O-5S quantitative real-time PCR: a new diagnostic tool for laboratory confirmation of human onchocerciasis
Mekonnen et al. Parasites & Vectors
O-5S quantitative real-time PCR: a new diagnostic tool for laboratory confirmation of human onchocerciasis
Equal contributors 1 3
Department of Medical Laboratory Sciences 1
College of 1
0 Department Microbial, Cellular and Molecular Biology, Addis Ababa University , Addis Ababa , Ethiopia
1 Health Sciences, Jimma University , Jimma , Ethiopia
2 Department of Infectious Diseases and Tropical Medicine, Medical Center of the University of Munich , Munich , Germany
3 Department of Medical Laboratory Sciences and Pathology, College of Health Sciences, Jimma University , Jimma , Ethiopia
4 German Centre for Infection Research (DZIF), partner site Munich , Munich , Germany
5 University Medical Center Hamburg-Eppendorf, University of Hamburg , Hamburg , Germany
6 Dr. Battke SCIENTIA GmbH , Taufkirchen , Germany
Background: Onchocerciasis is a parasitic disease caused by the filarial nematode Onchocerca volvulus. In endemic areas, the diagnosis is commonly confirmed by microscopic examination of skin snip samples, though this technique is considered to have low sensitivity. The available melting-curve based quantitative real-time PCR (qPCR) using degenerated primers targeting the O-150 repeat of O. volvulus was considered insufficient for confirming the individual diagnosis, especially in elimination studies. This study aimed to improve detection of O. volvulus DNA in clinical samples through the development of a highly sensitive qPCR assay. Methods: A novel hydrolysis probe based qPCR assay was designed targeting the specific sequence of the O. volvulus O-5S rRNA gene. A total of 200 clinically suspected onchocerciasis cases were included from Goma district in South-west Ethiopia, from October 2012 through May 2013. Skin snip samples were collected and subjected to microscopy, O-150 qPCR, and the novel O-5S qPCR. Results: Among the 200 individuals, 133 patients tested positive (positivity rate of 66.5%) and 67 negative by O-5S qPCR, 74 tested positive by microscopy (37.0%) and 78 tested positive by O-150 qPCR (39.0%). Among the 133 O-5S qPCR positive individuals, microscopy and O-150 qPCR detected 55.6 and 59.4% patients, respectively, implying a higher sensitivity of O-5S qPCR than microscopy and O-150 qPCR. None of the 67 individuals who tested negative by O-5S qPCR tested positive by microscopy or O-150 qPCR, implying 100% specificity of the newly designed O-5S qPCR assay. Conclusions: The novel O-5S qPCR assay is more sensitive than both microscopic examination and the existing O-150 qPCR for the detection of O. volvulus from skin snip samples. The newly designed assay is an important step towards appropriate individual diagnosis and control of onchocerciasis.
Confirmation; Microscopy; Molecular diagnosis; Onchocerciasis; O-5S qPCR; O-150 qPCR; Ethiopia
Onchocerciasis, also referred to as “river blindness,” is
caused by the filarial nematode Onchocerca volvulus, which
is transmitted to humans by black flies (Simulium spp.) in
endemic countries of Africa, including Ethiopia, three
countries in Latin America, and in Yemen. This parasitic
disease was listed by the World Health Organization
(WHO) as one of the eighteen neglected tropical diseases
]. Onchocerciasis constitutes a major public health
problem in affected regions, as an estimated 37 million
persons are infected with O. volvulus worldwide, with the
vast majority of them living in East and West Africa. As this
disease is endemic in some of the world’s poorest areas, it
had a major impact on the economic and social aspects of
affected communities [
]. Ethiopia is one of the countries
with a high burden of onchocerciasis, where the disease has
spread widely to regions that were previously non-endemic
. In 1998, the “African Program for Onchocerciasis
Control” sponsored the first nationwide epidemiological
mapping of onchocerciasis in Ethiopia. Following the
results of that study, most areas of Central, South, and West
Ethiopia were defined endemic [
To avoid severe progression of the disease, early detection
and treatment are essential. A clinically suspected O.
