Application of a real time Polymerase Chain Reaction (PCR) assay for the early diagnosis of human leptospirosis in Sri Lanka.

Leptospirosis has a major impact on health in Sri Lanka but is probably grossly under-recognized due to difficulties in clinical diagnosis and lack of diagnostic laboratory services. The objective of this study was to establish and evaluate a SYBR Green-based real-time Polymerase Chain Reaction (rt-PCR) assay for early, rapid and definitive laboratory diagnosis of leptospirosis in Sri Lanka. The rt-PCR assay was established and analytical specificity and sensitivity were determined using reference DNA samples. Evaluation of the assay for diagnosis of clinical samples was performed using two panels of serum samples obtained from 111 clinically suspected adult patients. Patients were confirmed as leptospirosis (n = 65) and non-leptospirosis (n = 30) by the Patoc - MAT. Other 16 samples gave ambiguous results. The analytical sensitivity of the rt-PCR was approximately 60 genome copies and no cross-reactivity was observed with saprophytic Leptospira spp. and other pathogenic microorganisms. Based on confirmation with Patoc-MAT on paired samples this corresponds to a diagnostic sensitivity and specificity of 67.7% (44/65) and 90.0% (27/30), respectively. This study showed that rt-PCR has the potential to facilitate rapid and definitive diagnosis of leptospirosis during early phase of infection in Sri Lanka.

'Leptorapide' - a one-step assay for rapid diagnosis of human leptospirosis.

SUMMARY: Leptospirosis is a globally important zoonotic infection caused by spirochaetes of the genus Leptospira. It is transmitted to humans by direct contact with infected animals or indirectly via contaminated water. It is mainly a problem of the resource-poor developing countries of the tropical and sub-tropical regions of the world but outbreaks due to an increase in travel and recreational activities have been reported in developed and more industrialized areas of the world. Current methods of diagnosis are costly, time-consuming and require the use of specialized laboratory equipment and personnel. The purpose of this paper is to report the validation of the 'Leptorapide®' test (Linnodee Ltd, Northern Ireland) for the diagnosis of human leptospirosis. It is a simple one-step latex agglutination assay performed using equal volumes of serum sample and antigen-bound latex beads. Evidence of leptospiral antibodies is determined within minutes. Agglutination is scored on a scale of 1-5 and the results interpreted using a score card provided with the kit. Validation has been performed with a large sample size obtained from individuals originating from various parts of the world including Brazil and India. The test has shown sensitivity and specificity values of 97·1% and 94·0%, respectively, relative to the microscopic agglutination test. The results demonstrate that Leptorapide offers a cost-effective and accurate alternative to the more historical methods of antibody detection.

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.

Classification of Leptospira genomospecies 1, 3, 4 and 5 as Leptospira alstonii sp. nov., Leptospira vanthielii sp. nov., Leptospira terpstrae sp. nov. and Leptospira yanagawae sp. nov., respectively.

The genus Leptospira currently comprises 16 named species. In addition, four unnamed hybridization groups were designated Leptospira genomospecies 1, 3, 4 and 5. These groups represent valid species-level taxa, but were not assigned names in the original description by Brenner et al. [Int J Syst Bacteriol 49, 839-858 (1999)]. To rectify this situation, it is proposed that Leptospira genomospecies 1, genomospecies 3, genomospecies 4 and genomospecies 5 should be classified as Leptospira alstonii sp. nov., Leptospira vanthielii sp. nov., Leptospira terpstrae sp. nov. and Leptospira yanagawae sp. nov., respectively, with strains L. alstonii 79601(T) ( = ATCC BAA-2439(T)), L. vanthielii WaZ Holland(T) ( = ATCC 700522(T)), L. terpstrae LT 11-33(T) ( = ATCC 700639(T)) and L. yanagawae Sao Paulo(T) ( = ATCC 700523(T)) as the type strains. The type strains are also available from the culture collections of the WHO Collaborating Centres in Amsterdam, The Netherlands, and Brisbane, Australia.

