The hanta hunting study: underdiagnosis of Puumala hantavirus infections in symptomatic non-travelling leptospirosis-suspected patients in the Netherlands, in 2010 and April to November 2011.

Leptospirosis and haemorrhagic fever with renal syndrome (HFRS) are hard to distinguish clinically since these two important rodent-borne zoonoses share hallmark symptoms such as renal failure and haemorrhage. Leptospirosis is caused by infection with a spirochete while HFRS is the result of an infection with certain hantaviruses. Both diseases are relatively rare in the Netherlands. Increased incidence of HFRS has been observed since 2007 in countries that border the Netherlands. Since a similar rise in incidence has not been registered in the Netherlands, we hypothesise that due to overlapping clinical manifestations, hantavirus infections may be confused with leptospirosis, leading to underdiagnosis. Therefore, we tested a cohort of non-travelling Dutch patients with symptoms compatible with leptospirosis, but with a negative diagnosis, during 2010 and from April to November 2011. Sera were screened with pan-hantavirus IgG and IgM enzyme-linked immunosorbent assays (ELISAs). Sera with IgM reactivity were tested by immunofluorescence assay (IFA). ELISA (IgM positive) and IFA results were confirmed using focus reduction neutralisation tests (FRNTs). We found hantavirus-specific IgG and/or IgM antibodies in 4.3% (11/255) of samples taken in 2010 and in 4.1% (6/146) of the samples during the 2011 period. After FRNT confirmation, seven patients were classed as having acute Puumala virus infections. A review of hantavirus diagnostic requests revealed that at least three of the seven confirmed acute cases as well as seven probable acute cases of hantavirus infection were missed in the Netherlands during the study period.

Coagulation disorders in patients with severe leptospirosis are associated with severe bleeding and mortality.

OBJECTIVE: To determine the involvement of coagulation in bleeding and poor outcome in patients with severe leptospirosis. METHODS: In a prospective study, parameters of the coagulation system were measured on admission and during follow-up in 52 consecutive patients with severe leptospirosis. RESULTS: All patients showed coagulation disorders, such as prolonged prothrombin time (PT) and activated partial thromboplastin time, marked procoagulant activity [thrombin-antithrombin (TAT) complexes, prothrombin fragment 1+2, D-dimer], reduced levels of anticoagulant markers (protein C, antithrombin) and increased (anti-) fibrinolytic activity [plasmin-antiplasmin (PAP) complexes, plasminogen activator inhibitor-1]. These disorders were more pronounced in patients who died eventually. PT prolongation was associated with mortality (OR 1.4, 95% CI: 1.0-1.8, P = 0.04). Bleeding occurred in 31 subjects (60%). Of these, 24 had mild bleeding and seven had severe haemorrhages. Thrombocytopenia (platelets

An outbreak of leptospirosis in seals (Phoca vitulina) in captivity.

An outbreak of leptospirosis in seals (Phoca vitulina) in captivity is described. In a zoo in The Netherlands 5 adult seals died within 12 days. At necropsy all animals showed signs of acute septicaemia, consistent with acute leptospirosis. Serological examination of one animal was positive for antibodies against Leptospira interrogans serovar Icterohaemorrhagiae and the serologically closely related serovar Copenhageni. Polymerase chain reaction was positive in one other animal. 8 nutria (Myocastor coypus) were examined, serologically, through bacteriological culture and PCR. 81,8% (9/11) were serologically positive for Leptospira. The seals and nutria were housed in the same water system.

Preliminary Investigations on the Distribution of Leptospira Serovars in Domestic Animals in North-west Morocco.

