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Leptospiral culture, direct immunofluorescence, and the polymerase chain reaction (PCR) were used to detect leptospiral material in postmortem specimens collected from eight patients who died of leptospirosis. Diagnosis of leptospiral infection was based on clinical summary (premortem) and confirmed by serological analysis and/or culture of leptospires. Leptospiral culture was the least sensitive technique, yielding two isolates (3%) from 65 samples. Both isolates were from the aqueous humour and cerebrospinal fluid of the same patient. Direct immunofluorescence was of intermediate sensitivity for detection of leptospires, confirming the presence of leptospires in 11% (2 of 18) of tissue samples from three patients. PCR analysis was the most sensitive technique for detection of leptospiral material in tissue samples, being positive in 20% (11 of 56) of samples from eight patients. Both samples (cerebellum and liver) positive by immunofluorescence were also positive by PCR. The sensitivity of the PCR assay was 1-10 leptospires ml(-1) sample, and the assay was specific for Leptospira pathogenic species. Multi-system involvement was indicated based on successful amplification of leptospiral DNA from more than one tissue sample, which corroborated with the clinical and pathologic findings. The results suggest that in acute and/or fatal leptospirosis, the pathogenesis of the pathologic features are related to the presence of the organisms in the tissues. In conclusion, PCR combined with serology appears to be a useful tool for diagnosis of leptospirosis and may be invaluable in epidemiological studies.

Early diagnosis of leptospirosis is important because severe leptospiral infection can run a fulminant course. The polymerase chain reaction (PCR) was evaluated for the detection of leptospires in clinical samples from patients with acute leptospiral infection. Blood and urine samples from 71 patients with leptospirosis were examined by PCR, culture or serology. Samples from 44 (62%) patients with the diagnosis of leptospirosis were positive by PCR as compared to 34 (48%) by culture. The presence of leptospires was demonstrated by PCR in 13 patients before the development of antibodies, as well as in two patients who were seronegative during their illness and at autopsy. Samples from 16 patients without leptospirosis were seronegative and culture negative, and also negative by PCR. We conclude that PCR is a rapid, sensitive and specific means of diagnosing leptospiral infection, especially during the first few days of the disease.

Leptospirosis was confirmed by Microscopic Agglutination Test (MAT) and/or ELISA in 57 patients admitted to the Government General Hospital, Madras, India, during November and December of 1990 and 1991 with symptomatology suggestive of the disease. Fifty (88%) of the 57 cases were males; the mean age of all the cases was 39.6 years (range 17-72). The main clinical features were: fever 100% jaundice 84%, Myalgia 82%, acute renal failure 72% and conjunctival suffusion 58%. Non-azotemic jaundice occurred in 19% of cases. Renal failure was non-oliguric in 24% of cases. 3.5% of patients died. 23 patients underwent peritoneal and/or hemodialysis. ELISA IgM titres ranged from 1:80 to 1:10240 (geometric mean tire 911). MAT titres > or = 1:1600 and > or = 1:800 occurred in 39 of 54 and 51 of 54 cases respectively. Autumnalis was the serogroup most commonly recorded serologically, and Leptospira interrogans serovar autumnalis was isolated from one patient. This study shows that leptospirosis is a significant health problem in Madras, though normally grossly underestimated due to the absence of routine laboratory diagnostic facilities for the disease. Gross under-reporting is also likely in other high rainfall third world areas.

Between November 1979 and December 1991, 398 cases of severe leptospirosis were confirmed on Barbados (range for 1980-1991 23-56; mean 32.7; incidence 13.3/100,000/year). For the six-year periods 1980-1985 and 1986-1991 there was no significant change in incidence with time. Incidence is unlikely to change significantly in the next decade. Monthly average case numbers ranged from 1.4 (July) to 4.3 (November). The average (2.8) for June to December (the 7 wetter months) was not significantly higher than that (2.5) for January to May (the 5 drier months). The age range was 7-86. There were three times as many male cases (302) as female (96), and nearly 10 times as many in those < 35. Although the highest number of cases (69) was in males aged 15-24, the highest incidence was in the older age groups, particularly the male 65-74 year-olds, and the female 55-64 year-olds. Leptospirosis was the proven cause of death in 55 (13.8%) hospital patients (annual range 0-13, mean 4.5). Some of a further 39 fatalities might have been cases. Death from leptospirosis was nearly twice as common among the women as among the men. Only one patient under 20 years of age died. Leptospira were isolated and identified from 117 (29.4%) of the 398 sick patients. The infecting organisms were bim (serogroup Autumnalis--75), copenhageni (Icterohaemorrhagiae-26), arborea (Ballum-14) and bajan (Australis-2). These infecting serovars could not be distinguished clinically, but infection was milder in children than in adults. Despite its predominance in surveyed children, serogroup Panama was virtually absent in this study. Rainfall is the major factor affecting the distribution of cases; not surprisingly, sanitation workers and agricultural workers appear to be the groups at highest risk. The general lack of clear-cut risk factors reflects the ubiquity of leptospires in the environment and the fact that the disease is not entirely occupational.

