ABSTRACT
Nelson medium and modified PYNFH medium were used for the axenic culture of the Naegleria fowleri clinical strain LDL to compare the effects of different temperatures on the organism's growth. In addition, Nelson medium supplemented with 1% peptone (N + pep) and modified PYNFH medium without peptone (PYNFH - pep), without yeast extract (PYNFH - yext), without folic acid (PYNFH - folac), and without yeast nucleic acid (PYNFH - yna) were used in order to compare the various effects of these medium components. In general, N. fowleri grew best at 37 C. The highest trophozoite densities per 10,000 µm2 were observed when N + pep and PYNFH - yext were used. At 25, 37, and 43 C, the growth density profile values were 50.5 ± 6.36 vs. 58 ± 1.41; 2,550 ± 494.97 vs. 2,100 ± 141.42; and 1,735 ± 21.21 vs. 1,800 ± 14.14, respectively. On the other hand, PYNFH - pep gave the lowest growth with its highest cell densities being 9 ± 1.41 at 25 C, 108 ± 7.07 at 37 C, and 169 ± 15.55 at 43 C. When the various medium components were compared, supplementation with peptone promoted parasite growth. Besides, yeast extract had an inhibitory effect and was able to swamp the growth promoting effect of peptone. Thus N + pep and PYNFH - yext are recommended as the best media for in vitro culture of N. fowleri.
Subject(s)
Culture Media/metabolism , Naegleria fowleri/growth & development , Azure Stains , Coloring Agents , Culture Media/chemistry , Folic Acid , Nucleic Acids , Peptones , Temperature , Yeasts/chemistryABSTRACT
Vittaforma corneae is an obligate intracellular fungus and can cause human ocular microsporidiosis. Although accumulating reports of V. corneae causing keratoconjunctivitis in both healthy and immunocompromised persons have been published, little is known about the organism's occurrence in aquatic environments. Limitations in detection sensitivity have meant a large sampling volume is required to detect the pathogen up to now, which is problematic. A recent study in Taiwan has shown that some individuals suffering from microsporidial keratitis (MK) were infected after exposure to the pathogen at a hot spring. As a consequence of this, a survey and analysis of environmental V. corneae present in hot springs became an urgent need. In this study, sixty water samples from six hot spring recreation areas around Taiwan were analyzed. One liter of water from each sample site was filtered to harvest the fungi. The positive samples were detected using a modified nested PCR approach followed by sequencing using specific SSU rRNA gene primer pairs for V. corneae. In total fifteen V. corneae-like isolates were identified (25.0% of sites). Among them, six isolates, which were collected from recreational areas B, C and D, were highly similar to known V. corneae keratitis strains from Taiwan and other countries. Furthermore, five isolates, which were collected from recreation areas A, C, E and F, were very similar to Vittaforma-like diarrhea strains isolated in Portugal. Cold spring water tubs and public foot bath pools had the highest detection rate (50%), suggesting that hot springs might be contaminated via untreated water sources. Comparing the detection rate across different regions of Taiwan, Taitung, which is in the east of the island, gave the highest positive rate (37.5%). Statistical analysis showed that outdoor/soil exposure and a high heterotrophic plate count (HPC) were risk factors for the occurrence of V. corneae. Our findings provide empirical evidence supporting the need for proper control and regulations at hot spring recreational waters in order to avoid health risks from this pathogen. Finally, we have developed a small volume procedure for detecting V. corneae in water samples and this has proved to be very useful.
