JCM Accepts, published online ahead of print on 2 April 2014 J. Clin. Microbiol. doi:10.1128/JCM.00525-14 Copyright © 2014, American Society for Microbiology. All Rights Reserved.
1
Loop-Mediated Isothermal Amplification (LAMP) for the Rapid and Semi-
2
Quantitative Detection of Loa loa Infection
3 4
Papa M. Dramea, Doran L. Finka*, Joseph Kamgnob,c, Jesica A. Herricka*, Thomas B.
5
Nutmana#
6 7
Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases,
8
National Institutes of Health, Bethesda, Maryland, USAa; Centre for Research on
9
Filariasis and other Tropical Diseases Yaounde, Cameroonb; Faculty of Medicine and
10
Biomedical Sciences, University of Yaounde I, Yaounde, Cameroonc.
11 12
Running Title:
13
Rapid and Semi-Quantitative Detection of Loa loa
14 15
# Address correspondence to Thomas B. Nutman: [email protected]
16
*Present address: Doran L. Fink, Center for Biologics Evaluation and Research, Food
17
and Drug Administration, Silver Spring, Maryland, USA; Jesica A. Herrick, Section of
18
Infectious Diseases, Immunology, and International Medicine, Department of Medicine,
19
University of Illinois at Chicago, Chicago, Illinois, USA.
20 21 22 23
1
24
Abstract:
25
Rapid and accurate tests are currently needed to identify individuals with high levels of
26
Loa loa (L. loa) microfilaria, so that they may be excluded from mass ivermectin
27
administration campaigns against onchocerciasis and lymphatic filariasis being conducted
28
in co-endemic areas. To address this need, colorimetric LAMP assays targeting the L.
29
loa-specific gene sequences LLMF72 and LLMF342 were developed for the detection
30
and quantification of L. loa microfilaremia.
31
Both LAMP assays were highly specific (100%) for L. loa infection compared to absence
32
of infection or infection with related filarial pathogens. The LLMF72 assay showed
33
greater analytic sensitivity (limit of detection: 0.1 pg/ml of gDNA and/or 5 microfilariae
34
(mf)/ml) compared to LLMF342-based LAMP (10 pg/ml of gDNA and/or 50 mf/ml), and
35
similar analytic sensitivity to LLMF72-based qPCR. A high level of correlation was
36
observed between microfilaria counts as determined by LLMF72-based qPCR and time
37
to positivity by LAMP, and performance measures of sensitivity, specificity, and positive
38
and negative predictive values were similar for both assays when applied to field-
39
collected clinical samples. By simply varying the run time, the LAMP assay was able to
40
accurately distinguish individuals at risk for post-ivermectin serious adverse events
41
(SAEs), using thresholds of >5,000 mf/ml and >30,000 mf/ml as indicators of increasing
42
levels of risk.
43
In summary, LLMF72 LAMP represents a new molecular diagnostic tool that is readily
44
applicable as a point-of-care method for L. loa microfilarial detection and quantification
45
in resource-limited endemic countries.
46
2
47
Background
48
L. loa, the causative agent of loiasis, is a parasitic nematode transmitted to humans by
49
the tabanid Chrysops fly (1), with transmission confined to the rainforest and some
50
savannah areas of West and Central Africa (2). Although the overwhelming majority of
51
L. loa-infected individuals are clinically asymptomatic, Calabar swelling (transient,
52
localized angioedema) and the subconjunctival migration of an adult worm (“eyeworm”)
53
are the most common clinical manifestations (3-5). Rarely, nephropathy,
54
cardiomyopathy, retinopathy, neuropsychiatric complications and encephalopathy (6-12)
55
can occur as a consequence of chronic infection. Among the 8 filarial infections of
56
humans, L. loa had been largely neglected as a public health concern in Africa, though it
57
has gained prominence of late because of the neurologic serious adverse events (SAEs)
58
that have occurred, occasionally leading to death, in highly microfilaremic individuals
59
following exposure to ivermectin (IVM) given during mass drug administration (MDA)
60
for control of Onchocerca volvulus and/or Wuchereria bancrofti in regions where they
61
are co-endemic with L. loa (13, 14).
62
Although the pathogenesis of the neurologic SAEs are still not well understood
63
(8, 15), data clearly demonstrate a relationship between the pre-treatment L. loa
64
microfilariae (mf) density (14, 16) and the risk of SAEs, with levels of 5,000 mf/ml and
65
30,000 mf/ml felt to be the most significant thresholds (13), though other factors may
66
also be involved (17-19). Consequently, some IVM-based MDA for onchocerciasis (Ov)
67
have been delayed, and lymphatic filariasis (LF) control programs (using
68
IVM/albendazole) have been put on hold in large parts of Central Africa because of co-
69
endemic L. loa infection (20).
