A unique strain of Leptospira isolated from a patient with pulmonary haemorrhages in the Andaman Islands: a proposal of serovar portblairi of serogroup Sehgali.

Leptospirosis is endemic in the Andaman Islands, often occurring as outbreaks during the post-monsoon period. Pulmonary involvement is common and associated with high morbidity and mortality. During the investigation of an outbreak in North Andaman in 1996 an isolate was recovered from the blood of a patient with fever, headache, body aches and haemoptysis with respiratory distress as presenting symptoms. The isolate was characterized using the cross-agglutination absorption test (CAAT) and monoclonal antibodies (mAbs). The isolate showed typical morphology and characteristic motility of the genus Leptospira. Growth was inhibited at 13 degrees C and in the presence of 8-azaguanine. The isolate could not be identified with grouping sera representing 25 serogroups, CAAT and mAbs. A new serovar of a new serogroup is proposed. Genetic characterization using polymerase chain reaction (PCR) followed by sequencing of the PCR product and randomly amplified polymorphic DNA fingerprinting (RAPD) showed that the isolate was genetically similar to L. interrogans sensu stricto.

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.

Analysis of Leptospira isolates from mainland Portugal and the Azores islands.

From 228 recent Leptospira isolates from mainland Portugal and Azorean wild mammals, 149 were characterized at the serovar level by monoclonal antibodies (MAbs), a quick serological method in epidemiological studies. In order to compare this antigenic information with that from new genetic techniques, a sample of isolates was analyzed through pulsed-field agarose gel electrophoresis (PFGE) (n=71), mapped restriction site polymorphisms (MRSPs) in PCR-amplified rRNA genes (n=45, including 13 saprophytes) and arbitrarily primed polymerase chain reaction (AP-PCR) fingerprinting (n=32). MRSP and AP-PCR lead to species identification of the studied 32 pathogenic isolates: Leptospira interrogans (n=3), Leptospira kirschneri (n=8) and Leptospira borgpetersenii (n=21). MAbs and PFGE characterized pathogenic isolates at the serovar level and resulted mainly in agreement (64%) although many discrepancies (35%) were observed.

International multi-centre evaluation of a dipstick assay for human leptospirosis.

A dipstick assay for the detection of Leptospira-specific immunoglobulin M (IgM) antibodies in human sera was evaluated in 27 laboratories in 23 countries. 873 serum samples from 711 patients including 329 laboratory-confirmed leptospirosis case patients, 239 noncase patients and 69 patients with viral infections causing heamorrhagic fever were tested. Relative to the results of the reference leptospirosis test, the sensitivity of the dipstick assay was 84.5% for serum samples collected during the first 10 days of the disease and 92.1% for serum samples collected 10-30 days after the onset of disease. The specificity was 87.5% and 94.4%, respectively. Similar to viral haemorrhagic fevers, leptospirosis may cause bleeding. A small number of serum samples from patients with haemorrhagic viral infections gave a weak (1 +) stain. All other samples were negative. In conclusion, the dipstick assay is sensitive and specific and reacts well with serum samples from patients infected with a range of leptospiral strains. It is also easy to use and does not require special equipment or refrigeration. Therefore the assay is ideal for use in developing countries and rural settings.

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.

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.

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.

First epidemiological data on pathogenic leptospires isolated on the Azorean islands.

Insectivores (Erinaceus europaeus) and rodents (Rattus rattus, R. norvegicus and Mus musculus) from different islands of the Azores Archipelago were found to carry three distinct Leptospira interrogans s.l. serovars (copenhageni, icterohaemorrhagiae and ballum) which have never been previously investigated there. The house mouse and the black rat were the major Leptospira reservoirs showing isolation rates ranging from 0% for both species (in Graciosa) to 88% and 33%, respectively (in Sãao Miguel). This study also showed that the majority of the animals with positive kidney cultures exhibited specific agglutinins against the isolated strains of Leptospira. The observed isolation rates in the different islands, with a very interesting island variation in prevalence, suggest that small mammals, serving as sylvatic reservoirs of pathogenic leptospires, may represent an important risk to the health of humans and livestock, particularly in the islands of Terceira and Sãao Miguel.

[Characterication of reference strains of L. interrogans in China by ISSP-PCR].

The gene polymorphism of the DNAs extracted from reference strains of L. interrogans in China was characterized by LSSP-PCR primered by G1 or G2, which were a pair of specific primers for L. interrogans. The fingerprinting produced by LSSP-PCR showed that serovar lai, serovar canicola, serovar pyrogens, serovar autumnalis, serovar australis, serovar linhai, serovar wolffi and serovar haemolytic have similar patterns, but serovar hebdomadis, serovar javanica, serovar ballum, serovar pomona, serovar spaidjin, serovar tarassov and serovar manahao I have different profiles. This result agreed with the classification of genetic species by Yasuda. The utilization of LSSP-PCR banding patterns in the identification of leptospries in blood samples also gained encouraging results. Compared with the routine methods in genetic species classification, LSSP-PCR has the advantages of rapidity, simplicity and low-cost. It appears to be a promising tool in studying such slowly growing bacteria as leptospires. Further exploration of LSSP-PCR in classification and identification of Leptospires is worthwhile.

[Analysis on speciems of serum and urine in 66 cases of early leptospirosis by PCR and biotin-AMPPD hybridization].

