Scott B. Craig

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.

A single multilocus sequence typing (MLST) scheme for seven pathogenic Leptospira species.

Background The available Leptospira multilocus sequence typing (MLST) scheme supported by a MLST website is limited to L. interrogans and L. kirschneri. Our aim was to broaden the utility of this scheme to incorporate a total of seven pathogenic species. Methodology and Findings We modified the existing scheme by replacing one of the seven MLST loci (fadD was changed to caiB), as the former gene did not appear to be present in some pathogenic species. Comparison of the original and modified schemes using data for L. interrogans and L. kirschneri demonstrated that the discriminatory power of the two schemes was not significantly different. The modified scheme was used to further characterize 325 isolates (L. alexanderi [nu200a=u200a5], L. borgpetersenii [nu200a=u200a34], L. interrogans [nu200a=u200a222], L. kirschneri [nu200a=u200a29], L. noguchii [nu200a=u200a9], L. santarosai [nu200a=u200a10], and L. weilii [nu200a=u200a16]). Phylogenetic analysis using concatenated sequences of the 7 loci demonstrated that each species corresponded to a discrete clade, and that no strains were misclassified at the species level. Comparison between genotype and serovar was possible for 254 isolates. Of the 31 sequence types (STs) represented by at least two isolates, 18 STs included isolates assigned to two or three different serovars. Conversely, 14 serovars were identified that contained between 2 to 10 different STs. New observations were made on the global phylogeography of Leptospira spp., and the utility of MLST in making associations between human disease and specific maintenance hosts was demonstrated. Conclusion The new MLST scheme, supported by an updated MLST website, allows the characterization and species assignment of isolates of the seven major pathogenic species associated with leptospirosis.

Leptospira kmetyi sp. nov., isolated from an environmental source in Malaysia

A single Leptospira strain (designated Bejo-Iso9(T)) was isolated from a soil sample taken in Johor, Malaysia. The isolate showed motility and morphology typical of the genus Leptospira under dark-field microscopy. Cells were found to be 10-13 microm in length and 0.2 microm in diameter, with a wavelength of 0.5 microm and an amplitude of approximately 0.2 microm. Phenotypically, strain Bejo-Iso9(T) grew in Ellinghausen-McCullough-Johnson-Harris medium at 13, 30 and 37 degrees C, and also in the presence of 8-azaguanine. Serologically, strain Bejo-Iso9(T) produced titres towards several members of the Tarassovi serogroup, but was found to be serologically unique by cross-agglutinin absorption test and thus represented a novel serovar. The proposed name for this serovar is Malaysia. Phylogenetic analysis of 16S rRNA gene sequences placed this novel strain within the radiation of the genus Leptospira, with sequence similarities within the range 90.4-99.5% with respect to recognized Leptospira species. DNA-DNA hybridization against the three most closely related Leptospira species was used to confirm the results of the 16S rRNA gene sequence analysis. The G+C content of the genome of strain Bejo-Iso9(T) was 36.2 mol%. On the basis of phenotypic, serological and phylogenetic data, strain Bejo-Iso9(T) represents a novel species of the genus Leptospira, for which the name Leptospira kmetyi sp. nov. is proposed. The type strain is Bejo-Iso9(T) (=WHO LT1101(T)=KIT Bejo-Iso9(T)).

Leptospira wolffii sp. nov., isolated from a human with suspected leptospirosis in Thailand

A single Leptospira strain (designated Khorat-H2(T)) was isolated from the urine of an adult male patient with suspected leptospirosis from the province of Nakornrachasima, Thailand. The isolate showed typical Leptospira motility and morphology under dark-field microscopy. Cells were 10-13 mum long and 0.2 mum in diameter, with a wavelength of 0.5 mum and an amplitude of approximately 0.3 mum. Phenotypically, strain Khorat-H2(T) did not grow at 13 degrees C but grew at 30 and 37 degrees C and in the presence of 8-azaguanine. Serological identification using the microscopic agglutination test revealed that strain Khorat-H2(T) had no cross-reaction with any recognized Leptospira serogroups. Phylogenetic analysis of the 16S rRNA gene sequence placed the novel strain within the radiation of the genus Leptospira, with sequence similarities of 88.1-97.7 % to recognized Leptospira species. DNA-DNA hybridization against the type strains of the three most closely related Leptospira species was used to confirm the results of the 16S rRNA sequence analysis. The G+C content of strain Khorat-H2(T) was 41.8 mol%. On the basis of phenotypic, serological and phylogenetic data, strain Khorat-H2(T) represents a novel species of the genus Leptospira, for which the name Leptospira wolffii sp. nov. is proposed. The type strain is Khorat-H2(T) (=WHO LT1686(T) =KIT Khorat-H2(T)).

