A molecular beacon biosensor for viral RNA detection based on HyCaSD strategy.

In the present study, a molecular beacon biosensor was developed to enable efficient detection of the viral RNAs using a previously described HyCaSD platform. The HyCaSD molecular beacon probes were labeled with the Cy5 and BHQ3 at each end of the hairpin probes. The fluorescent signal was detected immediately only when the molecular beacon probes specifically hybridized to the target sequence and unfolded their hairpin structures. This combination greatly improved the sensitivity with LOD of 100 copy equivalents per reaction (around 20-fold greater than the original HyCaSD). In addition, our MB-based HyCaSD demonstrated a single-step, single-tube and actual-time RNA detection procedure, thereby bringing it a major step closer to point-of-care diagnostic applications for viral infectious diseases.

Hybridization Cascade Plus Strand-Displacement Isothermal Amplification of RNA Target with Secondary Structure Motifs and Its Application for Detecting Dengue and Zika Viruses.

Biological RNA generally comprises secondary structure motifs which cause a problem for target RNA detection by isothermal amplification methods. The complexity of the secondary structures makes RNA targets inaccessible for probe hybridization, resulting in decreased sensitivity and selectivity. This is particularly important because the hybridization step of the isothermal amplification method requires a limited temperature range. A strand-displacement strategy can enhance the hybridization efficiency between the probe and target RNA with secondary structure motifs. A short, single-stranded segment within the secondary structure can be used as a toehold for initiating strand displacement. The strategy has been used to establish a highly sensitive isothermal amplification by a combination of a hairpin probe hybridization and strand-displacement amplification. The hairpin probe is placed on the single-stranded segment of the target RNA's secondary structure to initiate strand displacement. The probe's hybridization cascade provides a template for exponential amplification in two directions by strand-displacement amplification, designated hybridization cascade plus strand-displacement isothermal amplification (HyCaSD). The method requires no reverse transcription step. HyCaSD showed an excellent sensitivity with the limit of detection in the femtomolar (fM) range for synthetic targets as well as viral RNAs. Discrimination between DENV/ZIKV and JEV/CHIKV was successfully demonstrated using real viruses. Therefore, HyCaSD is a promising platform that can be further developed for diagnostic applications.

Differentiation between non-virulent and virulent Burkholderia pseudomallei with monoclonal antibodies to the Ara+ or Ara- biotypes.

Burkholderia pseudomallei is the causative agent of melioidosis, a fatal tropical infectious disease endemic in Southeast Asia. Environmental isolates of B. pseudomallei have two distinctive biotypes. Some soil isolates are arabinose-assimilators (Ara+ biotype) and are non-virulent in experimental animals. The others cannot assimilate arabinose (Ara- biotype) and are virulent in experimental animals. The Ara- biotype is found in almost all B. pseudomallei clinical isolates. In the present study, a panel of eight monoclonal antibodies that agglutinate the bacteria were produced and tested. The first group, Bps-D2, -D3, -D5, -L1, and -L2 agglutinated 100% of Ara+ clinical and soil isolates of B. pseudomallei. Another group Bps-A1, -A2, and -D1 agglutinated 92.9% and 90.9% of Ara- clinical and soil isolates, respectively. This panel of monoclonal antibodies may be useful for rapid differentiation between non-virulent Ara+ and virulent Ara- B. pseudomallei.

Burkholderia pseudomallei-specific recombinant protein and its potential in the diagnosis of melioidosis.

Melioidosis is an important public health problem in Southeast Asia and Northern Australia. This disease is caused by the gram-negative bacilli, Burkholderia pseudomallei. Wide spectra of clinical manifestations are observed in melioidosis ranging from asymptomatic to septicemic infection. Although serodiagnostic methods of melioidosis have been improved significantly in recent years, a highly specific diagnostic test that can differentiate asymptomatic seropositive individuals and melioidosis patients remains to be the subject of current investigations. In this study, a B. pseudomallei-specific gene, pBps-1, expressing a novel 18.7 kDa recombinant protein was selected from genomic libraries of two B. pseudomallei virulent isolates by using pooled sera from septicemic melioidosis patients. Nucleotide sequence analysis demonstrated that this gene is unique and does not show substantial similarity with any known genes in the Genbank database. The Bps-1 recombinant protein was evaluated for its potential in serodiagnosis of melioidosis by Western blot analysis. A high degree of specificity was demonstrated using sera from healthy individuals in the endemic (98.5%) and non-endemic areas (100%), with moderate sensitivity (69.7%) in melioidosis patients. The study demonstrated that this approach can be used to obtain highly specific recombinant antigens such as that described in the present report. A combination of such antigens should provide materials for successful serodiagnosis of melioidosis in the endemic areas.