Subject(s)
Environmental Monitoring , Hot Springs , Vittaforma , Humans , Microsporidiosis/prevention & control , Portugal , TaiwanABSTRACT
BACKGROUND: A reliable and simple test for the detection of malaria parasite is crucial in providing effective treatment and therapeutic follow-up, especially in malaria elimination programmes. A comparison of four methods, including nested polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) were used for the malaria diagnosis and treatment follow-up in São Tomé and Príncipe, during a successful pre-elimination campaign. METHOD: During the period September to November 2009, blood samples from 128 children (five to 14 years old) with temperature ≥38°C (tympanic) in the District of Agua Grande were examined using four different methods, i.e., histidine-rich protein 2 (HRP-2) based rapid diagnostic tests (HRP-2-RDTs), optical microscopy, nested PCR, and LAMP. First-line treatment with artesunate-amodiaquine was given for uncomplicated malaria and intravenous quinine was given for complicated malaria. Children with persistent positivity for malaria by microscopy, or either by nested PCR, or by LAMP on day 7 were given second-line treatment with artemether-lumefantrine. Treatment follow-up was made weekly, for up to four weeks. RESULTS: On day 0, positive results for HRP-2-RDTs, microscopy, nested PCR, and LAMP, were 68(53%), 47(37%), 64(50%), and 65(51%), respectively. When nested PCR was used as a reference standard, only LAMP was comparable; both HRP-2-RDTs and microscopy had moderate sensitivity; HRP-2-RDTs had poor positive predictive value (PPV) and a moderate negative predictive value (NPV) for the treatment follow-up. Seventy-one children with uncomplicated malaria and eight children with complicated falciparum malaria were diagnosed based on at least one positive result from the four tests as well as clinical criteria. Twelve of the 79 children receiving first-line treatment had positive results by nested PCR on day 7 (nested PCR-corrected day 7 cure rate was 85%). After the second-line treatment, nested PCR/LAMP-corrected day 28 cure rate was 83% for these 12 children. CONCLUSIONS: HRP-2-RDTs have similar sensitivity as microscopy but less specificity. However, as compared to nested PCR, the poor sensitivity of HRP-2-RDTs indicates that low parasitaemia may not be detected after treatment, as well as the low specificity of HRP-2-RDTs indicates it cannot be applied for treatment follow-up. LAMP has similar sensitivity and specificity to nested PCR. With high PPV and NPV, LAMP is simpler and faster as compared to nested PCR with the advantage of detecting low parasitaemia becoming a potential point-of-care test for treatment follow-up.
Subject(s)
Drug Monitoring/methods , Malaria/diagnosis , Malaria/drug therapy , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , Adolescent , Antimalarials/administration & dosage , Atlantic Islands , Child , Child, Preschool , Female , Humans , Male , Sensitivity and SpecificityABSTRACT
AIMS: The main focus of this study is to illustrate the importance of the statistical analysis in the evaluation of the accuracy of malaria diagnostic tests, without admitting a reference test, exploring a dataset (n=3317) collected in São Tomé and Príncipe. METHODS: Bayesian Latent Class Models (without and with constraints) are used to estimate the malaria infection prevalence, together with sensitivities, specificities, and predictive values of three diagnostic tests (RDT, Microscopy and PCR), in four subpopulations simultaneously based on a stratified analysis by age groups (< 5, ≥ 5 years old) and fever status (febrile, afebrile). RESULTS: In the afebrile individuals with at least five years old, the posterior mean of the malaria infection prevalence is 3.2% with a highest posterior density interval of [2.3-4.1]. The other three subpopulations (febrile ≥ 5 years, afebrile or febrile children less than 5 years) present a higher prevalence around 10.3% [8.8-11.7]. In afebrile children under-five years old, the sensitivity of microscopy is 50.5% [37.7-63.2]. In children under-five, the estimated sensitivities/specificities of RDT are 95.4% [90.3-99.5]/93.8% [91.6-96.0]--afebrile--and 94.1% [87.5-99.4]/97.5% [95.5-99.3]--febrile. In individuals with at least five years old are 96.0% [91.5-99.7]/98.7% [98.1-99.2]--afebrile--and 97.9% [95.3-99.8]/97.7% [96.6-98.6]--febrile. The PCR yields the most reliable results in four subpopulations. CONCLUSIONS: The utility of this RDT in the field seems to be relevant. However, in all subpopulations, data provide enough evidence to suggest caution with the positive predictive values of the RDT. Microscopy has poor sensitivity compared to the other tests, particularly, in the afebrile children less than 5 years. This type of findings reveals the danger of statistical analysis based on microscopy as a reference test. Bayesian Latent Class Models provide a powerful tool to evaluate malaria diagnostic tests, taking into account different groups of interest.
