Parasitology Research

https://doi.org/10.1007/s00436-017-5720-7

ORIGINAL PAPER

Highly sensitive and specific detection of Giardia duodenalis,

Entamoeba histolytica, and Cryptosporidium spp. in human stool
samples by the BD MAX™ Enteric Parasite Panel
Marijo Parčina 1 & Ingrid Reiter-Owona 1 & Frank P. Mockenhaupt 2 & Valerija Vojvoda 1 & Jean Bosco Gahutu 3 &
Achim Hoerauf 1 & Ralf Ignatius 4,5

Received: 7 June 2017 / Accepted: 11 December 2017

# Springer-Verlag GmbH Germany, part of Springer Nature 2017

Abstract
Detection of intestinal protozoan parasites by light microscopy is cumbersome, needs experienced personnel, and may lack
sensitivity and/or specificity as compared with molecular-based stool assays. Here, we evaluated the BD MAX™ Enteric Parasite
Panel, i.e., a multiplex real-time PCR assay for simultaneous detection of Giardia duodenalis, Entamoeba histolytica, and
cryptosporidia (Cryptosporidium parvum and C. hominis), by examining 200 positive human stool samples (138 ×
G. duodenalis, 27 × E. histolytica, 35 × Cryptosporidium spp.) and 119 controls including 18 samples with E. dispar. The
majority of the samples, i.e., 153/200 (76.5%) positive samples and 66/119 (55.5%) controls, were confirmed by multiplex in-
house PCR detecting the same parasites as the BD MAX™ Enteric Parasite Panel. The BD MAX™ assay did not yield false-
positive results. Sensitivity and specificity were 97.8% (95% CI, 93.3–99.4%) and 100% (95% CI, 97.4–100%) for G.
duodenalis, 100% (95% CI, 84.5–100%) and 100% (95% CI, 98.4–100%) for E. histolytica, and 100% (95% CI, 87.7–100%)
and 100% (95% CI, 98.3–100%) for cryptosporidia, and similar data were obtained when only the 219 PCR-confirmed samples
were analyzed. Thus, the BD MAX™ Enteric Parasite Panel provides a highly sensitive and specific tool for the laboratory
diagnosis of three predominant protozoan parasites causing enteritis.

Keywords Intestinal protozoa . Real-time PCR . Detection . Sensitivity . Specificity

Introduction protozoa and helminths (van Lieshout and Roestenberg 2015;

In clinical parasitology, novel highly sensitive real-time PCR
(qPCR) assays may facilitate the detection of various enteric
Marijo Parčina and Ingrid Reiter-Owona contributed equally to this work.
* Ingrid Reiter-Owona
Ingrid.Reiter-Owona@ukbonn.de
1
Institute of Medical Microbiology, Immunology and Parasitology,
University Hospital Bonn, Bonn, Germany
2
Institute of Tropical Medicine and International Health,
Charité-Universitätsmedizin Berlin, Berlin, Germany
3
University Teaching Hospital of Butare and School of Medicine and
Pharmacy, University of Rwanda, Butare, Rwanda
4
MVZ Labor 28, Berlin, Germany
5
Department of Microbiology and Hygiene,
Charité-Universitätsmedizin Berlin, Berlin, Germany
Verweij and Stensvold 2014). Although dependent on techno-
logically advanced equipment and still expensive, they may
be of particular interest in endemic areas because repeatedly or
chronically infected individuals living in these areas may shed
parasites at considerably lower numbers than acutely infected
travelers, which may lead to decreased sensitivity of light
microscopy and assays based on antigen detection (Ignatius
et al. 2014). Recently, various novel commercial qPCR assays
have been developed. Some of these assays detect parasites
among bacterial and viral pathogens (Buss et al. 2015; Khare
et al. 2014; McAuliffe et al. 2013; Navidad et al. 2013;
Wessels et al. 2014), and might be beneficial for the microbi-
ological diagnosis of traveler’s diarrhea (Zboromyrska et al.
2014). In contrast, others are designed to exclusively detect
enteric protozoa (Laude et al. 2016; Stark et al. 2014). A major
problem with the clinical evaluation of these assays, however,
may arise when some of the pathogens are absent or present at
a very low prevalence in the study population, which