Human leptospira isolates circulating in Mayotte (Indian Ocean) have unique serological and molecular features.

Leptospirosis is one of the most widespread zoonoses in the world. However, there is a lack of information on circulating Leptospira strains in remote parts of the world. We describe the serological and molecular features of leptospires isolated from 94 leptospirosis patients in Mayotte, a French department located in the Comoros archipelago, between 2007 and 2010. Multilocus sequence typing identified these isolates as Leptospira interrogans, L. kirschneri, L. borgpetersenii, and members of a previously undefined phylogenetic group. This group, consisting of 15 strains, could represent a novel species. Serological typing revealed that 70% of the isolates belonged to the serogroup complex Mini/Sejroe/Hebdomadis, followed by the serogroups Pyrogenes, Grippotyphosa, and Pomona. However, unambiguous typing at the serovar level was not possible for most of the strains because the isolate could belong to more than one serovar or because serovar and species did not match the original classification. Our results indicate that the serovar and genotype distribution in Mayotte differs from what is observed in other regions, thus suggesting a high degree of diversity of circulating isolates worldwide. These results are essential for the improvement of current diagnostic tools and provide a starting point for a better understanding of the epidemiology of leptospirosis in this area of endemicity.

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

[Post-mortem diagnosis of leptospirosis in two dogs]. / Twee postmortaal vastgestelde gevallen van leptospirose bij de hond.

Leptospirosis was diagnosed post-mortem in a 2-year-old male Dogo Argentino and a 7-week-old male Foxhound puppy. The two cases were unrelated. Clinical symptoms were mainly confined to the gastro-intestinal tract. Pathological lesions were suggestive of acute leptospirosis. Leptospires infection was confirmed by serological (indirect IgM/Ig6 ELISA and MAT) and immunohistochemical techniques.

Identification of cattle, buffaloes and rodents as reservoir animals of Leptospira in the District of Gampaha, Sri Lanka.

BACKGROUND: Leptospirosis is an important emerging infectious disease in Sri Lanka. Rats are the most important reservoir of Leptospira but domestic and wild mammals may also act as important maintenance or accidental hosts. In Sri Lanka, knowledge of reservoir animals of leptospires is poor. The objective of this study was to identify potential reservoir animals of Leptospira in the District of Gampaha, Sri Lanka. FINDINGS: Blood and kidney samples were collected from 38 rodents and mid-stream urine samples were randomly collected from 45 cattle and five buffaloes in the District of Gampaha. Kidney and urine samples were tested by real-time polymerase chain reaction (PCR) and serum samples were tested by the microscopic agglutination test (MAT). Of the 38 rodent kidney samples, 11% (4/38) were positive by real-time PCR. The prevalence of leptospiral carriage was 11% (3/26) and 8% (1/12) in female and male rodents, respectively. Three rodent serum samples were positive by MAT. Of the 50 cattle/buffalo urine samples tested, 10% (5/50) were positive by real-time PCR. The prevalence of leptospiral carriage was 9% (4/45) and 20% (1/5) in cattle and buffaloes, respectively. CONCLUSION: Results of PCR and MAT showed that Leptospira were present in a significant proportion of the rodents and farm animals tested in this study and suggest that these (semi-) domestic animals form an infection reservoir for Leptospira. Therefore, there is a potential zoonotic risk to public health, most notably to farmers in this area.

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.

Diagnosis of infections caused by Enterocytozoon bieneusi and Encephalitozoon intestinalis using polymerase chain reaction in stool specimens.