Subject(s)
Malaria/diagnosis , Models, Statistical , Bayes Theorem , Child , Child, Preschool , Humans , Malaria/epidemiology , Predictive Value of TestsABSTRACT
BACKGROUND: Plasmodium falciparum is the major cause of malaria infection in the island of São Tomé, in the Republic of São Tomé and Príncipe (STP), with an incidence of 40 - 50% before 2004. Since 2004, through the coordination of the Ministry of Health of STP and their Centro Nacional de Endemias (CNE), an integrated malaria control programme has been intensively deployed on the island of São Tomé. Malaria morbidity and mortality decreased by 95% after three years of effective intervention. In the low transmission settings, however, malaria seasonal fluctuation can be a potential problem directly related to epidemics if ongoing control measures are interrupted. Studies on a number of associated factors with malaria epidemics and the measures taken to respond to outbreaks are presented. METHODS: The integrated malaria control programme included indoor residual spraying (IRS), long-lasting insecticidal nets (LLINs), intermittent preventive therapy for pregnant women, as well as early diagnosis and prompt treatment with artemisinin-based combination therapy (ACT). Regular implementation of an island-wide IRS programme was carried out yearly in 2004-2007, and enhanced throughout the island in 2009. Malaria incidence and prevalence were estimated based on passive case detection and mass screening, respectively. Slide positivity rates were used for monitoring the beginning of a malaria epidemic or a seasonal peak. RESULTS: A steep decline of ca. 95% of malaria morbidity and mortality was observed between 2004 and 2008 with use of the combined control methods. Malaria incidence was 2.0%, 1.5%, and 3.0% for 2007, 2008, and 2009, respectively. In April 2008, a cross-sectional country-wide surveillance showed malaria prevalence of 3.5%, of which 95% cases were asymptomatic carriers. Only 50% of asymptomatic carriers were cured with ACT treatment, while 90% of the symptomatic patients were cured by ACT treatment as confirmed with a follow up study. Malaria morbidity increased by three-fold during the first half of 2009 as compared to the same period in 2008. Over this period of six months, severe malaria was also noted in all age groups and malaria mortality increased by two-fold in children less than five years old. After an emergency IRS was deployed, with increased use of LLINs, and an active search of asymptomatic carriers was followed and given complete ACT treatment, malaria incidence decreased to less than 1% in the second half of 2009. CONCLUSION: At the initial stage of the integrated malaria control programme, IRS contributed to the visible effect on the rapid reduction of malaria morbidity and mortality, while this programme highlights an urgent demand for the improvement of other measures, particularly promotion of LLINs usage, with close monitoring of asymptomatic carriers and with ACT treatment in malaria transmission hotspots. In addition, both daily reports and a regular active surveillance to prevent malaria outbreaks should be established permanently, so that a fast response to epidemics can be effectively made when necessary.
