Evaluation of SD BIOLINE rapid antibody test for diagnosis of

Vol. 4(5), pp. 62-66, November 2013

DOI: 10.5897/JMLD2013-0078

http://www.academicjournals.org/JMLD

Journal of Medical Laboratory

and Diagnosis

Full Length Research Paper

Evaluation of SD BIOLINE rapid antibody test for

diagnosis of Helicobacter pylori infection

Endale Tadesse

, Gebru Mulugeta

and Techalew Shimelis

Department of Medical Laboratory Science, College of Medicine and Health Sciences, Hawassa University,

Hawassa, Ethiopia.

School of Medical Laboratory Science, College of Health Sciences, Addis Ababa University, Addis Ababa, Ethiopia.

Accepted 6 November, 2013

Helicobacter pylori infection is most prevalent and known to cause chronic gastritis and peptic ulcer

disease in Ethiopia. To date, simple and rapid point-of-care tests are commercially available; however,

information is limited regarding their diagnostic significance. This cross-sectional study was conducted

to evaluate the diagnostic performance of SD BIOLINE H. pylori rapid antibody test. A consecutive 203

enzyme linked immunosorbent assay (ELISA) confirmed sera (148 H. pylori positive and 55 negative)

from dyspeptic patients were tested using SD BIOLINE H. pylori kit (Standard Diagnostic Inc, Korea) at

Hawassa Teaching and Referral Hospital, southern Ethiopia from October, 2012 to January, 2013.

Individuals under 15 years old, who were on anti- H. pylori treatment during the month prior to the

study, those with discordant ELISA results, and refused to participate, were excluded. The sensitivity,

specificity, positive predictive value, negative predictive value, and accuracy of the SD BIOLINE H.

pylori test kit were 95.3, 94.5, 97.9, 88.1 and 95.1%, respectively. Therefore, this point-of-care test could

be used as alternative to ELISA testing and best fit our context.

Key words: Helicobacter pylori, point-of-care, enzyme linked immunosorbent assay (ELISA), SD BIOLINE.

INTRODUCTION

Helicobacter pylorus is a small, spiral, gram-negative

bacillus that appears to inhabit the mucous layer

overlying the gastric epithelial cells in humans. It was

formerly known as Campylobacter pyloridis then

Campylobacter pylori (Engstrand and Lindberg, 2013).

An estimated 50% of the world’s population is infected;

yet, its principal mode of transmission remains largely

unknown. It might be due to poverty related factors that

the prevalence of H. pylori infection is markedly higher in

developing countries than in developed nations (Khalifa

et al., 2010). In Ethiopia (Addis Ababa), an investigation

using different diagnostic methods showed H. pylori

prevalence varying between 69 and 91% among adult

dyspeptic patients (Asrat et al., 2004). Similar high

prevalence of H. pylori infection (70%) was also shown in

dyspeptic patients in northwest Ethiopia (Bahir-Dar)

(Tadege et al., 2005). The bacterium is known as the

most common cause of chronic gastritis and peptic ulcer

disease. Also, infection is associated with gastric adeno-

carcinoma, which is the deadliest cancer (McColl and El-

Omar, 2002). It has been shown that eradicating H. pylori

infection reduces ulcer relapse (Arkkila et al., 2005) and

the risk of gastric cancer development among patients

with or without precancerous lesion (Wong et al., 2004,

2012).

H. pylori can be diagnosed using invasive and non-

invasive methods; but a combination of at least two

methods has been recommended to improve accuracy of

*Corresponding author. E-mail: techalew03@yahoo.com. Tel: +251468209290.

its detection (Ramis et al., 2012). The invasive methods

such as culture, histology and rapid urease testing of

endoscopic biopsy from gastric mucosa are commonly

used. But these methods are tedious and time consuming

and require sampling procedure that cause discomfort to

patients (Cirak et al., 2007). The non-invasive methods

include

C and

C urea breath tests (UBTs) and

serology. UBTs are specific for active infection; however,

they are more expensive or have safety concern, limiting

their diagnostic suitability (Mansour-Ghanaei et al., 2011;

Kato et al., 2002). In contrast, serological tests are

inexpensive and widely available even though enzyme

linked immunosorbent assay (ELISA) based tests require

costly laboratory equipment, trained personnel and

electric supply to establish diagnosis in resource-

constrained settings (Leal et al., 2007).