consequently hampers the ascertainment of the sensitivity of
the assay.
Here, we evaluated the BD MAX™ Enteric Parasite
Panel, which includes DNA extraction and qPCR for si-
multaneous detection of the major enteric protozoan para-
sites, Giardia duodenalis, Entamoeba histolytica, and
Cryptosporidium spp. (C. parvum and C. hominis), in hu-
man stool samples. To overcome the above-mentioned ob-
stacle of possibly low numbers of positive samples within
the routinely examined patient population, we decided to
use a panel of stool samples with known parasitological
results, similar to the design of a recent French study
(Laude et al. 2016; Stark et al. 2014). Since a previous
study indicated some problems of this assay with the de-
tection of G. duodenalis (Mölling et al. 2016), we includ-
ed a large number of G. duodenalis-positive samples in
our study. Notably, 53 of 138 G. duodenalis-positive sam-
ples had been collected from chronically/repeatedly infect-
ed Rwandan children known to shed fewer parasites than
acutely infected individuals (Ignatius et al. 2014).
Methods
Patients’ samples
Stool samples included in this study were collected from
two different patient populations. Two-hundred forty-three
samples from 243 patients had been submitted to the lab-
oratories in Bonn or Berlin, both cities of Germany, for
routine ova and parasite examination by light microscopy
following formol-ethyl acetate concentration. Additionally,
76 samples had been collected from Rwandan children <
5 years of age. Details of this study including the ethic
statement have been published before (Ignatius et al.
2012). All samples were anonymized and kept without in-
termediate thawing at − 20 °C until use. The characteristics
of all samples are summarized in Table 1.
Positive samples collected in Germany G. duodenalis had
been detected in 85 samples (Table 1); in 30 samples, the
pathogen had been found by light microscopy only (no
PCR had been performed), in 48 samples by both light mi-
croscopy and in-house multiplex PCR detecting G.
duodenalis, Entamoeba histolytica, and cryptosporidia
(Verweij et al. 2004), and in seven samples by in-house mul-
tiplex PCR only (light microscopy negative). In 22 samples,
typical cysts of E. histolytica/E. dispar/E. moshkovskii had
been detected microscopically, and the species E. histolytica
had been identified subsequently by PCR (Hamzah et al.
2006). Finally, 17 samples had yielded positive results for
Cryptosporidium spp. oocysts by a direct fluorescent-
antibody assay (DFA) (MeriFluor Crypto&Giardia;
Meridian Bioscience, Cincinnati, OH, USA).
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Positive samples collected in Rwanda
In 53 of the Rwandan samples, G. duodenalis had been de-
tected (microscopy and in-house multiplex PCR, 27 samples;
in-house multiplex PCR only, 26 samples), five were positive
for E. histolytica (microscopy and in-house multiplex PCR,
two samples; in-house multiplex PCR only, three samples),
and 18 samples were positive for Cryptosporidium parvum
by in-house multiplex PCR (Verweij et al. 2004).
Control samples
Eighteen of 28 samples negative for G. duodenalis, E.
histolytica, or cryptosporidia by in-house multiplex PCR but
microscopically positive for cysts of E. histolytica/E. dispar/
E. moshkovskii were positive by PCR for E. dispar (Verweij
et al. 2003). Additionally, 91 microscopically negative sam-
ples, 38 of them confirmed by in-house multiplex PCR, were
included as controls (Table 1).
DNA extraction and real-time PCR
Samples were processed according to the manufacturer’s in-
structions. Briefly, all 319 frozen native stool samples were
thawed and directly aliquoted (one aliquot was used immedi-
ately and additional one to two aliquots were refrozen at −
20 °C to avoid repeated freeze-thaw processes of single sam-
ples), and 10 μL of each aliquot (alternatively 10 μL of a dark
brown liquid received after vigorous shaking of a solid stool
aliquot in sample buffer) was transferred to the BD MAX™
sample buffer (SB) tube. Following vigorous vortexing, the
samples were transferred to the BD heating station to facilitate
cell lysis. This pre-treatment lasted 52 min including a rise of
the temperature to 121 °C, which was maintained for 9 min
followed by cooling to room temperature. Samples were then
loaded into the BD MAX™ system where sample lysis con-
tinued. DNA extraction and isolation from specimens and the
sample processing control (SPC) plasmid were automatically
performed via magnetic affinity beads as well as the subse-
quent real-time PCR. The amplified targets were detected
using hydrolysis of double-labeled probes. According to the
manufacturer’s information, probes and primers target SSU
rRNA genes from E. histolytica and G. duodenalis, and a
specific DNA region of C. hominis and C. parvum.
Valid results were reported as positive or negative for all
three targets. Samples unresolved due to an invalid SPC
(inhibition) or undetermined due to process error were repeat-
ed (n = 7).
Repetition was first done with the 1:2 diluted sample from
the respective sample buffer tube (stored at 2–8 °C) and in
case of repeated inhibition with a second aliquot of the origi-
nal sample if available. Five samples gave valid results upon
repeat testing from the original sample material, and two
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Table 1 Sample characteristics

