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Recent developments in laboratory diagnosis of melioidosis
Acta Tropica 74 (2000) 235 – 245 www.elsevier.com/locate/actatropica
Recent developments in laboratory diagnosis of melioidosis Stitaya Sirisinha a,b,*, Narisara Anuntagool a, Tararaj Dharakul c, Pattama Ekpo c, Surasakdi Wongratanacheewin d, Pimjai Naigowit e, Benja Petchclai f, Visanu Thamlikitkul g, Yupin Suputtamongkol g a
Laboratory of Immunology, Chulabhorn Research Institute, Bangkok 10210, Thailand Department of Microbiology, Faculty of Science, Mahidol Uni6ersity, Rama VI Road, Bangkok 10400, Thailand c Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol Uni6ersity, Bangkok, Thailand d Department of Microbiology, Faculty of Medicine, Khon Kaen Uni6ersity, Khon Kaen, Thailand e National Institute of Health, Department of Medical Ser6ices, Ministry of Public Health, Nonthaburi, Thailand f Department of Pathology, Faculty of Medicine Ramathibodi Hospital, Mahidol Uni6ersity, Bangkok, Thailand g Department of Internal Medicine, Faculty of Medicine Siriraj Hospital, Mahidol Uni6ersity, Bangkok, Thailand
b
1. Introduction In tropical countries, melioidosis remains an important public health problem, particularly with regard to diagnosis and treatment. Clinical manifestations of the disease vary considerably, from fatal septicemia to chronic localized infection. Subclinical infection is not uncommon in people residing in the endemic areas of infection, e.g. the northeastern part of Thailand. If improperly handled, the mortality rate of acute septicemic cases can be as high as 70 – 80%. Even when diagnosis can be made early and antibiotic therapy is initiated soon after admission, the mortality rate is still high. In the hands of experienced clinicians, the septicemia caused by Burkholderia pseudomallei is still difficult to differentiate from that caused by other organisms. Laboratory diagnosis is thus crucial for successful patient management. Bacterial identification by culture is now the only accepted ‘gold standard’. A number of * Corresponding author. Fax: + 66-2-6445411 and +66-2247122. E-mail address: [email protected] (S. Sirisinha)
immunological and molecular approaches, e.g. PCR, have been developed but to date they cannot yet replace the more cumbersome and time consuming culture method. This article includes a short review of recent developments in the laboratory methods for the diagnosis of melioidosis and description of our recent multi-center collaborative project on laboratory diagnosis here in Thailand, with the main objective of having the most suitable and reliable diagnostic kits for the detection of specific B. pseudomallei antibody, antigen and genetic material.
1.1. Bacterial culture Isolation and identification of B. pseudomallei by the culture method is still the method of choice for definitive diagnosis of melioidosis. It is relatively simple and economical to perform. It needs no elaborate and expensive equipment but requires experienced personnel, particularly in the interpretation of the results. Its main drawback is that it takes at least 3–4 days to obtain the results and by that time it may be too late for successful management, as a high percentage of patients
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admitted for acute septicemia die within 24 – 48 h of admission. It is recently reported that using an automatic BacT/Alert® nonradiomatric blood culture system, about 62% B. pseudomallei positive culture could be detected within 24 h of incubation and more than 90% within 48 h (Tiangpitayakorn et al., 1997). It is interesting to note that case fatality varied indirectly with the time of ‘alarm’ in this automatic system; fatalities also occurred more often in those in which the bacterial growth (alarm) was detected within the first 24 h. However, for definitive diagnosis this positive culture needs to be subcultured for biochemical characterization, which takes at least another 24 – 36 h for species identification. Although the use of a sensitive automated system has considerably cut down the incubation time normally required for the less sensitive semiautomatic or manual systems, its main drawback is the running cost and its availability only in large hospitals. This factor is rather crucial, as for this disease, particularly in acute sepsis, rapid and early diagnosis is necessary and one needs to cut down unnecessary time loss as much as possible, e.g. transportation time to transfer clinical specimens from small health centers to the regional hospitals where the automated machine is available. In some clinical specimens, such as throat swab and sputum, which are generally heavily contaminated by other organisms, B. pseudomallei can often be overgrown by contaminating flora, as the generation time for B. pseudomallei is considerably longer than that of other common bacteria. However, this problem can be minimized with the use of selective media such as the Ashdown, which contains various dyes and gentamicin. The use of a modified Ashdown medium containing colistin further helps to increase the efficiency of isolation (Dance et al., 1989). Availability of a costly API 20NE test panel has considerably simplified the identification of B. pseudomallei. However, this API panel of tests has been reported to misidentify B. pseudomallei for Chromobacterium 6iolacium and others (Inglis et al., 1998). Several groups of investigators have attempted to speed up the bacterial identification step which currently involves subculturing and biochemical characterization. This includes the use of specific
B. pseudomallei antibodies in combination with bacterial culture. If it is proven to be reliable, this procedure can cut down the time needed by at least 24–36 h when compared with the classical bacterial identification procedure now in use in all diagnostic laboratories. This possibility will be discussed in more detail in a later section of this article.
1.2. Immunological methods Several immunological assays have been developed and used for the detection of either B. pseudomallei antibodies or antigens in the clinical specimens. Many of these assays, i.e. indirect hemagglutination (IHA) for the detection of antibody, are simple to perform with minimum experience and equipment (Appassakij et al., 1990; Naigowit et al., 1992). The results can be obtained more rapidly than the bacterial culture mentioned earlier. However, regardless of the detection of antibodies or antigens, with the immunological assays one needs highly specific reagents. Even so, the interpretation of the data, particularly in the antibody detection, is still a problem due to the presence of seropositive subclinical infection in apparently healthy individuals residing in the endemic area of infection.
1.3. Detection of antibodies A number of serological tests for the detection of specific B. pseudomallei antibodies have been developed, all of which need improvement one way or another (Ashdown et al., 1989; Sirisinha et al., 1996). Some of the older techniques including complement fixation are not used much any more, but others e.g., indirect hemagglutination (IHA) test are still being widely used in many endemic areas of infection (Appassakij et al., 1990). One of the main drawbacks of these antibody assays that limits their value in clinical situations is the presence of background antibody in some healthy individuals in the endemic area. This is largely attributable to unrecognized subclinical infection in these individuals. However, this should not pose a great problem in non-endemic areas, e.g. countries in the temperate zone, where only spo-
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radic cases occur. To minimize misinterpretation of the results, the measurement of specific IgM antibody has been developed (Ashdown, 1981) but its usefulness has not yet been evaluated in a large-scale field test. In addition to the problem of sensitivity, some patients with acute sepsis do not exhibit significant IHA antibody levels, thus resulting in false negative reports (Appassakij et al., 1990). It is possible that the elevated background antibody level in individuals in the endemic areas of infection is attributable to existing antibodies against other Gram-negative organisms that happen to cross react with the B. pseudomallei antigens used in the testing systems. The latter include lipopolysaccharide (LPS), which represents a major component in the crude B. pseudomallei antigens used in the IHA test. Although early reports (Pitt et al., 1992) suggested that B. pseudomallei LPS was rather homogeneous, more recent reports including those from our group (Perry et al., 1995; Anuntagool et al., 1998) showed contradicting results. We found the LPS from B. pseudomallei isolates from a few patients among the 200 – 300 examined to possess atypical ladder pattern in SDS-PAGE analysis and the sera from these patients failed to react with the typical LPS and vice versa (Anuntagool et al., 1998). With this in mind, it is reasonable to expect that a few septicemic patients who may be infected with the B. pseudomallei possessing atypical LPS will give low IHA antibody titer against typical B. pseudomallei LPS. Other B. pseudomallei specific antigens that have been identified and characterized include 36-kDa exotoxin (Ismail et al., 1987), 19.5 kDa (Anuntagool et al., 1993) and 40-kDa proteins (Wongratanacheewin et al., 1993) and LPS (Petkanjanapong et al., 1992). However, these antigens have yet to be evaluated for their clinical usefulness in a large-scale field trial. We recently characterized and purified a 200-kDa surface antigen of B. pseudomallei by affinity chromatography using monoclonal antibody (5F8) to this specific component (Rugdech et al., 1995). In the indirect ELISA test, this antigen was found to be highly specific for the detection of B. pseudomallei antibody (Rugdech et al., 1995). The diagnostic value of this test was evaluated in an actual
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clinical situation in an area endemic for melioidosis (Dharakul et al., 1997). The results showed that the detection of specific IgG antibody against this immunoaffinity purified antigen is clinically useful for the diagnosis of acute melioidosis in an endemic area. Using the same assay system, the detection of IgM antibody is less reliable. This is in contrast with the results reported earlier by other groups suggesting the IgM antibody detection to be more specific and reliable than the IgG antibody for the diagnosis of acute septicemic melioidosis (Ashdown, 1981; Khupulsup and Petchclai, 1986; Kunakorn et al., 1990, 1991). More recently, recombinant antigens specific for B. pseudomallei have been produced (Dharakul, T., personal communication; Wongratanacheewin, S., personal communication). One such antigen, to be described in detail in a later section, appears to be highly specific and useful for the diagnosis of melioidosis.
