Establishment and evaluation of a qPCR method for the detection of pfmdr1 mutations in Plasmodium falciparum, the causal agent of fatal malaria.

Drug resistance surveillance is a major integral part of malaria control programs. Molecular methods play a pivotal role in drug resistance detection and related molecular research. This study aimed to develop a rapid and accurate detection method for drug resistance of Plasmodium falciparum (P. falciparum). A quantitative real-time PCR (qPCR) assay has been developed that identifies the mutation at locus A256T in the P.falciparum multi-drug resistance(pfmdr1) gene producing amino acid change at position 86. The results of 198 samples detected by qPCR were consistent with nested PCR and sequencing, giving an accuracy of 94.3%. The sensitivity, specificity, positive and negative predictive value of qPCR were 85.7%, 97.6%, 90.0% and 96.4%, respectively. The results of qPCR are basically consistent with the nested PCR, which is expected to replace the nested PCR as a new molecular biological method for drug resistance detection, providing reliable technical support for global malaria prevention and control.

Additive Therapy of Plasmodium berghei-Induced Experimental Cerebral Malaria via Dihydroartemisinin Combined with Rapamycin and Atorvastatin.

Cerebral malaria (CM), caused by Plasmodium falciparum, is the primary cause of death from severe malaria. Even after immediate parenteral therapy with antimalarial drugs, the mortality rate remains 15 to 25%. Currently, no effective therapeutic agents are available for the radical treatment of CM. Thus, further in-depth explorations of adjuvant therapies in combination with antimalarial drugs are urgently needed. The experimental cerebral malaria (ECM) model was established by infecting C57BL/6 mice with Plasmodium berghei ANKA. Subsequently, infected mice were continuously treated with dihydroartemisinin (DHA) in combination with rapamycin (RAP) and atorvastatin (AVA) for 5 days at different time points, including day 0, day 3, and day 6 postinfection (p.i.). Treatment efficacy was evaluated by comparing behavioral scores, body weight, parasitemia, survival rate, blood-brain barrier (BBB) integrity, and histopathology. The optimal combination therapy of DHA, RAP, and AVA on day 3 p.i. was selected for ECM. This strategy significantly improved survival rate, reduced parasitemia, improved the rapid murine coma and behavioral scale scores and permeability of the BBB, attenuated cerebrovascular and hepatic central venous obstruction and hemozoin deposition in the liver, and decreased the red pulp area of the spleen, which effectively ameliorated neurological damage in ECM. It also improved histopathology and neurological damage caused by ECM. In this study, the optimal therapeutic strategy for ECM was selected, which is expected to be a potential therapy for human CM. IMPORTANCE Although artemisinin-based combination therapies (ACTs) have greatly improved the clinical outcome of cerebral malaria (CM) as a fatal disease that can permanently disable a significant proportion of children even if they survive, new treatment options are needed as Plasmodium falciparum develops resistance to antimalarial drugs. Recent reports suggest that basal treatment with artemisinin derivatives often fails to protect against cell death, neurological damage, and cognitive deficits. In this study, the combination of dihydroartemisinin with rapamycin and atorvastatin improved the current antimalarial outcomes by overcoming the limitations of current antimalarials for CM morbidity and neurological sequelae. This combination offers a new adjunctive treatment for the clinical treatment of human CM in susceptible populations, including children under 5 years old and pregnant women.

Detection of Antimalarial Resistance-Associated Mutations in Plasmodium falciparum via a Platform of Allele-Specific PCR Combined with a Gold Nanoparticle-Based Lateral Flow Assay.

Since single nucleotide polymorphisms (SNPs) have attracted attention, there have been many explorations and improvements in screening and detection methods for SNPs. Traditional methods are complex and time-consuming and rely on expensive instruments. Therefore, there is an urgent need for a low-cost, simple, and accurate method that is convenient for use in resource-poor areas. Thus, a platform based on allele-specific PCR (AS-PCR) and a gold nanoparticle-based lateral flow assay (LFA) was developed, optimized, and used to detect the SNPs of the drug resistance gene pfmdr1. Subsequently, the system was assessed on clinical isolates and compared with nested PCR followed by Sanger sequencing. The sensitivity and specificity of the AS-PCR-LFA platform were up to 99.43% and 100%, respectively, based on the clinical isolates. The limit of detection is approximately 150 fg/µL for plasmid DNA as the template and 50 parasites/µL for dried filter blood spots from clinical isolates. The established and optimized AS-PCR-LFA system is more adaptable and rapidly translated to SNP analysis of other drug resistance genes and genetic diseases. In addition, while actively responding to the point-of-care testing policy, it also contributes to the Global Malaria Eradication Program. IMPORTANCE Rapid detection of single nucleotide polymorphisms (SNPs) is essential for malaria treatment. Based on the techniques of allele-specific PCR (AS-PCR) and lateral flow assay (LFA), an accurate and powerful platform for SNP detection of pfmdr1 was developed and evaluated with plasmid and clinical isolates. It offers a useful tool to identify antimalarial drug resistance and can support the effort to eliminate malaria globally.

Cerebral malaria induced by plasmodium falciparum: clinical features, pathogenesis, diagnosis, and treatment.

