Diagnostic accuracy of automation and non-automation techniques for identifying Burkholderia pseudomallei: A systematic review and meta-analysis.

BACKGROUND: Burkholderia pseudomallei, a Gram-negative pathogen, causes melioidosis. Although various clinical laboratory identification methods exist, culture-based techniques lack comprehensive evaluation. Thus, this systematic review and meta-analysis aimed to assess the diagnostic accuracy of culture-based automation and non-automation methods. METHODS: Data were collected via PubMed/MEDLINE, EMBASE, and Scopus using specific search strategies. Selected studies underwent bias assessment using QUADAS-2. Sensitivity and specificity were computed, generating pooled estimates. Heterogeneity was assessed using I2 statistics. RESULTS: The review encompassed 20 studies with 2988 B. pseudomallei samples and 753 non-B. pseudomallei samples. Automation-based methods, particularly with updating databases, exhibited high pooled sensitivity (82.79%; 95% CI 64.44-95.85%) and specificity (99.94%; 95% CI 98.93-100.00%). Subgroup analysis highlighted superior sensitivity for updating-database automation (96.42%, 95% CI 90.01-99.87%) compared to non-updating (3.31%, 95% CI 0.00-10.28%), while specificity remained high at 99.94% (95% CI 98.93-100%). Non-automation methods displayed varying sensitivity and specificity. In-house latex agglutination demonstrated the highest sensitivity (100%; 95% CI 98.49-100%), followed by commercial latex agglutination (99.24%; 95% CI 96.64-100%). However, API 20E had the lowest sensitivity (19.42%; 95% CI 12.94-28.10%). Overall, non-automation tools showed sensitivity of 88.34% (95% CI 77.30-96.25%) and specificity of 90.76% (95% CI 78.45-98.57%). CONCLUSION: The study underscores automation's crucial role in accurately identifying B. pseudomallei, supporting evidence-based melioidosis management decisions. Automation technologies, especially those with updating databases, provide reliable and efficient identification.

A systematic review and meta-analysis of the global prevalence and relationships among Burkholderia pseudomallei sequence types isolated from humans, animals, and the environment.

Background and Aim: Burkholderia pseudomallei, a highly pathogenic bacterium responsible for melioidosis, exhibits ecological ubiquity and thrives within soil and water reservoirs, posing significant infection risks to humans and animals through direct contact. The aim of this study was to elucidate the genetic diversity and prevalence patterns of B. pseudomallei sequence types (STs) across a global spectrum and to understand the relationships between strains isolated from different sources. Materials and Methods: We performed a systematic review and meta-analysis in this study. Extensive research was carried out across three comprehensive databases, including PubMed, Scopus, and ScienceDirect with data collected from 1924 to 2023. Results: A total of 40 carefully selected articles contributed 2737 B. pseudomallei isolates attributed to 729 distinct STs and were incorporated into the systematic review. Among these, ST46 emerged as the most prominent, featuring in 35% of the articles and demonstrating a dominant prevalence, particularly within Southeast Asia. Moreover, ST51 consistently appeared across human, animal, and environmental studies. Subsequently, we performed a meta-analysis, focusing on nine specific STs: ST46, ST51, ST54, ST70, ST84, ST109, ST289, ST325, and ST376. Surprisingly, no statistically significant differences in their pooled prevalence proportions were observed across these compartments for ST46, ST70, ST289, ST325, and ST376 (all p > 0.69). Conversely, the remaining STs, including ST51, ST54, ST84, and ST109, displayed notable variations in their prevalence among the three domains (all p < 0.04). Notably, the pooled prevalence of ST51 in animals and environmental samples surpassed that found in human isolates (p < 0.01). Conclusion: To the best of our knowledge, this study is the first systematic review and meta-analysis to investigate the intricate relationships between STs and their sources and contributes significantly to our understanding of B. pseudomallei diversity within the One Health framework.

First isolation of verocytotoxin-producing Escherichia coli O157:H7 from sports animals in Southern Thailand.

