JCM Accepts, published online ahead of print on 11 December 2013 J. Clin. Microbiol. doi:10.1128/JCM.02085-13 Copyright © 2013, American Society for Microbiology. All Rights Reserved.
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A Rapid Quantitative Serological Test To Detect Infection with Mycobacterium leprae, the
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Causative Agent of Leprosy.
3 4
By Malcolm S. Duthie1, Marivic F. Balagon2, Armi Maghanoy2, Florenda M. Orcullo2, Marjorie
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Cang2, Ronaldo Ferreira Dias3, Marco Collovati3 and Steven G. Reed1
6 7 8 9
From the 1Infectious Disease Research Institute, 1616 Eastlake Ave E, Seattle, WA 98102, 2
Leonard Wood Memorial Center for Leprosy Research, Cebu City, Philippines and
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OrangeLife, Rio de Janeiro, Brazil.
10 11
This work was supported by the American Leprosy Missions and the Renaissance Health Service
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Corporation.
13 14
Address correspondence and reprint requests:
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Infectious Disease Research Institute, 1616 Eastlake Ave E, Seattle, WA 98102
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Tel: 206-858-6012; Fax: 206-381-3678; Email address: [email protected]
17 18
Abbreviations used in this paper:
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EC, endemic control
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HHC, healthy household contact
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LID-1, Leprosy IDRI Diagnostic-1
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MB, multibacillary
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MDT, multi-drug therapy
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NEC, non-endemic control
25
PB, paucibacillary
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PGL-I, phenolic glycolipid-I
27
TB, tuberculosis
28 29
Running title: Rapid serology test for leprosy.
30 31 32
Keywords for submission: leprosy; diagnosis; mycobacteria; rapid; quantitative
33 34
Abstract Leprosy remains an important health problem in a number of regions. Early detection of
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infection, followed by effective treatment, is critical to reduce disease progression. New sensitive
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and specific tools for early detection of infection will be a critical component of an effective
37
leprosy elimination campaign. Diagnosis is made by recognizing clinical signs and symptoms
38
but few clinicians are able to confidently identify these. Simple tests to facilitate referral to
39
leprosy experts are not widely available and the correct diagnosis of leprosy is often delayed.
40
Within this report we evaluated the performance of a new leprosy serological test (NDO-LID®).
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As expected, the test readily detected clinically-confirmed MB patients and the rate of positive
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results declined with bacterial burden. The NDO-LID® detected larger proportions of MB and
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PB patients than the alternative SD Leprosy test (87.0% versus 81.7% and 32.3% versus 6.5%,
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respectively) while also demonstrating improved specificity (97.4% versus 90.4%). Coupled
45
with a new cell phone-based test reader platform (Smart Reader®), the NDO-LID® tests
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provided consistent, objective test interpretation that could facilitate wider use in non-specialized
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settings. In addition, results obtained from sera at the time of diagnosis versus the end of
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treatment, indicated that the quantifiable nature of this system can also be used to monitor
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treatment efficacy. Taken together, these data indicate that the NDO-LID®/ Smart Reader®
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system can assist in the diagnosis and monitoring of MB leprosy, as well as detect a significant
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number of earlier stage infections.
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53 54
Introduction Despite advances toward the elimination of leprosy over the last two decades, new case
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detection rates have stabilized over the last decade and leprosy remains an important health
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problem in many regions (36). Like the number of registered cases, however, the number of
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clinicians who can reliably diagnose leprosy has waned, and delays in correct diagnosis are
58
common (6, 30). Efforts to develop and improve surveillance and referral systems appear
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necessary to achieve the early detection required to ensure that prompt and appropriate treatment
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can be provided to limit disabilities. The development of simple and practical tools to facilitate
61
diagnosis would appear prudent.
62
Currently the diagnosis of leprosy is dependent on the appearance and recognition of
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clinical signs and symptoms. When skin-smear or pathology services are available, leprologists
64
utilize the Ridley-Jopling scale to characterize five forms (27, 28). In practice, however, most field
65
programs lack such services and the clinical system suggested by WHO to classify individual
66
patients and to select their treatment regimen is used (35). The WHO system uses skin lesions,
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bacterial positivity by skin smear and the number of involved nerves to group leprosy patients
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into one of two simplified categories; multibacillary (MB) leprosy (typically smear positive or
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more than one nerve involvement or more than 5 lesions) and paucibacillary (PB) leprosy (smear
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negative, none to one nerve involvement, maximum of 5 lesions). In many settings, particularly
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the poorest rural settings where access to experienced leprologists is limited or entirely absent,
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primary care providers cannot identify the signs of leprosy and patients are commonly
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misdiagnosed and mistreated (5, 8, 9). With the delays in diagnosis of MB leprosy, transmission of
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M. leprae from infected individuals to their contacts continues and, in many cases, irreversible
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nerve damage has occurred before they are registered as patients (7, 18, 34). Simple tools that can
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facilitate leprosy diagnosis could help to address this deficit.