volvulus infection can be confirmed by different laboratory
methods: microscopic detection of microfilariae (mf ) from
skin snip samples (SSS) following incubation in normal
saline; detection of the major sperm protein 2 (MSP2) by
dipstick test; detection of recombinant antigens by means of a
luciferase immune precipitation system (QLIPS); and rapid
DNA detection test strips; and polymerase chain reaction
]. The microscopic examination is cheap and
easy to perform. However, this method has several
limitations, as its sensitivity depends on the location where skin
snips were taken, mf density, stage of disease, previous
treatment, and especially on the qualifications of staff
performing tests [
Various PCR-based assays were described for the
detection of different larval stages of O. volvulus [
more than a decade, a conventional gel-based PCR applying
degenerated primers amplifying the O. volvulus-specific
O150 tandem repeat [
] was often used to confirm
onchocerciasis from SSS [
14, 26, 27
]. In 2011, Fink et al.
described a melting curve based quantitative real-time PCR
(qPCR) which also used degenerated primers to detect a
154 bp amplicon of the O-150 repeat (O-150 qPCR) .
However, the long PCR amplicon may lead to limited
sensitivity, and the lack of a hybridization probe may reduce the
specificity of this assay [
]. In recent years, Lloyd et al.
] and Golden et al. [
] showed the importance of PCR
for the detection of O. volvulus.
In Ethiopia, cases of onchocerciasis are routinely
diagnosed by microscopic examination of SSS, which has
limited sensitivity especially in samples with low mf density
and is therefore not sufficiently reliable for programmatic
monitoring and evaluation of the effects of the WHO
recommended mass drug administration [
]. The objective
of this study was to develop and validate a novel
hydrolysis probe-based qPCR targeting the 5S rRNA gene of O.
volvulus (O-5S qPCR) with enhanced sensitivity for the
detection of O. volvulus DNA from SSS of clinically
diagnosed patients from Ethiopia. The diagnostic validity of
the novel qPCR assay was compared with direct
microscopy and the established O-150 qPCR. The study
furthermore compared socio-demographic data, potential risk
factors for onchocerciasis, clinical symptoms and medical
histories with the laboratory results.
one of the 13 districts in Jimma zone, situated in Oromia
region in Ethiopia [
]. Goma district has 36 rural and three
urban “kebeles” (i.e. smallest administrative unit in Ethiopia).
The number of study participants was calculated in
proportion to the total number of inhabitants in each of these
kebeles. The following numbers of study participants were
included from four randomly chosen kebeles: Dedessa
(n = 28), Omo Gobu (n = 52), Belfo Konche (n = 84) and
Kilole Qirqir (n = 36). Individuals living in the study area
with any form of the following clinical manifestation(s) were
included in the present study: papular rash, scarring
“leopard skin” (i.e. spotted depigmentation of the skin) and skin
itching around the buttocks. Patients who received
ivermectin treatment during the past six months were excluded.
Study design and data collection
In this cross-sectional study, data from 200 clinically
suspected onchocerciasis individuals were collected from
October 2012 through May 2013. After written informed
consent was obtained, study participants were interviewed
by trained study nurses to record socio-demographic data,
potential risk factors for onchocerciasis and medical
history by pre-designed and pre-tested questionnaires.
