on the taxonomy, diagnosis, epidemiology
Krepel, H.P.Citation
Krepel, H. P. (1994, June 28). Oesophagostomum bifurcum infection in man. A study on the taxonomy, diagnosis, epidemiology. Retrieved from https://hdl.handle.net/1887/13885
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SEROLOGICAL DIAGNOSIS OF OESOPHAGOSTOMUM INFECTIONS
A.M. Polderman, H.P. Krepel, J.J. Verwey, S. Baeta, and J.P. Rotmans.
Published in the Transactions of the Royal Society
Abstract
A sensitive and specific enzyme-linked immunosorbent assay (ELISA) is described to diagnose human infection with Oesophagostomum bijurcum. In an ELISA using crude soluble antigen, prepared from adult O. bifurcum, many cross reactions occured when measuring IgG titres in patients with other helminth infections. An ELISA based on the detection of specific IgG4, however, had a specificity of over 95%. The sensitivity of the IgG4-ELISA was
difficult to assess because a reliable parasitological diagnosis is not available. The IgG4
Introduction
Until recently human infections with Oesophagostomum sp. were considered as rare zoonotic events. The species involved, mostly O. bifurcum and more rarely O. aculeatum and O.
stephanostomum, are parasites of monkeys. After (probably) oral infection with the infective
larvae, further development takes place in small tumour-like nodules in the serosa of the intestinal wall or in the omentum. The fifth stage larvae or young adult worms emerge from these nodules to return to the intestinal lumen, mate and start egg production.
In the northern tip of Togo and Ghana more cases of clinical oesophagostomiasis have been described than anywhere else [1,2]. These cases were seen in the surgical wards of the hospitals of Bawku and Dapaong and the infections were diagnosed by finding the typical nodules containing developing larval stages. In current studies in that area it has been shown that not only surgical cases can be regularly seen, but also that in many villages asymptomatic infections are common in the resident population [3]. In an area-wide survey prevalences of 30% and over were found in 15 of the 43 villages examined [4]. Diagnosis of the infection and knowledge of the geographical distribution of the parasite are hampered by the fact that the eggs cannot be differentiated morphologically from those of hookworms. Stool cultures are required to provide the characteristic third stage larvae. The stool cultures have to be inoculated with fresh specimens and the results are sometimes unreliable: fungal infections and the development of fly larvae may have a detrimental effect. As a result, the infection long remained unrecognized in humans and the geographical distribution and the abundance of the parasite remain unknown. Oesophagostomum may be prevalent in areas where many subjects are also infected with hookworm. Surveys using stool cultures should be carried out or, alternatively, other diagnostic methods need to be developed.
We therefore attempted to develop a serological test for the diagnosis of Oesophagostomum infections. However, many cross reactions are known to occur among infections with various nematodes. The cross-reactivity is mainly due to antibodies directed against phosphorylcholine and carbohydrate epitopes. Phosphorylcholine is widely distributed in nature; it is present in many micro-organisms and several parasites [5-7]. In the sera of almost all patients infected with Wuchereria bancrofti and in half of those infected with
Strongyloides stercoralis circulating antigens bearing phosphorylcholine were found [8].
Antibodies directed against phosphorylcholine and these carbohydrate epitopes are mainly present in the immunoglobulin (Ig) G, subclass in young children and in the IgG2 subclass
in adults [9]. IgG4 antibodies to these epitopes, however, are generally absent from serum
of healthy children and adults [10]. It has therefore been possible to improve the specificity of the enzyme-linked immunosorbent assay (ELISA) for the immunodiagnosis of Onchocerca
subclass, and/or by removing phosphorylcholine epitopes from crude antigens [11-14]. In this study we have investigated whether the specificity of the Oesophagostomum-speciüc ELISA can be increased, by restricting the measurement of IgG antibodies to antibodies of the IgG4
class. The antibody response to antigens of O. bifurcum has been examined both at total IgG and IgG4 level using antigens prepared from adult O. bifurcum and a panel of different
human sera from clinically defined cases of oesophagostomiasis.
The present study is a preliminary one. The antibody response to various components of the antigens, and comparisons of the response to different antigenic preparations, are now being investigated. In this paper the observations will be limited to the total IgG and the IgG4
respons to adult worm antigens in patients with O. bifurcum infections and patients with a variety of other helminth infections.
Table 1. Origin of the sera used.
Group Origin of sera (*) No. Description
1(**) Dassoute, Togo
2(**) Tami, Togo 3(**) Tami, Togo; school 4(1) Ghana
5(§) Maniema, Zaire
6(A) Schistosomiasis patients 7(A) Echinococcosis patients 8(A) Strongyloides patients
9 Blood donors
24 Highly endemic area for Oesophagostomum
bifurcum and Necator americanus
25 As Dassoute 19 As Dassoute
25 Patients with proven O. volvulus from mid Ghana, at least 10 also with hookworm
10 High prevalence of 5". mansoni; A. lumbricoides,
Loa Loa and O. volvulus
10 Schistosomiasis patients diagnosed at Leiden 10 Echinococcosis patients diagnosed at Leiden 14 Dutch travellers with confirmed Strongyloides
infection
20 Healthy Dutch blood donors
(*) (**)
(5) ( A )
Group 3 aged 10-17 years; other groups mainly adults.