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While PB leprosy patients have absent bacterial indices (BI; a measure of the number of
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acid-fast bacilli in the dermis expressed in a logarithmic scale) and generally have low or absent
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anti-M. leprae antibody responses, MB patients do not control bacterial replication and
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demonstrate titers of anti-M. leprae antibodies that correlate with their burdens (27). Phenolic
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glycolipid (PGL)-I is well recognized as a target of the antibody response of leprosy patients, with
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the magnitude of the response strongly correlating with bacterial index (BI) (14). To date, leprosy
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rapid diagnostic tests have comprised of only PGL-I mimetics as the antigenic target (ML Flow
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and Standard Diagnostic, containing tri- or disaccharides NTP and NDO, respectively)(1-3, 33).
85
Using laboratory-based assays our group and others have identified numerous native and
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recombinant proteins recognized by antibodies in MB patient serum (10, 11, 13, 20-22, 31, 32).
87
One example is the chimeric fusion protein Leprosy IDRI Diagnostic (LID)-1, an antigen
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specifically recognized by sera from leprosy patients from geographically and ethnically diverse
89
populations with a direct correlation between seroreactivity and BI (10, 15, 24, 25).
90
Complementary detection of antibodies against PGL-I or the components of LID-1could lead to
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improved sensitivity within tests.
92
In conjunction with OrangeLife, Rio de Janeiro, we have created simple
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immunochromatographic lateral flow tests with the capacity to detect PGL-I and LID-1 specific
94
antibodies. Within this report we assessed the performance of this new rapid diagnostic test
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against that of a previously available test using newly acquired and archived serum samples from
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clinically confirmed leprosy patients in Cebu, Philippines.
97 98 99 100 101 102
103
Materials and Methods
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Study site and participants. Blood samples were collected from Cebu Skin Clinic attendees
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after signing informed consent forms. Patients were fully characterized within the Ridley-Jopling
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scale by slit skin smear and biopsy (27). Healthy household contacts of MB patients were
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enrolled as individuals at elevated risk of developing leprosy (17). Individuals presenting with
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other skin conditions/diseases were also enrolled as endemic controls. Serum samples from a
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total of 208 newly diagnosed MB patients, 62 newly diagnosed PB patients, 51 healthy
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household contacts of MB patients and 63 endemic controls were evaluated in distinct panels
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(Tables 1, 2 and 3). Sera were prepared by centrifugation. Following diagnosis, each leprosy
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patient was provided standard MDT regimen as recommended by WHO (MB: Rifampicin: 600
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mg once a month; Dapsone: 100 mg daily; Clofazimine: 300 mg once a month and 50 mg daily;
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for 12 months. PB: Rifampicin: 600 mg once a month; Dapsone: 100 mg daily; for six months).
115 116
Rapid diagnostic tests. Sera were either tested within 2 hours of collection or after thawing
117
following storage at -20oC for up to 6 years. Two rapid diagnostic tests were evaluated; SD
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Leprosy tests were purchased from Standard Diagnostics (Yongin, South Korea) and the NDO-
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LID® was fabricated by OrangeLife (Rio de Janeiro, Brazil). Each are simple
120
immunochromatographic (lateral flow) tests with the purpose of detecting circulating antibodies.
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The SD test detects IgM antibodies to M. leprae-specific PGL-I, through the use of NDO-BSA (a
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synthetic mimetic of PGL-I conjugated to BSA), while NDO-LID® detects IgM antibodies to
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PGL-I and IgG antibodies specific to LID-1 (the synthetic mimetic conjugated to the
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recombinant fusion protein product of the M. leprae genes ML0405 and ML2331)(10).
125
Evaluations with each rapid diagnostic test involved the addition of undiluted serum (10 µl) and
126
running buffer (2-3 drops; ~100 µl) to a sample well, followed by readings of line development
127
in the detection window after 10 minutes. Validation of the results required the visualization of a
128
colored control line. A positive result was defined by the staining of both the control line and the
129
test line; faint or no staining was considered as a negative result. Visual readings were performed
130
by a minimum of two independent readers.
131
132
Objective measurement of NDO-LID®. NDO-LID® have been adapted such that they can be
133
read electronically by a Smart Reader®, an Android-based smartphone rapid test reader platform
134
mechanically attached to the existing camera unit (Figure 1). This reader collected test images
135
and objectively quantified the signal intensity of control and test lines in each NDO-LID® test.
136
The calculation of Smart Reader® cut-off values was based on the Receiver Operating Curve,
137
taking into account the visual results of the tests obtained with a panel of Brazilian MB leprosy
138
patient samples and control samples. Assuming a sensitivity of 87%, as determined by visual
139
readings, the Smart Reader® cut-off was calculated as 9.99. For data analysis the cut-off for
140
positive results by Smart Reader® was therefore considered as 10.0. Assuming this cut-off, the
141
sensitivity of the test among the registration cohort of Brazilian MB leprosy patients was 87%
142
(95% confidence interval (CI): 79.2 to 92.7%) and the specificity was 96.1% (CI 95%: 91.7 to
143
98.6%), with an area under the curve (AUC) of 0.96 (standard deviation, sd 0.01; p < 0.0001)
144
(4).
