Quantitative Polymerase Chain Reaction: Filling the Gap for Early Leptospirosis Diagnosis

(See the Major Article by Agampodi et al, on pages 1249–55.) Leptospirosis is considered to be the most globally widespread bacterial zoonotic illness. It has been classified as an emerging or reemerging infectious disease by the Centers for Disease Control and Prevention and the World Health Organization (WHO) [1, 2]. Because its greatest impact is on resource-poor, developing countries, affecting rural subsistence farmers and urban slum dwellers with limited access to basic medical services, the WHO also considers leptospirosis to be a neglected zoonotic disease [3, 4]. The nonspecific and protean manifestations of leptospirosis mimic those of many other infections. Common signs and symptoms include fever, headache, myalgia, arthralgia, nausea, vomiting, and diarrhea, but patients may also present with jaundice, oliguria or anuria, nuchal rigidity, myocarditis, or pulmonary hemorrhage [1, 5–8]. The differential diagnosis is broad and includes influenza, aseptic meningitis, encephalitis, gastroenteritis, hepatitis, dengue, malaria, and hantavirus infection [1, 6–9]. Early recognition and prompt administration of appropriate antimicrobial therapy can ameliorate symptoms and shorten the duration of illness [1, 7]. However, early diagnosis has been a perennial challenge. Leptospires are detectable in the blood during the first week (acute leptospiremic phase) of illness, but concurrent with an increase in immunoglobulin M (IgM) concentration, the leptospires are cleared and become nondetectable. Culture and isolation are difficult because leptospires are fastidious, slow-growing organisms that require special medium, and culture is labor-intensive and requires weekly dark-field microscopic examinations for up to 16 weeks before specimens may be discarded as negative [6, 8, 9]. Serology has been the most common diagnostic approach to leptospirosis. The reference standard serologic test, the microscopic agglutination test (MAT), is technically challenging and relies on the maintenance of a battery of live test antigens including all locally common enzootic strains. Laboratory confirmation generally requires demonstration of seroconversion or a ≥4-fold MAT titer increase between paired acute- and convalescent-phase serum specimens [1, 6–9]. The need for paired specimens only allows for delayed results. Because of the complexity of the MAT, its use is limited to a finite number of laboratories including WHO reference laboratories [7, 8]. Rapid serologic tests are available, but they are primarily IgM detection assays, and because IgM is not detectable until days 5–7 after symptom onset, these assays have demonstrated low sensitivity in the early acute phase of illness [9, 10]. The development of accurate molecular-based detection methods has been a major advance in enabling early diagnosis. Real-time quantitative polymerase chain reaction (qPCR) assays have redefined the reference standard as their sensitivity greatly exceeds that of culture and they can detect low levels of leptospires in the acute leptospiremic phase of illness, when diagnosis is most needed. In this issue of Clinical Infectious Diseases, Agampodi and colleagues have convincingly demonstrated the clinical strengths of the qPCR assay and documented inherent weaknesses with the MAT [11]. Although the MAT has been considered the reference serologic test for leptospirosis, it is well known that false-negative results may occur if the infecting serovar is missing from the antigen test battery [12] or in cases of delayed seroconversion [6]. The antibody response can also be affected by early treatment [13]. Hence, the approach taken by Agampodi and colleagues to use the MAT as the reference test to assess the sensitivity but not the specificity of the qPCR test was an epidemiologically sound methodologic decision. The authors recognize that MAT documentation of seroconversion or increasing antibody titers in paired serum specimens provides strong evidence for the presence of a true infection, whereas the lack thereof does not equate to absence of infection. Indeed, the authors clearly documented false-negative MAT results for 6 patients who tested positive by qPCR and single-tube nested PCR with sequencing confirmation. Agampodi and colleagues calculated the sensitivity of the qPCR test (when applied to serum) to be 51%, using as the reference standard a diagnostic MAT result (defined as paired serum specimens demonstrating either seroconversion or a ≥4-fold titer increase) when the acute-phase serum specimen was collected within 10 days after symptom onset, the convalescent-phase serum specimen was collected after day 14, and the 2 specimens were collected at least 7 days apart. Although a sensitivity of 51% is not optimal, it is an improvement over existing acute-phase diagnostic tests and should be seen in perspective. Cultures are not useful for early diagnosis for reasons mentioned above. Although diagnostic MAT results generally depend on paired serum specimens and hence are also not useful for early diagnosis, laboratory case definitions have been developed for single (unpaired) MAT results. The WHO Leptospirosis Burden Epidemiology Reference Group recently defined an MAT titer of ≥1:400 in a single serum specimen as evidence supporting laboratory confirmation [4]. In the present study, Agampodi and colleagues found the sensitivity of a single acute-phase serum specimen with an MAT titer of ≥1:400, collected within the first 15 days after symptom onset, to be only 17%. Rapid serologic screening tests have also demonstrated low sensitivities when applied during the acute phase of disease. A recent study assessing the performance of 8 rapid serologic screening tests showed all but 1 to have sensitivities of <25% for specimens collected during the first week of illness. The single exception had a specificity of only 10% and hence was considered to be unacceptable for clinical use [10]. In addition to improved sensitivity, the qPCR assay was demonstrated to have a wider window of positivity to detect leptospirosis cases. Although qPCR positivity peaked from day 4 to day 8 after symptom onset, cases were detected up to 15 days after symptom onset with no significant difference in qPCR sensitivity for specimens collected before day 10 after symptom onset. The finding of Agampodi and colleagues that quantitative leptospiremic loads do not seem to be associated with disease severity is interesting, but it may be explained by the small sample sizes: of 58 patients for whom bacterial loads were assessed, only 18 had cases classified as “complicated.” The patients without complications did indeed show lower leptospiremic loads than those with complications, but this finding was statistically insignificant. In addition, as recognized by the authors, explanations for their findings might be related to the timing of specimen collection or variability in the virulence of different serovars. Future studies should assess bacterial load in relationship to illness severity to confirm these results. The finding that male patients had significantly higher Leptospira bacterial loads was another interesting finding. Most leptospirosis surveillance reports have demonstrated a consistent male predominance. This has been attributed to sex differences in occupational and recreational exposures. However, male predominance has not been demonstrated in population-based seroprevalence studies. Jansen and colleagues explain this finding as being due to a sex difference in disease severity rather than a sex difference in exposure or infection probability. They note that most surveillance systems are based on passive reporting and will be biased toward inclusion of more severe cases, as mild or subclinical cases may not present to a healthcare provider and hence will go unrecognized and unreported [14]. The findings of Agampodi and colleagues provide a biological explanation for sex differences in disease severity and help explain the male predominance observed in leptospirosis case series and surveillance reports. In conclusion, Agampodi and colleagues definitively show the qPCR assay to offer substantive improvements for early leptospirosis diagnosis. Solly Faine, in the seminal text on leptospirosis, noted: “Better bedside tests are required, that will recognize an illness as leptospirosis in humans or animals earlier than serological methods” [13]. The study by Agampodi and colleagues has clearly documented the potential for qPCR to fill this gap. However, the cost of and requirement for special equipment and technical expertise currently remain as barriers limiting the wide-scale use of qPCR in poor underresourced areas such as Sri Lanka, where leptospirosis is endemic and the need for early diagnosis is greatest. The author certifies no potential conflicts of interest. The author has submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.