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Usanee Anukool,1,2 Ponrut Phunpae,1,2 Chayada Sitthidet Tharinjaroen,1,2 Bordin Butr-Indr,1,2 Sukanya Saikaew,1– 3 Nathiprada Netirat,4 Sorasak Intorasoot,1,2 Vorasak Suthachai,4 Khajornsak Tragoolpua,1,2 Angkana Chaiprasert5 1Division of Clinical Microbiology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Muang District, Chiang Mai 50200, Thailand; 2Infectious Diseases Research Unit (IDRU), Faculty of Associated Medical Sciences, Chiang Mai University, Muang District, Chiang Mai 50200, Thailand; 3Faculty of Public Health Chiang Mai University, Muang District, Chiang Mai 50200, Thailand; 4Office of Disease Prevention and Control, 1 (ODPC 1) Chiang Mai, Department of Disease Control, Ministry of Public Health Thailand, Muang District, Chiang Mai 50000, Thailand; 5Office for Research and Development Affairs, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, ThailandCorrespondence: Usanee AnukoolDivision of Clinical Microbiology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, 110 Intawaroros Road, Sripoom, Muang District, Chiang Mai 50200, ThailandTel +66 53935068 ext. 15Fax +66 53936042Email usanee.anukool@cmu.ac.thIntroduction: Knowledge of the prevalence and distribution of multidrug-resistant tuberculosis (MDR-TB) genotypes in northern Thailand is still limited. An accurate, rapid, and cost-effective diagnostic of MDR-TB is crucial to improve treatment and control of increased MDR-TB.Materials and Methods: The molecular diagnostic assays named “RIF-RD” and “INH-RD” were designed to detect rifampicin (RIF) and isoniazid (INH) resistance based on real-time PCR and high-resolution melting curve analysis. Applying the ∆Tm cutoff values, the RIF-RD and INH-RD were evaluated against the standard drug susceptibility testing (DST) using 107 and 103 clinical Mycobacterium tuberculosis (Mtb) isolates from northern Thailand. DNA sequence analysis of partial rpoB, katG, and inhA promoter of 73 Mtb isolates, which included 30 MDR-TB, was performed to elucidate the mutations involved with RIF and INH resistance.Results: When compared with the phenotypic DST, RIF-RD targeting rpoB showed sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of 83.9, 98.6, 96.9, and 92.0%, respectively. The multiplex reaction of the INH-RD targeted both katG and inhA promoter showed high sensitivity, specificity, PPV, and NPV of 97.1, 94.2, 89.2, and 98.5%, respectively. Six patterns of rpoB mutation, predominately at codons 531 (50%) and 526 (40%) along with a rare S522L (3.33%) and D516V (3.33%), were detected. A single pattern of katG mutation (S315T) (63.3%) and four patterns of inhA promoter mutation, predominately − 15 (C>T), were found. Approximately, 17% of MDR-TB strains possessed double mutations within the katG and inhA promoter.Conclusion: Up to 86.7% and 96.7% of MDR-TB could be accurately detected by RIF-RD and INH-RD, emphasizing its usefulness as a low unit price assay for rapid screening of MDR-TB, with confirmation of INH resistance in low and middle-income countries. The MDR-TB genotypes provided will be beneficial for TB control and the development of drug-resistant TB diagnostic technology in the future.Keywords: multidrug-resistant tuberculosis, Mycobacterium tuberculosis, high-resolution melting curve analysis, diagnosis, genotype

Tuberculosis (TB) therapy requires long-course multidrug regimens leading to the emergence of drug-resistant TB and increased public health burden worldwide. As the treatment strategy is more challenging, seeking a potent non-antibiotic agent has been raised. Propolis serve as a natural source of bioactive molecules. It has been evidenced to eliminate various microbial pathogens including Mycobacterium tuberculosis (Mtb). In this study, we fabricated the niosome-based drug delivery platform for ethanolic extract of propolis (EEP) using thin film hydration method with Ag85A aptamer surface modification (Apt-PEGNio/EEP) to target Mtb. Physicochemical characterization of PEGNio/EEP indicated approximately -20 mV of zeta potential, 180 nm of spherical nanoparticles, 80% of entrapment efficiency, and the sustained release profile. The Apt-PEGNio/EEP and PEGNio/EEP showed no difference in these characteristics. The chemical composition in the nanostructure was confirmed by Fourier transform infrared spectrometry. Apt-PEGNio/EEP showed specific binding to Mycobacterium expressing Ag85 membrane-bound protein by confocal laser scanning microscope. It strongly inhibited Mtb in vitro and exhibited non-toxicity on alveolar macrophages. These findings indicate that the Apt-PEGNio/EEP acts as an antimycobacterial nanoparticle and might be a promising innovative targeted treatment. Further application of this smart nano-delivery system will lead to effective TB management.