145 146
Statistical analysis. Statistical significance was assessed using unpaired t-test for comparison
147
between two groups. Results were considered statistically significant when p values < 0.05 were
148
obtained.
149 150 151
152
Results
153
Comparison of two rapid diagnostic tests for leprosy. We analyzed sera using both SD
154
leprosy (based on the detection of IgM antibodies against the PGL-I mimetic NDO antigen) and
155
NDO-LID® rapid diagnostic tests (based on the detection of IgM antibodies against NDO and
156
IgG antibodies against the LID-1 protein) to permit direct comparison between these tests. In an
157
initial study, subjective interpretation indicated that when developed with sera from MB patients
158
NDO-LID® tests produced a significantly stronger band than that observed in SD Leprosy tests
159
(Figure 2A). This was true for both fresh and frozen samples (p-values = 0.011 and 0.003,
160
respectively). Sensitivity for MB patients in this initial study was found to be 93.8% (45 of 48) in
161
NDO-LID® versus 77.1% (37 of 48) in SD Leprosy (Table 1). While previous storage did not
162
affect performance in the NDO-LID® tests, the signal intensity of SD Leprosy tests was
163
surprisingly lower when freshly prepared sera were added (Figure 2A). These results were
164
verified against another panel of sera (Figure 2B, p-value = 0.02).
165
PB patients have low or absent antibody responses and are not well recognized in rapid
166
diagnostic test containing only PGL-I mimetics (1, 3). In agreement, when NDO-LID® and SD
167
Leprosy rapid diagnostic tests were developed with PB patient sera, only a subset of PB patients
168
could be distinguished. A stronger signal was, however, observed in the NDO-LID® tests than in
169
the SD Leprosy tests such that a greater proportion of PB patients were positive (Figure 2A, p-
170
value < 0.0001 and Table 1; 52.6% positive in NDO-LID® versus 0.0% in SD Leprosy). Despite
171
returning stronger results with patient samples, the NDO-LID® tests were less likely to be
172
positive than SD Leprosy tests when developed with sera from control individuals (Figure 2A
173
and Table 1; 0.0% positive in NDO-LID® versus 25.0% in SD Leprosy). An independent
174
follow-up study using only fresh sera confirmed that NDO-LID® tests had a greater band
175
intensity when developed with leprosy patient sera (Figure 2B) and that a greater proportion of
176
patients could be discriminated (Table 2). Together, these data indicate that the NDO-LID®
177
provide a greater differential of positive and negative results than SD Leprosy tests, improving
178
upon the discrimination of patients from healthy individuals.
179
180
Objective and quantitative evaluation by NDO-LID®. Visual interpretation of results is
181
highly subjective and represents an important limitation when performed by personnel lacking
182
expertise, limiting their scope of use. Results from any diagnostic test would ideally also be
183
blinded from the clinical evaluation but this is difficult to achieve in rural settings with limited
184
resources. To address this deficit, a simple test reader (Smart Reader®) can be used to permit
185
objective scrutiny of data following the subjective evaluation of each NDO-LID®. While
186
readings on the control line were relatively consistent, a wide range of values were obtained
187
when tests developed with MB sera were analyzed (Figure 3A). The Smart Reader® identified
188
an additional 1 and 5 samples, respectively, increasing sensitivity to 95.8% as positive in study A
189
(46 of 48) and 97.5% in study B (39 of 40). When signal intensities were compared, there was a
190
highly significant correlation of readings (Spearman r = 0.967; Figure 3A). Repetitive Smart
191
Reader® quantification of the same test, and repeat testing of the same sample, yielded highly
192
consistent results (data not shown). In addition, although out with the test recommendations, only
193
a minor but consistent drop in signal intensity was measured when values were re-evaluated
194
approximately one month later (Spearman r = 0.980; Figure 3B). Thus, when coupled with a
195
Smart Reader®, NDO-LID® tests provide rapid, consistent, robust and objective quantification
196
of seroreactivity.
197 198
Correlation of rapid diagnostic test results with BI. We then evaluated rapid diagnostic test
199
performance across sera from MB patients identified to have either high (> 4.0), medium (2.0-
200
3.9) or low (4.0.
323 324 325
n
NDO-LID® (n, %)
SD Leprosy (n, %)
MB
208
181 (87.0)
170 (81.7)
PB
62
20 (32.3)
4 (6.5)
HHC
51
2 (3.9)
5 (9.8)
EC
63
1 (1.6)
6 (9.5)
(leprosy)
270
201 (74.4)
174 (64.4)
(not leprosy)
114
3 (2.6)
11 (9.6)
326 327
Table 3. Cumulative performance of rapid diagnostic tests following subjective
328
interpretation. Tests were determined as rendering a positive result when a distinct band was
329
observed (scored as 1 or greater).