1.4. Detection of antigens This approach is more logical and is superior to antibody detection because it indicates active disease. This is particularly valid when used in the endemic area of infection, where the background antibodies often interfere with the interpretation in the antibody assay. Many immunological methods have been developed for the detection of B. pseudomallei antigen, including the detection of soluble secreted product in blood and urine, and the detection of whole organisms in, for example, pus, wound, sputum and throat swabs. This approach was first undertaken by Ismail et al. (1987) when they developed a monoclonal antibodybased assay for the quantitation of exotoxin. This assay could detect toxin in the range of 16 ng/ml of the culture supernatant fluid. However, it has never been evaluated in real clinical situations. Similarly, Wongratanacheewin et al. (1990) have developed a polyclonal antibody-based avidin–biotin ELISA which could detect B. pseudomallei antigens in the culture filtrate at a concentration as low as 4 ng/ml. The main component detected by this ELISA system appeared to be a 40-kDa protein secreted into the protein-free culture medium (Wongratanacheewin et al., 1993). How-
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ever, like the exotoxin situation, this assay has not yet been evaluated in clinical situations. More recently, a highly sensitive assay was developed by Desakorn et al. (1994) for the detection of antigen, most likely the LPS, in the urine of patients with systemic and localized infection. This ELISA system was based on the use of an additional amplification step to increase sensitivity. Briefly, it was a sandwich ELISA in which the amplified detecting system consisted of FITC conjugated rabbit antibody to B. pseudomallei antigens and horseradish peroxidase conjugated mouse monoclonal antibody to FITC. With this assay system, it was possible to detect B. pseudomallei LPS at a concentration of 12.2 ng/ml. When applied to clinical specimens, a sensitivity of 81% and a specificity of 96% were achieved. Although some non-specific reaction was noted in the urine of patients with other Gram-negative infection of the urinary tract, it has one practical advantage over other assays that have been described in that it can be used with unconcentrated urine. The assay is, however, more complicated and more costly to perform and thus may not be very practical for routine use in smaller health centers with a limited health budget. Recently we described another antigen-detecting assay based on the use of our monoclonal antibody reactive against the 200kDa antigen secreted by B. pseudomallei (Anuntagool et al., 1996). This sandwich ELISA was shown to be highly reliable in detecting B. pseudomallei antigens in different clinical specimens tested including pus, sputum, urine and pleural fluid. The results showed the sensitivity and specificity of the test to be 75 and 98%, respectively. Other assay systems that have been developed and evaluated including latex agglutination (Smith et al., 1993, 1995) and immunofluorescence (Walsh et al., 1994). The former is not sensitive enough for routine use and needs prior concentration while the latter requires a fluorescent microscope which is not readily available in most laboratories in the endemic area of infection.
1.5. Molecular approach The detection of genetic materials in different clinical specimens has been used successfully for
the diagnosis of a large number of infectious diseases of man and animals, and more recently evaluated for its reliability in melioidosis. With the latter, both the hybridization technique using specific DNA probes and the PCR using a different set of DNA primers have been developed by investigators in many research and diagnostic laboratories. The hybridization technique did not have sufficient sensitivity for B. pseudomallei infection (Sermswan et al., 1994) and, therefore, in more recent years, the PCR approach has been more commonly performed with satisfactory results (Lee and Desmarchelier, 1994; Kunakorn and Markham, 1995; Dharakul et al., 1996; Brook et al., 1997; Rattanathongkom et al., 1997; Sura et al., 1997). Different sets of primers that have been evaluated including the regions in the 23S ribosomal RNA, 16S RNA, the junction between 16S and 23S RNA and specific sequences designed from a specific DNA probe. Using these different primers, as few as a single bacterium present in the clinical specimens can be successfully amplified and detected. However, with the exception of the more recent report by Haase et al. (1998), none of these has been subjected to critical evaluation in the real clinical situation. Based on the use of 16S RNA as the PCR primer, these investigators reported a sensitivity approaching 100%. However, based on this small scale clinical testing, the specificity was still far from satisfactory, as approximately one-third of the patients with other diagnoses were positive by this PCR system.
1.6. Comparati6e study of the efficacy of 6arious laboratory diagnostic methods Because the reports on the use of various immunological methods from the same or different groups performed at different times often give inconsistent results and have never been critically and simultaneously evaluated in a well-controlled clinical setting, we have, therefore, set up a multicenter collaborative project, hoping to settle this point once and for all. The project is a large-scale, double-blind case-controlled study carried out in a real clinical setting. It consisted of nine subprojects designed to detect antibodies, antigens
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and genetic materials, all of which employed clinical specimens from the same melioidosis and control patients. These specimens were given codes by laboratory and clinical personnel not involved in the development of the assays and testing of the samples. Only after laboratory work was finished and all the data were obtained were the codes opened and the results from different groups compared.
2. Materials and methods
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demic northeastern part of Thailand, i.e. at Ubon Ratchatani (Sapprasithiprasong Hospital), Srisaket, Surin and Khon Kaen (Srinakarind Hospital) provinces (Fig. 1). Only patients with clinical manifestations compatible with melioidosis who were subsequently proved to be culture-positive for B. pseudomallei and those with community-acquired septicemia whose hemocultures were positive for other organisms were included in this series. The latter, serving as control for each septicemic case, were those admitted at the same hospital not more than 14 days prior to or after the admission of the septicemic patients.
