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    Diagnostic tests by sample type.

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    Area under the receiver operating characteristic curve of the Panbio Leptospira IgM ELISA vs. the PCR assay.

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Evaluation of the Panbio Leptospira IgM ELISA among Outpatients Attending Primary Care in Southeast Asia

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  • 1 Mahidol-Oxford Tropical Research Medicine Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand;
  • 2 Nuffield Department of Medicine, Nuffield Department of Medicine Research Building, Centre for Tropical Medicine and Global Health, University of Oxford, Oxford, United Kingdom;
  • 3 Department of Livestock Development, National Institute of Animal Health, Ministry of Agriculture and Cooperatives, Bangkok, Thailand;
  • 4 Lao-Oxford-Mahosot Hospital-Wellcome Trust Research Unit, Microbiology Laboratory, Mahosot Hospital, Vientiane, Lao People’s Democratic Republic

ABSTRACT

Despite estimates suggesting Leptospira spp. being endemic in Southeast Asia, evidence remains limited. Diagnostic accuracy evaluations based on Leptospira ELISA mainly rely on hospitalized and severe patients; therefore, studies measuring the pathogen burden may be inaccurate in the community. We evaluated the Panbio Leptospira ELISA IgM among 656 febrile outpatients attending primary care in Chiangrai, Thailand, and Hlaing Tha Yar, Yangon, Myanmar. ELISA demonstrated limited diagnostic accuracy for the detection of acute leptospiral infection using the manufacturer recommended cutoff, with a sensitivity of 71.4% and specificity of 36.4%, and an area under the receiver operator characteristic curve value of 0.65 (95% CI: 0.41–0.89), compared with our reference test, the PCR assay. ELISA also performed poorly as a screening tool for detecting recent exposure to Leptospira spp. compared with the “gold-standard” microscopic agglutination test, with a specificity of 42.7%. We conclude that the utility of the Leptospira IgM ELISA for both serodiagnosis and seroprevalence is limited in our setting.

INTRODUCTION

Leptospirosis is a widespread but neglected zoonotic disease caused by a bacterium of the genus Leptospira.1,2 Leptospirosis contributes to approximately 48,000 annual deaths globally,35 with 500,000 cases estimated worldwide,6 and remains a major public health concern in tropical and subtropical regions.7 Agriculturers and animal farmers are at a higher risk, but also in case of poor sanitation,2 with evidence of pathogenic Leptospira recently reported in urban poor areas.2,710 The endemicity of leptospirosis is primarily localized to Southeast Asia, where the burden of the disease is concentrated.11,12 In Thailand, it is a major public health concern with several outbreaks reported annually,11,13 and a background prevalence persisting at five cases per 100,000 population per year according to the CDC.13 Moreover, a recent study at the Thai–Myanmar border established leptospirosis as the second most common cause of undifferentiated febrile illness in this region,14 suggesting that the disease may be endemic in Myanmar as well.

However, underdiagnosis and underreporting of leptospirosis remain considerable problems. Clinical presentation is not specific, overlapping with common tropical pathogens such as dengue virus or other hemorrhagic febrile illnesses.6 Furthermore, Leptospira spp. is inconsistently integrated in Southeast Asian national surveillance, without standardized laboratory data collection nor reporting systems, explaining gaps in the disease true burden estimate.12

The serological “gold standard” for identifying pathogenic Leptospira is the microscopic agglutination test (MAT); however, it requires significant technical expertise and the maintenance of viable Leptospira serovars. Microscopic agglutination test is often not beneficial for acute patient management, mainly because it relies on immunoglobulins which react 5 days after the exposure. This is illustrated by a sensitivity of approximately 50%.7,15,16 On the other hand, MAT is serovar specific, with specificity ranging from 94 to 97%.1619 Microscopic agglutination test is also considered the “gold standard” for seroprevalence studies and epidemiological surveys, where exposure to Leptospira spp. is measured instead of overt disease.20,21 Given the high specificity of MAT, estimates of exposure are likely to be very accurate.