330 331 332 333
334
Figure Legends
335
Figure 1. Representative images and subjective scoring system for NDO-LID®. Tests were
336
developed and examples of each scoring group that was subjectively assigned are shown. The
337
NDO-LID® have been adapted such that they can be read electronically by a Smart Reader®
338
application, an Android-based cell phone rapid test reader platform mechanically attached to the
339
existing camera unit.
340 341
Figure 2. Improved performance of NDO-LID® over SD Leprosy. In an initial study (A), stored
342
(MB only, n = 38) or fresh (MB, n = 10; PB, n = 19; and EC, n = 12) sera were added to either
343
SD Leprosy and NDO-LID® rapid diagnostic tests. The strength of the test band was then
344
subjectively interpreted on a scale of 0-4 (negative to strong positive). In a follow-on study (B),
345
fresh sera (MB, n = 40; PB, n = 13; HHC, n = 26; and EC, n = 11) were evaluated in the same
346
manner, with the exception that the scoring scale had a maximum value of 3. Results from one
347
interpreter are shown, and were verified/ corroborated by the additional interpreter. Strength of
348
the NDO-LID® test and control bands was then objectively measured using the Smart Reader®
349
(C indicates the objective measurement of the tests that were subjectively assessed in A, and D
350
depicts the objective measurement of the tests subjectively assessed in B). * = p-value < 0.05, **
351
= p-value < 0.01, *** = p-value < 0.001, and **** = p-value < 0.0001 between the indicated
352
groups.
353 354
Figure 3. NDO-LID®/ Smart Reader provide a robust system for evaluation. In A, NDO-LID®
355
were developed and the subjectively assigned values plotted versus objective Smart Reader®
356
measurements to determine correlation. In B, Smart Reader® values obtained 10 minutes after
357
test development (initial) are plotted versus values obtained by reading the same tests one month
358
later. The solid line represents the best fit linear regression and the Spearman r is shown.
359 360
Figure 4. Measurement of patient response to treatment by NDO-LID®. Archived sera were
361
selected based upon patient BI at time of collection then evaluated in NDO-LID(R) and SD
362
Leprosy (MB; high BI, n = 40; medium BI, n = 40 and low BI, n= 40; PB, n = 30; HHC, n = 30
363
and; EC, n = 25). In A, the strength of the test band in each rapid diagnostic test was subjectively
364
interpreted on a scale of 0-3 (negative to strong positive). Results are shown as mean and SEM
365
for each group. In B, the objective NDO-LID®/ Smart Reader® of sera collected from patients at
366
either time of diagnosis or end of MDT are shown. The values for ‘at diagnosis’ samples were
367
generated from the tests depicted in A, while the same number of ‘after treatment’ samples. * =
368
p-value < 0.05 and ** = p-value < 0.01 between indicated groups.
369 370 371
372
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Siddiqui, M. R., N. R. Velidi, S. Pati, N. Rath, A. K. Kanungo, A. K. Bhanjadeo, B. B. Rao, B. M. Ojha, K. Krishna Moorthy, D. Soutar, J. D. Porter, and P. V. Ranganadha Rao. 2009. Integration of leprosy elimination into primary health care in orissa, India. PLoS One 4:e8351. Spencer, J., M. S. Duthie, A. Geluk, M. Balagon, H. Kim, W. H. Wheat, D. Chatterjee, M. Jackson, W. Li, J. Kurihara, A. Maghanoy, I. Mallari, P. Saunderson, P. Brennan, and D. H. Dockrell. 2012. Identification of Serological Biomarkers of Infection, Disease Progression and Treatment Efficacy for Leprosy. Mem Inst Oswaldo Cruz 107, suppl.1:79-89. Spencer, J. S., H. J. Kim, W. H. Wheat, D. Chatterjee, M. V. Balagon, R. V. Cellona, E. V. Tan, R. Gelber, P. Saunderson, M. S. Duthie, S. T. Reece, W. Burman, R. Belknap, W. R. Mac Kenzie, A. Geluk, L. Oskam, H. M. Dockrell, and P. J. Brennan. 2011. Analysis of antibody responses to Mycobacterium leprae phenolic glycolipid I, lipoarabinomannan, and recombinant proteins to define disease subtype-specific antigenic profiles in leprosy. Clinical and vaccine immunology : CVI 18:260-267. Stefani, M. M., A. B. Grassi, L. H. Sampaio, A. L. de Sousa, M. B. Costa, P. Scheelbeek, K. D. Neupane, D. A. Hagge, M. Macdonald, S. N. Cho, L. Oskam, and S. Buhrer-Sekula. 2012. Comparison of two rapid tests for anti-phenolic glycolipid-I serology in Brazil and Nepal. Memorias do Instituto Oswaldo Cruz 107 Suppl 1:124-131. Van Veen, N. H., A. Meima, and J. H. Richardus. 2006. The relationship between detection delay and impairment in leprosy control: a comparison of patient cohorts from Bangladesh and Ethiopia. Lepr Rev 77:356-365. WHO. 2010. Enhanced Global Strategy for Further Reducing the Disease Burden Due to Leprosy: 2011–2015. World Health Organization WHO. 2010. Global leprosy situation, 2010. Wkly Epidemiol Rec 85:337-348.
