TRAFfic signals: High-throughput CAR discovery in NK cells reveals novel TRAF-binding endodomains that drive enhanced persistence and cytotoxicity.

Natural killer (NK) cells are a promising alternative therapeutic platform to CAR T cells given their favorable safety profile and potent killing ability. However, CAR NK cells suffer from limited persistence in vivo , which is, in part, thought to be the consequence of limited cytokine signaling. To address this challenge, we developed an innovative high-throughput screening strategy to identify CAR endodomains that could drive enhanced persistence while maintaining potent cytotoxicity. We uncovered a family of TRAF-binding endodomains that outperform benchmarks in primary NK cells along dimensions of persistence and cytotoxicity, even in low IL-2 conditions. This work highlights the importance of cell-type-specific cell therapy engineering and unlocks a wide range of high-throughput molecular engineering avenues in NK cells.

Staphylococcus Lugdunensis Endocarditis and Cerebrovascular Accident: A Systemic Review of Risk Factors and Clinical outcome.

Infective endocarditis (IE) secondary to Staphylococcus lugdunensis has been increasingly recognized since 1988. IE-related thromboembolism represents an associated complication of the disease and carries a dismal prognosis. However, the incidence of cerebrovascular accident secondary to S. lugdunensis IE is relatively uncommon and its treatment has not been clearly elucidated yet. We performed an extensive literature search using Pubmed, Medline, Scopus, and Google Scholar to identify the articles using the following keywords: 'Staphylococcus lugdunensis', 'infective endocarditis', 'stroke', and 'cerebrovascular accident.' Patient characteristics, risk factors, severity of neurological deficit, echocardiographic findings, medical management, required surgical intervention, complications and mortality rate were reviewed in detail. Eighteen cases (mean age of 47.8 years, 55% male) from 17 publications with S. lugdunensis-related cerebrovascular accident (CVA) were identified. Of these, 16 (87%) cases were left-sided endocarditis and 10 (61%) cases experienced right-sided neurological deficit. The source of infection was documented in eight cases (50%) in which four cases (50%) were related to groin-related procedures and the mitral valve (52.5%) was mostly infected followed by aortic valve (37%). Surgical valve replacement was done in 61% of patients and overall mortality rate was 22%. S. lugdunensis endocarditis is associated with high mortality and morbidity, including a higher prevalence of CVA. Early disease identification with aggressive intervention is crucial for better outcomes.

The role of the C-domain of bacteriophage T4 gene 32 protein in ssDNA binding and dsDNA helix-destabilization: Kinetic, single-molecule, and cross-linking studies.

The model single-stranded DNA binding protein of bacteriophage T4, gene 32 protein (gp32) has well-established roles in DNA replication, recombination, and repair. gp32 is a single-chain polypeptide consisting of three domains. Based on thermodynamics and kinetics measurements, we have proposed that gp32 can undergo a conformational change where the acidic C-terminal domain binds internally to or near the single-stranded (ss) DNA binding surface in the core (central) domain, blocking ssDNA interaction. To test this model, we have employed a variety of experimental approaches and gp32 variants to characterize this conformational change. Utilizing stopped-flow methods, the association kinetics of wild type and truncated forms of gp32 with ssDNA were measured. When the C-domain is present, the log-log plot of k vs. [NaCl] shows a positive slope, whereas when it is absent (*I protein), there is little rate change with salt concentration, as expected for this model.A gp32 variant lacking residues 292-296 within the C-domain, ΔPR201, displays kinetic properties intermediate between gp32 and *I. The single molecule force-induced DNA helix-destabilizing activitiesas well as the single- and double-stranded DNA affinities of ΔPR201 and gp32 truncated at residue 295 also fall between full-length protein and *I. Finally, chemical cross-linking of recombinant C-domain and gp32 lacking both N- and C-terminal domains is inhibited by increasing concentrations of a short single-stranded oligonucleotide, and the salt dependence of cross-linking mirrors that expected for the model. Taken together, these results provide the first evidence in support of this model that have been obtained through structural probes.

