A simple fluorescence-based lateral flow test platform for rapid influenza B virus screening.

A simple fluorescence-based lateral flow test platform for rapid influenza B virus screening as a model target molecule was successfully developed. In this work, Cy5-loaded silica nanoparticles were directly conjugated to monoclonal antibodies, specific to the influenza B nucleoprotein, via a direct physisorption method and used as detector probes. Using this approach, the signal response to the detection was further determined using a fluorescent signal intensity measurement method via a portable reader, in combination with fluorescence imaging analysis. The degree to which the fluorescence signal response is detected is proportional to the amount of the target virus protein present in the system, reflected by the accumulation of the formed particle-antibody conjugates within the test system. Under optimized conditions, the system is capable of detecting the influenza B virus protein at a level of 0.55 µg per test within 30 min, using small sample volumes as low as 100 µL (R2 = 0.9544). In addition to its simplicity, further application of the system in detecting the influenza B virus protein was demonstrated using the viral transport media as specimen matrices. It was also shown that the system can perform the detection without cross-reactivity to other closely related respiratory viruses.

Pre-clinically evaluated visual lateral flow platform using influenza A and B nucleoprotein as a model and its potential applications.

A visual colorimetric rapid screening system based on a lateral flow device for simultaneous detection and differentiation between influenza A and B nucleoprotein as a model was developed. Monoclonal antibodies, specific for either influenza A or B nucleoproteins, were evaluated for their reactivities and were used as targeting ligands. With the best antibody pairs selected, the system exhibited good specificity to both viruses without cross reactivity to other closely related respiratory viruses. Further semi-quantitative analysis using a strip reader revealed that the system is capable of detecting influenza A and B protein content as low as 0.04 and 1 ng per test, respectively, using a sample volume as low as 100 µL, within 10 minutes (R 2 = 0.9652 and 0.9718). With a performance comparison to the commercial tests, the system demonstrated a four-to-eight-fold higher sensitivity. Pre-clinical evaluation with 101 nasopharyngeal swabs reveals correlated results with a standard molecular approach, with 89% and 83% sensitivity towards influenza A and B viruses, and 100% specificity for both viruses.

HIGH LEVEL OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI AND OCCURRENCE OF STX-NEGATIVE E. COLI O157 FROM RAW MEATS: CHARACTERIZATION OF VIRULENCE PROFILE AND GENETIC RELATEDNESS.

Raw meats, especially beef, are particularly prone to Shiga toxinproducing Escherichia coli (STEC)/enterohemorrhagic E. coli (EHEC) contamination. However, data regarding their quantity in raw meats are seldom reported in Thailand. Among four common meat types, beef possessed highest value of stx1-producing E. coli (STEC1) contamination in February 2015 [> 1,100 most probable number (MPN)/g] and stx2-producing E. coli (STEC2) highest MPN/g (460) in March of the same year. STEC2 was found, for the first time, in shrimp samples in March and April, 2015 with MPN/g value of 6.6 and 9.3, respectively. EHEC at 3 MPN/g was detected in only one (2%) beef sample. Even though stx-negative E. coli O157 from beef has rarely been reported in Thailand, isolation of E. coli O157 using immunomagnetic separation method revealed that four strains (PSVX-1, PSVX-2, PSVX-3, and PSVX-4) from three (8%) beef samples were shown to be stx-negative E. coli O157. These strains were members of phylogenetic group A and were multi-drug resistant. Genetic relatedness as determined by polytrinucleotide (GTG)5-PCR and BOX-PCR showed identical DNA profiles of PSVX-2 and PSVX-4, which by BOX-PCR were 90% to a clinical isolate, O157 strain PSU120, from Hat-Yai Hospital in 2014. The presence of these environment stx-negative E. coli O157 strains with the ability to acquire additional virulence properties could pose a potential public health problem particularly in this region of Thailand.

ENTEROTOXIGENIC ESCHERICHIA COLI O169:HUT FROM A DIARRHEAL PATIENT: PHYLOGENETIC GROUP AND ANTIMICROBIAL SUSCEPTIBILITY.

Enterotoxigenic Escherichia coli (ETEC) is one of the most common pathogenic E. coli pathotypes causing diarrhea in children worldwide. Its enterotoxins, LT and ST, including colonization factors mainly are responsible for human pathogenesis. From 239 rectal swabs of diarrheal patients at Hat Yai and Pattani Hospitals during August 2013 and May 2014, five isolates from only a single E. coli sample demonstrated the possession of estA1, encoding porcine heat-stable enterotoxin (STp). These isolates all belonged to serotype 0169:H Untypeable (HUT) and carried astA, encoding enteroaggregative heat-stable enterotoxin 1. A PCR-based phylogenetic group investigation classified them as members of the virulent E. coli phylogenetic group D. The isolates were resistant to cephalothin, penicillin G, streptomycin, tetracycline and vancomycin. Confirmation of their clonality was conducted by enterobacterial repetitive intergenic consensus sequence PCR typing, which revealed that these ETEC were derived from the same clone. This is the first report of ETEC O169:HUT in southern Thailand.

MOST PROBABLE NUMBER-POLYMERASE CHAIN REACTION-BASED QUANTIFICATION OF ENTEROTOXIGENIC ESCHERICHIA COLI FROM RAW MEATS IN SOUTHERN THAILAND.

The detection of enterotoxigenic Escherichia coli (ETEC) in food, especially raw meat, has rarely been documented in Thailand, although the presence of this bacterial pathogen is considered of important public health concern. The quantity of ETEC in 150 meat samples collected from fresh food markets in southern Thailand were determined using a most probable number (MPN)-PCR-based quantification approach. ETEC contamination of raw chicken, pork and beef samples was 42%, 25% and 12%, respectively (a significant difference between chicken and beef, p < 0.05). The maximum MPN/g value for enterotoxin gene est-positive ETEC from pork and elt-positive ETEC from chicken were > 1,100 MPN/g, but the range of MPN/g values was greater for ETEC from chicken than from pork or beef. ETEC from raw chicken meat contained significantly more elt- than est-positives (p < 0.05). Thus, a significant proportion of raw meat, in particular chicken, sold in fresh food markets in southern Thailand harbors ETEC and poses a potential threat to consumer health.