2.1. Patient selection
2.2. Collection of clinical specimens
Patients enrolled in this project were selected from those admitted between January and December, 1997 to four provincial hospitals in the en-
These samples including blood, urine, throat and wound swabs, pus and sputum were subjected to bacterial culture and biochemical characterization for species identification as described elsewhere. Specimens from clinically suspected melioidosis patients, both septicemic and non-septicemic, were all collected at the time of admission. Blood, serum and urine specimens from those with culture positive for B. pseudomallei were processed further for subsequent laboratory study. Similar specimens from community-acquired septicemic patients (clinically not compatible with melioidosis) serving as control for this study were collected 3–4 days after admission when the results from the bacterial cultures were known. Therefore, in this group of patients, only those with other bacterial infections (culture negative for B. pseudomallei ) served as non-melioidosis controls. However, a small proportion of patients in this group were later found to have positive blood culture for B. pseudomallei and were, therefore, included in the melioidosis group. These additional melioidosis patients differed from the ones with clinical melioidosis in that they were initially clinically not compatible with melioidosis and the specimens from these patients, like those of non-melioidosis controls, were collected 3–4 days after admission when the culture results were known. During this short time interval before a definitive diagnosis could be made, they were routinely treated with antibiotics and other medications which might interfere with the antigen detection and PCR analysis.
Fig. 1. Map of Thailand showing the endemic northeastern region where the study was undertaken. Patients were from hospitals in the four provinces.
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240 Table 1 Antibody detection assays in serum Method
Antigen
Indirect ELISA Indirect ELISA Indirect ELISA Western blot
Affinity purified 200-kDa component LPS 30-kDa protein (CCF) 16-kDa recombinant B. pseudomallei antigen 40-kDa protein (CF)
Dot immunoassay IHA (M) IHA (P) IHA (L) Immunochromatography
Melioidin Protein fraction of crude culture filtrate LPS Bacterial cell suspension
1992) and the one used for IHA was a hot phenol–water extract (Westphal and Jan, 1965). Recombinant protein from recombinant E. coli was partially purified by electroelution and was found to contain a predominant 16-kDa-protein component (Dharakul, T., unpublished). The crude culture filtrate (CCF) antigen for the ELISA was prepared by culturing B. pseudomallei in proteinfree synthetic broth containing 1% glycine, 0.25% disodium phosphate, 0.5% sodium chloride and 0.2% dextrose (Ekpo, P., unpublished). The supernatant fluid was centrifuged, filter-sterilized and then concentrated by membrane ultrafiltration (PM10 Diaflow®). It contained predominantly a 30-kDa protein (Fig. 3). The culture-filtrate antigen (CF) for dot immunoassay was prepared by culturing the organisms at 37°C in a protein-free modified Proskauer and Beck Medium (PBM) for 2 weeks without shaking (Wongratanacheewin et al., 1993). The supernatant fluid obtained after centrifugation and filter sterilization was dialyzed and lyophilized. It contained a 40-kDa protein as its major component (Fig. 3).
2.4. Monoclonal antibodies
Fig. 2. Immunoblotting profiles of the affinity-purified 200kDa antigen (5F8) and the LPS preparations used for the antibody assays. The latter was developed with three different MAbs specific for B. pseudomallei LPS.
2.3. Antigen preparations Details of the preparation of antigens used for the antibody detection shown in Table 1 have been or will be described elsewhere. In short, the affinity-purified antigen was prepared using MAb 5F8 specific for a 200-kDa component of B. pseudomallei (Fig. 2). The LPS used for indirect ELISA (Fig. 2) was prepared by a modified phenol-chloroform petroleum ether extraction method (Galanos et al., 1969; Kawahara et al.,
The mouse MAbs used for antigen detection in the urine included: (1) MAb 5F8 which was an agglutinating IgM antibody specific for a 200-kDa surface antigen (Fig. 2) which was also secreted in soluble form in the culture fluid (Rugdech et al., 1995; Sirisinha et al., 1998). (2) MAbs specific for LPS component. Three such clones were available, all gave a typical ladder profile in Western blot using purified LPS. The Bps L1 and Bps L2 were agglutinating IgM (Dharakul, unpublished) while the 9D5 (Anuntagool et al., this issue) was non-agglutinating IgG antibody (Fig. 2).
2.5. Immunological assays The assays shown in Tables 1 and 2 were performed as described elsewhere. Horseradish peroxidase or biotin conjugated rabbit anti-mouse immunoglobulins were used to detect the reaction in the indirect ELISA and dot immunoassay. For
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241
Fig. 3. SDS-PAGE profiles of 40-kDa protein (CF) used in the dot immunoassay and of 30-kDa protein (CCF) used in the indirect ELISA.
the antigen detection, the MAbs, i.e. Bps L1, Bps L2 and 9D5, were coated directly on the microtiter plates while the MAb 5F8 used in the sandwich ELISA had to be captured with rabbit anti-mouse immunoglobulins. The sensitivity of the immunochromatography was increased by using the blocking principle.
3. Results and discussion This was the first collaborative study to develop alternative laboratory methods and to evaluate their efficacies for the diagnosis of melioidosis in a real clinical situation. A very strict criterion was used for the selection of melioidosis and ‘control’ cases and the evaluation was carried out using an unbiased double-blind protocol. The most purified and specific B. pseudomallei antigens and specific MAbs currently available in different laboratories in Thailand were used for the development of the methods for the detection of antibodies and antigens respectively. The detection of genetic materials by PCR will only be briefly mentioned for the purpose of comparative discussion. Full details of the latter will be dis-
cussed by Kunakorn and his colleagues (this issue).
3.1. Antibody detection The efficacy of different antibody detection assays is summarized in Table 3. The use of a CCF antigen containing predominantly a 30-kDa protein and a recombinant antigen produced in E. coli seemed to give the most reliable results. However, the reliabilities of these two assay systems were still below the expected minimum values of 90% when compared with the culture method used as gold standard. The use of a combination Table 2 Antigen detection assays in urine Method
Antibody
Sandwich ELISA
IgM Monoclonal antibody against 200-kDa antigen (5F8) IgG MAb against LPS (9D5) IgM MAb against LPS (BpsL2) IgM MAb against LPS (Bps L2) IgM MAb against 200-kDa antigen (5F8)
Sandwich ELISA Sandwich ELISA Latex agglutination Competitive immunochromatography
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242
Table 3 Performance of various antibody detection assays in 101 patientsa Method
Indirect ELISA Indirect ELISA Indirect ELISA Western blot Dot immunoassay IHA IHA IHA Immunochromatographyb
Antigen
%
Affinity-purified (200 kDa) LPS CCF (30 kDa) Recombinant CF (40 kDa) Melioidin Secreted protein LPS Crude antigen
Sensitivity
Specificity
Positive predictive
Negative predictive
74 70 82 70 72 93 54 46 23
75 77 80 91 64 34 61 82 89
79 80 84 91 72 65 65 76 76
69 67 78 70 64 79 51 54 47
a 57 Patients were culture positive for B. pseudomallei and 44 patients were culture positive for organisms other than B. pseudomallei. b Detecting only IgM antibody.
Table 4 Performance of antigen detection assays in 120 patientsa Method
Sandwich ELISA Latex agglutination Immunochromatography
Antibody
MAb 5F8 Bps L2 MAb 5F8
% Sensitivity
Specificity
Positive predictive
Negative predictive
59 23 23
56 80 95
58 54 82
57 50 54
a 61 Patients were culture positive for B. pseudomallei and 59 patients were culture positive for organisms other than B. pseudomallei.
of two or three antibody detection assays did not significantly improve performance. There are several possible explanations for this unexpected result. Firstly, subclinical infection is known to occur among healthy individuals residing in the endemic area of infection and the antibody response to B. pseudomallei infection is known to be long-lasting. The elevated antibody levels are thus maintained by continuous exposure. Secondly, it is possible that other closely related Gram-negative organisms might possess components that share similar antigenic epitopes which can stimulate antibodies that cross react with the B. pseudomallei antigens used. Lastly, it is possible that the identification of B. pseudomallei by the culture method was not 100% reliable as there were a few
cases in which all serological tests gave results which agreed with one another but totally disagreed with the culture results used as gold standard.