To date, rapid diagnostic tests (RDTs) detecting

antibodies against H. pylori infection are commercially

available and widely applied in clinical practice in our

settings. RDTs are simple to perform, has short test time

and do not require laboratory support. However, studies

reported that RDTs show varying level of diagnostic

performance, which is influenced by H. pylori prevalence,

genetic diversity and study population (Chen et al., 1997;

Elitsur et al., 1997; Pelerito et al., 2006). Thus, it

highlights the need to assess the diagnostic usefulness of

RDTs in our context where H. pylori is prevalent; but little

is known regarding RDT performance characteristics.

This study was conducted to evaluate the diagnostic per-

formance of SD BIOLINE H. pylori rapid antibody test so

that its diagnostic value to serve as alternative to ELISA

would be elucidated.

MATERIALS AND METHODS

Study subjects

A cross-sectional study was conducted in consecutive dyspeptic

patients who provide blood for H. pylori serological testing at

Hawassa Teaching and Referral Hospital, southern Ethiopia from

October, 2012 to January, 2013. Two hundred and three ELISA

confirmed sera (148 H. pylori positive and 55 negative) were used.

There were 11 patients that were excluded: 3 were on anti-H. pylori

treatment during the month prior to the study; 1 refused to

participate; 4 had discordant ELISA results; 3 were children under

15 years old. The study obtained ethical clearance from the

Institutional Review Board of College of Medicine and Health

Sciences, Hawassa University.

Serology

Venous blood was collected from the study participants and sera

were stored at -20°C until analyzed. All sera were tested for anti-H.

pylori immunoglobulin G (IgG) antibody using two different ELISA

kits: Pyloriset EIA-G III (Orion Diagnostica, Germany) and H. pylori

IgG ELISA (IBL International, Hamburg, Germany). According to the

manufacturers’ claim, the respective sensitivity and specificity were

100 and 94.3% for Pyloriset EIA-G III and > 95 for H. pylori IgG

ELISA. In this study, H. pylori infection status was determined on

Tadesse et al. 63

the bases of the same positive or negative results obtained by both

ELISA tests.

ELISAs

Pyloriset EIA-G III testing

A 100 µl of each calibrator sera (labeled 1 to 4) and 100 µl of each

1 to 201 diluted patient sera were pipetted to microtiter wells coated

with the specific H. pylori antigen. Plates were incubated on a plate

shaker (1000 rpm) at 25°C for 30 min. After washing 3 times, 100 µl

of enzyme conjugate was added into the wells and incubated again

at 25°C for 30 min. Following washing step, 100 µl of substrate

solution was added and plates were incubated at 25°C for 10 min.

The reaction was stopped by adding 100 µl of 0.5 M sulfuric acid

and the optical density (OD) of each well was read at 450 nm within

10 min after stopping. As specified by the manufacturer, samples

were defined as positive when their OD values are equal to or

higher than that of the calibrator serum 2 OD.

H. pylori IgG ELISA testing

A 100 µl of each standard (labeled A to D) and 100 µl of each 1 to

101 diluted patient sera were added into the respective microtiter

wells. Plates were covered with adhesive foil and incubated at 25°C

for 60 min. After washing 3 times with washing buffer, 100 µl of

enzyme conjugate was added into each well and incubated at 25°C

for 30 min. Following similar washing steps, 100 µl of TMB

substrate solution was added into each well and incubated at 25°C

for 20 min. The reaction was stopped by adding 0.5 M sulfuric acid,

and the OD of each well was read at 450/630 nm within 60 minutes

after stopping. As per the instruction of the manufacturer, the OD of

each sample was divided by the cut-off standard (standard B) value

to obtain a cut-off index (COI). A respective COI value of < 0.8, 0.8

to 1.2 and > 1.2 were interpreted as negative, equivocal and

positive.