Tuberculosis is a highly contagious disease caused by the Mycobacterium tuberculosis complex (MTBC). Although TB is treatable, multidrug-resistant, extensively drug-resistant, and totally drug-resistant forms of M. tuberculosis have become a new life-threatening concern. New anti-TB drugs that are capable of curing these drug-resistant strains are urgently needed. The purpose of this study is to determine the antimycobacterial activity of D-enantiomer human lactoferricin 1-11 (D-hLF 1-11) against mycobacteria in vitro using a 3-(4,5-dimethylthiazol-2-yl)-2,5-dephenyltetrazolium bromide colorimetric assay, resazurin microplate assay, and microscopic observation drug susceptibility assay. Three previously described antimicrobial peptides, protegrin-1, AK 15-6, and melittin, with potent anti-TB activity, were included in this study. The findings suggest that D-hLF 1-11 can inhibit the growth of M. tuberculosis with a minimum inhibitory concentration of 100−200 µg/mL in susceptible, isoniazid (INH)-monoresistant, rifampicin (RF)-monoresistant, and MDR strains. The peptide can also inhibit some nontuberculous mycobacteria and other MTBC in similar concentrations. The antibiofilm activity of D-hLF 1-11 against the biofilm-forming M. abscessus was determined by crystal violet staining, and no significant difference is observed between the treated and untreated biofilm control. The checkerboard assay was subsequently carried out with M. tuberculosis H37Rv and the results indicate that D-hLF 1-11 displays an additive effect when combined with INH and a synergistic effect when combined with RF, with fractional inhibitory concentration indices of 0.730 and 0.312, respectively. The red blood cell hemolytic assay was initially applied for the toxicity determination of D-hLF 1-11, and negligible hemolysis (<1%) was observed, despite a concentration of up to 4 mg/mL being evaluated. Overall, D-hLF 1-11 has potential as a novel antimycobacterial agent for the future treatment of drug-sensitive and drug-resistant M. tuberculosis infections.

Rifampicin-resistant tuberculosis (RR-TB) has become a major threat globally. This study aims to develop a new assay, RIF-RDp, to enhance the detection of RR-TB based on combined locked nucleic acid (LNA) probes with high-resolution melting curve analysis (HRM). Two new LNA probes were designed to target the class-III and IV mutations of rpoB , H526D, and D516V. LNA probes showed 100% specificity in the detection of mutant targets among characterized and blinded Mycobacterium tuberculosis (Mtb) isolates. The performance of RIF-RDp was evaluated using 110 blinded clinical Mtb isolates in northern Thailand against drug-susceptibility testing (DST), DNA sequencing, and a commercial real-time PCR kit. This assay showed sensitivity and specificity of 94.55% and 98.18% compared to DST, and 96.36% and 100% compared to DNA sequencing. The efficacy of RIF-RDp was comparable to the commercial kit and DNA sequencing. The Cohen's Kappa statistic showed almost perfect agreement between RIF-RDp and the commercial kit (κ = 0.95), and RIF-RDp and DNA sequencing (κ = 0.96). Furthermore, this is the first report of the rare mutation profiles, S531W, and a triple codon deletion (510-512) in northern Thailand. According to high accuracy, the RIF-RDp assay may render an easy-to-use, low-cost, and promising diagnostics of RR-TB in the future.

Streptococcus suis , a Gram-positive bacterium, is an important swine and human pathogen, with serotype 2 being the most prevalent strain found worldwide. Deafness, meningitis, and death (in severe cases) are observed in S. suis -infected cases. Development of the ligands that can bind to S. suis with high affinity and specificity could be beneficial for the diagnosis and treatment of S. suis infection. Herein, the nuclease-resistant RNA aptamers based on 2'-fluoropyrimidine modification against S. suis serotype 2, strain P1/7, were established using the cell- Systematic Evolution of Ligands by Exponential enrichment (SELEX) technique. One of the aptamers, R8-su12, could bind to the S. suis target strain as well as other S. suis serotypes, i.e., 1, 1/2, 9, and 14, but not to other bacteria tested, i.e., S. pneumoniae ATCC 49619, Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, and Pseudomonas aeruginosa ATCC 27853. Moreover, the R8-su12 RNA aptamer was also capable of inhibiting the biofilm formation of the S. suis target strain, making it potentially useful for the study of biofilm formation and the treatment of S. suis infection in humans and pigs in the future.