Duthie et al., Figure 1
Duthie et al., Figure 2 A
Study A
4
*
subje ective value
3
**
B
Study B
3
**** 2
*
2
1
0.17
MB
MB
SD
NDO--LID
SD
PB
0
EC
MB
fresh
stored
C
NDO--LID
SD
NDO--LID
SD
0
NDO--LID
1
D
**** ****
***
**** ***
*
PB
HHC
EC
Duthie et al., Figure 3
A
B r = 0.967
r = 0.980
Duthie et al., Figure 4
A
3
**
B
2
1
* 0
high
medium MB (BI)
low
PB
HHC
EC
**
1
A Rapid Quantitative Serological Test To Detect Infection with Mycobacterium leprae, the
2
Causative Agent of Leprosy.
3 4
By Malcolm S. Duthie1, Marivic F. Balagon2, Armi Maghanoy2, Florenda M. Orcullo2, Marjorie
5
Cang2, Ronaldo Ferreira Dias3, Marco Collovati3 and Steven G. Reed1
6 7 8 9
From the 1Infectious Disease Research Institute, 1616 Eastlake Ave E, Seattle, WA 98102, 2
Leonard Wood Memorial Center for Leprosy Research, Cebu City, Philippines and
2
OrangeLife, Rio de Janeiro, Brazil.
10 11
This work was supported by the American Leprosy Missions and the Renaissance Health Service
12
Corporation.
13 14
Address correspondence and reprint requests:
15
Infectious Disease Research Institute, 1616 Eastlake Ave E, Seattle, WA 98102
16
Tel: 206-858-6012; Fax: 206-381-3678; Email address: [email protected]
17 18
Abbreviations used in this paper:
19
EC, endemic control
20
HHC, healthy household contact
21
LID-1, Leprosy IDRI Diagnostic-1
22
MB, multibacillary
23
MDT, multi-drug therapy
24
NEC, non-endemic control
25
PB, paucibacillary
26
PGL-I, phenolic glycolipid-I
27
TB, tuberculosis
28 29
Running title: Rapid serology test for leprosy.
30 31 32
Keywords for submission: leprosy; diagnosis; mycobacteria; rapid; quantitative
33 34
Abstract Leprosy remains an important health problem in a number of regions. Early detection of
35
infection, followed by effective treatment, is critical to reduce disease progression. New sensitive
36
and specific tools for early detection of infection will be a critical component of an effective
37
leprosy elimination campaign. Diagnosis is made by recognizing clinical signs and symptoms
38
but few clinicians are able to confidently identify these. Simple tests to facilitate referral to
39
leprosy experts are not widely available and the correct diagnosis of leprosy is often delayed.
40
Within this report we evaluated the performance of a new leprosy serological test (NDO-LID®).
41
As expected, the test readily detected clinically-confirmed MB patients and the rate of positive
42
results declined with bacterial burden. The NDO-LID® detected larger proportions of MB and
43
PB patients than the alternative SD Leprosy test (87.0% versus 81.7% and 32.3% versus 6.5%,
44
respectively) while also demonstrating improved specificity (97.4% versus 90.4%). Coupled
45
with a new cell phone-based test reader platform (Smart Reader®), the NDO-LID® tests
46
provided consistent, objective test interpretation that could facilitate wider use in non-specialized
47
settings. In addition, results obtained from sera at the time of diagnosis versus the end of
48
treatment, indicated that the quantifiable nature of this system can also be used to monitor
49
treatment efficacy. Taken together, these data indicate that the NDO-LID®/ Smart Reader®
50
system can assist in the diagnosis and monitoring of MB leprosy, as well as detect a significant
51
number of earlier stage infections.
52
53 54
Introduction Despite advances toward the elimination of leprosy over the last two decades, new case
55
detection rates have stabilized over the last decade and leprosy remains an important health
56
problem in many regions (36). Like the number of registered cases, however, the number of
57
clinicians who can reliably diagnose leprosy has waned, and delays in correct diagnosis are
58
common (6, 30). Efforts to develop and improve surveillance and referral systems appear
59
necessary to achieve the early detection required to ensure that prompt and appropriate treatment
60
can be provided to limit disabilities. The development of simple and practical tools to facilitate
61
diagnosis would appear prudent.