Subject(s)
Malaria/epidemiology , Malaria/prevention & control , Plasmodium/isolation & purification , Adolescent , Adult , Aged , Aged, 80 and over , Antimalarials/therapeutic use , Artemisinins/therapeutic use , Atlantic Islands/epidemiology , Child , Child, Preschool , Female , Humans , Incidence , Infant , Infant, Newborn , Insecticide-Treated Bednets , Lactones/therapeutic use , Male , Microscopy/methods , Middle Aged , Mosquito Control/methods , Parasitology/methods , Pregnancy , Seasons , Young AdultABSTRACT
BACKGROUND: Plasmodium falciparum is the major species responsible for malaria transmission on the island of Príncipe, in the Republic of São Tomé and Príncipe (STP). Indoor residual spraying (IRS) has been intensively deployed on the island, since 2003. Other measures included intermittent preventive therapy (IPT), since 2004, as well as artemisinin-based therapy (ACT) and long-lasting insecticidal nets (LLINs) from 2005. The work was coordinated by the Ministry of Health of STP through their Centro Nacional de Endemias (CNE) and the impact of such an integrated control programme on the prevalence and epidemiology of malaria in Príncipe was evaluated. METHODS: The scaling-up of preventive strategies included IRS, LLINs, IPT for pregnant women, as well as early diagnosis and prompt treatment with ACT. Regular implementation of an island-wide IRS programme was carried out yearly in 2003-2005, and later in 2008. Malaria incidence and prevalence were estimated based on passive case detection and active case detection, respectively. Slide positivity rate (SPR) was used as an indicator of any increase of malaria cases during and after the control programme was initiated. RESULTS: Regular IRS achieved a coverage of 85-90% for each of the four annual cycles (2003-2005, annually and one spraying in 2008) while usage of LLINs was never superior to 50% from 2006-2009. Coverage of IPT steadily increased from 50% in 2004 to 80% in 2008. Since 2006, over 90% of uncomplicated malaria patients received ACT treatment. Severe malaria cases were hospitalized and treated with quinine. Monthly trends of SPR were constantly over 50% in 2003, but steadily decreased below 10% in 2006. SPR has been below 5% since 2007, but an increase to up to 15% was noted in June 2009 when 16 imported cases were detected. A steep decline by 99% of malaria incidence was observed between 2003 and 2008, with an incidence risk of the population of five per thousand, in 2008. No malaria mortality has been reported since 2005. Species shift from falciparum to non-falciparum malaria was noted after a five-year intensive control programme. Cross-sectional country-wide active surveillances showed malaria prevalences of 1.1%, 0.7%, and 0.9% in June 2006, Oct 2007, and July 2009, respectively, of which over 90% were asymptomatic. CONCLUSION: The effective measures of the combination of four major control methods have produced a rapid decline in malaria morbidity and mortality on the island of Príncipe. The combination of IRS, IPT, and active surveillance with ACT treatment seemed to have played important roles to achieve a present status of low and stable malaria on the island. In low transmission settings, any increase of malaria morbidity indicates potential epidemics and assumes that current control strategies were interrupted. Active surveillance should be reinforced to follow and monitor all asymptomatic carriers and imported cases. Consolidation and a shift to elimination phase demands the sustainability of such integrated programmes.
Subject(s)
Antimalarials/therapeutic use , Insect Vectors/parasitology , Malaria, Falciparum/prevention & control , Mosquito Control/methods , Animals , Atlantic Islands/epidemiology , Bedding and Linens/statistics & numerical data , Cross-Sectional Studies , Drug Therapy, Combination , Female , Humans , Incidence , Insecticides , Malaria, Falciparum/drug therapy , Malaria, Falciparum/epidemiology , Malaria, Falciparum/parasitology , Male , Parasitemia/drug therapy , Parasitemia/epidemiology , Parasitemia/parasitology , Plasmodium falciparum/isolation & purification , Pregnancy , Pregnancy Complications/parasitology , Prevalence , Surveys and QuestionnairesABSTRACT
The oxacillinase gene was reported to confer limited resistance to carbapenem in Acinetobacter baumannii. In this study, we have demonstrated that an A. baumannii clinical isolate harboring a plasmid, pTVICU53, has 11,037 bp encoding 13 open reading frames. A bla(OXA-58) gene with an upstream insertion of truncated ISAba3 (DeltaISAba3) and IS1008 was found in this plasmid. DeltaISAba3and IS1008 provided two independent promoters for the transcription control of the bla(OXA-58) gene. The transformation of pTVICU53 or a shuttle vector bearing IS1008-DeltaISAba3-bla(OXA-58) to different A. baumannii recipients can increase their MICs of carbapenem 64- to 256-fold. The deletion of promoters provided by IS1008 resulted in dramatic decreases in bla(OXA-58) transcription and a 32- to 64-fold reduction in the carbapenem MIC. These findings highlight that A. baumannii might develop carbapenem resistance with a single transformation step, taking up a plasmid containing a genetic construct with a potentially high level of transcription of the bla(OXA-58) gene.
