Optimization and prospective evaluation of sensitive real-time PCR assays with an internal control for the diagnosis of melioidosis in Thailand.

IMPORTANCE: Melioidosis is a serious infectious disease caused by Burkholderia pseudomallei, an environmental Gram-negative bacterium. Early detection of B. pseudomallei infection is crucial for successful antibiotic treatment and reducing mortality rates associated with melioidosis. Bacteria culture is currently used to identify B. pseudomallei in clinical samples, but the method is slow. Therefore, there is a need for more accurate and sensitive molecular-based diagnostic methods that can detect B. pseudomallei in all sample types, including samples from blood. We developed an optimal DNA extraction method for B. pseudomallei from plasma samples and used an internal control for real-time PCR. We evaluated six PCR target genes and identified the most effective target for the early detection of B. pseudomallei infection in patients. To prevent delays in the treatment of melioidosis that can lead to fatal outcomes, we recommend implementing this new approach for routine early detection of B. pseudomallei in clinical settings.

Identification and function of a novel human memory-like NK cell population expressing CD160 in melioidosis.

NK cells are endowed with immunological memory to a range of pathogens but the development of NK cell memory in bacterial infections remains elusive. Here, we establish an assay inducing memory-like NK cell response to Burkholderia pseudomallei, the causative agent of the severe bacterial disease called melioidosis, and explore NK cell memory in a melioidosis patient cohort. We show that NK cells require bacteria-primed monocytes to acquire memory-like properties, demonstrated by bacteria-specific responses, features that strongly associate with CD160 expression. Induction of this memory-like NK cell is partly dependent on CD160 and IL-12R. Importantly, CD160 expression identifies memory-like NK cells in a cohort of recovered melioidosis patients with heightened responses maintained at least 3 months post hospital admission and reduced numbers of this cell population independently correlate with recurrent melioidosis. These newly identified memory-like NK cells are a promising target for future vaccine design and for monitoring protection against infection.

Prospective Analysis of Antibody Diagnostic Tests and TTS1 Real-Time PCR for Diagnosis of Melioidosis in Areas Where It Is Endemic.

Melioidosis is a tropical infectious disease caused by Burkholderia pseudomallei. Melioidosis is associated with diverse clinical manifestations and high mortality. Early diagnosis is needed for appropriate treatment, but it takes several days to obtain bacterial culture results. We previously developed a rapid immunochromatography test (ICT) based on hemolysin coregulated protein 1 (Hcp1) and two enzyme-linked immunosorbent assays (ELISAs) based on Hcp1 (Hcp1-ELISA) and O-polysaccharide (OPS-ELISA) for serodiagnosis of melioidosis. This study prospectively validated the diagnostic accuracy of the Hcp1-ICT in suspected melioidosis cases and determined its potential use for identifying occult melioidosis cases. Patients were enrolled and grouped by culture results, including 55 melioidosis cases, 49 other infection patients, and 69 patients with no pathogen detected. The results of the Hcp1-ICT were compared with culture, a real-time PCR test based on type 3 secretion system 1 genes (TTS1-PCR), and ELISAs. Patients in the no-pathogen-detected group were followed for subsequent culture results. Using bacterial culture as a gold standard, the sensitivity and specificity of Hcp1-ICT were 74.5% and 89.8%, respectively. The sensitivity and specificity of TTS1-PCR were 78.2% and 100%, respectively. The diagnostic accuracy was markedly improved if the Hcp1-ICT results were combined with TTS1-PCR results (sensitivity and specificity were 98.2% and 89.8%, respectively). Among patients with initially negative cultures, Hcp1-ICT was positive in 16/73 (21.9%). Five of the 16 patients (31.3%) were subsequently confirmed to have melioidosis by repeat culture. The combined Hcp1-ICT and TTS1-PCR test results are useful for diagnosis, and Hcp1-ICT may help identify occult cases of melioidosis.

Melioidosis Patient Survival Correlates With Strong IFN-γ Secreting T Cell Responses Against Hcp1 and TssM.

Melioidosis, caused by the Gram-negative bacterium Burkholderia pseudomallei, is a serious infectious disease with diverse clinical manifestations. The morbidity and mortality of melioidosis is high in Southeast Asia and no licensed vaccines currently exist. This study was aimed at evaluating human cellular and humoral immune responses in Thai adults against four melioidosis vaccine candidate antigens. Blood samples from 91 melioidosis patients and 100 healthy donors from northeast Thailand were examined for immune responses against B. pseudomallei Hcp1, AhpC, TssM and LolC using a variety of cellular and humoral immune assays including IFN-γ ELISpot assays, flow cytometry and ELISA. PHA and a CPI peptide pool were also used as control stimuli in the ELISpot assays. Hcp1 and TssM stimulated strong IFN-γ secreting T cell responses in acute melioidosis patients which correlated with survival. High IFN-γ secreting CD4+ T cell responses were observed during acute melioidosis. Interestingly, while T cell responses of melioidosis patients against the CPI peptide pool were low at the time of enrollment, the levels increased to the same as in healthy donors by day 28. Although high IgG levels against Hcp1 and AhpC were detected in acute melioidosis patients, no significant differences between survivors and non-survivors were observed. Collectively, these studies help to further our understanding of immunity against disease following natural exposure of humans to B. pseudomallei as well as provide important insights for the selection of candidate antigens for use in the development of safe and effective melioidosis subunit vaccines.

Rapid Clinical Screening of Burkholderia pseudomallei Colonies by a Bacteriophage Tail Fiber-Based Latex Agglutination Assay.