Pathogen No. Patient information Previous methods of detection

Microscopy In-house PCR

Positive samples
G. duodenalis 48 Routine diagnostics Positive Positive
30 Positive n.d.a
7 Negative Positive
27 Rwandan children Positive Positive
26 Negative Positive
E. histolytica 22 Routine diagnostics Positive Positive
3 Rwandan children Negative Positive
2 Positive Positive
Cryptosporidium 18 Rwandan children n.d. Positive
spp. 17 Routine diagnostics Positive (DFA*) n.d.
Control samples
E. histolytica/E. dispar group 28 Routine diagnostics Positive Negative
(18 × E. dispar)
No pathogen 38 Routine diagnostics Negative Negative
53 Negative n.d.
a
Not done
*Direct fluorescent-antibody assay

samples had to be excluded from the study due to repeated
inhibition (original samples were no more available). The rate
of unresolved samples was 2.2% before and 0.6% after repeat
testing.
Data analysis
To determine the accuracy of the BD MAX™ Enteric Parasite
Panel, we calculated sensitivity, specificity, and positive and
negative predictive values (PPV, NPV) with 95% confidence
intervals (95% CI) in comparison with the previous results
obtained by microscopy, in-house multiplex PCR, or DFA
with the same panel of stool samples.
Results and discussion
In total, 319 frozen human stool samples, i.e., 200 samples
with known parasitological results (138 × G. duodenalis, 27 ×
E. histolytica, and 35 × Cryptosporidium spp.) and 119 con-
trol samples (38 negative by both microscopy and in-house
multiplex PCR, 53 negative by microscopy only, and 28 mi-
croscopically positive for E. histolytica/E. dispar cysts but
negative by in-house multiplex PCR for G. duodenalis,
Entamoeba histolytica, and cryptosporidia) were examined
by using the BD MAX™ Enteric Parasite Panel. All three
parasites were detected by the assay with high accuracy
(Table 2), and similar data were obtained when only the 219
PCR-confirmed samples were analyzed. This is in accordance
with two recently published studies evaluating the same assay
(Batra et al. 2016; Madison-Antenucci et al. 2016), as well as
with data published recently for the G-DiaParaTrio assay,
which targets the same parasite species (Laude et al. 2016).
All 119 controls including 18 E. dispar-positive samples were
confirmed as true negatives by the BD MAX™. In contrast,
discrepant results have been reported by others for the analysis
of stool samples from Egyptian patients by using an in-house
PCR assay (Nazeer et al. 2013).
All 28 stool samples with microscopically detected E.
histolytica/E. dispar/E. moshkovskii cysts (including the 18
E. dispar-positive samples) yielded negative results with the
BD panel confirming the data obtained by in-house multiplex
PCR. The absence of E. histolytica in this sample subset cor-
responds with the very low detection rate of this parasite out-
side of endemic areas. The data confirm, however, that the BD
MAX™ assay detects the pathogenic E. histolytica only but
none of the apathogenic species of this genus in contrast to the
EasyScreen Enteric Parasite Detection kit (Stark et al. 2008).
Notably, the BD MAX™ panel did not yield false-positive
results for E. histolytica, which have been reported for the
Luminex assay by some authors (Navidad et al. 2013;
Wessels et al. 2014). Thus, the assay may be better suited to
discern an infection with E. histolytica from intestinal coloni-
zation with apathogenic Entamoeba spp. than antigen detec-
tion by EIA (Stark et al. 2008).
The sensitivity of the BD MAX™ assay for G. duodenalis-
confirmed samples was 97.8% (Table 2). In contrast to our
data, the sensitivity of the same assay for the detection of G.

