Absence of association between Pfnfs1 mutation and in vitro susceptibility to lumefantrine in Plasmodium falciparum.

Artemether-lumefantrine (AL) is the most widely used antimalarial drug for treating uncomplicated falciparum malaria. This study evaluated whether the K65Q mutation in the Plasmodium falciparum cysteine desulfurase IscS (Pfnfs1) gene was associated with alternated susceptibility to lumefantrine using clinical parasite samples from Ghana and the China-Myanmar border area. Parasite isolates from the China-Myanmar border had significantly higher IC50 values to lumefantrine than parasites from Ghana. In addition, the K65 allele was significantly more prevalent in the Ghanaian parasites (34.5%) than in the China-Myanmar border samples (6.8%). However, no difference was observed in the lumefantrine IC50 value between the Pfnfs1 reference K65 allele and the non reference 65Q allele in parasites from the two regions. These data suggest that the Pfnfs1 K65Q mutation may not be a reliable marker for reduced susceptibility to lumefantrine.

ITPR1-AS1 promotes small cell lung cancer metastasis by facilitating P21HRAS splicing and stabilizing DDX3X to activate the cRaf-MEK-ERK cascade.

The mechanisms underlying the involvement of long non-coding RNAs (lncRNAs) in the metastasis of small cell lung cancer (SCLC) remain largely unknown. Here, we identified that the lncRNA ITPR1-AS1 was upregulated in SCLC and lymph node metastasis tissues and positively correlated with SCLC malignant features. The overexpression of ITPR1-AS1 in SCLC was an independent risk factor for the overall survival of patients with SCLC. Our data confirmed that ITPR1-AS1 induces SCLC cell metastasis both in vitro and in vivo. Mechanistically, ITPR1-AS1 acts as a scaffold to enhance the interaction between SRC-associated in mitosis 68 kDa and heterogeneous nuclear ribonucleoprotein A1, which facilitates the alternative splicing of the H-Ras proto-oncogene (HRAS) pre-mRNA (P21HRAS). Moreover, we observed that ITPR1-AS1 could associate in a complex with and maintain the stability of DEAD-box polypeptide 3 (DDX3X), which inhibited the latter's ubiquitination and degradation. Our data provide evidence that ITPR1-AS1 activates the cRaf-MEK-ERK cascade by upregulating P21HRAS production and stabilizing DDX3X, to promote SCLC metastasis.

Genetic diversity of Plasmodium vivax populations from the China-Myanmar border identified by genotyping merozoite surface protein markers.

BACKGROUND: Parasite diversity and population structure influence malaria control measures. Malaria transmission at international borders affects indigenous residents and migrants, defying management efforts and resulting in malaria re-introduction. Here we aimed to determine the extent and distribution of genetic variations in Plasmodium vivax populations and the complexity of infections along the China-Myanmar border. METHODS: We collected clinical P. vivax samples from local and migrant malaria patients from Laiza and Myitsone, Kachin State, Myanmar, respectively. We characterized the polymorphisms in two P. vivax merozoite surface protein markers, Pvmsp-3α and Pvmsp-3ß, by PCR-restriction fragment length polymorphism (PCR-RFLP) analysis. We sought to determine whether these genetic markers could differentiate these two neighboring parasite populations. RESULTS: PCR revealed three major size variants for Pvmsp-3α and four for Pvmsp-3ß among the 370 and 378 samples, respectively. PCR-RFLP resolved 26 fragment-size alleles by digesting Pvmsp-3α with Alu I and Hha I and 28 alleles by digesting Pvmsp-3ß with Pst I. PCR-RFLP analysis of Pvmsp-3α found that infections in migrant laborers from Myitsone bore more alleles than did infections in residents of Laiza, while such difference was not evident from genotyping Pvmsp-3ß. Infections originating from these two places contained distinct but overlapping subpopulations of P. vivax. Infections from Myitsone had a higher multiplicity of infection as judged by the size of the Pvmsp-3α amplicons and alleles after Alu I/Hha I digestion. CONCLUSIONS: Migrant laborers from Myitsone and indigenous residents from Laiza harbored overlapping but genetically distinct P. vivax parasite populations. The results suggested a more diverse P. vivax population in Myitsone than in the border town of Laiza. PCR-RFLP of Pvmsp-3α offers a convenient method to determine the complexity of P. vivax infections and differentiate parasite populations.