Cerebral malaria (CM) caused by Plasmodium falciparum is a fatal neurological complication of malaria, resulting in coma and death, and even survivors may suffer long-term neurological sequelae. In sub-Saharan Africa, CM occurs mainly in children under five years of age. Although intravenous artesunate is considered the preferred treatment for CM, the clinical efficacy is still far from satisfactory. The neurological damage induced by CM is irreversible and lethal, and it is therefore of great significance to unravel the exact etiology of CM, which may be beneficial for the effective management of this severe disease. Here, we review the clinical characteristics, pathogenesis, diagnosis, and clinical therapy of CM, with the aim of providing insights into the development of novel tools for improved CM treatments.

Observation of the Gut Microbiota Profile in BALB/c Mice Induced by Plasmodium yoelii 17XL Infection.

Rodent malaria caused by Plasmodium yoelii 17XL (Py 17XL) is an ideal animal model for human malaria studies. Although the gut microbiota plays an important role in the occurrence and development of infectious diseases, the gut microbiota associated with Py 17XL infection remains unclear. In the current study, the gut microbiota composition of infected BALB/c mice was surveyed. Mouse fecal samples were collected at 0, 2, 5 days post-infection (dpi), and the gut microbiota was characterized by 16S rRNA sequencing. Operational taxonomic units (OTUs) were 634 ± 26 on average. Firmicutes and Bacteroidetes were typically predominant in the gut microbiota composition at the phylum level. Compared with the Ctrl, Firmicutes was significantly decreased after infection, while Bacteroidetes was notably increased. The most dominant family was Lactobacillaceae in all samples. The alpha diversity index showed that compared with that of the Ctrl, the observed OTU number was decreased at 2 dpi and then slightly increased at 5 dpi. LEfSe analysis revealed several bacterial taxa were notably related to Py-infected mice at the phylogenetic level. Several bacterial genera, such as Lactobacillus, were overrepresented in the Py-infected fecal microbiota at 2 dpi, while Muribaculaceae was overrepresented at 5 dpi. Moreover, Alistipes and Helicobacter were overrepresented at 5 dpi compared with 2 dpi. The results indicated Py infection could alter the gut microbiota composition of mice. Besides, biomarkers could serve as direct targets to elucidate their roles in the progression and pathogenesis of malaria and provide insights into studies of antimalarial drugs and malaria vaccines.

A Rapid and Specific Genotyping Platform for Plasmodium falciparum Chloroquine Resistance via Allele-Specific PCR with a Lateral Flow Assay.

Single-nucleotide polymorphisms and genotyping related to genetic detection are several of the focuses of contemporary biotechnology development. Traditional methods are complex, take a long time, and rely on expensive instruments. Therefore, there is an urgent need for a rapid, simple, and accurate method convenient for use in resource-poor areas. Thus, a platform based on allele-specific PCR (AS-PCR) combined with a lateral flow assay (LFA) was developed, optimized, and used to detect the genotype of the Plasmodium falciparum chloroquine transporter gene (pfcrt). Subsequently, the system was assessed by clinical isolates and compared with Sanger sequencing. The sensitivity and specificity of the AS-PCR-LFA platform were 95.83% (115/120) and 100% (120/120), respectively, based on the clinical isolates. The detection limit of plasmid DNA was approximately 3.38 × 105 copies/µL. In addition, 100 parasites/µL were used for the dried filter blood spots from clinical isolates. The established rapid genotyping technique is not limited to antimalarial drug resistance genes but can also be applied to genetic diseases and other infectious diseases. Thus, it has realized the leap and transformation from scientific research theory to practical application and actively responds to the point-of-care testing policy. IMPORTANCE Accurate recognition of the mutation and genotype of genes are essential for the treatment of infectious diseases and genetic diseases. Based on the techniques of allele-specific PCR (AS-PCR) and a lateral flow assay (LFA), a rapid and useful platform for mutation detection was developed and assessed with clinical samples. It offers a powerful tool to identify antimalarial drug resistance and can support malaria control and elimination globally.

The application of enhanced recovery after surgery and negative-pressure wound therapy in the perioperative period of elderly patients with colorectal cancer.

OBJECTIVE: To Explore the perioperative application of enhanced recovery after surgery (ERAS) and negative-pressure wound therapy in the elderly patients with colorectal cancer. METHODS: A retrospective clinical data were studied in the patients with colorectal cancer in Department of General Surgery in Shanghai Fourth People,s Hospital (from March, 2017 to March, 2019), One hundred and fifty patients with undergoing radical surgery for colorectal cancer were divided into two groups: ERAS group (n = 76 cases, accepting ERAS management) and Conventional treatment(CT) group (n = 74 cases, accepting traditional treatment), Bleeding in operation, the time of postoperative anal flatus, number of wound dressing changing, time of wound healing, the length of postoperative hospital stay, readmission rate, postoperative complication, were compared between the two groups. RESULTS: ERAS was associated with less bleeding in operation, less Wound fat liquefaction, less wound dressing changing, less time of wound healing, less time of postoperative anal flatus compare to CT group (P < 0.05); anastomotic fistula, readmission rate is similar in two groups (P > 0.05). CONCLUSION: The modified ERAS can be safely applied to the perioperative period of elderly colorectal cancer patients and promote recovery; negative-pressure wound therapy is helpful for wound healing and promoting rehabilitation.