Between November 1979 and the end of December 1986 (7.17 years), 248 cases of leptospirosis were confirmed among hospital patients on Barbados (mean 35 per year; range 25-57). Considering the 235 who were greater than or equal to 15 years of age, the annual incidence of leptospirosis was 19.2/100,000 population (14.0 for all age groups). There were 173 males and 62 females, and for cases aged 15-34 leptospirosis was 9.6 times more common in men than women. Among men, incidence increased fairly steadily with age, and an even steadier increase was apparent in women up to age 64, with some decline in later years. The incidence of disease was much higher among agricultural than other workers and the non-employed. Highest case numbers were recorded in the parishes of St Michael (65 or 28%) and Christ Church (36 or 15%), though the incidence was lowest in these two parishes (13.1/100,000 and 17.4/100,000, respectively). The highest incidence rates were in St Andrew and St Joseph (50.2 and 36.1/100,000, respectively). The incidence in areas with rainfall greater than or equal to 1600 mm (32.6/100,000) was nearly twice that in areas with rainfall less than 1600 mm (17.3/100,000). There is a clear link between cases of severe disease and recent rainfall. Using 134 patients greater than or equal to 15 years of age with fever due to other illnesses as controls, a higher proportion of cases than controls came from rural areas. The risk of contracting leptospirosis was increased for all categories of manual workers relative to the group at lowest risk (non-manual indoor workers). Sugar-cane workers were five times more likely to contract leptospirosis than were non-manual indoor workers, while those whose families minded livestock were 2.5 times more likely, and those with rodents in their garden/yard were 1.8 times more likely to do so. Other risk factors examined did not show significant associations with the disease. Despite increasing mechanization and the use of more protective clothing, agricultural workers are still at high risk from leptospirosis. The annual range of cases is likely to stay much as it is in the foreseeable future.

In a study of 21 wild-caught Barbadian vervet monkeys (Cercopithecus aethiops sabaeus), naturally-acquired leptospiral agglutinins were found to persist for over five years. Groups of seropositive as well as seronegative vervets were given a placebo, or full-strength monoclonal antibodies MCA F12C3 (Icterohaemorrhagiae copenhageni), or diluted F12C3 MCAs. They were challenged 24 hours later with a suspension of highly virulent leptospires (copenhageni) administered intraperitoneally. Immunoprotection was evident in animals receiving full strength MCAs as measured by their failure to develop any substantial antibody response and by their lower geometric mean titres over a period of 142 weeks (maximum GMT of 113 compared with a maximum of 1280 in the placebo group). Diluted MCAs had little or no protective value. The serological response of the monkeys which were seropositive at capture to challenge with virulent copenhageni antigen was strongly anamnestic both in those given MCAs and those given placebo. None of the naturally or experimentally infected vervets showed clinical signs of leptospiral illness.

Sera from 1,419 patients who attended a Barbadian general practice for a variety of complaints between 1 April 1984 and 30 April 1988 were examined for leptospiral agglutinins by the microscopic agglutination test (MAT). Sera from the 42 patients with pyrexia of unknown origin, jaundice or kidney involvement, and the 26 patients with titres greater than or equal to 1:400 in the MAT, were also examined by the ELISA for IgM and IgG antibodies. Current or recent mild leptospiral infection was diagnosed in four of the patients, but some mild cases may have been missed. Two further cases of leptospirosis were recorded after the study period ended; both were very ill but responded well to doxycycline therapy. Altogether 177 of the 1,419 patients (12.5%) had agglutination titres greater than or equal to 1:50. These were 104/739 males (14%) and 73/680 females (11%). Seropositivity tended to increase with age (p less than 0.01), and the highest rates were in agricultural workers (35%), labourers (24%) and non-manual outdoor workers (19%). The difference in seropositivity between the main occupational groups was highly significant (p less than 0.001). The serogroups most commonly recorded among the seropositive patients were Autumnalis (31%), Panama (26%), Australis (24%) and Pyrogenes (20%). Autumnalis predominated in each of the main occupational groups except indoor non-manual workers where Panama, Pyrogenes and Australis occurred more frequently. 95% of the positive titres ranged between 1:50 and 1:400. Titres tended to increase with age, but there was no obvious association between higher titres and particular occupations.(ABSTRACT TRUNCATED AT 250 WORDS)

Two groups of whistling frogs (Eleutherodactylus johnstonei) comprising 99 and 117 animals were examined for leptospiral infection. Group I animals were caught in 14 areas of Barbados, and Group II animals in seven areas of suburban Bridgetown. Leptospires were isolated from the kidneys or body fluid of six frogs in Group I and the kidneys of 3 frogs in Group II. Two of the Group I isolates died out; the others were identified as bajan (a new serovar in the Australis serogroup) (6) and bim (Autumnalis) (1). The macerated body tissues and fluid of Group I frogs were put into phosphate buffered saline and examined by the microscopic agglutination test using 22 antigens. The results were all negative. For the Group II frogs the methodology was altered; blood was collected onto filter paper discs and allowed to dry out before being agitated in PBS and examined by the MAT. 15/117 (12.8%) animals were positive at greater than or equal to 1:100 and 19 (16.2%) at greater than or equal to 1:50. The geometric mean titre was 179. Seventeen of the sera reacted predominantly to antigens in the Australis serogroup, and two to Pyrogenes on its own. The serological results reflected the identity of the isolates. Serovars of Australis are not known to cause illness on Barbados, but bim is the commonest cause of severe leptospirosis on the island.

A monoclonal antibody (mAb) was prepared by hybridoma technology in BALB/c mice immunized to Leptospira interrogans serovar copenhageni. This mAb agglutinated serovars copenhageni and icterohaemorrhagiae to high titres and protected hamsters, dogs and monkeys against challenge with a virulent strain of serovar copenhageni. The mAb gave protection to hamsters at dilutions up to 1 in 1000; at a 1 in 10 dilution the protective effect lasted for at least two weeks. Biochemical analysis by SDS-PAGE and Western blotting indicated that this mAb reacted with an epitope of a carbohydrate nature.