Comparison of preservation media and freezing conditions for storage of specimens of faeces

To evaluate the long-term recoverability of bacterial enteropathogens, two freezing conditions (deep-freezing at -70 degrees C and liquid nitrogen) and three preservation media (Cary-Blair, Amies, and buffered glycerol-saline) were tested. These were compared with storage in containers with no preservation medium and refrigeration at 4 degrees C. At 4 degrees C, viability of organisms could not be consistently maintained beyond one month; Cary-Blair medium generally gave the best results and storage without preservation medium was the least efficient. Storage in liquid nitrogen and deep-freezing effectively preserved all organisms except Campylobacter jejuni for the entire period of study (12 months). There was no difference between the various preservation media, or between storage with or without medium. Storage in preservation medium was superior to storage without such supplement for C. jejuni. We conclude that most enteropathogens survive in faecal specimens for as long as 12 months when stored at very low temperatures (-70 degrees C) whether or not preservation media are used.     

Performance of six culture media for isolation ofSalmonella species from stool samples

The relative performance of five plating media [Rambach agar; salmonella-shigella (SS) agar, novobiocin-brilliant green-glycerol-lactose (NBGL), modified semisolid Rappaport-Vassiliadis medium (MSRV), andSalmonella Detection and Identification-2 (SM2)] and selenite broth (SB) subcultured in SS agar in the recovery ofSalmonella spp. from 500 human stool specimens was evaluated. On Rambach agar and SS agar, the C₈-esterase test was also used for selection of suspicious colonies. Eighty-one samples were positive for salmonellae on at least one of the six media. Sensitivities and specificities of MSRV, SB, NBGL, SS, Rambach agar, and SM2 were 95.1 and 98.1%, 87.6 and 99.8%, 79 and 91.9%, 69.1 and 99.3%, 56.8 and 96.9%, and 54.3 and 92.4%, respectively. There were statistically significant differences between MSRV and NBGL, Rambach agar, SS agar, and SM2 (p < 0.005), between SB and SS agar, Rambach agar, and SM2 (p < 0.05), and between NBGL and SM2 and Rambach agar (p < 0.005). The greatest number of isolates was recovered with MSRV, whose performance surpassed that of enrichment in selenite broth, probably because the subcultures were not repeated on MSRV. This hypothesis is now under investigation. The specificity of each of the five solid media was greater than 90%.     

Polymerase chain reaction for detection of adenoviruses in stool samples.

The usefulness of the polymerase chain reaction (PCR) method for diagnosing adenovirus infections was investigated. Several primers, including primers specific for the hexon-coding region and for enteric adenovirus types 40 and 41, were evaluated. The PCR method was validated against cell culturing in routine diagnostic work and against restriction enzyme analysis of viral DNA. Sixty diagnostic specimens were selected for evaluation by the PCR method. Twenty of the 60 specimens were found positive on the basis of cytopathic effects and latex agglutination (Adenolex [Orion Diagnostica, Helsinki, Finland]), and 16 were identified and typed as adenoviruses by polyacrylamide gel electrophoresis. PCR was performed on all 60 specimens in parallel directly on diluted stool samples and on viral DNA extracted from cells inoculated with the same stool samples. When the general hexon primers were used 51 of the 60 specimens from infected cell cultures were found positive by PCR, whereas only 13 specimens were found positive when PCR was performed directly on stool samples. With the use of selective primers for enteric adenoviruses 16 of the 60 cell cultures were found to exhibit amplification products by PCR, whereas 4 were detected in stool samples. None of the 60 specimens were found positive by PCR when an adenovirus type 40-specific primer pair was used. PCR was found to be a fast, sensitive, and reliable method for the detection of adenoviruses in diarrheal disease, provided the amplifications were performed directly on diluted stool samples.     

Evaluation of Streck tissue fixative, a nonformalin fixative for preservation of stool samples and subsequent parasitologic examination

We undertook a study to evaluate Streck tissue fixative (STF) as a substitute for formalin and polyvinyl alcohol (PVA) in fecal preservation. A comparison of formalin, PVA, (mercuric chloride based), and STF was done by aliquoting fecal samples into each fixative. Stool specimens were collected in Haiti, and parasites included Cyclospora cayetanensis, Giardia intestinalis, Entamoeba coli, Iodamoeba butschlii, Endolimax nana, Ascaris lumbricoides, Trichuris trichiura, Strongyloides stercoralis, and Necator americanus. Preserved stools were examined at various predetermined times (1 week, 1 month, and 3 months) to establish the quality of the initial preservation as well as the suitability of the fixative for long-term storage. At each time point, stool samples in fixatives were examined microscopically as follows: (i) in wet mounts (with bright-field and epifluorescence microscopy), (ii) in modified acid-fast-, trichrome-, and safranin-stained smears, and (iii) with two commercial test kits. At the time points examined, morphologic features remained comparable for samples fixed with 10% formalin and STF. For comparisons of STF- and 10% formalin-fixed samples, specific findings showed that Cyclospora oocysts retained full fluorescence, modified acid-fast- and safranin-stained smears of Cryptosporidium and Cyclospora oocysts were equal in staining quality, and results were comparable in the immunofluorescence assay and enzyme immunoassay commercial kits. Stool fixed in STF and stained with trichrome showed less-than-acceptable staining quality compared with stool fixed in PVA. STF provides an excellent substitute for formalin as a fixative in routine examination of stool samples for parasites. However, modifications to the trichrome staining procedures will be necessary to improve the staining quality for protozoal cysts fixed in STF to a level comparable to that with PVA.     