Background and Aim: Escherichia coli O157:H7 is enterohemorrhagic E. coli, which produces verocytotoxin or Shiga toxin. It is a well-known cause of severe diseases in humans worldwide. Cattle and other ruminants are the main reservoirs of this organism. Sports animals, such as fighting bulls, riding horses, and fighting cocks, are economic animals in Southern Thailand. This study aimed to identify E. coli O157:H7 from the rectal swabs of these sports animals and determine the antimicrobial susceptibility patterns of isolated bacteria. Materials and Methods: The rectal swabs were collected from 34 fighting bulls, 32 riding horses, and 31 fighting cocks. The swabs were cultured on MacConkey (MAC) Agar; the suspected colonies were then identified by VITEK® 2 GN card, and the antimicrobial susceptibility was tested by VITEK® 2 AST N194 in VITEK® 2 Compact automation. Escherichia coli O157:H7 was confirmed by culturing on sorbitol MAC agar, the ability to grow at 44°C, and the presence of H7 antigen. In addition, the eaeA (E. coli attaching and effacing), along with stx1 and stx2 (Shiga cytotoxins) genes, were determined using polymerase chain reaction. Finally, the cytotoxicity of Shiga toxin was confirmed using the Vero cytotoxicity test. Results: Fifty-five suspected isolates (56.70%), which were collected from 19 fighting bulls (55.88%), 13 riding horses (40.63%), and 23 fighting cocks (71.13%), were identified as E. coli. However, one sample (Bull H9/1) from fighting bulls had an equal confidence level (50%) for E. coli and E. coli O157. The confirmation of this isolate demonstrated that it was sorbitol non-fermenter, could assimilate L-lactate, was unable to grow well at 44°C, and reacted with anti-serum to H7 antigen. In addition, it was positive with stx2 and eaeA genes, and the toxin affected Vero cells by a dose-dependent response. The antimicrobial susceptibility test revealed that five out of 55 (9.09%) E. coli isolates were resistant to antimicrobial agents. All five isolates (21.74%) were collected from fighting cocks. Escherichia coli Cock H4/3 was only one of the five isolates resistant to three antimicrobial agents (ciprofloxacin, moxifloxacin, and trimethoprim/sulfamethoxazole). Fortunately, it was not multidrug-resistant bacteria. Conclusion: This is the first report on detection of E. coli O157:H7 in fighting bulls and antibiotic-resistant characteristic of E. coli in fighting cocks in Southern Thailand. This research is beneficial in preventing the dissemination of E. coli O157:H7 or antimicrobial agent-resistant E. coli in sports animals and humans.

High prevalence and genetic diversity of a single ancestral origin azole-resistant Aspergillus fumigatus in indoor environments at Walailak University, Southern Thailand.

This study explored the prevalence, genetic diversity, and population structure of azole-resistant Aspergillus fumigatus (ARAf) at Walailak University in Southern Thailand. Three hundred samples were collected from dwellings and workplaces, screened for azole resistance, and tested for drug susceptibility. Molecular detection of alterations in the cyp51A gene and CSP1 typing was performed. Nucleotide polymorphism and haplotype diversity were calculated, and selective neutrality tests were performed. In total, 62 A. fumigatus isolates were identified, with 17 isolates displaying resistance to medical azoles. The prevalence of ARAf in the A. fumigatus isolates was 27.4%. Almost all azole-resistant isolates harboured an amino acid substitution in the hotspot region of the cyp51A gene, especially at or nearby the G54 residue that has been reported as a cause of azole resistance arising from long-term azole treatment. Moreover, some of the ARAf isolates harboured tandem repeats in the promoter region, which have been reported as a cause of resistance arising from the use of azole fungicides in crop protection. Finally, selective neutrality testing also suggested an impact of natural selection on DNA diversity. Therefore, we hypothesize that the factors causing the high prevalence of ARAf in this area are both in vivo- and ex vivo-acquired resistance.

Evaluation of Susceptibility of the Human Pathogen Helicobacter pylori to the Antibiotic Capreomycin.

Helicobacter pylori infection causes gastritis, peptic ulcer disease, mucosa-associated lymphoid tissue lymphoma, and gastric cancer and can also promote thrombosis. It is estimated that approximately 4.5 billion individuals are infected, thus rendering H. pylori the most prevalent microbial pathogen. Currently established regimes for antibiotic treatment are massively challenged by increasing drug resistance and the development of novel antimicrobial therapies is urgently required. The antibiotic capreomycin is clinically used against multiple drug-resistant strains of Mycobacterium tuberculosis. It targets the complex between TlyA, a hemolysin- and RNA-binding protein, and the bacterial rRNA. In this study we have explored the possible antibacterial effects of capreomycin against several strains of H. pylori and found only moderate activity which was comparable to metronidazole-resistant strains. Molecular docking of capreomycin to TlyA proteins from H. pylori and M. tuberculosis identified several residues within TlyA which interact with the drug; however, binding affinities of H. pylori- TlyA for capreomycin appear to be higher than those of Mycobacterium- TlyA. The data suggest that capreomycin may warrant further investigations into its potential use as antibiotic against H. pylori.