62
Currently the diagnosis of leprosy is dependent on the appearance and recognition of
63
clinical signs and symptoms. When skin-smear or pathology services are available, leprologists
64
utilize the Ridley-Jopling scale to characterize five forms (27, 28). In practice, however, most field
65
programs lack such services and the clinical system suggested by WHO to classify individual
66
patients and to select their treatment regimen is used (35). The WHO system uses skin lesions,
67
bacterial positivity by skin smear and the number of involved nerves to group leprosy patients
68
into one of two simplified categories; multibacillary (MB) leprosy (typically smear positive or
69
more than one nerve involvement or more than 5 lesions) and paucibacillary (PB) leprosy (smear
70
negative, none to one nerve involvement, maximum of 5 lesions). In many settings, particularly
71
the poorest rural settings where access to experienced leprologists is limited or entirely absent,
72
primary care providers cannot identify the signs of leprosy and patients are commonly
73
misdiagnosed and mistreated (5, 8, 9). With the delays in diagnosis of MB leprosy, transmission of
74
M. leprae from infected individuals to their contacts continues and, in many cases, irreversible
75
nerve damage has occurred before they are registered as patients (7, 18, 34). Simple tools that can
76
facilitate leprosy diagnosis could help to address this deficit.
77
While PB leprosy patients have absent bacterial indices (BI; a measure of the number of
78
acid-fast bacilli in the dermis expressed in a logarithmic scale) and generally have low or absent
79
anti-M. leprae antibody responses, MB patients do not control bacterial replication and
80
demonstrate titers of anti-M. leprae antibodies that correlate with their burdens (27). Phenolic
81
glycolipid (PGL)-I is well recognized as a target of the antibody response of leprosy patients, with
82
the magnitude of the response strongly correlating with bacterial index (BI) (14). To date, leprosy
83
rapid diagnostic tests have comprised of only PGL-I mimetics as the antigenic target (ML Flow
84
and Standard Diagnostic, containing tri- or disaccharides NTP and NDO, respectively)(1-3, 33).
85
Using laboratory-based assays our group and others have identified numerous native and
86
recombinant proteins recognized by antibodies in MB patient serum (10, 11, 13, 20-22, 31, 32).
87
One example is the chimeric fusion protein Leprosy IDRI Diagnostic (LID)-1, an antigen
88
specifically recognized by sera from leprosy patients from geographically and ethnically diverse
89
populations with a direct correlation between seroreactivity and BI (10, 15, 24, 25).
90
Complementary detection of antibodies against PGL-I or the components of LID-1could lead to
91
improved sensitivity within tests.
92
In conjunction with OrangeLife, Rio de Janeiro, we have created simple
93
immunochromatographic lateral flow tests with the capacity to detect PGL-I and LID-1 specific
94
antibodies. Within this report we assessed the performance of this new rapid diagnostic test
95
against that of a previously available test using newly acquired and archived serum samples from
96
clinically confirmed leprosy patients in Cebu, Philippines.
97 98 99 100 101 102
103
Materials and Methods
104
Study site and participants. Blood samples were collected from Cebu Skin Clinic attendees
105
after signing informed consent forms. Patients were fully characterized within the Ridley-Jopling
106
scale by slit skin smear and biopsy (27). Healthy household contacts of MB patients were
107
enrolled as individuals at elevated risk of developing leprosy (17). Individuals presenting with
108
other skin conditions/diseases were also enrolled as endemic controls. Serum samples from a
109
total of 208 newly diagnosed MB patients, 62 newly diagnosed PB patients, 51 healthy
110
household contacts of MB patients and 63 endemic controls were evaluated in distinct panels
111
(Tables 1, 2 and 3). Sera were prepared by centrifugation. Following diagnosis, each leprosy
112
patient was provided standard MDT regimen as recommended by WHO (MB: Rifampicin: 600
113
mg once a month; Dapsone: 100 mg daily; Clofazimine: 300 mg once a month and 50 mg daily;
114
for 12 months. PB: Rifampicin: 600 mg once a month; Dapsone: 100 mg daily; for six months).
115 116
Rapid diagnostic tests. Sera were either tested within 2 hours of collection or after thawing
117
following storage at -20oC for up to 6 years. Two rapid diagnostic tests were evaluated; SD
118
Leprosy tests were purchased from Standard Diagnostics (Yongin, South Korea) and the NDO-
119
LID® was fabricated by OrangeLife (Rio de Janeiro, Brazil). Each are simple
120
immunochromatographic (lateral flow) tests with the purpose of detecting circulating antibodies.
121
The SD test detects IgM antibodies to M. leprae-specific PGL-I, through the use of NDO-BSA (a
122
synthetic mimetic of PGL-I conjugated to BSA), while NDO-LID® detects IgM antibodies to
123
PGL-I and IgG antibodies specific to LID-1 (the synthetic mimetic conjugated to the
124
recombinant fusion protein product of the M. leprae genes ML0405 and ML2331)(10).