Rapid diagnostic test

Those ELISA characterized sera were tested using a commercial

rapid diagnostic kit, SD BIOLINE H. pylori kit (Standard Diagnostic

Inc, Korea). A single investigator blinded to the results of ELISA

read all the rapid test results. Tests were carried out according to

instruction of the manufacturer. The SD BIOLINE H. pylori antibody

test is a simple one step immune-chromatographic assay for

qualitative detection of antibodies of all isotopes to H. pylori. The

test procedure is to add 10 µl of serum, plasma or whole blood to a

sample well, and then adding 3 drops of assay diluent to the same

well. The result was read at 10 min. Positive test result is indicated

when control (C) and test line (T) are visible, and negative result is

when only control line (C) is visible. The test is invalid when the

control line is invisible. Data entry and analysis was performed

using STATA Version-10. Results are summarized using mean and

proportion as appropriate. Parameters including sensitivity,

specificity, positive predictive value (PPV), negative predictive value

(NPV) and accuracy were calculated considering concordant ELISA

results as true results.

RESULTS

Sera from 203 patients (102 male, 101 female) were cha-

racterized for the H. pylori status by ELISA testing. They

64 J. Med. Lab. Diagn.

Table 1. Serology results of SD BIOLINE H. pylori

antibody test kit, southern Ethiopia, 2012-2013.

RDT

ELISA Testing

Total

Positive

Negative

SD BIOLINE H. pylori

Positive

141

144

Negative

Total

148

203

RDT, rapid diagnostic test; ELISA, enzyme-linked

immunosorbent assay

Table 2. The diagnostic performance characteristics of SD BIOLINE H. pylori antibody test kit, southern

Ethiopia, 2012-2013.

RDT

Sensitivity (%)

Specificity (%)

PPV (%)

NPV (%)

SD BIOLINE H. pylori kit

95.3 (90.5-98.1)

94.5 (84.9-98.9)

97.9 (94-99.6)

88.1 (77.1-95.1)

RDT, rapid diagnostic test; PPV, positive predictive value; NPV, negative predictive value.

were aged between 15 to 78 years, with a mean age of

36.9 years and standard deviation 14.8 years. One

hundred and forty eight (72.9%) sera were positive for H.

pylori IgG antibody and 55 (27.1%) were negative. There

was an agreement between the SD BIOLINE H. pylori

and ELISA results in 193 of 203 sera (overall accuracy

95.1%). As presented in Table 1, 141 true-positive, 52

true-negative, 3 false-positive and 7 false-negative

results was obtained by SD BIOLINE H. pylori test kit.

Out of 144 positive results, 8 were faint positive (Figure

1). The sensitivity, specificity, positive predictive value

(PPV), and negative predictive value (NPV) of SD

BIOLINE H. pylori rapid test were 95.3, 94.5, 97.9 and

88.1%, respectively (Table 2).

DISCUSSION

In our context where lack of resources and expertise limit

regular evaluations of diagnostic methods, many new

tests are marketed and used directly without prior

evaluation for their appropriateness. Rapid serological

kits for H. pylori are among the most widely used tests as

these products are easily applicable and other diagnostic

methods are limited. However, the need to determine

their diagnostic value through evaluation should be

emphasized for the improved management and control of

H. pylori infection. In attempt to find a reliable point-of-

care test, we evaluated the diagnostic performance of SD

BIOLINE H. pylori rapid antibody test. The sensitivity,

specificity, PPV and NPV of the test kit were 95.3, 94.5,

97.9 and 88.1%, respectively. The kit’s manufacturer

claimed similar performance characteristics: sensitivity

(95.9%), specificity (89.6%), PPV (93.4%), and NPV

(93.5%). Also, the overall accuracy (95.1%) we reported

was in accordance with that of the manufacturer (93.4%).

The various diagnostic products, dissimilar

methodologies and diverse studied population may limit

direct comparison between findings coming out of valida-

tion studies. In agreement with our findings, a previous

evaluation of the Assure H. pylori rapid serological test

(Genelabs Diagnostics, Singapore) reported a respective

sensitivity and specificity of 90.1 and 80.9% in

Bangladeshi adult patients (Pelerito et al., 2006).

Moreover, evaluation of the Assure H. pylori in

Portuguese pediatric population showed compatible

specificity (95%) to that of the SD BIOLINE H. pylori kit,

although its sensitivity (75.7%) was inferior (Rahman et

al., 2008). However, unlike our evaluation, those studies

included methods such as culture, rapid urease test, and

histology to confirm patients’ H. pylori status.

