Genetic diversity analysis of PvCSP and its application in tracking of Plasmodium vivax.

BACKGROUND: Plasmodium vivax remains a potential cause of morbidity and mortality for people living where it is endemic. Understanding the regional genetic diversity of P. vivax is valuable for studying population dynamics and tracing the origins of parasites. The Plasmodium vivax circumsporozoite gene (PvCSP) is highly polymorphic and has been used previously as a marker in P. vivax population studies. The aim of this study is to investigate the genetic diversity of the PvCSP, to provide more genetic polymorphism data for further studies on P. vivax population structure, and tracking of the origin of clinical cases. METHODS: Nested PCR and DNA sequencing of the PvCSP were performed to obtain nucleotide sequences of P. vivax isolates collected from Zhejiang province, China, between 2006 and 2014. To investigate the genetic diversity of PvCSP, the nucleotide sequences and amino acid sequences of the PvCSP were analyzed using DNAstar, Mega software and the phylogenetic tree constructed. The relatedness between the polymorphism and infection source were also analyzed using the SPSS software. RESULTS: The 66 P. vivax isolates collected from Zhejiang province were either indigenous cases or cases imported from different regions of the world. All 66 P. vivax isolates belonged to the VK210 variant. Fourteen different Peptide Repeat Motifs (PRMs) were detected in the Central Repeat Region (CRR) of PvCSP, among which, two PRMs of GDRADGQPA and GDRAAGQPA were widely distributed in all isolates. Several polymorphic characteristics of the VK210 variant were observed, including the insertion sequence of 12 peptides, the frequency of the GGNA repeat, the frequency of the PRMs repeat in CRR, and the frequency of the PRM of GNGAGGQAA repeat, which were indicative for tracking the parasite. CONCLUSION: This study presents abundant genetic diversity in the PvCSP marker among P. vivax strains around the world. The genetic data are valuable to expand the polymorphism information on P. vivax, which could be helpful for further study on population dynamics and tracking the origin of P. vivax.

[Identification of Trypanosoma Species in Rattus flavipectus and R. losea using Microscopy and PCR Method].

OBJECTIVE: To identify two suspected Trypanosoma species in Rattus found in Zhejiang Province using microscopy and PCR method. METHODS: Blood samples were collected from Rattus losea and R. flavipectus. Blood smears were prepared, and observed under microscope. The morphological indices of trypanosomes were measured and calculated. The genomic DNA was extracted from the trypanosomes, and the specific fragment of Trypanosoma 18S rRNA gene was amplified using PCR. The products were further sequenced and submitted to GenBank. Blast analysis was performed on line in NCBI. RESULTS: Blood samples from Ratts flavipectus and R. losea were collected from Lucheng District and Wencheng County of Wenzhou, respectively. The parasites from R. losea and R. flavipectus were found to possess the characteristic features of Trypanosoma species, such as nucleus, free flagellum, and kinetoplast, etc. The body length was 27.50 µm and 23.80 µm, and the free flagellum length was 9.60 im and 9.20 jim, respectively. The nucleus index was 0.74 and 1.05, the kinetoplast Index was 1.40 and 1.57, respectively. Based on the morphological characteristics and host specificity, the parasites from R. losea and R. flavipectus were identified as Herpetosoma species, mainly found in rodents. The amplified products were about 700 bp by 18S rRNA gene PCR with the DNA isolated from the trypanosomes. The products were further sequenced, and the resulting sequences were submitted to GenBank with assession numbers of KP098535 (from R. losea) and KP098536 (from R. flavipectus). Blast analysis showed that KP098535 was completely homologous with the sequences from Herpetosoma subgenus (AY491765.1, AY491764.1, and AJ223568.1), and KP098536 was completely homologous with Trypanosoma lewisi (AB242273.1, AJ009156.1). CONCLUSION: The Trypanosoma species found from Rattus flavipectus is Trypanosoma lewisi, and the other one belongs to Herpetosoma subgenus, which may be named as Trypanosoma lewisi-like trypanosome.

[Iditification of five imported cases of Plasmodium ovale wallikeri infection in Zhejiang Province].

OBJECTIVE: To identify and analyze Plasmodium ovale wallikeri in 5 imported malaria cases, who were detected positive by microscopy and negative by conventional PCR. METHODS: Epidemiological information and blood samples were collected from the five patients. The detection was conducted by microscopy, Rapid Diagnostic Test (RDT) and nested PCR with Plasmodium genus-specific, species-specific and Plasmodium ovale wallikeri-specific primers. The amplified products were sequenced and Blast analysis was performed on line in NCBI. RESULTS: The five patients returned from Africa, and all had a history of malaria. They were microscopically positive for Plasmodium sp., and two cases showed Pan positive RDT result. All blood samples were negative for four Plasmodium spp. by conventional nested PCR, but positive by nested PCR with Plasmodium ovale wallikeri-specific primers. Blast analysis showed that the amplified sequences of the five cases had complete homology with P. ovale wallikeri clone RSH10 18S ribosomal RNA gene (Accession No. KF219561.1). CONCLUSION: The five cases which classified as positive by microscopy while negative by conventional PCR have been confirmed as Plasmodium ovale wallikeri infection by nested PCR with P. ovale wallikeri-specific primers.

