Diagnostic procedures in the investigation of infectious diarrhoea

Diagnostic procedures in the investigation of infectious diarrhoea G. C. COOK

The laboratory diagnosis of a gastrointestinal infection-whether bacterial, viral, protozoan, or helminthic-has depended historically on visualization of the causative agent in a faecal sample, either before or after culture. This ‘traditional’ approach allows identification of the organism, its isolation and further testing, and, in the case of bacterial pathogens, determination of its antibiotic-susceptibility pattern; several selective media and enrichment techniques have improved efficiency for the isolation of certain pathogens, e.g. Clostridium difficile, Yersinia spp., Campylobacter spp., enterohaemorrhagic Escherichia coli (EHEC) 0157:H7 and non-cholera vibrios (Martin, 1986). However, a major drawback with this approach is that several days are required before a diagnosis can be achieved. Reviews concerning recent developments in diagnosis have been provided by Martin (1986), Thorne (1988, 1990) and Gracey and Burke (1993). Improved laboratory diagnosis has been greatly facilitated by the introduction of several newer techniques (Rose, 1992)) in particular gene probes, the enzyme-linked immunoassay (ELISA), and the polymerase chain reaction (PCR) (White et al, 1989). These developments in molecular biology have been especially valuable in the diagnosis of infections in individual patients, and also in survey and epidemiological work concerning diarrhoeal disease. In addition, rapid techniques and bacterial toxin detection systems have aided the identification of many gastrointestinal pathogens. RECENT

ADVANCES

IN DIAGNOSTIC

TECHNIQUES

The last decade has seen important advances in laboratory techniques; those involving the bacterial enteropathogens are undoubtedly the most impressive (Ashkenazi and Pickering, 1989). Also, several ‘new’ aetiological agents have been added to the list of intestinal pathogens, some pathogenic mechanisms have been delineated, and various diagnostic methods have been perfected. Baillit?re’s Clinical GastroenterologyVol. 7, No. 2, June 1993 ISBN 0-702@1749-3

421 Copyright 0 1993, by Baillikre Tindall All rights of reproduction in any form reserved

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Bacteria produce a range of toxins (Table 1): ‘bacterial products that act on the mucosal epithelium of the small intestine, causing fluid secretion and profuse watery diarrhoea, without damage to the intestinal mucosa’ (Ashkenazi and Pickering, 1989). Fluid secretion is related to the enzymatic effect of enterotoxin on the enterocyte, often via a specific receptor, together with an increased cyclic nucleotide concentration; in the case of Vibrio cholerue, ADP ribosylation of an adenylate cyclase regulatory protein leads to an increase in intracellular CAMP. Table 1 lists several other bacteria which produce either a heat-labile (LT) or heat-stable (ST) enterotoxin. Cytotoxins consist of a distinct group of diarrhoea-related compounds which have the property of killing mammalian cells, usually by inhibiting protein synthesis; cytotoxic activity can be demonstrated in vitro in several tissue culture cell systems. The prototype is that produced by ShigeZla dysenteriae 1; functionally, immunologically and structurally closely related toxins are produced by certain strains of E. coli, e.g. EHEC serotypes 0157:H7 and 026:Hll, which have been implicated in haemorrhagic colitis and haemolytic uraemic syndrome. In vivo, they cause enterocyte damage and interference with the normal absorptive mechanisms; they do not (unlike the enterotoxins) however, cause active fluid secretion, and nor do they leave the enterocyte morphologically intact. The mode of action of Clostridium difficile toxin, which produces antibiotic-associated pseudomembranous colitis (see below), remains unclear. Adherence is a prerequisite for many infections; enterotoxigenic E. coli (ETEC) has been studied most extensively in this context. Adherence is usually mediated by the binding of bacterial surface proteins (adhesins or lectins) to receptors, which are usually sugar residues. Several adherenceassociated surface structures have been isolated from human and animal strains of ETEC: human colonization factor antigens, porcine K88 antigen Table

1.

Pathogenic mechanisms in bacterial diarrhoea (Ashkenazi and Pickering, 1989).

Pathogenic mechanism

Enteric pathogen

Adherence

Escherichia

Toxin production Heat-labile enterotoxin

Vibrio

coli (ETEC, EPEC, others?), Salmonella spp.?, Shigella spp.? cholerae, Escherichia coli (ETEC), Salmonella spp., Campylobacter spp., Clostridium perfringens, Vibrio spp., Aeromonas spp., Bacillus cereus, Plesiomonas

shigelloides

Heat-stable enterotoxin

Escherichia coli (ETEC), Yersinia enterocolitica, Staphylococcus aureus, Salmonella spp.?, Aeromonas spp.?, Plesiomonas shigelloides?, Vibrio spp.?

Cytotoxin

Shigella spp., Escherichia coli (mainly EHEC), Clostridium diftkile, Salmonella spp., Campylobacter spp., Aeromonas spp., Plesiomonas shigelloides, Vibrio

SPP. Invasiveness

Shigella spp., Salmonella Yersinia spp., Escherichia Plesiomonas shigelloides?

spp., Campylobacter coli

(EIEC), Vibrio

spp.,

spp.,

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and bovine K99 antigen. By causing destruction and effacement of small intestinal microvilli, some enteropathogenic E. coli (EPEC) can cause significant disease via adherence alone. The exact importance of adherence in Shigella spp, and Salmonella spp. remains to be delineated. The archetypal invasive organism of the colorectum is Shigella dysenteriae 1, which has the ability to produce keratoconjunctivitis in the guinea-pig (the SerCny test) (Se&y, 1955). Invasiveness is related to a 120-140-MDa plasmid which encodes for five virulence-associated polypeptides; bacterial lipopolysaccharide and chromosomally encoded products are required for full pathogenicity. The properties so far summarized are crucially important in the context of rapid methods for the aetiological diagnosis of bacterial diarrhoea (Table 2); they are beginning to replace some of the traditional techniques (see above). Their introduction is especially important because the list of known enteropathogens is steadily lengthening. Whilst representing an exciting development in applied molecular genetics (Eisenstein and Engleberg, 1986), they continue to present certain limitations (Tenover, 1988); in many instances, sensitivity, specificity and predictive values have not yet been precisely determined. No single method is available for the diagnosis of all of the common causes of bacterial diarrhoea; therefore, the introduction of a new technique will not eliminate the necessity for routine microbiological testing of faecal samples. Also, and very importantly, antibiotic sensitivity is not possible with the rapid techniques; and furthermore, the isolate is not available for serotyping or epidemiological study. Table 2. Rapid methods Pickering, 1989).

for the aetiological

Method Toxin

of bacterial

diarrhoea

(Ashkenazi

and

Details detection

Serological bacterial DNA

diagnosis

detection antigens

hybridization

Biological assays DNA hybridization studies Immunological methods Serological methods of

Agglutination Salmonella di’cile

tests for Vibrio cholerae, Shigella spp., spp., Campylobacter spp., Clostridium

Detection of: Salmonella spp., Campylobacter spp. Toxin production Invasive plasmid (Shigella spp.) Enteroadherent factor (Escherichia coli)

Toxin production Toxin detection is essential for the diagnosis of ‘toxin-related’ diarrhoea; there are no biochemical markers to distinguish toxin-producing from nontoxin-producing strains of bacteria. Biological assays are based on either animal models or cell culture systems. ELISA or radio-immunoassay

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methods are available for the detection of E. coli enterotoxins and cytotoxins, Shigella dysenteriae 1 and staphylococcal enterotoxins, and Clostridium dijffkile cytotoxin. Knowledge of the binding sites of various toxins has led to the development of assays utilizing the specific binding of toxins to microtitre plates coated with their receptors: GM1 and Gb3 have been used to detect E. coli LT enterotoxin and S. dysenteriae 1 toxin. Although confined mainly to research settings, a commercial ‘kit’ is currently available for the biological detection of Clostridium dificile cytotoxin (Ashkenazi and Pickering, 1989). Clostridium perfringens and staphylococcal enterotoxins can be detected by serological agglutination assays. Bacterial antigen detection

