A comparative study of leptospirosis and dengue in Thai children

Background

Leptospirosis is an emerging zoonosis that is often under-recognized in children and commonly confused with dengue in tropical settings. An enhanced ability to distinguish leptospirosis from dengue in children would guide clinicians and public health personnel in the appropriate use of limited healthcare resources.

Methodology/Principal Findings

We conducted a prospective, hospital-based, study of children with acute febrile illnesses and dengue in Thailand. Among the children without dengue, we identified those with leptospirosis using anti-leptospira IgM and microscopic agglutination titers in paired acute and convalescent blood samples. We then performed a case-control comparison of symptoms, signs, and clinical laboratory values between children with leptospirosis and dengue.In a semi-rural region of Thailand, leptospirosis accounted for 19% of the non-dengue acute febrile illnesses among children presenting during the rainy season. None of the children with leptospirosis were correctly diagnosed at the time of hospital discharge, and one third (33%) were erroneously diagnosed as dengue or scrub typhus. A predictive model to distinguish pediatric leptospirosis from dengue was generated using three variables: the absolute neutrophil count, plasma albumin, and aspartate aminotransferase levels in the first 72 hours of illness.

Conclusions/Significance

Unrecognized leptospirosis can be a significant cause of “dengue-like” febrile illness in children. Increased awareness of pediatric leptospirosis, and an enhanced ability to discriminate between leptospirosis and dengue early in illness, will help guide the appropriate use of healthcare resources in often resource-limited settings.     

Crystal structures of an oxygen-binding cytochrome c from Rhodobacter sphaeroides

The photosynthetic bacterium Rhodobacter sphaeroides produces a heme protein (SHP), which is an unusual c-type cytochrome capable of transiently binding oxygen during autooxidation. Similar proteins have not only been observed in other photosynthetic bacteria but also in the obligate methylotroph Methylophilus methylotrophus and the metal reducing bacterium Shewanella putrefaciens. A three-dimensional structure of SHP was derived using the multiple isomorphous replacement phasing method. Besides a model for the oxidized state (to 1.82 A resolution), models for the reduced state (2.1 A resolution), the oxidized molecule liganded with cyanide (1. 90 A resolution), and the reduced molecule liganded with nitric oxide (2.20 A resolution) could be derived. The SHP structure represents a new variation of the class I cytochrome c fold. The oxidized state reveals a novel sixth heme ligand, Asn(88), which moves away from the iron upon reduction or when small molecules bind. The distal side of the heme has a striking resemblance to other heme proteins that bind gaseous compounds. In SHP the liberated amide group of Asn(88) stabilizes solvent-shielded ligands through a hydrogen bond.     

Primary structure characterization of a Rhodocyclus tenuis diheme cytochrome c reveals the existence of two different classes of low-potential diheme cytochromes c in purple phototropic bacteria

The complete amino acid sequence of a 26-kDa low redox potential cytochrome c-551 from Rhodocyclus tenuis was determined by a combination of Edman degradation and mass spectrometry. There are 240 residues including two heme binding sites at positions 41, 44, 128, and 132. There is no evidence for gene doubling. The only known homolog of Rc. tenuis cytochrome c-551 is the diheme cytochrome c-552 from Pseudomonas stutzeri which contains 268 residues and heme binding sites at nearly identical positions. There is 44% overall identity between the Rc. tenuis and Ps. stutzeri cytochromes with 10 internal insertions and deletions. The Ps. stutzeri cytochrome is part of a denitrification gene cluster, whereas Rc. tenuis is incapable of denitrification, suggesting different functional roles for the cytochromes. Histidines at positions 45 and 133 are the fifth heme ligands and conserved histidines at positions 29, 209, and 218 and conserved methionines at positions 114 and 139 are potential sixth heme ligands. There is no obvious homology to the low-potential diheme cytochromes characterized from other purple bacterial species such as Rhodobacter sphaeroides. There are therefore at least two classes of low-potential diheme cytochromes c found in phototrophic bacteria. There is no more than 11% helical secondary structure in Rc. tenuis cytochrome c-551 suggesting that there is no relationship to class I or class II c-type cytochromes.     

Predominance of HLA-Restricted Cytotoxic T-Lymphocyte Responses to Serotype-Cross-Reactive Epitopes on Nonstructural Proteins following Natural Secondary Dengue Virus Infection

We examined the memory cytotoxic T-lymphocytic (CTL) responses of peripheral blood mononuclear cells (PBMC) obtained from patients in Thailand 12 months after natural symptomatic secondary dengue virus infection. In all four patients analyzed, CTLs were detected in bulk culture PBMC against nonstructural dengue virus proteins. Numerous CD4⁺ and CD8⁺ CTL lines were generated from the bulk cultures of two patients, KPP94-037 and KPP94-024, which were specific for NS1.2a (NS1 and NS2a collectively) and NS3 proteins, respectively. All CTL lines derived from both patients were cross-reactive with other serotypes of dengue virus. The CD8⁺ NS1.2a-specific lines from patient KPP94-037 were HLA B57 restricted, and the CD8⁺ NS3-specific lines from patient KPP94-024 were HLA B7 restricted. The CD4⁺ CTL lines from patient KPP94-037 were HLA DR7 restricted. A majority of the CD8⁺ CTLs isolated from patient KPP94-024 were found to recognize amino acids 221 to 232 on NS3. These results demonstrate that in Thai patients after symptomatic secondary natural dengue infections, CTLs are mainly directed against nonstructural proteins and are broadly cross-reactive.     