Table 2 Detection of parasites in
200 pre-characterized positive Pathogen
samples and 119 control samples
by the BD MAX™ Enteric G. duodenalis
Parasite Panel E. histolytica
Cryptosporidium spp.
*Value plus 95% CI in parentheses
duodenalis in PCR-positive samples was only 66.7% in a
previous study (Mölling et al. 2016). This discrepancy might
be due to different numbers of samples, which led to a rather
large 95% CI in that study (40.0–93.4%) as compared to our
data (93.3–99.4%), or to samples with very low parasite
counts in the previous study. But even if we calculate the
sensitivity for those samples only, which were negative by
light microscopy but PCR-positive (n = 33, Table 1), the sen-
sitivity of the assay was 93.9% (95% CI, 79.4–99.3%) in our
study.
All 35 Cryptosporidium sp.-positive samples yielded pos-
itive results by the BD panel (sensitivity, 100%). Although we
did not analyze the Cryptosporidium species present in these
samples, the result underlines the dominance of the two spe-
cies, C. parvum and C. hominis, which are targeted by the BD
MAX™ assay as specified by the manufacturer. It still re-
mains to be determined whether the assay may additionally
identify other, less prevalent zoonotic Cryptosporidium spp.
Considering the positive samples as associated with disease
in all individuals, PPV and NPV were 100% (95% CI, 96.6–
100%) and 98.4% (95% CI, 94.9–99.6%) for G. duodenalis,
100% (95% CI, 84.5–100%) and 100% (95% CI, 98.4–100%)
for E. histolytica, and 100% (95% CI, 87.7–100%) and 100%
(95% CI, 98.3–100%) for cryptosporidia. Since PPVand NPV
depend on the prevalence of the disease, however, these data
were influenced by the study design, i.e., the selection of large
numbers of positive samples.
Microscopic detection of enteric protozoan parasites is
time-consuming and requires extensive training of a staff in
order to achieve a high-quality diagnostic standard.
Furthermore, E. histolytica cysts cannot be distinguished mi-
croscopically from those of other non-pathogenic Entamoeba
spp., which, however, constitute the majority of all
Entamoeba spp. cysts detected. The multiplex assay tested
by us detects and correctly identifies, with high sensitivity,
three of the clinically most important intestinal pathogens.
Still, Btraditional^ parasitological diagnosis based on the de-
tection of enteric parasites by light microscopy will remain
essential in the future for the many other parasites that cannot
be detected by the assay evaluated here. Limitations of our
study are the retrospective study design (sensitivities and
specificities might be different when the assay is used under
routine conditions, e.g., with parasites being less prevalent),
the lack of stool samples containing other parasites (except E.
dispar), the unfeasibility to confirm the results for all 319
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Positive Sensitivity Specificity
135/138 97.8%* (93.3–99.4%) 100% (97.4–100%)
27/27 100% (84.5–100%) 100% (98.4–100%)
35/35 100% (87.7–100%) 100% (98.3–100%)
samples by in-house multiplex PCR, and the difficulty to de-
termine the species in all 28 microscopically Entamoeba sp.-
positive, by PCR E. histolytica-negative samples.
In conclusion, the BD MAX™ provides a highly sensitive
and specific assay for the detection of the three most important
enteric protozoan parasites independently of the parasitologi-
cal experience of the laboratory staff. The inclusion of addi-
