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.

[Diagnosis and treatment of a local case of Taenia saginata infection in Zhejiang Province].

Objective: To diagnose and provide treatment for a local case of taenia infection in Zhejiang Province and identify the species of the worm. Methods: The information of disease onset, clinical feature and therapeutic process was collected and epidemiological investigation was carried out. The anal cellophane swab was used to detect the eggs. Areca and pumpkin seeds were used for deworming. Morphological observation, PCR amplification and sequencing of cytochrome C oxidase 1(COX1) gene were performed for the discharged worm. Results: The epidemiological results showed that the patient did not go outside Pujiang County in the past two years, and had no history of eating raw pork, beef or animal offal. But she often had barbecues and hot-pot food, occasionally with raw vegetables. Taenia eggs were found on her perianal skin. The discharged worm was suspected to be Taenia saginata or Taenia asiatica by morphological observation. PCR amplification of COX1 resulted in a band of 832 bp, which was 99%, 96% and 88% homologous to COX1 of Taenia saginata (GenBank accession number: AB107239.1), Taenia asiatica (GenBank accession number: AB107235.1) and Taenia solium (GenBank accession number: AB066485.1), respectively. Conclusion: According to the clinical feature, epidemiological information and sequencing results, this case is confirmed to be a local infection of Taenia saginata.

[An Investigation on a Case of Family-clustered Paragonimiasis in Wenzhou City].

Paragonimus infection was reported in a family of 8 members after consuming wine-preserved raw crabs. Seven members developed symptoms of fever, cough, sputum, fatigueness, chest pain, and abdominal pain during 2-3 months after crab feeding, while one member was normal. Serum samples were collected from the 7 members for anti-paragonimus antibody test, with 5 members showing positive, 1 weakly positive and 1 negative. In view of the epidemiological history, clinical symptoms, imaging and laboratory test results, this case was diagnosed to be a family-clustered paragonimus infection.

[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.

Development and application of an AllGlo probe-based qPCR assay for detecting knockdown resistance (kdr) mutations in Anopheles sinensis.

BACKGROUND: Anopheles sinensis is one of the most important malaria vectors in China and other Southeast Asian countries. High levels of resistance have been reported in this species due to the long-term use of insecticides, especially pyrethroids, for public health and agricultural purposes. Knockdown resistance (kdr) caused by a single base pair mutation in the gene encoding the sodium channel is strongly associated with pyrethroid insecticide resistance in many Anopheles mosquitoes. There are few methods currently available for detecting kdr mutations in An. sinensis. METHODS: A novel AllGlo probe-based qPCR (AllGlo-qPCR) method was developed to screen for the predominant kdr mutations in An. sinensis mosquitoes from the Jiangsu Province. The results from AllGlo-qPCR, allele-specific PCR (AS-PCR), and TaqMan-MGB probe-based qPCR (TaqMan-qPCR) were compared. A comparative analysis of the equipment required, ease of use and cost of the available methods was also performed. Finally, the AllGlo-qPCR method was used to detect the frequencies of kdr mutations from the other four provinces in central China. RESULTS: Six kdr genotypes were detected in An. sinensis from the Jiangsu Province by DNA sequencing. The AllGlo-qPCR method detected all of the kdr genotypes with a high level of accuracy (97% sensitivity and 98% specificity). AllGlo-qPCR correctly determined the kdr genotypes of 98.73% of 158 An. sinensis samples, whereas TaqMan-qPCR and AS-PCR correctly identified 96.84% and 88.61% of mutations, respectively. Furthermore, the AllGlo-qPCR method is simpler to perform, requires less equipment, and exhibits a moderate expense cost comparing with the other tested methods of kdr mutation detection. Samples collected from four of the other provinces in central China showed a high frequency of kdr mutation in An. sinensis, as detected by the established AllGlo-qPCR method. CONCLUSION: The novel AllGlo-qPCR method developed for kdr mutation detection in An. sinensis exhibits greater specificity and sensitivity than currently available methods and is more cost-effective; therefore, it represents a useful tool for entomological surveillance.