Isolation and characterization of a novel human bladder cancer cell line: BK10

Summary Molecular studies of bladder carcinomas have aided in determining causative genetic events and the prognosis of cancers endowed with certain abnormalities. In vitro bladder cancer characterization of key cytogenetic alterations is useful for study of molecular changes that may promote oncogenic events. In our laboratory, a novel human bladder cancer cell line, BK10, has been established in vitro and passaged for more than 20 mo. This new bladder cancer cell line (BK10) was derived from bladder tissue containing grade III-IV/IV transitional cell carcinoma. Bladder cancer tissue was obtained at the time of radical cystoprostatectomy extirpation. Cell cultures derived from this surgical sample exhibited an epithelial morphology and expressed epithelial cytokeratins. Immunostains of BK10 were negative for prostate specific antigen (PSA), fibronectin, smooth muscle actin alpha, and desmin. Karyotypic analysis revealed an aneuploid chromosomal content 〈4n〉 with many numerical and structural abnormalities previously linked to bladder oncogenesis. Translocations occurred in chromosomes 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 13, 14, 15, 16, 17, 19, 20, 21, 22, X and Y. G-banding analysis revealed rearrangements involving chromosomes 9q and 17p, and the location of the ab11 oncogene and the p53 gene, respectively. The availability of this bladder cancer cell line will provide a useful too for the further study of bladder carcinoma oncogenesis and gene therapy.     

SHIP2 interaction with the cytoskeletal protein Vinexin

The src homology 2 (SH2) domain-containing inositol 5-phosphatase 2 (SHIP2) catalyses the dephosphorylation of phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] to phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P2]. We report the identification of the cytoskeletal protein Vinexin as a protein interacting with SHIP2. This was achieved by yeast two-hybrid screening using the C-terminal region of SHIP2 as bait. Vinexin has previously been identified as a vinculin-binding protein that plays a key role in cell spreading and cytoskeletal organization. The interaction between SHIP2 and Vinexin was confirmed in lysates of both COS-7 cells and mouse embryonic fibroblasts (MEF). The C-terminus was involved in the interaction, as shown by the transfection of a truncated C-terminus mutant of SHIP2. In addition, we showed the colocalization between Vinexin alpha and SHIP2 at the periphery of transfected COS-7 cells. When added in vitro to SHIP2, Vinexin did not affect the PtdIns(3,4,5)P3 5-phosphatase activity of SHIP2. Enhanced cell adhesion to collagen-I-coated dishes was shown upon transfection of either SHIP2 or Vinexin to COS-7 cells. This effect was no longer observed with either a catalytic mutant or the C-terminus mutant of SHIP2. It also appears SHIP2 specific; this was not seen with SHIP1. Adhesion to the same matrix was decreased in SHIP2-/- MEF cells compared with MEF+/+ cells. Our data suggest that SHIP2 interaction with Vinexin promotes the localization of SHIP2 at the periphery of the cells leaving its catalytic site intact. The complex formation between Vinexin and SHIP2 may increase cellular adhesion. The data reinforce the concept that SHIP2 is active both as a PtdIns(3,4,5)P3 5-phosphatase and as a modulator of focal contact formation.     

The influence of anionic lipids on SHIP2 phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase activity

The SH2 domain containing inositol 5-phosphatase 2 (SHIP2) catalyzes the dephosphorylation of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P₃) to phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P₂) and participates in the insulin signalling pathway in vivo. In a comparative study of SHIP2 and the phosphatase and tensin homologue deleted on chromosome 10 (PTEN), we found that their lipid phosphatase activity was influenced by the presence of vesicles of phosphatidylserine (PtdSer). SHIP2 PtdIns(3,4,5)P₃ 5-phosphatase activity was greatly stimulated in the presence of vesicles of PtdSer. This effect appears to be specific for di-C8 and di-C16 fatty acids of PtdIns(3,4,5)P₃ as substrate. It was not observed with inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P₄) another in vitro substrate of SHIP2, nor with Type I Ins(1,4,5)P₃/Ins(1,3,4,5)P₄ 5-phosphatase activity, an enzyme which acts on soluble inositol phosphates. Vesicles of phosphatidylcholine (PtdCho) stimulated only twofold PtdIns(3,4,5)P₃ 5-phosphatase activity of SHIP2. Both a minimal catalytic construct and the full length SHIP2 were sensitive to the stimulation by PtdSer. In contrast, PtdIns(3,4,5)P₃ 5-phosphatase activity of the Skeletal muscle and Kidney enriched Inositol Phosphatase (SKIP), another member of the mammaliam Type II phosphoinositide 5-phosphatases, was not sensitive to PtdSer. Our enzymatic data establish a specificity in the control of SHIP2 lipid phosphatase activity with PtdIns(3,4,5)P₃ as substrate which is depending on the fatty acid composition of the substrate.     