3.2. Antigen detection Three laboratories using five different assay systems participated in this evaluation (Table 2). The results shown in Table 4 were, in general, below our expectation. The performance of all tests needs improvement. Two assays using the same MAb 5F8, namely ELISA and immunochromatography, gave results markedly different from one another. The results from immunochromatography assay were acceptable
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3.4. Detection of genetic materials by PCR
regarding specificity but were rather insensitive. It appears possible to increase the sensitivity of this assay, but it is to be expected that the specificity has to be slightly compromised. In fact, this MAb was subsequently used for the development of other antigen detection assays in the urine specimens, i.e. latex agglutination and dot immunoassay, all of which did not give any better results (data not shown) than the ones reported in Table 4 in this communication. Moreover, using this same MAb 5F8 to replace the polyclonal antibody in the amplified ELISA system previously reported by Desakorn et al. (1994) also failed to give satisfactory results.
Three groups, each using different sets of PCR primers, participated in this project. None of them gave satisfactory performance compared with the culture method. Again, like the immunological approach, both false positives and false negatives were noted for this molecular approach and will be discussed in detail in a subsequent report. When immunological methods (both antibody and antigen detections) and molecular assays were paired in all possible permutations, no significant improvement in performance was noted.
3.3. E6aluation of a combined result from the assays of serum and urine from the same patients
4. Conclusion
Because the assay systems shown in Tables 3 and 4 did not give reliable results that can be used in real clinical settings when compared with the culture method, an attempt was made to improve the results by combining the results from antigen and antibody detection assays. The data shown in Table 5 again failed to support our expectation as no significant improvement in the performance could be detected from any of the three laboratories participating in this experiment. Pairings of the assays from different laboratories did not give any better results.
With the technologies and reagents currently available, it seems that the culture method remains the method of choice, not only in its reliability but also in its simplicity and cost. Its only drawback is the time needed before a definitive diagnosis can be made. To help speed up the time needed for bacterial identification, a combination of the culture method with any one of the immunological and molecular assays discussed in this article could be used and the results evaluated. For instance, to improve the sensitivity, an overnight hemoculture can be carried out to in-
Table 5 Performance of a combined antibody and antigen detection assays of specimens from the same patients No of patients
83a
83a 82b
Antibody detection
Affinity purified 200-kDa antigen (ELISA) Recombinant antigen (Western blot) Bacterial cell (IM)
Antigen detection
%
Sensitivity
Specificity
Positive predictive
Negative predictive
5F8 (ELISA)
80
51
65
69
Bps L2 (LA)
84
59
70
77
5F8 (IM)
48
90
83
62
a 44 Patients were culture positive for B. pseudomallei . and 39 patients were culture positive for organisms other than B. pseudomallei. b 42 Patients were culture positive for B. pseudomallei and 40 patients were culture positive for organisms other than B. pseudomallei. IM, immunochromatography.
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crease the number of bacteria and then the bacterial identification in the culture can be made using any of the specific monoclonal antibodies or PCR primers currently available. Our limited and preliminary data on this approach compares very favorably with the classical bacterial culture approach. If this proves reliable, then the time required to reach a definitive diagnosis can be reduced from 3 to 4 days to within 18 – 36 h.
Acknowledgements This collaborative project was supported by the Thailand Research Fund, Chulabhorn Research Institute, and National Science and Technology Development Agency of Thailand. We thank Maurice Broughton for his advice during the preparation of this manuscript.
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Dance, D.A.B., Wuthiekanun, V., Naigowit, P., White, N.J., 1989. Identification of Pseudomonas pseudomallei in clinical practice: use of simple screening tests and API 20NE. J. Clin. Pathol. 42, 645 – 648. Desakorn, V., Smith, M.D., Wuthiekanun, V., Dance, D.A.B., Aucken, H., Suntharasamai, P., Rajchanuwong, A., White, N.J., 1994. Detection of Pseudomonas pseudomallei antigen in urine for the diagnosis of melioidosis. Am. J. Trop. Med. Hyg. 51, 627 – 633. Dharakul, T., Songsivilai, S., Anuntagool, N., Chaowagul, W., Wongbunnate, S., Intachote, P., Sirisinha, S., 1997. Diagnostic value of an antibody enzyme-linked immunosorbent assay using affinity-purified antigen in an area endemic of melioidosis. Am. J. Trop. Med. Hyg. 56, 418 – 423. Dharakul, T., Songsivilai, S., Viriyachitra, S., Luangwedchakarn, V., Tassaneetritap, B., Chaowakul, W., 1996. Detection of Burkholderia pseudomallei DNA in patients with septicemic melioidosis. J. Clin. Microbiol. 34, 609 – 614. Galanos, C., Luderitz, O., Westphal, O., 1969. A new method for the extraction of R lipopolysaccharides. Eur. J. Biochem. 9, 245 – 249. Haase, A., Brennan, M., Barrett, S., Wood, Y., Huffam, S., O’Brien, D., Currie, B., 1998. Evaluation of PCR for diagnosis of melioidosis. J. Clin. Microibol. 36, 1039 – 1041. Inglis, T.J., Chiang, D., Lee, G.S., Chor-Kiang, L., 1998. Potential misidentification of Burkholderia pseudomallei by API 2ONE. Pathology 30, 62 – 64. Ismail, G., Embi, M.N., Omar, O., Allen, J.C., Smith, C.L., 1987. A competitive immunosorbent assay for detection of Pseudomonas pseudomallei exotoxin. J. Clin. Microibol. 23, 353 – 357. Lee, A.E., Desmarchelier, P.M., 1994. Detection of Pseudomonas pseudomallei by PCR and hybridization. J. Clin. Microbiol. 32, 1326 – 1332. Kawahara, K., Dejsirilert, S., Danbasa, H., Ezaki, T., 1992. Extraction and characterization of lipopolysaccharide from Pseudomonas pseudomallei. FEMS Microbiol. Lett. 96, 123 – 133. Khupulsup, K., Petchclai, B., 1986. Application of indirect hemagglutination test and indirect fluorescent antibody test for IgM antibody for diagnosis of melioidosis in Thailand. Am. J. Trop. Med. Hyg. 35, 366 – 369. Kunakorn, M., Boonma, P., Khulpulsup, K., Petchclai, B., 1990. Enzyme-linked immunosorbent assay for immunoglobulin M specific antibody for the diagnosis of melioidosis. J. Clin. Microbiol. 28, 1249 – 1253. Kunakorn, M., Markham, R.B., 1995. Clinically practical seminested PCR for Burkholderia pseudomallei with and without solution hybridization. J. Clin. Microbiol. 33, 2131 – 2135. Kunakorn, M., Petchclai, B., Khupulsup, K., Naigowit, P., 1991. Gold blot for detection of immunoglobulin M (IgM)and IgG-specific antibodies for rapid serodiagnosis of melioidosis. J. Clin. Microbiol. 29, 2065 – 2067.