Subject(s)
Acinetobacter baumannii/drug effects , Acinetobacter baumannii/genetics , Carbapenems/pharmacology , Genes, Bacterial , beta-Lactam Resistance/genetics , Acinetobacter Infections/drug therapy , Acinetobacter Infections/microbiology , Acinetobacter baumannii/enzymology , Base Sequence , Cross Infection/drug therapy , Cross Infection/microbiology , DNA Primers/genetics , DNA Transposable Elements/genetics , DNA, Bacterial/genetics , Humans , Microbial Sensitivity Tests , Molecular Sequence Data , Plasmids/genetics , Promoter Regions, Genetic , Transformation, Genetic , beta-Lactamases/biosynthesis , beta-Lactamases/geneticsABSTRACT
Rickettsioses are emerging infectious diseases caused by rickettsiae in association with arthropods. We report the detection of spotted fever group rickettsiae (SFGR) in Taiwan using molecular methods. Phylogenetic analyses of the 17-kd protein and citrate synthase (gltA) genes showed that SFGR TwKM01 detected in Rhipicephalus haemaphysaloides ticks was most similar to Rickettsia rhipicephali. Three TwKM01 isolates were obtained from three individual R. haemaphysaloides ticks. Small, intracellular, coccobacillary bacteria were found in infected L929 cells using immunofluorescence antibody testing and transmission electron microscopy. Two other SFGRs, TwKM02 and TwKM03, identified in Leptotrombidium chigger mites, were closely related to R. australis and R. felis URRWXCal(2), respectively. The TwKM03 strain was also detected in Ixodes granulatus ticks and widely distributed in Hualien, Kinmen, and Lienchiang counties in Taiwan. The endonucleases MaeII and HhaI selected for restriction fragment length polymorphism analysis of the gltA and 17-kd polymerase chain reaction products, respectively, were useful for genotyping Rickettsia species TwKM01, TwKM02, TwKM03, and other SFGRs. Although their infectivity and pathogenicity for vertebrates are unknown, the finding of SFGRs raises the possibility that bacteria other than Orientia tsutsugamushi, Coxiella burnetii, and R. typhi may be involved in rickettsial diseases in Taiwan.
Subject(s)
Rickettsia Infections/microbiology , Rickettsia/isolation & purification , Animals , Ixodes/microbiology , Phylogeny , Rats , Rhipicephalus/microbiology , Rickettsia/genetics , Rickettsia/ultrastructure , Rodentia/parasitology , Taiwan , Trombiculidae/microbiology , Voltage-Dependent Anion ChannelsABSTRACT
In a study comparing the virus load and immune reaction between patients with primary and secondary dengue-2 (DEN-2) infections in a hospital-based analysis, we found that 40.7% (55/135) of the 135 patients had secondary DEN-2 infection following a DEN-2 outbreak in southern Taiwan. Most of the secondary infections had subclinical primary dengue infections (78.2%; 43/55). Patients with secondary DEN-2 infections had lower platelet counts, and blood interferon-alpha and virus load, but significantly higher interleukin-10 (P=0.030) and anti-DEN-1 neutralization titers (P=0.013) than those with primary infection. Patients with secondary DEN-2 infection also had a higher rate of dengue hemorrhagic fever (DHF) (61.7% vs. 36.3%). A previous subclinical dengue infection is involved in the secondary DEN-2 infection associated with altered immune reaction and higher DHF rate, but lower blood virus load.