These antigens can be detected serologically by rapid agglutination techniques; they are available for Vibrio cholerae 01, Clostridium dijjicile, Campylobacter spp., Shigella spp. and Salmonella spp. (see below). DNA hybridization

assays

These are based on labelled DNA probes (segments)-most of them radiolabelled-which are examined for hybridization (binding) with a complementary nucleic acid sequence. Such assays have been used to detect genes which encode for virulence factors (e.g. toxin production, adherence and invasiveness), or sequences which characterize certain bacterial pathogens (e.g. Salmonella spp. and Campylobacter spp.). The PCR (see above) has been developed for the in vitro amplification of microbial DNA or RNA, and provides the potential advantage of rapidly establishing the aetiology of an episode of infectious diarrhoea. However, it is premature to even consider the replacement of traditional methods (see above) with these rapid techniques, which are still undergoing development; one or more method(s) should be considered for a given laboratory, and on an individual basis-reliability, cost and frequency of the particular pathogen in the aetiology of diarrhoea at the location under consideration should be carefully considered (Ashkenazi and Pickering, 1989). ANCILLARY TECHNIQUES WHICH DEMONSTRATION OF A SPECIFIC

DO NOT INVOLVE PATHOGEN

Although non-specific, several simple techniques can assist in the diagnostic approach to infectious diarrhoea. Faecal leukocyte examination

A fresh faecal sample (preferably mucus) is mixed with two drops of dilute Loeffler’s methylene blue and examined microscopically under a coverslip. Polymorphonuclear leukocytes (PL) (when present) can be readily identi-

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fied. A high concentration strongly suggests invasive colonic disease, i.e. tissue invasion of either bacterial or, less commonly, protozoan origin, or alternatively inflammatory bowel disease (IBD). Absence of PL suggests small intestinal diarrhoea, probably of toxigenic or protozoan origin. In a study in Punjab, India, the value of faecal PL examination was assessed in 400 infants with acute diarrhoea and 40 normal healthy infants (Jindal and Arora, 1991); 16 (57.1%) out of 28 infected with Salmonella spp., 4 (66.6%) out of 6 with Shigella jlexneri, and 4 (66.6%) out of 6 with enteroinvasive (EIEC) Escherichia coli had >lO faecal PLs per high-power field; in contrast, only 6 (18.7%) out of 32 with EPEC, 3 (10.7%) out of 28 with ETEC, and 35 (12.3%) out of 285 without a gastrointestinal pathogen had a high faecal leukocyte count. These authors considered that the major value of this technique (in that geographical setting) lies in providing a clue to the patient(s) in whom a faecal culture might yield a significant result. In another large study-involving 11358 diarrhoeic patients-carried out in Bangladesh (Hossain and Albert, 1991), comparable results were obtained; 2681 (72.3%) out of 3895 patients in whom Shigellu spp. was the sole pathogen harboured both PLs and erythrocytes in a faecal sample, whilst the remainder only had the former (P < 0.001). The presence of both types of cell proved to be a good predictor of shigellosis, as was >25 PLs per high-power field without erythrocytes. However, the best predictor was ~25 PLs together with erythrocytes-whatever their number. In Thailand, where clinical illness associated with Shigellu spp. and EIEC infections is similar, faecal microscopy proved useful (Echeverria et al, 1991); fewer children with an EIEC infection compared with those with a Shigellu spp. infection had >lO PLs per high-power field (36% versus 67%), and, furthermore, fewer had a positive occult blood test (36% versus 82%). Peripheral blood platelet count in infectious diarrhoea and inflammatory bowel disease (IBD)

A raised platelet count (~450 x 109/1) is more common in patients suffering from IBD compared with infectious diarrhoea (Harries et al, 1991); 16 (59%) out of 27 patients suffering from IBD and 3 (1.6%) out of 188 with infectious diarrhoea had an elevated count (P < 0.001). Other differencesbut with a lower diagnostic probability-were: length of duration of diarrhoea, presence of faecal blood, anaemia, leukocytosis, raised ESR and reduced serum albumin concentration (P < 0.05 to < 0.001) in those with IBD. COLLECTION, TRANSPORT FAECAL SPECIMENS

AND EXAMINATION

OF

Appropriate and correct specimen collection is the ‘key element in the microbiologic diagnosis of infectious disease’ (Washington, 1992). Faecal samples should be obtained as early as possible in the course of the disease (when the causative agent is likely to be present at a high concentration), and before antibiotic chemotherapy has been commenced (Martin, 1986). A

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C. COOK

rectal swab is generally less satisfactory, although invasive pathogens, e.g. Shigellu spp., are sometimes isolated more readily by this method. The sample should be collected in a clear, non-absorbent container and processed as soon as possible, and certainly within 2 h; after this time interval it should be refrigerated at 4°C or placed in transport (e.g. Cary-Blair) medium; if held for longer periods, freezing at -70°C is preferable. In acute gastroenteritis, examination of a faecal sample by dark-field or phase-contrast microsocpy may reveal motile Cumpylobacter spp. or Vibrio spp. Gram-staining of a faecal smear is of value in some diseases, e.g. staphylococcal enteritis (see below). Methods for detection of eggs, cysts and parasites are described below. When both faecal sample and rectal swab are cultured, the likelihood of detection of a pathogen increases by 11% (Adkins and Santiago, 1987). However, owing to the length of time involved, routine faecal culture cannot be justified on cost-effective grounds. If faecal leukocytes are present (see above), an invasive organism should certainly be sought. Specimens should be plated immediately they are brought to the laboratory; when this is impossible they should be stored at 4°C; several selective/differential media are widely used (Thorne, 1988; Gracey and Burke, 1993). When Vibrio spp., Yersinia spp., Campylobacter spp. or a viral aetiology seem likely on clinical grounds, an appropriate culture medium should be used. Media with low selectivity should be inoculated with a light loopful and those with high selectivity with several loopfuls of faecal material. Optimal use of diagnostic facilities Optimal results can only be obtained if and when the laboratory is used intelligently. Table 1 summarizes the pathogenic mechanisms underlying diarrhoea caused by some bacteria. Enterotoxigenic diarrhoea usually (but not always) originates in the small intestine. Invasive diarrhoeas (which usually involve the colorectum) have been defined as ‘the response of the human gastrointestinal tract to enteric pathogens . . . that have the capacity to invade the mucosa of the small and/or large intestine’ (Mathan and Mathan, 1991). Invasion goes beyond the epithelial layer and is associated with an inflammatory response in the lamina propria of the intestinal mucosa. Bacterial causes include Shigella spp., Salmonella spp., Campylobatter spp. and EIEC; viruses include rotavirus; examples of parasitic causes are Entamoeba histolytica and Trichuris trichiura (usually in children). Overall, Shigella spp. is the most common cause and clearly most emphasis on diagnosis should be concentrated on a search for this group of organisms. Although several other ‘enteric’ organisms invade, they do not produce dysentery (bloody, urgent, frequent stools associated with rectal tenesmus); examples are Salmonella typhi (which invades and proliferates in the Peyer’s patches-especially the ileum-and produces systemic infection involving the reticuloendothelial system), rotavirus (which multiplies in, and causes desquamation of enterocytes in the duodenum and upper jejunum) and Strongyloides stercoralis, which causes widespread enteritis in the upper small intestine.

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427

A clinical diagnosis is impossible in some cases and it is here that laboratory techniques must be applied (Mathan and Mathan, 1991); for example, in up to one-third of patients from whom Shigella spp. is isolated, there is no blood in the stool, and the relevance of mucus alone is difficult to assess. Much has been written on the inappropriate use of laboratory facilities in the diagnosis of infectious diarrhoea. A retrospective study on hospital inpatients in Philadelphia, USA, was designed to assessthe extent to which routine faecal culture, a search for eggs and parasites, and Clostridium difJiciZe toxin assay, were inappropriately requested (Siegel et al, 1990). During a 3-year period, only one out of 191 faecal cultures, and none out of 90 egg/parasite examinations, proved positive in a group of inpatients whose specimens had been submitted when they had been in hospital 3 days or more. Analysis of the laboratory workload for a l-year period also demonstrated that specimens from this inpatient group constituted nearly 50% of the >3000 samples received. In contrast, approximately 25% of samples, regardless of the length of hospitalization, were positive for Clostridium dijjkile toxin, this being the most likely organism to account for nosocomial diarrhoea. The authors concluded that elimination of routine faecal culture and egg/parasite examinations on inpatients would ‘significantly reduce hospital and patient costs without altering patient care’; resultant national cost-saving would amount to $20-30 million annually (Siegel et al, 1990). BACTERIAL

PATHOGENS

Shigella spp.

