Additional cell damage after transpupillary thermotherapy in choroidal malignant melanoma.

The aim of this study was to evaluate the effectiveness of transpupillary thermotherapy (TTT) in generating tumour necrosis by light and electron microscopy, as well as to evaluate additional cell damage in the area directly adherent to the necrotic zone. Four eyes of four patients diagnosed with intraocular malignant melanoma of the uvea were treated experimentally with diode laser TTT. In all cases a standard technique was used. All eyes were enucleated: one eye the day after TTT, two eyes 2 days after TTT, and one eye 6 weeks after TTT. Immediately after enucleation the eyes were immersed in standard Karnovsky's fixative with cocodylate buffer and prepared for light and electron microscopy. In the treated area of all four melanomas we found a dense band of necrotic tissue (zone A) consisting of an amorphous mass of dead cells sharply demarcated from the rest of the neoplastic tissue. Next to this zone was a more eosinophilic and also sharply demarcated band (zone B) that consisted of similar but less intensive changes. In the next band (zone C), marked injury to the cellular membrane and subcellular structures were seen on electron microscopy. The next band (zone D) consisted of changes mainly observed only within the cytoplasm of neoplastic cells and significantly less intensive than those in zone C. Outside zone D tumour cells that were normal in appearance were seen. No scleral alterations induced by heat were found. We concluded that after TTT the cytotoxic effect gradually decreases in proportion to the distance from the central point of the diode laser spot, with additional cell damage in the area adjacent to the necrotic zone. The interval between TTT and enucleation had no influence on the histological results.

Temperature dependence of thermal damage to the sclera: exploring the heat tolerance of the sclera for transscleral thermotherapy.

Thermal damage to the human sclera in relation to temperature and duration of exposure was studied in order to determine the heat tolerance of the sclera with respect to transscleral thermotherapy of choroidal melanoma. Samples of human sclera were submerged in saline for 10 sec to 10 min at temperatures of 37-100 degrees C. The effects of heat on the shape, weight and size of the samples were studied. Thermal damage of scleral collagen was examined by polarized light microscopy (LM) and electron microscopy (EM). The sclera was embedded in epoxy resin and stained with toluidine blue for LM and with uranyl acetate and lead citrate for EM. Thermal damage of scleral collagen on polarized LM was graded on a five point scale. Scleral damage was visible on macroscopic examination and on LM and EM in sclera heated at 65 degrees C for 20 sec or longer, at 70 degrees C for 10 sec or longer, and at higher temperatures. A sigmoidal function was used to define the relation between temperature and changes in diameter, thickness, and weight of scleral samples. Using fitted functions, the threshold temperature for thermal damage was estimated to be 59-61 degrees C when samples were heated for 10 min, 62-63 degrees C when heated for 1 min, and 66-67 degrees C when heated for 10 sec; the threshold exposure time at 60 degrees C was estimated to be 7-12 min. These results indicate a temperature of 60 degrees C for 1 min is well tolerated by human donor sclera; information of in vivo studies is required to validate whether this setting can be used in transscleral thermotherapy (TSTT) for choroidal melanoma.

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.

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.

Identification of a serogroup bataviae Leptospira strain isolated from an ox in Zimbabwe.

A strain belonging to the genus Leptospira serogroup Bataviae, isolated from an ox at slaughter in Zimbabwe, was identified by using cross-agglutinin absorption, the monoclonal antibody, restriction fragment length polymorphism, and polymerase chain reaction analyses. Results of both serological tests used showed that the isolate (strain SBF 37) was antigenically similar to reference strain Paidjan and therefore belongs to serovar paidjan. Strain SBF 37 was, however, genetically different from strain Paidjan since their chromosomal DNA had different restriction fragment length polymorphism patterns. The Zimbabwe paidjan strain was identified as belonging to the species Leptospira kirschneri while strain Paidjan reacted as a member of one of the non-kirschneri species in the polymerase chain reaction. The Zimbabwe isolate therefore belongs to Leptospira kirschneri serovar paidjan.

[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.

A new leptospiral serovar, ngavi, in the Tarassovi serogroup isolated from Zimbabwe oxen.

Two strains of the genus Leptospira, belonging to serogroup Tarassovi, were isolated from kidneys of apparently healthy oxen slaughtered at an abattoir in Zimbabwe. Both strains belonged to the same serovar but could not be assigned to previously known serovars using the cross-agglutinin absorption test. The name ngavi is proposed for the new serovar containing these two strains; strain SBF 16 is the reference strain. The Zimbabwe isolates showed some antigenic similarity to serovar gatuni when analyses were carried out using eight monoclonal antibodies, and had restriction patterns similar to those of serovars tarassovi, tunis, moldaviae and guidae when their chromosomal DNAs were analysed using RFLP analysis. The restriction patterns of the two strains could be distinguished from each other and from those of the four serovars when their Southern blots were hybridized with a probe synthesized from a repetitive sequence element cloned from serovar hardjo strain Hardjo-bovis.

Transpupillary thermotherapy: results in 50 patients with choroidal melanoma.

OBJECTIVE: To evaluate the efficacy and safety of transpupillary thermotherapy in treating choroidal melanoma. METHODS AND PATIENTS: To perform transpupillary thermotherapy, infrared diode laser energy at 810 nm was used with a beam diameter of about 3 mm and 1-minute exposure time. All 50 patients had choroidal melanoma. We performed transpupillary thermotherapy in 21 tumors that had responded insufficiently to 800 Gy ruthenium 106 brachytherapy; it was combined with 800 Gy106Ru brachytherapy for 10 tumors greater than 5 mm in height and with 600 Gy for 19 tumors 5 mm or less in height. RESULTS: All but 1 tumor exhibited reduction in tumor height within a mean follow-up of 20.5 months (range, 6-49 months). In 41 eyes (82%), the tumor flattened completely. Visual acuity was 20/60 or better in 43 eyes (86%) before treatment and in 14 eyes (28%) at the last examination because of radiation vasculopathy. Neovascular glaucoma developed in 1 eye, and total retinal detachment developed in 2 eyes. Tumor recurrence was observed in 1 patient. CONCLUSIONS: Although long-term results are necessary to properly appraise this new therapy, transpupillary thermotherapy may be useful as a complementary modality to brachytherapy.

