Persistence of anti-leptospiral IgM, IgG and agglutinating antibodies in patients presenting with acute febrile illness in Barbados 1979-1989.

The persistence of anti-leptospiral IgM and IgG antibodies and agglutinating antibodies was studied in serologically confirmed cases of severe leptospirosis during the acute illness and over periods of several years after recovery. The antibody response in non-leptospirosis patients presenting to hospital with similar symptoms over the same period of time was used to estimate the background antibody level to leptospirosis in the community. All patients enrolled in the study had blood samples collected twice in the acute stage of illness, once during convalescence and then annually from the time of initial hospitalisation until the end of the study period. Six hundred and thirty-eight patients presented to hospital with acute febrile illness, of whom 321 were diagnosed with leptospirosis. Patients who had severe leptospirosis commonly remained seropositive, with IgM, IgG and agglutinating antibodies detectable for several years after infection. A significant proportion of cases had high titres of agglutinating antibody detectable by the microscopic agglutination test (> or = 800). There were marked differences in the magnitude and duration of persistence of agglutinating antibodies directed against different serogroups. More than 20% of cases with evidence of infection with serogroup Autumnalis retained titres of >800, 4 years after the acute illness. In one case a titre of 800 was detected 11 years after infection. Persistence of agglutinating antibody titres can create problems in interpretation of serological results and make it impossible to estimate the time of infection, given a specific titre. This study demonstrates that in endemic areas where seroprevalence is high, use of a single elevated titre is not reliable to define a current infection.

Assessment of the efficacy of an IgM-elisa and microscopic agglutination test (MAT) in the diagnosis of acute leptospirosis.

In a prospective study in Barbados between 1979 and 1989, 321 cases were diagnosed in 638 patients presenting at a hospital with symptoms of leptospirosis. Initial diagnosis was based on patient history and characteristic signs and symptoms. In 92 cases (29%), diagnosis was confirmed by isolation of organisms from the blood, urine, or dialysate fluid; in the remaining 229 cases (71%) diagnosis was confirmed by serology alone. Results of an IgM-ELISA and microscopic agglutination test (MAT) in cases with isolates and in non-leptospirosis cases were used to assess the sensitivity and specificity of the tests. The sensitivity of IgM detection by ELISA was 52% in the first acute-phase specimen, increasing to 89% and 93% in the second acute-phase and convalescent specimens, respectively. The specificity of the IgM-ELISA was high (> or = 94%) in all specimens. The sensitivity of the MAT was low (30%) in the first acute-phase specimen, increasing to 63% in the second acute-phase specimen and 76% in the convalescent specimen. The specificity of the MAT was > or = 97% in all specimens.

Surveillance of leptospiral carriage by feral rats in Barbados.

Rodents, particularly rats, are widely held to be the source of most human cases of leptospirosis. Feral rats were trapped at sites throughout Barbados during two six month surveys: from October to March 1986/87 and from October to March 1994/95. During the first survey, 63 rats were trapped, of which 26 (41%) were identified as Rattus rattus and 37 (59%) as Rattus norvegicus. In the second study, 100 rats were trapped, of which R. rattus comprised 24% (24) and R. norvegicus 76% (76). Cultures of blood, urine and kidney were made in EMJH medium. Leptospires were isolated from 12/63 (19%) and from 16/100 (16%) of the rats during 1986/87 and 1994/95, respectively; 27/28 isolates were recovered from the kidneys or urine or both, while only one isolate was recovered from the blood. During the first study, isolates were identified as serovars copenhageni (11) and arborea (1), while in the second study, serovars copenhageni (9), arborea (5) and bim (1) were identified; one isolate was lost before it could be identified. In the first study, antibodies were detected by microscopic agglutination at a titre of > or = 100 in 26/62 (42%) of rats tested, while in the second survey, 5/100 (5%) of rats had similar titres. In two surveys, conducted eight years apart, we confirmed that rats in Barbados are commonly infected with leptospires, and that viable organisms are found in the kidneys and urine, evidence of chronic infection and thus excretion of leptospires in rodent urine. Moreover, the predominant serovar isolated was copenhageni, of which Rattus spp. are the worldwide reservoir. There was little evidence that rats act as a reservoir for the serovar bim, the most common cause of human leptospirosis in Barbados.