Different In Vitro Drug Susceptibility Profile of Plasmodium falciparum Isolates from Two Adjacent Areas of Northeast Myanmar and Molecular Markers for Drug Resistance.

The Greater Mekong Subregion (GMS) is the epicenter of antimalarial drug resistance. We determined in vitro susceptibilities to 11 drugs of culture-adapted Plasmodium falciparum isolates from adjacent areas (Laiza and Muse) along the China−Myanmar border. Parasites from this region were highly resistant to chloroquine and pyrimethamine but relatively sensitive to other antimalarial drugs. Consistently, the Dd2-like pfcrt mutations were fixed or almost fixed in both parasite populations, and new mutations mediating piperaquine resistance were not identified. Similarly, several mutations related to pfdhfr and pfdhps were also highly prevalent. Despite their geographical proximity, malaria parasites from Laiza showed significantly higher in vitro resistance to artemisinin derivatives, naphthoquine, pyronaridine, lumefantrine, and pyrimethamine than parasites from Muse. Likewise, the pfdhfr N51I, pfdhps A581G, pfmrp1 H785N, and pfk13 F446I mutations were significantly more frequent in Laiza than in Muse (p < 0.05). For the pfmdr1 mutations, Y184F was found only in Laiza (70%), whereas F1226Y was identified only in Muse (31.8%). Parasite isolates from Laiza showed a median RSA value of 5.0%, significantly higher than the 2.4% in Muse. Altogether, P. falciparum parasite populations from neighboring regions in the GMS may diverge substantially in their resistance to several antimalarial drugs. This information about different parasite populations will guide antimalarial treatment policies to effectively manage drug resistance during malaria elimination.

PvMSP-3α and PvMSP-3ß genotyping reveals higher genetic diversity in Plasmodium vivax parasites from migrant workers than residents at the China-Myanmar border.

BACKGROUND: The genetic diversity of malaria parasites traces the origin and spread of new variants and can be used to evaluate the effectiveness of malaria control measures. Therefore, this study aims to improve the understanding of the molecular epidemiology of Plasmodium vivax malaria at the China-Myanmar border by genotyping the PvMSP-3α and PvMSP-3ß genes. METHODS: Blood samples were collected from P. vivax malaria patients along the China-Myanmar border. The PvMSP-3α and PvMSP-3ß genes were amplified by polymerase chain reaction (PCR) and the genetic polymorphism and haplotype of the two genes were analyzed. RESULTS: A total of 422 blood samples were used for this study, of which 224 were analyzed at PvMSP-3α and 126 at PvMSP-3ß. Samples mainly were from young adults aged 18-45 years, although local patients were significantly younger than migrant laborers crossing the border at Tengchong (P < 0.0001). Molecular evolutionary analysis revealed that PvMSP-3α and PvMSP-3ß underwent diversifying natural selection, and intragenic recombination contributed to the diversity of the isolates. Based on the length of the genes, we identified three types of PvMSP-3α [1.9-2.0 kb (Type-A), 1.4-1.5 kb (Type-B), and 1.1-1.3 kb (Type-C)] and two types of PvMSP-3ß [1.7-2.2 kb (Type-A) and 1.4-1.5 kb (Type-B)]. Migrant laborers returning to China through Tengchong bore P. vivax infections displaying significantly higher genetic diversity than local residents. CONCLUSIONS: Both PvMSP-3 paralogs were subjected to diversifying selection in each sample population. Clustering of alleles supports ephemeral endemic differentiation of alleles, but the broader phylogeny suggests that alleles transit the globe, perhaps accelerated by movements of migrants such as those transiting Tengchong.