Multiple replicons constitute the 6.5-megabase genome of Burkholderia pseudomallei.

Burkholderia pseudomallei is a causative agent of melioidosis, a fatal tropical infectious disease endemic in Southeast Asia and Northern Australia. In order to determine the size and characteristics of the bacterial genome, the B. pseudomallei genome and genes were analyzed by pulsed field gel electrophoresis of the undigested, intact megabase DNA, and by computational analysis of nucleotide sequences of B. pseudomallei genes which have been sequenced by several investigators and already deposited in a public database. The results showed that the B. pseudomallei genome consists of two large replicons, and that both contain ribosomal RNA gene sequences, indicating the presence of two chromosomes. The classical arabinose-negative B. pseudomallei isolate K96243 has chromosomes of approximately 3563 +/- 73 and 2974 +/- 40 kilobase-pairs in size, giving a total genome size of about 6.5 million base-pairs. The arabinose-positive nonvirulent biotype of B. pseudomallei also has two replicons which are smaller than those of the arabinose-negative biotype. Analysis of the publicly-available nucleotide sequences showed that the average B. pseudomallei gene is approximately 1031 base-pairs in size, with an average G + C content of 65.7%. The genome is gene-rich and about 89% of the coding capacity is used as coding sequences. It can therefore be estimated that the entire B. pseudomallei genome encodes about 5600 genes.

Comparison of three PCR primer sets for diagnosis of septicemic melioidosis.

Several sets of PCR primers have recently been developed for detection of Burkholderia pseudomallei. In this report, the performance of 16S rRNA gene primers (16S), rRNA spacer gene primers (spacer), and 'LPS' primers (LPS) were compared. All primer sets were tested by PCR amplification of the same DNA samples extracted from blood specimens of 46 patients from northeastern Thailand, of which 29 had melioidosis based on blood culture as a gold standard. The sensitivities were 41, 35.7, and 31% while the specificities were 47, 59, and 100% for the 16S, spacer, and LPS primers, respectively. The positive predictive values were 60, 59, and 100%, while negative predictive values were 35, 34, and 46%, for these primers. The low sensitivity of PCR was suspected to be because of small numbers of bacteria in the samples. In addition, one primer set could not detect all B. pseudomallei strains. To make PCR for melioidosis more practical, bacterial concentration steps must be added. Lastly, mixed infection of patients in endemic areas may be the cause of controversial false positive PCR results, and should be further investigated.

HLA association with hepatitis C virus infection.

Hepatitis is one of the most important infectious diseases in Thailand. The knowledge of host factors that influence the course of the disease is still limited. In this study, the HLA class I and class II phenotypes were analyzed in the 2 groups of HCV-infected Thai populations. The first group included 43 individuals with transient HCV infection (HCV antibody positive, HCV RNA PCR negative), and the second included 57 individuals with persistent chronic HCV infection (HCV antibody positive, PCR positive). HLA class I typing was performed by 2-stage microlymphocytotoxicity test, and HLA class II typing, by PCR-SSO. No significant difference in the frequencies of HLA-A and -B antigens was observed between the 2 groups of HCV-infected individuals. The frequency of DRB1*0301 and DQB1*0201 was significantly higher in the persistent-infection group than in the transient-infection group (Pc = 0.03, Pc = 0.04, respectively). In addition, DRB1*0701 and DQA1*0201 were significantly decreased in all the HCV-infected patients compared with levels in the normal controls (Pc = 0.003, Pc = 0.001, respectively). This study demonstrated that DRB1*0301 and DQB1*0201 are associated with persistent HCV infection, whereas DRB1*0701 and DQA*0201 are associated with protection against HCV infection.

Dengue virus specific T cell responses to live attenuated monovalent dengue-2 and tetravalent dengue vaccines.