OBJECTIVE: To study the usefulness of polymerase chain reaction (PCR) for the species identification of microsporidia in stool specimens obtained from HIV-infected patients with Enterocytozoon bieneusi or Encephalitozoon intestinalis infections. SETTING: Infectious disease clinic in a university hospital. PATIENTS: Thirty-seven stool specimens from 29 HIV-infected patients with microsporidiosis were tested. The diagnosis of microsporidian infection was made by light microscopy of stool specimens and species identification was made by transmission electron microscopy of duodenal biopsies. Sixty-one stool specimens from 45 HIV-infected patients without microsporidiosis served as controls. METHODS: PCR was performed using DNA extracted from stools with two primers sets, one specific for E. bieneusi and one specific for E. intestinalis. RESULTS: A 1265 base-pair fragment of the small subunit ribosomal RNA (rrs) gene could be amplified from all 31 stool specimens infected with E. bieneusi. In addition, a 930 base-pair fragment of the rrs gene could be amplified from all six stool specimens infected with E. intestinalis. The 61 control stools were negative with both primers. CONCLUSIONS: These results suggest that a PCR based assay using species-specific primers sets can be used successfully for microsporidian species differentiation from stool specimens, thus obviating the need for invasive biopsy procedures.

Effect of selective chemical modification and CNBr-cleavage at methionine20 in catalytic activity and substrate binding properties of porcine pancreatic phospholipase A2.

Porcine pancreatic phospholipase A2 contains 2 methionine (Met) residues located at positions 8 and 20, respectively. Reaction of the enzyme with methyliodide and iodoacetic acid resulted in the selective methylation and carboxymethylation, respectively, of Met20. It was found that porcine pancreatic iso-phospholipase A2, possessing only Met8, was not affected by either modification. Reaction of porcine phospholipase A2 with cyanogen bromide in 0.1 N hydrochloric acid gave rise to cleavage only at Met20. The enhanced reactivity of Met20 compared to that of Met8 is in agreement with the known X-ray structure of phospholipase A2 which shows that Met8 is located in the interior of the protein, while Met20 is at the surface. Both methylation and carboxymethylation of Met20 do not significantly affect catalytic and substrate binding properties of the enzyme. In contrast, the more rigorous cleavage at Met20 by CNBr resulted in the loss of catalytic activity, while substrate and Ca2+ binding was diminished only to a limited extent. Most likely CNBr cleavage at Met20 perturbs the active site despite the fact that the N-terminal fragment Ala1-Hse20 is still bound via the disulfide bridge Cys11-Cys77 to the remainder of the protein. The results obtained strongly suggest that the conformation of the sequences Ala1-Hse20 and/or Asp21-Gly26 are important for the maintenance of the special microenvironment of the active site cleft.

Use of nondenaturing silver-stained polyacrylamide gel analysis of polymerase chain reaction amplification products for the differential diagnosis of Leptospira interrogans infection.

A 285-bp DNA fragment was amplified using the polymerase chain reaction from 38 Leptospira serovars of six different genomic species. The fragments amplified exhibited differential mobilities on nondenaturing polyacrylamide gels resulting from sequence-dependent conformational alterations. Leptospira interrogans serovars could be distinguished from those of other species on this basis.

Characterization of the Leptospira interrogans S10-spc-alpha operon.

A ribosomal protein gene cluster from the spirochaete Leptospira interrogans was characterized. This locus is homologous to the Escherichia coli S10, spc, and alpha operons. Analysis of L. interrogans RNA showed that the ribosomal protein genes within this cluster are co-transcribed, thus forming an operon. Two transcription initiation sites were mapped by primer extension, upstream of fus, the first gene in this cluster, and sequences from this region provided promoter activity in E. coli. Transcription terminates near a predicted stem-loop structure following rplQ, the last gene in the cluster. These data suggest that two promoters upstream of fus direct transcription of this 17.5-kb ribosomal protein gene cluster. Comparison of the L. interrogans S10-spc-alpha cluster to homologous loci from Borrelia burgdorferi and Treponema pallidum provided evidence that this region of the genome underwent several rearrangements during spirochaete evolution.

Leptospira interrogans serovar Valbuzzi: a cause of severe pulmonary haemorrhages in the Andaman Islands.