This group of organisms is responsible for classical invasive colorectal diarrhoea-‘bacillary dysentery’. After invasion, bacteria multiply within the colonocyte (and less often enterocyte), causing host-cell destruction; however, they rarely progress beyond the mucosa. The 39 serotypes or subtypes are divided on the basis of 0 antigens, into four groups: S. dysenteriae, S. JEexneri, S. boydii and S. sonnei; these are often referred to as groups A-D. Currently available methods for diagnosis of Shigella spp. have been reviewed (Echeverria et al, 1991); standard bacteriological methods and testing of E. coli isolates for hybridization with the ShigeZL-EIEC probe are at present the most sensitive means of diagnosis. Shigella spp. can be detected as lactose-negative colonies on eosinmethylene blue (EMB), MacConkey, Salmonella-Shigella (NS) agar and Kligler’s iron agar (KIA)-an alkaline red slant and acid yellow butt being produced (Thorne, 1988). The organisms are non-motile and ureasenegative; they do not produce H$, and only rarely produce gas during sugar fermentation. There is a resemblance to EIEC; they are non-lactosefermenters and express identical surface antigenic properties; both also carry a 120-140-MDa plasmid (see above) which encodes the genes necessary for virulence. Slide agglutination tests for serotyping are simple and rapid, but are heavily dependent on quality of the antisera; commercially produced

428

G. C.

COOK

preparations are not always satisfactory (Thorne, 1990). In a study carried out at Charkov, USSR, the presence of specific soluble Shigellu spp., Salmonella spp. and Yersinia enterocolitica antigens was determined by using a coagglutination reaction (COA) on coprofiltrate, saliva and urine from 268 patients with a ‘diarrhoea syndrome’ (Belaya et al, 1989); COA was diagnostically valuable in 79 ‘54% of patients with a Shigella spp. or Salmonella spp. infection’; Shigella spp., Salmonella spp. and Yersinia enterocolitica antigens were detected in 47.7%, 23.4% and 10.8%, respectively, of patients in whom bacterial excretion was not confirmed. Enzyme immunoassays (EIAs) which utilize O-antigen-containing lipopolysaccharides extracted with phenol-water from S. dysenteriae 1, S. Jtexneri (serotypes la-5b) and S. sonnei have been assessed using serum samples from 175 Vietnamese and 47 Swedish patients from whom Shigellu spp. had been isolated from a faecal sample (Lindberg et al, 1991). Sensitivity of the EIA ranged from 78 to 100%) and the authors concluded that increased IgA (in the early phase) and IgG (in the convalescent phase)-as determined by EIA-were the most reliable indicators of Shigellu infection. DNA probes have been developed by cloning fragments from invasive (1~) plasmids of S. jlexneri and an EIEC strain, respectively. All SerCny-positive strains hybridize with both DNA probes; however, there is some evidence of false-positive probe reactivity. Recently, good sensitivity and specificity for Shigella spp. and EIEC has been demonstrated using three specific oligonucleotide probes. Other detection methods-using EIAs-can be developed incorporating mouse monoclonals selected for their recognition of the invasive plasmid antigens B (&pa B) and C (Zpa C) (Thorne, 1990), which are involved in expression of the invasive phenotype in all species of Shigella and EIEC. However, Venkatesan et al (1989) have indicated that the S. JZexneri Ipa H probe is more effective. In a study carried out in Delhi, India, a non-radioactive alkaline phosphatase-conjugated oligonucleotide DNA probe was compared with the Ser6ny test in a determination of the sensitivity and specificity of the probe in detection of virulent Shigella spp. strains (Panda et al, 1990); the probe hybridized with all 52 SerCny-positive strains (sensitivity 100%) and 4 out of 21 Serkny-negative strains (specificity 81%); it did not however hybridize with any of the Serkny-negative S. dysenteriae 1 strains. The authors consider that this technique will ‘contribute to an improved understanding of the epidemiologic patterns of shigellosis in developing countries’. A rapid diagnostic technique which employed the PCR procedure has been used to identify Shigellu spp. and EIEC (Frankel et al, 1990); a 21-base oligonucleotide corresponding to the invasive-associated locus (ial) gene sequence has been shown to hybridize specifically with EIEC strains and Shigellu spp. This PCR was 105- and lo*-fold more sensitive than standard biochemical tests and a macrocolony hybridization assay, respectively, in the identification of Shigella spp. and EIEC. Shigella strains resistant to several antibiotics are now a major problem in chemotherapy of this disease, especially in the Indian subcontinent and South East Asia (Salam and Bannish, 1991). Recently, increasing S. jlexneri resistance to trimethoprim-sulphamethoxazole has been recorded in Thai

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429

children (Thisyakorn and Reinprayoon, 1992), and in Saudi Arabia, a study of 234 children infected with Shigella spp. indicated that 54% of isolates were resistant to ampicillin, 72% to trimethoprim-sulphamethoxazole, and 77% to tetracycline (Kagalwalla et al, 1992); 80% were resistant to two or more antimicrobials. Of the cheaper chemotherapeutic agents, nalidixic acid is now the most appropriate, but there seems no doubt that when available the fluoroquinolones are the most effective agents; however, a lingering doubt exists about safety in young children, cartilage damage having been demonstrated in young experimental animals. Escherichia coli

ETEC, EAEC, EIEC, EHEC and EPEC cannot be reliably differentiated using currently available biochemical techniques; furthermore, they cannot be separated from non-pathogenic E. coli. When both 0- and H-antigen typing is performed they can be differentiated by their distinctive serotypes (Levine, 1987; Thorne, 1988); also, DNA probes targeted towards plasmid-encoded virulence factors of EIEC, EPEC and ETEC have proved valuable. ETEC This organism is usually transmitted by contaminated food or water; it is a common cause of diarrhoea in developing countries, especially in children, and is the most common aetiological agent in travellers’ diarrhoea. Resultant diarrhoea is watery (‘cholera-like’). A study in South Africa, using a PCR technique (see below), has shown that LT-producing ETEC account for 9% of previously undiagnosed episodes of diarrhoea. Colonies can be taken from primary isolation plates and stored on nutrient slants until tested for one or both of two enterotoxins: LT (a heat-labile enterotoxin which is destroyed by heating at 60°C for 15 min), and ST (a heat-stable toxin which resists heating at 100°C for 15 min). The former (LT) is a high molecular weight protein which is structurally and functionally related to cholera toxin (see below). Specific binding to GM1 ganglioside has been utilized in the design of serological detection systems. Several assays exist. Three simple/rapid methods consist of: a modified GMi-horseradish peroxidase-ELISA (Svennerholm et al, 1983), the modified Biken (Elek) test, and the modified staphylococcal coagglutination test (Ronnberg and Wadstrom, 1983). LT detection using a reverse passive agglutination (RPLA) test is now available commercially in kit form (Scotland et al, 1989). Recently, PCR has been used for detection of the LT gene (Olive, 1989; Victor et al, 1991); amplification of specific target DNA sequences is highly sensitive and specific in the detection of pathogenic strains in clinical specimens; prior isolation is unnecessary. Fewer tests exist for detecting ST; those available include a GMi ELISA, which might prove valuable when combined with the GM1 ELISA for LT. A probe which hybridizes with LT, ST-Ia and ST-IIb has been described (Abe et al, 1990).