Mindfulness-Based Therapy for Military Populations with Chronic Pain: A Systematic Review.

Background: Due to the limited role of chronic pain medication in military personnel and the distress caused to the military population, mindfulness-based therapy has been considered for the follow-up treatment of military personnel with chronic pain. The purpose of this review is to explore the effect and the implementation of mindfulness-based therapy for the military population with chronic pain. Methods: The keywords for the search included "mindfulness" AND ("pain" OR "chronic pain") AND ("military" OR "veteran"). The PubMed, Embase, and Cochrane Library databases were searched. The Cochrane Collaboration tool was used to independently assess the risk of bias of the included randomized controlled trials, and the Newcastle-Ottawa Scale was used to independently assess the risk of bias of the included case-control studies. Results: A total of 175 papers were identified; 65 duplicates were excluded, and 59 papers that did not meet the inclusion criteria were excluded after reading the titles and abstracts. The remaining 51 papers were read in full, 42 of which did not meet the inclusion criteria. Nine papers met the inclusion criteria and were included in the study. The nine studies included 507 veterans and 56 active-duty female military personnel. All pain interventions were mindfulness-based therapy, and all of them were integrated into or adapted from standard mindfulness courses. The results all showed that after mindfulness-based therapy, the relevant indicators improved. Conclusions: Mindfulness-based therapy is an effective treatment method for the military population with chronic pain. The review indicates that future research should focus on the best setting for mindfulness-based therapy, including the course content and time.

[Species identification in 5 imported cases previously diagnosed as Vivax malaria by parasitological and nested PCR techniques].

OBJECTIVE: To identify the species of malaria parasites in 5 imported cases previously diagnosed as vivax malaria. METHODS: Epidemiological information and blood samples were collected from five patients who returned from Africa and were diagnosed as vivax malaria. The detection was conducted by microscopy, right VIEW rapid malaria test (RDTs) and nested PCR with Plasmodium genus-specific and species-specific primers. The amplified products were sequenced and Blast analysis was performed. RESULTS: Three of the 5 cases had a history of malaria attack. Microscopically, 4 cases were confirmed as Plasmodium ovale infection, 1 (case 1) was co-infected with P. vivax and P. ovale. All 5 cases showed negative RDT results. Nested PCR detection revealed that the 5 cases had a P. ovale-specific fragment (800 bp), while case 1 had a P. vivax-specific fragment (120 bp) concurrently. Blast analysis showed that the amplified sequence of the 5 cases had a high sequence homology (99%) with P. ovale gene for small subunit ribosomal RNA from GenBank, and that of case 1 also shared 99% homology with P. vivax isolate SV5 18S ribosomal RNA gene (GenBank accession number: JQ627157.1). CONCLUSION: Among the five cases, four were infected by Plasmodium ovale, and one was co-infected with both P. vivax and P. ovale.

SARS-CoV-2 and Plasmodium falciparum Co-Infection in a Returning Traveler.

Since December 2019, the Coronavirus Disease 2019 (COVID-19) pandemic has become a non-neglectable context for the whole healthcare system. Under the background of COVID-19, the detection and diagnosis of malaria cases are under challenge. Here, we reported a COVID-19 and malaria co-infection traveler who has a long living history in Cameroon. The case was administered with dihydroartemisinin and piperaquine tablets for malaria, Lopinavir and Ritonavir tablets, Arbidol, recombinant human interferon α-2b and Compound Maxing Yifei mixture for COVID-19, and Zolpidem Tartrate tablets, Diazepam, Paroxetine Hydrochloride tablets, Thymosin α1, and Lianhua Qinwen Jiaonang during the second hospitalization of the patient since the patient has a certain level of anxiety and insomnia with no evidence of inflammatory reactions. After being tested negative two times for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 48 h, the patient met China's COVID-19 discharge standards and was discharged with stable vital signs and mental state. Since most countries in the sub-Saharan region have a fragile health system, co-infection for both Plasmodium and SARS-CoV-2 may not be uncommon, and raise a challenge in diagnosis, treatment, and prevention for both diseases. We add to the literature on co-infection of P. falciparum malaria and COVID-19 and offer operational advice on diagnosis, prevention, and treatment for the co-infection.

Classification of Early-Onset and Late-Onset Idiopathic Chronic Pancreatitis Needs Reconsideration.