3
70
In endemic areas, the routine method for diagnosis and quantification of L. loa is
71
based on calibrated microscopic examination of mid-day blood for microfilariae, a
72
process that is neither point-of-care nor high-throughput. Indeed, it is felt to be
73
impractical for use as a widespread screening tool (21). Serological (22, 23) and
74
molecular (24-26) alternative tests have been developed, but only real-time PCR
75
methodology to date has been able to combine a high degree of sensitivity and specificity
76
with the ability to accurately quantify L. loa mf levels (26, 27). These methods, however,
77
require a centralized, well-equipped laboratory and relatively expensive reagents.
78
Loop-mediated isothermal amplification (LAMP) has emerged as a potential
79
alternative to DNA amplification techniques requiring thermocycling and automated
80
fluorescence description (28-30). LAMP relies on an auto cycling strand displacement
81
DNA synthesis performed at a single temperature (30, 31). The byproduct of the LAMP
82
reaction is magnesium pyrophosphate that accumulates as the reaction progresses and
83
that can be monitored using turbidity measurements or visually using a variety of
84
intercalating dyes (e.g., SYBR Green I, calcein, hydroxy naphthol blue (HNB)). In
85
particular, adding HNB dye to the reaction tube before the amplification has improved
86
the capacity of the LAMP method to be used as a point-of-care diagnostic test, since it
87
has made the assay easier to use in resource-limited settings (32, 33). Recently, LAMP
88
technology has been applied with high accuracy to detect infection with a wide array of
89
pathogens including bacteria (34, 35), viruses (36) and parasites (28, 37, 38).
90
In the present study, we have used two L. loa-specific single copy targets,
91
LLMF72 and LLMF342, to design LAMP primers for detection of L. loa mf in human
92
blood and have developed methods that provide similar sensitivity and specificity as the
4
93
previously described qPCR assay targeting the LLMF72 gene (26). Furthermore, we have
94
identified LAMP conditions that allow for colorimetric determination of microfilarial
95
levels above or below specified thresholds such that our LAMP method now has the
96
potential to be a practical point-of-care tool for identifying people at risk for SAEs when
97
given IVM.
98 99
Methods
100 101
Samples
102
The source of genomic DNA from L. loa, Wuchereria bancrofti, Brugia malayi and
103
Mansonella perstans mf has been described previously (26). For some experiments,
104
100,000 purified L. loa mf were placed in a fixed volume (1 ml) of distilled water and
105
serially diluted to create standards for quantification. DNA was then extracted and used
106
for assessment of the sensitivity of the assays. Finally, to evaluate the performance of the
107
L. loa LAMP assay compared to the standard qPCR method, 93 mid-day dried blood-
108
spots on filter paper (Whatman 1mm) previously collected in Cameroon (26) were
109
assessed. These blood spots were obtained by fingerprick from volunteers living in a
110
region endemic for L. loa infection. Briefly, capillary blood was spotted onto Whatman
111
filter paper, dried and stored at 4°C until their use for DNA extraction.
112 113
DNA extraction for spiked mf and field-blood spot samples
114
Blood spots were punched using a disposable 6mm biopsy punch (Acuderm, Inc., Ft.
115
Lauderdale, FL, USA). Two punched blood spots (10µL dried blood each) were
5
116
immersed in 400µL of water in a Precellys® lysing tube (Peqlab, Wilmington, USA)
117
containing glass and ceramic beads. Tubes were homogenized at 6,500 rcf for 5 minutes
118
using a Precellys24® bead beating machine (Peqlab, Wilmington, USA). These
119
homogenized samples were then heated for 30 minutes at 99°C while shaking in a
120
thermomixer (Eppendorf, North America, USA). Finally, the samples were centrifuged at
121
15,700 rcf for 10 minutes, and the supernatant was collected for use. DNA was also
122
isolated from water spiked with 100,000 purified L. loa mf/ml using the method
123
described above.
124 125
Primer design
126
L. loa mf-specific LAMP primers were designed for LLMF72 (GenBank: HM753552.1)
127
and LLMF342 (intronic region 1,900-2,200 of a L. loa contig 3.498; GenBank:
128
ADBU02000498.1) using Primer Explorer v4
129
(http://primerexplorer.jp/elamp4.0.0/index.html). A set of six specific primers comprising
130
two outer, two inner or internal, and two loop primers were designed for each targeted
131
gene. Details of the sequences and targets of the designed primers are listed in Table 1.
