Serum Levels of Copper in Human Leptospirosis Cases

Background: Copper deficiency can weaken immunity and increase the incidence of infections or infections may reduce the serum copper levels. Objectives: The present investigation assessed the serum level of copper in the cases of leptospirosis. Methods: The study involved 55 patients of whom had either icteric or non-icteric type of leptospirosis, 25 as other febrile and 25 as healthy controls. Serum copper was evaluated using commercially available kits. All the 55 patients are serologically confirmed for leptospirosis by using both ELISA and MAT. Twenty five age-matched febrile cases other than leptospirosis and twenty five healthy normal individuals were taken as control. The serum copper level was estimated by calorimetric method of Di-Br-PAESA. Results: The maximum and minimum age of patients was 54 and 18 years respectively; males dominated with 47 cases in this study. Out of 55 cases, 45 cases are considered as high risk groups of having minimum of 10 years of occupational exposure. There was a significant decrease of serum copper level among 55 leptospirosis confirmed cases (ranged from 26 to 63 μg/dl) when compared with controls (ranged from 114.12 to 126.32 μg/dl); whereas in febrile cases other than leptospirosis showed maximum of serum decrease upto 52.7 μg/dl. Conclusion: In all cases including non leptospirosis febrile cases also, the serum copper levels are significantly decreased after treatment with doxycycline and other antibiotics. This indicates that serum copper can be used as a biochemical marker for screening leptospirosis, as a valuable prognostic indicator for monitoring disease

Paediatric leptospirosis: A population based case–control study from Chennai, India.

The surveillance in Chennai identified 134 children and 443 adults clinically suspected for leptospirosis. Of these, 35 (26.1%) children and 118 (26.6%) adults had laboratory confirmed diagnosis for leptospirosis. The paediatric leptospirosis exhibited a higher frequency of classic features of Weil’s disease. The prevalent serovar encountered was Icterohaemorrhagiae with no difference in the pattern of infecting serovars between the two groups. Further, confirmation of diagnosis was achieved by polymerase chain reaction (PCR) with a positivity of 28.4% (specificity 96%). Univariate analysis showed significant association of paediatric leptospirosis with rat infestation (odds ratio 87.4). Thus, PCR facilitates early diagnosis of febrile illness among paediatric cases.

Risk factors associated with leptospirosis during an outbreak in Middle Andaman, India.

Background & objectives: Leptospirosis outbreaks occur frequently in North and South Andaman Islands but not in Middle Andaman. In 2002, an outbreak appeared in Middle Andaman for the first time. Although a study on risk factors was conducted in North Andaman, it used seropositivity to define leptospirosis. Since seropositivity might not indicate current leptospiral infection and as no study on risk factors was conducted in Middle Andaman, we carried out this study to identify the risk factors during the outbreak. Methods: A suspected outbreak of leptospirosis occurred in Rangat of Middle Andaman during October - November 2002. Suspected cases were screened for leptospirosis using microscopic agglutination test (MAT). Fifty two patients confirmed to have leptospirosis based on rising titres in MAT on paired sera, and 104 age, sex and neighbourhood seronegative matched controls, were included in the study. A conditional multiple regression by backward elimination process was carried out with acute leptospirosis as the dependent factor and various environmental, occupational and behavioural factors as independent factors. A stratified analysis was also carried out. Results: The presence of cattle in the house, drinking stream water, contact with garbage, walking barefoot and standing in water while working were identified as significant factors associated with leptospirosis. Stratified analysis showed a dose response relationship between number of cattle in the house and the risk of leptospiral infection suugesting that cattle could be a source of infection. Interpretation & conclusions: Identification of the potential risk factors would help understand the transmission dynamics of the disease and formulate public health interventions.

Phenotypic & genotypic conservation of ompL1 & lipL41 among leptospiral isolates of Andaman Islands.

BACKGROUND & OBJECTIVES: The leptospiral antigens that are conserved among the diverse pathogenic leptospires have potential importance in the development of new serodiagnostic and immunoprotective strategies. The present study was therefore carried out to find out the phenotypic conservation of the leptospiral proteins OmpL1 and LipL41, and the genetic conservation of ompL1 and lipL41 genes among the leptospiral isolates of Andaman Islands and among the reference strains. METHODS: In one dimensional SDS-PAGE the leptospiral samples prepared from strains of various leptospiral serovars were run and transferred on to nitrocellulose paper and probed with pooled convalescent phase human sera to find out the phenotypic conservation of the protein fragments at 31 and 41 kDa. Further, the proteins were indirectly confirmed as OmpL1 and LipL41 by using specific rabbit hyperimmune sera. Specific primers were utilized to amplify the fragments to study the genetic conservation of ompL1 and lipL41. Further, these two fragments were sequenced and BLAST analysis was done with the whole genome of Leptospira interrogans serovar Lai for comparison. RESULTS: Analysis of individual immunoblots using patient sera showed that the OmpL1 and LipL41 were conserved among all the isolates used in the study. Further, these two proteins were probed with specific rabbit hyperimmune sera of OmpL1 and LipL41 for confirming the fragments and it was found to be conserved among all the isolates. The PCR based amplification further showed that the genes ompL1 and lipL41 were conserved among the leptospiral isolates studied. Sequencing followed by BLAST analysis of these showed 97 per cent similarity with the whole genome sequence and low score values in comparison with other bacterial species. INTERPRETATION & CONCLUSION: The antigenic and genetic conservation of the two proteins, OmpL1 and LipL41, indicated that these could be potential candidates for development of diagnostic test systems for leptospirosis.