Application of a low-cost, specific, and sensitive loop-mediated isothermal amplification (LAMP) assay to detect Plasmodium falciparum imported from Africa.

BACKGROUND: Chinese citizens traveling abroad bring back imported malaria cases to China. Current malaria diagnostic tests, including microscopy and antigen-detecting rapid tests, cannot reliably detect low-density infections. To complement existing diagnostic methods, we aimed to develop a new loop-mediated isothermal amplification (LAMP) assay to detect and identify Plasmodium falciparum in Chinese travelers returning from Africa. METHODS: We developed a miniaturized LAMP assay to amplify the actin I gene of P. falciparum. Each reaction consumed only 25% of the reagents used in a conventional LAMP assay and the same amount of DNA templates used in nested PCR. We evaluated this LAMP assay's performance and compared it to microscopy and a nested PCR assay using 466 suspected malaria cases imported from Africa. We assessed the sensitivity of the new LAMP assay using cultured P. falciparum, clinical samples, and a plasmid construct, allowing unprecedented precision when quantifying the limit of detection. RESULTS: The new LAMP assay was highly sensitive and detected two more malaria cases than nested PCR. Compared to nested PCR, the sensitivity and specificity of the novel LAMP assay were 100% [95% confidence interval (CI) 98.5-100%] and 99.1% (95% CI 96.7-99.9%), respectively. When evaluated using serial dilutions of the plasmid construct, the detection limit of the new LAMP was as low as 102 copies/µL, 10-fold lower than PCR. The LAMP assay detected 0.01 parasites/µL of blood (equal to 0.04 parasites/µL of DNA) using cultured P. falciparum and 1-7 parasites/µL of blood (4-28 parasites/µL of DNA) in clinical samples, which is as good as or better than previously reported and commercially licensed assays. CONCLUSION: The novel LAMP assay based on the P. falciparum actin I gene was specific, sensitive, and cost-effective, as it consumes 1/4 of the reagents in a typical LAMP reaction.

In vitro susceptibility profile of Plasmodium falciparum clinical isolates from Ghana to antimalarial drugs and polymorphisms in resistance markers.

Drug resistance in Plasmodium falciparum compromises the effectiveness of antimalarial therapy. This study aimed to evaluate the extent of drug resistance in parasites obtained from international travelers returning from Ghana to guide the management of malaria cases. Eighty-two clinical parasite isolates were obtained from patients returning from Ghana in 2016-2018, of which 29 were adapted to continuous in vitro culture. Their geometric mean IC50 values to a panel of 11 antimalarial drugs, assessed using the standard SYBR Green-I drug sensitivity assay, were 2.1, 3.8, 1.0, 2.7, 17.2, 4.6, 8.3, 8.3, 19.6, 55.1, and 11,555 nM for artemether, artesunate, dihydroartemisinin, lumefantrine, mefloquine, piperaquine, naphthoquine, pyronaridine, chloroquine, quinine, and pyrimethamine, respectively. Except for chloroquine and pyrimethamine, the IC50 values for other tested drugs were below the resistance threshold. The mean ring-stage survival assay value was 0.8%, with four isolates exceeding 1%. The mean piperaquine survival assay value was 2.1%, all below 10%. Mutations associated with chloroquine resistance (pfcrt K76T and pfmdr1 N86Y) were scarce, consistent with the discontinuation of chloroquine a decade ago. Instead, the pfmdr1 86N-184F-1246D haplotype was predominant, suggesting selection by the extensive use of artemether-lumefantrine. No mutations in the pfk13 propeller domain were detected. The pfdhfr/pfdhps quadruple mutant IRNGK associated with resistance to sulfadoxine-pyrimethamine reached an 82% prevalence. In addition, five isolates had pfgch1 gene amplification but, intriguingly, increased susceptibilities to pyrimethamine. This study showed that parasites originating from Ghana were susceptible to artemisinins and the partner drugs of artemisinin-based combination therapies. Genotyping drug resistance genes identified the signature of selection by artemether-lumefantrine. Parasites showed substantial levels of resistance to the antifolate drugs. Continuous resistance surveillance is necessary to guide timely changes in drug policy.