132
All of the primers (Eurofins MWG Operon, Huntsville, AL, USA) were HPLC purified.
133
Their specificity was further confirmed using the BLAST algorithm
134
(http://blast.ncbi.nlm.nih.gov/Blast.cgi).
135 136
LAMP assay
137
The LAMP reaction was performed using a kit designed by Eiken Chemical Company
138
(Eiken Chemical Co., Ltd., Tokyo, Japan). Briefly, the reaction was carried out in a total
6
139
volume of 25 µL (23 µL of the reaction mixture and 2 µL of the DNA template) in PCR
140
micro-tubes (United Scientific Products, San Leandro, CA, USA). The reaction mixture
141
contained 50 pmol each of the FIP and BIP primers, 5 pmol each of the F3 and B3
142
primers, 25 pmol each of the LB and LF primers in 12.5 µL of a 2x buffer. Ultrapure
143
water (7 µL), 1 µL of 1 mM of HNB (Sigma-Aldrich, Inc., St. Louis, MO, USA), 1 µL of
144
the large fragment of Bst DNA polymerase (Eiken Chemical Co., Ltd., Tokyo, Japan) and
145
2 µL of the DNA template were added to complete the reaction mixture. Positive (L. loa
146
mf genomic DNA: 100 pg/ml) and negative (water) controls were included in each
147
experiment, and precautions were taken to prevent cross-contamination (all experiments
148
performed in a AirClean® 600 PCR workstation (AirClean® Systems, Creedmoor, NC,
149
USA) with UV radiation for sterilization). All reactions were performed at 65°C
150
(isothermal condition) for up to 60 min. The LAMP assay was stopped by incubation at
151
80°C for 5 min, inactivating the Bst DNA polymerase. The amplification efficiency was
152
measured by the HNB color change or by the turbidity using a real-time turbidimeter
153
(Eiken Chemical Co., Ltd., Tokyo, Japan). A sample was considered positive for L. loa
154
mf DNA if an obvious color change from purple to blue or increase in turbidity was
155
observed, compared to the negative control. All samples were run in duplicate. If the
156
duplicates varied (one positive, one negative), the samples were re-run in triplicate.
157 158
Real-time PCR assay
159
The qPCR was performed as previously described (26); all samples were also were tested
160
for the ability to amplify a control plasmid DNA that was spiked into the DNA samples
161
prior to DNA extraction to ensure a lack of inhibition (27).
7
162
Statistical Analysis of results
163
Statistical analyses including specificity and sensitivity calculations and correlations
164
(Spearman Rank) were performed using GraphPad Prism 6.0 (GraphPad Software, Inc.,
165
San Diego, CA, USA).
166 167
Results
168 169
Analytical specificity and sensitivity of Loa loa LAMP assay
170
Species specificity of the LAMP assays was assessed using a real time turbidimeter
171
(Figure 1A) with the LLMF72 set of primers. As can be seen, these primers fail to
172
amplify genomic DNA of B. malayi, W. bancrofti, O.volvulus, M. perstans whereas they
173
amplified genomic DNA of L. loa easily and did so in a dose dependent manner. In data
174
not shown, the primers derived from LLMF72 and LLMF342 failed to amplify genomic
175
DNA of any of the 5 Plasmodium species capable of infecting humans either.
176
To allow for a simplified detection method that could be visualized by the naked
177
eye, colorimetric detection of the LAMP assays using a HNB dye was performed next
178
(Figure 1B). For both LLMF72- and LLMF342-based (not shown) assays, specificity for
179
L. loa could be seen in that only the L. loa DNA samples were blue (positive), whereas
180
samples with each of the related other filarial species were purple (negative).
181
To assess the analytic sensitivity of the LAMP assays, the limit of DNA detection
182
was determined by testing serial dilutions of genomic DNA obtained from varying
183
numbers of L. loa mf (Figure 2). With the LLMF72 assay, the lowest amount of
184
detectable gDNA was 0.1 pg/ml (Fig. 2A, top panel) whereas for LLMF342 (Fig 2A,
8
185
middle panel) the limit of detection was 10 pg/ml. When both sets of primers were used
186
together (Fig 2A, bottom panel) in an attempt to improve even further the sensitivity, we
187
found little to no improvement over the analytic sensitivity of LLMF72 alone. Similarly,
188
when these assays were performed on DNA extracted from varying concentrations of mf,
189
we found that the limit of detection was 5 mf/ml for LLMF72 (Fig. 2B, top panel), 50
190
mf/ml for LLMF342 (Fig. 2B, middle panel), and 5 mf/ml for the two sets of primers
191
together (Fig. 2B, lower panel).
