A comparison of two molecular methods for diagnosing leptospirosis from three different sample types in patients presenting with fever in Laos.

OBJECTIVES: To compare two molecular assays (rrs quantitative PCR (qPCR) versus a combined 16SrRNA and LipL32 qPCR) on different sample types for diagnosing leptospirosis in febrile patients presenting to Mahosot Hospital, Vientiane, Laos. METHODS: Serum, buffy coat and urine samples were collected on admission, and follow-up serum ∼10 days later. Leptospira spp. culture and microscopic agglutination tests (MAT) were performed as reference standards. Bayesian latent class modelling was performed to estimate sensitivity and specificity of each diagnostic test. RESULTS: In all, 787 patients were included in the analysis: 4/787 (0.5%) were Leptospira culture positive, 30/787 (3.8%) were MAT positive, 76/787 (9.7%) were rrs qPCR positive and 20/787 (2.5%) were 16SrRNA/LipL32 qPCR positive for pathogenic Leptospira spp. in at least one sample. Estimated sensitivity and specificity (with 95% CI) of 16SrRNA/LipL32 qPCR on serum (53.9% (33.3%-81.8%); 99.6% (99.2%-100%)), buffy coat (58.8% (34.4%-90.9%); 99.9% (99.6%-100%)) and urine samples (45.0% (27.0%-66.7%); 99.6% (99.3%-100%)) were comparable with those of rrs qPCR, except specificity of 16SrRNA/LipL32 qPCR on urine samples was significantly higher (99.6% (99.3%-100%) vs. 92.5% (92.3%-92.8%), p <0.001). Sensitivities of MAT (16% (95% CI 6.3%-29.4%)) and culture (25% (95% CI 13.3%-44.4%)) were low. Mean positive Cq values showed that buffy coat samples were more frequently inhibitory to qPCR than either serum or urine (p <0.001). CONCLUSIONS: Serum and urine are better samples for qPCR than buffy coat, and 16SrRNA/LipL32 qPCR performs better than rrs qPCR on urine. Quantitative PCR on admission is a reliable rapid diagnostic tool, performing better than MAT or culture, with significant implications for clinical and epidemiological investigations of this global neglected disease.

Serological diagnosis of Leptospirosis in bovine serum samples using a microsphere immunoassay.

Leptospirosis causes significant economic loss within the cattle industry worldwide. Current diagnostic methods are generally inadequate for dealing with large numbers of samples, are outdated, and provide little useful diagnostic and epidemiological information. This aim of this study was to apply a microsphere immunoassay (MIA), utilising Luminex xMap technology, to 200 bovine serum samples to determine this method's usefulness in leptospirosis diagnosis in comparison with the current gold standard, the microscopic agglutination test (MAT). Although MAT is the most widely used laboratory test for the diagnosis of leptospirosis, its reliance on live cultures, subjective interpretation of results and an inability to differentiate between antibody classes, suggest MAT is no longer the best method for the diagnosis of leptospirosis. The results presented in this paper show that MIA was able to determine reactive from non-reactive samples when compared with MAT, and was able to differentiate IgG and IgM classes of antibody. The results suggest increased sensitivity in MIA and the ability to multiplex up to 500 antigens at one time allows for significant improvements in cost-effectiveness as well as a reduced dependency on live cultures. The relatively low cost, high throughput platform and differentiation of antibody class, as shown in previous research, make this assay worthy of consideration for the diagnosis of leptospirosis in small-scale or large-scale bovine populations.

The emergence of Leptospira borgpetersenii serovar Arborea as the dominant infecting serovar following the summer of natural disasters in Queensland, Australia 2011.

The following research reports the emergence of Leptospira borgpetersenii serovar Arborea as the dominant infecting serovar following the summer of disasters and the ensuing clean up in Queensland, Australia during 2011. For the 12 month period (1 January to 31 December) L. borgpetersenii serovar Arborea accounted for over 49% of infections. In response to a flooding event public health officials need to issue community wide announcements warning the population about the dangers of leptospirosis and other water borne diseases. Communication with physicians working in the affected community should also be increased to update physicians with information such as clinical presentation of leptospirosis and other waterborne diseases. These recommendations will furnish public health officials with considerations for disease management when dealing with future disaster management programs.

Neutrophil counts in leptospirosis patients infected with different serovars.

