Diagnostic performance of direct wet mount microscopy in detecting intestinal helminths among pregnant women attending ante-natal care (ANC) in East Wollega, Oromia, Ethiopia
Mengist et al. BMC Res Notes
Diagnostic performance of direct wet mount microscopy in detecting intestinal helminths among pregnant women attending ante-natal care (ANC) in East Wollega, Oromia, Ethiopia
Hylemariam Mihiretie Mengist 0 1
Gebreselassie Demeke 1
Olifan Zewdie 0
Adugna Belew 0
0 Department of Medical Laboratory Sciences, College of Health Sciences, Wollega University , P.O. Box: 395, Nekemte , Ethiopia
1 Department of Medical Laboratory Sciences, College of Health Sciences, Debre Markos University , P.O. Box: 269, Debre Markos , Ethiopia
Objective: The aim of this study was to evaluate the diagnostic performance of direct wet mount microscopy compared to formalin ether concentration (FEC) technique in detecting intestinal helminths in pregnant women. Results: The total prevalence of intestinal helminths was 18.8% (70/372) by direct wet mount microscopy and 24.7% (92/372) by FEC technique (P < 0.001). The sensitivity, negative predictive value (NPV) and test efficiency (TE) of direct wet mount microscopy in diagnosing intestinal helminths was 76, 92.7 and 94%, respectively. The sensitivity of direct w et mount microscopy was very low in detecting ova of Hymenolepis nana. The two methods showed excellent agreement in detecting ova of Hook worm and Ascaris lumbricoides (Kappa > 0.81) but they fairly agreed in detecting ova of Hymenolepis nana (Kappa = 0.39). Intestinal helminths were underdiagnosed and the total diagnostic performance of direct wet mount microscopy was significantly poor in detecting intestinal helminths as compared to FEC technique. Routine use of FEC method is recommended for the diagnosis of intestinal helminths in pregnant women.
Diagnostic performance; Direct microscopy; Helminths; Pregnant women
Intestinal parasitic infections, especially due to
helminths, increase anemia in pregnant women. The results
of this are low pregnancy weight gain and intra uterine
growth retardation, followed by low birth weight, with its
associated greater risks of infection and higher prenatal
mortality rates. An estimated 44 million pregnant women
have hookworm infections which can cause chronic loss
of blood from the intestines and predisposes the women
to developing iron deficiency anemia [
Although several diagnostic tools are available to
diagnose intestinal helminths, direct wet mount microscopy
is commonly used for the diagnosis of intestinal parasitic
infections generally in Africa and particularly in Ethiopia
]. However, low sensitivity of the direct wet mount
technique has been reported to have poor performance
in the detection of low intensity infection elsewhere
which shows that the use of direct wet mount
microscopy will significantly increase misdiagnosis of intestinal
helminthic infections .
Different studies showed that formol-ether
concentration technique (FEC) is more sensitive than the
conventional direct wet mount microscopy. Therefore, the
employment of FEC techniques as a confirmatory test in
routine laboratory examination of stool will significantly
aid in accurate determination and management of
parasitic infections [
In Ethiopia, especially in health center and
hospital laboratories, diagnosis of intestinal helminths solely
depends on direct wet mount microscopy which is not
]. For better follow up and make morbidity free
pregnancy, screening of pregnant women for intestinal
helminths should be at most sensitive. The present study
is, therefore, aimed to evaluate the diagnostic
performance of direct wet mount microscopy among pregnant
women using FEC technique as a gold standard.
Materials and methods
Study setting and context
A cross sectional study was conducted in selected five
health centers of East Wollega Zone of Oromia region,
Ethiopia namely Jimma- Arjo health center, Arjo-Gudatu
health center, Sire health center, Gute health center and
Nekemte health center between November 2015 and
Sample size and sampling technique
Sample size was calculated using single population
proportion formula considering 95% CI and a marginal error
of 0.05 as follows;
n = Z(a/2)2 P(1 − P)/d2
where n is sample size which is 372; P is prevalence of
intestinal helminths in pregnant women from
previous similar study which was 0.41 [
]; d is marginal error
which is 0.05.