In this study, we describe a nano-carrier system for propolis that is able to cross an in vitro model of the blood-brain barrier (BBB) and effectively reduce the virulence of Cryptococcus neoformans in animal models. Antimicrobial properties of propolis have been widely studied. However, propolis applications are limited by its low water solubility and poor bioavailability. Therefore, we recently formulated novel poly (n-butyl cyanoacrylate) nanoparticles (PBCA-NP) containing propolis. PBCA-NP are biocompatible, biodegradable and have been shown to effectively cross the BBB using apolipoprotein E (ApoE) as a ligand. Prepared nanoparticles were characterized for particle size, zeta potential, propolis entrapment efficiency and in vitro release. Additionally, the PBCA-NP were functionalized with polysorbate 80, which then specifically adsorbs ApoE. Using an in vitro BBB model of human brain microvascular endothelial cells hCMEC/D3, it was shown that fluorescence labelled ApoE-functionalized PBCA-NP were internalized by the cells and translocated across the cell monolayer. Propolis-loaded PBCA-NP had in vitro , antifungal activity against C. neoformans , which causes meningitis. To utilize the invertebrate model, Galleria mellonella larvae were infected with C. neoformans and treated with propolis-loaded PBCA-NP. The larvae exhibited normal behavior in toxicity testing, and treatment with propolis-loaded PBCA-NP increased survival in the C. neoformans -infected larvae group. In addition, following cryptococcal infection and then 7 days of treatment, the tissue fungal burden of mice treated with propolis-loaded PBCA-NP was significantly lower than control groups. Therefore, our ApoE-functionalized propolis-loaded PBCA-NP can be deemed as a potential targeted nanoparticle in the therapeutic treatment of cerebral cryptococcosis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Thammasit, Tharinjaroen, Tragoolpua, Rickerts, Georgieva, Bäumler and Tragoolpua.)

Streptococcus suis is an emerging zoonotic bacterium causing septicemia and meningitis in humans. Due to rapid disease progression, high mortality rate, and many underdiagnosed cases by time-consuming routine identification methods, alternative diagnostic testing is essential. Among 29 broadly accepted S. suis serotypes, serotypes 2 and 14 are high prevalent; however, many PCR assays showed an inability to differentiate serotype 2 from 1/2, and 1 from 14. In this study, we developed and validated a new multiplex PCR assay that facilitates the identification of only the 29 true serotypes of S. suis and simultaneously differentiates serotypes 1, 1/2, 2, and 14 within a single reaction. Importantly, the multiplex PCR could detect S. suis directly from positive hemocultures and CSF. The results revealed high sensitivity, specificity, and 100% accuracy with almost perfect agreement (κ = 1.0) compared to culture and serotyping methods. Direct detection enables a decrease in overall diagnosis time, rapid and efficient treatment, reduced fatality rates, and proficient disease control. This multiplex PCR offers a rapid, easy, and cost-effective method that can be applied in a routine laboratory. Furthermore, it is promising for developing point-of-care testing (POCT) for S. suis detection in the future.

The Burkholderia pseudomallei phylogenetic cluster includes B. pseudomallei, B. mallei, B. thailandensis, B. oklahomensis, B. humptydooensis and B. singularis. Regarded as the only pathogenic members of this group, B. pseudomallei and B. mallei cause the diseases melioidosis and glanders, respectively. Additionally, variant strains of B. pseudomallei and B. thailandensis exist that include the geographically restricted B. pseudomallei that express a B. mallei-like BimA protein (BPBM), and B. thailandensis that express a B. pseudomallei-like capsular polysaccharide (BTCV). To establish a PCR-based assay for the detection of pathogenic Burkholderia species or their variants, five PCR primers were designed to amplify species-specific sequences within the bimA (Burkholderia intracellular motility A) gene. Our multiplex PCR assay could distinguish pathogenic B. pseudomallei and BPBM from the non-pathogenic B. thailandensis and the BTCV strains. A second singleplex PCR successfully discriminated the BTCV from B. thailandensis. Apart from B. humptydooensis, specificity testing against other Burkholderia spp., as well as other Gram-negative and Gram-positive bacteria produced a negative result. The detection limit of the multiplex PCR in soil samples artificially spiked with known quantities of B. pseudomallei and B. thailandensis were 5 and 6 CFU/g soil, respectively. Furthermore, comparison between standard bacterial culture and the multiplex PCR to detect B. pseudomallei from 34 soil samples, collected from an endemic area of melioidosis, showed high sensitivity and specificity. This robust, sensitive, and specific PCR assay will be a useful tool for epidemiological study of B. pseudomallei and closely related members with pathogenic potential in soil., Competing Interests: The authors have declared that no competing interests exist.