192 193
Time dependent threshold assessments for performing semi-quantitative LAMP
194
assays
195
Because the goal of this project was to provide a potentially point-of-care method of
196
amplification, detection and quantitation, we next assessed whether our LAMP assay
197
could be used in a semi-quantitative manner by exploring how varying the reaction time
198
affected the results of the colorimetric LAMP assays when performed on DNA obtained
199
from varying concentrations of L. loa mf. For each of the LLMF72, LLMF342 and
200
LLMF72-LLMF342 LAMP assays, the color change in the reaction tubes from purple
201
(negative) to blue (positive) was monitored every 5 minutes. The time to positivity was
202
then plotted as a function of mf concentration (Figure 3). For DNA samples prepared
203
from L. loa mf concentrations of 30,000 mf/ml and above, the earliest color change was
204
observed at 15 minutes after initiation for LLMF72-based assays, at 25 minutes for
205
LLMF342, and at 20 minutes for combined LLMF72-LLMF342-based assays. At the
206
limits of detection for L. loa mf concentration (5 mf/ml in LLMF72 and LLMF72-
207
LLMF342 assays and 10 mf/ml in LLMF342 LAMP), a color change occurred after 30
9
208
minutes for LLMF72 assay and after 40 minutes for LLMF342 and the combined
209
LLMF72-LLMF342 assays (Figure 3). In addition, over the range of mf concentration
210
dilutions there was a high degree of correlation observed between the time to LAMP
211
reaction positivity (minutes) and the cycle number (Ct value) obtained by qPCR for both
212
the LLMF72 assays (Figure 4A; r = 0.96 and p5,000 mf/ml), and 25
220
minutes for a threshold of (100 mf/ml). This difference in time to positivity based on the
221
amount of DNA was also observed in the LLMF342 and LLMF72-LLMF342 assays,
222
though the run time for which a positive test corresponded to the specified threshold
223
concentrations of 30,000, 5,000 and 100 mf/ml differed slightly between the various
224
assays.
225 226
LLMF72 Loa loa LAMP performance compared to qPCR
227
To further assess the performance of the LAMP assay, a formal comparison was made
228
between LLMF72-based qPCR and colorimetric LAMP using field-collected dried blood
229
spots obtained from 93 individuals living in L. loa-endemic regions of Cameroon (Table
230
2). Of the 93 samples tested, 50 were positive and 43 were negative by qPCR. All qPCR
10
231
positive samples were also positive by LAMP, and there were 3 additional qPCR negative
232
samples that were positive or indeterminate (not purple but not completely blue) in the
233
LAMP assay (presumably false positives). Thus, considering the qPCR to be the “gold
234
standard”, the LAMP assay has a sensitivity of 100.0% (95% CI, 92.9%–100.0%), a
235
specificity of 93.0% (95% CI, 80.9%–98.5%), a positive predictive value of 94.3% (95%
236
CI, 94.3%–98.8%), and a negative predictive value of 100.0% (95% CI, 91.2%–100.0%).
237 238
Discussion:
239
Because co-incident Loa loa infection has had a severely negative impact on control
240
(MDA) programs for onchocerciasis and lymphatic filariasis in West and Central Africa,
241
there is a consensus that point-of-care diagnostics for L. loa infection in co-endemic
242
regions of the world are needed to achieve Ov and LF global elimination goals. Current
243
qPCR-based methods are highly sensitive, specific and quantitative but require
244
sophisticated equipment and are costly. Thus, we developed a LAMP assay and
245
demonstrated its potential for use as a point-of-care diagnosis tool in field settings.
246
Both LLMF72 and LLMF342-based LAMP assays were highly L. loa-specific with
247
respect to other related filarial parasites (W. bancrofti, M. perstans, B. malayi and O.
248
volvulus). This result was expected based on de novo bioinformatics assessments
249
previously performed in a slightly different context (26). Moreover, the analytic
250
sensitivity was equally high when compared to qPCR (Figure 4). The LAMP assay was
251
also highly comparable to the “gold standard” qPCR in terms of clinical sensitivity and
252
specificity. In addition, its predictive positive (94.3%) and negative (100.0%) values
253
support its use for clinical diagnostic purposes.