Finally, 372 pregnant women were consecutively
enrolled from the five health centers using proportional
Study population and data collection
Pregnant women taking anti-helminthic/anti-protozoan
drugs or received mass drug administration within the
past 2 weeks were excluded. Medical laboratory
professionals were trained on data collection for this particular
study to attain standardization and reliability. All
reagents used were checked for their expiry date and
prepared according to the manufacturer’s instructions. We
obtained all the reagents from Pharmaceuticals Fund and
Supply Agency of Ethiopia.
A single stool specimen was collected from each
participant. Freshly voided stool specimens were directly
examined microscopically and preserved with 10% formalin
for further analysis. Then preserved specimens were
processed using formalin-ether concentration technique
and examined microscopically for ova and larvae of
Direct wet mount analysis A singles stool specimen was
obtained from all study participants. Then a direct saline
wet mount microscopy of each sample was used to detect
intestinal parasites microscopically. Briefly, one drop of
normal saline was added on a clean slide and then a stool
equivalent to a match stick head (2 mg) was mixed with it.
Then, the wet mounts were examined under light
microscope under 100X and 400X magnifications [
Formol–Ether concentration (FEC) method A portion of
each preserved stool specimen was taken and processed
following standard procedures. Briefly, 1 gm stool was
placed in a clean conical centrifuge tube containing 7 mL
10% formol water by using applicator stick. The resulting
suspension was filtered through a sieve into another
conical tube. After adding 3-4 ml of diethyl ether to the
formalin solution, the content was centrifuged at 3200 rpm
for 1 min. The supernatant was discarded; smear was
prepared from these sediment and observed under light
microscope with a magnification of 100X and 400X after
air dried [
Data quality assurance
All laboratory analyses were carried out using standard
operating procedures. Only one medical laboratory
technologist performed the direct wet mount microscopy in
each health center and one senior medical laboratory
technologist performed the FEC technique blindly for all
False positive (FP)–the individual does not have the
condition but tests positive for the condition.
Sensitivity—is the probability that a truly infected
individual will test positive (sensitivity = TP/
(TP + FN)).
Specificity—is the ability of a method to identify
non-infected individuals correctly (specificity = TN/
(TN + FP)).
Positive predictive value (PPV): the probability
that those testing positive by the method are truly
infected (PPV = TP/(TP/FP)).
Negative predictive value (NPV)–the probability
that those testing negative by the test are truly
uninfected (NPV = TN/(TN + FN)).
Test efficiency (TE)–the overall ability of the test
to correctly identify positives from negatives and
implies the absence of false positives& false
negatives (TE = (TP + TN)/(TN + TP + FN + FP)).
Accuracy: closeness of the tests to a true value (a
true positive or a true negative).
Data entry and analysis were done using SPSS version 20
statistical software for descriptive and inferential
statistics. Prevalence, sensitivity, specificity, positive and
negative predictive values of direct wet mount microscopy
was determined by using FEC as a gold standard
technique. Agreement of the two methods in detecting
intestinal helminths was determined by Kappa test. Kappa
values were interpreted as follows; from 0.01 to 0.20 as
slight agreement, from 0.21 to 0.40 as fair agreement,
0.41–0.60 as moderate agreement, 0.61–0.80 as
substantial agreement and 0.81–0.99 as perfect agreement [
Prevalence of intestinal helminths
The total prevalence of intestinal helminths using direct
wet mount microscopy and formol-ether concentration
(FEC) method was 18.8% (70/372) and 24.7% (92/372),
respectively with the difference rate of 5.9% (P < 0.001)
Regarding the detection of specific intestinal
helminths, direct wet mount microscopy reported 12.9%
(48/372) Hookworm, 5.37% (20/372) Ascaris
lumbricoides, and 0.54% (2/372) Hymenolepis nana. But Taenia
species and Strogyloides stercoralis were not detected
by this method. Mixed infection was reported in two
(0.54%) of study participants using this method unlike the
FEC technique which detected mixed infections in four
(1.08%) of pregnant women. Based on FEC technique,
the prevalence rate of Hookworm, Ascaris lumbricoides,
Hymenolepis nana, Taenia species and Strogyloides
stercoralis was 15.1% (56/372), 6.5% (24/372), 1.6% (6/372),
1.34% (5/372) and 0.27% (1/372), respectively. The two
methods showed significantly different diagnosing
ability in detecting intestinal helminths (P < 0.001) (Table 1).