Dengue virus-reactive CD8+ T cells display quantitative and qualitative differences in their response to variant epitopes of heterologous viral serotypes

Reactivation of serotype cross-reactive CD8+ memory T lymphocytes is thought to contribute to the immunopathogenesis of dengue disease during secondary infection by a heterologous serotype. Using cytokine flow cytometry, we have defined four novel HLA-A*02-restricted dengue viral epitopes recognized by up to 1.5% of circulating CD8+ T cells in four donors after primary vaccination. All four donors had the highest cytokine response to the epitope NS4b 2353. We also studied the effect of sequence differences in heterologous dengue serotypes on dengue-reactive CD8+ memory T cell cytokine and proliferative responses. The D3 variant of a different NS4b epitope 2423 and the D2 variant of the NS4a epitope 2148 induced the largest cytokine response, compared with their respective heterologous sequences in all donors regardless of the primary vaccination serotype. Stimulation with variant peptides also altered the relative frequencies of the various subsets of cells that expressed IFN-gamma, TNF-alpha, MIP-1beta, and combinations of these cytokines. These results indicate that the prior infection history of the individual as well as the serotypes of the primary and heterologous secondary viruses influence the nature of the secondary response. These differences in the effector functions of serotype cross-reactive memory T cells induced by heterologous variant epitopes, which are both quantitative and qualitative, may contribute to the clinical outcome of secondary dengue infection.     

The implementation and impact of a pilot hydrocele surgery camp for LF-endemic communities in Ethiopia

BackgroundEthiopia aims to eliminate lymphatic filariasis by 2020, through a dual approach of mass drug administration to interrupt transmission and morbidity control which includes making hydrocele surgery available in all endemic areas. Locating patients requiring surgery, providing high quality surgeries, and following up patients are all formidable challenges for many resource-challenged or difficult-to-reach communities. To date, hydrocele surgery in Ethiopia has only occurred when a patient has the knowledge, time and resources to travel to regional hospitals. Ethiopia tested the novel approach of using a surgical camp, defined as mobilizing, transporting, providing surgery at a static site, and following up of a large cohort of hydrocele patients within a hospital’s catchment area, to address delays in seeking and receiving care.Methodology and resultsHealth extension workers mobilized 252 patients with scrotal swelling from a list of 385 suspected hydrocele cases from seven endemic districts in the region of Beneshangul-Gumuz. Clinical health workers and surgeons confirmed 119 as eligible for surgery. Of 70 additional patients who self-referred, 56 were eligible for surgery. Over a two-week period at a regional hospital, 175 hydrocele excision surgeries were conducted. After discharge three days after surgery, trained clinical health workers followed up with the patients on Day 5, Day 8, Day 14 and 1st-month benchmarks with a randomized follow-up of a selection of patients conducted at 9–12 months. There were no post-operative complications upon discharge at Day 3 and 22, while minor complications occurred (12.6%) between Day 3 and one month. The 9–12 month follow-up found patients self-reported an improvement in quality of life, health and economic status.ConclusionA hydrocele surgery camp was effective at providing a large number of quality surgeries in a short time. Using peripheral health workers to mobilize and follow up patients helped address delays in seeking and receiving quality care. Mainstreaming patient mobilization and follow-up into a community health system could be effective in other countries. The camp’s results also influenced two regions in Ethiopia to change their policies in order to offer free hydrocele surgery (including patient transport, consultation, surgery, diagnostic tests and necessary medications).     

Prediction of Dengue Disease Severity among Pediatric Thai Patients Using Early Clinical Laboratory Indicators

Background

Dengue virus is endemic in tropical and sub-tropical resource-poor countries. Dengue illness can range from a nonspecific febrile illness to a severe disease, Dengue Shock Syndrome (DSS), in which patients develop circulatory failure. Earlier diagnosis of severe dengue illnesses would have a substantial impact on the allocation of health resources in endemic countries.

Methods and Findings

We compared clinical laboratory findings collected within 72 hours of fever onset from a prospective cohort children presenting to one of two hospitals (one urban and one rural) in Thailand. Classification and regression tree analysis was used to develop diagnostic algorithms using different categories of dengue disease severity to distinguish between patients at elevated risk of developing a severe dengue illness and those at low risk. A diagnostic algorithm using WBC count, percent monocytes, platelet count, and hematocrit achieved 97% sensitivity to identify patients who went on to develop DSS while correctly excluding 48% of non-severe cases. Addition of an indicator of severe plasma leakage to the WHO definition led to 99% sensitivity using WBC count, percent neutrophils, AST, platelet count, and age.

Conclusions

This study identified two easily applicable diagnostic algorithms using early clinical indicators obtained within the first 72 hours of illness onset. The algorithms have high sensitivity to distinguish patients at elevated risk of developing severe dengue illness from patients at low risk, which included patients with mild dengue and other non-dengue febrile illnesses. Although these algorithms need to be validated in other populations, this study highlights the potential usefulness of specific clinical indicators early in illness.