ABSTRACT
BACKGROUND: Leptospirosis is a re-emerging infectious disease that is under-recognized due to low-sensitivity and cumbersome serological tests. MAT is the gold standard test and it is the only serogroup specific test used till date. Rapid reliable alternative serogroup specific tests are needed for surveillance studies to identify locally circulating serogroups in the study area. METHODS/PRINCIPAL FINDINGS: In the present investigation the serological specificity of leptospiral lipopolysaccharides (LPS) was evaluated by enzyme linked immunosorbent assay (ELISA), dot blot assay and rapid immunochromatography based lateral flow assay (ICG-LFA). Sera samples from 120 MAT positive cases, 174 cases with febrile illness other than leptospirosis, and 121 seronegative healthy controls were evaluated for the diagnostic sensitivity and specificity of the developed assays. LPS was extracted from five locally predominant circulating serogroups including: Australis (27.5%), Autumnalis (11.7%), Ballum (25.8%), Grippotyphosa (12.5%), Pomona (10%) and were used as antigens in the diagnostics to detect IgM antibodies in patients' sera. The sensitivity observed by IgM ELISA and dot blot assay using various leptospiral LPS was >90% for homologous sera. Except for Ballum LPS, no other LPS showed cross-reactivity to heterologous sera. An attempt was made to develop LPS based ICG-LFA for rapid and sensitive serogroup specific diagnostics of leptospirosis. The developed ICG-LFA showed sensitivity in the range between 93 and 100% for homologous sera. The Wilcoxon analysis showed LPS based ICG-LFA did not differ significantly from the gold standard MAT (P>0.05). CONCLUSION: The application of single array of LPS for serogroup specific diagnosis is first of its kind. The developed assay could potentially be evaluated and employed for as MAT alternative.
Subject(s)
Antibodies, Bacterial/blood , Biological Assay/methods , Immunoglobulin M/blood , Leptospira/isolation & purification , Leptospirosis/diagnosis , Lipopolysaccharides/analysis , Adult , Agglutination Tests , Biological Assay/standards , Chromatography, Affinity , Enzyme-Linked Immunosorbent Assay , Female , Humans , Immunoblotting , Leptospira/immunology , Leptospirosis/blood , Leptospirosis/immunology , Leptospirosis/microbiology , Lipopolysaccharides/immunology , Male , Middle Aged , Sensitivity and Specificity , SerogroupABSTRACT
Successful treatment of leptospirosis is heavily dependent on early diagnosis and prompt initiation of antibiotic therapy. An ELISA test to detect specific IgM antibodies against LipL32 for early diagnosis of leptospirosis is described and evaluated here. One thousand one hundred and eighty sera from clinically suspected leptospirosis cases were enrolled together with 109 healthy volunteers selected from an endemic area between October 2007 and January 2010. Patients were categorized based on their clinical signs and symptoms. Sera were screened for leptospiral antibodies by the microscopic agglutination test (MAT) using a panel of locally circulating serovars followed by enzyme-linked immunosorbent assay (ELISA) based on recombinant LipL32 from Leptospira interrogans serovar Autumnalis strain N2. The sensitivity and specificity of the ELISA test were determined to establish its diagnostic efficiency. The cut-off value was determined to be 0·205. Overall sensitivity and specificity compared to the MAT were found to be 96·4 and 90·4%, respectively. The LipL32-specific IgM ELISA had good sensitivity and acceptable specificity and may be a candidate for the early serodiagnosis of human leptospirosis.
Subject(s)
Antibodies, Bacterial/blood , Antigens, Bacterial , Bacterial Outer Membrane Proteins , Clinical Laboratory Techniques/methods , Immunoglobulin M/blood , Leptospirosis/diagnosis , Lipoproteins , Adolescent , Antigens, Bacterial/genetics , Antigens, Bacterial/isolation & purification , Bacterial Outer Membrane Proteins/genetics , Bacterial Outer Membrane Proteins/isolation & purification , Child , Child, Preschool , Early Diagnosis , Enzyme-Linked Immunosorbent Assay/methods , Female , Humans , Infant , Infant, Newborn , Leptospira interrogans/genetics , Leptospira interrogans/immunology , Lipoproteins/genetics , Lipoproteins/isolation & purification , Male , Pregnancy , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Sensitivity and SpecificityABSTRACT
The main goal of this study was to evaluate the prevalence of leptospirosis among field rodents of Tiruchirappalli district, Tamil Nadu, India. In total 35 field rats were trapped and tested for seroprevalence by the microscopic agglutination test (MAT). Isolation of leptospires was performed from blood and kidney tissues and characterized to serovar level. Genomospecies identification was carried out using 16S rRNA and lipL32 gene sequencing. The molecular phylogeny was constructed to find out species segregation. Seroprevalence was about 51.4 percent, and the predominant serovars were Autumnalis, Javanica, Icterohaemorrhagiae and Pomona. Two isolates from the kidneys were identified as serovar Javanica of Serogroup Javanica, and sequence based molecular phylogeny indicated these two isolates were Leptospira borgpetersenii.
Subject(s)
Animals , Rats , Base Sequence , Leptospirosis , Leptospira interrogans serovar autumnalis/isolation & purification , Leptospira interrogans serovar icterohaemorrhagiae/isolation & purification , Leptospira interrogans serovar pomona/isolation & purification , Phylogeny , Agglutination Tests , Methods , Prevalence , Seroepidemiologic Studies , SerotypingABSTRACT
The main goal of this study was to evaluate the prevalence of leptospirosis among field rodents of Tiruchirappalli district, Tamil Nadu, India. In total 35 field rats were trapped and tested for seroprevalence by the microscopic agglutination test (MAT). Isolation of leptospires was performed from blood and kidney tissues and characterized to serovar level. Genomospecies identification was carried out using 16S rRNA and lipL32 gene sequencing. The molecular phylogeny was constructed to find out species segregation. Seroprevalence was about 51.4 %, and the predominant serovars were Autumnalis, Javanica, Icterohaemorrhagiae and Pomona. Two isolates from the kidneys were identified as serovar Javanica of Serogroup Javanica, and sequence based molecular phylogeny indicated these two isolates were Leptospira borgpetersenii.