RNA helicase, DDX27 regulates skeletal muscle growth and regeneration by modulation of translational processes.

Gene expression in a tissue-specific context depends on the combined efforts of epigenetic, transcriptional and post-transcriptional processes that lead to the production of specific proteins that are important determinants of cellular identity. Ribosomes are a central component of the protein biosynthesis machinery in cells; however, their regulatory roles in the translational control of gene expression in skeletal muscle remain to be defined. In a genetic screen to identify critical regulators of myogenesis, we identified a DEAD-Box RNA helicase, DDX27, that is required for skeletal muscle growth and regeneration. We demonstrate that DDX27 regulates ribosomal RNA (rRNA) maturation, and thereby the ribosome biogenesis and the translation of specific transcripts during myogenesis. These findings provide insight into the translational regulation of gene expression in myogenesis and suggest novel functions for ribosomes in regulating gene expression in skeletal muscles.

Ccdc94 protects cells from ionizing radiation by inhibiting the expression of p53.

DNA double-strand breaks (DSBs) represent one of the most deleterious forms of DNA damage to a cell. In cancer therapy, induction of cell death by DNA DSBs by ionizing radiation (IR) and certain chemotherapies is thought to mediate the successful elimination of cancer cells. However, cancer cells often evolve to evade the cytotoxicity induced by DNA DSBs, thereby forming the basis for treatment resistance. As such, a better understanding of the DSB DNA damage response (DSB-DDR) pathway will facilitate the design of more effective strategies to overcome chemo- and radioresistance. To identify novel mechanisms that protect cells from the cytotoxic effects of DNA DSBs, we performed a forward genetic screen in zebrafish for recessive mutations that enhance the IR-induced apoptotic response. Here, we describe radiosensitizing mutation 7 (rs7), which causes a severe sensitivity of zebrafish embryonic neurons to IR-induced apoptosis and is required for the proper development of the central nervous system. The rs7 mutation disrupts the coding sequence of ccdc94, a highly conserved gene that has no previous links to the DSB-DDR pathway. We demonstrate that Ccdc94 is a functional member of the Prp19 complex and that genetic knockdown of core members of this complex causes increased sensitivity to IR-induced apoptosis. We further show that Ccdc94 and the Prp19 complex protect cells from IR-induced apoptosis by repressing the expression of p53 mRNA. In summary, we have identified a new gene regulating a dosage-sensitive response to DNA DSBs during embryonic development. Future studies in human cancer cells will determine whether pharmacological inactivation of CCDC94 reduces the threshold of the cancer cell apoptotic response.

A splice site mutation in laminin-α2 results in a severe muscular dystrophy and growth abnormalities in zebrafish.

Congenital muscular dystrophy (CMD) is a clinically and genetically heterogeneous group of inherited muscle disorders. In patients, muscle weakness is usually present at or shortly after birth and is progressive in nature. Merosin deficient congenital muscular dystrophy (MDC1A) is a form of CMD caused by a defect in the laminin-α2 gene (LAMA2). Laminin-α2 is an extracellular matrix protein that interacts with the dystrophin-dystroglycan (DGC) complex in membranes providing stability to muscle fibers. In an N-ethyl-N-nitrosourea mutagenesis screen to develop zebrafish models of neuromuscular diseases, we identified a mutant fish that exhibits severe muscular dystrophy early in development. Genetic mapping identified a splice site mutation in the lama2 gene. This splice site is highly conserved in humans and this mutation results in mis-splicing of RNA and a loss of protein function. Homozygous lama2 mutant zebrafish, designated lama2(cl501/cl501), exhibited reduced motor function and progressive degeneration of skeletal muscles and died at 8-15 days post fertilization. The skeletal muscles exhibited damaged myosepta and detachment of myofibers in the affected fish. Laminin-α2 deficiency also resulted in growth defects in the brain and eye of the mutant fish. This laminin-α2 deficient mutant fish represents a novel disease model to develop therapies for modulating splicing defects in congenital muscular dystrophies and to restore the muscle function in human patients with CMD.

cpsf1 is required for definitive HSC survival in zebrafish.