[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.

[Tracing investigation and diagnosis of one transfusion-transmitted malaria case in Zhejiang Province].

OBJECTIVE: To identify the sources of infection and the mode of transmission of a malaria case with unknown origin. METHODS: Clinical data of the case were collected and the epidemiological investigation was conducted. The blood samples of the patient and the suspected infection source (blood donor) were detected by microscopy, rapid diagnostic test strip (RDT) and nested PCR. RESULTS: The patient did not visited malaria endemic areas. After a blood transfusion, the patient had chills and fever, and was confirmed as falciparum malaria by microscopy with bone marrow and peripheral blood smears and RDT. The blood donor was a worker returned from Africa. Before blood donation she was sick like malaria carrier, and took anti-malarial drug. She was then confirmed as falciparum malaria by RDT and microscopy. The blood samples from the patient and the blood donor were diagnosed as falciparum malaria by nested PCR, and the similarity of the small subunit rRNA (SSU rRNA) sequence was 100%, showing they were mix-infected with K1 and MAD20 genotypes of Plasmodium falciparum. CONCLUSION: This patient is confirmed P. falciparum infection via blood transfusion from a donor who returned from Africa.

Sodium hydrosulfide alleviates lung inflammation and cell apoptosis following resuscitated hemorrhagic shock in rats.

AIM: To investigate the protective effects of hydrogen sulfide (H2S) against inflammation, oxidative stress and apoptosis in a rat model of resuscitated hemorrhagic shock. METHODS: Hemorrhagic shock was induced in adult male SD rats by drawing blood from the femoral artery for 10 min. The mean arterial pressure was maintained at 35-40 mmHg for 1.5 h. After resuscitation the animals were observed for 200 min, and then killed. The lungs were harvested and bronchoalveolar lavage fluid was prepared. The levels of relevant proteins were examined using Western blotting and immunohistochemical analyses. NaHS (28 µmol/kg, ip) was injected before the resuscitation. RESULTS: Resuscitated hemorrhagic shock induced lung inflammatory responses and significantly increased the levels of inflammatory cytokines IL-6, TNF-α, and HMGB1 in bronchoalveolar lavage fluid. Furthermore, resuscitated hemorrhagic shock caused marked oxidative stress in lung tissue as shown by significant increases in the production of reactive oxygen species H2O2 and ·OH, the translocation of Nrf2, an important regulator of antioxidant expression, into nucleus, and the decrease of thioredoxin 1 expression. Moreover, resuscitated hemorrhagic shock markedly increased the expression of death receptor Fas and Fas-ligand and the number apoptotic cells in lung tissue, as well as the expression of pro-apoptotic proteins FADD, active-caspase 3, active-caspase 8, Bax, and decreased the expression of Bcl-2. Injection with NaHS significantly attenuated these pathophysiological abnormalities induced by the resuscitated hemorrhagic shock. CONCLUSION: NaHS administration protects rat lungs against inflammatory responses induced by resuscitated hemorrhagic shock via suppressing oxidative stress and the Fas/FasL apoptotic signaling pathway.

[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.

[Diagnosis and analysis of the first imported ovale malaria case in Wenzhou City].

The first imported case of Plasmodium ovale infection in Wenzhou City was confirmed by microscopy and PCR test. The patient returned from the People's Republic of Congo to Wenzhou for three and a half months presented a history of fever with chills and rigors on April 30, 2012. The results from peripheral blood smear examination and PCR analysis confirmed a diagnosis of P. ovale infection. The patient was treated with chloroquine plus primaquine for eight days and the symptoms improved.

Exogenous hydrogen sulfide protects against traumatic hemorrhagic shock via attenuation of oxidative stress.