tional parasites, e.g., Dientamoeba fragilis where the diagno-
sis also has been shown to be facilitated by the use of in-house
PCRs (Stark et al. 2011; Stensvold and Nielsen 2012) in the
BD MAX™ assay may be worth considering for the future.
References
Batra R, Judd E, Eling J, Newsholme W, Goldenberg SD (2016)
Molecular detection of common intestinal parasites: a performance
evaluation of the BD Max Enteric Parasite Panel. Eur J Clin
Microbiol Infect Dis 35(11):1753–1757. https://doi.org/10.1007/
s10096-016-2722-9
Buss SN, Leber A, Chapin K, Fey PD, Bankowski MJ, Jones MK,
Rogatcheva M, Kanack KJ, Bourzac KM (2015) Multicenter eval-
uation of the BioFire FilmArray gastrointestinal panel for etiologic
diagnosis of infectious gastroenteritis. J Clin Microbiol 53(3):915–
925. https://doi.org/10.1128/JCM.02674-14
Hamzah Z, Petmitr S, Mungthin M, Leelayoova S, Chavalitshewinkoon-
Petmitr P (2006) Differential detection of Entamoeba histolytica,
Entamoeba dispar, and Entamoeba moshkovskii by a single-round
PCR assay. J Clin Microbiol 44(9):3196–3200. https://doi.org/10.
1128/JCM.00778-06
Ignatius R, Gahutu JB, Klotz C, Musemakweri A, Aebischer T,
Mockenhaupt FP (2014) Detection of Giardia duodenalis assem-
blage A and B isolates by immunochromatography in stool samples
from Rwandan children. Clin Microbiol Infect 20(10):O783–O785.
https://doi.org/10.1111/1469-0691.12596
Ignatius R, Gahutu JB, Klotz C, Steininger C, Shyirambere C, Lyng M,
Musemakweri A, Aebischer T, Martus P, Harms G, Mockenhaupt
FP (2012) High prevalence of Giardia duodenalis assemblage B
infection and association with underweight in Rwandan children.
PLoS Negl Trop Dis 6(6):e1677. https://doi.org/10.1371/journal.
pntd.0001677
Khare R, Espy MJ, Cebelinski E, Boxrud D, Sloan LM, Cunningham SA,
Pritt BS, Patel R, Binnicker MJ (2014) Comparative evaluation of
two commercial multiplex panels for detection of gastrointestinal
pathogens by use of clinical stool specimens. J Clin Microbiol
52(10):3667–3673. https://doi.org/10.1128/JCM.01637-14
Laude A, Valot S, Desoubeaux G, Argy N, Nourrisson C, Pomares C,
Machouart M, Le Govic Y, Dalle F, Botterel F, Bourgeois N, Cateau
E, Leterrier M, Le Pape P, Morio F (2016) Is real-time PCR-based
diagnosis similar in performance to routine parasitological
Parasitol Res
examination for the identification of Giardia intestinalis,
Cryptosporidium parvum/Cryptosporidium hominis and
Entamoeba histolytica from stool samples? Evaluation of a new
commercial multiplex PCR assay and literature review. Clin
Microbiol Infect 22(2):190 e1–190 e8
Madison-Antenucci S, Relich RF, Doyle L, Espina N, Fuller D, Karchmer
T, Lainesse A, Mortensen JE, Pancholi P, Veros W, Harrington SM
(2016) Multi-center evaluation of the BD MAX Enteric Parasite RT-
PC R as say for t he det ecti on of G i a rd i a du od e n al i s ,
Cryptosporidium hominis and C. parvum and Entamoeba
histolytica. J Clin Microbiol 54(11):2681–2688. https://doi.org/10.
1128/JCM.00765-16
McAuliffe GN, Anderson TP, Stevens M, Adams J, Coleman R,
Mahagamasekera P, Young S, Henderson T, Hofmann M, Jennings
LC, Murdoch DR (2013) Systematic application of multiplex PCR
enhances the detection of bacteria, parasites, and viruses in stool
samples. J Inf Secur 67(2):122–129
Mölling P, Nilsson P, Ennefors T, Ögren J, Florén K, Thulin Hedberg S,
Sundqvist M (2016) Evaluation of the BD Max Enteric Parasite
Panel for clinical diagnostics. J Clin Microbiol 54(2):443–444.