A comprehensive understanding of the genes and pathways regulating hematopoiesis is needed to identify genes causally related to bone marrow failure syndromes, myelodysplastic syndromes, and hematopoietic neoplasms. To identify novel genes involved in hematopoiesis, we performed an ethyl-nitrosourea mutagenesis screen in zebrafish (Danio rerio) to search for mutants with defective definitive hematopoiesis. We report the recovery and analysis of the grechetto mutant, which harbors an inactivating mutation in cleavage and polyadenylation specificity factor 1 (cpsf1), a gene ubiquitously expressed and required for 3' untranslated region processing of a subset of pre-mRNAs. grechetto mutants undergo normal primitive hematopoiesis and specify appropriate numbers of definitive HSCs at 36 hours postfertilization. However, when HSCs migrate to the caudal hematopoietic tissue at 3 days postfertilization, their numbers start decreasing as a result of apoptotic cell death. Consistent with Cpsf1 function, c-myb:EGFP(+) cells in grechetto mutants also show defective polyadenylation of snrnp70, a gene required for HSC development. By 5 days postfertilization, definitive hematopoiesis is compromised and severely decreased blood cell numbers are observed across the myeloid, erythroid, and lymphoid cell lineages. These studies show that cpsf1 is essential for HSC survival and differentiation in caudal hematopoietic tissue.

The zebrafish dag1 mutant: a novel genetic model for dystroglycanopathies.

In a forward genetic approach to identify novel genes for congenital muscle diseases, a zebrafish mutant, designated patchytail, was identified that exhibits degenerating muscle fibers with impaired motility behavior. Genetic mapping identified a genomic locus containing the zebrafish ortholog of the dystroglycan gene (DAG1). Patchytail fish contain a point mutation (c.1700T>A) in dag1, resulting in a missense change p.V567D. This change is associated with reduced transcripts and a complete absence of protein. The absence of α-dystroglycan and ß-dystroglycan caused destabilization of dystroglycan complex, resulting in membrane damages. Membrane damage was localized on the extracellular matrix at myosepta as well as basement membrane between adjacent myofibers. These studies also identified structural abnormalities in triads at 3 days post fertilization (dpf) of dystroglycan-deficient muscles, significantly preceding sarcolemmal damage that becomes evident at 7 dpf. Immunofluorescence studies identified a subpopulation of dystroglycan that is expressed at t-tubules in normal skeletal muscles. In dag1-mutated fish, smaller and irregular-shaped t-tubule vesicles, as well as highly disorganized terminal cisternae of sarcoplasmic reticulum, were common. In addition to skeletal muscle defects, dag1-mutated fish have brain abnormalities and ocular defects in posterior as well as anterior chambers. These phenotypes of dystroglycan-deficient fish are highly reminiscent of the phenotypes observed in the human conditions muscle-eye-brain disease and Walker-Warburg syndrome. This animal model will provide unique opportunities in the understanding of biological functions of dystroglycan in a wide range of dystroglycanopathies, as disruption of this gene in higher vertebrates results in early embryonic lethality.

Characterization of immune-matched hematopoietic transplantation in zebrafish.

Evaluating hematopoietic stem cell (HSC) function in vivo requires a long-term transplantation assay. Although zebrafish are a powerful model for discovering the genetics of hematopoiesis, hematopoietic transplantation approaches have been underdeveloped. Here we established a long-term reconstitution assay in adult zebrafish. Primary and secondary recipients showed multilineage engraftment at 3 months after transplantation. Limiting dilution data suggest that at least 1 in 65 000 zebrafish marrow cells contain repopulating activity, consistent with mammalian HSC frequencies. We defined zebrafish haplotypes at the proposed major histocompatibility complex locus on chromosome 19 and tested functional significance through hematopoietic transplantation. Matching donors and recipients dramatically increased engraftment and percentage donor chimerism compared with unmatched fish. These data constitute the first functional test of zebrafish histocompatibility genes, enabling the development of matched hematopoietic transplantations. This lays the foundation for competitive transplantation experiments with mutant zebrafish HSCs and chemicals to test for effects on engraftment, thereby providing a model for human hematopoietic diseases and treatments not previously available.