Data were completely pseudonymized and transferred to
an Excel-based database (Microsoft, Redmond, WA,
USA). SSS were collected from either side of the buttocks
by senior medical laboratory technologists according to
routine standardized procedures [
The collected SSS was transferred to one cavity of a
96well plate, leaving blank cavities between samples to avoid
cross contamination. Three drops of phosphate buffered
saline (PBS, Sigma-Aldrich, Bangalore, India) were added
to allow complete release of mf during a four h incubation
period. The samples were sealed by Titer Tops sealing
film for microplates (Sigma-Aldrich) and transported to a
laboratory of health centers nearby the study areas for wet
mount microscopic examination. There, PBS from SSS
was transferred to microscopic slides for the detection of
O. volvulus mf with light microscopy under 10- and
40fold objectives by experienced senior laboratory
technologists. The remaining samples were stored in the respective
96-well plates, transported within 24 h to the Department
of Medical Laboratory Sciences and Pathology of Jimma
University, and there immediately deep-frozen at -20 °C
until shipment by courier service to the Department for
Infectious Diseases and Tropical Medicine (DITM) of the
Ludwig-Maximilians University in Munich, Germany.
Study area and inclusion criteria
The study was conducted in Goma district, which has a
population of approximately 213,000 inhabitants. Goma is
O-5S qPCR assay
Target sequence and primer design
A qPCR assay was developed for the detection of O.
volvulus from human SSS. After an extensive database search
(GenBank [PubMed, NCBI]) and sequence analyses
(DNASIS Max software [MiraiBio, San Francisco, CA]), an
intergenic spacer region of the O. volvulus 5S rRNA gene was
chosen as a target for PCR amplification. Primers and a
hydrolysis probe (MWG Eurofins, Ebersberg, Germany) for
amplification of this O. volvulus-specific intergenic spacer
region of the 5S rRNA gene were designed by alignment of
5S rRNA gene sequences (DNASIS Max) retrieved from
] from closely related filarial nematodes
potentially contaminating the SSS from human skin by primer 3.
Specificity of the primers for O. volvulus was confirmed in
silico using the basic local alignment search tool (BLAST,
GenBank, NCBI). The selected 5S rRNA target, primers
and hydrolysis probe, and alignments of the target region
from O. volvulus with Wuchereria bancrofti, Brugyia
malayi, Loa loa, Mansonella streptocerca and Plasmodium
falciparum are provided in Additional file 1.
O-5S qPCR standard
A PCR standard template was generated by gene synthesis
of the target sequence and subsequent cloning to the
pEXA2 vector (MWG, Ebersberg, Germany). The vector was
transformed into E. coli (JM109, Zymo, Freiburg, Germany)
and the transformation mix was plated on LB agar plates
with 100 mg/l ampicillin (Carl Roth, Karlsruhe, Germany)
and cultivated at 37 °C overnight. By selection of one
suitable colony, a master cryo culture was obtained and stored
in RotiStore tubes (Carl Roth). From this master cryo
culture, a 3 ml LB liquid culture with 100 mg/l ampicillin (Carl
Roth) was grown over night. The plasmid DNA was
extracted with a commercial plasmid extraction kit, according
to the manufacturer’s instruction (HiYield Plasmid Mini kit;
Süd-Laborbedarf, Gauting, Germany). The cloned plasmid
sequence was confirmed by direct DNA sequencing as
previously described [
]. The purity of extracted plasmid
DNA was assessed by photometry on a BioPhotometer plus
(Eppendorf, Wesseling-Berzdorf, Germany) and agarose
gel-electrophoresis on a 1% TAE gel. The plasmid solution
was quantified by a dsDNA fluorescence quantification kit
(Qubit, Life Technologies, Karlsruhe, Germany), according
to the manufacturer’s instruction. The number of fragments
per μl was calculated and aliquots of the plasmid solution,
diluted to the desired fragment numbers, were used for
determination of the lower limit of detection (LOD, i.e.
lowest template concentration rendering positive
amplification of 95% of samples) [
O-5S qPCR protocol
The primers for O-5S qPCR were as described in Table 1.