S. Sirisinha et al. / Acta Tropica 74 (2000) 235–245 Naigowit, P., Maneebooyong, W., Wangroonsub, P., Chaowagul, W., Kanai, K., 1992. Serosurviellance for Pseudomonas pseudomallei infection in Thailand. Jpn. J. Med. Sci. Biol. 45, 215–230. Perry, M.B., Maclean, L.L., Schollaardt, T., Bryan, L.E., Ho, M., 1995. Structural characterization of the lipopolysaccharide O antigens of Burkholderia pseudomallei. Infect. Immun. 63, 3348 – 3352. Petkanjanapong, V., Naigowit, P., Kondo, E., Kanai, K., 1992. Use of endotoxin antigens in enzyme-linked immunosorbent assay for the diagnosis of P. pseudomallei infections (melioidosis). Asian Pacific J. Allergy Immunol. 10, 145 – 150. Pitt, T.L., Aucken, H., Dance, D.A.B., 1992. Homogeneity of lipopolysaccharide antigens in Pseudomonas pseudomallei. J. Infect. 25, 139 – 146. Rattanathongkom, A., Sermswan, R.W., Wongratanacheewin, S., 1997. Detection of Burkholderia pseudomallei in blood samples using polymerase chain reaction. Mol. Cell. Probes 11, 25 – 31. Rugdech, P., Anuntagool, N., Sirisinha, S., 1995. Monoclonal antibodies to Pseudomonas pseudomallei and their potential for diagnosis of melioidosis. Am. J. Trop. Med. Hyg. 52, 231 – 235. Sermswan, R.W., Wongratanacheewin, S., Tattawasart, U., Wongwajana, S., 1994. Construction of a specific DNA probe for diagnosis of melioidosis and use as an epidemiological marker of Pseudomonas pseudomallei. Mol. Cell Probes 8, 1 – 9. Sirisinha, S., Anuntagool, N., Intachote, P., 1996. Improved methods for diagnosis of melioidosis. J. Sci. Soc. Thailand 22, 131 – 142. Sirisinha, S., Anuntagool, N., Intachote, P., Wuthiekanun, V., Puthucheary, S.D., Vadivelu, J., White, N.J., 1998. Antigenic difference between clinical and environmental isolates
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of Burkholderia pseudomallei. Microbiol. Immunol. 42, 731 – 737. Smith, D.M., Wuthiekanun, V., Walsh, A.L., Pitt, T.L., 1993. Latex agglutination test for identification of Pseudomonas pseudomallei. J. Clin. Pathol. 46, 374 – 375. Smith, D.M., Wuthiekanun, V., Walsh, A.L., Teerawattanasook, N., Desakorn, V., Suputtamongkol, Y., Pitt, T.L., White, N.J., 1995. Latex agglutination for rapid detection of Pseudomonas pseudomallei antigen in urine of patients with melioidosis. J. Clin. Pathol. 48, 174 – 176. Sura, T., Smith, D.M., Cowan, G.M., Walsh, A.L., White, N.J., Krishna, S., 1997. Polymerase chain reaction for the detection of Burkholderia pseudomallei. Diagn. Microbiol. Infect. Dis. 29, 121 – 127. Tiangpitayakorn, C., Songsivilai, S., Piyasangthong, N., Dharakul, T., 1997. Speed of detection of Burkholderia pseudomallei in blood cultures and its correlation with the clinical outcome. Am. J. Trop. Med. Hyg. 57, 96 – 99. Walsh, A.L., Smith, D.M., Wuthiekanun, V., Suputtamongkol, Y., Desakorn, V., Chaowagul, W., White, N.J., 1994. Immunofluorescence microscopy for the rapid diagnosis of melioidosis. J. Clin. Pathol. 47, 377 – 379. Westphal, O., Jan, K., 1965. Bacterial lipopolysaccharides; extraction with phenol – water and further applications of the procedures. Methods Carbohydr. Chem. 5, 83 – 91. Wongratanacheewin, S., Tattawasart, U., Lulitanond, V., 1990. An avidin-biotin enzyme-linked immunosorbent assay for the detection of Pseudomonas pseudomallei antigens. Trans. R. Soc. Trop. Med. Hyg. 84, 429 – 430. Wongratanacheewin, S., Tattawasart, U., Lulitanond, V., Wongwajana, S., Sermswan, R., Sookpranee, M., Nuntirooj, K., 1993. Characterization of Pseudomonas pseudomallei antigens by SDS-polyacrylamide gel electrophoresis and Western blot. Southeast Asian J. Trop. Med. Pub. Hlth. 24, 107 – 113.
Recent developments in laboratory diagnosis of melioidosis Stitaya Sirisinha a,b,*, Narisara Anuntagool a, Tararaj Dharakul c, Pattama Ekpo c, Surasakdi Wongratanacheewin d, Pimjai Naigowit e, Benja Petchclai f, Visanu Thamlikitkul g, Yupin Suputtamongkol g a
Laboratory of Immunology, Chulabhorn Research Institute, Bangkok 10210, Thailand Department of Microbiology, Faculty of Science, Mahidol Uni6ersity, Rama VI Road, Bangkok 10400, Thailand c Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol Uni6ersity, Bangkok, Thailand d Department of Microbiology, Faculty of Medicine, Khon Kaen Uni6ersity, Khon Kaen, Thailand e National Institute of Health, Department of Medical Ser6ices, Ministry of Public Health, Nonthaburi, Thailand f Department of Pathology, Faculty of Medicine Ramathibodi Hospital, Mahidol Uni6ersity, Bangkok, Thailand g Department of Internal Medicine, Faculty of Medicine Siriraj Hospital, Mahidol Uni6ersity, Bangkok, Thailand
b
1. Introduction In tropical countries, melioidosis remains an important public health problem, particularly with regard to diagnosis and treatment. Clinical manifestations of the disease vary considerably, from fatal septicemia to chronic localized infection. Subclinical infection is not uncommon in people residing in the endemic areas of infection, e.g. the northeastern part of Thailand. If improperly handled, the mortality rate of acute septicemic cases can be as high as 70 – 80%. Even when diagnosis can be made early and antibiotic therapy is initiated soon after admission, the mortality rate is still high. In the hands of experienced clinicians, the septicemia caused by Burkholderia pseudomallei is still difficult to differentiate from that caused by other organisms. Laboratory diagnosis is thus crucial for successful patient management. Bacterial identification by culture is now the only accepted ‘gold standard’. A number of * Corresponding author. Fax: + 66-2-6445411 and +66-2247122. E-mail address: [email protected] (S. Sirisinha)
immunological and molecular approaches, e.g. PCR, have been developed but to date they cannot yet replace the more cumbersome and time consuming culture method. This article includes a short review of recent developments in the laboratory methods for the diagnosis of melioidosis and description of our recent multi-center collaborative project on laboratory diagnosis here in Thailand, with the main objective of having the most suitable and reliable diagnostic kits for the detection of specific B. pseudomallei antibody, antigen and genetic material.