OBJECTIVE: This study was designed to investigate the protective effects of exogenous hydrogen sulfide (H(2)S) on trauma-hemorrhagic shock (T-H). MATERIALS AND METHODS: Forty-eight male Sprague-Dawley rats were anesthetized, while 32 were subjected to both midline laparotomy and hemorrhagic shock (35-40 mmHg for 90 min) by bleeding them from the femoral artery. One hour later, resuscitation was initiated with Ringer lactate. NaHS (28 µmol/kg) or vehicle alone was administered intraperitoneally at the onset of resuscitation. Two hours later, eight animals from each group were re-anesthetized to determine cardiac function, blood gas concentrations, and hepatic and renal function. Superoxide dismutase activity (SOD), malondialdehyde concentrations (MDA), and the activity of myeloperoxidase (MPO) in the serum were measured and pulmonary wet/dry (W/D) ratio and histopathologic evaluations performed. RESULTS: NaHS resulted in an increase in mean arterial blood pressure, left ventricular pressure and positive (+dP/dt(max)) and negative (-dP/dt(max)) first derivatives of pressure as compared with the vehicle only group. The pH, PaO(2) and base excess (BE) were increased in the NaHS-treated group compared with the vehicle-treated group. Aspartate aminotransferase, alanine aminotransferase, blood urea nitrogen, and serum creatinine were reduced in the NaHS-treated group. NaHS also significantly reduced the high mortality rate at 24 h otherwise caused by T-H. The NaHS-treated group showed a remarkable decrease in MDA and MPO concentrations in plasma and an increase in SOD as compared with the vehicle-treated group. Histopathologic analysis indicated less edema, congestion, inflammatory cell infiltration and necrosis in heart, lung, liver and kidney tissue in NaHS-treated group. CONCLUSIONS: The present study demonstrates that exogenous H(2)S administered at an appropriate dose confers protective effects after T-H and resuscitation, by preventing a decrease in the antioxidant defense system.

An exogenous hydrogen sulphide donor, NaHS, inhibits the nuclear factor κB inhibitor kinase/nuclear factor κb inhibitor/nuclear factor-κB signaling pathway and exerts cardioprotective effects in a rat hemorrhagic shock model.

Hemorrhagic shock (HS) is a common condition and leading cause of death in trauma patients universally. Severe inflammatory responses during HS finally lead to multiple-organ failure. Hydrogen sulphide (H2S) is increasingly recognized as an important signaling molecule with various protective effects. In the present study, we investigated the antiinflammatory and cardioprotective effects of an exogenous H2S donor, sodium hydrosulfide (NaHS), in an HS rat model. Male Sprague-Dawley rats were randomly divided into the sham-operated, sham-operated treated with NaHS (28 µmol/kg, intraperitoneally (i.p.)), HS, and HS treated with NaHS (28 µmol/kg, i.p.) groups. The HS groups were subjected to mimicked HS for 1 h and then treated with NaHS or left untreated. The rats were then resuscitated with Ringer lactate solution for 1 h. Myocardial enzymes and inflammatory cytokines were evaluated. Morphologic changes in cardiac tissue and ultrastructural injury were also analyzed. HS resulted in significant hemodynamic deterioration and increased myocardial enzyme and inflammatory cytokine levels. Intraperitoneal administration of NaHS significantly prevented hemodynamic deterioration and decreased the elevation of myocardial enzymes. NaHS also inhibited the nuclear factor κB inhibitor kinase (IKK)/nuclear factor κB inhibitor (IκB)/nuclear factor κB (NF-κB) signaling pathway. The results suggest that NaHS exerts cardioprotective effects against HS. The protective effects of NaHS may occur via down-regulation of the IKK/IκB/NF-κB signaling pathway.

[Pathogen identification and clinical diagnosis for one case infected with Babesia].

OBJECTIVE: To identify the pathogen and make diagnosis on a case who was misdiagnosed as malaria. METHODS: Clinical and epidemiological information of the suspected case was collected. Blood samples during hospitalization were collected and examined microscopically. Genomic DNA from the blood samples was amplified by Babesia 18S RNA genus- and species- specific primers, respectively, and the amplified products were used in sequencing and BLAST sequence analysis. RESULTS: The case had a fever over 20 days repeatedly with anaemia (RBC 2.59 x 10(12), HB 5.5 g/L) and hepatosplenomegaly. The unidentified parasites were found in the bone marrow and blood smear after Giemsa staining. Epidemiological information revealed that this case had a history of blood transfusion and tick bites. 1 625 bp and 449 bp band generated by PCR amplification from blood sample using Babesia genus- and species-specific primers, and the sequence homology was 99% in comparison to Babesia microti (AB241632) with BLAST analysis. CONCLUSION: The clinical information, epidemiological history, and the PCR identification confirm the diagnosis of Babesia microti infection.