The reaction mixture (total volume: 20 μl) contained 8.6 μl
molecular grade H2O (Carl Roth), 4 μl 5-fold PCR buffer
qPCR Mix Plus (Solis BioDyne, Tartu, Estonia), 2 μl 10-fold
Exo IPC Mix (TaqMan exogenous internal positive control
reagent; ThermoFisher Scientific, Darmstadt, Germany),
1 μl of each primer and probe (10 μM), 0.4 μl 50-fold Exo
IPC DNA (ThermoFisher Scientific) and 2 μl DNA
template. The amplification was performed at 95 °C for 15 min,
followed by 45 cycles of 95 °C for 15 s, 56 °C for 20 s and
detection at 72 °C for 20 s on a Bio-Rad CFX-96 real-time
PCR machine (Bio-Rad, Munich, Germany) and included
negative extraction, negative “no template”, positive run
controls as well as the internal positive controls (IPC).
Specificity and sensitivity of O-5S qPCR
Specificity of the assay was assessed by using 20 O-150
PCR positive (“must detect”) and 20 O-150 PCR negative
(“must not detect”) O. volvulus clinical DNA extracts from
SSS collected in a previous study [
], confirmed DNA
extracts from Dirofilaria immitis (n = 3), D. repens (n = 1),
Loa loa (n = 1) and Mansonella streptocerca (n = 1). The
analytical sensitivity of the O-5S qPCR assay was
determined as LOD by using 10-fold serial dilutions of O-5S
plasmid standards with known copy numbers. Intra- and
inter-assay variability were assessed by testing each sample
in quadruplicate within one 96-well plate, repeated on
three different days. Variability was judged low if the
maximum cycle threshold variation range (Cq-rangemax; i.e.
range of Cq-values of samples tested in the same dilution)
was ≤0.5 (intra-assay) and ≤1.0 (inter-assay).
O-150 melting curve based qPCR
The melting curve based O-150 qPCR assay was conducted
according to the protocol described by Fink et al. [
] on a
Bio-Rad CFX-96 real-time PCR machine (Bio-Rad) and
included negative extraction, negative “no template” and
positive run controls.
O-150 hybridization probe based qPCR assay
The O-150 hybridization probe based qPCR assay was
performed as described by Golden et al. 2016 [
] with minor
modifications as the original qPCR protocol did not lead to
any amplicons. Briefly, the sequences of primers and probe
were: OvFWD 5′-TGT GGA AAT TCA CCT AAA TAT
G3′, OvREV 5′-AAT AAC TGA TGA CCT ATG ACC-3′
(Eurofins Genomics, Ebersberg, Germany), and OvProbe
5′6FAM-TAG GAC CCA ATT CGA ATG TAT GTA
CCCMGBNFQ-3′ (minor groove binding TaqMan® Probe
#5208995 P/N 4316033, Applied Biosystems, Inchinnan,
UK). The qPCR assay was designed to amplify the O-150
repeat of O. volvulus using 7.5 μl SuperHot Master Mix (2×)
(BIORON, Ludwigshafen, Germany), 0.1 μl of 100 μM
forward primer, 0.1 μl of 100 μM reverse primer, 0.1 μl of
100 μM probe, and 7.2 μl DNA extract/negative control
(nuclease free water), positive control (O150 plasmid standard).
Finally, the O-150 qPCR was run in a total volume of 15 μl
with an initial denaturation at 94 °C for 2 min, [denaturation
at 94 °C for 15 s, hybridization at 49 °C for 30 s, and
elongation at 60 °C for 2 min] × 45 cycles. All test or control
samples were run in a single well and analyzed using an Illumina
Eco Real-time PCR system. An O-150 plasmid standard
(Genbank accession number J04659.1) was generated by gene
synthesis (Eurofins Genomics). The maximum quantification
cycle for a positive result was set to 37 according to the
lowest detectable plasmid standard concentration. This assay
was performed in an observer-blinded way without
knowledge of microscopy or other PCR results.
Comparison of O-150 and O-5S qPCR assays
The SSS were thawed and transferred into 2 ml screw
cap tubes (Sarstedt, Nümbrecht, Germany) containing
700 μl cell lysis buffer (CLS, Qiagen, Hilden, Germany)
to allow transport at ambient temperature without DNA
degradation by courier service to DITM.
