M.-A. Burns

Causes of non-malarial fever in Laos: a prospective study

Summary Background Because of reductions in the incidence of Plasmodium falciparum malaria in Laos, identification of the causes of fever in people without malaria, and discussion of the best empirical treatment options, are urgently needed. We aimed to identify the causes of non-malarial acute fever in patients in rural Laos. Methods For this prospective study, we recruited 1938 febrile patients, between May, 2008, and December, 2010, at Luang Namtha provincial hospital in northwest Laos (n=1390), and between September, 2008, and December, 2010, at Salavan provincial hospital in southern Laos (n=548). Eligible participants were aged 5–49 years with fever (≥38°C) lasting 8 days or less and were eligible for malaria testing by national guidelines. Findings With conservative definitions of cause, we assigned 799 (41%) patients a diagnosis. With exclusion of influenza, the top five diagnoses when only one aetiological agent per patient was identified were dengue (156 [8%] of 1927 patients), scrub typhus (122 [7%] of 1871), Japanese encephalitis virus (112 [6%] of 1924), leptospirosis (109 [6%] of 1934), and bacteraemia (43 [2%] of 1938). 115 (32%) of 358 patients at Luang Namtha hospital tested influenza PCR-positive between June and December, 2010, of which influenza B was the most frequently detected strain (n=121 [87%]). Disease frequency differed significantly between the two sites: Japanese encephalitis virus infection (p=0·04), typhoid (p=0·006), and leptospirosis (p=0·001) were more common at Luang Namtha, whereas dengue and malaria were more common at Salavan (all p<0·0001). With use of evidence from southeast Asia when possible, we estimated that azithromycin, doxycycline, ceftriaxone, and ofloxacin would have had significant efficacy for 258 (13%), 240 (12%), 154 (8%), and 41 (2%) of patients, respectively. Interpretation Our findings suggest that a wide range of treatable or preventable pathogens are implicated in non-malarial febrile illness in Laos. Empirical treatment with doxycycline for patients with undifferentiated fever and negative rapid diagnostic tests for malaria and dengue could be an appropriate strategy for rural health workers in Laos. Funding Wellcome Trust, WHO–Western Pacific Region, Foundation for Innovative New Diagnostics, US Centers for Disease Control and Prevention.

The role of fruit bats in the transmission of pathogenic leptospires in Australia

Abstract Although antileptospiral antibodies and leptospiral DNA have been detected in Australian fruit bats, the role of such bats as infectious hosts for the leptospires found in rodents and humans remains unconfirmed. A cohort‐design, replicated survey was recently conducted in Far North Queensland, Australia, to determine if the abundance and leptospiral status of rodents were affected by association with colonies of fruit bats (Pteropus conspicillatus spp.) via rodent contact with potentially infectious fruit‐bat urine. In each of four study areas, a ‘colony site’ that included a fruit‐bat colony and the land within 1500 m of the colony was compared with a ‘control site’ that held no fruit‐bat colonies and was >2000 m from the nearest edge of the colony site. Rodents were surveyed, for a total of 2400 trap‐nights, over six sampling sessions between September 2007 and September 2008. A low abundance of rodents but a high carriage of leptospires in the rodents present were found to be associated with proximity to a fruit‐bat colony. For example, means of 0·4 and 2·3 fawn‐footed melomys (Melomys cervinipes) were collected/100 trap‐nights at sites with and without fruit‐bat colonies, respectively (P<0·001), but the corresponding prevalences of leptospiral carriage were 100% and 3·6% (P<0·001). Such trends were consistent across all of the sampling sessions but not across all of the sampling sites. Leptospires were not isolated from fruit bats by culture, and the role of such bats in the transmission of leptospires to rodents cannot be confirmed. The data collected do, however, indicate the existence of a potential pathway for transmission of leptospires from fruit bats to rodents, via rodent contact with infectious fruit‐bat urine. Fruit bats may possibly be involved in the ecology of leptospires (including emergent serovars), as disseminators of pathogens to rodent populations. Stringent quantitative risk analysis of the present and similar data, to explore their implications in terms of disease prevalence and wildlife population dynamics, is recommended.

Causes of Fever in Rural Southern Laos

The etiology of fever in rural Lao Peoples Democratic Republic (Laos) has remained obscure until recently owing to the lack of laboratory facilities. We conducted a study to determine the causes of fever among 229 patients without malaria in Savannakhet Province, southern Laos; 52% had evidence of at least one diagnosis (45% with single and 7% with apparent multiple infections). Among patients with only one diagnosis, dengue (30.1%) was the most common, followed by leptospirosis (7.0%), Japanese encephalitis virus infection (3.5%), scrub typhus (2.6%), spotted fever group infection (0.9%), unspecified flavivirus infection (0.9%), and murine typhus (0.4%). We discuss the empirical treatment of fever in relation to these findings.

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.

Validation of a microsphere immunoassay for serological leptospirosis diagnosis in human serum by comparison to the current gold standard.