125
Evaluations with each rapid diagnostic test involved the addition of undiluted serum (10 µl) and
126
running buffer (2-3 drops; ~100 µl) to a sample well, followed by readings of line development
127
in the detection window after 10 minutes. Validation of the results required the visualization of a
128
colored control line. A positive result was defined by the staining of both the control line and the
129
test line; faint or no staining was considered as a negative result. Visual readings were performed
130
by a minimum of two independent readers.
131
132
Objective measurement of NDO-LID®. NDO-LID® have been adapted such that they can be
133
read electronically by a Smart Reader®, an Android-based smartphone rapid test reader platform
134
mechanically attached to the existing camera unit (Figure 1). This reader collected test images
135
and objectively quantified the signal intensity of control and test lines in each NDO-LID® test.
136
The calculation of Smart Reader® cut-off values was based on the Receiver Operating Curve,
137
taking into account the visual results of the tests obtained with a panel of Brazilian MB leprosy
138
patient samples and control samples. Assuming a sensitivity of 87%, as determined by visual
139
readings, the Smart Reader® cut-off was calculated as 9.99. For data analysis the cut-off for
140
positive results by Smart Reader® was therefore considered as 10.0. Assuming this cut-off, the
141
sensitivity of the test among the registration cohort of Brazilian MB leprosy patients was 87%
142
(95% confidence interval (CI): 79.2 to 92.7%) and the specificity was 96.1% (CI 95%: 91.7 to
143
98.6%), with an area under the curve (AUC) of 0.96 (standard deviation, sd 0.01; p < 0.0001)
144
(4).
145 146
Statistical analysis. Statistical significance was assessed using unpaired t-test for comparison
147
between two groups. Results were considered statistically significant when p values < 0.05 were
148
obtained.
149 150 151
152
Results
153
Comparison of two rapid diagnostic tests for leprosy. We analyzed sera using both SD
154
leprosy (based on the detection of IgM antibodies against the PGL-I mimetic NDO antigen) and
155
NDO-LID® rapid diagnostic tests (based on the detection of IgM antibodies against NDO and
156
IgG antibodies against the LID-1 protein) to permit direct comparison between these tests. In an
157
initial study, subjective interpretation indicated that when developed with sera from MB patients
158
NDO-LID® tests produced a significantly stronger band than that observed in SD Leprosy tests
159
(Figure 2A). This was true for both fresh and frozen samples (p-values = 0.011 and 0.003,
160
respectively). Sensitivity for MB patients in this initial study was found to be 93.8% (45 of 48) in
161
NDO-LID® versus 77.1% (37 of 48) in SD Leprosy (Table 1). While previous storage did not
162
affect performance in the NDO-LID® tests, the signal intensity of SD Leprosy tests was
163
surprisingly lower when freshly prepared sera were added (Figure 2A). These results were
164
verified against another panel of sera (Figure 2B, p-value = 0.02).
165
PB patients have low or absent antibody responses and are not well recognized in rapid
166
diagnostic test containing only PGL-I mimetics (1, 3). In agreement, when NDO-LID® and SD
167
Leprosy rapid diagnostic tests were developed with PB patient sera, only a subset of PB patients
168
could be distinguished. A stronger signal was, however, observed in the NDO-LID® tests than in
169
the SD Leprosy tests such that a greater proportion of PB patients were positive (Figure 2A, p-
170
value < 0.0001 and Table 1; 52.6% positive in NDO-LID® versus 0.0% in SD Leprosy). Despite
171
returning stronger results with patient samples, the NDO-LID® tests were less likely to be
172
positive than SD Leprosy tests when developed with sera from control individuals (Figure 2A
173
and Table 1; 0.0% positive in NDO-LID® versus 25.0% in SD Leprosy). An independent
174
follow-up study using only fresh sera confirmed that NDO-LID® tests had a greater band
175
intensity when developed with leprosy patient sera (Figure 2B) and that a greater proportion of
176
patients could be discriminated (Table 2). Together, these data indicate that the NDO-LID®
177
provide a greater differential of positive and negative results than SD Leprosy tests, improving
178
upon the discrimination of patients from healthy individuals.
179
180
Objective and quantitative evaluation by NDO-LID®. Visual interpretation of results is
181
highly subjective and represents an important limitation when performed by personnel lacking
182
expertise, limiting their scope of use. Results from any diagnostic test would ideally also be
183
blinded from the clinical evaluation but this is difficult to achieve in rural settings with limited
184
resources. To address this deficit, a simple test reader (Smart Reader®) can be used to permit
185
objective scrutiny of data following the subjective evaluation of each NDO-LID®. While
186
readings on the control line were relatively consistent, a wide range of values were obtained
187
when tests developed with MB sera were analyzed (Figure 3A). The Smart Reader® identified
188
an additional 1 and 5 samples, respectively, increasing sensitivity to 95.8% as positive in study A
189
(46 of 48) and 97.5% in study B (39 of 40). When signal intensities were compared, there was a
190
highly significant correlation of readings (Spearman r = 0.967; Figure 3A). Repetitive Smart
191
Reader® quantification of the same test, and repeat testing of the same sample, yielded highly
192
consistent results (data not shown). In addition, although out with the test recommendations, only
193
a minor but consistent drop in signal intensity was measured when values were re-evaluated
194
approximately one month later (Spearman r = 0.980; Figure 3B). Thus, when coupled with a
195
Smart Reader®, NDO-LID® tests provide rapid, consistent, robust and objective quantification
196
of seroreactivity.