11
254
Although LAMP assays (like qPCR) can be quantitated in a real-time using a
255
sophisticated turbidimeter (38), having the ability to perform the assays at the point-of-
256
care or with minimal instrumentation led us to monitor our assays using a colorimetric
257
readout through the use of HNB dye, previously shown to be useful in endpoint LAMP
258
assays (32). Thus, the HNB-based LAMP assay provides a potential point-of-care method
259
of rapid amplification and easy detection of L. loa DNA that, when standardized, can
260
accurately distinguish levels of mf that are correlated with increased risk for SAEs
261
(>30,000 mf/ml: LAMP assay positive at 15 minutes) from those who might not be at
262
risk (
1
Loop-Mediated Isothermal Amplification (LAMP) for the Rapid and Semi-
2
Quantitative Detection of Loa loa Infection
3 4
Papa M. Dramea, Doran L. Finka*, Joseph Kamgnob,c, Jesica A. Herricka*, Thomas B.
5
Nutmana#
6 7
Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases,
8
National Institutes of Health, Bethesda, Maryland, USAa; Centre for Research on
9
Filariasis and other Tropical Diseases Yaounde, Cameroonb; Faculty of Medicine and
10
Biomedical Sciences, University of Yaounde I, Yaounde, Cameroonc.
11 12
Running Title:
13
Rapid and Semi-Quantitative Detection of Loa loa
14 15
# Address correspondence to Thomas B. Nutman: [email protected]
16
*Present address: Doran L. Fink, Center for Biologics Evaluation and Research, Food
17
and Drug Administration, Silver Spring, Maryland, USA; Jesica A. Herrick, Section of
18
Infectious Diseases, Immunology, and International Medicine, Department of Medicine,
19
University of Illinois at Chicago, Chicago, Illinois, USA.
20 21 22 23
1
24
Abstract:
25
Rapid and accurate tests are currently needed to identify individuals with high levels of
26
Loa loa (L. loa) microfilaria, so that they may be excluded from mass ivermectin
27
administration campaigns against onchocerciasis and lymphatic filariasis being conducted
28
in co-endemic areas. To address this need, colorimetric LAMP assays targeting the L.
29
loa-specific gene sequences LLMF72 and LLMF342 were developed for the detection
30
and quantification of L. loa microfilaremia.
31
Both LAMP assays were highly specific (100%) for L. loa infection compared to absence
32
of infection or infection with related filarial pathogens. The LLMF72 assay showed
33
greater analytic sensitivity (limit of detection: 0.1 pg/ml of gDNA and/or 5 microfilariae
34
(mf)/ml) compared to LLMF342-based LAMP (10 pg/ml of gDNA and/or 50 mf/ml), and
35
similar analytic sensitivity to LLMF72-based qPCR. A high level of correlation was
36
observed between microfilaria counts as determined by LLMF72-based qPCR and time
37
to positivity by LAMP, and performance measures of sensitivity, specificity, and positive
38
and negative predictive values were similar for both assays when applied to field-
39
collected clinical samples. By simply varying the run time, the LAMP assay was able to
40
accurately distinguish individuals at risk for post-ivermectin serious adverse events
41
(SAEs), using thresholds of >5,000 mf/ml and >30,000 mf/ml as indicators of increasing
42
levels of risk.
43
In summary, LLMF72 LAMP represents a new molecular diagnostic tool that is readily
44
applicable as a point-of-care method for L. loa microfilarial detection and quantification
45
in resource-limited endemic countries.
46
2
47
Background
48
L. loa, the causative agent of loiasis, is a parasitic nematode transmitted to humans by
49
the tabanid Chrysops fly (1), with transmission confined to the rainforest and some
50
savannah areas of West and Central Africa (2). Although the overwhelming majority of
51
L. loa-infected individuals are clinically asymptomatic, Calabar swelling (transient,
52
localized angioedema) and the subconjunctival migration of an adult worm (“eyeworm”)
53
are the most common clinical manifestations (3-5). Rarely, nephropathy,
54
cardiomyopathy, retinopathy, neuropsychiatric complications and encephalopathy (6-12)
55
can occur as a consequence of chronic infection. Among the 8 filarial infections of
56
humans, L. loa had been largely neglected as a public health concern in Africa, though it
57
has gained prominence of late because of the neurologic serious adverse events (SAEs)
58
that have occurred, occasionally leading to death, in highly microfilaremic individuals
59
following exposure to ivermectin (IVM) given during mass drug administration (MDA)
60
for control of Onchocerca volvulus and/or Wuchereria bancrofti in regions where they
61
are co-endemic with L. loa (13, 14).
62
Although the pathogenesis of the neurologic SAEs are still not well understood
63
(8, 15), data clearly demonstrate a relationship between the pre-treatment L. loa
64
microfilariae (mf) density (14, 16) and the risk of SAEs, with levels of 5,000 mf/ml and
65
30,000 mf/ml felt to be the most significant thresholds (13), though other factors may
66
also be involved (17-19). Consequently, some IVM-based MDA for onchocerciasis (Ov)
67
have been delayed, and lymphatic filariasis (LF) control programs (using
68
IVM/albendazole) have been put on hold in large parts of Central Africa because of co-
69
endemic L. loa infection (20).