The NPV of SD BIOLINE H. pylori kit suggests that

negative results could be used to rule out H. pylori infec-

tion. Also, the observed high PPV of the test kit indicates

the reliability of positive results to detect H. pylori expo-

sure, truly. Nonetheless, the faint positive results may

increase inter-observer variation and the risk of reporting

false-negative result. However, since the predictive

values of a test depend on the prevalence of H. pylori

infection (Banoo et al., 2010), the test kit may demon-

strate different performance elsewhere if the prevalence

is lower.

Serology is a non-invasive method detecting antibody

against H. pylori infection and it is the only test unaffected

by changes in the stomach that could lead to a low

bacterial load and to false-negative results of the other

Tadesse et al. 65

Figure 1. Photograph of SD BIOLINE H. pylori rapid antibody test

results.

Left: faint positive; Middle: Positive; Right: Negative (61, 94, and 96:

Patients’ identification numbers).

tests (Malfertheiner et al., 2012). However, serology is

unable to distinguish between active infection and past

exposure due to the fact that antibodies against H. pylori

remain elevated for longer period after the disappearance

of the infection (Ekstrom et al., 2001). In Ethiopian setting

with a high prevalence of H. pylori infection (Asrat et al.,

2004; Tadege et al., 2005), ELISA was shown to be

highly accurate in diagnosing dyspeptic patients (Asrat et

al., 2007). However, ELISA-based H. pylori serological

test takes longer time, requires trained staff and a

laboratory set-up; thus, it has application difficulties in

resource-limited developing countries (Leal et al., 2008).

The SD BIOLINE H. pylori rapid antibody test addresses

challenges related to ELISA as it is simple to test, has

short test time (less than 30 min), does not require

laboratory equipment or electricity supply, and use whole

blood, plasma or serum as diagnostic sample.

However, the results of this study should be interpreted

in light of its methodological limitation as the tests

employed for H. pylori diagnosis did not include methods

of choice such as culture, UBTs and histology against

those comparisons of the serological tests would have

been imperative.

Conclusion

The SD BIOLINE H. pylori rapid antibody test was found

sensitive and specific for screening of H. pylori infection

in Ethiopian populations. Therefore, this point-of-care test

best fit our context and could be used as alternative to

ELISA testing.

ACKNOWLEDGEMENTS

The authors would like to thank the Hawassa University

for finical support of the study; and the Standard

Diagnostics, Inc. Ethiopia for providing kits free of charge.

We extend our appreciation to Ms. Sara Tadesse for

technical assistance during laboratory work and finally to

the study subjects who voluntarily participated in the

study.

REFERENCES

Arkkila PE, Seppala K, Kosunen TU, Sipponen P, Makinen J, Rautelin

H, Farkkila M (2005). Helicobacter pylori eradication as the sole

66 J. Med. Lab. Diagn.

treatment for gastric and duodenal ulcers. Eur. J. Gastroenterol.

Hepatol. 17:93-101.

Asrat D, Kassa E, Mengistu Y, Ashenafi S, Ayenew K, Abu-Al-Soud W,

Wadstrom T, Nilsson I (2007). Comparison of diagnostic methods for

detection of Helicobacter pyloriinfection in different clinical samples of

Ethiopian dyspeptic patients. Austral-Asian. J. Cancer 6:231-237.

Asrat D, Nilsson I, Mengistu Y, Ashenafi S, Ayenew K, Abu-Al-Soud W,

Wadstrom T, Kassa E (2004). Prevalence of Helicobacter pylori

infection among adult dyspeptic patients in Ethiopia. Ann. Trop. Med.

Parasitol. 98:181-189.

Banoo S, Bell D, Bossuyt P, Herring A, Mabey D, Poole F, Smith PG,

Sriram N, Wongsrichanalai C, Linke R, O'Brien R, Perkins M,

Cunningham J, Matsoso P, Nathanson CM, Olliaro P, Peeling RW,

Ramsay A (2010). Evaluation of diagnostic tests for infectious

diseases: general principles. Nat. Rev. Microbiol. 12:17-29.

Chen TS, Chang FY, Lee SD (1997). Serodiagnosis of Helicobacter

pylori infection: comparison and correlation between enzyme-linked

immunosorbent assay and rapid serological test results. J. Clin.

Microbiol. 35:184-186.