[Correlation between conventional ultrasound features and BRAFV600E gene mutation and central lymph node metastasis in thyroid papillary carcinoma].

Objective:To investigate whether central lymph node metastasis（CLNM） in the central region of thyroid papillary carcinoma（PTC） is related to conventional ultrasound features of the primary lesion and BRAFV600E gene mutation. Methods:A total of 300 patients with PTC confirmed by surgical pathology and central lymph node dissection in the First Affiliated Hospital of Jinzhou Medical University from October 2019 to June 2021 were collected. The subjects were divided into the metastatic group and the non-metastatic group according to whether CLNM occurred. The correlation was determined by analyzing the conventional ultrasound characteristics and BRAFV600E gene test results of the two groups of patients. Results:Among 300 PTC patients, 120（40%） had CLNM. Univariate analysis showed that there were statistically significant differences between groups in gender, nodule maximum diameter line, number of lesions, boundaries, morphology, aspect ratio, proximity to the membrane, calcification and BRAFV600E gene mutation（P<0.05）. Logistic multivariate regression analysis showed that gender, maximum diameter line, aspect ratio, proximity to the membrane, microcalcification and BRAFV600E were the risk factors for CLNM in PTC patients（P<0.05）. ROC curve showed that when the maximum diameter was 8.5 mm, the Yooden index was the maximum. Conclusion:When the risk factors of male, maximum diameter ≥8.5 mm, aspect ratio ≥1, microcalcification, proximity to capsule and BRAFV600E（+） appear in PTC patients, high attention should be paid to preventive CLN dissection as soon as possible.

[Clinical and ultrasonic features of papillary thyroid microcarcinoma and risk factors for central lymph node metastasis].

Objective:To investigate the clinical and ultrasonic features of papillary thyroid microcarcinoma （PTMC） and the risk factors of central lymph node metastasis （CLNM）, to provide help for individualized treatment. Methods:One hundred and eighty-five patients with PTMC confirmed by surgery and pathology and underwent prophylactic CLN dissection were retrospectively analyzed. According to lymph node metastasis, patients were divided into metastasis group and non metastasis group. The size, shape, echo, aspect ratio, edge, protruding capsule, nodule location, calcification and calcification were analyzed. The relationship between clnm and ultrasonographic features, sex, age, single/multiple, whether or not with Hashimoto thyroiditis was analyzed. Results:Among 185 patients with PTMC, 55 cases had lymph node metastasis, and the metastasis rate was 30.0%. In univariate analysis, clnm of PTMC was significantly correlated with gender, age, tumor size, protruding capsule, type of calcification and single / multiple （all P<0.05）. In multivariate logistic regression analysis, tumor size, single/multiple, protruding capsule, gender and age were the risk factors of clnm. According to the ROC curve, when the age was 49.5 years old and the mass size was 4.8 mm, the diagnostic test yoden index reached the maximum value. Conclusion:When the risk factors of male, ≤49.5 years old, tumor size≥4.8 mm, multiple and protruding capsule appeared in PTMC patients, preventive CLN clearance should be taken actively.

[MEDIUM-TERM EFFECTIVENESS OF BOTH SIDE STEREO SUTURE FOR MENISCUS BUCKET HANDLE TEAR UNDER ARTHROSCOPY].

