We describe the direct clinical application of molecular based techniques to the diagnosis of acute leptospirosis in the resource-poor setting of rural Sri Lanka. The data and analysis here allow us to describe several important advances: (1) that a new qPCR protocol to diagnose acute leptospirosis is sensitive (often more than acute or retrospective gold-standard MAT serology); (2) that the window of positivity for qPCR in human clinical samples is longer than previously known (up to 15 days); and (3) that serum may be better than whole blood as a specimen type for the molecular diagnosis of acute human leptospirosis. These results have important implications not only for the diagnosis of acute leptospirosis but also for making new inferences into the pathogenesis of this disease.
Evidence suggests that although the MAT is considered to be the gold standard diagnostic test for leptospirosis, MAT sensitivity and specificity are often <100% because of limitations of knowing the regionally specific serovars [18
]. Our initial comparison data have confirmed these findings; we show through sequencing that 2 patients with a negative MAT and detected by qPCR and STNPCR were confirmed to have had leptospirosis despite negative MAT. Indeed, 6 MAT-negative serum samples later proved positive by qPCR and STNPCR and were identified as L. interrogans
. A likely explanation could be that local serovars or serogroups in Sri Lanka are not represented in the panel used in the diagnosis, although it contained the strains isolated from Sri Lanka as well as common strains to represent a broad panel of serovars. Alternatively, some patients may simply be unable to make detectable antibody responses to leptospiral lipopolysaccharide. Nonetheless, regional differences in leptospiral biodiversity will always present a problem for serological diagnosis in geographically disparate regions. On the other hand, by their nature structural RNAs and essential core genes are less variable and are therefore more amenable to broad-range strain detection. Indeed Weisburg et al [20
] have described several generic 16S primers that amplify several bacterial genera. Because MAT has been the standard reference laboratory confirmation procedure, our study findings raise the question about actual sensitivity of MAT [21
]. This observation further confirms the need for more sensitive diagnostic tests that are available at point of care [22
]. We found, during the acute phase of leptospirosis (up to 15 days in this study), the diagnostic sensitivity of qPCR was nearly 3 times higher than acute phase MAT test, considering 400 as the cutoff. This finding strengthens the validity of qPCR as a valid diagnostic tool over serology-based methods. However, the sensitivity and specificity of qPCR are still suboptimal, and further work is needed to improve the application of this technique to the diagnosis of leptospirosis.
Contrary to the previous reports that the leptospiral load is a main predictive factor of severity of illness, our study showed that high levels of leptospiremia—comparable with previous reports of severe/fatal leptospirosis—could occur without such complications. Previous studies [24
] observed that >104
organisms in bloodstream was a critical factor associated with severe pulmonary manifestations and death. In our study, 17 patients had bacterial load of >104
organisms/mL in whole blood/serum yet did not have severe complications. The difference in our observation from 2 previously published studies may be due to variability of virulence among different leptospiral species/serovars/strains. Even within our study sample, complications with low bacterial load and not having complications with high level of bacterial load could be due to different serovars that manifest in different ways. On the other hand, complications with a bacterial load of 103
per mL is difficult to interpret because the timing of sample might not been at peak leptospiremia. However, the number of patients in each complication category was small, and further studies are needed to strengthen this observation.
In this study, serum was a better specimen type than whole blood for obtaining DNA for diagnostic qPCR. However, rather than reflecting intrinsic differences between samples, this observation could reflect the fact that that there could be 2–3 times as much leptospiral DNA per microliter of serum compared with whole blood, assuming Leptospira
are only found in serum. However, this is an unlikely explanation because phagocytosed Leptospira
appear to be concentrated in the buffy coat [5
]. Moreover, most serum-positive/whole blood-negative samples were well above the theoretical limit of detection of the qPCR (3 × 102
per mL under the conditions used). Thus, it is more likely that PCR inhibitors in blood such as heme/heme derivatives [26
], host leukocyte DNA [27
], or added anticoagulants [28
] present in whole blood samples reduced the efficiency of qPCR amplification of leptospiral DNA. In this comparison we used only sensitivity but not specificity due to the limitation of study design and leptospirosis diagnostics we used for comparison. First, the control group was not ideal “controls” but febrile patients who could be “possible” cases of leptospirosis. Although these patients showed negative results in paired sample MAT, it is difficult to exclude the diagnosis because MAT is not 100% sensitive, as confirmed in this study. Further, the ideal test for assessing sensitivity of any diagnostic test would be a direct demonstration of the presence of Leptospira
in acute blood samples such as by a positive blood culture. However, in this study, because of limitations of resources, we were unable to perform blood cultures on patient samples; isolation of Leptospira
is nonetheless recognized to be insensitive itself because of the difficulty in isolating fastidious Leptospira
A notable observation made during this work was significantly high leptospiremia among male patients. Epidemiological evidence has consistently reported that males are more often affected by leptospirosis than females; whether there is a biological explanation of this observation or males are simply more often exposed to Leptospira
remains unclear. However, in animal populations where the exposure bias is minimal, males have shown higher seropositivity rate [29
]. Our finding of higher leptospiremia in human males provides evidence for a probable higher biological susceptibility of men for leptospirosis. This preliminary observation needs further investigation.
Limitations of the data from this study should be recognized. First, culture isolation of infecting serovars was not performed. Quantification and clinical correlation of leptospiremia should ideally be performed with systematic serial sampling to determine the peak leptospiremia and correlate it with clinical outcome. It is difficult to do this because it would require withholding of antibiotic treatment. Nonetheless, the diagnostic utility of qPCR was done using acute phase blood sample taken on admission, which reflects clinical practice. The molecular testing we performed did not differentiate infecting Leptospira beyond the species level. We did attempt to carry out multilocus sequence typing (MLST) on the qPCR-positive specimens in this study, 12 of which preliminarily suggested the presence of a limited number of sequence types (ST) similar to ST 1 (n = 11) and ST44 (n = 1) (unpublished observations). Finally, the samples for this study were obtained in 2008–2009, whereas the qPCR was done in 2011. During this period, samples were thawed at least 3–4 times, which might have reduced the sensitivity of qPCR.
The most important conclusion that we draw from this study is that qPCR is promising as a rapid diagnostic tool in the diagnosis of acute leptospirosis with a wide window of positivity. However, the technical expertise and cost needed for qPCR still impede its use in resource-poor settings.