Glenn Graham

Establishing a public health analytical service based on chemical methods for detecting and quantifying Pacific ciguatoxin in fish samples

A referee analysis method for the detection and quantification of Pacific ciguatoxins in fish flesh has recently been established by the public health analytical laboratory for the State of Queensland, Australia. Fifty-six fish samples were analysed, which included 10 fillets purchased as negative controls. P-CTX-1 was identified in 27 samples, and P-CTX-2 and P-CTX-3 were found in 26 of those samples. The range of P-CTX-1 concentrations was 0.04-11.4 microg/kg fish flesh; coefficient of variation from 90 replicate analyses was 7.4%. A liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) method utilising a rapid methanol extraction and clean-up is reliable and reproducible, with the detection limit at 0.03 microg/kg fish flesh. Some matrix effects are evident, with fish oil content a likely signal suppression factor. Species identification of samples by DNA sequence analysis revealed some evidence of fish substitution or inadvertent misidentification, which may have implications for the management and prevention of ciguatera poisoning. Blinded inspection of case notes from suspect ciguatera poisoning cases showed that reporting of ciguatera-related paraesthesias was highly predictable for the presence of ciguatoxins in analysed fish, with 13 of 14 expected cases having consumed fish that contained P-CTX-1 (p<0.001, Fishers Exact Test).

Haematological and clinical-chemistry markers in patients presenting with leptospirosis: a comparison of the findings from uncomplicated cases with those seen in the severe disease

Abstract In a retrospective study, the laboratory findings from the first blood samples taken following hospital presentation in patients with uncomplicated leptospirosis have been compared with the corresponding data for patients admitted, to a high-dependency medical ward or intensive-care unit, with severe leptospirosis. The aim was to identify those laboratory markers that differentiate the two clinical groups upon initial presentation. Marked differences were observed, in some of the haematological and clinical-chemistry markers, between the patients with severe leptospirosis and those with the uncomplicated disease. Statistically significant differences were found in haemoglobin concentrations, haematocrits, counts of erythrocytes, leucocytes, neutrophils and platelets, and serum concentrations of creatinine, urea, protein and albumin. These markers may therefore be useful in the assessment and early detection of disease severity in patients with suspected leptospirosis. Investigations into the use of albumin treatments, which might significantly improve the clinical care of patients with acute leptospirosis, appear to be justified.

Lymphopenia in leptospirosis

Leptospires, the aetiological agents of leptospirosis, are tightly coiled spirochaetes that are approximately 6–20 mm in length and 0.1–0.2 mm in diameter. They have optimal growth at 30uC, are obligate aerobes, and utilize long-chain fatty acids as their carbon source (Levett, 2001). The species of the genus Leptospira are traditionally classified by serological means, with the formal taxonomic units of the genus broken into serovars, each of which is identified by agglutination after cross-absorption with the homologous antigen. Although it has no official taxonomic standing, and more contemporary classifications make use of DNAbased methodologies (Yasuda et al., 1987; Brenner et al., 1999), serotyping continues to be used for most epidemiological investigations of leptospirosis (Levett, 2001). The anti-lymphoid activity of leptospiral exoproducts was first observed more than 30 years ago. Oravec and Kmety (1978) reported that Syrian hamsters given intraperitoneal injections of the supernatant fluid from a culture of L. biflexa serovar Patoc 1 subsequently showed a pronounced and transient fall in their peripheral lymphocyte counts. Much later, in their retrospective review of patients with leptospirosis who had been admitted to Pontchaillou Hospital, in Rennes, north–western France, Jauréguiberry et al. (2005) found that 29 (85%) of the 34 cases they investigated were lymphopenic, and concluded that lymphopenia is a common feature of leptospirosis. In response to this finding, however, Lopes et al. (2005) reviewed 253 leptospirosis cases in Salvador, north–eastern Brazil, and found that only 17% of these patients were lymphopenic on admission. Lopes et al. (2005) suggested that differences in environmental factors and in the leptospiral serovars involved may account for the differences seen in the frequency of lymphopenia in Salvador and Rennes. The most common serovar detected in Salvador is L. interrogans serovar Copenhageni (Ko et al., 1999; Tucunduva de Faria et al., 2008) whereas Jauréguiberry et al. (2005) found serovar Grippotyphosa (of L. interrogans or L. kischneri) to be the most common serovar among the patients attending Pontchaillou Hospital. In neither the French study nor in the Brazilian investigation, however, was any attempt made to determine the frequency of lymphopenia according to infecting serovar. In the present, retrospective study, in an attempt to see if lymphopenia in leptospirosis may, at least in part, be serovar-dependant, the prevalence of lymphopenia (defined as a lymphocyte count of ,1.2610/litre) in 258 patients diagnosed with leptospirosis at the WHO/FAO/OIE Collaborating Centre for Reference and Research on Leptospirosis, in Queensland, Australia, was assessed, separately for each infecting serovar.