Outbreaks of leptospirosis that present with predominant pulmonary signs and symptoms have been occurring in the Andaman Islands since the late 1980s. Before this, pulmonary haemorrhage had not been observed as a common complication of leptospirosis in India. During an outbreak on North Andaman in 1997, four leptospire isolates were obtained from blood of a fatal case and three other patients who recovered. These isolates were characterized using serological and molecular techniques. Cross-agglutination absorption tests and microscopic agglutination tests using mAbs were used for serological characterization. Genetic typing was done using DNA sequencing of PCR products. Serologically, the isolates were closely related to strain Valbuzzi serovar Valbuzzi of serogroup Grippotyphosa. The sequences of PCR products from these isolates were compared with those of 45 strains belonging to seven species. The isolates showed 97.5-100 % sequence similarity to reference strains belonging to Leptospira interrogans, indicating that the isolates belong to L. interrogans. Serogroups Icterohaemorrhagiae and Australis have been incriminated as the cause of pulmonary haemorrhage in China, Korea and Australia. The four isolates characterized in the present study were obtained from patients with similar symptoms. However, they belonged to serovar Valbuzzi of serogroup Grippotyphosa, indicating that serogroups other than Icterohaemorrhagiae and Australis can also cause pulmonary haemorrhage.

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.

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.

Lai-like leptospira from the Andaman Islands.

BACKGROUND & OBJECTIVES: Leptospirosis has been an important public health problem in the Andaman Islands since 1988. As information about the exact etiological agent is not available, the present study was undertaken to isolate and identify Leptospira from human patients. METHODS: An isolate coded AF61 was recovered from the blood of a patient clinically suspected to have leptospirosis, with fever, headache and body aches as the main symptoms. The isolation was done using Ellinghausen-McCullough-Johnson-Harris (EMJH) medium following standard procedure. The isolate was identified using microscopic agglutination test (MAT) with 'groupsera', cross agglutination absorption test (CAAT) and monoclonal antibodies. RESULTS: Agglutination tests with rabbit antisera revealed that the isolate belonged to the serogroup icterohaemorrhagiae. The CAAT results showed that it was closely related to the serovar lai. Analysis of AF61 with monoclonal antibodies confirms our observation with CAAT that it is closely related to the reference strain Lai serovar lai. INTERPRETATION & CONCLUSIONS: Serovar lai, has been associated with pulmonary haemorrhage in China and Korea. However, the strain AF61 was not isolated from a patient with pulmonary symptoms. Further studies are needed to understand the possible relationship between serovars and clinical patterns and the distribution of serovar lai and lai-like strains in Asia.

[Leptospirosis in the Netherlands, 1991-1995]. / Leptospirose in Nederland 1991-1995.

OBJECTIVE: To determine all cases of leptospirosis in the Netherlands in 1991-1995 that were confirmed by serological investigation or culturing. DESIGN: Descriptive, retrospective. SETTING: Department of Biomedical Research, Royal Tropical Institute, Amsterdam, the Netherlands, and Department of Internal Medicine, Academic Medical Centre, Amsterdam, the Netherlands. METHOD: Using data of the Reference Laboratory for Leptospirosis, the number of leptospirosis cases in 1991-1995 was determined and compared with the data of 1986-1990. Additional information was obtained about patients with confirmed leptospirosis or who had been hospitalized because of leptospirosis. RESULTS: The number of confirmed cases dropped from 229 in 1986-1990 to 159 in 1991-1995. This decrease could be attributed mainly to a marked decrease of the number of dairy farm fever (hardjo) cases. There was a clear increase of the number of infections acquired during travel in foreign countries, notably outside Europe. Thus, leptospirosis in the Netherlands shifted from an occupational disease towards a disease due to recreational activities. In about 10% of the patients the disease ran a severe course with Weil's syndrome (icterus, renal failure, and haemorrhages). Eight patients (5%) died. Clinical data of 5 of these 8 patients indicated that they had suffered from Weil's syndrome.