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

Leptospirosis is an infectious disease with multi-organ-system involvement in which the clinical spectrum ranges from mild, flu-like illness to severe and fatal forms (Faine et al., 1999). The clinical presentation varies from patient to patient and, depending upon the predominant organ-system involvement, the manifestations hepato-renal failure, myocarditis and severe pulmonary haemorrhage leading to respiratory distress or central nervous system involvement are observed.

The earliest report of confirmed cases of leptospirosis in India came from the Andaman Islands in 1929. These cases showed signs and symptoms typical of Weil's syndrome, with predominant hepato-renal involvement. Twenty-eight isolates were recovered from patients. Twenty-four of them belonged to serogroup Andamana, previously called Andamans A (Taylor & Goyle, 1931), and the remaining four to serogroup Grippotyphosa. Between 1929 and 1988, there were no reports of leptospirosis from these islands.

Since 1988, post-monsoon outbreaks of febrile illness with haemorrhagic manifestations and high case-fatality rates have been occurring in these islands. As the aetiology of the disease could not be established, it was named Andaman haemorrhagic fever (AHF). In 1993, AHF was proved to be leptospirosis (Sehgal et al., 1995). In the recent outbreaks, pulmonary involvement has been the predominant complication, with haemoptysis as the common symptom. The reason behind this shift in clinical presentation has not been studied in detail. It is not clearly understood whether the variation in clinical presentation in leptospirosis is caused by differences in the infecting agent or host- and environment-related factors.

Pulmonary involvement in leptospirosis was first observed in India during the recent outbreaks in Andaman Islands. It has been observed previously in China and Korea and, recently, in other countries such as Australia and Nicaragua. During the past few years, this form of presentation has also been observed occasionally in mainland India. In countries like China and Korea, the occurrence of pulmonary haemorrhage has been linked to infection with serovar Lai of serogroup Icterohaemorrhagiae (Oh et al., 1991). In Australia, pulmonary haemorrhage has been reported in patients infected with serovar Australis (Simpson et al., 1998).

No extensive studies have been carried out on the infecting serovars in the Andaman Islands. The existence of a serovar resembling Lai has been established in the islands; however, the patient from whom this isolate was recovered had only mild illness (Sehgal et al., 2000). Hence, the question of agent factors being responsible for the observed shift in clinical presentation of leptospirosis in the Andaman Islands is still unresolved. Hence, we undertook a study on the serological and genetic characteristics of leptospiral isolates recovered during an outbreak of leptospirosis in which the predominant clinical presentation was that of pulmonary involvement.

Patients and isolates.
An outbreak of leptospirosis occurred at Diglipur on North Andaman during OctoberNovember 1997. Investigations were carried out during the outbreak and four leptospire isolates were recovered from blood samples of patients, three males and one female. All four patients were from rural areas and their ages ranged from 18 to 28 years. All of them presented with fever, headache, generalized body ache, cough and haemoptysis. The female patient died due to massive pulmonary haemorrhage and respiratory distress.

Determination of pathogenicity.
The four isolates, designated DS15, DS18, DCCH30 and BL10, were tested in duplicate for their ability to grow at 13 °C and in the presence of 8-azaguanine (Sigma) following standard procedures (Johnson & Rogers, 1964; Johnson & Harris, 1967). As controls, a pathogenic strain (Wijnberg) and a saprophytic strain (Patoc 1) were also included in the test.

Serotyping
Microscopic agglutination test (MAT) with group sera. MAT was performed following standard procedures (Wolff, 1954) using a panel of 36 group-specific rabbit antisera (group sera) representing all pathogenic serogroups. An isolate was considered to belong to the serogroup of the group serum that gave the highest titre (Dikken & Kmety, 1978).

Cross-agglutination and absorption test (CAAT). Each isolate was tested by MAT against all reference antisera of the identified serogroup to establish cross-reactivity patterns. CAAT was performed following a standard procedure (Dikken & Kmety, 1978) to identify the serovar status of the isolates.

Typing with mAbs. MAT was performed with mouse mAbs F71C2, F71C3, F71C9, F71C13, F71C16, F71C17, F164C1, F165C1, F165C2, F165C3, F165C7, 165C8 and 165C12 (WHO/FAO Collaborating Centre for Reference and Research, KIT Biomedical Research, Amsterdam, The Netherlands) to generate characteristic agglutination profiles, as described previously (Desmecht et al., 1991; Korver et al., 1988). For identification at the serovar level, agglutination profiles generated against the isolates were compared with profiles of all reference strains belonging to the identified serogroup.