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G. C. COOK

EAEC These strains of E. coli (EA-ggEC) are defined as those which exhibit mannose-resistant adherence to HeLa or HEp-2 tissue culture cells or both, do nol produce conventional enterotoxins, and are not enteroinvasive; both EPEC (see below) and non-EPEC strains probably exist. HEp-Zadherent , non-EPEC strains cause diarrhoea, but the symptom is of ‘lower frequency’ than that caused by traditional EPEC isolates (Thorne, 1988). The relationship of non-EPEC strains to adherent EPEC is unclear (Nataro et al, 1985). Probes have now been described for both diffuse (Levine et al, 1988) and enteroaggregative (Baudry et al, 1990) adherence. Rapid methods for diagnosis of this group of mild diarrhoea-producing E. coli are required. EIEC Antigen sharing between EIEC and ShigeZZaspp. is widespread (see above). These strains of E. coli-which are readily confused with Shigella spp.-are often delayed or non-lactose fermenters, and are anaerobic, non-motile and lysine decarboxylase-negative. Although there are several serotypes, only three can be delineated with appropriate antisera: 0112, 0124 and 0126. Tests for EIEC are beyond the scope of the routine clinical laboratory; Congo red binding has been tested and found unsatisfactory (Albert and Leach, 1989). When suspected (in a patient suffering from dysenteric (colonit) diarrhoea), 5-10 colonies should be screened by the SerCny test (Se&y, 1955), and/or sent to a centre where either biological testing and/or DNA hybridization (Small, 1988) techniques are available. Taylor et al (1988) have used a single DNA probe (17 kb) to detect EIEC and Shigella spp. in Thai children with and without diarrhoea; EIEC and Shigellu spp. were isolated from 17% and 23 % of 410 children with diarrhoea and 1% and 3% of 410 controls. All colonies found to hybridize with the probe were confirmed to be EIEC by the SerCny test, presence of virulence marker antigen by ELISA, and by serotyping. PCR amplification using a faecal sample has been used (Frankel et al, 1990). A rapid faecal blot method has a sensitivity of 76% for EIEC and 45% for Shigella spp. (Thorne, 1990). EHEC There are more than 30 serotypes of E. coli which do not possess classical LT and ST enterotoxins but contain verotoxin or ‘Shiga-like’ toxin; this is produced by all strains of E. coli 0157:H7, the major causative agent of haemorrhagic colitis (in the UK, North America and South America), haemolytic uraemic syndrome and thrombotic thrombocytopenic purpura. EHEC are rapidly shed during the course of the illness and can rarely be cultured after the fifth day; therefore, if they are suspected to be the cause of bloody diarrhoea (‘dysentery’), a faecal swab should be kept at -70°C in 20% gylcerol with this diagnosis in mind (Thorne, 1988). It is readily identified on D-sorbitol (1%) MacConkey medium; unlike most strains of E. coli, it does not ferment (or only very slowly ferments) sorbitol. Alternatively, an H7 antiserum-sorbitol medium can be used, and the organism

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identified by serotyping with 0157 antiserum as for EPEC. A simple and reliable agglutination test for serotype 0157:H7 is commercially available (March and Ratnam, 1989; Thorne, 1990). Commercial 0:H antisera are also available, and H-typing should be performed when possible. Several other E. coli serotypes-which produce Shiga-like cytotoxinhave also been incriminated in haemorrhagic colitis/haemolytic uraemic syndrome; these include serotypes 04:NM, 026:Hll, 045:H2, Olll:H& 0113, 0121:H19, 0125:NM, 0126:Hll and 0145:NM. Other strains isolated from cases of colitis can only be recognized by screening for increased Verotoxin I (‘Shiga-like’ cytotoxin I) (SLT-I) production; this is virtually identical to the potent cytotoxin-neurotoxin-enterotoxin of S. dysenteriae I and is neutralized by antibody to Shiga toxin. Many strains also produce a second potent cytotoxin, Verotoxin II (‘Shiga-like’ toxin II) (SLT-II), which is not neutralized by antisera against Shiga toxin. Strains producing one or both of the Shiga-like cytotoxins I and II are pathogenic. Both Shiga-like cytotoxins can be rapidly detected using Vero cell or HeLa cell cytotoxicity assays (Thorne, 1990). A receptor-specific ELISA is available for the rapid and specific detection of SLT-I (Basta et al, 1989); this utilizes the deacylated Gba as the capture molecule for the toxin. Shiga-like toxin is produced by several other bacteria, including Vibrio cholerae 01, V. cholerae non-01, and V. paruhaemolyticus. It might constitute an important virulence factor in these organisms. EPEC

These organisms constitute an important cause of paediatric diarrhoea and in Santiago, Chile, have been shown to be the most common aetiological agent (Levine et al, 1988). They can be detected by the EPEC adherence factor (EAF) probe and show a localized adherence pattern to human laryngeal carcinoma (HEp-2) cells; in contrast, EAF-negative strains show diffuse adherence patterns to HEp-2 cells, and have been shown to be equally common in a group of controls compared with infants with diarrhoea (Levine et al, 1988). Salmonella spp.

This group comprises many different organisms, ranging from those responsible for the zoonotic salmonelloses to enteric fever (S. typhi and S. paratyphi A and B). They are typically urease-negative and produce a K-A, G reaction (alkaline (K) slant, acid (A) butt and gas (G), usually with H$S) on KIA (Thorne, 1988). When presumptive tests suggest typical Salmonella spp., the isolate should be tested with Salmonella O-group A-E antisera. A DNA probe allowing rapid identification of Salmonella spp. has been described (Scholl et al, 1990), but is not yet commercially available. Campylobacter jejuni

This organism can cause gastritis, enteritis

and enterocolitis.

It remains

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G. C. COOK

viable in a faecal sample for several hours at 25°C and for 2 weeks or more at 4°C in either a faecal sample, urine, water or milk (Thorne, 1988); a filtration technique has proved effective. Several commercial systems are available for growing C. jejuni. Identification and differentiation is based on a limited number of morphological and biochemical rections (Thorne, 1988). In future, nucleic acid probes are likely to assist in early detection of this group of enteric pathogens. A commercially available probe system (Syngene, San Diego, California) linked to alkaline phosphatase is available and seems to give accurate identification of CumpyZobacter spp. (Olive et al, 1990; Thorne et al, 1990). Yersinia enterocolitica

Food- and water-borne outbreaks occur as well as sporadic cases. Pathogenicity varies with the species and biotype. Acute gastroenteritis or an appendicitis-like syndrome are usual; less commonly, extraintestinal infection occurs. Virulence is associated with the presence of plasmids (4082 MDa), and its expression is temperature-dependent. Yersinia spp. grow more rapidly at 22-29°C than at 37°C. Details of suitable culture and staining techniques have been summarized (Thorne, 1988). DNA colony hybridization is one of the more reliable methods for diagnosis (Robins-Browne et al, 1989); the most useful is based on probes prepared from the virulence plasmid (calcium-dependent region) which identifies all virulent Yersinia spp. A PCR assay for the pYV virulence plasmid has given promising results (Wren and Tabaqchali, 1990). Vibrio cholerae and other vibrios Vibrio choZerae 01 is a water-borne enterotoxigenic bacterium, which after a l-4 day incubation period causes severe, large-volume watery stools. Non-cholera vibrios (NCVs) are biochemically identical to V. choZerue but they are not agglutinable in Vibrio 0 group 1 antiserum; some can, however, cause a similar illness. When this is suspected, a faecal sample should be plated on thiosulphate-citrate-bile salts-sucrose (TCBS) medium, which is selective and differential; however, several commercial sources with variable efficacy of this medium exist. Typical V. cholerae colonies should be subcultured on nutrient agar before a slide agglutination test with O-group 1 serum is performed. An inoculum can be transferred to KIA; following overnight incubation, a K-A reaction without gas or H2S production is indicative of V. cholerae. A rapid coagglutination test, using 4-h faecal enrichment culture in bile-peptone broth control, gives very high sensitivity and specificity (Rahman et al, 1987). A trivalent probe developed for recognizing ETEC (Abe et al, 1990) (see above) is also of value in the detection of cholera toxin. V. parahaemolyticus forms part of the normal flora of estuarine waters during the warmer months of the year; it is therefore common in seafood. It causes abdominal cramps, nausea, vomiting and diarrhoea after 4-96h; a dysenteric form also exists. Suspect colonies from TCBS should be