Subject(s)
Antibodies, Viral/blood , Dengue Virus/immunology , Dengue/immunology , Severe Dengue/immunology , Viral Load , Cytokines/blood , Dengue/epidemiology , Dengue/etiology , Dengue/virology , Dengue Virus/classification , Dengue Virus/physiology , Humans , Interleukin-10/blood , Severe Dengue/blood , Severe Dengue/epidemiology , Taiwan/epidemiologyABSTRACT
Dengue viruses (DV) infection is an important public health issue all over the world. Although the pathogenesis remains unclear, the overwhelmingly triggered immune responses have been consistently observed. Recently, we and other researchers demonstrated that the natural hosts for DV are dendritic cells (DC), the primary sentinels of immune system. In light of the significance of T cells in dengue virus pathogenesis, here, we examine the possible consequences of DC-T cell interaction that is supposed to be happening in lymphoid tissues after infection. We showed that DV-infected DC induced the interacting T cells to proliferate, to produce interleukin-2 as well as to express activation markers on cell surface. Compared to mock-infected DC, the infection of DC by DV also induced T cells to produce interleukin-4, interleukin-10 and interferon-gamma, a cytokine pattern suggesting Th0 phenotype. Such an effect was either totally abolished or greatly reduced when DV were pre-inactivated with heat or ultraviolet before infection. In addition, we demonstrated that such a Th0 phenotype shift of T cells was affected neither by different dosages of viruses that infected DC nor by different durations of DC-T cell interaction. Our results provide a basic support for clinical observations and may be of help in understanding the pathogenesis of DV infection.
Subject(s)
Cytokines/immunology , Dendritic Cells/immunology , Dendritic Cells/virology , Dengue Virus/immunology , Flavivirus Infections/immunology , Lymphocyte Activation/immunology , T-Lymphocytes, Helper-Inducer/immunology , Cell Differentiation , Cell Proliferation , Cells, Cultured , Cytokines/biosynthesis , Flavivirus Infections/virology , Humans , Phenotype , T-Lymphocytes, Helper-Inducer/cytology , Th1 Cells/cytology , Th1 Cells/immunology , Th2 Cells/cytology , Th2 Cells/immunologyABSTRACT
To survey the antibiotic susceptibility patterns of some commercially available Lactobacillus, we collected four commercial products that contain active Lactobacillus. We incubated individual product and identified these colonies by the methods of API50 CH test kit and RAPID ID 32A kit. Strains of Streptococcus thermophilus, Bifidobacterium infantis, Lactobacillus acidophilus and Lactobacillus casei were collected. By agar dilution method, each identified strain was inoculated to Brucella blood agar-MIC plates. Each plate contained one of the following antibiotics with different concentrations: amoxicillin, cephalothin, gentamicin, vancomycin, erythromycin, rifampin, tetracyclin and penicillin G, clindamycin, chloramphenicol, cefmetazole, metronidazole, ampicillin/sulbactum, cefoxtin, etc. After incubation, the growth condition of each Brucella blood agar-MIC plate was observed and the breakpoint of each antibiotic to different Lactobacillus products determined. The MICs of amoxicillin, ampicillin/sulbactum and penicillin-G to all identified strains were < or =2 microg/ml and those of vancomycin, clindamycin, erythromycin, metronidazole, cefmetazole and cefoxtin for L. casei were >32 microg/ml. L. casei was more resistant to all the testing antibiotics than the other strains. According to the MICs of the above antibiotics, proper active lactobacillus products could be chosen to prevent antibiotic-associated diarrhea in the pediatric field.