Rat neonatal beta cells lack the specialised metabolic phenotype of mature beta cells.

AIMS/HYPOTHESIS: Fetal and neonatal beta cells have poor glucose-induced insulin secretion and only gain robust glucose responsiveness several weeks after birth. We hypothesise that this unresponsiveness is due to a generalised immaturity of the metabolic pathways normally found in beta cells rather than to a specific defect. METHODS: Using laser-capture microdissection we excised beta cell-enriched cores of pancreatic islets from day 1 (P1) neonatal and young adult Sprague-Dawley rats in order to compare their gene-expression profiles using Affymetrix U34A microarrays (neonatal, n = 4; adult, n = 3). RESULTS: Using dChip software for analysis, 217 probe sets for genes/38 expressed sequence tags (ESTs) were significantly higher and 345 probe sets for genes/33 ESTs significantly lower in beta cell-enriched cores of neonatal islets compared with those of adult islets. Among the genes lower in the neonatal beta cells were key metabolic genes including mitochondrial shuttles (malate dehydrogenase, glycerol-3-phosphate dehydrogenase and glutamate oxalacetate transaminase), pyruvate carboxylase and carnitine palmitoyl transferase 2. Differential expression of these enzyme genes was confirmed by quantitative PCR on RNA from isolated neonatal (P2 until P28) and adult islets and with immunostaining of pancreas. Even by 28 days of age some of these genes were still expressed at lower levels than in adults. CONCLUSIONS/INTERPRETATION: The lack of glucose responsiveness in neonatal islets is likely to be due to a generalised immaturity of the metabolic specialisation of pancreatic beta cells.

Proteomics of human cerebrospinal fluid: discovery and verification of biomarker candidates in neurodegenerative diseases using quantitative proteomics.

There is an urgent need for novel biomarkers that can be used to improve the diagnosis, predict the disease progression, improve our understanding of the pathology or serve as therapeutic targets for neurodegenerative diseases. Cerebrospinal fluid (CSF) is in direct contact with the CNS and reflects the biochemical state of the CNS under different physiological and pathological settings. Because of this, CSF is regarded as an excellent source for identifying biomarkers for neurological diseases and other diseases affecting the CNS. Quantitative proteomics and sophisticated computational software applied to analyze the protein content of CSF has been fronted as an attractive approach to find novel biomarkers for neurological diseases. This review will focus on some of the potential pitfalls in biomarker studies using CSF, summarize the status of the field of CSF proteomics in general, and discuss some of the most promising proteomics biomarker study approaches. A brief status of the biomarker discovery efforts in multiple sclerosis, Alzheimer's disease, and Parkinson's disease is also given.

High-throughput cell transplantation establishes that tumor-initiating cells are abundant in zebrafish T-cell acute lymphoblastic leukemia.

Self-renewal is a feature of cancer and can be assessed by cell transplantation into immune-compromised or immune-matched animals. However, studies in zebrafish have been severely limited by lack of these reagents. Here, Myc-induced T-cell acute lymphoblastic leukemias (T-ALLs) have been made in syngeneic, clonal zebrafish and can be transplanted into sibling animals without the need for immune suppression. These studies show that self-renewing cells are abundant in T-ALL and comprise 0.1% to 15.9% of the T-ALL mass. Large-scale single-cell transplantation experiments established that T-ALLs can be initiated from a single cell and that leukemias exhibit wide differences in tumor-initiating potential. T-ALLs also can be introduced into clonal-outcrossed animals, and T-ALLs arising in mixed genetic backgrounds can be transplanted into clonal recipients without the need for major histocompatibility complex matching. Finally, high-throughput imaging methods are described that allow large numbers of fluorescent transgenic animals to be imaged simultaneously, facilitating the rapid screening of engrafted animals. Our experiments highlight the large numbers of zebrafish that can be experimentally assessed by cell transplantation and establish new high-throughput methods to functionally interrogate gene pathways involved in cancer self-renewal.