The dsDNA of O. volvulus was extracted using the
Puregene DNA extraction tissue kit (Qiagen) using enzymatic
lysis as described elsewhere [
] at DITM. Briefly, SSS
were inactivated at 95 °C for 15 min. Subsequently,
samples were incubated overnight at 55 °C in 700 μl CLS
enriched with proteinase K (Sigma-Aldrich) to a final
concentration of 300 μg/ml. The proteinase K was
inactivated at 95 °C for 15 min. After the specimens were
cooled to ambient temperature, lysozyme (Qiagen) was
added to a final concentration of 250 μg/ml and
specimens were incubated at 37 °C for one h. Finally, the
DNA pellets were resuspended in 200 μl DNA hydration
solution (Qiagen). DNA extracts were stored at 4 °C
(short-term, up to one week) or -20 °C (long-term
storage) until further procession by PCR.
At DITM DNA extracts from SSS were subjected to O-150
qPCR and to the novel O-5S qPCR assays by independent
and blinded laboratory technicians who were unaware of
the microscopic results.
All data were stored in a Microsoft Excel database
(Microsoft). Bivariate tests (chi-square tests including Fisher’s exact
test and McNemar chi-square test for matched pairs of
samples with categorical test results), multivariate logistic
regression, t-tests as parametric test, and estimation of standard
error of proportion (to calculate 95% confidence intervals,
95% CI, of categorical test results) were conducted using
EpiInfo, version 3.3.2. (Centers for Disease Control and
Prevention, Atlanta, GA) and Stata software, version 9.0. (Stata
Corporation, College Station, TX). Significant differences
were defined as p values below 0.05 or as non-overlapping
95% CI of proportions. The sensitivity and specificity of
microscopy and O-150 qPCR were calculated by comparing
these results with those of O-5S qPCR as a reference test.
Novel O-5S qPCR results
Out of 266 individuals with clinically suspected
onchocerciasis, 200 (75.19%) fulfilled the inclusion criteria and
composed the study population. Among these, 133
(66.50%) tested positive by the newly developed O-5S
qPCR assay. Interestingly, none of the individuals who
tested negative by the novel O-5S qPCR tested positive
by microscopy or by O-150 qPCR assay (Table 2).
Among the 200 individuals comprising the study
population, microscopic examination of SSS detected O. volvulus
mf in 74 individuals, corresponding to a positivity rate of
37.00%. These individuals tested positive by the newly
developed assay, O-5S qPCR, which corresponds to a
sensitivity of 55.64% (95% CI: 47.20–64.08%), and none of the
individuals who tested positive by microscopy tested
negative by O-5S qPCR, which corresponds to a specificity of
100%. The probability for positive microscopy results to
be true positive (positive predictive value) was 100%, and
for negative results to be true negative (negative predictive
value) was 53.17% (95% CI: 44.46–61.89) (Table 2).
O-150 qPCR results
Among the 200 individuals clinically suspected for
onchocerciasis, the O-150 qPCR assay by Fink et al. detected
DNA of O. volvulus in 78 patients, which corresponds to a
positivity rate of 39.00%. All these patients tested positive
by O-5S qPCR, which corresponds to a sensitivity of
59.40% (95% CI: 50.28–67.02%), with none of these
individuals testing negative by O-5S qPCR, which corresponds
to a specificity of 100%. The probability for positive O-150
qPCR results to be true positive (positive predictive value)
was 100%, and for negative results to be true negative
(negative predictive value) was 54.92% (95% CI: 46.09–
63.75) (Table 2).
aMcNemar’s chi-square test for matched pairs: χ2 = 59.00 (microscopy and O-5S qPCR); χ2 = 54.00 (O-150 qPCR and O-5S qPCR)
*Significant differences were defined as P-values < 0.05
Abbreviation: na not applicable
From the total of 200 SSS collected among individuals
clinically suspected for onchocerciasis, the O-150 qPCR
assay by Golden et al. detected DNA of O. volvulus in 33
of them, which corresponds to a positivity rate of 16.5%.