Molecular methods such as the PCR may be of greater performance for the diagnosis of acute leptospirosis, by improving the Leptospira spp. early phase detection with a sensitivity ranging from 51 to 100%.2,22,23 Based on PCR, a multicountry study based in Africa reported a sensitivity near 100% with 95% specificity using the TaqMan Array Card (TAC) assay, compared with MAT and single-plex PCR-confirmed samples,24 hence indisputable evidence of acute infection.25

An alternative diagnostic strategy relies on the ELISA, which is widely used for detecting Leptospira spp. antibodies. ELISA IgM antibodies are detectable 5 days after exposure and only persist for a few months, suggesting a potential application both in acute diagnosis and recent exposure to Leptospira spp., whereas the procedure is simple to perform with results available in around 2 hours.26

For serodiagnosis purpose, diagnostic accuracy evaluations have shown inconsistent sensitivity and specificity.5,23,27,28 This lack of consistency may be explained by the specific interaction between the background immunity of a given population and the exposure chronicity.15,29 In Southeast Asia, recent studies have confirmed broad variations in diagnostic accuracy, with a sensitivity ranging from 36.0% to 60.9% and a specificity ranging from 41% to 98.0%.15,2931 These variations also reflect imperfect reference methods, such as the MAT or blood culture: performances of which fluctuate depending on symptom onset and diagnostic cutoff.27 Furthermore, most samples included in ELISA diagnostic evaluations originate from severe hospitalized patients, implying particular immunological profiles and limited application to leptospirosis with a mild presentation. A single study recruited outpatients in southern Vietnam, and attributed the diagnosis of acute leptospirosis without any reference method.30 Therefore, the utility of commercial IgM ELISA kits for serodiagnosis of Leptospira spp. remains unclear, particularly among non-severe outpatients attending the primary levels of care.

ELISA has also been used for seroprevalence purposes, with the objective to screen for exposure to Leptospira spp.21,3237 Alarmingly, a single study evaluated the ELISA diagnostic accuracy for measuring such exposure in Southeast Asia, using MAT as a reference test and reporting 24.3% sensitivity and 93.5% specificity.20 In this Malaysian study, samples were exclusively taken from a particular subpopulation of market workers and food handlers, whereas children were excluded from the evaluation. Whether IgM ELISA could be an accurate screening tool for recent exposure to pathogenic Leptospira in the general population attending primary care is unknown.

In this study, we report the diagnostic accuracy of the commercial Leptospira IgM ELISA (Panbio Pty., Ltd., Queensland, Australia) compared with single-plex PCR and TAC assays among both children and adults attending primary care in Chiang Rai, Thailand, and Yangon, Myanmar. We also measure exposure to Leptospira spp. using the MAT, and calculate the corresponding IgM ELISA specificity as a screening tool for recent exposure.

METHODS

Study sites.

Samples were analyzed from the CRP Study, which was conducted in 10 primary care centers, of which six were located in Chiang Rai Province, northern Thailand and four in Hlaing Tha Yar, Yangon, Myanmar.38 Chiang Rai Province is located in the north of Thailand and borders Myanmar and Lao People’s Democratic Republic (PDR). The sites included in this study were within a 30-km radius from Chiang Rai city center, and included rural, peri-urban, mountainous, and plateau areas. Hlaing Tha Yar is a peri-urban slum in the west of Yangon Myanmar. All study sites are defined by their tropical climate, with a population composed by ethnic minorities.

Patient details.

Patients (≥ 1 year) were recruited between June 2016 and August 2017. Inclusion criterion was a documented fever (tympanic temperature > 37.5°C) or a complaint of acute fever (< 14 days). Patients were excluded if they presented with symptoms requiring hospital referral such as impaired consciousness, inability to take oral medication, etc. On enrollment, the patients’ demographic information was collected, and a routine clinical examination was performed.

Leptospira testing.

Of 2,392 primary care children and adults recruited in the original trial, 799 were randomized in a control group for whom various samples were collected.38 From these 799 outpatients, 740 had a blood sample collected. Samples were stored onsite at −80°C and then shipped to the Mahidol-Oxford Tropical Medicine Research Unit and National Institute of Animal Health for microbiological investigations. A commercial ELISA (Panbio Pty., Ltd.) was used for the detection of IgM antibodies against Leptospira spp. The Manufacturer’s specifications were followed with Panbio units of ≥ 11.0 considered positive. Positive Leptospira IgM ELISA samples were then tested by MAT, with titers of ≥ 1:100 used to classify past exposure to leptospirosis.20,21,3941 TaqMan Array Card and single-plex PCR assays were performed on 601 blood samples where sufficient plasma volumes were able to be extracted. The TAC assay targeted all pathogenic serovars of the Leptospira genus, whereas the single-plex PCR used real-time methodology to target the rss gene4245 (Figure 1).