Subject(s)
Anti-Bacterial Agents/pharmacology , Lactobacillus/drug effects , Amoxicillin/pharmacology , Cefoxitin/pharmacology , Cephalothin/pharmacology , Clindamycin/pharmacology , Drug Resistance, Bacterial , Drug Resistance, Multiple, Bacterial , Erythromycin/pharmacology , Food Microbiology , Gentamicins/pharmacology , Metronidazole/pharmacology , Microbial Sensitivity Tests , Rifampin/pharmacology , Tetracycline/pharmacology , Vancomycin/pharmacologyABSTRACT
Japanese encephalitis (JE) is the most common mosquito-borne encephalitis in the Asia-Pacific region. Patients with JE usually present neuronal involvement, but other organ involvement is relatively rare. Employing human neuroblast-derived (NB) cell lines and different blood cells (erythrocytes, lymphocytes, granulocytes and monocytes), the neurotropism and persistency of Japanese encephalitis virus (JEV) in human cells was investigated. It was found that JEV could not replicate in erythrocytes, granulocytes or lymphocytes. Monocytes and NB cell lines could support replication of JEV as demonstrated by expression of viral NS3 antigen and virus plaque-forming units (p.f.u.). JEV could replicate more efficiently in neuroblastoma (HTB-11) cells than in monocytes after infection for 48 h (2.1+/-1.2x10(7) vs 2.8+/-0.7x10(2) p.f.u. ml(-1)). Two different strains of JEV revealed a similar infectivity to different leukocytes and four NB cell lines. In a kinetic study, it was found that JEV-infected monocytes possessed a high viability (90 %) after infection for 5 days, while JEV-infected neuroblastoma cells suffered cell apoptosis in 2 days and decreased viability to less than 1 % in 5 days. Further studies showed that monocytes could take up JEV rapidly, displaying a log scale increase of intracellular JEV titres in 9 h after infection. Significantly, extracellular production of JEV by monocytes started in 12 h, peaked in 3 days and persisted for more than 3 weeks. These results suggest that JEV-infected monocytes may play an important role in harbouring JEV for eventual transmission to NB cells and that modulation of JEV-induced NB cell apoptosis may be useful in treating patients with JE.
Subject(s)
Encephalitis Virus, Japanese/physiology , Leukocytes/virology , Neuroblastoma/virology , Neurons/virology , Animals , Antigens, Viral/analysis , Cell Line, Tumor , Cell Survival , Culicidae , Encephalitis Virus, Japanese/pathogenicity , Encephalitis, Japanese/pathology , Encephalitis, Japanese/virology , Erythrocytes/virology , Fluorescent Antibody Technique, Indirect , Humans , Insect Vectors , Neuroblastoma/pathology , Neurons/pathology , Virus ReplicationABSTRACT
In this study, a serotype-specific monoclonal antibody (mAb), D(2) 16-1 (Ab4), against dengue virus type 2 (DEN-2) was generated. The specificity of Ab4, which recognized DEN-2 non-structural protein 1, was determined by ELISA, immunofluorescence and immunoblotting analyses. The serotype-specific B-cell epitope of Ab4 was identified further from a random phage-displayed peptide library; selected phage clones reacted specifically with Ab4 and did not react with other mAbs. Immunopositive phage clones displayed a consensus motif, His-Arg/Lys-Leu/Ile, and a synthetic peptide corresponding to the phage-displayed peptide bound specifically to Ab4. The His and Arg residues in this epitope were found to be crucial for peptide binding to Ab4 and binding activity decreased dramatically when these residues were changed to Leu. The epitope-based synthetic peptide not only identified serum samples from DEN-2-immunized mice and rabbits by ELISA but also differentiated clearly between serum samples from DEN-2- and Japanese encephalitis virus-immunized mice. This mAb and its epitope-based peptide antigen will be useful for serologic diagnosis of DEN-2 infection. Furthermore, DEN-2 epitope identification makes it feasible to dissect antibody responses to DEN and to address the role of antibodies in the pathogenesis of primary and secondary DEN-2 infections.