Surveillance of leptospiral carriage by feral rats in Barbados

Rodents, particularly rats, are widely held to be the source of most human cases of leptospirosis. Feral rats were trapped at sites throughout Barbados during two six month surveys: from October to March 1986/87 and from October to March 1994/95. During the first survey, 63 rats were trapped, of which 26 (41) were identified as Rattus rattus and 37 (59) as Rattus norvegicus. In the second study, 100 rats were trapped, of which R. rattus comprised 24(24) and R. norvegicus 76(76). Cultures of blood, urine and kidney were made in EMJH medium. Leptospires were isolated from 12/63 (19) and from 16/100 (16) of the rats during 1986/87 and 1994/95, respectively; 27/28 isolates were recovered from the kidneys or urine or both, while only one isolate was recovered from the blood. During the first study, isolates were identified as serovars copenhageni (11) and arborea (1), while in the second study, serovars copenhageni (9), arborea (5) and bim (1) were identified; one isolate was lost before it could be identified. In the first study, antibodies were detected by microscopic agglutination at a titre of > or = 100 in 26/62 (42) of rats tested, while in the second survey, 5/100 (5) of rats had similar titres. In two surveys, conducted eight years apart, we confirmed that rats in Barbados are commonly infected with leptospires, and that viable organisms are found in the kidneys and urine, evidence of chronic infection and thus excretion of leptospires in rodent urine. Moreover, the predominant serovar isolated was copenhageni, of which Rattus spp. are the worldwide reservoir. There was little evidence that rats act as a reservoir for the serovar bim, the most common cause of human leptospirosis in Barbados.

Seroepidemiology of canine leptospirosis on the island of Barbados.

Previous surveillance in Barbados documented the absence of infection with Leptospira serogroup Canicola in dogs. The aim of this study was to survey the current state of canine leptospirosis in Barbados, 10 years after the last survey. Sera from 78 unwanted dogs scheduled for euthanasia and 61 dogs suspected of having acute leptospirosis were tested by microscopic agglutination (MAT) and by an ELISA method adapted for canine IgM and IgG antibodies. The seroprevalence in unwanted dogs was 62% (48/78), at an MAT titre of > or = 100. The majority of animals had low titres, suggestive of previous infection. Serogroup Autumnalis was the most common reactor (45%), followed by serogroups Icterohaemorrhagiae and Australis (each 16%) and Pomona (13%). Serogroup Ballum was uncommon in this group. The seroprevalence determined by MAT in acutely-ill dogs was 75% (46/61). The most common predominant serogroup was Icterohaemorrhagiae (36%) followed by serogroup Australis (13%), while serogroups Autumnalis and Ballum were also of little significance. Paired specimens were available from eight acutely-ill dogs. One animal was seronegative while five dogs showed evidence of seroconversion. An IgM-ELISA titre of > or = 320 was used to confirm current infection in eight of these nine animals. Previous studies in Barbados showed a higher prevalence of serogroup Icterohaemorrhagiae than of Autumnalis, but the relative frequency of these two serogroups may be changing. The high seroprevalence in dogs is of public health concern because the close contact between dogs and man may provide the link between a reservoir in the environment and susceptible humans.

Evaluation of the polymerase chain reaction for early diagnosis of leptospirosis.

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 in Madras--a clinical and serological study.

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.

A twelve-year study of leptospirosis on Barbados.

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.

Detection of seven species of pathogenic leptospires by PCR using two sets of primers.

Two sets of primers derived from genomic DNA libraries of Leptospira serovars icterohaemorrhagiae (strain RGA) and bim (strain 1051) enabled the amplification by PCR of target DNA fragments from leptospiral reference strains belonging to all presently described pathogenic Leptospira species. The icterohaemorrhagiae-derived primers (G1/G2) enabled amplification of DNA from L. interrogans, L. borgpetersenii, L. weilii, L. noguchii, L. santarosai and L. meyeri, whereas the bim-derived primers (B64-I/B64-II) enabled the amplification of L. kirschneri. Southern blot and DNA sequence analysis revealed inter-species DNA polymorphism within the region spanned by primers G1 and G2 between L. interrogans and various other Leptospira species. Using a mixture of primer sets G1/G2 and B64-I/B64-II, leptospires of serovars icterohaemorrhagiae, copenhageni, hardjo, pomona, grippotyphosa and bim were detected in serum samples collected from patients during the first 10 days after the onset of illness.