The proliferative T cell responses to dengue vaccines were studied using the parental strains of dengue vaccines as antigens in 26 dengue immune individuals who resided in Bangkok which is the endemic area of dengue infection. The magnitude of the T cell responses in subjects with flavivirus cross-reactive neutralizing antibody was much higher and the cross-reactivity was broader than in those with dengue serotype-specific neutralizing antibodies, Japanese encephalitis (JE) specific antibodies or dengue cross-reactive antibodies. The T cell response in those with neutralizing antibody against a single serotype or in those who had dengue cross-reactive neutralizing antibody was relatively low, independent of the level or degree of cross-reactivity of the antibody. Evaluation of the proliferative T cell responses in 8 recipients of the monovalent dengue-2 (16681-PDK53) or the tetravalent dengue vaccines demonstrated that both vaccines induced high levels of neutralizing antibody as well as high levels of T cell responses to all serotypes of dengue virus. These results indicate that the evaluated dengue vaccines efficiently induced humoral and cell mediated immunity comparable to natural infection with dengue virus.

Rapid identification of Burkholderia pseudomallei in blood cultures by latex agglutination using lipopolysaccharide-specific monoclonal antibody.

Melioidosis, an infection caused by Burkholderia pseudomallei, is endemic in Southeast Asia. The septicemic form of melioidosis is the leading cause of death due to community-acquired bacteremia in the northeastern part of Thailand. The delay in isolation and identification of the causative organism is a major contributing factor to the high mortality. The present study describes the evaluation of a latex agglutination test for rapid identification of the bacteria directly from blood cultures. The Bps-L1 monoclonal antibody recognized the lipopolysaccharide antigen of 96.8% of B. pseudomallei clinical isolates and was highly specific for B. pseudomallei. The diagnostic value of the latex agglutination test based on Bps-L1 monoclonal antibody was prospectively evaluated in an area endemic for melioidosis. The agglutination test kit was evaluated in 88 blood cultures with gram-negative bacteria identified with Gram staining. The sensitivity and specificity of the test kit were both 100%. These results indicated that the detection of B. pseudomallei lipopolysaccharide by specific monoclonal antibody in a latex agglutination format is clinically useful for the rapid identification of the bacteria in blood cultures in areas endemic for melioidosis.

Polymorphism in the promoter region of tumor necrosis factor-alpha gene is associated with severe meliodosis.

Melioidosis is an important infectious disease endemic in Southeast Asia and the Northern territories of Australia. Septicemic melioidosis, is the leading cause of fatality from community acquired septicemia in northeastern part of Thailand where death often occurs within a few days after hospitalization. The present study was carried out to investigate the polymorphisms of the position -308 promoter region of the TNF-alpha gene, as well as of the intron 1 of the TNF-beta gene in patients with melioidosis compared with normal uninfected controls in the same endemic area. The gene frequency of TNF2 allele was significantly higher in melioidosis patients compared with control subjects (p = 0.0097, relative risk 2.32). The increase in TNF2 allele in melioidosis patients was found in both heterozygous and homozygous forms. In addition, the increase in TNF2 allele was most apparent in patients who had fatal outcome from septicemic melioidosis (p = 0.017), but was also observed with lesser degree in other groups of melioidosis patients. However, no difference in the frequency of TNF-beta polymorphism the melioidosis patients was observed.

Phylogenetic analysis of Ara+ and Ara- Burkholderia pseudomallei isolates and development of a multiplex PCR procedure for rapid discrimination between the two biotypes.

A Burkholderia pseudomallei-like organism has recently been identified among some soil isolates of B. pseudomallei in an area with endemic melioidosis. This organism is almost identical to B. pseudomallei in terms of morphological and biochemical profiles, except that it differs in ability to assimilate L-arabinose. These Ara+ isolates are also less virulent than the Ara- isolates in animal models. In addition, clinical isolates of B. pseudomallei available to date are almost exclusively Ara-. These features suggested that these two organisms may belong to distinctive species. In this study, the 16S rRNA-encoding genes from five clinical (four Ara- and one Ara+) and nine soil isolates (five Ara- and four Ara+) of B. pseudomallei were sequenced. The nucleotide sequences and phylogenetic analysis indicated that the 16S rRNA-encoding gene of the Ara+ biotype was similar to but distinctively different from that of the Ara- soil isolates, which were identical to the classical clinical isolates of B. pseudomallei. The nucleotide sequence differences in the 16S rRNA-encoding gene appeared to be specific for the Ara+ or Ara- biotypes. The differences were, however, not sufficient for classification into a new species within the genus Burkholderia. A simple and rapid multiplex PCR procedure was developed to discriminate between Ara- and Ara+ B. pseudomallei isolates. This new method could also be incorporated into our previously reported nested PCR system for detecting B. pseudomallei in clinical specimens.