Of these 33 individuals, only 23 individuals tested
positive by microscopy, the O-150 qPCR assay by Fink et al.
assay and our newly developed O-5S qPCR assay,
respectively. Unexpectedly, ten individuals that tested
positive in the assay by Golden et al. tested negative with
all other three diagnostic methods (Additional file 2:
Table S1). Therefore, these results could not be used as
Performance characteristics of the novel O-5S qPCR assay
In silico analysis of the novel primers/probe revealed
100% specificity for O. volvulus among human pathogenic
filariae, which excluded amplification of human DNA.
Testing of “must detect O-5S samples” revealed positive
amplification results for 20/20 (100%) O. volvulus DNA
extracts, while all other “must not detect O-5S samples”
remained negative. Therefore, the specificity of the novel
O-5S qPCR was 100%. The LOD was six copies of the
target sequence of O. volvulus. Intra- and inter-assay
variabilities were low. The calibration curve is provided in
Additional file 3: Figure S1. Furthermore, the novel O-5S
qPCR assay does not amplify the DNA of Mansonella
streptocerca, a closely related filarial parasite (Fig. 1).
Among the 133 individuals tested positive by O-5S qPCR,
61 (45.86%) had a positive microscopy result. Out of the
78 individuals tested positive by O-150 qPCR, 13
individuals (9.77%) had a positive microscopy result, 17
individuals (12.78%) had a positive result only with O-150 qPCR,
and 42 (31.58%) had a negative result with both tests. The
concordance rate between microscopy and O-150 qPCR
was 77.44% (103/133) among individuals tested positive
by O-5S qPCR. Among all 67 individuals tested negative
by O-5S qPCRs, both tests resulted in negative results,
corresponding to a concordance rate between microscopy
and O-150 qPCR of 100% (67/67). In the study population
of 200 patients, the overall concordance rate between
microscopy and O-150 qPCR was 85% (170/200). Between
microscopy and O-5S qPCR, the concordance rate was
70.5% (141/200), and between O-150 qPCR and O-5S
qPCR, 72.5% (145/200) (Table 2). Of note, there is no
correlation between both assays (Additional file 4: Figure S2).
The introduction of the O-150 qPCR to the already widely
performed microscopy for the diagnosis of onchocerciasis
has been shown in this study to provide an additional
diagnostic yield of 13% (17/133 cases). By replacing
microscopy with O-150 qPCR, the additional diagnostic yield
would have been 3% (4/133 cases) in this study. Among
the 133 individuals tested positive by O-5S qPCR, 91
(68.42%) tested positive by microscopy or O-150 qPCR.
By performing the novel O-5S qPCR, an additional
diagnostic yield of 42 (31.58%) was achieved (Table 2).
Among the individuals who tested positive by the newly
designed O-5S qPCR assay, the proportion of females (22.56%)
was not significantly (χ2 = 0.07, df = 1, P = 0.79) different
from those tested negative (20.90%). The median age was
32 years among individuals who tested positive, whereas it
was 15 years among those tested negative. The proportion
of age group 30–72 years among those who tested positive
(56.39%) was significantly (χ2 = 9.85, df = 1, P < 0.01) higher
than among those who tested negative (32.84%). The
proportion of farmers among individuals tested positive
(70.68%) was significantly (χ2 = 22.14, df = 2, P < 0.01)
higher than among those tested negative (37.31%), however,
this association was highly confounded by age (Table 3).
Risk factors and symptoms
For the variables “distance of residence from river” (<
1 km: 84.96% / 74.63%; > 1 km: 15.04% / 25.37%) and
“fetching water from river nearby residence” (Yes: 96.24%
/ 94.03%), no significantly different proportions were
found among individuals tested positive and negative. For
the variable “collecting wood from nearby forest” (Yes:
72.93% / 55.22%), the proportion were significantly
(χ2 = 6.29, df = 1, P = 0.01) higher among individuals
tested positive than among those who tested negative.