Stool samples of patients 1-3 were examined by various methods: Kato smear (2x25 mg), Ridley's formol concentration, faecal culture on charcoal; blood films (22.00) were examined for malaria and microfilariae and skin-snips of groups 1 and 2 were examined for microfilariae of O. volvulus. From most patients 2-4 stool samples were received. A person was considered positive for O. bifurcum when the stool cultures of at least one of these samples were positive. No faecal culture performed.
No faecal culture performed. No specific stool examination for hookworms or Strongyloides. Results reported earlier [16].
Patients, Materials and Methods
Sera
Sera were obtained from volunteers in the villages Tami and Dassoute, north Togo. Faecal culture surveys had shown these villages to be highly endemic for O. bifurcum [4]. For assessment of the specificity of the test, negative control sera and sera from patients with other helminth infections were included in the study. Origin and parasitological background of the various groups of sera are summarized in Table 1.
Antigen and enzyme-linked immunosorbent assay
Following treatment with pyrantel pamoate and purgation, male and female O. bifurcum were isolated from the faeces of patients in Lotogou, a few kilometers from Dassoute, Togo. Washed worms were lyophilized, ground with a mortar and pestle and then extracted in 0.035 M phosphate-buffered saline (PBS;pH 7.6) overnight with gentle stirring at 4°C. MaxisorbR microtitration plates (Nunc) were coated with O. bifurcum antigen at 5 /*g protein
per mL in 0.1 M sodium carbonate buffer (pH 9.6) 4°C. Plates were incubated with a blocking solution of 5% bovine serum albumen in PBS for 1 h at 37°C. For assays of specific IgG, duplicate serum samples were incubated for 1 h at 37°C at a dilution of 1:1000 in PBS with foetal calf serum (2%) and TweenR (0.05%). The second incubation was carried
out with Fc-specific peroxidase-conjugated rabbit antibody to human IgG (Dako). For assays of specific IgG4, using serum samples diluted 1:4, a second incubation (1 h, 37°C) was
carried out with anti-human IgG4 mouse monoclonal antibody (Sigma) followed by incubation
(lh, 37°C) with peroxidase-conjugated rabbit anti-mouse antibody (Dako). After incubation with 2,2'-azino-bis-(2-ethylbenz-thiazoline-sulphonic acid (Sigma) plus hydrogen peroxide for 15 min at room temperature, absorbance was read at 414 nm. To correct for day to day assay variation, results were expressed as ratios between the absorbance values of the sample and a well defined control serum.
Results
Many non-specific reactions were seen in the IgG response (Fig.) The IgG4 response was
The results of the IgG4-ELISA in each of the 3 infected groups are given in Table 2. The
sensitivity in groups 1 and 2 was 86%; in group 3 it was 60%. The specificity was 97-99%.
positive culture negative culture D no culture done
1.4 1.0 -0.6 . 0.2 _ 3 4 1.0 0.6 0.2 • • • • ° A • • • • ' • 8 • •• i <4P> 1 ° m • ( • l IgG4 i 7 8 9 patient group
Figure. Absorbance values in enzyme-linked immunosorbent assays specific for IgG (upper panel) and IgG4 (lower panel): • , positive culture; o, negative culture; [], no culture made. The characteristics
Table 2. Serological results for culture-positive and culture-negative
patients from three groups of patients infected with O. bifurcum groups in Togo. Patients Dassoute Culture positive Culture negative Total Tami, adults Culture positive Culture negative Total Tami, schoolchildren Culture positive Culture negative Total ELISA Positive 17 3 20 11 3 14 6 4 10 Negative 4 0 4 1 2 3 4 5 9 Discussion
The test based on the detection if IgG clearly failed to detect O. bifurcum infection specifically.
Specificity and sensitivity of the test based on the detection of specific IgG4 subclass
antibodies to homologous crude adult worm antigen were difficult to assess reliably because the faecal cultures gave variable results and the true prevalence could not properly be established with cultures of only a few stool specimens. The absence of positive test results in the groups 4-9 (except the 2 patients in groups 4 and 5) and the positive results with the sera of most of the subjects of groups 1 and 2 with negative cultures, suggested that the latter subjects in Tami and Dassoute probably were, or had been infected.
Negative serological results were found more frequently among the schoolchildren in Tami, partly due to the fact that in infection rates are normally lower in children than in adults [4]. Several of the children with negative cultures and negative ELISA results were probably 'real' negatives. Furthermore it has been shown by Schur and others that the IgG4 response
Very little information is available on the geographical distribution of Oesophagostomum infection. It is possible that occasional infections may occur in mid Ghana, at a distance of only a few hundred kilometers. This might explain the positive test result in one of the Ghanaian patients. Since oesophagostomes are thought to be primarily parasites of monkeys, it should not be surprising to find cases among the population of the tin-mining area in Maniema, to the east of Kindu, in the rain forest belt of Kivu. Stool cultures have never been made in that area. The observation of one positive case from Maniema suggests the need to search more carefully for the presence of oesophagostomes in humans in this area of Zaire. Clearly, many relevant questions have not yet been adressed. Does the test identify active infections only, or will past infections from which no parasite survives remain serologically positive for many years? Many asymptomatic infections can be detected serologically, but what about the symptomatic case? Do such patients respond differently? Although a more thorough description is required of the antigens which initiated the response described in this paper, and although a great deal more experience with the use of this diagnostic technique is required before the test results can be properly interpreted, the IgG4 ELISA is thought to
be a helpful tool in preliminary screening of 'hookworm'-infected areas for the presence of
Oesophagostomum. In northern Togo and Ghana the infection is well known by the local
Acknowledgements
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