3
70
In endemic areas, the routine method for diagnosis and quantification of L. loa is
71
based on calibrated microscopic examination of mid-day blood for microfilariae, a
72
process that is neither point-of-care nor high-throughput. Indeed, it is felt to be
73
impractical for use as a widespread screening tool (21). Serological (22, 23) and
74
molecular (24-26) alternative tests have been developed, but only real-time PCR
75
methodology to date has been able to combine a high degree of sensitivity and specificity
76
with the ability to accurately quantify L. loa mf levels (26, 27). These methods, however,
77
require a centralized, well-equipped laboratory and relatively expensive reagents.
78
Loop-mediated isothermal amplification (LAMP) has emerged as a potential
79
alternative to DNA amplification techniques requiring thermocycling and automated
80
fluorescence description (28-30). LAMP relies on an auto cycling strand displacement
81
DNA synthesis performed at a single temperature (30, 31). The byproduct of the LAMP
82
reaction is magnesium pyrophosphate that accumulates as the reaction progresses and
83
that can be monitored using turbidity measurements or visually using a variety of
84
intercalating dyes (e.g., SYBR Green I, calcein, hydroxy naphthol blue (HNB)). In
85
particular, adding HNB dye to the reaction tube before the amplification has improved
86
the capacity of the LAMP method to be used as a point-of-care diagnostic test, since it
87
has made the assay easier to use in resource-limited settings (32, 33). Recently, LAMP
88
technology has been applied with high accuracy to detect infection with a wide array of
89
pathogens including bacteria (34, 35), viruses (36) and parasites (28, 37, 38).
90
In the present study, we have used two L. loa-specific single copy targets,
91
LLMF72 and LLMF342, to design LAMP primers for detection of L. loa mf in human
92
blood and have developed methods that provide similar sensitivity and specificity as the
4
93
previously described qPCR assay targeting the LLMF72 gene (26). Furthermore, we have
94
identified LAMP conditions that allow for colorimetric determination of microfilarial
95
levels above or below specified thresholds such that our LAMP method now has the
96
potential to be a practical point-of-care tool for identifying people at risk for SAEs when
97
given IVM.
98 99
Methods
100 101
Samples
102
The source of genomic DNA from L. loa, Wuchereria bancrofti, Brugia malayi and
103
Mansonella perstans mf has been described previously (26). For some experiments,
104
100,000 purified L. loa mf were placed in a fixed volume (1 ml) of distilled water and
105
serially diluted to create standards for quantification. DNA was then extracted and used
106
for assessment of the sensitivity of the assays. Finally, to evaluate the performance of the
107
L. loa LAMP assay compared to the standard qPCR method, 93 mid-day dried blood-
108
spots on filter paper (Whatman 1mm) previously collected in Cameroon (26) were
109
assessed. These blood spots were obtained by fingerprick from volunteers living in a
110
region endemic for L. loa infection. Briefly, capillary blood was spotted onto Whatman
111
filter paper, dried and stored at 4°C until their use for DNA extraction.
112 113
DNA extraction for spiked mf and field-blood spot samples
114
Blood spots were punched using a disposable 6mm biopsy punch (Acuderm, Inc., Ft.
115
Lauderdale, FL, USA). Two punched blood spots (10µL dried blood each) were
5
116
immersed in 400µL of water in a Precellys® lysing tube (Peqlab, Wilmington, USA)
117
containing glass and ceramic beads. Tubes were homogenized at 6,500 rcf for 5 minutes
118
using a Precellys24® bead beating machine (Peqlab, Wilmington, USA). These
119
homogenized samples were then heated for 30 minutes at 99°C while shaking in a
120
thermomixer (Eppendorf, North America, USA). Finally, the samples were centrifuged at
121
15,700 rcf for 10 minutes, and the supernatant was collected for use. DNA was also
122
isolated from water spiked with 100,000 purified L. loa mf/ml using the method
123
described above.
124 125
Primer design
126
L. loa mf-specific LAMP primers were designed for LLMF72 (GenBank: HM753552.1)
127
and LLMF342 (intronic region 1,900-2,200 of a L. loa contig 3.498; GenBank:
128
ADBU02000498.1) using Primer Explorer v4
129
(http://primerexplorer.jp/elamp4.0.0/index.html). A set of six specific primers comprising
130
two outer, two inner or internal, and two loop primers were designed for each targeted
131
gene. Details of the sequences and targets of the designed primers are listed in Table 1.