https://doi.org/10.1128/JCM.02100-15
Navidad JF, Griswold DJ, Gradus MS, Bhattacharyya S (2013)
Evaluation of Luminex xTAG gastrointestinal pathogen analyte-
specific reagents for high-throughput, simultaneous detection of
bacteria, viruses, and parasites of clinical and public health impor-
tance. J Clin Microbiol 51(9):3018–3024. https://doi.org/10.1128/
JCM.00896-13
Nazeer JT, El Sayed Khalifa K, von Thien H, El-Sibaei MM, Abdel-
Hamid MY, Tawfik RA, Tannich E (2013) Use of multiplex real-
time PCR for detection of common diarrhea causing protozoan par-
asites in Egypt. Parasitol Res 112(2):595–601. https://doi.org/10.
1007/s00436-012-3171-8
Stark D, Al-Qassab SE, Barratt JL, Stanley K, Roberts T, Marriott D,
Harkness J, Ellis JT (2011) Evaluation of multiplex tandem real-
time PCR for detection of Cryptosporidium spp., Dientamoeba
fragilis, Entamoeba histolytica, and Giardia intestinalis in clinical
stool samples. J Clin Microbiol 49(1):257–262. https://doi.org/10.
1128/JCM.01796-10
Stark D, Roberts T, Ellis JT, Marriott D, Harkness J (2014) Evaluation of
the EasyScreen enteric parasite detection kit for the detection of
Blastocystis spp., Cryptosporidium spp., Dientamoeba fragilis,
Entamoeba complex, and Giardia intestinalis from clinical stool
samples. Diagn Microbiol Infect Dis 78(2):149–152. https://doi.
org/10.1016/j.diagmicrobio.2013.10.013
Stark D, van Hal S, Fotedar R, Butcher A, Marriott D, Ellis J, Harkness J
(2008) Comparison of stool antigen detection kits to PCR for diag-
nosis of amebiasis. J Clin Microbiol 46(5):1678–1681. https://doi.
org/10.1128/JCM.02261-07
Stensvold CR, Nielsen HV (2012) Comparison of microscopy and PCR
for detection of intestinal parasites in Danish patients supports an
incentive for molecular screening platforms. J Clin Microbiol 50(2):
540–541. https://doi.org/10.1128/JCM.06012-11
van Lieshout L, Roestenberg M (2015) Clinical consequences of new
diagnostic tools for intestinal parasites. Clin Microbiol Infect
21(6):520–528. https://doi.org/10.1016/j.cmi.2015.03.015
Verweij JJ, Blange RA, Templeton K, Schinkel J, Brienen EA, van
Rooyen MA, van Lieshout L, Polderman AM (2004)
Simultaneous detection of Entamoeba histolytica, Giardia lamblia,
and Cryptosporidium parvum in fecal samples by using multiplex
real-time PCR. J Clin Microbiol 42(3):1220–1223. https://doi.org/
10.1128/JCM.42.3.1220-1223.2004
Verweij JJ, Oostvogel F, Brienen EA, Nang-Beifubah A, Ziem J,
Polderman AM (2003) Short communication: prevalence of
Entamoeba histolytica and Entamoeba dispar in northern Ghana.
Tropical Med Int Health 8(12):1153–1156. https://doi.org/10.1046/
j.1360-2276.2003.01145.x
Verweij JJ, Stensvold CR (2014) Molecular testing for clinical diagnosis
and epidemiological investigations of intestinal parasitic infections.
Clin Microbiol Rev 27(2):371–418. https://doi.org/10.1128/CMR.
00122-13
Wessels E, Rusman LG, van Bussel MJ, Claas EC (2014) Added value of
multiplex Luminex Gastrointestinal Pathogen Panel (xTAG(R)
GPP) testing in the diagnosis of infectious gastroenteritis. Clin
Microbiol Infect 20(3):O182–O187. https://doi.org/10.1111/1469-
0691.12364
Zboromyrska Y, Hurtado JC, Salvador P, Alvarez-Martínez MJ, Valls
ME, Mas J, Marcos MA, Gascón J, Vila J (2014) Aetiology of
traveller’s diarrhoea: evaluation of a multiplex PCR tool to detect
different enteropathogens. Clin Microbiol Infect 20(10):O753–
O759. https://doi.org/10.1111/1469-0691.12621