1.1. Bacterial culture Isolation and identification of B. pseudomallei by the culture method is still the method of choice for definitive diagnosis of melioidosis. It is relatively simple and economical to perform. It needs no elaborate and expensive equipment but requires experienced personnel, particularly in the interpretation of the results. Its main drawback is that it takes at least 3–4 days to obtain the results and by that time it may be too late for successful management, as a high percentage of patients
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admitted for acute septicemia die within 24 – 48 h of admission. It is recently reported that using an automatic BacT/Alert® nonradiomatric blood culture system, about 62% B. pseudomallei positive culture could be detected within 24 h of incubation and more than 90% within 48 h (Tiangpitayakorn et al., 1997). It is interesting to note that case fatality varied indirectly with the time of ‘alarm’ in this automatic system; fatalities also occurred more often in those in which the bacterial growth (alarm) was detected within the first 24 h. However, for definitive diagnosis this positive culture needs to be subcultured for biochemical characterization, which takes at least another 24 – 36 h for species identification. Although the use of a sensitive automated system has considerably cut down the incubation time normally required for the less sensitive semiautomatic or manual systems, its main drawback is the running cost and its availability only in large hospitals. This factor is rather crucial, as for this disease, particularly in acute sepsis, rapid and early diagnosis is necessary and one needs to cut down unnecessary time loss as much as possible, e.g. transportation time to transfer clinical specimens from small health centers to the regional hospitals where the automated machine is available. In some clinical specimens, such as throat swab and sputum, which are generally heavily contaminated by other organisms, B. pseudomallei can often be overgrown by contaminating flora, as the generation time for B. pseudomallei is considerably longer than that of other common bacteria. However, this problem can be minimized with the use of selective media such as the Ashdown, which contains various dyes and gentamicin. The use of a modified Ashdown medium containing colistin further helps to increase the efficiency of isolation (Dance et al., 1989). Availability of a costly API 20NE test panel has considerably simplified the identification of B. pseudomallei. However, this API panel of tests has been reported to misidentify B. pseudomallei for Chromobacterium 6iolacium and others (Inglis et al., 1998). Several groups of investigators have attempted to speed up the bacterial identification step which currently involves subculturing and biochemical characterization. This includes the use of specific
B. pseudomallei antibodies in combination with bacterial culture. If it is proven to be reliable, this procedure can cut down the time needed by at least 24–36 h when compared with the classical bacterial identification procedure now in use in all diagnostic laboratories. This possibility will be discussed in more detail in a later section of this article.
1.2. Immunological methods Several immunological assays have been developed and used for the detection of either B. pseudomallei antibodies or antigens in the clinical specimens. Many of these assays, i.e. indirect hemagglutination (IHA) for the detection of antibody, are simple to perform with minimum experience and equipment (Appassakij et al., 1990; Naigowit et al., 1992). The results can be obtained more rapidly than the bacterial culture mentioned earlier. However, regardless of the detection of antibodies or antigens, with the immunological assays one needs highly specific reagents. Even so, the interpretation of the data, particularly in the antibody detection, is still a problem due to the presence of seropositive subclinical infection in apparently healthy individuals residing in the endemic area of infection.
1.3. Detection of antibodies A number of serological tests for the detection of specific B. pseudomallei antibodies have been developed, all of which need improvement one way or another (Ashdown et al., 1989; Sirisinha et al., 1996). Some of the older techniques including complement fixation are not used much any more, but others e.g., indirect hemagglutination (IHA) test are still being widely used in many endemic areas of infection (Appassakij et al., 1990). One of the main drawbacks of these antibody assays that limits their value in clinical situations is the presence of background antibody in some healthy individuals in the endemic area. This is largely attributable to unrecognized subclinical infection in these individuals. However, this should not pose a great problem in non-endemic areas, e.g. countries in the temperate zone, where only spo-
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radic cases occur. To minimize misinterpretation of the results, the measurement of specific IgM antibody has been developed (Ashdown, 1981) but its usefulness has not yet been evaluated in a large-scale field test. In addition to the problem of sensitivity, some patients with acute sepsis do not exhibit significant IHA antibody levels, thus resulting in false negative reports (Appassakij et al., 1990). It is possible that the elevated background antibody level in individuals in the endemic areas of infection is attributable to existing antibodies against other Gram-negative organisms that happen to cross react with the B. pseudomallei antigens used in the testing systems. The latter include lipopolysaccharide (LPS), which represents a major component in the crude B. pseudomallei antigens used in the IHA test. Although early reports (Pitt et al., 1992) suggested that B. pseudomallei LPS was rather homogeneous, more recent reports including those from our group (Perry et al., 1995; Anuntagool et al., 1998) showed contradicting results. We found the LPS from B. pseudomallei isolates from a few patients among the 200 – 300 examined to possess atypical ladder pattern in SDS-PAGE analysis and the sera from these patients failed to react with the typical LPS and vice versa (Anuntagool et al., 1998). With this in mind, it is reasonable to expect that a few septicemic patients who may be infected with the B. pseudomallei possessing atypical LPS will give low IHA antibody titer against typical B. pseudomallei LPS. Other B. pseudomallei specific antigens that have been identified and characterized include 36-kDa exotoxin (Ismail et al., 1987), 19.5 kDa (Anuntagool et al., 1993) and 40-kDa proteins (Wongratanacheewin et al., 1993) and LPS (Petkanjanapong et al., 1992). However, these antigens have yet to be evaluated for their clinical usefulness in a large-scale field trial. We recently characterized and purified a 200-kDa surface antigen of B. pseudomallei by affinity chromatography using monoclonal antibody (5F8) to this specific component (Rugdech et al., 1995). In the indirect ELISA test, this antigen was found to be highly specific for the detection of B. pseudomallei antibody (Rugdech et al., 1995). The diagnostic value of this test was evaluated in an actual
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clinical situation in an area endemic for melioidosis (Dharakul et al., 1997). The results showed that the detection of specific IgG antibody against this immunoaffinity purified antigen is clinically useful for the diagnosis of acute melioidosis in an endemic area. Using the same assay system, the detection of IgM antibody is less reliable. This is in contrast with the results reported earlier by other groups suggesting the IgM antibody detection to be more specific and reliable than the IgG antibody for the diagnosis of acute septicemic melioidosis (Ashdown, 1981; Khupulsup and Petchclai, 1986; Kunakorn et al., 1990, 1991). More recently, recombinant antigens specific for B. pseudomallei have been produced (Dharakul, T., personal communication; Wongratanacheewin, S., personal communication). One such antigen, to be described in detail in a later section, appears to be highly specific and useful for the diagnosis of melioidosis.
1.4. Detection of antigens This approach is more logical and is superior to antibody detection because it indicates active disease. This is particularly valid when used in the endemic area of infection, where the background antibodies often interfere with the interpretation in the antibody assay. Many immunological methods have been developed for the detection of B. pseudomallei antigen, including the detection of soluble secreted product in blood and urine, and the detection of whole organisms in, for example, pus, wound, sputum and throat swabs. This approach was first undertaken by Ismail et al. (1987) when they developed a monoclonal antibodybased assay for the quantitation of exotoxin. This assay could detect toxin in the range of 16 ng/ml of the culture supernatant fluid. However, it has never been evaluated in real clinical situations. Similarly, Wongratanacheewin et al. (1990) have developed a polyclonal antibody-based avidin–biotin ELISA which could detect B. pseudomallei antigens in the culture filtrate at a concentration as low as 4 ng/ml. The main component detected by this ELISA system appeared to be a 40-kDa protein secreted into the protein-free culture medium (Wongratanacheewin et al., 1993). How-
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ever, like the exotoxin situation, this assay has not yet been evaluated in clinical situations. More recently, a highly sensitive assay was developed by Desakorn et al. (1994) for the detection of antigen, most likely the LPS, in the urine of patients with systemic and localized infection. This ELISA system was based on the use of an additional amplification step to increase sensitivity. Briefly, it was a sandwich ELISA in which the amplified detecting system consisted of FITC conjugated rabbit antibody to B. pseudomallei antigens and horseradish peroxidase conjugated mouse monoclonal antibody to FITC. With this assay system, it was possible to detect B. pseudomallei LPS at a concentration of 12.2 ng/ml. When applied to clinical specimens, a sensitivity of 81% and a specificity of 96% were achieved. Although some non-specific reaction was noted in the urine of patients with other Gram-negative infection of the urinary tract, it has one practical advantage over other assays that have been described in that it can be used with unconcentrated urine. The assay is, however, more complicated and more costly to perform and thus may not be very practical for routine use in smaller health centers with a limited health budget. Recently we described another antigen-detecting assay based on the use of our monoclonal antibody reactive against the 200kDa antigen secreted by B. pseudomallei (Anuntagool et al., 1996). This sandwich ELISA was shown to be highly reliable in detecting B. pseudomallei antigens in different clinical specimens tested including pus, sputum, urine and pleural fluid. The results showed the sensitivity and specificity of the test to be 75 and 98%, respectively. Other assay systems that have been developed and evaluated including latex agglutination (Smith et al., 1993, 1995) and immunofluorescence (Walsh et al., 1994). The former is not sensitive enough for routine use and needs prior concentration while the latter requires a fluorescent microscope which is not readily available in most laboratories in the endemic area of infection.