Peganum harmala Extract Has Antiamoebic Activity to Acanthamoeba triangularis Trophozoites and Changes Expression of Autophagy-Related Genes.

Peganum harmala, a well-known medicinal plant, has been used for several therapeutic purposes as it contains numerous pharmacological active compounds. Our study reported an anti-parasitic activity of P. harmala seed extract against Acanthamoeba triangularis. The stress induced by the extract on the surviving trophozoites for Acanthamoeba encystation and vacuolization was examined by microscopy, and transcriptional expression of Acanthamoeba autophagy-related genes was investigated by quantitative PCR. Our results showed that the surviving trophozoites were not transformed into cysts, and the number of trophozoites with enlarged vacuoles were not significantly different from that of untreated control. Molecular analysis data demonstrated that the mRNA expression of tested AcATG genes, i.e., ATG3, ATG8b, and ATG16, was at a basal level along the treatment. However, upregulation of AcATG16 at 24 h post treatment was observed, which may indicate an autophagic activity of this protein in response to the stress. Altogether, these data revealed the anti-Acanthamoeba activity of P. harmala extract and indicated the association of autophagy mRNA expression and cyst formation under the extract stress, representing a promising plant for future drug development. However, further identification of an active compound and a study of autophagy at the protein level are needed.

Use of Recombinant Escherichia coli Strains in Immunofluorescence Assays for Melioidosis Diagnosis.

Burkholderia pseudomallei is a Gram-negative bacterium and the causative agent of melioidosis in humans and animals in the tropics. The clinical manifestations of melioidosis are diverse, ranging from localized infections to whole-body sepsis. The effective serological method is crucial for the point-of-care diagnosis of melioidosis. The aim of this study was to develop indirect immunofluorescence assay (IFA)-based methods for detecting immunoglobulin G (IgG) antibodies in melioidosis patients. These methods use whole-cell antigens made from recombinant E. coli strains that express major B. pseudomallei antigens, including TssM, OmpH, AhpC, BimA, and Hcp1. A total of 271 serum samples from culture-confirmed melioidosis patients (n = 81), patients with other known infections (n = 70), and healthy donors (n = 120) were tested. Our study showed that the recombinant TssM strain had the highest performance, with 92.6% sensitivity, 100% specificity, 100% positive predictive value, 96.9% negative predictive value, 97.8% efficiency, 97.0% accuracy, and no cross-reactivity. The method agreement analysis based on k efficiency calculations showed that all five IFA methods perfectly agreed with the standard culturing method, while the traditional indirect hemagglutination (IHA) method moderately agreed with the culture. In summary, our investigations showed that the TssM-IFA method could be used for melioidosis diagnosis.

Amoebicidal activity of Cassia angustifolia extract and its effect on Acanthamoeba triangularis autophagy-related gene expression at the transcriptional level.

Cassia angustifolia Vahl. plant is used for many therapeutic purposes, for example, in people with constipation, skin diseases, including helminthic and parasitic infections. In our study, we demonstrated an amoebicidal activity of C. angustifolia extract against Acanthamoeba triangularis trophozoite at a micromolar level. Scanning electron microscopy (SEM) images displayed morphological changes in the Acanthamoeba trophozoite, which included the formation of pores in cell membrane and the membrane rupture. In addition to the amoebicidal activity, effects of the extract on surviving trophozoites were observed, which included cyst formation and vacuolization by a microscope and transcriptional expression of Acanthamoeba autophagy in response to the stress by quantitative polymerase chain reaction. Our data showed that the surviving trophozoites were not transformed into cysts and the trophozoite number with enlarged vacuole was not significantly different from that of untreated control. Molecular analysis data demonstrated that the mRNA expression of AcATG genes was slightly changed. Interestingly, AcATG16 decreased significantly at 12 h post treatment, which may indicate a transcriptional regulation by the extract or a balance of intracellular signalling pathways in response to the stress, whereas AcATG3 and AcATG8b remained unchanged. Altogether, these data reveal the anti-Acanthamoeba activity of C. angustifolia extract and the autophagic response in the surviving trophozoites under the plant extract pressure, along with data on the formation of cysts. These represent a promising plant for future drug development. However, further isolation and purification of an active compound and cytotoxicity against human cells are needed, including a study on the autophagic response at the protein level.