PCR.
PCR was performed on all isolates using two sets of primers, G1/G2 and B64-I/B64-II, described by Gravekamp et al. (1993). Primer set G1/G2 amplifies DNA from all pathogenic species except Leptospira kirschneri and primer set B64-I/B64-II amplifies DNA from genomospecies L. kirschneri.

DNA sequencing.
One strand of the G1/G2-generated PCR product was sequenced on an ABI PRISM model 377 automatic sequencer, giving 99 % sequence accuracy. DNASIS software (Pharmacia LKB) was used for comparison.

Isolates DS15, DS18, DCCH30 and BL10 did not grow at 13 °C or in the presence of 8-azaguanine, in either of the two experiments performed, suggesting a pathogenic status of the isolates. MAT with 36 serogroup-specific sera revealed that all four isolates belonged to the serogroup Grippotyphosa. In the CAAT procedure, all isolates cross-reacted strongly with reference rabbit sera against the all of the serovars of the Grippotyphosa serogroup except serovar Huanuco. Rabbit anti-sera raised against the isolates also showed the same reactivity patterns against all reference serovars of serogroup Grippotyphosa. The residual titre obtained after absorption was less than 10 % of the titre obtained before absorption in the case of serovars Ratnapura, Valbuzzi, Grippotyphosa and Mülleri. The results were consistent in repeated experiments done independently (Table 1). In the case of serovars Canalzonae, Huanuco and Vanderhoedeni, the residual titre after absorption was always 50100 % of the titre before absorption (data not shown). Thus, CAAT excluded these serovars. However, it was not able to resolve between Ratnapura, Valbuzzi, Grippotyphosa and Mülleri.

Table 1. CAAT results for isolates DS15, DS18, DCCH30 and BL10

It is known that the antigenic nature of leptospires is complex, and it is still not well understood. Strong antigenic similarities between some serovars of the serogroup Grippotyphosa are documented and these similarities sometimes interfere with the interpretation of CAAT results and assignment of isolates to serovars (Terpstra, 1992).

Since the serovar status of these isolates could not be ascertained by CAAT, we used a panel of 13 mAbs to identify the isolates by comparing their antigenic profiles. All isolates showed moderate to strong reactions with 11 of 13 mAbs against serovars of the Grippotyphosa group. Histograms depicting the agglutination titre profiles of the isolates were similar (Fig. 1), suggesting that these isolates belong to one serovar. These histograms had no similarity to histograms obtained with serovars Mülleri and Grippotyphosa, excluding them from the list of possible serovars. The histograms of the isolates were similar to those of serovars Ratnapura and Valbuzzi. In fact, the histogram patterns of Valbuzzi and Ratnapura were quite close except for mAb F71C3, against which Valbuzzi gave a titre of 1 : 640, while Ratnapura showed no reaction at all. All the isolates tested showed some reaction against this mAb. Otherwise, the patterns were close to those of both Ratnapura and Valbuzzi. The presence of reactivity against this mAb, though much lower than that shown by the reference strain of Valbuzzi, was a factor in favour of categorizing these isolates as serovar Valbuzzi. Thus, mAbs are useful in such situations, allowing a detailed analysis of the antigenic nature of closely similar serovars and resolving ambiguities.

(50K): Fig. 1. Comparison of antigenic profiles of isolates D15, BL10, DS18 and DCCH30 with serovars of serogroup Grippotyphosa. mAbs are identified as: 1, F71C2; 2, F71C3; 3, F71C9; 4, F71C13; 5, F71C16; 6, F71C17; 7, F164C1; 8, F165C1; 9, F165C2; 10, F165C3; 11, F165C7; 12, 165C8; 13, 165C12.

Serovar Ratnapura belongs to genospecies L. kirschneri, whereas the reference strain Valbuzzi of serovar Valbuzzi belongs to genospecies Leptospira interrogans sensu stricto. The DNA of isolates DS15, DDS18, DCCH30 and BL10 did not amplify in a PCR using primers B64-I/B64-II (Gravekamp et al., 1993), which are specific for L. kirschneri, whereas primer set G1/G2, specific for other species, amplified the correct sequence from these isolates, giving a characteristic band pattern (not shown). This indicates that the isolates do not belong to the genospecies L. kirschneri, thereby excluding the possibility that they belonged to serovar Ratnapura. Although CAAT was not able to resolve the serovar status of these isolates conclusively, the antigenic pattern obtained against a panel of mAbs and the PCR results indicate that the isolates probably belong to serovar Valbuzzi.

