OBJECTIVE: A sensitive method for the estimation of CRM in plasma, as well as fecal matter-based solid self-nano emulsifying drug delivery system (S-SNEDDS), has been reported for the first time.
METHODS: A bioanalytical method was optimized using Box-Behnken Design having 13 runs and 3 responses. The optimized method was developed using methanol and water (70:30 v/v) with a flow rate of 1 mL/min. Quercetin was used as an internal standard. A specificity test was also performed for the developed CRM solid self-nano emulsifying drug delivery system.
RESULTS: The retention time of CRM was found to be 14.18 minutes. The developed method was validated and found to be linear in the range of 50-250 ng/mL with an R2 of 0.999. Accuracy studies indicated that CRM had a percentage recovery of less than 105% and more than 95%, respectively. Precision studies were carried out for inter, intraday, and inter-analyst precision, and the %RSD was found to be less than 2%. The limit of detection (LOD) and limit of quantification (LOQ) were found to be 3.37 ng/mL and 10.23 ng/mL, respectively. Stability studies for shortterm, long term and freeze-thaw cycles showed a %RSD of less than 2%, indicating the stability of CRM in the plasma matrix. Moreover, the blank fecal microbiota extract slurry did not show any peak at the retention time of CRM in a CRM-loaded solid nanoemulsifying drug delivery system containing fecal microbiota extract indicating its specificity.
CONCLUSION: Hence, the developed method can have clinical implications as it helps estimate CRM in blood samples and also provides a simple and sensitive method for the estimation of plant-based flavonoids along with fecal microbiota extract formulations.
METHODS: Ten RDTs were evaluated: nine to detect clinical P. knowlesi infections from Malaysia, and nine assessing limit of detection (LoD) for P. knowlesi (PkA1-H.1) and P. falciparum (Pf3D7) cultures. Targets included Plasmodium-genus parasite lactate dehydrogenase (pan-pLDH) and P. vivax (Pv)-pLDH.
RESULTS: Samples were collected prior to antimalarial treatment from 127 patients with microscopy-positive PCR-confirmed P. knowlesi mono-infections. Median parasitaemia was 788/µL (IQR 247-5,565/µL). Pan-pLDH sensitivities ranged from 50.6% (95% CI 39.6-61.5) (SD BIOLINE) to 87.0% (95% CI 75.1-94.6) (First Response® and CareStart™ PAN) compared to reference PCR. Pv-pLDH RDTs detected P. knowlesi with up to 92.0% (95% CI 84.3-96.7%) sensitivity (Biocredit™). For parasite counts ≥200/µL, pan-pLDH (Standard Q) and Pv-pLDH RDTs exceeded 95% sensitivity. Specificity of RDTs against 26 PCR-confirmed negative controls was 100%. Sensitivity of six highest performing RDTs were not significantly different when comparing samples taken before and after (median 3 hours) antimalarial treatment. Parasite ring stages were present in 30% of pre-treatment samples, with ring stage proportions (mean 1.9%) demonstrating inverse correlation with test positivity of Biocredit™ and two CareStart™ RDTs.For cultured P. knowlesi, CareStart™ PAN demonstrated the lowest LoD at 25 parasites/µL; LoDs of other pan-pLDH ranged from 98 to >2000 parasites/µL. Pv-pLDH LoD for P. knowlesi was 49 parasites/µL. No false-positive results were observed in either P. falciparum-pLDH or histidine-rich-protein-2 channels.
CONCLUSION: Selected RDTs demonstrate sufficient performance for detection of major human malaria species including P. knowlesi in co-endemic areas where microscopy is not available, particularly for higher parasite counts, although cannot reliably differentiate among non-falciparum malaria.