was used. Reconcentration of water samples by a speedVac concentrator showed significantly higher rotavirus recovery than polyethylene glycol precipitation through both negatively and positively charged filters (p-value <0.001). In addition, speedVac concentration using negatively charged filtration resulted in greater rotavirus recovery than that using positively charged filtration (p-value = 0.004). Thirty eight environmental water samples were collected from river, domestic sewage, canals receiving raw sewage drains, and tap water collected in containers for domestic use, all from congested areas of Bangkok. In addition, several samples of commercial drinking water were analyzed. All samples were concentrated and examined for rotavirus antigen. Coliforms and fecal coliforms (0->1,800 MPN/100 ml) were observed but rotavirus was not detected in any sample. This study suggests that the speedVac reconcentration method gives the most efficient rotavirus recovery from water samples.

Determination of tumor necrosis factor-alpha levels in dengue virus infected patients by sensitive biotin-streptavidin enzyme-linked immunosorbent assay.

A modified sandwich enzyme-linked immunosorbent assay using biotin-streptavidin system (BS-ELISA) was developed to determine levels of tumor necrosis factor-alpha (TNF-alpha) in serum samples of children infected with dengue virus (n=99) and healthy controls (n=41). The minimum detectable concentration of TNF-alpha by the BS-ELISA was 3.3 pg/ml. The mean TNF-alpha level was highest in those patients with dengue shock syndrome (DSS) or dengue hemorrhagic fever (DHF) grade III (37.44+/-42.0 pg/ml). Lower levels were found in DHF grade I (28.44+/-42.7 pg/ml), DHF grade II (24. 21+/-25.4 pg/ml) and dengue fever (DF) (14.10+/-24.0 pg/ml). TNF-alpha in the sera of DF and DHF patients could be detected on days 2-6 after the onset of fever, the high level occurring on day 5. TNF-alpha was detected in 41.4% (24.01+/-35.2 pg/ml) of dengue virus infected patients and 7.3% (4.2+/-15.6 pg/ml) of control subjects. The sera of patients contained significantly higher levels of TNF-alpha than the sera of controls, P-value<0.001. DHF patients had significantly higher levels of TNF-alpha than DF patients (P-value=0.020) but no difference in the TNF-alpha levels from sera of DHF grades I-III patients was observed (P-value=0.295). The results indicate that the BS-ELISA is a very sensitive method for determining TNF-alpha in serum samples of DF and DHF patients. The TNF-alpha levels might be associated with dengue virus infection and related to disease severity of DHF.

Hepatitis B sero-prevalence and risk factors among school-age children in a low socioeconomic community, Bangkok.

A cross-sectional study of 165 school-age children who had no history of HBV vaccination was carried out in a low socioeconomic community of Din-Daeng, Bangkok. Blood specimens were collected for determination of HBV seromarkers (HBsAg, Anti-HBs and Anti-HBc) by EIA commercial kits. The results showed that the prevalence of HBV seromarkers was 24.85%, the HBsAg carrier rate was 3.64%, the anti-HBs positive rate was 15.15%, and the prevalence of only anti-HBc was 6.06%. To investigate factors associated with the positivity of HBV seromarkers, children were divided into two groups-the first group consisted of 41 children with HBV seromarkers and the second consisted of 124 children without HBV seromarkers. The study variables between the two groups were compared and analysed. The results revealed that factors associated with HBV positivity were (a) child factors: childs age, childs sex, ear piercing in female, sharing blade during haircutting, contact wound from other persons, using wares with other persons, searching things in garbage, and (b) family factors: older parent, low education in parent, low family income per month, low parents knowledge and attitude about HBV infection and vaccination, (P < 0.05). After using stepwise regression analysis, the factor of ear piercing in female was only one significant variable (P = 0.007).

Detection and characterization of hepatitis A virus in water samples in Thailand

Aims:  Outbreaks of hepatitis A in Thailand have been reported continuely and associated with water supply. However, the genetic analysis of hepatitis A virus (HAV) in water is limited. This study described the application of virus concentration method and reverse transcriptase‐nested polymerase chain reaction (RT‐nested PCR) to detect HAV RNA and analyse the genetic sequence of the virus in environmental water samples.

Development of a method for concentrating and detecting rotavirus in oysters

Identification of enteric viruses in outbreak-implicated bivalve shellfish is difficult because of low levels of contamination and natural inhibitors present in shellfish tissue. In this study, the acid adsorption-alkaline elution method developed in our laboratory was proposed for the detection of rotavirus from oyster samples. The acid adsorption-alkaline elution process included the following steps: acid adsorption at pH 4.8, elution with 2.9% tryptose phosphate broth containing 6% glycine, pH 9.0, two polyethylene glycol precipitations, chloroform extraction and reconcentration using speedVac centrifugation. Oyster concentrates were extracted for RNA and examined for rotavirus using reverse transcription-nested polymerase chain reaction (RT-nested PCR). A comparison of SuperScript One-Step RT-PCR system and RT followed by PCR before the nested PCR reaction showed the former detecting four-fold lower concentration of rotavirus (78.12 plaque forming units [PFU]/ml or 0.26 PFU/assay) than the latter (3.12 x 10(2) PFU/ml or 1.04 PFU/assay). In the seeding experiment, the developed acid adsorption-alkaline elution gave high sensitivity of rotavirus detection (125 PFU/g of oyster). From August 2005 to February 2006, 120 oyster samples (Crassostrea belcheri) were collected from local markets and oyster farms, concentrated, and tested for naturally occurring rotaviruses. Four oyster samples were group A rotavirus-positive. Based on phylogenetic analysis of rotavirus DNA sequences in those positive samples, the oyster samples contained the sequences associated with human rotavirus G9 (two samples), G3 (one sample), and G1 (one sample). The present study demonstrates the successful application of developed virus concentration method and RT-nested PCR for the detection of rotaviruses in naturally contaminated oyster samples. The method might be used as a tool for evaluating the presence of enteric viruses in shellfish for monitoring and control of public health.