Prevalence of leptospiral agglutinins among conservancy workers in Madras City, India.

In a study of 584 Corporation conservancy (sanitation) workers who lived mostly in slums, and who worked in four Corporation Circles of Madras City, India, 192 (32.9%) were found to be positive for agglutinins to Leptospira interrogans. Seropositivity prevalence increased with age, but was similar in males and females except in the youngest age group, where males predominated. Prevalence in the four study areas ranged between 17.8 and 40.5% (P < 0.01). Among 152 sera in which one serogroup predominated, Autumnalis was the most commonly recorded (33.6%), followed by Icterohaemorrhagiae (15.1%), Panama (15.1%), Sejroe (14.5%) and others (21.7%). Forty sera reacted to two or more serogroups at the same (highest) titre, most frequently to the first three serogroups above. The titre range was 1:50-1:3200 (geometric mean titre 149). Among a group of 46 male automobile industry workers who lived in middle-class housing, seropositivity prevalence (17.4%) was approximately half that of the sanitation workers (P < 0.05), and the titre range was lower (1:50-1:200, GMT 84). The predominating serogroups were those found in the sanitation workers. Bearing in mind that sanitation workers are the urban group probably at highest risk of leptospiral infection, the prevalence rate (< 33%) found in our study is not considered to be particularly high.

Mongoose rabies in the Caribbean.

Mongooses (Herpestes auropunctatus) have been introduced into most of the larger Caribbean islands, some notable exceptions being Dominica, Tobago, and Montserrat. Rabies in Caribbean mongooses is present in Puerto Rico, Cuba, the Dominican Republic (and presumably Haiti), and Grenada. Bat rabies is known on Cuba, Grenada, and Trinidad, although mongooses found on Trinidad are free of the disease. None of the other islands is known to have rabies, although it could be present in sequestered bat populations. All reported case numbers of mongoose rabies in the Caribbean are underestimates, and available information is at best incomplete and at times fragmentary. Nevertheless, data are presented from the four affected islands. Mongoose reduction campaigns have been undertaken on Cuba and Grenada. In Cuba strychnine sulfate inoculated into labeled eggs is used, whereas in Grenada sodium fluoroacetate (1080) has been used in boiled cowhide baits. Mongoose poisoning is unsatisfactory and ineffective in the long-term. Because many mongooses naturally exposed to rabies virus develop serum neutralizing antibodies and are considered to be immunized, possibly for life, vaccination in the wild has been under consideration since the mid-1970s. Early attempts to produce a pill coated with ERA vaccine for enteric absorption in mongooses were not very successful, but new modified vaccines and recombinant techniques hold considerable promise.

An investigation of some risk factors for severe leptospirosis on Barbados.

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.

Leptospires isolated from toads and frogs on the Island of Barbados.

Four pathogenic strains of leptospires were isolated from the kidneys of toads (Bufo marinus) and seven from frogs (Eleutherodactylus johnstonei). Isolates from two toads and one frog belonged to serovar bim, the causative agent of most cases of severe leptospirosis on Barbados. The other eight strains belonged to a new serovar within the Australis serogroup. The name bajan is proposed for this new serovar of Leptospira interrogans.

Antigens recognized by the human immune response to severe leptospirosis in Barbados.