Zebrafish blood stem cells.

Within the past two decades, the zebrafish (Danio rerio) has become an excellent model to study the development of hematopoietic stem cells (HSCs). All vertebrates including zebrafish have primitive and definitive waves of hematopoiesis, but self-renewing pluripotent HSCs are only produced by the definitive wave. The primitive wave occurs in two intraembryonic locations called the intermediate cell mass (ICM) and the anterior lateral mesoderm (ALM). Primitive erythropoiesis is in the ICM, whereas myelopoiesis initiates in the ALM. After circulation starts at 24 h post-fertilization, hematopoiesis shifts to the posterior blood island (PBI) for a brief period. The definitive wave starts in the aorta-gonad-mesonephros (AGM). There are three different HSC migration and colonization events that begin 2 days post-fertilization: AGM progenitor cells migrate to (1) the caudal hematopoietic tissue (CHT), which is an intermediate site of blood development; (2) the thymus, which is a site of lymphocyte maturation; and (3) the developing kidney marrow, which is the larval and adult location for production of all hematopoietic cell types, and is comparable to the bone marrow of mammals. Many of the transcription factors and signaling pathways that regulate the formation of HSCs in a zebrafish are conserved with mammals. Large-scale forward and reverse genetic screens have identified zebrafish blood and HSC mutants that represent models for known human diseases. Along with the technological advancements in the field of zebrafish research, future HSC studies in zebrafish will help us illuminate the genetic network controlling the development and function of stem cells in all vertebrates.

Fine-needle aspiration cytology of metaplastic carcinoma of the breast.

BACKGROUND: Metaplastic carcinoma of the breast encompasses a heterogeneous group of tumours with variable components of sarcomatoid, squamous or poorly differentiated carcinomas. AIM: To review a series of 19 cytological preparations of metaplastic carcinomas to assess diagnostic cytological features. METHODS: 17 cases of fine-needle aspirates of histologically proven metaplastic carcinomas (4 monophasic spindle cell carcinomas, 4 squamous cell carcinomas and 11 biphasic tumours) were reviewed, with an emphasis on the presence of poorly differentiated carcinoma, squamous cell carcinoma, atypical spindle cells, benign stromal fragments and necrosis. RESULTS: All cases were diagnosed as malignant, with 68% of cases showing moderate to high cellularity, and 47% showing necrosis. If the tumours were analysed according to the constituting components histologically, 7, 15 and 8 cases, respectively, possess poorly differentiated carcinoma cells, sarcomatoid malignant cells and squamous carcinoma cells, whereas these components were cytologically identified in 11, 10 and 7 cases, respectively. Dual tumour populations were identified in only 5 of the 11 biphasic carcinomas in the cytological preparations; and the stromal material was cytologically identified in the only case with chondroid stroma. CONCLUSIONS: Identification of metaplastic carcinoma in cytology remains problematic. There seems to be morphological overlap between various components. The identification of dual components, unequivocal squamous carcinoma cells and chondroid stroma is helpful for diagnosis, but it is uncommon. The presence of poorly differentiated carcinoma cells with a suggestion of focal spindle morphology is another clue to the suggestion of metaplastic carcinoma.

Private GPs contribute to TB control in Myanmar: evaluation of a PPM initiative in Mandalay Division.