197 198
Correlation of rapid diagnostic test results with BI. We then evaluated rapid diagnostic test
199
performance across sera from MB patients identified to have either high (> 4.0), medium (2.0-
200
3.9) or low (4.0.
323 324 325
n
NDO-LID® (n, %)
SD Leprosy (n, %)
MB
208
181 (87.0)
170 (81.7)
PB
62
20 (32.3)
4 (6.5)
HHC
51
2 (3.9)
5 (9.8)
EC
63
1 (1.6)
6 (9.5)
(leprosy)
270
201 (74.4)
174 (64.4)
(not leprosy)
114
3 (2.6)
11 (9.6)
326 327
Table 3. Cumulative performance of rapid diagnostic tests following subjective
328
interpretation. Tests were determined as rendering a positive result when a distinct band was
329
observed (scored as 1 or greater).
330 331 332 333
334
Figure Legends
335
Figure 1. Representative images and subjective scoring system for NDO-LID®. Tests were
336
developed and examples of each scoring group that was subjectively assigned are shown. The
337
NDO-LID® have been adapted such that they can be read electronically by a Smart Reader®
338
application, an Android-based cell phone rapid test reader platform mechanically attached to the
339
existing camera unit.
340 341
Figure 2. Improved performance of NDO-LID® over SD Leprosy. In an initial study (A), stored
342
(MB only, n = 38) or fresh (MB, n = 10; PB, n = 19; and EC, n = 12) sera were added to either
343
SD Leprosy and NDO-LID® rapid diagnostic tests. The strength of the test band was then
344
subjectively interpreted on a scale of 0-4 (negative to strong positive). In a follow-on study (B),
345
fresh sera (MB, n = 40; PB, n = 13; HHC, n = 26; and EC, n = 11) were evaluated in the same
346
manner, with the exception that the scoring scale had a maximum value of 3. Results from one
347
interpreter are shown, and were verified/ corroborated by the additional interpreter. Strength of
348
the NDO-LID® test and control bands was then objectively measured using the Smart Reader®
349
(C indicates the objective measurement of the tests that were subjectively assessed in A, and D
350
depicts the objective measurement of the tests subjectively assessed in B). * = p-value < 0.05, **
351
= p-value < 0.01, *** = p-value < 0.001, and **** = p-value < 0.0001 between the indicated
352
groups.
353 354
Figure 3. NDO-LID®/ Smart Reader provide a robust system for evaluation. In A, NDO-LID®
355
were developed and the subjectively assigned values plotted versus objective Smart Reader®
356
measurements to determine correlation. In B, Smart Reader® values obtained 10 minutes after
357
test development (initial) are plotted versus values obtained by reading the same tests one month
358
later. The solid line represents the best fit linear regression and the Spearman r is shown.
359 360
Figure 4. Measurement of patient response to treatment by NDO-LID®. Archived sera were
361
selected based upon patient BI at time of collection then evaluated in NDO-LID(R) and SD
362
Leprosy (MB; high BI, n = 40; medium BI, n = 40 and low BI, n= 40; PB, n = 30; HHC, n = 30
363
and; EC, n = 25). In A, the strength of the test band in each rapid diagnostic test was subjectively
364
interpreted on a scale of 0-3 (negative to strong positive). Results are shown as mean and SEM
365
for each group. In B, the objective NDO-LID®/ Smart Reader® of sera collected from patients at
366
either time of diagnosis or end of MDT are shown. The values for ‘at diagnosis’ samples were
367
generated from the tests depicted in A, while the same number of ‘after treatment’ samples. * =
368
p-value < 0.05 and ** = p-value < 0.01 between indicated groups.
369 370 371
372
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Duthie et al., Figure 1
Duthie et al., Figure 2 A
Study A
4
*
subje ective value
3
**
B
Study B
3
**** 2
*
2
1
0.17
MB
MB
SD
NDO--LID
SD
PB
0
EC
MB
fresh
stored
C
NDO--LID
SD
NDO--LID
SD
0
NDO--LID
1
D
**** ****
***
**** ***
*
PB
HHC
EC
Duthie et al., Figure 3
A
B r = 0.967
r = 0.980
Duthie et al., Figure 4
A
3
**
B
2
1
* 0
high
medium MB (BI)
low
PB
HHC
EC
**