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.

Two new leptospiral serovars in the Hebdomadis serogroup isolated from Zimbabwe cattle.

Four strains belonging to the genus Leptospira serogroup Hebdomadis were isolated from Zimbabwe cattle at slaughter. These isolates were subjected to cross-agglutinin absorption tests and to restriction fragment length polymorphism and pulsed-field gel electrophoresis analyses of their genomic DNAs. One of these strains represents a new serovar, for which the name mhou is proposed; strain SBF 40 is the reference strain of this serovar. The other three strains belong to a second new serovar, for which the name marondera is proposed; the reference strain of this serovar is strain SBF 5. The three strains of serovar marondera could be differentiated by their restriction fragment polymorphism and pulsed-field gel electrophoretic patterns.

Identification of leptospires of the Pomona and Grippotyphosa serogroups isolated from cattle in Zimbabwe.

Two strains of the genus Leptospira, isolated from kidneys of oxen slaughtered in Zimbabwe, one belonging to serogroup Pomona (strain SBF 8) and the other to serogroup Grippotyphosa (strain SBF 32), were identified by using cross-agglutinin absorption, monoclonal antibody, restriction fragment length polymorphism and polymerase chain reaction analyses. The identification of the two strains was equivocal. Strain SBF 8 showed a close similarity to both serovars mozdok and proechimys by cross-agglutinin absorption tests and to serovar pomona by monoclonal antibody analysis, but had a distinct DNA restriction pattern. Strain SBF 32 showed a close antigenic similarity to serovars ratnapura, grippotyphosa and valbuzzi by the cross-agglutinin absorption test, and to serovar ratnapura by monoclonal antibody analysis but also had a distinct DNA restriction pattern. Both strains SBF 8 and SBF 32 reacted as members of species Leptospira kirschneri by the polymerase chain reaction. It is concluded that strains SBF 8 and SBF 32 represent new genetic strains in the Pomona and Grippotyphosa groups, respectively.

Transpupillary thermotherapy in choroidal melanomas.

OBJECTIVE: To determine safety and efficacy of transpupillary thermotherapy (TTT) as a new treatment for choroidal melanoma. METHODS AND PATIENTS: To perform TTT, diode laser energy at 810 nm was used with a beam diameter of 1.5 to 4.5 mm for a 1-minute exposure. All 12 patients had choroidal melanoma. Six had had insufficient response to ruthenium 106 (106Ru) brachytherapy. Three patients with tumors more than 5 mm in height were treated simultaneously with 106Ru and TTT. Three patients with juxtapapillary or macular tumors were treated by TTT only. RESULTS: All but one tumor exhibited a reduction of tumor height in a follow-up period of 3 to 14 months. Side effects were minimal. Severe visual loss occurred in two patients due to radiation retinopathy, in two patients whose foveas were included in the TTT area, and in one patient resulting from a serous retinal detachment that extended over the posterior pole. CONCLUSIONS: Treatment with TTT may be useful as a complementary modality to brachytherapy. A longer follow-up period is required for final evaluation.

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.

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.

A new serovar in the Grippotyphosa serogroup comprising leptospiral isolates from different regions.

Pulsed-field gel electrophoresis (PFGE) studies performed with leptospiral isolates led us to suspect the existence of a new serovar in the Grippotyphosa serogroup. The results obtained with reference serological techniques used in leptospiral identification, including cross-agglutination absorption and monoclonal antibody techniques, confirmed the existence of a new serovar exemplified by strain Dadas I. Four other isolates from different regions of the world were submitted for identification by PFGE and produced NotI restriction patterns similar to that of strain Dadas I. Our data demonstrate the power of PFGE for identifying leptospiral isolates. The name dadas is proposed for the new serovar.

A new leptospiral serovar in the Icterohaemorrhagiae serogroup isolated from an ox in Zimbabwe.

A strain of Leptospira interrogans that was isolated from an ox slaughtered in Zimbabwe and belonged to serogroup Icterohaemorrhagiae could not be identified when we compared it with 18 reference strains belonging to this serogroup by using cross-agglutinin absorption, monoclonal antibody, and restriction endonuclease DNA analyses. The name zimbabwe is proposed for the new serovar containing this strain; the type strain of this serovar is strain SBF 23.

Muskrats as carriers of pathogenic leptospires in The Netherlands.

Leptospires were isolated from 24 of 327 (7%) muskrats (Ondatra zibethicus) caught in The Netherlands. All isolates were identified as Leptospira interrogans. One isolate was typed as serovar copenhageni in the Icterohaemorrhagiae serogroup, one as serovar lora in the Australis serogroup. Twenty-one isolates showed a close relationship with serovars grippotyphosa, valbuzzi, muelleri and ratnapura from the Grippotyphosa serogroup. One isolate was lost. Sera from 196 muskrats were examined by the microscopic agglutination test. Forty-five (23%) sera reacted positively (titers greater than or equal to 1:160), 42 (21%) of these 45 sera to Grippotyphosa and 3 (2%) to Sejroe serogroup antigens. This is the first report of serological and cultural evidence of leptospira infection in muskrats in The Netherlands.

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.

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. (AU)

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.