In leptospirosis patients, haematological abnormalities have been reported. The aim of this study was to determine if neutrophil counts were different between patients known to be infected with a range of leptospiral serovars. The study retrospectively compared the neutrophil counts from the first blood samples taken from 210 leptospirosis patients at first presentation to a Queensland Health hospital. Significant differences (p <0.001) were observed in neutrophil counts across the 11 different infecting serovars. These findings suggest that neutrophil counts may be useful in the development of an algorithm determining the infecting serovar in suspected leptospirosis patients. Further studies are required to delineate host cytokine responses which may suggest the underlying aetiology of the observed differences in neutrophil counts. Such studies would also provide valuable therapeutic insights into treating the disease.

Equivalent metabolic acidosis with four colloids and saline on ex vivo haemodilution.

Colloid infusions can cause metabolic acidosis. Mechanisms and relative severity with different colloids are incompletely understood. We compared haemodilution acid-base effects of 4% albumin, 3.5% polygeline, 4% succinylated gelatin (all weak acid colloids, strong ion difference 12 mEq/l, 17.6 mEq/l and 34 mEq/l respectively), 6% hetastarch (non-weak acid colloid, strong ion difference zero) and 0.9% saline (crystalloid, strong ion difference zero). Gelatin weak acid properties were tracked via the strong ion gap. Four-step ex vivo dilutions of pre-oxygenated human venous blood were performed to a final [Hb] near 50% baseline. With each fluid, base excess fell to approximately -13 mEq/l. Base excess/[Hb] relationships across dilution were linear and direct (R2 > or = 0.96), slopes and intercepts closely resembling saline. Baseline strong ion gap was -0.3 (2.1) mEq/l. Post-dilution increases occurred in three groups: small with saline, hetastarch and albumin (to 3.5 (02) mEq/l, 4.3 (0.3) mEq/l, 3.3 (1.4) mEq/l respectively), intermediate with polygeline (to 12.2 (0.9) mEq/l) and greatest with succinylated gelatin (to 20.8 (1.4) mEq/l). We conclude that, despite colloid weak acid activity ranging from zero (hydroxyethyl starch) to greater than that of albumin with both gelatin preparations, ex vivo dilution causes a metabolic acidosis of identical severity to saline in each case. This uniformity reflects modifications to the albumin and gelatin saline vehicles, in part aimed at pH correction. By proportionally increasing the strong ion difference, these modifications counter deviations from pure saline effects caused by colloid weak acid activity. Extrapolation in vivo requires further investigation.

Stability of the strong ion gap versus the anion gap over extremes of PCO2 and pH.

The strong ion gap (SIG) is under evaluation as a scanning tool for unmeasured ions. SIG is calculated by subtracting [buffer base], which is ([A-]+[HCO3-), from the apparent strong ion difference, which is ([Na+]+[K+]+[Ca++]+[Mg++]-[Cl-]-[L-lactate]). A- is the negative charge on albumin and phosphate. We compared the pH stability of the SIG with that of the anion gap (AG). In normal and hypoalbuminaemic hyperlactaemic blood, PCO2 was reduced stepwise in vitro from >200 mmHg to <20 mmHg, with serial blood gas and electrolyte analyses, and [albumin] and [phosphate] measurement on completion. Respective [haemoglobin], [albumin], [phosphate] and [lactate] in normal blood were 156 (0.9) g/l, 44 (2) g/l, 1.14 (0.06) mmol/l and 1.7 (0.8) mEq/l, and in hypoalbuminaemic blood 116 (0.9) g/l, 24 (2) g/l, 0.78 (0.06) mmol/l and 8.5 (0.5) mEq/l. pH increased from < 6.85 to > 7.55, causing significant falls in [Na+] and elevations in [Cl-]. Initial and final SIG values did not differ, showing no correlation with pH. Mean SIG was 0.5 +/- 1.5 mEq/l. AG values were directly correlated with pH (normal: R2 = 0.51, hypoalbuminaemic: R2 = 0.65). Final AG values significantly exceeded initial values (normal blood: 15.9 (1.7) mEq/l versus 8.9 (1.8) mEq/l, P < 0.01; hypoalbuminaemic blood: 16.5 (0.8) mEq/l versus 11.8 (2.0) mEq/l, P < 0.05). We conclude that, unlike the AG, the SIG is not affected by severe respiratory acidosis and alkalosis, enhancing its utility in acid-base disturbances.