We used polymerase chain reaction (PCR) and Biotin-AMPPD hybridization to detect leptospiral DNA in blood and urine samples in 66 patients at the early stage of leptospirosis. The results showed that PCR and Biotin-AMPPD hybridization not only ruled out the non-specific PCR amplification and increased the reliability for clinical specineus of leptospirosis but also raised the detecting sensibility (from 71.3% to 86.1%). The positive rates of PCR resulted from serum and urine showed no statistical difference; therefore urine sample is worthy of application and dissemination for detecting leptospires at the early stage of leptospirosis. Urine sample is easier for one to collect, preserve and has less intervention. The primers G1, G2 are optimal opplication of PCR in epdemic areas in China.

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.

Dog-DAT: a direct agglutination test using stabilized, freeze-dried antigen for the serodiagnosis of canine visceral leishmaniasis.

We have evaluated the use of an improved direct agglutination test (DAT) based on stable, freeze-dried antigen for the detection of anti-Leishmania antibodies in canine serum samples. With a cut-off value of 1:640, the sensitivity of the DAT was shown to be 100% and the specificity of the test was 98.8%.

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.

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.

Genetic and immunological characterization of the microsporidian Septata intestinalis Cali, Kotler and Orenstein, 1993: reclassification to Encephalitozoon intestinalis.

The relationships between the Encephalitozoon-like Septata intestinalis and other microsporidia that occur in humans; notably Encephalitozoon cuniculi and Encephalitozoon hellem, is insufficiently documented using morphological descriptions alone. To assess mutual relationships, we have examined other phenotypic as well as genetic aspects of S. intestinalis, obtained both from tissue culture and clinical specimens, in comparison with a number of other microsporidia. Phenotypic characterization was performed by analysis of the protein composition and antigenic structure of various microsporidian spores by SDS-PAGE and Western blotting. The genetic characterization consisted of the determination of the sequence of the S. intestinalis rrs gene encoding the small subunit ribosomal RNA (srRNA), restriction fragment length polymorphism (RFLP) analysis of amplified rrs genes and establishment of the degree of sequence identity between rrs genes of various microsporidian species. The unique sequence of rrs of S. intestinalis as well as the distinct RFLP and SDS-PAGE profiles indicate that S. intestinalis is clearly different from other human microsporidian species. However, its rrs gene shared about 90% sequence identity with rrs of both Encephalitozoon spp., E. cuniculi and E. hellem. This is remarkably higher than the about 70% identity observed between rrs of microsporidian species which belong to different genera and thus suggests that S. intestinalis should be regarded as a species of the genus Encephalitozoon.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of leptospires in urine by PCR for early diagnosis of leptospirosis.

We tested urine samples from patients at different stages of current leptospirosis and thereafter to determine whether use of the PCR for detection of leptospires in urine can be a valuable alternative to culturing. The procedure of DNA extraction and subsequent PCR applied to 15 freshly voided urine samples proved to be twice as sensitive as culturing. Overall, we were able to detect leptospires in approximately 90% (26 of 29) of the urine samples. Urine and serum samples were obtained from seven patients, before the eighth day of illness. Although it is generally assumed that leptospiruria starts approximately in the second week of illness, we were able to detect leptospires in all of these early urine samples. In contrast, only two of seven corresponding serum samples gave positive PCR results, which suggests that PCR analysis of urine can be more successful for early diagnosis of leptospirosis than PCR analysis of serum. Urine samples from six patients who had been treated with antibiotics at the time of illness were positive by PCR, implying that the patients were still shedding leptospires in their urine despite treatment. Some of these samples were even taken years after the infection, indicating that shedding of leptospires in urine may last much longer than is generally assumed. We conclude that detection of leptospires in urine with PCR is a promising approach for early diagnosis of leptospirosis and may also be useful in studying long-term shedding.

Leptospirosis in travelers.

Between 1987 and 1991 leptospirosis in 32 Dutch travelers was diagnosed. Infections were acquired predominantly in Thailand and other Southeast Asian countries. Contact with surface waters could be confirmed in all but one case. Fever, headache, and myalgia were the most common complaints. Signs included conjunctival injection and lymphadenopathy in 11 patients each, jaundice in 8, and nuchal rigidity in 3; renal function was impaired in 8. Leptospires were isolated from the blood or urine of nine patients. Thirty-one patients developed an antibody response. Classification of strains identified a variety of serogroups. Although only 14 patients received adequate treatment, all patients recovered completely. Since the number of patients with imported leptospirosis is increasing and the signs and symptoms of the disease are not specific, leptospirosis should be included in the differential diagnosis when a traveler returns from the Tropics with fever.

Classification of leptospires of the pyrogenes serogroup isolated from cattle in Zimbabwe by cross-agglutinin absorption and restriction fragment length polymorphism analysis.

Five strains of the genus Leptospira belonging to serogroup Pyrogenes were isolated from cattle slaughtered in Zimbabwe and subjected to cross-agglutinin absorption and restriction fragment length polymorphism analysis. One strain, SBF 2, represents a new genetic strain of serovar kwale, while another strain, SBF 49, is a new genetic strain closely related to serovar nigeria. Three strains belong to a new serovar for which the name mombe with reference strain SBF 20 is proposed. Restriction fragment length polymorphism analysis indicated that each of these three strains represents a different restriction polymorphism pattern group.