Also for the variables “palpable inguinal lymph nodes”
(43.61% / 20.90%) and “skin itching around buttock”
(74.44% / 46.27%), the proportions were significantly
(χ2 = 9.77, df = 1, and χ2 = 15.46, df = 1, respectively,
P < 0.01 each) higher among individuals who tested
positive than those who tested negative (Table 4).
Multivariate logistic regression has shown association
only for age and itching of the skin with positive results of
O-5S qPCR. Among those who tested positive, individuals
were more likely 30 years or older than among individuals
tested negative (adjusted OR = 2.17; 95% CI: 1.11–4.23).
Among those who tested positive, individuals reported
itching around buttocks more often than individuals tested
negative (adjusted OR = 2.13; 95% CI: 1.04–4.47) (Table 4).
This is the first study assessing a novel O-5S quantitative
real-time PCR for the molecular confirmation of O.
bFisher exact chi-square test
*Significant differences were defined as P-value < 0.05
Abbreviation: na not applicable
Age group: 3–29
Age group: 30–72
volvulus infections in Goma district, Southwest Ethiopia,
which is an endemic region for onchocerciasis. The present
study showed that the newly developed assay is more
sensitive than both microscopic examination of SSS and the
conventional O-150 qPCR assay for the diagnosis of human
onchocerciasis. This might be attributable to the
implementation of a very stable hydrolysis probe that is 100% specific
for O. volvulus from clinical samples. Moreover, the data
presented in this study showed that the O-5S qPCR assay
for the diagnosis of onchocerciasis from SSS can detect
low-density DNA of O. volvulus. This is especially needed
for reliable programmatic monitoring and evaluations
regarding national elimination campaigns [
the novel assay was highly sensitive, approaching a lower
limit of detection of six templates of the target sequence,
which increased the detection limit by half in comparison
to the O-150 qPCR [
]. This might be due to the careful
choice of the target sequence with only moderate secondary
structure forming primers, which prevent the formation of
primer-primer dimers [
The results of the present study did not show any
significant differences between microscopy and O-150 qPCR.
These two tests had comparable positivity rates of 37% and
39%, respectively. Furthermore, the concordance rate
between microscopy and O-150 qPCR was high: 77%
among individuals tested positive by O-5S qPCR, 100%
among individuals tested negative by O-5S qPCR, and 85%
of all clinically suspected patients. These results indicate
that the additional introduction of the O-150 qPCR would
not substantially increase the diagnostic yield (13%).
Completely replacing microscopy by O-150 qPCR would have
even decreased the diagnostic yield by 3% in this study. In
contrast, application of the novel O-5S qPCR resulted in an
additional diagnostic yield of 32%. The O-5S qPCR had a
much higher sensitivity in comparison to microscopy (56%)
or O-150 qPCR (59%), whereas the two conventional tests
had limited concordance rates of 71% and 73%,
respectively, compared to O-5S qPCR. Although the sensitivity
of the two conventional tests is limited, the present
study showed that both tests reached a specificity of
100% for O. volvulus.
Previously, different studies were conducted in various
regions of Ethiopia to determine the prevalence of
onchocerciasis using direct microscopy from SSS. In the present
study, the positivity rate for microscopic detection of
onchocerciasis was 37%, a finding comparable to positivity
rates as described in other studies from Ethiopia 31% in
] and 34% in Blue Nile valley [
]. On the other
hand, the positivity rate of onchocerciasis in the present
study was higher than reports from other locations in
Ethiopia 17% Gilgel Ghibe river valley [
], 23% Kaso Hixi
] and 22% in Teppi area [
] and lower than a study
conducted in Anfilo district (75%) [
]. Of note, our finding
has initiated the Zonal health bureau to launch ivermectin
mass treatment which benefits not only the study
participant but also other individuals living in the study area
which was not included in the present study.