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All of the primers (Eurofins MWG Operon, Huntsville, AL, USA) were HPLC purified.
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Their specificity was further confirmed using the BLAST algorithm
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(http://blast.ncbi.nlm.nih.gov/Blast.cgi).
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LAMP assay
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The LAMP reaction was performed using a kit designed by Eiken Chemical Company
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(Eiken Chemical Co., Ltd., Tokyo, Japan). Briefly, the reaction was carried out in a total
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volume of 25 µL (23 µL of the reaction mixture and 2 µL of the DNA template) in PCR
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micro-tubes (United Scientific Products, San Leandro, CA, USA). The reaction mixture
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contained 50 pmol each of the FIP and BIP primers, 5 pmol each of the F3 and B3
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primers, 25 pmol each of the LB and LF primers in 12.5 µL of a 2x buffer. Ultrapure
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water (7 µL), 1 µL of 1 mM of HNB (Sigma-Aldrich, Inc., St. Louis, MO, USA), 1 µL of
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the large fragment of Bst DNA polymerase (Eiken Chemical Co., Ltd., Tokyo, Japan) and
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2 µL of the DNA template were added to complete the reaction mixture. Positive (L. loa
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mf genomic DNA: 100 pg/ml) and negative (water) controls were included in each
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experiment, and precautions were taken to prevent cross-contamination (all experiments
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performed in a AirClean® 600 PCR workstation (AirClean® Systems, Creedmoor, NC,
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USA) with UV radiation for sterilization). All reactions were performed at 65°C
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(isothermal condition) for up to 60 min. The LAMP assay was stopped by incubation at
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80°C for 5 min, inactivating the Bst DNA polymerase. The amplification efficiency was
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measured by the HNB color change or by the turbidity using a real-time turbidimeter
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(Eiken Chemical Co., Ltd., Tokyo, Japan). A sample was considered positive for L. loa
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mf DNA if an obvious color change from purple to blue or increase in turbidity was
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observed, compared to the negative control. All samples were run in duplicate. If the
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duplicates varied (one positive, one negative), the samples were re-run in triplicate.
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Real-time PCR assay
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The qPCR was performed as previously described (26); all samples were also were tested
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for the ability to amplify a control plasmid DNA that was spiked into the DNA samples
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prior to DNA extraction to ensure a lack of inhibition (27).
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Statistical Analysis of results
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Statistical analyses including specificity and sensitivity calculations and correlations
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(Spearman Rank) were performed using GraphPad Prism 6.0 (GraphPad Software, Inc.,
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San Diego, CA, USA).
166 167
Results
168 169
Analytical specificity and sensitivity of Loa loa LAMP assay
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Species specificity of the LAMP assays was assessed using a real time turbidimeter
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(Figure 1A) with the LLMF72 set of primers. As can be seen, these primers fail to
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amplify genomic DNA of B. malayi, W. bancrofti, O.volvulus, M. perstans whereas they
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amplified genomic DNA of L. loa easily and did so in a dose dependent manner. In data
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not shown, the primers derived from LLMF72 and LLMF342 failed to amplify genomic
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DNA of any of the 5 Plasmodium species capable of infecting humans either.
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To allow for a simplified detection method that could be visualized by the naked
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eye, colorimetric detection of the LAMP assays using a HNB dye was performed next
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(Figure 1B). For both LLMF72- and LLMF342-based (not shown) assays, specificity for
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L. loa could be seen in that only the L. loa DNA samples were blue (positive), whereas
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samples with each of the related other filarial species were purple (negative).
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To assess the analytic sensitivity of the LAMP assays, the limit of DNA detection
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was determined by testing serial dilutions of genomic DNA obtained from varying
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numbers of L. loa mf (Figure 2). With the LLMF72 assay, the lowest amount of
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detectable gDNA was 0.1 pg/ml (Fig. 2A, top panel) whereas for LLMF342 (Fig 2A,
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185
middle panel) the limit of detection was 10 pg/ml. When both sets of primers were used
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together (Fig 2A, bottom panel) in an attempt to improve even further the sensitivity, we
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found little to no improvement over the analytic sensitivity of LLMF72 alone. Similarly,
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when these assays were performed on DNA extracted from varying concentrations of mf,
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we found that the limit of detection was 5 mf/ml for LLMF72 (Fig. 2B, top panel), 50
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mf/ml for LLMF342 (Fig. 2B, middle panel), and 5 mf/ml for the two sets of primers
191
together (Fig. 2B, lower panel).