Comparatively the detecting ability of direct wet mount
microscopy was poor in diagnosing Hookworm,
Hymenolepis nana and Taenia species specifically.
Sensitivity and negative predictive value (NPV) of direct wet mount microscopy
The sensitivity of direct wet mount method in
detecting total intestinal helminths, Hookworm, Ascaris
lumbricoides and Hymenolepis nana was 76, 85.7, 83.3 and
33.3%, respectively. Taenia species and Strongyloides
stercoralis were not detected by the direct wet mount
microscopy (Table 2). This result indicates that direct wet
mount microscopy is not enough to rule out absence of
intestinal helminths in a single stool specimen in general.
The specificity and positive predictive value of direct
wet mount microscopy was 100% due to absence of
falsely positive result. The ability of direct wet mount
to rule out true negatives as negative was better (above
92.7%) in general. The probability of truly negative
pregnant women to be negative by direct wet mount
microscopy for the presence of Hookworm, Ascaris
lumbricoides, Hymenolepis nana, Taenia species and
Strongyloides stercoralis was 97.5, 98.8, 98.9, 98.6 and 99.7%,
respectively (Table 2).
Test efficiency (TE) of direct wet mount microscopy
The overall ability of direct wet mount microscopy to
correctly diagnose intestinal helminths (TE) was 94%.
The test efficiency of this method in diagnosing
specifically Hookworm was comparatively low (97.8%). Direct
wet mount microscopy showed similar efficiency in
NPF no parasite found, NA not available, ND not determined
diagnosing Ascaris lumbricoides and Hymenolepis nana
(98.9%) (Table 2).
Agreement of the test methods
The two methods perfectly agreed in diagnosing total
intestinal helminths, ova of Hookworm and Ascaris
lumbricoides (Kappa > 0.81). But these methods fairly agreed
in detecting ova of Hymenolepis nana (Kappa = 0.39)
World Health Organization (WHO) recommends the use
of Kato-Katz method in duplicate slides and other
techniques like FEC and McMaster methods for the detection
of human soil transmitted helminths (STH) like Ascaris
lumbricoides, Trichuris trichiura and the Hookworms.
All of these techniques rely on visual examination of
a small sample of stool to determine the presence and
number of STH eggs with different sensitivities especially
in low transmission areas [
The traditional direct wet mount microscopy has
lower sensitivity in detecting intestinal helminths
when compared to the FEC method. Therefore, the
use of FEC technique as a confirmatory test in routine
laboratory examination of stool will significantly aid in
accurate determination and management of parasitic
The findings of the present study showed that direct
wet mount microscopy has showed lower prevalence
rate of intestinal helminths when compared to the
FEC method (18.8% versus 24.7%) (P < 0.001) which is
similar to a study done in Nigeria [
differences in diagnostic performance might be due the low
sensitivity of the direct wet mount technique as
indicated by other studies, inter personal skill variations or
could be due to the technical errors of the two
methods. This means direct wet mount is not enough to rule
out the absence of intestinal helminths as reported by
other studies [
]. This low detection ability of direct
wet mount microscopy in single stool specimen could
be due to intermittent shading of intestinal helminths
in a single stool and low ova production ability of some
Direct wet mount microscopy showed significantly
low detecting ability specifically in diagnosing
Hookworm, Ascaris lumbricoides, Hymenolepis nana and
Taenia species in the present study. This is similar with
reports from other studies which stated as direct wet
mount has lower sensitivity in detecting Ascaris
lumbricoides, Schistosoma mansoni, Trichuris trichiura
and Hookworms [
]. The lower sensitivity in this study
could be due to inclusion of only a single stool
specimen which impedes the identification of intermittently
shading intestinal helminths.