SETTING: Mandalay Division, Myanmar. AIM: To assess the effect of an initiative to involve private general practitioners (GPs) in the National Tuberculosis Programme (NTP) and to identify lessons learnt for public-private mix scale-up. METHODS: Source of referral/diagnosis and place of treatment were included in the routine recording and reporting systems to enable disaggregated analysis of the contribution of GPs to case notification and treatment outcomes. Case notification trends were compared between the intervention and control areas over a 4-year period. RESULTS: Private GPs contributed 44% of new smear-positive cases registered during the study period (July 2002-December 2004). The notification of new sputum smear-positive TB in the study area increased by 85% between the year prior to the GP involvement and 2 years after (from 46 to 85/100,000). Case notification increased by 57% in the control townships and by 42% in all of Mandalay Division. The treatment success rate for new smear-positive cases treated by GPs was 90%. CONCLUSIONS: The involvement of private GPs substantially increased TB case notification, while a high treatment success rate was maintained. Success factors include a well-developed local medical association branch, strong managerial support, training and supervision by the public sector and provision of drugs and consumables free of charge by the NTP.

Leptospirosis among patients presenting with dengue-like illness in Puerto Rico.

Leptospirosis is difficult to distinguish from dengue fever without laboratory confirmation. Sporadic cases/clusters of leptospirosis occur in Puerto Rico, but surveillance is passive and laboratory confirmation is rare. We tested for leptospirosis using an IgM ELISA on sera testing negative for dengue virus IgM antibody and conducted a case-control study assessing risk factors for leptospirosis, comparing clinical/laboratory findings between leptospirosis (case-patients) and dengue patients (controls). Among 730 dengue-negative sera, 36 (5%) were positive for leptospirosis. We performed post mortem testing for leptospirosis on 12 available specimens from suspected dengue-related fatalities; 10 (83%) tested positive. Among these 10 fatal cases, pulmonary hemorrhage and renal failure were the most common causes of death. We enrolled 42 case-patients and 84 controls. Jaundice, elevated BUN, hyperbilirubinemia, anemia, and leukocytosis were associated with leptospirosis (p < .01 for all). Male sex, walking in puddles, rural habitation, and owning horses were independently associated with leptospirosis. Epidemiological, clinical, and laboratory criteria may help distinguish leptospirosis from dengue and identify patients who would benefit from early antibiotic treatment.

Evaluation of the indirect hemagglutination assay for diagnosis of acute leptospirosis in Hawaii.

Timely diagnosis of leptospirosis is important to ensure a favorable clinical outcome. The definitive serologic assay, the microscopic agglutination test (MAT), requires paired sera and is not useful for guiding early clinical management. The only screening test approved for use in the United States, the indirect hemagglutination assay (IHA), has not undergone extensive field evaluation. To assess the performance of the leptospirosis IHA in Hawaii, serum from patients evaluated for leptospirosis between 1992 and 1997 were tested with the IHA at the Hawaii State Laboratories Division and with the MAT at the Centers for Disease Control and Prevention. Leptospirosis was considered confirmed by a fourfold rise in MAT titer and/or a positive culture. A total of 92 (41%) of 226 specimens from 114 persons with confirmed leptospirosis were found positive by IHA. Only 18 (15%) of 119 specimens obtained within 14 days of onset were IHA positive, compared to 74 (69%) of 107 specimens collected more than 14 days after onset (P <0.001). Repeat testing ultimately resulted in 78 (68%) of the confirmed cases having at least one specimen found positive by IHA. Thirteen different presumptive infecting serogroups were identified among 251 specimens with an MAT titer of >/=200 and obtained from persons with confirmed or probable leptospirosis. Fifty (68%) of 73 specimens with Icterohaemorrhagiae as the presumptive infecting serogroup were found positive by IHA, compared to 44 (47%) of 93 specimens with Australis as the presumptive infecting serogroup (P, 0.01). The IHA test was positive for 3 (1%) of 236 specimens from 154 persons without leptospirosis. The sensitivity of the leptospirosis IHA in Hawaii was substantially below figures reported previously, particularly early in the course of illness, limiting its usefulness for diagnosing acute infection. Since the presumptive infecting serogroup affected IHA results and the prevalence of serovars varies with geography, the performance of the IHA should be assessed locally. More sensitive leptospirosis screening tests are needed in Hawaii.