Figure 1.
Figure 1.

Diagnostic tests by sample type.

Citation: The American Journal of Tropical Medicine and Hygiene 104, 5; 10.4269/ajtmh.20-0818

Statistical analysis.

Diagnostic accuracy was calculated for ELISA IgM to the single-plex PCR and TAC assays. Microscopic agglutination test was only performed on samples with a positive IgM ELISA reading, with specificity calculated for seroprevalence accuracy measurement.

Standard diagnostic accuracy indices of sensitivity, specificity with exact 95% CIs, as well as area under the receiver operator characteristic curves (AUROCCs) were calculated using Stata/IC 15.0 (StataCorp LP, College Station, TX). An area of > 0.8 was considered good, 0.7–0.8 was considered fair, 0.5–0.6 was considered poor, and ≤ 0.5 was considered diagnostically uninformative.4648

RESULTS

Blood samples were collected from children and adults in Thailand and Myanmar and their baseline characteristics are presented in Supplemental Table 1. Of the 740 blood samples collected, 656 (88.6%) had a sufficient volume for the current analysis. The proportion of patients with a confirmed acute leptospirosis infection as defined by either single-plex or TAC PCR assay was 1.1% (7/656), whereas 23.5% (154/656) were seropositive by IgM ELISA. The AUROCC analysis was 0.65 (95% CI: 0.41–0.89) (Supplemental Table 2), suggesting the IgM ELISA to be diagnostically poor (Figure 2).

Figure 2.
Figure 2.

Area under the receiver operating characteristic curve of the Panbio Leptospira IgM ELISA vs. the PCR assay.

Citation: The American Journal of Tropical Medicine and Hygiene 104, 5; 10.4269/ajtmh.20-0818

The IgM ELISA cutoff of 11 Panbio units (as per manufacturer’s specifications) showed sensitivity and specificity of 71.4% and 36.4%, respectively. Increasing the percentage of patients correctly classified to > 95% resulted in an improved specificity of 96.0% at a cutoff optical density (OD) of 1.56; however, the sensitivity was reduced to 14.3% (Supplemental Table 2).

Of the 154 positive IgM ELISA samples, all had a sufficient blood volume to be tested for the MAT. Of these, exposure as defined by MAT ≥ 1:100 was confirmed in 47 (30.5%) samples, including 30 (63.8%, 30/47) from Thailand and 17 (36.2%, 17/47) from Myanmar, and this difference was significant (P-value 0.030). A titer of ≥ 1:100 was seen in 35 samples (74.5%, 35/47), 10 samples (23.8%, 10/42) showed a titer ≥ 1:200, and two (4.3%, 2/47) showed a titer of ≥ 1:800.

The specificity of IgM ELISA as a screening tool for recent exposure against MAT (defined as ≥ 1:100) was 39.9%, using the manufacturer’s recommendations. The highest specificity was measured at 98.0%, using a cutoff OD of 1.73.

DISCUSSION

We measured poor serodiagnostic performance from the Leptospira IgM ELISA compared with PCR assays on a single acute sample, considering outpatient children and adults attending primary care in Thailand and Myanmar. A similar finding was reported in Vietnamese clinics among non-severe patients; however, authors used an in-house ELISA as a reference method, challenging the interpretation of serodiagnostic accuracy.30 Similar performance has been reported in Lao PDR, with sensitivity of 95% and specificity of 41%.29 However, samples originated from hospitalized and severe patients and relied on a suboptimal MAT dilution titer (i.e., ≥ 1:400). Other factors may explain such poor specificity for serodiagnosis, including the persistence of Leptospira IgM antibody for many months post-recovery,18 especially when PCR assays are used as a reference method. Nonetheless, cross-reactions to nonpathogenic Leptospira have been reported using the ELISA.30

We also detected a substantial exposure in the community to pathogenic Leptospira using MAT (30.5%). This indicates Leptospira to be an ubiquitous pathogen in Southeast Asia, which should be particularly highlighted in Myanmar where seroprevalence data were limited to animals before our study.12 There was a significant difference in exposure between Thailand and Myanmar, confirming the prevalence of Leptospira in rural environments.4