The sensitivity of direct wet mount microscopy was
generally low in diagnosing total intestinal helminths
(76%) and very low in detecting ova of Hymenolepis
nana (33.3%) in the present study. The sensitivity of
direct wet mount in detecting intestinal helminths in
this study is higher than a study done in Ethiopia [
which showed a sensitivity of 48.9%. This could be due
to the inclusion of all intestinal parasites in the
previous study and inter personal skill variations.
Although the two methods had a perfect agreement
in detecting total intestinal helminths similar to other
], they fairly agreed in detecting Hymenolepis
nana which means that direct wet mount microscopy is
poor in detecting low ova producing helminths from a
single stool specimen.
Our findings showed that direct wet mount
microscopy exhibited low sensitivity for the detection of
intestinal helminths as compared to the FEC technique
which is similar with another study done in Ethiopia
]. This suggested that the use of direct wet mount
microscopy alone in diagnosing intestinal helminthic
infections is insufficient and may lead to false negative
The prevalence of intestinal helminths was
underreported by direct wet mount microscopy. The total
diagnostic performance of direct wet mount microscopy for
the diagnosis of intestinal helminths in pregnant women
was significantly low compared to FEC technique in this
study. Therefore, the FEC method should be used as a
routine technique in health center or hospital
laboratories for the diagnosis of intestinal helminths especially in
pregnant women who need special emphasis.
This study has limitations regarding using sensitive
techniques as a true gold standard to compare laboratory
techniques and collection of only a single stool specimen
which may impede the diagnostic performance of the
ANC: antenatal care; FEC: formalin ether concentration; TP: true positive; TN:
true negative; FP: false positive; FN: false negative; NPV: negative predictive
value; PPV: positive predictive value; TE: test efficiency.
Conceived and designed the experiments: HMM. Performed the experiments:
HMM, OZ, AB. Analyzed the data: HMM. Contributed
reagents/materials/analysis tools: HMM, OZ, AB. Wrote the paper: HMM, GD. Assisted with design,
analysis, and interpretation of data: GD, OZ, AB. A critical review of the manuscript:
HMM, GD, OZ, AB. Read and approved the final manuscript: HMM, GD, OZ, AB.
Critical appraisal of the manuscript: HMM, GD, OZ, AB.
We would like to thank all the administrative and laboratory staffs of the
respective health centers for all their support given to carry out this study.
Moreover, we would like to thank the data collectors and study participants
without whom the research would not be a reality. I duly acknowledge my
wife (Desta Temesgen) for her patience and motivation.
All authors declare that they have no conflict of interest associated with the
publication of this manuscript.
Availability of data and materials
The data sets during and/or analyzed during the current study are available
from the corresponding author on reasonable request.
Consent to publish
Ethics approval and consent to participate
The study was conducted after it was ethically reviewed and approved by
the Institutional review board (IRB) of the research directorate of Wollega
University. Then a letter informing the respective health center an
administrator was written from Wollega University and permission obtained. All the
information obtained from the study participants was coded to maintain
confidentiality. Data were collected after participants consented. The
IRB approved the use of oral consent documented by a witness after the
objectives of the study had been explained. The positive results were timely
reported to the clinicians for appropriate intervention.
The source of funding for the research was Wollega University research
directorate office. The funder has no role in the design of the study and
collection, analysis, and interpretation of data and in writing the manuscript.
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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