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inoculated into KIA; a K-A reaction without gas or H2S production confirms its identity. Other Vibrio species which can account for human disease include: V. jluvialis, V. hollisae, V. mimicus and V. furnissi. Clostridium difficile

A wide range of clinical disease has been attributed to this organism, ranging from a mild antibiotic-associated (occasionally not associated) diarrhoea to pseudomembranous colitis. These entities have been described in association with gastrointestinal surgery, staphylococcal infection, intestinal obstruction, vascular insufficiency and uraemia; they can occur over a wide age-range from infancy and childhood to adult years (Bartlett et al, 1977; Viscidi and Bartlett, 1981; Jarvis and Feldman, 1984). C. dijj‘kile is rarely present in the colonic flora of healthy adults, but when it is, certain antibiotics-notably clindamycin and lincomycin-set the scenario for overgrowth; this is followed by toxin production and pseudomembranous colitis. Toxin A is an established enterotoxin; while possessing only minimal enterotoxic activity, toxin B is the primary cytotoxin (1000 times more potent than A); in addition there is a ‘motility factor’. There is no ideal technique for the isolation and identification of C. difjicile and/or detection of its toxins. Available methods include isolation of C. difficile and detection of A and B toxins; the cytotoxicity assay developed by Chang and coworkers (Chang et al, 1979a,b) is most widely used. This depends on detection of a cytopathic effect on one of a variety of cell lines; specific neutralization assays using C. sordellii or specific C. difjLicile antitoxins usually yield a positive result within 6-8 h (Thorne, 1988). A rapid method for detecting C. difficile toxin has been developed by Chang and Gorbach (1982), but a simple and efficient test to replace culture of a faecal sample followed by tissue culture cytotoxicity assay would obviously be an advantage. Various other assays (including countercurrent immunoelectrophoresis (CIE), ELISA, and latex agglutination (LA)) have been used to detect toxins A and B. An LA method, although not completely specific, seems suitable for rapid screening, but is best used in conjunction with a cytotoxicity assay or bacteriological culture. A rapid LA test for C. diflcile has now been shown to react with a non-toxic 43-kD protein distinct from toxins A and B; a positive test result should be confirmed by faecal cytotoxin assay (Bennett et al, 1989). Isolation of C. difficile from a faecal sample became possible with the development of a selective medium which consisted of an egg yolk-fructose base with cycloserine + cefoxitin (Iwen et al, 1989); this medium is significantly more effective than either cycloserine + mannitol agar, or cycloserine + mannitol + blood agar. Isolation has yielded a higher diagnostic rate for C. dijfkile from faecal samples of patients with antibiotic-associated colitis than has toxin detection; however, procedures for complete identification of the organism remain time-consuming and require gas-liquid chromatography (GLC). Therefore, until the relative contributions of toxins A and B and the motility factor, together with the physiological control of toxin production,

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G. C. COOK

are more completely understood, the diagnosis of C. difficile infection must rest on detection of one or both toxins, coupled with culture of the organism and/or demonstration of pseudomembranes by colonoscopy. Aeromonas spp. and Plesiomonas shigelloides

The role(s) of these two organisms in the causation of gastrointestinal disease remains controversial; there is no consistent pattern of biochemical reaction(s) or virulence properties which can at present predict the pathogenicity of a particular strain (Thorne, 1988). A range of symptoms has been attributed to Aeromonas spp., ranging from mild self-limiting diarrhoea, which lasts a few days, to acute dysentery or chronic watery diarrhoea persisting for several weeks or months (Gracey et al, 1982; Burke et al, 1983; Freij, 1984; Burke and Gracey, 1986; San Joaquin and Pickett, 1988). Aeromonas spp. occur widely in soil and natural surface waters and many foodstuffs, e.g. pork, fish, shellfish, poultry and unpasteurized milk. There might be a range of strains with different virulence patterns, as occurs in E. coli (Burke and Gracey, 1986). One screening test consists of flooding an area of a blood agar plate with several drops of oxidase reagent; colonies become dark purple, but enterobacteria remain colourless. The identity of Aeromonas spp. can be confirmed by the results of various reactions: lysine decarboxylase negative (or only weakly positive), ornithine decarboxylase negative, arginine dihydrolase positive, negative growth in 6.5% NaCl, and fermentation positive in oxidative fermentation medium. Gene probe technology might assist in unravelling the role and pathogenic characteristics of this group of organisms. Plesiomonas shigelloides grows in most enteric isolation media, but not on thiosulphate-citrate-bile salts-sucrose (TCBS) or bismuth sulphite agar. On MacConkey agar it is a late or non-lactose fermenter. It is also anaerobic, H2S negative and oxidase positive, and produces a K-A or A-A reaction in KIA. Organisms responsible for bacterial ‘food poisoning’ Various strains of Staphylococcus aureus produce six serologically distinct extracellular proteins-A, B, C, C2, D and E. One to six hours after ingestion of preformed toxin (which is highly heat-stable), nausea, vomiting, abdominal pain and diarrhoea ensue; fever is very unusual and the disease is usually self-limiting within 24 h. Contaminated foods are usually prepared ones, e.g. custard, cream dessert, ice-cream or cooked meat(s); a food handler who is a staphylococcal carrier constitutes an important source. Diagnosis is by detection of enterotoxin in contaminated food, or its production by S. aureus cultured therefrom; phage-typing is important epidemiologically. Growth of S. aureus (on blood agar) in almost pure culture from a faecal sample or specimen of vomit is adequate evidence for diagnosis. ELISA and reverse passive latex agglutination (RPLA) (Oxoid, Basingstoke, UK) techniques are available for toxin detection (Thorne, 1988).

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435

Some strains of Clostridium perfringens type A produce heat-resistant spores which cause ‘food poisoning’. These are present in raw meat (e.g. meat, poultry and gravy), animal faeces and faeces of healthy humans. Diarrhoea and abdominal cramps commence 10-24 h after ingestation and can last for 12-24h. (Enteritis necroticans (pig-be1 disease), which is potentially fatal, and is characterized by severe abdominal pain, vomiting, prostration and shock, is caused by C. perfringens type C (Cook, 1991). It was formerly common in Papua New Guinean infants and children and is now amenable to control with a vaccine (Lawrence et al, 1990). Confirmation of diagnosis is dependent on a qualitative assay (1 x lo5 colonyforming units/g) of the food suspected to be contaminated. Also, quantitative faecal cultures (lo6 spores/g) of ill and healthy individuals-with serological confirmation-are necessary for differentiation from normal bowel flora and other infecting organisms. A rapid enterotoxin test on a faecal sample involves an RPLA (Oxoid, Basingstoke, UK) (see above). BaciEZus ceYeUScauses two syndromes: (a) diarrhoea and abdominal cramps, some 8-16h after eating contaminated food (meats, vegetable dishes, milk, sauce, pasta and desserts have been implicated); and (b) vomiting after an incubation period of less than l-5 h (usually following a rice dish). The first entity is considered to be caused by two proteinaceous moieties (MW 50000) which have loop fluid inducing-skin test permeability-necrotic activity. The aetiology of the second syndrome is unclear. Diagnosis is by isolation of 10’ or more colony-forming units/g in contaminated food, faeces or vomit (Thorne, 1988). B. cereus is, however, widely distributed in nature and can be isolated at low concentration from numerous sources. Confirmatory tests for B. cereus include: positivity on glucose, and negativity on mannitol, xylose and arabinose ammonium salt sugar slants. However, there is currently no satisfactory test for B. cereus which can be used in a routine laboratory. Staphylococcal enterocolitis When the normal