OBJECTIVE: To investigate the medium-term effectiveness of both side stereo suture for meniscus bucket handle tear under arthroscopy. METHODS: Between January 2009 and December 2012, 38 patients with combined injury of meniscus bucket handle tear underwent arthroscope surgery by the method of both side stereo suture. There were 26 males and 12 females with an average age of 32 years (range, 19-42 years). The injury causes included sports injury in 21 cases, traffic accident injury in 11 cases, and other trauma in 6 cases. The left knee was involved in 15 cases and the right knee in 23 cases. The interval between injury and operation was 2 days to 6 months (mean, 2.5 months). MRI examination showed meniscus injury at grade III, including 22 cases of internal injury and 16 cases of lateral injury. The Barrett standard, Lysholm score of knee joint, and joint mobility were used to assess the function recovery of the knee joint. RESULTS: All incisions healed primarily with no complication. All cases were followed up 24.5 months on average (range, 18-36 months). Based on the Barrett standard, meniscus bucket handle tear was successfully repaired in all cases. The Lysholm scores and range of motion of the knee at 6 months, 12 months, and last follow-up were significantly better than preoperative ones (P < 0.05). CONCLUSION: The both side stereo suture of meniscus bucket handle tear under arthroscopy has an excellent medium-term effectiveness, with the advantages of firm suture and high healing rate.

[Study on correlation between the pathological changes under arthroscopy and the cytokine levels in the knee osteoarthritis of the Blood Stasis type].

OBJECTIVE: To explore the correlation between cytokine levels and the pathological changes under arthroscopy in knee osteoarthritis of Blood Stasis type. METHODS: From 2009.2 to 2010.3, 90 patients with knee osteoarthritis were reviewed. Among the patients, 17 patients were male and 73 patients were female, ranging in age from 40 to 70 years, averaged 57.2 years, the duration of the disease ranged from 1 month to 10 years, with a mean of 3.4 years. Thirty-one patients had osteoarthritis in left knee, and 59 patients in right knee. The patients had the syndrome of blood stasis. All the patients had pain and morning stiffness; most patients had joint interlocking; and all the patients didn't have joint swelling. The synovial fluid was collected before surgery, and ELISA was used to detect the contents of interleukin-1beta and transforming growth factor-beta1. At the same time, the pathological changes of the joint were observed under the arthroscopy. Based on the above datum analysis, the severity of knee osteoarthritis of blood stasis type was studied, and the correlation between different types of pathological changes under arthroscopy and cytokine levels was analyzed. RESULTS: The contents of IL-1beta and TGF-beta1 in synovial fluid were (28.18 +/- 5.57) pg/ml and (51.69 +/- 6.56) pg/ml respectively. The level of IL-1beta of grade III-IV cartilage degeneration was (30.65 +/- 3.48) pg/ml, which was significantly higher than (20.55 +/- 3.50) pg/ml of grade I-II cartilage degeneration group; the level of TGF-beta1 of grade I-II cartilage degeneration was (58.18 +/- 3.98) pg/ml,which was significantly higher than (49.59 +/- 5.83) pg/ml of grade II-IV cartilage degeneration group. IL-1beta and cartilage degeneration was positively correlated, the correlation coefficient was 0.744; TGF-beta1 and cartilage degeneration was negatively correlated, the correlation coefficient was -0.563. The level of IL-1beta of grade II-III synovial hyperplasia was (33.48 +/- 2.95) pg/ml, which was significantly higher than (25.40 +/- 4.50) pg/ml of grade I synovial hyperplasia group; IL-beta was positively correlated with synovial hyperplasia, the cor- relation coefficient was 0.801. The levels of IL-1beta of grade I osteophyte formation was (34.18 +/- 2.69) pg/ml, which was significantly higher than (25.74 +/- 4.48) pg/ml of grade 0 osteophyte formation group; the level of TGF-beta 1 of grade 0 osteophyte formation was (53.11 +/- 6.78) pg/ml, which was higher than (48.21 +/- 4.47) pg/ml of grade I osteophyte formation group. IL-1beta was positively correlated with osteophyte formation, the correlation coefficient was 0.762; TGF-beta1 was negatively correlated with osteophyte formation, the correlation coefficient was - 0.340. CONCLUSION: All the patients with knee osteoarthritis identified as blood stasis syndrome have pathological changes such as articular cartilage degeneration and synovial hyperplasia. The level of IL-1beta has important reference value to estimate the severity of cartilage degeneration, synovial hyperplasia and osteophyte proliferation.