Serum samples obtained from patients hospitalized in Barbados with severe leptospirosis were tested by the microscopic agglutination test (MAT), enzyme immunoassay (EIA) and immunoblotting with leptospires that had been isolated from these patients. While serum samples taken a few days after onset of symptoms often showed no apparent correlation between MAT and EIA, later sequential serum samples produced similar profiles in both tests during the course of infection. Immunoblotting sonicate from Leptospira interrogans serovars arborea, copenhageni and bim with patients' sera, revealed reactions with a number of bands that corresponded with outer envelope components. These components included lipopolysaccharide (LPS), flagella and other outer membrane proteins, in addition to a low-molecular-weight (MW) carbohydrate cross-reactive with members of the Leptospiraceae. IgM antibodies elicited in the first to second week after infection reacted mainly with LPS and the low-MW cross-reactive carbohydrate. Comparative analysis of isolates of the same serovar by sodium dodecyl sulphate polyacrylamide gel electrophoresis and immunoblotting showed that while two serovar arborea isolates were identical, serovar bim isolates differed significantly from each other. This difference was also observed in comparative MAT testing.

Retention of leptospiral agglutinins and long-term response to administration of monoclonal antibodies in vervet monkeys (Cercopithecus aethiops) on Barbados.

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.

Absence of epidemicity of severe leptospirosis in Barbados.

The possibility of micro-epidemics of severe leptospirosis occurring on the island of Barbados was investigated by examining the space-time clustering of the disease in 212 laboratory-confirmed cases admitted to Queen Elizabeth Hospital, Bridgetown, over a 7-year period. A series of 109 patients with symptoms compatible with leptospirosis but shown to be otherwise by laboratory examination were also examined for comparison. No significant space-time clustering was found among the leptospirosis cases, indicating no evidence for micro-epidemics. By comparison, statistically significant clustering was apparent among the smaller non-leptospirosis series. Possible explanations for the absence of observed micro-epidemics of leptospirosis are discussed.

Leptospiral antibodies in patients from a Barbadian general practice.

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)

Leptospiral infection: a household serosurvey in urban and rural communities in Barbados and Trinidad.

A longitudinal study of leptospiral agglutinins in subjects five years of age and over was undertaken in Trinidad and Barbados between 1980 and 1982. Households were sampled randomly from one urban and two rural communities on each island, giving a total of 576 eligible individuals in Barbados and 524 in Trinidad. Participants were examined three times at approximately annual intervals. The prevalence of seropositivity at a titre of 1:50 using the microscopic agglutination test was 18.5% in Barbados and 21.9% in Trinidad. Prevalence increased steeply with age in both sexes and was higher in males than females on both islands. There was a marked difference in predominating serogroups on the two islands--Autumnalis (42% of positive cases) predominated in Barbados while Bataviae (29% of positive cases) predominated in Trinidad. Estimates of incidence rates for seroconversion were 2.9% per annum for Barbados and 3.5% per annum for Trinidad. Occupational risk varied between the islands, but in both cases highest seropositivity rates (greater than 50%) were found in outdoor labourers and lowest were found in indoor non-manual workers and urban homeworkers. In Barbados seroprevalence was higher among persons who cleared drains or who had contact with livestock. Lack of an inside toilet was associated with an increase in seropositivity on both islands. There was little evidence of household clustering of seropositive cases.

Leptospires in the whistling frog (Eleutherodactylus johnstonei) on Barbados.

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.

Leptospirosis in Barbados. A clinical study.

A 39-month clinical study of leptospirosis was undertaken at the Queen Elizabeth Hospital, Barbados. Eighty-eight patients had a confirmed diagnosis of the disease during the period. The major serogroups identified were autumnalis (including a new serovar bim), icterohaemorrhagiae, ballum and canicola. The majority of patients presented with jaundice (95%,) anorexia and headaches (85%), fever (76%) and conjunctival suffusion (54%). While abnormal creatinine levels were seen in 49% of patients on admission, only 16% were judged to have had renal failure. The urine to plasma urea ratio showed high sensitivity and specificity in the diagnosis of pre-renal azotemia. Cardiac arrhythmias and myocarditis occurred in 18% of patients and pericarditis in 6%. An elevated serum amylase was found in 65% of cases. The bilirubin level took 5.5 weeks to return to normal. Thrombocytopenia was shown not to be due to a disseminated intravascular coagulation, and a randomised trial of high dose penicillin did not reveal any benefit to jaundiced patients. The overall mortality during the study was 5.7%.