colonic flora is suppressed by antimicrobial agents, the predominant aerobic faecal organism; diarrhoea is the usual sequel. Underlying antibiotic usage of, for example, chloramphenicol, tetracycline and neomycin is usual; penicillin, streptomycin and sulphonamides have also been held responsible. However, only a small minority of individuals receiving one of these agents develops the syndrome and the underlying pathogenesis remains unclear. In a severe episode, mucosal lesions progress to mucosal necrosis and formation of pseudomembranes. S. aureus can be isolated-at a high concentration-from a faecal sample. Individuals who experience diarrhoea whilst receiving broad-spectrum antibiotics sometimes produce a pure or predominant growth of other organisms in a faecal sample, e.g. Streptococcus faecalis, Pseudomonas aeruginosa, Proteus mirabilis or Candida albicans. Overgrowth of a single ‘resistant’ organism with suppression of the normal faecal flora therefore seems important in the production of enterocolitis. Staphylococcus

aureas may become

436 VIRAL

G. C. COOK

PATHOGENS

Prompt identification of viruses is necessary in the management of an individual patient, and it is also necessary in the containment of an outbreak (Yolken et al, 1992); on a larger scale, accurate diagnosis is essential in monitoring the course of an epidemic in order to prevent transmission within a susceptible population. In infants and young children (especially in developing countries), rotavirus is a common and important cause of childhood enterocolitis; it has also been implicated in older children and adults (Echeverria et al, 1983). The organism was first detected by direct electron microscopy in 1974 (Bishop et al, 1974). The virus is difficult to grow in a simple culture system and a variety of diagnostic methods has therefore been introduced. Immunoelectronmicroscopy depends on the interaction between virus and antibody; other direct detection systems involve immunological assays in order to detect virus-associated antigen (Thorne, 1988). Dennehy et al (1990) concluded that, although very specific, ‘EM . . . is relatively insensitive compared with a highly sensitive monoclonal antibody-based specific enzyme immunoassay (EIA)‘; EM had a sensitivity of only 80%) although it is highly specific. Several rapid tests are now available in commercial kit form, notably EIA, which utilize polyclonal or monoclonal antibodies. Nine such products designed to detect rotavirus have been compared in the investigation of 100 faecal samples (Dennehy et al, 1988); four EIA products gave satisfactory sensitivity and specificity (95-lOO%), whilst LA was less sensitive and specific, though more rapid. Brooks et al (1989) tested a commercially available rapid qualitative test (see below) for the detection of rotavirus; this was significantly more sensitive (95%) than a standard immunoassay (84%); however, specificity was .only 90% compared with 98% for the latter technique. In India, Chakravarti et al (1991) compared ELISA, LA (using a commercially available kit (‘Ranbaxy Diagnostic’)), and a polyacrylamide gel electrophoresis (PAGE) technique; 28 out of 145 faecal samples produced a positive result using all three systems: sensitivity of LA and PAGE was 32/35 (91.4%) and 28/35 (80%) respectively, and specificity 108/110 (98.2%) and llO/llO (100%). The authors concluded that LA was ‘the least complex, required the least amount of apparatus and provided a result within a short time. It showed a high specificity and reasonable amount of sensitivity and the results corresponded well with ELISA and PAGE’. In Egypt, also, LA (which is simple and inexpensive) was shown to be valuable for the diagnosis of rotavirus diarrhoea (Amer et al, 1990); 68 out of 200 faecal samples gave a positive result compared with 79 using an ELISA. Not all commercially available kits give satisfactory results (Lipson et al, 1990); when tested against three reference assays, the Abbott ‘TestPack’ enzyme immunoassay kit (Brooks et al, 1989) displayed (in their hands) a performance specificity of 83%-which the authors considered precluded ‘incorporation of this antigen detection kit into the routine regimen of diagnostic virologic testing’. An alkaline-phosphatase-conjugated synthetic oligodeoxyribonucleotide probe has been compared to PAGE and ELISA

DIAGNOSTIC

437

PROCEDURES

techniques for the detection of rotavirus in faecal samples from Kuwaiti children suffering from gastroenteritis (Olive and Sethi, 1989); these authors concluded that ‘the probe assay coupled with chromatographic purification of rotavirus RNA is an effective method for detecting rotavirus and compares favourably with PAGE analysis and ELISA’. Working at Riyadh, Saudi Arabia, Rahman (1990) concluded that where appropriate laboratory support was absent (current methods for diagnosis are both costly and unavailable), assessment of faecal neutral fat (although providing only modest sensitivity) was of value in the diagnosis of rotavirus diarrhoea in children under 5 years of age. PROTOZOAN

PARASITIC

INFECTIONS

Entamoeba histolytica

Diagnosis of E. histolytica colitis remains dependent upon detection of active motile trophozoites in a fresh faecal sample (Healy, 1988a) (see below). With the recognition that this protozoan consists of invasive and non-invasive zymodemes, it seems, overall, unlikely that an ideal rapid immunological technique will be available in the near future. Bhattacharya et al (1988) have documented DNA probes for the specific detection of E. histolytica. A diagnostic probe (clones contained 14.5 base-pair sequences tandemly repeated in the genome) which binds to highly repeated and species-specific DNA sequences in E. histolytica has also been reported by Samuelson et al (1989); this rapid technique was able to detect as few as eight cultured amoebae per sample in a dot-blot hybridization format, and when applied to clinical samples identified 2.5 out of 2.5 microscopically positive faecal samples correctly. Overall, the probe gave a specificity of 93% in 98 specimens, but was not able to distinguish between pathogenic and nonpathogenic zymodemes. A claim that these strains can be separated has been made by Garfinkel et al (1989); however, the molecular basis for pathogenicity and its relationship to the different zymodemes remains unclear and this work clearly requires confirmation. (A great deal of work has centred on the differentiation of pathogenic and non-pathogenic zymodemes of E. histoZytica (Mirelman, 1988; Sargeaunt, 1988). Although exceedingly interesting, this work remains at a research level and the possibility of it being widely available in the routine laboratory seems at present remote.) Further development of ELISA and DNA hybridization technology will provide a valuable addition to faecal microscopy. Numerous serological techniques are available for detection of E. histoZytica antibody in peripheral blood (Healy, 1988b); however, such techniques are of value only when active tissue invasion (usually colonic or hepatic) has occurred. Results remain negative both in an individual with non-invasive disease, and in the cyst-carrier state. The fluorescent antibody test (FAT) remains most widely used; however, seroconversion takes up to 10 days and positivity remains long after satisfactory chemotherapy, Countercurrent immunoelectrophoresis (CIE) and cellulose acetate membrane precipitation (CAP) techniques usually give positive results early in

438

G. C. COOK

invasive disease; furthermore, they become negative more rapidly after successful treatment. A dot-ELISA technique has been used at Chandigarh, India, for the serological diagnosis of intestinal and extraintestinal E. histolytica infection (Mahajan et al, 1989); 51 (94%) out of 54 patients with an amoebic liver ‘abscess’ and seven (87%) out of eight with colonic disease had a positive result; one control also had a positive result. This test, which is easy to perform, was considered by the authors ‘to have great potential for the specific diagnosis of amoebiasis in peripheral hospitals and also at the field level where sophisticated immunodiagnostic facilities are not available’. Comparable results were obtained in New Delhi, India (Baveja et al, 1991); in 34 patients with a liver ‘abscess’ the dot immunobinding assay was 100% specific and 92% sensitive in detecting anti-amoebic antibodies and this compared with an 89% sensitivity using an ELISA; this technique was, however, less sensitive in detecting amoebic antigen. Two recent reports document positive serological results in the asymptomatic cyst-carrier state. Thus Takeuchi et al (1988) demonstrated antiamoebic antibodies in more than 80% of serum samples obtained from such patients, and Gonc.alves et al (1990), working in Brazil, have recorded a 15% positivity rate. Giardia lamblia