Bimodal classification of idiopathic chronic pancreatitis (ICP) into early-onset (<35 years) and late-onset (>35 years) ICP was proposed in 1994 based on a study of 66 patients. However, bimodal distribution wasn't sufficiently demonstrated. Our objective was to examine the validity and relevance of the age-based bimodal classification of ICP. We analyzed the distribution of age at onset of ICP in our cohort of 1633 patients admitted to our center from January 2000 to December 2013. Classify ICP patients into early-onset ICP(a) and late-onset ICP(a) according to different cut-off values (cut-off value, a = 15, 25, 35, 45, 55, 65 years old) for age at onset. Compare clinical characteristics of early-onset ICP(a) and late-onset ICP(a). We found slightly right skewed distribution of age at onset for ICP in our cohort. There were differences between early-onset and late-onset ICP with respect to basic clinical characteristics and development of key clinical events regardless of the cut off age at onset i.e. 15, 25, 35, 45 or even higher. The validity of the bimodal classification of early-onset and late-onset ICP could not be established in our large patient cohort and therefore such a classification needs to be reconsidered.

Identification and molecular typing of Naegleria fowleri from a patient with primary amebic meningoencephalitis in China.

Naegleria fowleri is the only Naegleria spp. known to cause an acute, fulminant, and rapidly fatal central nervous system infection in humans called primary amebic meningoencephalitis (PAM). In 2016, a patient with suspected PAM was found in Zhejiang Province of China. The pathogen was identified by microscopic examination and PCR. The positive PCR products were sequenced and the sequences were aligned using the NCBI BLAST program. The homologous and phylogenetic analysis was conducted using MEGA 6 program. On microscopy of direct smears, motile cells with pseudopodia were observed, and the motion characteristics of the pseudopodia as well as the cell morphology suggested that the pathogens were amoeba trophozoites. Wright-Giemsa-stained smears showed amoeba trophozoites of various shapes, which measured 10-25µm in size; these were characterized by a prominent, centrally placed nucleolus and a vacuolated cytoplasm. PCR was negative for Entamoeba histolytica and Entamoeba dispar, but positive for Naegleria spp. and N. fowleri. The nucleotide sequences acquired in this study have been submitted to GenBank with accession numbers KX909928 and KX909927, respectively. The BLAST analysis revealed that the sequences of KX909928 and KX909927 had 100% similarity with the sequence of the N. fowleri gene (KT375442.1). Sequence alignment and the phylogenetic tree revealed that the N. fowleri collected in this study was classified as genotype 2 and was most closely related to Naegleria lovaniensis. This study confirmed N. fowleri as the agent responsible for the infection in this patient. PAM normally progresses rapidly and is generally universally fatal within a week. Unfortunately this patient died at 2 weeks after the onset of symptoms.

Genetic diversity of Plasmodium Vivax revealed by the merozoite surface protein-1 icb5-6 fragment.

BACKGROUND: Plasmodium vivax remains a potential cause of morbidity and mortality for people living in its endemic areas. Understanding the genetic diversity of P. vivax from different regions is valuable for studying population dynamics and tracing the origins of parasites. The PvMSP-1 gene is highly polymorphic and has been used as a marker in many P. vivax population studies. The aim of this study was to investigate the genetic diversity of the PvMSP-1 gene icb5-6 fragment and to provide more genetic polymorphism data for further studies on P. vivax population structure and tracking of the origin of clinical cases. METHODS: Nested PCR and sequencing of the PvMSP-1 icb5-6 marker were performed to obtain the nucleotide sequences of 95 P. vivax isolates collected from Zhejiang province, China. To investigate the genetic diversity of PvMSP-1, the 95 nucleotide sequences of the PvMSP-1 icb5-6 fragment were genotyped and analyzed using DnaSP v5, MEGA software. RESULTS: The 95 P. vivax isolates collected from Zhejiang province were either indigenous cases or imported cases from different regions around the world. A total of 95 sequences ranging from 390 to 460 bp were obtained. The 95 sequences were genotyped into four allele-types (Sal I, Belem, R-III and R-IV) and 17 unique haplotypes. R-III and Sal I were the predominant allele-types. The haplotype diversity (Hd) and nucleotide diversity (Pi) were estimated to be 0.729 and 0.062, indicating that the PvMSP-1 icb5-6 fragment had the highest level of polymorphism due to frequent recombination processes and single nucleotide polymorphism. The values of dN/dS and Tajima's D both suggested neutral selection for the PvMSP-1icb5-6 fragment. In addition, a rare recombinant style of R-IV type was identified. CONCLUSIONS: This study presented high genetic diversity in the PvMSP-1 marker among P. vivax strains from around the world. The genetic data is valuable for expanding the polymorphism information on P. vivax, which could be helpful for further study on population dynamics and tracking the origin of P. vivax.