A microsphere immunoassay (MIA) utilising Luminex xMap technology that is capable of determining leptospirosis IgG and IgM independently was developed. The MIA was validated using 200 human samples submitted for routine leptospirosis serology testing. The traditional microscopic agglutination (MAT) method (now 100 years old) suffers from a significant range of technical problems including a dependence on antisera which is difficult to source and produce, false positive reactions due to auto-agglutination and an inability to differentiate between IgG and IgM antibodies. A comparative validation method of the MIA against the MAT was performed and used to determine the ability of the MIA to detect leptospiral antibodies when compared with the MAT. The assay was able to determine samples in the reactive, equivocal and non-reactive ranges when compared to the MAT and was able to differentiate leptospiral IgG antibodies from leptospiral IgM antibodies. The MIA is more sensitive than the MAT and in true infections was able to detect low levels of antibody in the later stages of the acute phase as well as detect higher levels of IgM antibody earlier in the immune phase of the infection. The relatively low cost, high throughput platform and significantly reduced dependency on large volumes of rabbit antisera make this assay worthy of consideration for any microbiological assay that currently uses agglutination assays.

High-resolution melt-curve analysis of random amplified polymorphic DNA (RAPD-HRM) for the characterisation of pathogenic leptospires: intra-serovar divergence, interserovar convergence, and evidence of attenuation in Leptospira reference collections.

Abstract High-resolution melt-curve analysis of random amplified polymorphic DNA (RAPD–HRM) is a novel technology that has emerged as a possible method to characterise leptospires to serovar level. RAPD–HRM has recently been used to measure intra-serovar convergence between strains of the same serovar as well as inter-serovar divergence between strains of different serovars. The results indicate that intra-serovar heterogeneity and inter-serovar homogeneity may limit the application of RAPD–HRM in routine diagnostics. They also indicate that genetic attenuation of aged, high-passage-number isolates could undermine the use of RAPD–HRM or any other molecular technology. Such genetic attenuation may account for a general decrease seen in titres of rabbit hyperimmune antibodies over time. Before RAPD–HRM can be further advanced as a routine diagnostic tool, strains more representative of the wild-type serovars of a given region need to be identified. Further, RAPD–HRM analysis of reference strains indicates that the routine renewal of reference collections, with new isolates, may be needed to maintain the genetic integrity of the collections.

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.

Maximizing the chances of detecting pathogenic leptospires in mammals: the evaluation of field samples and a multi-sample-per-mammal, multi-test approach.

Abstract Identification of wild animals that harbour the causative leptospires, and the identification of the most important of these ‘wild reservoirs’ (in terms of threat to human health), are key factors in the epidemiology of human leptospirosis. In an epidemiological investigation in the Australian state of Queensland, in 2007–2008, samples were collected from fruit bats (Pteropus conspicillatus) and rodents (to investigate the potential role of fruit bats in the maintenance and transmission of leptospires to ground‐dwelling rodents) and checked for pathogenic leptospires. The results of these studies have now been carefully analysed in attempts to see which method of detection and type of test sample were best. The effects of pentobarbitone sodium used to euthanize wild mammals before collection of necropsy samples, on the survival and detection of leptospires in vitro, were also explored. In the earlier field investigation, serum, renal tissue and urine were collected from wild mammals, for the detection of pathogenic leptospires by culture, the microscopic agglutination test (MAT), real‐time PCR and silver impregnation of smears. Although 27·6% of the rodents investigated were found leptospire‐positive, culture only yielded four isolates, probably because many cultures were contaminated. The main aims of the present study were to quantify the performance of the individual diagnostic tests and examine the reasons behind the high incidence of culture contamination. The results of sensitivity and specificity analyses for the different diagnostic tests indicated that isolation by culture (the definitive diagnostic test for leptospiral shedding) had perfect (100%) sensitivity when compared with the results of the PCR but a low specificity (40%). The MAT performed poorly, with a sensitivity of 50% when compared against the results of culture. The prevalence of leptospiral carriage revealed by the PCR‐based investigation of kidney and urine samples (59·2%) was higher than that revealed using any other method and far higher than the 2·0% revealed by culture. The results of the culture of renal tissue agreed fairly well with those of the PCR‐based investigation of such tissue, with a Cohen’s unweighted kappa coefficient (κ) of 0·5 (P = 0·04). The levels of agreement between other pairs of tests were generally poor. The presence of pentobarbitone sodium, at final concentrations of 27·8 or 167 mg/ml, did not affect the viability or the detection of leptospires in culture, and is therefore unlikely to reduce the chances of isolating leptospires from an animal that has been euthanized with the compound. It appears that collecting multiple samples from each mammal being checked will improve the chances of detecting leptospires (and reduce the chances of reporting an inconclusive result for any of the mammals). For the identification of a leptospiral carrier, however, the use of just two detection methods (culture and PCR) and one type of sample (renal tissue) may give adequate sensitivity and specificity. Given the robustness of PCR to contamination and its high sensitivity (it can give a positive result when DNA from just two leptospiral cells is present in the sample), a PCR‐based serotyping method, to allow the combined detection and characterisation of leptospires from field isolates, would be extremely useful.

Haemoglobin and red cell counts in leptospirosis patients infected with different serovars

INTRODUCTION The aim of the study was to compare haemoglobin and red cell counts between patients known to be infected with a range of leptospiral serovars. METHODS The study retrospectively compared the haemoglobin and red cell count results from the first blood samples taken from 207 patients at presentation to a Queensland Health hospital. RESULTS Significant differences were observed in haemoglobin and red cell counts in those infected with Leptospira interrogans serovars Szwajizak and Canicola when compared with most of the other serovars. CONCLUSIONS These findings suggest that haemoglobin and red cell counts may be useful in differentiating leptospiral serovars in leptospirosis patients.