Leptospirosis and Goodpasture's syndrome: testing the aetiological hypothesis

Abstract Leptospiral pathogens have a world-wide distribution and cause a spectrum of disease ranging from a mild, influenza-like illness to Weils disease, which manifests itself in multi-organ failure. Recently, Leptospira-reactive sera from 40 leptospirosis patients were investigated in an ELISA designed to detect antibodies to the human glomerular basement membrane (GBM). The aim was to determine if host-derived leptospiral immunoglobulins cross-react with proteins in the human GBM, so facilitating the development of Goodpastures syndrome. As all 40 sera were found negative in the anti-GBM ELISA, the hypothesis that, during the immune phase of leptospirosis, patients are at risk of developing Goodpastures syndrome was not supported. Further work is required to determine if leptospirosis is a risk factor in the development of any other pulmonary–renal syndromes that are associated with auto-immune diseases, such as Wegeners granulomatosis, microscopic polyangiitis, Churg–Strauss syndrome, Behçets disease, IgA nephropathy and systemic lupus erythematosus.

Hypomagnesaemia in the first 10 days of severe leptospirosis.

Abstract Magnesium imbalance in leptospirosis has, for the most part, been neglected by the medical and leptospirosis communities. In a recent, retrospective study, serum concentrations of magnesium were followed in 15 patients with severe leptospirosis. The results revealed that 14 of the 15 patients developed hypomagnesaemia at some time during the first 10 days of their illness. In severely ill patients, such magnesium deficiency can worsen clinical outcome. Magnesium concentrations may affect a number of organ systems and mental status. Since altered mental status in leptospirosis is a poor prognostic indicator, it is suggested that serum concentrations of magnesium be monitored closely in patients with leptospirosis. Any hypomagnesaemia can then be treated promptly, in an effort to reduce the morbidity and mortality attributable to the disease.

Lymphopenia is observed regularly in the acute (leptospiraemic) phase but not the immune phase of leptospirosis

Lymphocyte counts in patients with leptospirosis have been shown to be variable. This study retrospectively compared lymphocyte counts from the first blood samples taken following hospital presentation in patients with leptospirosis who were either (i) IgM non-reactive, (ii) IgM reactive and microscopic agglutination test (MAT) non-reactive or (iii) IgM and MAT reactive in an effort to determine whether differences in lymphocyte counts are observed in the acute and immune phase of leptospirosis. Statistical differences in lymphocyte counts were observed between the three groups. In conclusion, this study has shown that the phase of leptospiral infection may affect patient lymphocyte counts.

A case of 'original antigenic sin' or just a paradoxical reaction in leptospirosis?

Leptospirosis is an important zoonotic disease caused by members of the genus Leptospira. In humans, infections with pathogenic leptospires result in a spectrum of disease ranging from a mild, influenzalike illness to Weils’ disease. The pulmonary haemorrhage and renal failure seen in the latter are associated with high mortality (Levett, 2001). Although the disease may be detected by serology, cross-reactions between serogroups and paradoxical reactions, where the initial immune response is directed toward a heterologous serovar, can confound the diagnostic process (Levett, 2003). Another confounding variable is ‘original antigenic sin’, a concept first posited by Francis (1953) and subsequently supported by Hennessy et al. (1955) and Fazekas de St Groth and Webster (1966). In serological investigations, according to this theory, the serotype with the highest neutralising titre observed after a secondary infection corresponds to the serotype that caused the primary infection. The laboratory findings on a patient who experienced two episodes of leptospirosis (from two different serovars) over a period of 3 months and displayed peculiar serological results are reported below. The patient may have had a paradoxical reaction or, alternatively, had an additional leptospiral infection prior to presenting with the other two infections.