A recent modelling study estimated that there are 2800 deaths due to melioidosis in Thailand yearly. The Thailand Melioidosis Network (formed in 2012) has been working closely with the Ministry of Public Health (MoPH) to investigate and reduce the burden of this disease. Based on updated data, the incidence of melioidosis is still high in Northeast Thailand. More than 2000 culture-confirmed cases of melioidosis are diagnosed in general hospitals with microbiology laboratories in this region each year. The mortality rate is around 35%. Melioidosis is endemic throughout Thailand, but it is still not uncommon that microbiological facilities misidentify Burkholderia pseudomallei as a contaminant or another organism. Disease awareness is low, and people in rural areas neither wear boots nor boil water before drinking to protect themselves from acquiring B. pseudomallei . Previously, about 10 melioidosis deaths were formally reported to the National Notifiable Disease Surveillance System (Report 506) each year, thus limiting priority setting by the MoPH. In 2015, the formally reported number of melioidosis deaths rose to 112, solely because Sunpasithiprasong Hospital, Ubon Ratchathani province, reported its own data ( n = 107). Melioidosis is truly an important cause of death in Thailand, and currently reported cases (Report 506) and cases diagnosed at research centers reflect the tip of the iceberg. Laboratory training and communication between clinicians and laboratory personnel are required to improve diagnosis and treatment of melioidosis countrywide. Implementation of rapid diagnostic tests, such as a lateral flow antigen detection assay, with high accuracy even in melioidosis-endemic countries such as Thailand, is critically needed. Reporting of all culture-confirmed melioidosis cases from every hospital with a microbiology laboratory, together with final outcome data, is mandated under the Communicable Diseases Act B.E.2558. By enforcing this legislation, the MoPH could raise the priority of this disease, and should consider implementing a campaign to raise awareness and melioidosis prevention countrywide., Competing Interests: Conflicts of Interest: The authors declare no conflict of interest.

Aims: To exploit immunomagnetic separation combined with PCR with confronting two-pair primers (IMS-PCR-CTPP) as a rapid method for detection of Mycobacterium tuberculosis complex (MTC) and identification of Mycobacterium bovis from sputum specimens., Methods and Results: Monoclonal antibody (mAb) against the mycobacterial antigen, 85B (Ag85B), was coupled with magnetic particles for specific immunomagnetic separation (IMS) of Mycobacterium spp. Immunofluorescence assay indicated the capability of mAb to bind to Ag85B in both the recombinant and the native form. The IMS combined with PCR-CTPP targeting the mycobacterial lep B gene was further implemented using 133 sputum samples with acid-fast bacilli grading from negative to 3+. The results showed the sensitivity and specificity of IMS-PCR-CTPP vs gold standard culture method were 89·9 and 88·6% respectively., Conclusions: The IMS-PCR-CTPP method shortens the time for tuberculosis (TB) diagnosis from months to a day. This method is also suitable for investigation of MTC and epidemiological study of Myco. bovis in sputum specimens., Significance and Impact of the Study: This study is the first report emphasizing the combination of IMS and PCR-CTPP for the detection of MTC and simultaneous identification of Myco. bovis from sputum. It could be used for TB diagnosis in resource-limited countries with high TB burden., (© 2016 The Society for Applied Microbiology.)

Background: The study of the biology, transmission and pathogenesis of Plasmodium vivax is hindered due to the lack of a robustly propagating, continuous culture of this parasite. The current culture system for P. vivax parasites still suffered from consistency and difficulties in long-term maintenance of parasites in culture and for providing sufficient biological materials for studying parasite biology. Therefore, further improvement of culture conditions for P. vivax is needed., Methods: Clinical samples were collected from patients diagnosed with P. vivax in western Thailand. Leukocyte-depleted P. vivax infected blood samples were cultured in a modified McCoy's 5A medium at 5% haematocrit under hypoxic condition (5% O2, 5% CO2, and 90% N2). Reticulocytes purified from adult peripheral blood were added daily to maintain 4% reticulocytes. Parasites were detected by microscopic examination of Giemsa-stained smears and molecular methods., Results: The effects of culture variables were first analysed in order to improve the culture conditions for P. vivax. Through analysis of the sources of host reticulocytes and nutrients of culture medium, the culture conditions better supporting in vitro growth and maturation of the parasites were identified. Using this system, three of 30 isolates could be maintained in vitro for over 26 months albeit parasite density is low., Conclusions: Based on the analysis of different culture variables, an improved and feasible protocol for continuous culture of P. vivax was developed.