Comparison of immunoreactive proteins of commonly circulating serogroups of Leptospira in Andaman Islands, India.

BACKGROUND & OBJECTIVE: Early diagnosis is the key to the treatment of leptospirosis. For development of rapid diagnostic kits, a thorough knowledge about the nature of the proteins expressed by the pathogen during infection is necessary. The present study was undertaken to understand the nature of immunoreactive proteins from commonly circulating serogroups of Leptospira in the endemic Andaman and Nicobar Islands, India. METHODS: Proteins were extracted from six strains of Leptospira representing five different serogroups following four different preparation methods, viz., whole cell lysis by sonication, detergent solubilization, outer and inner membrane isolations, and were subsequently characterized on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Immunoblots were made from the sonicated proteins using hyperimmune rabbit antisera, homologous and heterologous patient sera separately. RESULTS: The 67, 65, 45, 43, 35, 32 and 18 kDa major proteins in the whole cell lysate were common among all the five serogroups of Leptospira. The 67, 41, 35, 32, 28 and 22 kDa were the major outer membrane proteins, while 94, 32, 25 and 18 kDa protein were in inner membrane. Immunoblots with hyperimmune rabbit antisera detected 67, 65, 60, 45, 43, 41 and 32 kDa common proteins from the whole cell lysates of all strains while homologous and heterologous patient sera detected 32 kDa as the major immunoreactive protein in all pathogenic serogroups. This protein reacted against specific LipL32 antisera indicating that this protein was LipL32. INTERPRETATION & CONCLUSION: The circulating serogroups of Leptospira have common nature of expression of proteins during human infection. Among several immunoreactive proteins, three (67, 45 and 32 kDa) were recognized as major antigens by both rabbit hyperimmune sera and patients sera while the 32 kDa protein was recognized as the major immunoreactive protein by homologous and heterologous patient sera. These conserved immunoreactive proteins could be utilized in developing indigenous diagnostic tests for leptospirosis.

Leptospiral proteins expressed during acute & convalescent phases of human leptospirosis.

BACKGROUND & OBJECTIVES: The available serological techniques for the diagnosis of leptospirosis have less sensitivity during the early stage of the disease. Understanding of leptospiral proteins expressed during acute and convalescent phases of leptospirosis, would be help the develop of new serodiagnostic strategies. Therefore, the present study was carried out to identify (i) an antigen that is conserved among the various pathogenic leptospira; (ii) best protein antigen to which immune response can be identified in the acute phase; and (iii) best protein antigen which is present in convalescent sera which can be used for seroepidemiological studies. METHODS: Quantitative immunoblot analysis was performed using acute and convalescent phase human sera along with sera from normal healthy individuals and from patients with typhoid, malaria and hepatitis as the controls. All the samples were analyzed for the leptospiral protein recognition by using IgM and IgG immunoblots. Leptospiral cell fractionation was performed using triton X-114 and lysozyme and further the conservation of leptospiral proteins was also performed. RESULTS: In confirmed cases of leptospirosis, the IgG recognition in acute phase sera was 30.2, 39.5, 27.9, 55.8 and 27.9 per cent for the leptospiral proteins p32, p41/42, p58, p62 and p82 respectively. The IgG has considerably increased to 65.1, 55.8, 46.5, 67.4 and 48.8 per cent against the same proteins during convalescent phase. The IgM recognition was 32.6 , 32.6, 30.2 and 37.2 per cent for acute phase sera and 32.6, 37.2, 44.2 and 41.9 per cent for convalescent phase sera for the leptospiral proteins p14, p25, p32 and p41/42, respectively. Leptospiral proteins like p62 and p82 were recognized among all the control groups with 3.3-15.3 per cent for IgG recognition. INTERPRETATION & CONCLUSION: Leptospiral protein p32 was found to be highly sensitive and specific and could be useful for the development of newer techniques for diagnosis and seroepidemiological studies. Combination of p32 and p41/42 for IgG and p14, p25, p32, p41/42 for IgM would increase the sensitivity of these techniques further.

Leptospiral antibodies in patients with recurrent ophthalmic involvement.

Leptospiral antibodies could be demonstrated by microscopic agglutination test in 14 of 15 (93%) patients with acute panuveitis and retinal vasculitis in a preliminary study undertaken during the postmonsoon period at Madurai in Tamilnadu, India. The predominant serogroup was Pomona followed by Autumnalis, Australis and Javanica, the titres being between 1:160 and 1:10240. Titres in the normal controls were 1:20 to 1:80 in 8 of 20 mostly to the endemic serogroup Autumnalis. The involvement of leptospires particularly Pomona as a cause of ophthalmic complications in the patients studied is likely.