HLA-DR and -DQ associations with melioidosis.

Melioidosis is an important infectious disease of southeast Asia caused by an intracellular bacterium, Burkholderia pseudomallei. Cellular immunity is postulated to play important roles in immunity to melioidosis that may influence the severity and clinical outcome of the disease. The present study was undertaken to investigate possible associations of melioidosis with HLA class II alleles. HLA typing of HLA-DRB1, -DQA1, and -DQB1 was performed using polymerase chain reaction and sequence-specific oligonucleotide hybridization (PCR-SSO). Seventy-nine melioidosis patients and 105 healthy, ethnically and geographically matched controls were studied. Among 24 DRB1 alleles, 7 DQA1 alleles, and 13 DQB1 alleles identified in this population, an association with melioidosis was observed with DRB1*1602 which was increased in melioidosis patients (10.1%) compared to normal controls (4.8%), p = 0.047 (odds ratio (OR) = 2.25). In addition, significant increase of DRB1*1602 allele frequency and decrease of DQA1*03 were also observed in septicemic melioidosis patients, the most severe form of the disease (p = 0.01, OR = 3.10; and p = 0.047, respectively). Furthermore, a trend of association of DRB1*0701, DQA1*0201, and DQB1*0201 with relapse cases of melioidosis was also noted. In contrast, no HLA association was observed in localized melioidosis or melioidosis with diabetes mellitus. These findings provide the suggestive evidence of an immunogenetic basis of certain aspects of melioidosis.

A serotyping assay for hepatitis C virus in Southeast Asia.

A serotyping assay for hepatitis C virus (HCV) was evaluated with samples from Thailand, where the distribution of HCV genotypes was different from that in Western countries where the assay was designed and validated. The sensitivity of the assay was low (58%) for HCV RNA-positive samples compared to that of the genotyping assay (95%, P < 0.01). In addition, only 36% of anti-HCV-positive but HCV RNA-negative samples could be serotyped. The serotypes and genotypes were identical in 96% of the samples that could be typed by both methods. Most of the samples with genotype 6, which was common in Southeast Asia, were nontypeable by this serotyping assay.

Genetically engineered single-chain Fvs of human immunoglobulin against hepatitis C virus nucleocapsid protein derived from universal phage display library.

Specific single-chain Fvs (scFvs) of human immunoglobulin that specifically recognized the recombinant hepatitis C virus (HCV) nucleocapsid protein were isolated from a large phage display antibody library. This universal library of genetically engineered filamentous phagemids displayed random pairings of the variable regions of both human heavy and light chain immunoglobulin in the scFv format. Specific clones were isolated by affinity selection with purified recombinant HCV protein fused to glutathione-S-transferase (GST). The GST-specific clones were excluded by blocking the phagemid library with GST prior to the selection. After 4 rounds of selection, the HCV-reactive clones were enriched by a factor of 100,000. About 4% and 9% of the clones from rounds 4 and 5, respectively, specifically reacted to the HCV portion of the fusion protein in an enzyme immunoassay. The specificity was confirmed by specific binding inhibition with plasma from an HCV-infected individual. Nucleotide sequence analysis of 3 HCV-specific clones indicated that all 3 clones contained an almost identical VH gene sequence which was derived from the VH3 germline gene family. These clones had different VL gene sequences of the lambda type. There were some differences between nucleotide and amino acid sequences of the HCV-specific scFv genes and those of the closest matched germline genes, indicating the presence of somatic mutation. This study illustrated the feasibility of using antibody engineering technology with the universal phage display library to isolate human antibodies with predefined specificity to important microbial pathogen which may be useful for future therapeutic purpose.

High prevalence of hepatitis C infection among blood donors in northeastern Thailand.