Cross-reactivity of latex agglutination assay complicates the identification of Burkholderia pseudomallei from soil.

The monoclonal antibody-based latex agglutination tests targeting a high molecular weight exopolysaccharide antigen of Burkholderia pseudomallei are commercially available. The tests are primarily used in routine diagnosis of melioidosis in major hospitals in Thailand and some endemic countries. Being a rapid test, this technique was employed as a presumptive test to identify colonies of B. pseudomallei among many others grown from soil specimens collected from southern Thailand. Cross-reactivity of these tests with other soil bacteria was a concern since it complicated the identification of B. pseudomallei. Here, we describe the cross-reactivity of two commercial latex agglutination tests for melioidosis with B. territorii, B. pseudomultivorans, B. multivorans and B. cenocepacia isolates from soil.

A new biphasic test for the detection of Helicobacter pylori in gastric biopsies.

The objective of this study was to create a biphasic cultural method for the detection of Helicobacter pylori in gastric biopsy specimens. The biphasic systems were made by using a urea agar slant with overlaying broth in a single vessel. Initially, three different liquid media including brain-heart infusion broth, Brucella broth, and Bolton broth were tested for their ability to support the growth of H. pylori. Bolton broth with 10% defibrinated horse blood demonstrated a significant increase in the numbers of H. pylori (p<0.05). The result showed that positive urease was used to concentrate viable H. pylori cells where the numbers of bacteria were 10(5)cfu. In addition, the reliable incubation time was at least 36h. In total, 55 biopsies were comparatively studied using commercial rapid urease test and PCR. Seven samples (12.72%) were positive with H. pylori by the biphasic test. With the CLOtest, 6 (10.91%) samples were positive. In conclusion, the Hp biphasic test achieved more positive samples than did the commercial rapid urease test.

Enhancement of viable Campylobacter detection by chemotactic stimuli.

The effects of chemotactic stimuli on motility ability of viable Campylobacter to pass through a 0.45 microm pore size filter in viscous condition were investigated. Reference strains including C. jejuni ATCC 33291, C. coli MUMT 18407, C. lari ATCC 43675, and C. upsaliensis DMST 19055 were used. The initial numbers of artificially-inoculated viable cells per g of chicken meat were approximately 10 to 10(4). Constituents of mucin plus bile (1:1), varieties of amino acids, and sodium salts were added into a soft-agar-coated membrane filter and incubated at both 37 degrees C and 42 degrees C for 24h. The drop plate method was used to determine numbers of viable Campylobacter at 6, 12, 18, and 24h. After 6h, constituents of mucin plus bile at the concentrations of 1, 5, and 10% demonstrated significant increases in numbers of viable cells (p<0.05). The numbers of the organisms at 42 degrees C were higher than those at 37 degrees C. In contrast, no significant difference in cell numbers was observed by adding amino acids or sodium salts. In addition, the role of starvation on chemotactic responses was also studied. Starved cells showed lower chemotactic response than non-starved cells. This method permitted rapid detection of viable thermophilic Campylobacter.

A novel method and simple apparatus for the detection of thermophilic Campylobacter spp. in chicken meat products.

Conventional procedures for isolation of thermophilic Campylobacter spp. from chicken are complex, labor intensive, and time-consuming. The objective of this study was to create a novel Campylobacter culturing apparatus. A main concept of the device was based on the ability of Campylobacter to pass through a 0.45 microm pore size filter in viscous media. Preliminary study demonstrated that only viable Campylobacter moved through the membrane filter and could multiply in the enrichment culture. C. jejuni, C. coli, C. lari, and C. upsaliensis in the chicken samples were detected at cell concentrations as low as 10 cfu/g, after 24 h incubation at 42 degrees C. In total, 84 retail chicken samples were comparatively studied using both conventional method and apparatus. Sixteen samples (19.05%) were positive by the apparatus method; 14 (16.66%) of these positive samples contained C. coli and 2 (2.38%) contained C. jejuni. With the conventional method, 7 (8.33%) samples were positive 7 (8.33%) with C. coli. In conclusion, the apparatus detected more positive samples than did the conventional culture method.