Leptospirosis is a neglected zoonosis of global importance with a complex epidemiology that affects humans, domestic and wild mammals. However, due to the diversity of clinical signs and difficulties of establishing a confirmatory laboratory diagnosis, the disease remains poorly investigated, particularly in the developing world. In Morocco, a descriptive study of the seroprevalence of Leptospira infection in animals has never been undertaken. To fill this gap, the current study was conducted on a subset of animals in north-west Morocco as a preliminary step towards understanding the epidemiological patterns of animal leptospirosis in the country. The study was conducted on 289 serum samples collected between January and April 2012 from dogs, cattle, sheep, goats and donkeys in the areas of Rabat-Temara, Sidi Kacem and Oulmes. All serum samples were tested by the MAT with 14 reference strains of the most prevalent pathogenic serovars of Leptospira and two serovars of non-pathogenic Leptospira. The overall seroprevalence of Leptospira in cattle, sheep, goats, dogs and donkeys was 15%, 18%, 20%, 21% and 20%, respectively. The most prevalent serogroups found in each species were Ballum, Sejroe, and Australis in cattle, Ballum, Australis and Sejroe in sheep, Australis and Ballum in goats, Javanica and Australis in donkey and Australis, Ballum and Canicola in dogs. Of all the serogroups tested in this study, Icterohaemorrhagiae, the only serogroup which has been previously reported in humans in Morocco, was rarely reactive. The majority of reactive sera were collected from low land areas. A large number of sera samples classified as seronegative when tested against pathogenic leptospires were positive when tested against non-pathogenic leptospires; this is suggestive of possible novel, as yet unclassified, Leptospira serovars in Morocco. Eleven of thirteen sheep urine samples were positive by real-time PCR confirming their role as Leptospira carriers in Morocco.

Serological and molecular characterization of leptospira serovar Kenya from captive African giant pouched rats (Cricetomys gambianus) from Morogoro Tanzania.

Two identical leptospiral isolates coded Sh9 and Sh25 obtained from the urine of captive African giant pouched rats (Cricetomys gambianus), destined for use as biodetector of antipersonnel landmines were typed as serovar Kenya using cross-agglutination absorption test and DNA fingerprinting with the insertion element sequences IS1533 and IS1500 derived primers. The two isolates were previously characterized using cultural and serological-microagglutination test as pathogenic leptospires of the serogroup Ballum, closely related to serovars Kenya and Peru. To our knowledge, this is the first reported in-depth characterization of leptospira isolates from Tanzania.

New serovars of Leptospira isolated from patients in Costa Rica: implications for public health.

Leptospira strains JICH 05 and INCIENSA 04 were isolated from hospitalized leptospirosis patients in the province of Puntarenas, Costa Rica. The isolates produced agglutination titres notably against members of serogroups Pyrogenes and Tarassovi, respectively, but appeared serologically unique in the cross agglutinin absorption test (CAAT). Therefore, JICH 05 and INCIENSA 04 were considered to represent two new serovars, designated Corredores and Costa Rica of the serogroups Pyrogenes and Tarassovi, respectively. Multilocus sequence genotyping revealed that both strain INCIENSA 04 and strain JICH 05 belong to Leptospira santarosai. These two new serovars are in addition to various other recently identified highly virulent serovars, including the new L. santarosai, serovar Arenal. Considering the fact that isolation and typing of leptospires from patients has only recently been introduced in Costa Rica, these findings suggest that various known and unknown virulent serovars of Leptospira are circulating in this country and probably beyond, thus posing a severe threat to public and probably veterinary health in the region.

What role do coagulation disorders play in the pathogenesis of leptospirosis?

Leptospirosis is a zoonosis of worldwide distribution, spread by the urine of infected animals. It is a major public health problem, especially in developing countries, where circumstances for transmission are most favourable. The clinical picture varies from mild disease to a severe illness with haemostatic derangements and multiorgan failure eventually leading to death. Although the haemorrhagic complications of severe disease are serious, the pathophysiology is scarcely elucidated. The complex mechanisms involved in inflammation-induced coagulation activation are extensively studied in various infectious diseases, i.e. Gram-negative sepsis. Tissue factor-mediated coagulation activation, impairment of anticoagulant and fibrinolytic pathways in close concert with the cytokine network are thought to be important. But for human leptospirosis, data are limited. Because of the growing interest in this field, the impact of leptospirosis, and the availability of new therapeutic strategies, we reviewed the evidence regarding the role of coagulation in leptospirosis and provide suggestions for future research.

New Leptospira serovar Sokoine of serogroup Icterohaemorrhagiae from cattle in Tanzania.

The prevalence of leptospirosis is generally high in domestic animals and rodents in Tanzania. Identification of Leptospira isolates from cattle was carried out to establish prevalent Leptospira serovars. Serological typing was done based on monoclonal antibodies and the standard cross-agglutination absorption test. Molecular typing involved pathogenic- and saprophytic-specific PCRs and a PCR specifically amplifying DNA from the species Leptospira kirschneri. DNA fingerprinting with primers derived from sequences of insertion elements IS1500 and IS1533 was carried out. Both serological and molecular characterization indicated that one of the Leptospira isolates, coded RM1, represents a new serovar of the species L. kirschneri of serogroup Icterohaemorrhagiae. The serovar name Sokoine is proposed for this new Leptospira isolate.