Previous studies on the prevalence of hepatitis C virus (HCV) infection in Asian countries reported an average prevalence of less than 1.5%. In this study a combination of second- and third-generation enzyme immunoassays (EIAs), immunoblot analysis, and polymerase chain reaction was used to evaluate the prevalence of HCV infection in 3,255 volunteer blood donors in northeastern Thailand. Antibodies to HCV were detected in 6.5% of male blood donors and 0.9% of female blood donors, giving an overall prevalence of 5.6% in this population (gender-adjusted prevalence of 3.7%). The prevalence was higher in males than in females (P < 0.0001) and increased with age, reaching a peak at 31-40 years of age. More than 90% of the EIA-positive samples tested positive by immunoblot analysis, giving an estimated minimal prevalence of antibodies to HCV in the blood donors of 5.2%. Approximately 80% of the EIA-positive blood donors were viremic as determined by the presence of HCV RNA detected by the polymerase chain reaction, indicating that at least 4.5% of volunteer blood donors had detectable HCV RNA and were considered potentially infectious. The prevalence of HCV infection in this population was higher than that in previous reports for central and northern Thailand, while the prevalence of HBV infection was similar to that in other regions of the country. This study clearly demonstrated a very high prevalence of HCV infection in northeastern Thailand, especially in the male population.

Speed of detection of Burkholderia pseudomallei in blood cultures and its correlation with the clinical outcome.

Burkholderia pseudomallei is a major cause of fatalities from nonhospital-acquired, gram-negative bacterial septicemia in northeastern part of Thailand. Rapid isolation of the bacterium is critical for diagnosis and treatment. Bacterial culture is currently the gold standard method for laboratory diagnosis of melioidosis. The present study describes the time to detection of B. pseudomallei in blood cultures using a BacT/Alert automated blood culture system, and the correlation between the speed of detection and the clinical outcome of the patients. Of 813 consecutive positive blood cultures, 75 blood cultures from 71 patients were positive for B. pseudomallei. The mean +/- SD time to detection of growth of B. pseudomallei was 23.9 +/- 14.9 hr (95% confidence interval = 20.4-27.5 hr). A total of 62.5% of the B. pseudomallei-positive cultures was detected within 24 hr of incubation, and 93.1% within 48 hr. Interestingly, fatalities occurred in 73.7% of those in which the bacterial growth was detected within the first 24 hr, as compared with only 40.9% in those with a time to detection of culture more than 24 hr (P = 0.012). The shorter time of detection of the bacterial growth in blood cultures may reflect a higher bacterial level in the patient at the time blood was taken, and may be responsible for the poor clinical outcome.

Genotypic distribution of hepatitis C virus in different regions of Thailand.

The genotypic distribution of hepatitis C virus (HCV) isolated from blood donors from four major regions of Thailand was studied by reverse hybridization assays. PCR-amplified products from the 5' noncoding and core regions of the viral genome were hybridized to genotype- and subtype-specific probes which were immobilized on the nitrocellulose membrane. Of 332 anti-HCV-positive plasma samples studied, 71% contained HCV RNA. HCV genotype 3a was the most prevalent genotype (39%), followed by genotype 1b (20%) and genotype 6 group variants (18%). HCV genotype 1a was identified among 9% of all isolates. Other genotypes (genotype 1 which was neither 1a nor 1b, genotype 3b, and an unclassified genotype) were uncommon. There was no difference in the mean age of the donors infected with different HCV genotypes. The genotypic distribution pattern of HCV was similar among HCV isolates from different regions of Thailand.

Diagnostic value of an antibody enzyme-linked immunosorbent assay using affinity-purified antigen in an area endemic for melioidosis.

Melioidosis, an infection caused by Burkholderia pseudomallei, is endemic in southeast Asia. The septicemic form of melioidosis is the leading cause of death from nonhospital-acquired septicemia in the northeastern part of Thailand. A major factor that contributes to the high mortality is the delay in isolation and identification of the causative organism. The present study was undertaken to evaluate the use of enzyme-linked immunosorbent assays based on an immunoaffinity-purified antigen for detecting specific IgG and IgM antibodies to this organism as a rapid serodiagnostic method for melioidosis. The diagnostic value of these tests was evaluated in an actual clinical situation in an area endemic for melioidosis. The specificity of specific IgG test (82.5%) and the specific IgM test (81.8%) were significantly better than that of the indirect hemagglutination (IHA) test (74.7%). The sensitivity of the specific IgG assay (85.7%) was higher than that of the IHA test (71.0%) and the specific IgM test (63.5%). Specific IgG antibody was detected in a majority of septicemic melioidosis (87.8%), as well as in localized forms (82.6%). The specific IgG test was also better than the specific IgM test and the IHA test in identifying acute melioidosis cases in the first five days after admission. In addition, the IgG antibody level to this antigen remained high over a period of more than five years in those who had recovered from melioidosis and remained clinically free of the disease. These results indicate that the detection of specific IgG antibody is clinically useful for the diagnosis of acute melioidosis in an endemic area.