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Time dependent threshold assessments for performing semi-quantitative LAMP
194
assays
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Because the goal of this project was to provide a potentially point-of-care method of
196
amplification, detection and quantitation, we next assessed whether our LAMP assay
197
could be used in a semi-quantitative manner by exploring how varying the reaction time
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affected the results of the colorimetric LAMP assays when performed on DNA obtained
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from varying concentrations of L. loa mf. For each of the LLMF72, LLMF342 and
200
LLMF72-LLMF342 LAMP assays, the color change in the reaction tubes from purple
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(negative) to blue (positive) was monitored every 5 minutes. The time to positivity was
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then plotted as a function of mf concentration (Figure 3). For DNA samples prepared
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from L. loa mf concentrations of 30,000 mf/ml and above, the earliest color change was
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observed at 15 minutes after initiation for LLMF72-based assays, at 25 minutes for
205
LLMF342, and at 20 minutes for combined LLMF72-LLMF342-based assays. At the
206
limits of detection for L. loa mf concentration (5 mf/ml in LLMF72 and LLMF72-
207
LLMF342 assays and 10 mf/ml in LLMF342 LAMP), a color change occurred after 30
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208
minutes for LLMF72 assay and after 40 minutes for LLMF342 and the combined
209
LLMF72-LLMF342 assays (Figure 3). In addition, over the range of mf concentration
210
dilutions there was a high degree of correlation observed between the time to LAMP
211
reaction positivity (minutes) and the cycle number (Ct value) obtained by qPCR for both
212
the LLMF72 assays (Figure 4A; r = 0.96 and p5,000 mf/ml), and 25
220
minutes for a threshold of (100 mf/ml). This difference in time to positivity based on the
221
amount of DNA was also observed in the LLMF342 and LLMF72-LLMF342 assays,
222
though the run time for which a positive test corresponded to the specified threshold
223
concentrations of 30,000, 5,000 and 100 mf/ml differed slightly between the various
224
assays.
225 226
LLMF72 Loa loa LAMP performance compared to qPCR
227
To further assess the performance of the LAMP assay, a formal comparison was made
228
between LLMF72-based qPCR and colorimetric LAMP using field-collected dried blood
229
spots obtained from 93 individuals living in L. loa-endemic regions of Cameroon (Table
230
2). Of the 93 samples tested, 50 were positive and 43 were negative by qPCR. All qPCR
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231
positive samples were also positive by LAMP, and there were 3 additional qPCR negative
232
samples that were positive or indeterminate (not purple but not completely blue) in the
233
LAMP assay (presumably false positives). Thus, considering the qPCR to be the “gold
234
standard”, the LAMP assay has a sensitivity of 100.0% (95% CI, 92.9%–100.0%), a
235
specificity of 93.0% (95% CI, 80.9%–98.5%), a positive predictive value of 94.3% (95%
236
CI, 94.3%–98.8%), and a negative predictive value of 100.0% (95% CI, 91.2%–100.0%).
237 238
Discussion:
239
Because co-incident Loa loa infection has had a severely negative impact on control
240
(MDA) programs for onchocerciasis and lymphatic filariasis in West and Central Africa,
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there is a consensus that point-of-care diagnostics for L. loa infection in co-endemic
242
regions of the world are needed to achieve Ov and LF global elimination goals. Current
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qPCR-based methods are highly sensitive, specific and quantitative but require
244
sophisticated equipment and are costly. Thus, we developed a LAMP assay and
245
demonstrated its potential for use as a point-of-care diagnosis tool in field settings.
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Both LLMF72 and LLMF342-based LAMP assays were highly L. loa-specific with
247
respect to other related filarial parasites (W. bancrofti, M. perstans, B. malayi and O.
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volvulus). This result was expected based on de novo bioinformatics assessments
249
previously performed in a slightly different context (26). Moreover, the analytic
250
sensitivity was equally high when compared to qPCR (Figure 4). The LAMP assay was
251
also highly comparable to the “gold standard” qPCR in terms of clinical sensitivity and
252
specificity. In addition, its predictive positive (94.3%) and negative (100.0%) values
253
support its use for clinical diagnostic purposes.
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Although LAMP assays (like qPCR) can be quantitated in a real-time using a
255
sophisticated turbidimeter (38), having the ability to perform the assays at the point-of-
256
care or with minimal instrumentation led us to monitor our assays using a colorimetric
257
readout through the use of HNB dye, previously shown to be useful in endpoint LAMP
258
assays (32). Thus, the HNB-based LAMP assay provides a potential point-of-care method
259
of rapid amplification and easy detection of L. loa DNA that, when standardized, can
260
accurately distinguish levels of mf that are correlated with increased risk for SAEs
261
(>30,000 mf/ml: LAMP assay positive at 15 minutes) from those who might not be at
262
risk (