Long-Lasting Protective Immune Response to the 19-Kilodalton Carboxy-Terminal Fragment of Plasmodium yoelii Merozoite Surface Protein 1 in Mice

ABSTRACT Merozoite surface protein 1 (MSP1) is the major protein on the surface of the plasmodial merozoite, and its carboxy terminus, the 19-kDa fragment (MSP119), is highly conserved and effective in induction of a protective immune response against malaria parasite infection in mice and monkeys. However, the duration of the immune response has not been elucidated. As such, we immunized BALB/c mice with a standard four-dose injection of recombinant Plasmodium yoelii MSP119 formulated with Montanide ISA51 and CpG oligodeoxynucleotide (ODN) and monitored the MSP119-specific antibody levels for up to 12 months. The antibody titers persisted constantly over the period of time without significant waning, in contrast to the antibody levels induced by immunization with Freunds adjuvant, where the antibody levels gradually declined to significantly lower levels 12 months after immunization. Investigation of immunoglobulin G (IgG) subclass longevity revealed that only the IgG1 antibody level (Th2 type-driven response) decreased significantly by 6 months, while the IgG2a antibody level (Th1 type-driven response) did not change over the 12 months after immunization, but the boosting effect was seen in the IgG1 antibody responses but not in the IgG2a antibody responses. After challenge infection, all immunized mice survived with negligibly patent parasitemia. These findings suggest that protective immune responses to MSP119 following immunization using oil-based Montanide ISA51 and CpG ODN as an adjuvant are very long-lasting and encourage clinical trials for malaria vaccine development.

A comparison of virus concentration methods for molecular detection and characterization of rotavirus in bivalve shellfish species

The objectives of this study were to develop a method for concentrating rotavirus, to assess the detection rate, and to characterize the genotype of naturally occurring rotavirus in bivalve shellfish species; including oysters (Saccostrea forskali), cockles (Anadara nodifera), and mussels (Perna viridis). The results demonstrated that an adsorption-twice elution-extraction method was less-time consuming method of concentrating the spiked rotavirus, yielding high sensitivity of 1.14 genome copies/g of digestive tissues from all three shellfish species, as detected using an RT-nested PCR. In seeding experiments, rotavirus as low as 1.39 genome copies was able to be detected in 4 g of digestive tissues or per sample. In the period of August 2011 to July 2012, of the 300 bivalve shellfish samples collected and tested, 24 (8.0%) were found to be contaminated with rotavirus, the figures being: oysters, 13/100 samples; mussels, 10/100 samples; and cockles, 1/100 samples. By DNA sequencing of the RT-nested PCR products and phylogenetic analysis, the rotaviruses detected were classified into G1, lineage II (4 samples); G3 (10 samples): lineage I (3 samples), lineage IIIc (3 samples), lineage IIId (3 samples), lineage IV (1 sample); G9 (6 samples); and G12, lineage III (1 sample). These findings suggest that this virus concentration method provides high sensitivity for the detection of rotavirus from the three bivalve shellfish species. The prevalence of rotavirus and the identified genotypes contribute to the molecular epidemiology of rotavirus in different shellfish species.

Genetic Diversity of Rotavirus Strains Circulating in Environmental Water and Bivalve Shellfish in Thailand

Rotavirus is a common cause of acute diarrhea in young children worldwide. This study investigated the prevalence and molecular characterization of rotavirus in environmental water and oyster samples in Thailand. A total of 114 water samples and 110 oyster samples were collected and tested for group A rotavirus using RT-nested PCR. Rotavirus genotype was identified by phylogenetic analysis of the VP7 genetic sequences. Group A rotavirus was detected in 21 water samples (18.4%) and six oyster samples (5.4%). Twenty five rotavirus strains were successfully sequenced and classified into four genotypes; G1, G2, G3, and G9. Rotavirus G1 (three strains), G2 (three strains), and G9 (two strains) demonstrated the genetic sequences similar to human strains (90%–99% nucleotide identity), whereas G3 (17 strains) was closely related to animal strains (84%–98% nucleotide identity). G1 strains belonged to lineages I (sub-lineage c) and II. G2 strains belonged to lineage II. G9 strains belonged to lineages III (sub-lineage b) and IV. G3 strains belonged to lineages I, III (sub-lineage c), and IV with a predominance of lineage I. The present study provides important information on the rotavirus strains circulating in the environment.