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.

Leptospirosis from water sources

Abstract Leptospirosis outbreaks have been associated with many common water events including water consumption, water sports, environmental disasters, and occupational exposure. The ability of leptospires to survive in moist environments makes them a high-risk agent for infection following contact with any contaminated water source. Water treatment processes reduce the likelihood of leptospirosis or other microbial agents causing infection provided that they do not malfunction and the distribution networks are maintained. Notably, there are many differences in water treatment systems around the world, particularly between developing and developed countries. Detection of leptospirosis in water samples is uncommonly performed by molecular methods.

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.

The Aetiologies and Impact of Fever in Pregnant Inpatients in Vientiane, Laos

Introduction Laos has the highest maternal mortality ratio in mainland Southeast Asia and a high incidence of infectious diseases. Globally, malaria has been the pathogen most intensively investigated in relation to impact on pregnancy, but there has been relatively little research on the aetiology and impact of other diseases. We therefore aimed to determine the causes and impact of fever in pregnant women admitted to two central hospitals in Vientiane City, Lao PDR (Laos). Materials and Methods This hospital-based prospective study was conducted in Mahosot Hospital and the Mother and Child Hospital, Vientiane, between 2006 and 2010, with the aim to recruit 250 consenting pregnant women admitted with tympanic temperature ≥37.5°C. Primary outcome was the cause of fever and secondary outcomes were pregnancy outcomes. Specific investigations (culture, antigen, molecular and serological tests) were performed to investigate causes of fever. After discharge, all pregnant women were asked to return for review and convalescence serum on day 10–14 and were monitored until delivery. Principle findings 250 pregnant women were recruited to this study between February 2006 and November 2010. Fifty percent were pregnant for the first time. Their median (range) gestational age on admission was 24 (4–43) weeks. The median (range) tympanic admission temperature was 38.5°C (37.5–40.5°C). Fifteen percent of patients stated that they had taken antibiotics before admission. Headache, myalgia, back pain and arthralgia were described by >60% of patients and 149 (60%) were given a laboratory diagnosis. Of those with confirmed diagnoses, 132 (53%) had a single disease and 17 (7%) had apparent mixed diseases. Among those who had a single disease, dengue fever was the most common diagnosis, followed by pyelonephritis, scrub typhus, murine typhus and typhoid. Patients were also diagnosed with tuberculosis, appendicitis, Staphylococcus aureus septicemia, leptospirosis, Japanese encephalitis virus infection and Plasmodium falciparum malaria. Severe consequences, including maternal death, miscarriage, stillbirth, low birth weight and preterm birth, were found among 28 (78%) mothers with dengue fever, rickettsioses and typhoid. Conclusion Fevers other than malaria, such as dengue, pyelonephritis, rickettsioses and typhoid are common causes of fever during pregnancy in the Asian tropics. Further investigations of their impact in the community on maternal death, fetal loss, vertical transmission, low birth weight and preterm birth are needed.

A Large Leptospirosis Outbreak following Successive Severe Floods in Fiji, 2012

Severe flooding has been linked to outbreaks of leptospirosis. Two sequential extreme flood events in Western Fiji caused the largest outbreak of leptospirosis recorded in the South Pacific, with 1,217 total suspected cases, of which 314 were probable and confirmed. Most (83%) cases occurred within 6 weeks of the flood events, displaying a biphasic epidemic curve associated with the floods. Given the temporal proximity of cases to flooding events, most of the transmission appeared to occur during or immediately after the floods; therefore, prevention of exposure to contaminated environments is a priority in the immediate flood and post-flood period. In addition, genotyping studies suggest that multiple animal reservoirs were implicated in the outbreak, reaffirming the importance of integrated human and animal health strategies for leptospirosis control.