Subject(s)
Dengue Virus/classification , Dengue Virus/immunology , Epitope Mapping , Epitopes, B-Lymphocyte/immunology , Amino Acid Sequence , Animals , Antibodies, Monoclonal/immunology , Antibodies, Viral/blood , Antibody Specificity , Antigens, Viral/chemistry , Antigens, Viral/immunology , Cell Line , Cricetinae , Dengue/diagnosis , Dengue/immunology , Dengue/prevention & control , Enzyme-Linked Immunosorbent Assay , Female , Humans , Immunization , Mice , Mice, Inbred BALB C , Peptide Library , Peptides/chemical synthesis , Peptides/chemistry , Peptides/immunology , Serotyping , Viral Vaccines/administration & dosage , Viral Vaccines/immunologyABSTRACT
To evaluate the potential of DNA vaccine against dengue (DEN) infection, we characterize the protective efficacy and immune responses of mice intramuscularly injected with plasmid encoding DEN-2 non-structural protein 1 (NS1). Intravenously challenged by lethal DEN-2, mice vaccinated with NS1-DNA exhibited a delay onset of paralysis, a marked decrease of morbidity, and a significant enhancement of survival. In addition to a moderate increase of NS1-specific antibody titer from immunized mice measured by ELISA, a strong priming effect on anti-NS1 response was also noticed in plasmid NS1-vaccinated mice by radioimmunoprecipitation (RIP) or immunoblot analysis. Interestingly, newborn mice from NS1-DNA-immunized dam showed stronger resistance to viral challenge, as compared to those from vector DNA or PBS-immunized dams, indicating the protective role of NS1-specific antibody. In contrast to humoral immune response, DNA immunization can elicit strong cellular immune responses, including NS1-specific T cell proliferation and cytolytic activity. The NS1-DNA-induced protection can be further augmented by co-injection of plasmid encoding interleukin 12 (IL-12), suggesting an effector role of Th1 immunity against DEN infection. In summary, our results suggest the potential of NS1-DNA vaccine against DEN infection, and indicate both NS1-specific humoral and cellular immune responses contribute to the protection.
Subject(s)
Dengue Virus/immunology , Dengue/prevention & control , Vaccines, DNA/immunology , Viral Nonstructural Proteins/immunology , Viral Vaccines/immunology , Animals , Animals, Newborn , Antibody Formation/immunology , Cell Division/drug effects , Cell Line , Cytokines/biosynthesis , Cytokines/genetics , DNA, Bacterial/genetics , DNA, Bacterial/immunology , Dengue/immunology , Enzyme-Linked Immunosorbent Assay , Female , Immunity, Cellular/immunology , Immunoblotting , Indicators and Reagents , Interleukin-12/physiology , Lymphocyte Count , Male , Mice , Mice, Inbred C3H , Plasmids/genetics , Plasmids/immunology , Precipitin Tests , Reverse Transcriptase Polymerase Chain Reaction , Survival Analysis , T-Lymphocytes/immunology , Th1 Cells/immunologyABSTRACT
Glutamine is the most abundant amino acid in the body. A decrease of plasma glutamine concentrations is found in catabolic stress and is related to susceptibility to infections. Glutamine is known to modulate lymphocyte activation; however, little is known about glutamine modulation of cell death of activated human T cells. Using Jurkat T cells, we investigated glutamine modulation of T-cell apoptosis activated by PMA plus ionomycin. We found that glutamine at various concentrations significantly enhanced IL-2 production, cell proliferation, and cell viability of Jurkat T cells. Glutamine also decreased the number of apoptotic cells stimulated with PMA plus ionomycin as demonstrated by flow cytometry. Meanwhile, glutamine down-regulated CD95 and CD95L expression, but up-regulated CD45RO and Bcl-2 expression in activated T cells. Further investigation of CD95-mediated caspase activities revealed that supplementation of glutamine significantly decreased caspase-3 and caspase-8 activities in activated T cells. Since oxidative stress is closely associated with induction of lymphocyte apoptosis, we found that glutamine significantly increased glutathione (GSH), but decreased reactive oxygen species levels in activated T cells. Blockade of intracellular GSH formation enhanced, but exogenous GSH supplementation decreased, activated T-cell apoptosis. Studying normal peripheral lymphoproliferation, we also found that the presence of glutamine increased lymphoproliferation as well as Bcl-2 and CD95 expression; but decreased CD95L and activation-induced T-cell death. Taken together, glutamine appeared to augment lymphoproliferation but suppressed activation-induced T-cell death in both Jurkat T cells and human peripheral T lymphocytes.