Cirak MY, Akyon Y, Megraud F (2007). Diagnosis of Helicobacter pylori.

Helicobacter 12:4-9.

Ekstrom AM, Held M, Hansson LE, Engstrand L, Nyren O (2001).

Helicobacter pylori in gastric cancer established by CagA immunoblot

as a marker of past infection. Gastroenterology. 121:784-791.

Elitsur Y, Neace C, Triest WE (1997). Comparison between a rapid

office-based and ELISA serologic test in screening for Helicobacter

pylori in children. Helicobacter 2:180-184.

Engstrand L, Lindberg M (2013). Helicobacter pylori and the gastric

microbiota. Best Pract. Res. Clin. Gastroenterol. 27:39-45.

Kato S, Ozawa K, Konno M, Tajiri H, Yoshimura N, Shimizu T, Fujisawa

T, Abukawa D, Minoura T, Iinuma K (2002). Diagnostic accuracy of

the

C-urea breath test for childhood Helicobacter pylori infection: a

multicenter Japanese study. Am. J. Gastroenterol. 97:1668-1673.

Khalifa MM, Sharaf RR, Aziz KR (2010). Helicobacter pylori: a poor

man’s gut pathogen? Gut Pathogensis 2:2.

Leal YA, Flores LL, Garcia-Cortes LB, Cedillo-Rivera R, Torres J

(2008). Antibody-based detection tests for the diagnosis of

Helicobacter pylori infection in children: a meta-analysis. PLoS One

3(11):e3751.

Malfertheiner P, Megraud F, O'Morain CA, Atherton J, Axon AT, Bazzoli

F, Gensini GF, Gisbert JP, Graham DY, Rokkas T, El-Omar EM,

Kuipers EJ (2012). European Helicobacter Study Group:

Management of Helicobacter pylori infection-the Maastricht IV/

Florence Consensus Report. Gut 61:646-664.

Mansour-Ghanaei F, Sanaei O, Joukar F (2011). Clinical validation of

an office-based C-UBT (Heliprobe) for Helicobacter pylori diagnosis

in Iranian dyspeptic patients. Gastroenterol. Res. Pract.

2011:930941.

McColl KE, El-Omar E (2002). How does Helicobacter pylori infection

cause gastric cancer? Keio J. Med. 51:53-56.

Pelerito A, Oleastro M, Lopes AI, Ramalho P, Cabral J, Monteiro L

(2006). Evaluation of rapid test Assure Helicobacter pylori for

diagnosis of Helicobacter pylori in pediatric population. J. Microbiol.

Methods 66:331-335.

Rahman SH, Azam MG, Rahman MA, Arfin MS, Alam MM, Bhuiyan TM,

Ahmed N, Rahman M, Nahar S, Hassan MS (2008). Non-invasive

diagnosis of Helicobacter pylori infection: evaluation of serological

tests with and without current infection marker CIM. World J.

Gastroenterol. 14:1231-1236.

Ramis IB, de Moraes EP, Fernandes MS, Mendoza-Sassi R, Rodrigues

O, Juliano CR, Scaini CJ, da Silva PE (2012). Evaluation of

diagnostic methods for the detection of Helicobacter pylori in gastric

biopsy specimens of dyspeptic patients. Braz. J. Microbiol. 43:903-

908.

Tadege T, Mengistu Y, Desta K, Asrat D (2005). Seroprevalence of

Helicobacter pylori infection in and its relationship with ABO blood

groups. Ethiop. J. Health Dev.19:55-59.

Wong BC, Lam SK, Wong WM, Chen JS, Zheng TT, Feng RE, Lai KC,

Hu WH, Yuen ST, Leung SY, Fong DY, Ho J, Ching CK, Chen JS,

China (2004). Gastric Cancer Study Group: Helicobacter pylori

eradication to prevent gastric cancer in a high-risk region of China: a

randomized controlled trial. JAMA 291:187-94.

Wong BC, Zhang L, Ma JL, Pan KF, Li JY, Shen L, Liu WD, Feng GS,

Zhang XD, Li J, Lu AP, Xia HH, Lam S, You WC (2012). Effects of

selective COX-2 inhibitor and Helicobacter pylori eradication on

precancerous gastric lesions. Gut 61:812-818.