Identification of the isolates as serovar Valbuzzi was strongly supported by sequence analysis of the PCR products from two isolates, DS15 and BL10, generated with primer set G1/G2. Comparison of the sequences obtained from these isolates with those from 45 strains belonging to seven genospecies revealed the highest similarity (97.5100 %) to sequences from strains belonging to L. interrogans sensu stricto. Sequence similarity to other species ranged from 91.9 % for Leptospira noguchii (strain 1161 U, serovar Proecchimys) to 80.0 % to Leptospira alexanderi (strain M16901, serovar Nanding) (Brenner et al., 1999). The higher sequence similarity of the isolates to strains of L. interrogans sensu stricto is consistent with the identification of the isolates as serovar Valbuzzi. A sequence alignment of PCR products obtained with primers G1/G2 from Andaman isolate BL10 and members of seven pathogenic species is shown in Fig. 2.

(66K): Fig. 2. Sequence alignment of PCR products obtained with primers G1/G2 from Andaman isolate BL10 and members of seven pathogenic species, L. interrogans strains H. Utrecht IV and RGAT, L. noguchii strain CZ214T, Leptospira meyeri strain Iowa City FrogT (ICF), Leptospira santarosai strain CZ188, Leptospira borgpetersenii strain Poi, Leptospira weilii strain CellidoniT and L. alexanderi strain M6901.

Leptospirosis is known to have occurred in the Andaman Islands since the late 1920s. However, since 1988, it has been occurring as annual post-monsoon outbreaks, with a change in the clinical manifestation from the earlier hepato-renal form to a predominantly pulmonary form. The pulmonary form of leptospirosis, as it occurs in the Andaman Islands, has a significantly higher case-fatality rate than the classical Weil's disease, and the complications tend to occur earlier (Singh et al., 1999). In some countries where the pulmonary form of leptospirosis occurs, it is believed to be caused exclusively by serovar Lai (China and Korea). Serovar Canicola of serogroup Canicola and serovar Pomona of serogroup Pomona may have been involved in the 1995 outbreak in Nicaragua (Zuerner & Bolin, 1997; Trevejo et al., 1998). However, recently, some other serovars such as Australis have been found to be associated with this form of leptospirosis (Simpson et al. 1998). Although the presence of Lai-like serovars has been established in the Andamans, the patient from whom this isolate was recovered had only mild illness (Sehgal et al., 2000). The reasons for the variations in the clinical presentations of leptospirosis have not been fully understood. Agent-related factors such as the infecting serovar are important aspects to be studied.

Characterization of isolates recovered from patients during an outbreak in 1997 was attempted as a step in studying the role of agent-related factors in fatal pulmonary complications in leptospirosis. Characterization is also important from epidemiological and public health surveillance points of view, as different serovars/strains may exhibit different host specificities and information about serovar distribution would be useful for prevention and control. Identification of locally existing serovars is essential for the rational design of vaccines and for selecting the antigens for MAT, which is still the standard serodiagnostic test for leptospirosis.

Some of the original isolates from the Andamans belonged to serogroup Grippotyphosa serovar Grippotyphosa, with L. interrogans as the genomic species (Brenner et al., 1999), and caused mild non-icteric disease in the early part of the 20th century. The recent isolates belonging to same serogroup, Grippotyphosa, but to a different serovar, Valbuzzi, are genetically similar (L. interrogans sensu stricto) to earlier isolates recovered from severe or fatal cases. This leads us to believe that serogroup Grippotyphosa has been persisting in these islands throughout this period of more than 70 years, but that the organism has undergone an antigenic change leading to the origin of a different serovar. Although genetic homology has been preserved, a change in phenotypic characters might be responsible for the change in the clinical presentation and severity of the disease.

Serovar Lai, belonging to serogroup Icterohaemorrhagiae, had been incriminated as a cause of leptospirosis with haemoptysis as the predominant symptom in China and Korea (Oh et al., 1991), but serovars of serogroup Australis have also been isolated from similar cases in Australia (Simpson et al., 1998; WHO, 1999). Serovar Canicola of serogroup Canicola and serovar Pomona of serogroup Pomona were involved in the 1995 outbreak in Nicaragua (Zuerner & Bolin, 1997; Trevejo et al., 1998). Serogroup Canicola was also responsible for an outbreak of leptospirosis with pulmonary haemorrhage in Orissa, India, after a cyclone in 1999 (Sehgal et al., 2002). In the present study, we report the identification of serovar Valbuzzi of serogroup Grippotyphosa as responsible for similar clinical symptomatology. This observation further substantiates the view that there is no organ specificity for any serovar and that any serovar can cause any type of clinical illness.

The authors are grateful to the Medical Officers and staff of the community health centre Diglipur, North Andaman, for their help and co-operation and Dr Sameer Sharma, Technical Officer, Mr T. Umapathi, Laboratory Technician, and Mr Paritosh Dey, Laboratory Assistant, RMRC, Port Blair, for their assistance in the laboratory.