Melioidosis is a life-threatening disease in humans caused by the Gram-negative bacterium Burkholderia pseudomallei. As severe septicemic melioidosis can lead to death within 24 to 48 h, a rapid diagnosis of melioidosis is critical for ensuring that an optimal antibiotic course is prescribed to patients. Here, we report the development and evaluation of a bacteriophage tail fiber-based latex agglutination assay for rapid detection of B. pseudomallei infection. Burkholderia phage E094 was isolated from rice paddy fields in northeast Thailand, and the whole genome was sequenced to identify its tail fiber (94TF). The 94TF complex was structurally characterized, which involved identification of a tail assembly protein that forms an essential component of the mature fiber. Recombinant 94TF was conjugated to latex beads and developed into an agglutination-based assay (94TF-LAA). 94TF-LAA was initially tested against a large library of Burkholderia and other bacterial strains before a field evaluation was performed during routine clinical testing. The sensitivity and specificity of the 94TF-LAA were assessed alongside standard biochemical analyses on 300 patient specimens collected from an area of melioidosis endemicity over 11 months. The 94TF-LAA took less than 5 min to produce positive agglutination, demonstrating 98% (95% confidence interval [CI] of 94.2% to 99.59%) sensitivity and 83% (95% CI of 75.64% to 88.35%) specificity compared to biochemical-based detection. Overall, we show how a Burkholderia-specific phage tail fiber can be exploited for rapid detection of B. pseudomallei. The 94TF-LAA has the potential for further development as a supplementary diagnostic to assist in clinical identification of this life-threatening pathogen. IMPORTANCE Rapid diagnosis of melioidosis is essential for ensuring that optimal antibiotic courses are prescribed to patients and thus warrants the development of cost-effective and easy-to-use tests for implementation in underresourced areas such as northeastern Thailand and other tropical regions. Phage tail fibers are an interesting alternative to antibodies for use in various diagnostic assays for different pathogenic bacteria. As exposed appendages of phages, tail fibers are physically robust and easy to manufacture, with many tail fibers (such as 94TF investigated here) capable of targeting a given bacterial species with remarkable specificity. Here, we demonstrate the effectiveness of a latex agglutination assay using a Burkholderia-specific tail fiber 94TF against biochemical-based detection methods that are the standard diagnostic in many areas where melioidosis is endemic.

Nested polymerase chain reaction for detection of Helicobacter pylori in gastric biopsy specimens.

Sensitivity and specificity are important for tests used to defect Helicobacter pylori infection from gastric biopsy specimens. Molecular methods, such as PCR and nested PCR, are sensitive methods for H. pylori detection. The objective of this study was to evaluate the performance of PCR and nested PCR compared to culture, the rapid urease test (RUT) and histology for the diagnosis of H. pylori in 130 gastric biopsy specimens from symptomatic dyspeptic patients. Sensitivity and specificity with PCR were 91 and 100% and with nested PCR were 95 and 97%, respectively. H. pylori was detected by PCR and nested PCR at levels as low as 125 fg (70 cells) and 25 fg (14 cells), respectively. These results suggest nested PCR is a highly sensitive direct method to detect H. pylori infection from biopsy specimens.

Seroreactivity to specific antigens of Helicobacter pylori infection is associated with an increased risk of the dyspeptic gastrointestinal diseases.

OBJECTIVES: The correlation between seroreactivity to Helicobacter pylori-specific antigens and clinical outcomes in gastrointestinal disease remains unresolved. We investigated the anti-H. pylori antibody profile in northeast Thai dyspeptic patients with gastrointestinal disease in order to identify any H. pylori antigens that may be associated with an increased risk of gastrointestinal disease. PATIENTS AND METHODS: Eighty-nine H. pylori-infected dyspeptic patients (44 non-ulcer, 23 peptic ulcer, 22 gastric cancer) were included in the study. Patients were considered to have H. pylori infection when at least one invasive method (i.e., culture, rapid urease test, and histology on biopsy specimens) and serological tests including a commercial ELISA (Pyloriset EIA-GIII) and a commercial immunoblot (Helicoblot 2.1; Genelabs Diagnostics), were positive. In addition, the sera of 20 H. pylori-infected blood donors and 10 H. pylori-non-infected blood donors were also randomly collected and analyzed for H. pylori infection by ELISA and Helicoblot 2.1. RESULTS: Immunoreactive protein bands at 116-kDa, 89-kDa, 37-kDa, 35-kDa, 30-kDa, 19.5-kDa, and the current infection marker for H. pylori-infected patients had average frequencies of 97.8%, 77.5%, 36.0%, 25.8%, 79.8%, 58.4%, and 69.7%, respectively. The immunoreactive patterns obtained from the H. pylori-infected patients and H. pylori-infected blood donors were similar. The antibodies to VacA and CagA antigens were not significantly different among the H. pylori-infected gastroduodenal patient groups. The simultaneous presence of antibody to 19.5-kDa antigen and absence of antibody to 35-kDa antigen was associated with an increased risk of gastric cancer (p<0.05). The immunoreactive band to 35-kDa antigen was found at significantly higher levels in peptic ulcer patients, and the 37-kDa antigen was found at significantly higher levels in non-ulcer patients (both p<0.05). Significantly low levels of antibodies to 23-kDa and 85-kDa antigens were found associated with peptic ulcer (p<0.05). CONCLUSIONS: We confirm that the universal presence of CagA and VacA in H. pylori-infected patients in Thailand is independent of the gastroduodenal disease. The presence or absence of antibodies to H. pylori-specific antigens may be useful as indirect markers in the screening of H. pylori-infected patients, and may have specific protection roles in H. pylori-related gastroduodenal diseases.