Simple latex agglutination assay for rapid serodiagnosis of human leptospirosis.

A newly developed latex agglutination assay for the detection of genus-specific Leptospira antibodies in human sera was evaluated. The assay is performed by mixing, on an agglutination card, serum with equal volumes of stabilized antigen-coated, dyed test and control latex beads and is read within 2 min. The latex agglutination test was evaluated with groups of serum samples from patients with leptospirosis and control patients from Hawaii, the Seychelles, Thailand, and The Netherlands. The mean overall sensitivity was 82.3%, and the mean overall specificity was 94.6%. The assay is easy to perform and does not require special skills or equipment. The reagents have a long shelf life, even at tropical temperatures. Together, these factors make the assay suitable for use even at the peripheral level of a health care system as a rapid screening test for leptospirosis.

Leish-KIT, a stable direct agglutination test based on freeze-dried antigen for serodiagnosis of visceral leishmaniasis.

In order to increase the application potential of the direct agglutination test (DAT) for the detection of anti-Leishmania antibodies in human serum samples, we developed an antigen based on stained and freeze-dried Leishmania donovani promastigotes. We describe here the evaluation of the performance of the DAT based on this freeze-dried antigen. It was shown that the freeze-dried antigen remains fully active, even after storage at 56 degrees C for 18 months. With a cutoff value of 1:1,600, the sensitivity of the DAT was shown to be 92% and the specificity of the test was 99.7%, which were comparable with the results found for the DAT based on liquid antigen. The major advantages of the freeze-dried antigen are that the production of a large batch of this antigen allows reproducible results in the DAT over a long period of time and that the freeze-dried antigen can be stored at ambient temperature, which, as was shown, makes the test a valuable diagnostic tool for use in the field.

LEPTO dipstick, a dipstick assay for detection of Leptospira-specific immunoglobulin M antibodies in human sera.

We studied a dipstick assay for the detection of Leptospira-specific immunoglobulin M (IgM) antibodies in human serum samples. A high degree of concordance was observed between the results of the dipstick assay and an IgM enzyme-linked immunosorbent assay (ELISA). Application of the dipstick assay for the detection of acute leptospirosis enabled the accurate identification, early in the disease, of a high proportion of the cases of leptospirosis. Analysis of a second serum sample is recommended, in order to determine seroconversion or increased staining intensity. All serum samples from the patients who were confirmed to be positive for leptospirosis by either a positive microscopic agglutination test or a positive culture but were found to be negative by the dipstick assay were also judged to be negative by the IgM ELISA or revealed borderline titers by the IgM ELISA. Some cross-reactivity was observed for sera from patients with diseases other than leptospirosis, and this should be taken into account in the interpretation of test results. The dipstick assay is easy to perform, can be performed quickly, and requires no electricity or special equipment, and the assay components, a dipstick and a staining reagent, can be stored for a prolonged period without a loss of reactivity, even at elevated temperatures.

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.

Development and evaluation of a rapid dipstick assay for serodiagnosis of acute human brucellosis.

A dipstick assay for the detection of brucella-specific immunoglobulin M antibodies was evaluated with 707 sera from 247 laboratory-confirmed brucellosis patients and 342 control sera from brucellosis-free individuals. These sera were collected from six different countries. The assay was found to be highly sensitive and specific. In addition, the test is easy to use and does not require specialized training or equipment, and the components are stable without a requirement for refrigeration. All of these factors make the test ideal for developing countries and rural settings.

Latex based, rapid and easy assay for human leptospirosis in a single test format.

Leptospirosis is an often severe disease which requires prompt treatment. Laboratory testing is required to reach a valid diagnosis. An agglutination assay for the detection of Leptospira-specific antibodies consisting of individually wrapped agglutination cards containing a stable, dried detection reagent is evaluated. The assay is simply performed by suspending the dried reagent with a drop of serum. The result is obtained within 30 s. The sensitivity of the assay varied with the stage of the disease and was 72.3% for samples collected during the first 10 days of the illness and 88.2% for samples collected at a later stage. The specificity was 93.9% and 89.8%, respectively. These characteristics make the test ideal for use in areas where the disease is common and where laboratory support is not routinely available.