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.

The effect of ketamine on N-methyl-D-aspartate receptor subunit expression in neonatal rats.

BACKGROUND AND OBJECTIVE: Ketamine has been widely used in paediatric anaesthesia but its influence on development in infants and toddlers still remains unclear. In order to elucidate the influence of ketamine on brain development in neonatal rats, semiquantitative reverse transcriptase PCR, quantitative reverse transcriptase PCR and immunohistochemistry assays were performed to detect the expression of N-methyl-D-aspartate receptor subtypes expression. METHODS: Seven-day-old rats were divided into two random groups. All of them were injected with ketamine intraperitoneally at postnatal day (PND) 7; one group was sacrificed at PND 7, but the other group was sacrificed at PND 28. Each group was divided into five random subgroups. RESULTS: In the semiquantitative reverse transcriptase PCR and quantitative reverse transcriptase PCR experiments, ketamine treatment caused a marked increase in mRNA expression in all subtypes at PND 7 and in NR2A subtypes at PND 28. Immunohistochemistry results indicated that NR2A, 2B and 2C receptor protein increased significantly at PND 7, and NR2A receptor protein increased at PND 28. CONCLUSIONS: Exposure to ketamine resulted in an increase in N-methyl-D-aspartate receptor subunits at PND 7, and this increase persisted to PND 28 in NR2A.

[Epidemiological investigation on an outbreak of amoebic dysentery in Jiangshan City of Zhejiang Province].

The investigation was carried out from 15 May to 10 June 2006 among diarrhea patients of two schools and periphery residents in Hecun Town, Jiangshan City. Stool samples were examined for Entamoeba histolytica. Water samples were taken for microbial analysis. 31 cases with E. histolytica were found,with 74.2% (23 cases) of students and preschool children. 9 cases were found in Liuyi kindergarten and 8 cases in Hecun central primary school with a prevalence of 7.4% and 0.65%, respectively. Among 594 asymptomatic close contactors, 9 cases (1.5%) were carriers of cysts. Of the 31 cases, 22 were found with no habit of handwashing before eating or after defecation, and 14 cases had a close contact to the patients. No amoebic cysts or trophozoites were found from 12 water samples collected from schools or patient's houses, but the Escherichia coli level exceeded the national standard in 7 samples.

[The first national survey on natural nidi of Angiostrongylus cantonensis in China].

OBJECTIVE: To reveal the natural distribution of Angiostrongylus cantonensis in the mainland of China. METHODS: The potential areas of A. cantonensis and its main intermediate host Pomacea canaliculata were predicted based on degree-day models using GIS technique. A grid sampling was performed on the prediction map and 5% grids were randomly sampled. A total of 55 sampled sites were selected for the survey on A. cantonensis and its hosts in September-October of 2006. RESULTS: Nineteen Provinces/Municipalities/Autonomous Regions were found as potential habitats for P. canaliculata in the mainland of China. It was then confirmed that the snails distributed in the provinces of Fujian, Jiangxi, Zhejiang, Hunan, Guangdong, Guangxi, Hainan, and Yunnan. Higher prevalence of A. cantonensis in P. canaliculata was detected in Jianou of Fujian (36.6%), Xingguo of Jiangxi (19.9%), Rui'an of Zhejiang (16.0%), Rucheng of Hunan (5.0%), Huazhou of Guangdong (6.3%), Shangsi of Guangxi (39.1%) and Wuzhishan of Hainan (25.0%). CONCLUSION: Natural nidi of A. cantonensis have been found in seven provinces where natural infection in P. canaliculata has been detected.

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.