1.5. Molecular approach The detection of genetic materials in different clinical specimens has been used successfully for
the diagnosis of a large number of infectious diseases of man and animals, and more recently evaluated for its reliability in melioidosis. With the latter, both the hybridization technique using specific DNA probes and the PCR using a different set of DNA primers have been developed by investigators in many research and diagnostic laboratories. The hybridization technique did not have sufficient sensitivity for B. pseudomallei infection (Sermswan et al., 1994) and, therefore, in more recent years, the PCR approach has been more commonly performed with satisfactory results (Lee and Desmarchelier, 1994; Kunakorn and Markham, 1995; Dharakul et al., 1996; Brook et al., 1997; Rattanathongkom et al., 1997; Sura et al., 1997). Different sets of primers that have been evaluated including the regions in the 23S ribosomal RNA, 16S RNA, the junction between 16S and 23S RNA and specific sequences designed from a specific DNA probe. Using these different primers, as few as a single bacterium present in the clinical specimens can be successfully amplified and detected. However, with the exception of the more recent report by Haase et al. (1998), none of these has been subjected to critical evaluation in the real clinical situation. Based on the use of 16S RNA as the PCR primer, these investigators reported a sensitivity approaching 100%. However, based on this small scale clinical testing, the specificity was still far from satisfactory, as approximately one-third of the patients with other diagnoses were positive by this PCR system.
1.6. Comparati6e study of the efficacy of 6arious laboratory diagnostic methods Because the reports on the use of various immunological methods from the same or different groups performed at different times often give inconsistent results and have never been critically and simultaneously evaluated in a well-controlled clinical setting, we have, therefore, set up a multicenter collaborative project, hoping to settle this point once and for all. The project is a large-scale, double-blind case-controlled study carried out in a real clinical setting. It consisted of nine subprojects designed to detect antibodies, antigens
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and genetic materials, all of which employed clinical specimens from the same melioidosis and control patients. These specimens were given codes by laboratory and clinical personnel not involved in the development of the assays and testing of the samples. Only after laboratory work was finished and all the data were obtained were the codes opened and the results from different groups compared.
2. Materials and methods
239
demic northeastern part of Thailand, i.e. at Ubon Ratchatani (Sapprasithiprasong Hospital), Srisaket, Surin and Khon Kaen (Srinakarind Hospital) provinces (Fig. 1). Only patients with clinical manifestations compatible with melioidosis who were subsequently proved to be culture-positive for B. pseudomallei and those with community-acquired septicemia whose hemocultures were positive for other organisms were included in this series. The latter, serving as control for each septicemic case, were those admitted at the same hospital not more than 14 days prior to or after the admission of the septicemic patients.
2.1. Patient selection
2.2. Collection of clinical specimens
Patients enrolled in this project were selected from those admitted between January and December, 1997 to four provincial hospitals in the en-
These samples including blood, urine, throat and wound swabs, pus and sputum were subjected to bacterial culture and biochemical characterization for species identification as described elsewhere. Specimens from clinically suspected melioidosis patients, both septicemic and non-septicemic, were all collected at the time of admission. Blood, serum and urine specimens from those with culture positive for B. pseudomallei were processed further for subsequent laboratory study. Similar specimens from community-acquired septicemic patients (clinically not compatible with melioidosis) serving as control for this study were collected 3–4 days after admission when the results from the bacterial cultures were known. Therefore, in this group of patients, only those with other bacterial infections (culture negative for B. pseudomallei ) served as non-melioidosis controls. However, a small proportion of patients in this group were later found to have positive blood culture for B. pseudomallei and were, therefore, included in the melioidosis group. These additional melioidosis patients differed from the ones with clinical melioidosis in that they were initially clinically not compatible with melioidosis and the specimens from these patients, like those of non-melioidosis controls, were collected 3–4 days after admission when the culture results were known. During this short time interval before a definitive diagnosis could be made, they were routinely treated with antibiotics and other medications which might interfere with the antigen detection and PCR analysis.
Fig. 1. Map of Thailand showing the endemic northeastern region where the study was undertaken. Patients were from hospitals in the four provinces.
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240 Table 1 Antibody detection assays in serum Method
Antigen
Indirect ELISA Indirect ELISA Indirect ELISA Western blot
Affinity purified 200-kDa component LPS 30-kDa protein (CCF) 16-kDa recombinant B. pseudomallei antigen 40-kDa protein (CF)
Dot immunoassay IHA (M) IHA (P) IHA (L) Immunochromatography
Melioidin Protein fraction of crude culture filtrate LPS Bacterial cell suspension
1992) and the one used for IHA was a hot phenol–water extract (Westphal and Jan, 1965). Recombinant protein from recombinant E. coli was partially purified by electroelution and was found to contain a predominant 16-kDa-protein component (Dharakul, T., unpublished). The crude culture filtrate (CCF) antigen for the ELISA was prepared by culturing B. pseudomallei in proteinfree synthetic broth containing 1% glycine, 0.25% disodium phosphate, 0.5% sodium chloride and 0.2% dextrose (Ekpo, P., unpublished). The supernatant fluid was centrifuged, filter-sterilized and then concentrated by membrane ultrafiltration (PM10 Diaflow®). It contained predominantly a 30-kDa protein (Fig. 3). The culture-filtrate antigen (CF) for dot immunoassay was prepared by culturing the organisms at 37°C in a protein-free modified Proskauer and Beck Medium (PBM) for 2 weeks without shaking (Wongratanacheewin et al., 1993). The supernatant fluid obtained after centrifugation and filter sterilization was dialyzed and lyophilized. It contained a 40-kDa protein as its major component (Fig. 3).
2.4. Monoclonal antibodies
Fig. 2. Immunoblotting profiles of the affinity-purified 200kDa antigen (5F8) and the LPS preparations used for the antibody assays. The latter was developed with three different MAbs specific for B. pseudomallei LPS.
2.3. Antigen preparations Details of the preparation of antigens used for the antibody detection shown in Table 1 have been or will be described elsewhere. In short, the affinity-purified antigen was prepared using MAb 5F8 specific for a 200-kDa component of B. pseudomallei (Fig. 2). The LPS used for indirect ELISA (Fig. 2) was prepared by a modified phenol-chloroform petroleum ether extraction method (Galanos et al., 1969; Kawahara et al.,
The mouse MAbs used for antigen detection in the urine included: (1) MAb 5F8 which was an agglutinating IgM antibody specific for a 200-kDa surface antigen (Fig. 2) which was also secreted in soluble form in the culture fluid (Rugdech et al., 1995; Sirisinha et al., 1998). (2) MAbs specific for LPS component. Three such clones were available, all gave a typical ladder profile in Western blot using purified LPS. The Bps L1 and Bps L2 were agglutinating IgM (Dharakul, unpublished) while the 9D5 (Anuntagool et al., this issue) was non-agglutinating IgG antibody (Fig. 2).