Diagnosis of a G. lamblia infection is usually made by detection of cysts in a faecal sample (see below). Alternatively, trophozoites are demonstrated in duodenal or jejunal fluid or in a jejunal biopsy specimen. The ‘Enterotest’ technique, in which a nylon thread (containing a gelatin capsule at its distal end) is swallowed and positioned in the duodenum, is frequently of value; following removal, the distal end of the thread is smeared on a microscope slide and stained for G. lamb& trophozoites (Korman et al, 1990). Excretion of cysts is cyclical; therefore, serial faecal samples must be examined in any attempt to exclude this parasitosis. Rosoff et al (1989) have evaluated a proprietary diagnostic test (Pro Spect TIGiardia) which detects Giardia-specific antigen in an aqueous extract of faeces with monospecific polyclonal sera; 93 samples from symptomatic G. Eamblia-infected individuals proved positive and all had microscopic evidence of infection; 16 (94%) out of 17 microscopically positive samples and six which were microscopically negative also gave positive results. This technique, although sensitive, probably lacks specificity. A monoclonal-antibody-based antigen-capture enzyme immunoassay for detecting G. lamblia antigen gave a positive result in 30 (97%) out of 31 microscopically positive formalinized faecal samples, and 42 (82%) out of 60 unfixed specimens (Stibbs, 1989); this assay can detect five cysts per well, but is not able to detect antigen in trophozoites cultured in vitro. In an ideal immunological technique, antigen detection in both cyst and trophozoite is essential. Neither an ELISA (Isaac-Renton, 1991) nor a DNA probe (Butcher and Farthing, 1988) technique is in routine diagnostic use in the diagnosis of G. lamblia infection.

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PROCEDURES

Antibody is sometimes present in peripheral blood; however, most attempts at serological diagnosis have posed practical problems. It seems likely that a heavy infection with resultant small intestinal damage is required for a positive result. Coccidia

This group of organisms is assuming increasing importance because of the acquired immunodeficiency syndrome (AIDS), although they are also important aetiologically in travellers’ diarrhoea; Cryptosporidium spp., Isospora belli, Sarcocystis hominis and Microsporidium spp. are all involved. Detection is usually by light microscopy of a faecal sample; owing to small size, Microsporidium spp. is more easily detected by electron microscopy. These organisms stain poorly with routine staining techniques, and an acid-fast stain, e.g. Ziehl-Neelsen or Kinyoun, is required. An indirect, double antibody ELISA has been developed, using specifically produced goat and rabbit antisera, to detect Cryptosporidium antigen in a human faecal sample (Ungar, 1990). Fifty-one out of 62 microscopically proven specimens gave a positive result, and all negative ones were in samples with less than five oocysts per 0.01 ml concentrated faecal sample, examined after acid-fast or fluorescent antibody staining; 176 out of 182 specimens obtained from Cryptosporidium-negative individuals were also ELISA-negative. This assay was claimed to be 82.3% sensitive and 96.7% specific. An ELISA utilizing a monoclonal antibody is of comparable sensitivity compared with faecal microscopy, but has resulted in some falsepositive results (Chapman et al, 1990). A proprietary monoclonal antibody fluorescein-conjugated technique (Meridian Laboratories, Cincinnati, Ohio, USA) has been claimed to be 100% sensitive and specific (Garcia et al, 1989). Whether such sophisticated methods are, however, cost-effective in a low-risk population has been debated (Baron et al, 1989). A significant problem in diagnosis has recently been highlighted. An unusual type of Cryptosporidium oocysts has been recorded (Baxby and Blundell, 1988); these are fragile structures with a distinct outer coat and spherical sporozoites; they do not stain with safranine or acid-fast stains, and are non-reactive when a fluorescein-conjugated monoclonal antibody is used. Further observations are required. Blue-green algae have recently been recorded in faecal samples obtained from travellers (Shlim et al, 1991; Pollok et al, 1992) and immunocompromised individuals (Long et al, 1990); there is currently a great deal of controversy regarding their taxonomic position (Pollok et al, 1992). However, they may mimic oocysts of coccidia, from which they must be differentiated; they do not, however, stain with techniques used for those protozoa. PRACTICAL PROTOZOA

ASPECTS OF FAECAL AND HELMINTHS

EXAMINATION

FOR

Examination (both macro- and microscopically) of a faecal sample is of value in the detection of cysts and eggs and, in a minority of cases, adult

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G. C. COOK

parasites themselves. However, release of helminth eggs and protozoan cysts is erratic; therefore, three (and ideally six) samples should be examined on separate (preferably alternate) days. In some instances (e.g. when searching for Entumoeba histolytica trophozoites-see below), the sample should be examined as rapidly as possible (preferably
In clinical practice, the most important protozoan parasite of the gastrointestinal tract (the colorectum) is Entumoeba histolytica (see above). Figure 1 summarizes methods used for its identification (Cook, 1992). Table 3 summarizes some classical differences between E. histolytica and Entamoeba coli (and other non-pathogenic amoebae); trophozoites of E. histolytica are invasive (containing ingested host erythrocytes) only if they belong to an appropriate zymodeme. In amoebic ‘dysentery’, direct microscopy of afresh faecal sample (at 37°C) reveals motile trophozoites and cellular exudate. Presence of E. histolytica cysts in the absence of trophozoites is defined as the ‘cyst-carrier state’. A cellular exudate may coexist with both this and Balantidium coli (and also Shigella and Campylobacter) infection; presence of polymorphonuclear neutrophils, macrophages, erythrocytes and epithelial cells should be noted. Pus cells and macrophages are present in greater numbers in Shigella spp. compared with E. histolytica dysentery (see

441

DIAGNOSTIC PROCEDURES SPECIMEN

formed

stool

F-E cone*

unformed stool + blood/mucus

rectal

scrape

F-E cone + warm saline

warm

saline

stain with iodine, Burroughs or fluorescent technique

stain

CYSTS

*F-E

cone

= formol

with

trichrome

TROPHOZOITES

ether

concentration

Figure 1. Laboratory investigations used in the detection of Enfumoebn sample (Cook, 1992).

hisfolyfica

in a faecal

above). The pH of a faecal sample is usually more acid in the presence of an E. histolytica compared with a Shigella infection. Table 4 summarizes other

protozoan parasites which may be detected in a faecal sample, together with their site of origin in the gastrointestinal tract. Giardia Zumblia (a small intestinal protozoan parasite) (see above) can cause small intestinal malabsorption; therefore the faecal sample may contain a high concentration of fat. While the cyst form is often detectable (albeit intermittently) in a faecal sample, jejunal fluid or biopsy reveals flagellated trophozoites. Cryptosporidium spp. and Isospora belli (and possibly Sarcocystis hominis) can also be associated with malabsorption; these organisms are ‘opportunistic’ in AIDS. Oocysts are easily recognizable in a faecal sample, jejunal fluid or biopsy. Cryptosporidium spp. is an intracellular, extracytoplasmic organism; Isospora belli can be visualized within the enterocyte. There are several useful staining techniques for protozoa (Cheesbrough, 1987; Fleck and Moody, 1988). Temporary stains include: Lugol’s iodine, Burrough’s stain, acridine orange and eosin-saline. Permanent stains include: Giemsa, a modified rapid Field’s stain and ‘trichrome’ (a modified Gomori technique). When Cryptosporidium spp. and Isospora belli oocysts are being sought, it is essential to use an acid-fast technique, e.g. a modified Ziehl-Neelsen or the phenol-auramine method; routine stains do not allow easy identification.

442

G. C. COOK

Table 3. Some differentiating features between Entamoeba histolytica, Entamoeba coli and other non-pathogenic amoebae (Cheesbrough, 1987; Fleck and Moody, 1988).