Using our seroprevalence data from the MAT, we evaluated whether IgM ELISA could be used as a screening tool for recent exposure to pathogenic Leptospira.33,40,49 Before our study, a single seroprevalence survey assessed ELISA accuracy against the MAT in Southeast Asia, reporting a specificity of around 93%.20 In this Malaysian study, the target population was healthy wet market workers and food handlers. Our lower specificity may be explained by differences in clinical status: serodiagnosis studies enrolling ill patients clinically suspected of leptospirosis similarly described lower specificities than in Malaysia.16,30,32 Adding low specificity measured in hospital-based studies to our own primary care-based evaluation, it is therefore unlikely that IgM ELISA may be useful for measuring a recent exposure to pathogenic Leptospira among febrile patients, regardless of the level of care.

Our study also suggests the ELISA diagnostic accuracy to be higher when the IgM cutoff is optimized using ROC curve analysis rather than based on the manufacturer’s specifications. Further studies may aim to determine region-specific cutoffs in leptospirosis-endemic and non-endemic settings. However, our findings, combined with existing evidence, showed IgM ELISA to not be a promising candidate for diagnosing acute leptospirosis infection nor screening for recent leptospirosis exposure, regardless of the cutoff.

There were several limitations to our study. First, only acute samples were obtained, limiting the full serodiagnosis evaluation of Leptospira IgM ELISA. Second, the number of Leptospira spp.–confirmed cases using PCR assays was low, which may be attributed to the inclusion of non-severe patients with low bacterial loads, which lowered our detection sensitivity.50 Furthermore, the TAC assay, although highly specific, has shown low sensitivity in detecting bacterial pathogens.51,52 It has to be clarified that an IgM ELISA was used to test for seroprevalence and not IgG, whereas the MAT is composed of acute and chronic immunoglobulins. A peak in IgM is detected during early stages of infection and lasts for a few months, suggesting the measurement of a recent rather than past exposure.53 Finally, we did not test for MAT samples that were negative using IgM ELISA, and were therefore not able to calculate sensitivity.

CONCLUSION

The IgM ELISA, although simple and widely used, was found to be unsuitable for detecting acute leptospirosis infection among outpatient children and adults attending primary care, using PCR assays as a reference method. Its diagnostic accuracy may be improved by optimizing the cutoff based on a ROC curve instead of manufacturer’s specifications. Even so, it is unlikely that such test, used on an acute sample, may represent an attractive candidate for the diagnosis of acute leptospirosis. Similarly, the IgM ELISA was not specific as a screening method for recent exposure to pathogenic Leptospira. Using the MAT, we demonstrated a significant exposure to pathogenic Leptospira among Thai and Myanmar communities, including rural and semi-urban settings.

Supplemental tables

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Author Notes

Address correspondence to Stuart D. Blacksell, Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, Thailand. E-mail: stuart@tropmedres.ac

Disclosure: S. D. B. and Yoel Lubell are supported by the Wellcome Trust of the United Kingdom.

Authors’ addresses: Sandhya Dhawan, Mahidol-Oxford Tropical Research Medicine Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand, E-mail: sandhya@tropmedres.ac. Thomas Althaus and Yoel Lubell, Mahidol-Oxford Tropical Research Medicine Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand, and Nuffield Department of Medicine, Nuffield Department of Medicine Research Building, Centre for Tropical Medicine and Global Health, University of Oxford, Oxford, United Kingdom, E-mails: thomas.a@tropmedres.ac and yoel@tropmedres.ac. Duangjai Suwancharoen, Department of Livestock Development, National Institute of Animal Health, Ministry of Agriculture and Cooperatives, Thailand, E-mail: dj90705@hotmail.com. Stuart D. Blacksell, Mahidol-Oxford Tropical Research Medicine Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand, Nuffield Department of Medicine, Nuffield Department of Medicine Research Building, Centre for Tropical Medicine and Global Health, University of Oxford, Oxford, United Kingdom, and Lao-Oxford-Mahosot Hospital-Wellcome Trust Research Unit, Microbiology Laboratory, Mahosot Hospital, Vientiane, Lao People’s Democratic Republic, E-mail: stuart@tropmedres.ac.

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