Among individuals tested positive by O-5S qPCR, the
prevalence of males (77%) was higher than that of females
(23%). This finding is in line with previous studies that
reported a higher proportion of males among patients with
], which could be due to occupational
exposure and susceptibility of study participants. In the
rural parts of Ethiopia, males are more involved in outdoor
activities than females and prefer to wear short pants while
performing outdoor activities . Thus, outdoor activities
and wearing short pants may render males more prone to
black fly’s bites.
The newly designed assay for the detection of O. volvulus
DNA from SSS shows an improvement in performance and
an important step towards appropriate diagnosis of
onchocerciasis. This, in turn, can provide a crucial contribution to
disease progress monitoring including reliable programmatic
monitoring and evaluation of MDA, for example, to control
the elimination of onchocerciasis. Consequently, future
evaluation and demonstration trials are planned to apply the
novel O-5S qPCR in endemic regions of Ethiopia. Moreover,
the novel O-5S qPCR could serve as a valuable tool for
future drug trials, especially if detection and quantification
of low mf densities from tissue samples are required.
Additional file 1: Text. Selection of O. volvulus 5S sequence and
alignment of target sequence from O. volvulus with related nematode
sequences and Plasmodium falciparum. (DOCX 24 kb)
Additional file 2: Table S1. Additional file 2: Table S1. Socio-demographic,
clinical and diagnostic data of the study subjects. (XLSX 224 kb)
Additional file 3: Figure S1. Calibration curve of the novel O5-S qPCR.
(DOCX 22 kb)
Additional file 4: Figure S2. XY-graph showing the correlation
between O-150 qPCR and O-5S qPCR. (DOCX 19 kb)
DITM: Department for Infectious Diseases and Tropical Medicine;
ME: minimum evolution; mf: microfilariae; SSS: skin snip sample
The authors are very thankful for the study participants to take part in this study.
The authors would like to acknowledge the health extension workers at the site
of data collection for their nice mobilization of the community. Additionally, the
authors thank Drs Kifle Dagne and Haileyesus Adamu from Addis Ababa
University, Ethiopia, Dr. Bruktawit Kebede from Jimma University, Ethiopia, Mr.
Wondwossen M. Yeshaw from the University of Groningen, the Netherlands for
their constructive comments and fruitful discussions. Furthermore, the authors
thank Agata Rhomberg for technical assistance at DITM.
This work was supported by the Graduate School of Addis Ababa University (to
SAM), and by the Friedrich-Baur Foundation, the Ludwig-Maximilians University
of Munich (to MB).
Availability of data and materials
All data generated or analyzed during this study are included in this published
article as supplementary information files (Additional file 2: Table S1).
SAM, SA, KT, MB, TL, GB and KHH conceived the study. SAM, SA, AZ and
MGA collected samples. SAM, SA, MG, MB, MS and FB performed the
experiments. SAM, KT, MB, MS, SP and KHH analyzed the data. SAM, MB and
KHH wrote the manuscript with contributions from all authors. All authors
read and approved the final manuscript.
Ethics approval and consent to participate
The study protocol was reviewed by Jimma University, College of Public Health
and Medical Sciences Ethical Review Committees (RPGC/09/2012) and approved
by the Ministry of Science and Technology of Ethiopia (Ref. No 3–10/713/04).
Before the enrollment of study participants, written informed consent was
obtained from participants and parents in case of minors aged < 18 years. Given
illiteracy among rural populations, thumbprints were used as an alternative to
signatures in accordance with the recommendations of the national Ethics
Committee. Participants were fully free to withdraw from the study without any
consequences and without affecting the patients’ rights to health care, medical
advice, or treatment. Samples were shipped to the Department for Infectious
Diseases and Tropical Medicine at the Ludwig-Maximilians-University in Munich,
Germany based on an official “material transfer agreement.”
Consent for publication
The authors declare that they have no competing interests.
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