Increase of leptospirosis in dengue-negative patients after a hurricane in Puerto Rico in 1996 [correction of 1966].

Leptospirosis has rarely been reported in Puerto Rico, although in the period from 1948 to 1952, 208 cases of leptospirosis and an island-wide seroprevalence of antibody to Leptospira of 14% were documented. In Puerto Rico in October 1996, following rainfall and a period of flooding generated by Hurricane Hortense, serum specimens of 4 patients with suspected dengue fever that were negative for dengue tested positive for Leptospira-specific IgM antibodies in a dipstick assay. Subsequently, we used an island-wide dengue laboratory-based surveillance system to determine the increase in leptospirosis after hurricane-generated floods. All anti-dengue IgM-negative patients (n = 142) with disease onset from August 8 to October 6, 1996 from prehurricane and posthurricane groups were investigated for leptospirosis. Laboratory-confirmed leptospirosis cases were defined as microscopic agglutination test titers > or = 1 :400 to 1 or more serovars, or positive immunohistochemistry in autopsy tissues. Four (6%) of 72 prehurricane and 17 (24%) of 70 posthurricane patients had laboratory-confirmed cases of leptospirosis (relative risk [RR] = 4.4, 95% confidence interval [CI] = 1.6-12.4). The mean age of case-patients was 34 years (range = 13-64). Eighteen (86%) of 21 confirmed case-patients were males, including one patient who died (31 years old). Patients were located in 18 (38%) of 48 municipalities that submitted serum samples. Clinical features significantly associated with leptospirosis were eye pain (RR = 1.5, 95% CI = 1.3-1.9), joint pain (RR = 1.4, 95% CI = 1.1-1.6), diarrhea (RR = 1.7, 95% CI = 1.2-2.5), and jaundice (RR = 3.3, 95% CI = 1.5-7.2). This study demonstrates the utility of a dengue laboratory-based surveillance system for the detection of an increase of leptospirosis, which most likely would have gone unrecognized. Leptospirosis is treatable with antibacterial agents; knowledge of this diagnosis may significantly reduce morbidity and mortality.

International multicenter evaluation of the clinical utility of a dipstick assay for detection of Leptospira-specific immunoglobulin M antibodies in human serum specimens.

We performed a multicenter evaluation of a robust and easily performed dipstick assay for the serodiagnosis of human leptospirosis. The assay is aimed at the detection of Leptospira-specific immunoglobulin M (IgM) antibodies. The study involved 2,665 serum samples collected from 2,057 patients with suspected leptospirosis in 12 countries on five continents with different levels of endemicity and different surveillance systems. The patients were grouped as laboratory-confirmed leptospirosis case patients and noncase patients based on the results of culturing and the microscopic agglutination test. Paired samples from 27.7% of the subjects were tested. Of the 485 case patients, 87.4% had a positive dipstick result for one or more samples. Of the 1,513 noncase patients, only 7.2% had a positive result. Whereas most (88.4%) of the positive samples from the case patients showed moderate to strong (2+ to 4+) staining in the dipstick assay, most (68.1%) of the positive samples from the noncase patients showed weak (1+) staining. The sensitivity of the dipstick assay increased from 60.1% for acute-phase serum samples to 87.4% for convalescent-phase samples. The specificities for these two groups of samples were 94.1 and 92.7%, respectively. The dipstick assay detected a broad variety of serogroups. The results of the dipstick assay were concordant (observed agreement, 93.2%; kappa value, 0.76) with the results of an enzyme-linked immunosorbent assay for the detection of specific IgM antibodies, a test which is often used in the laboratory diagnosis of current or recent leptospirosis. This study demonstrated that this easily performed dipstick assay is a valuable and useful test for the quick screening for leptospirosis; has a wide applicability in different countries with different degrees of endemicity; can be used at all levels of the health care system, including the field; and will be useful for detecting and monitoring outbreaks of leptospirosis.