2.5. Immunological assays The assays shown in Tables 1 and 2 were performed as described elsewhere. Horseradish peroxidase or biotin conjugated rabbit anti-mouse immunoglobulins were used to detect the reaction in the indirect ELISA and dot immunoassay. For
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241
Fig. 3. SDS-PAGE profiles of 40-kDa protein (CF) used in the dot immunoassay and of 30-kDa protein (CCF) used in the indirect ELISA.
the antigen detection, the MAbs, i.e. Bps L1, Bps L2 and 9D5, were coated directly on the microtiter plates while the MAb 5F8 used in the sandwich ELISA had to be captured with rabbit anti-mouse immunoglobulins. The sensitivity of the immunochromatography was increased by using the blocking principle.
3. Results and discussion This was the first collaborative study to develop alternative laboratory methods and to evaluate their efficacies for the diagnosis of melioidosis in a real clinical situation. A very strict criterion was used for the selection of melioidosis and ‘control’ cases and the evaluation was carried out using an unbiased double-blind protocol. The most purified and specific B. pseudomallei antigens and specific MAbs currently available in different laboratories in Thailand were used for the development of the methods for the detection of antibodies and antigens respectively. The detection of genetic materials by PCR will only be briefly mentioned for the purpose of comparative discussion. Full details of the latter will be dis-
cussed by Kunakorn and his colleagues (this issue).
3.1. Antibody detection The efficacy of different antibody detection assays is summarized in Table 3. The use of a CCF antigen containing predominantly a 30-kDa protein and a recombinant antigen produced in E. coli seemed to give the most reliable results. However, the reliabilities of these two assay systems were still below the expected minimum values of 90% when compared with the culture method used as gold standard. The use of a combination Table 2 Antigen detection assays in urine Method
Antibody
Sandwich ELISA
IgM Monoclonal antibody against 200-kDa antigen (5F8) IgG MAb against LPS (9D5) IgM MAb against LPS (BpsL2) IgM MAb against LPS (Bps L2) IgM MAb against 200-kDa antigen (5F8)
Sandwich ELISA Sandwich ELISA Latex agglutination Competitive immunochromatography
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242
Table 3 Performance of various antibody detection assays in 101 patientsa Method
Indirect ELISA Indirect ELISA Indirect ELISA Western blot Dot immunoassay IHA IHA IHA Immunochromatographyb
Antigen
%
Affinity-purified (200 kDa) LPS CCF (30 kDa) Recombinant CF (40 kDa) Melioidin Secreted protein LPS Crude antigen
Sensitivity
Specificity
Positive predictive
Negative predictive
74 70 82 70 72 93 54 46 23
75 77 80 91 64 34 61 82 89
79 80 84 91 72 65 65 76 76
69 67 78 70 64 79 51 54 47
a 57 Patients were culture positive for B. pseudomallei and 44 patients were culture positive for organisms other than B. pseudomallei. b Detecting only IgM antibody.
Table 4 Performance of antigen detection assays in 120 patientsa Method
Sandwich ELISA Latex agglutination Immunochromatography
Antibody
MAb 5F8 Bps L2 MAb 5F8
% Sensitivity
Specificity
Positive predictive
Negative predictive
59 23 23
56 80 95
58 54 82
57 50 54
a 61 Patients were culture positive for B. pseudomallei and 59 patients were culture positive for organisms other than B. pseudomallei.
of two or three antibody detection assays did not significantly improve performance. There are several possible explanations for this unexpected result. Firstly, subclinical infection is known to occur among healthy individuals residing in the endemic area of infection and the antibody response to B. pseudomallei infection is known to be long-lasting. The elevated antibody levels are thus maintained by continuous exposure. Secondly, it is possible that other closely related Gram-negative organisms might possess components that share similar antigenic epitopes which can stimulate antibodies that cross react with the B. pseudomallei antigens used. Lastly, it is possible that the identification of B. pseudomallei by the culture method was not 100% reliable as there were a few
cases in which all serological tests gave results which agreed with one another but totally disagreed with the culture results used as gold standard.
3.2. Antigen detection Three laboratories using five different assay systems participated in this evaluation (Table 2). The results shown in Table 4 were, in general, below our expectation. The performance of all tests needs improvement. Two assays using the same MAb 5F8, namely ELISA and immunochromatography, gave results markedly different from one another. The results from immunochromatography assay were acceptable
S. Sirisinha et al. / Acta Tropica 74 (2000) 235–245
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3.4. Detection of genetic materials by PCR
regarding specificity but were rather insensitive. It appears possible to increase the sensitivity of this assay, but it is to be expected that the specificity has to be slightly compromised. In fact, this MAb was subsequently used for the development of other antigen detection assays in the urine specimens, i.e. latex agglutination and dot immunoassay, all of which did not give any better results (data not shown) than the ones reported in Table 4 in this communication. Moreover, using this same MAb 5F8 to replace the polyclonal antibody in the amplified ELISA system previously reported by Desakorn et al. (1994) also failed to give satisfactory results.
Three groups, each using different sets of PCR primers, participated in this project. None of them gave satisfactory performance compared with the culture method. Again, like the immunological approach, both false positives and false negatives were noted for this molecular approach and will be discussed in detail in a subsequent report. When immunological methods (both antibody and antigen detections) and molecular assays were paired in all possible permutations, no significant improvement in performance was noted.
3.3. E6aluation of a combined result from the assays of serum and urine from the same patients
4. Conclusion
Because the assay systems shown in Tables 3 and 4 did not give reliable results that can be used in real clinical settings when compared with the culture method, an attempt was made to improve the results by combining the results from antigen and antibody detection assays. The data shown in Table 5 again failed to support our expectation as no significant improvement in the performance could be detected from any of the three laboratories participating in this experiment. Pairings of the assays from different laboratories did not give any better results.
With the technologies and reagents currently available, it seems that the culture method remains the method of choice, not only in its reliability but also in its simplicity and cost. Its only drawback is the time needed before a definitive diagnosis can be made. To help speed up the time needed for bacterial identification, a combination of the culture method with any one of the immunological and molecular assays discussed in this article could be used and the results evaluated. For instance, to improve the sensitivity, an overnight hemoculture can be carried out to in-
Table 5 Performance of a combined antibody and antigen detection assays of specimens from the same patients No of patients
83a
83a 82b
Antibody detection
Affinity purified 200-kDa antigen (ELISA) Recombinant antigen (Western blot) Bacterial cell (IM)
Antigen detection
%
Sensitivity
Specificity
Positive predictive
Negative predictive
5F8 (ELISA)
80
51
65
69
Bps L2 (LA)
84
59
70
77
5F8 (IM)
48
90
83
62
a 44 Patients were culture positive for B. pseudomallei . and 39 patients were culture positive for organisms other than B. pseudomallei. b 42 Patients were culture positive for B. pseudomallei and 40 patients were culture positive for organisms other than B. pseudomallei. IM, immunochromatography.
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crease the number of bacteria and then the bacterial identification in the culture can be made using any of the specific monoclonal antibodies or PCR primers currently available. Our limited and preliminary data on this approach compares very favorably with the classical bacterial culture approach. If this proves reliable, then the time required to reach a definitive diagnosis can be reduced from 3 to 4 days to within 18 – 36 h.
Acknowledgements This collaborative project was supported by the Thailand Research Fund, Chulabhorn Research Institute, and National Science and Technology Development Agency of Thailand. We thank Maurice Broughton for his advice during the preparation of this manuscript.
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