A tiny sample rapid visual detection technology for imidacloprid resistance in Aphis gossypii by CRISPR/Cas12a.

Insecticide resistance monitoring is essential for guiding chemical pest control and resistance management policies. Currently, rapid and effective technology for monitoring the resistance of tiny insects in the field is absent. Aphis gossypii Glover is a typical tiny insect, and one of the most frequently reported insecticide-resistant pests. In this study, we established a novel CRISPR/Cas12a-based rapid visual detection approach for detecting the V62I and R81T mutations in the ß1 subunit of the nAChR in A. gossypii, to reflect target-site resistance to imidacloprid. Based on the nAChR ß1 subunit gene in A. gossypii, the V62I/R81T-specific RPA primers and crRNAs were designed, and the ratio of 10 µM/2 µM/10 µM for ssDNA/Cas12a/crRNA was selected as the optimal dosage for the CRISPR reaction, ensuring that Cas12a only accurately recognizes imidacloprid-resistance templates. Our data show that the field populations of resistant insects possessing V62I and R81T mutations to imidacloprid can be accurately identified within one hour using the RPA-CRISPR/Cas12a detection approach under visible blue light at 440-460 nm. The protocol for RPA-CRISPR detection necessitates a single less than 2 mm specimen of A. gossypii tissues to perform RPA-CRISPR detection, and the process only requires a container at 37 °C and a portable blue light at 440-460 nm. Our research represents the first application of RPA-CRISPR technology in insecticide resistance detection, offers a new method for the resistance monitoring of A. gossypii or other tiny insects, helps delay the development of resistance to imidacloprid, improves the sustainability of chemical control, and provides theoretical guidance for managing pest resistance.

Hospital-acquired neonatal infections in developing countries.

Hospital-born babies in developing countries are at increased risk of neonatal infections because of poor intrapartum and postnatal infection-control practices. We reviewed data from developing countries on rates of neonatal infections among hospital-born babies, range of pathogens, antimicrobial resistance, and infection-control interventions. Reported rates of neonatal infections were 3-20 times higher than those reported for hospital-born babies in industrialised countries. Klebsiella pneumoniae, other gram-negative rods (Escherichia coli, Pseudomonas spp, Acinetobacter spp), and Staphylococcus aureus were the major pathogens among 11,471 bloodstream isolates reported. These infections can often present soon after birth. About 70% would not be covered by an empiric regimen of ampicillin and gentamicin, and many might be untreatable in resource-constrained environments. The associated morbidity, mortality, costs, and adverse effect on future health-seeking behaviour by communities pose barriers to improvement of neonatal outcomes in developing countries. Low-cost, "bundled" interventions using systems quality improvement approaches for improved infection control are possible, but should be supported by evidence in developing country settings.

Nalidixic acid screening test in detection of decreased fluoroquinolone susceptibility in Salmonella typhi isolated from blood.

OBJECTIVE: To determine the validity of nalidixic acid screening test in the detection of high MICs of fluoroquinolone against Salmonella(S.) typhi isolated from blood and correlate zone diameters of ofloxacin with that of MIC value for nalidixic acid sensitive and resistant strains. DESIGN: Cross-sectional analytical study. PLACE AND DURATION OF STUDY: Clinical Microbiology Laboratory of the Aga Khan Hospital, Karachi from January 2002 to December 2003. PATIENTS AND METHODS: Two hundred S. typhi isolates from blood were included for nalidixic acid screening and ofloxacin susceptibility. Antibiotic susceptibilities for both the antibiotics were obtained by disc diffusion method whereas MICs were determined by standard agar dilution method as recommended by NCCLS guidelines. Sensitivity, specificity and correlation between both antimicrobial susceptibility methods were calculated and results expressed as scattergrams. RESULTS: The results broadly classify S. typhi isolates into nalidixic acid resistant strains with no zone of inhibition around 30 mug nalidixic acid disc and nalidixic acid sensitive strains with mean zone of inhibition of 24.9 mm. All S. typhi isolates with ofloxacin MIC of capital ZHE, Cyrillic 0.125 microg/ml were found to be nalidixic acid resistant (MIC capital ZHE, Cyrillic32 microg/ml) whereas the isolates with ofloxacin MIC 0.06 microg/ml were nalidixic acid sensitive (MIC 8 microg/ml). Screening for nalidixic acid resistance was found to be 100% sensitive and 97% specific in identifying S. typhi strains with reduced susceptibility to fluoroquinolone (MIC capital ZHE, Cyrillic 0.125 microg/ml). CONCLUSION: Nalidixic acid resistance as a screening method is proved to be significant in identifying S. typhi isolates with reduced susceptibility to fluoroquinolones. It is also suggested that inhibition zone of 25 mm around 5 microg ofloxacin disc is appropriate as a selection criterion to detect S. typhi isolates with reduced susceptibility to fluoroquinolones.

Antibiotic resistance among Salmonella enterica serovars Typhi and Paratyphi A in Pakistan (2001-2006).

OBJECTIVES: To compare antimicrobial resistance in S. Typhi and S. Paratyphi A isolates from Pakistan. METHODS: Blood samples were collected through > 175 laboratory collection points in major cities and towns across the country. The study included 3,671 S. Typhi and 1,475 S. Paratyphi A isolates (2001-2006). Multidrug resistance (MDR) was defined as resistance to first-line agents co-trimoxazole, chloramphenicol and ampicillin. RESULTS: In total, 79.3% S. Typhi and 59.9% S. Paratyphi A were isolated from patients under 15 years of age. During the study period, the MDR rate increased in S. Typhi (34.2 to 48.5% p<0.001). Quinolone resistance (MIC> 1 microg/ml) increased in both S. Typhi (1.6 to 64.1% p<0.001) and S. Paratyphi A (0 to 47% p<0.001). The increase in the proportion of strains showing high level quinolone resistance (MIC > 4 microg/ml) was greater in S. Paratyphi A when compared to S. Typhi. Resistance to first-line drugs was higher in those <15 years of age whereas quinolone resistance was higher in older patients. CONCLUSION: Differences between S. Typhi and S. Paratyphi A, in terms of evolution of resistance to first-line agents and to quinolones, are evident in this population. The rapid increase in quinolone resistance in S. Paratyphi A when compared to S. Typhi is concerning and requires further study.