Invasive Entamoeba histolytica Trophozoite Occurrence

Present at high concentration in dysenteric stool l&60 Pm

Size Mobility

cyst Size Nucleus (fewer in young) Cytoplasm (chromatoidal bars diminish as cyst matures) cyst wall

Other

Never at high concentration

Variable

20-40 pm (often larger Usually smaller, but than Entamoeba Balantidium coli histolytica) 50-200 Pm Sluggish; small Variable pseudopodia Never contains Variable erythrocytes

Active; large pseudopodia Ingested erythrocytes

Cytoplasm

E&amoeba coli

9-14.5 km (usually nearer 10 Pm)

14-30 I*rn (usually nearer 20 km)

Usually smaller; but Balantidium coli 5-60 Pm 1-4

4 when mature

8 when mature

Chromatoid bars present in fresh specimen. Diffuse glycogen Thin

Chromatoid bars rarely seen

Iodamoeba sp. has a compact glycogen vacuole

Thicker than Entamoeba histolytica

Variable

Table 4. Faecal protozoa (Cook, 1980). Usual anatomical site Amoebae Small intestine -

Flagellate

Ciliate

Giardia

-

lamblia*

Coccidia Cryptosporidium

sPP.*

Isospora belli* sarcocystis hominis* Blastocystis hominis*f

Colorectum

Entamoeba histolytica* E. coli E. hartmanni Iodamoebn biitschlei Dientamoeba fragilis

Chilomastix mesnili Trichomonas hominis Enteromonas spp. Retortamonas spp.

Balantidium

coli”

* Pathogenic organism. ? Classification remains undetermined.

Rectal scrapes often give a higher yield of positive results when an E. histolytica infection is suspected. Exudate should be obtained proctoscopically and immediately examined microscopically; active trophozoites, which are not necessarily visualized in a fresh faecal sample, may be demonstrable.

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PROCEDURES

443

Helminths Nematodes (roundworms)

These are cylindrical, pointed at both ends, and unsegmented-whatever the species. The male is usually smaller than the female. They contain a tough, smooth, outer cuticle and a body cavity containing a well-developed intestinal tract; the mouth may contain rudimentary teeth or cutting plates for attachment to the mucosal surface. The largest is Ascaris lumbricoides (male 15 cm; female 25 cm); it may be detected in either a faecal sample or vomit. (Differentiation from the earthworm-which is browner in colouris usually straightforward; this common environmental nematode is not infrequently delivered to the laboratory by an anxious patient after it has been noticed lying in a lavatory!) Both A. lumbricoides and hookworm are extremely common in most tropical countries, infecting a very high proportion of the indigenous population. Hookworms (Ancylostoma duodenale and Necator americanus) are much smaller than A. Zumbricoides (female approximately Smm); they contain two hook-like teeth at the top and two triangular cutting plates at the bottom. They are very important causes of iron-deficient, microcytic anaemia, which is very common in developing (tropical) countries. Enterobius vermicularis (the threadworm), which has a worldwide distribution, is extremely prevalent; it is arguably the most common intestinal helminth to afflict Homo sapiens. The female is S-12 and the male 2-4 mm in length; the latter dies rapidly after fertilization. The major clinical manifestation is pruritus ani. Adult worms and eggs can both be recovered from perianal skin; several methods are available. A saline-moistened swab can be gently rubbed around the perianal area, and afterwards agitated in a small tube of normal saline to dislodge the eggs; after centrifugation, the supernatant is decanted and the deposit examined microscopically. Alternatively, a ‘cellotape’ strip can be placed across the anus in the early morning, before defaecation; after removal this is placed (sticky side down) onto a microscope slide. Trichuris trichiura (the whipworm) is a caecal nematode which only causes symptoms in children when present at high concentration. Eggs are readily detectable in faecal samples. Strongyloides stercoralis is the most difficult enterocolitic nematode to detect; ova are not usually demonstrable in a faecal sample (the eggs hatch and produce larvae before reaching the anus). The larvae (200-250 pm in length) may, however, be demonstrable; a culture technique allows identification of these even when they are too scanty for detection by a concentration technique. (An ELISA is available and is of limited value in serological diagnosis; significant cross-reaction takes place with Filaria spp.) Cestodes (tapeworms)

Although a number of different species of flatworms can parasitize man, the two most common (and important) are Tuenia solium and T. suginatu. The importance of T. solium (the pork tapeworm) is that its larva is the causative

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G. C. COOK

agent in neurocysticercosis; human infection is acquired by ingestion of T. solium eggs, and not by ingesting ‘measly’ pork. Adult worms can reach a length of 4-8m; white segments are easily recognizable. After successful chemotherapy the entire worm (complete with head, neck and strobila) will be passed in the stool. Identification of the head (T. solium 1 mm and T. saginata l-2mm) is important; this is made difficult, however, after praziquantel or niclosamide chemotherapy, both of which dissolve this structure. For identification, a purged stool can be poured through a fine-mesh sieve and examined for the scolex (often detached from the rest of the body), about 0.5 cm of the neck remaining; when detected it can be squashed in a drop of saline under a coverslip and identified microscopically-it possesses four suckers and a rostellum (a double row of alternating hooks). Various techniques are used for fixation and staining of adult helminths (appropriate manuals of practical parasitology should be consulted); they are particularly useful for establishing a permanent record. The fish tapeworm Diphyllobothrium latum (present in Finland and Sweden and parts of the USA) may reach a length of 3 to 10m. A rare clinical manifestation is macrocytic (B12-deficient) anaemia. Characteristic eggs can be visualized in faecal samples.

Methods used for egg concentration (nematodes, cestodes and trematodes) are identical to those used for cysts (see above). Those of Schistosoma mansoni, S. intercalatum, S. matthei, S. japonicum and S. mekongi can usually be detected in a faecal sample or rectal biopsy specimen; S. haematobium (which causes urinary tract disease) is occasionally detected in the latter. When detected, identification of these should be straightforward; all microbiological laboratories should possess a reference collection. Potential difficulties in identification

of cysts, eggs and larvae

The greatest diagnostic problem lies in pronouncing a faecal sample ‘negative’! Once evidence of a parasitosis has been established, identification is relatively straightforward. However, misidentification of some nonpathogenic objects as parasites is a major practical problem; some of the difficulties are as follows (Fleck and Moody, 1988): 1. Protozoan cysts can be confused with air bubbles, fat globules or yeasts; if iodine is added to the wet preparation the internal structure of the cyst(s) can be visualized. 2. Trophozoites of pathogenic strains of E. histolytica are not always differentiated from those of non-pathogenic amoebic trophozoites and macrophages (see above). 3. Eggs of some helminths, especially those of Trichuris trichiura and Taenia spp., can be confused with pollen grains. 4. A. lumbricoides and Fasciola hepatica (the liver fluke) eggs can be mistaken for vegetable cells.

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5.

S. stercoralis and hookworm larvae can be confused with hairs or vegetable fibres; the latter are usually tapered at one end, whereas the former are blunt and have no internal structure. 6. Eggs of insects and mites can occasionally be found in a faecal sampleexamples of a ‘spurious’ infection(s). 7. Some other potential pitfalls in diagnosis are as follows. CharcotLeyden crystais [breakdown products of eosinophils) are sometimes found when an immune response has been initiated to a foreign substance. Undigested starch granules contain concentric rings, and stain blue with iodine, and red when partly digested. A cellular exudate and/or erythrocytes may be present, e.g. in an invasive E. histolytica infection. Macrophages (which possess a large nucleus compared with E. histolytica trophozoites) may be present in colonic amoebiasis. Polymorphonuclear leukocytes are often present in large numbers in bacillary dysentery (see above); these are scanty in E. histolytica dysentery. Epithelial cells can sometimes be visuahzed in faecal samples and frequently in material obtained at sigmoidoscopy. REFERENCES Abe A, Komase K, Bangtrakulnonth A et al (1990) Trivalent heat-labile and heat-stableenterotoxin probe conjugated with horseradish peroxidase for detection of enterotoxigenic Escherichia coli by hybridization. Journal of Clinical Microbiology 28: 26X2620. Adkins HJ & Santiago LT (1987) Increased recovery of enteric pathogens by use of both stool and rectal swab snecimens. Journal of Clinical Microbioloav 25: 158-159. Albert MJ & Leach A (1989) Lack of correlation between Congo red binding and enteroinvasiveness in Escherichia coli. Journal of infectious Diseases 160: 169-170. Amer AA, El-Mougi M, Hughes J et al (1990) Comparison of latex agglutination test with an ELISA to diagnose rotavirus-associated diarrhoea in infants and young children. Journal of Diarrhoeal

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