Direct detection of Vibrio cholerae in stool samples.

A direct method to detect Vibrio cholerae in stool samples was developed by using a PCR procedure that did not require a DNA purification step. Dilution (1/100) of stool samples prevented inhibition of the reaction by contaminants, and two consecutive PCRs, the second one with a nested primer, achieved the desired sensitivity. Comparison of the results obtained from stool swab samples processed by the two-step PCR and by an enzyme-linked immunosorbent assay using GM1 as the capture molecule showed that the former is more sensitive and gave positive results even when V. cholerae was not culturable or dead.     

ABC chromogenic agar: a cost-effective alternative to standard enteric media for Salmonella spp. isolation from routine stool samples

Salmonellosis is the second most common cause of bacterial gastroenteritis, yet the yield from routine stool culture is low. Commonly used selective enteric media have poor specificities for salmonella identification, resulting in a high laboratory workload. A special chromogenic medium, ABC agar, is a promising alternative but its cost-effectiveness has not been evaluated in diagnostic laboratories. A collaborative study is therefore undertaken in two district general hospitals laboratories. Routine stool samples (n=866) were subcultured onto ABC agar half plates after selective enrichment in selenite broth. Similarly, 246 and 620 stool samples were subcultured onto desoxycholate lactose sucrose (DCLS) and xylose lactose desoxycholate (XLD) whole agar plates, respectively. Salmonella spp. were isolated from only 14 (1.6%) of stool samples tested. Specificity was significantly higher for ABC (98%) than DCLS (67%) or XLD (78%) agars. Welcan workload units (ABC 4.8, XLD and DCLS both 7.3) and costs per specimen (ABC 1.26 pounds sterling, DCLS 3.81 pounds sterling and XLD 1.83 pounds sterling) were similarly lower with ABC agar. The results indicate that ABC chromogenic agar offers improvements in specificity, workload and costs over conventional enteric media for Salmonella spp. isolation.     

Filter paper for preservation, storage, and distribution of insect and pathogen DNA samples

Proper DNA storage is critical for studies involving genetic analysis of insects and for molecular diagnostics of pathogens carried by them. Molecular surveillance of pathogens carried by insects can involve screening of thousands of insect DNA samples. Problems with storage and degradation of these samples can arise. In this study, a simple filter paper-based method for storage and preservation of insect DNA was evaluated using polymerase chain reaction (PCR). DNA was isolated from individual house fly, Musca domestica L., adults by using a cell lysis technique. From 50 house flies known to carry Campylobacter spp., a portion of the DNA sample was stored frozen and another portion was pipetted onto filter paper. At monthly intervals for 7 mo, PCR was conducted using 1 microl of the frozen DNA sample and a 2.0-mm disk from the filter paper samples as the PCR template. Two markers were used, a 450-bp region of the insect mitochondrial DNA (mtDNA) ND5 gene and a 857-bp region of the Campylobacter spp. mtDNA 16S rDNA gene. PCR amplification was successful for all of the samples regardless of the storage method. The filter paper method is a simple and economical way to store, preserve, and distribute DNA samples for PCR analysis.     

Comparison of six media, including a semisolid agar, for the isolation of various Campylobacter species from stool specimens.

A recently described semisolid blood-free selective motility medium (SSM) (J. Goossens, L. Vlaes, I. Galand, C. Van den Borre, and J. P. Butzler, J. Clin. Microbiol. 27:1077-1080, 1989) was compared with two charcoal-based selective media (charcoal-based selective medium [CSM] and modified charcoal cefoperazone deoxycholate agar [CCDA]), two blood-based media (Skirrow medium [SKM] and CampyBAP), and a passive, 0.65-microns-pore-size cellulose acetate membrane filter technique for the recovery of campylobacters from stools of patients with diarrhea. A total of 1,980 specimens were tested, 161 of which were found to be positive for campylobacters. Campylobacter jejuni was isolated in 148 specimens (91.9%), C. coli was isolated in 27 (7.5%), and "C. upsaliensis" was isolated in 1 (0.6%). After 72 h of incubation with a single medium, the cumulative percentages of Campylobacter-positive specimens isolated on CSM, CCDA, SKM, and SSM were 87, 83, 80, and 72%, respectively. The filter method alone enabled us to recover 61% of all campylobacters. The "C. upsaliensis" strain was isolated by this method only. The highest isolation rates were observed when two media, including CSM, were combined. The combination of CSM and SSM yielded the highest rates (96%), but these were not statistically different from the rates observed with combinations of CSM and SKM (94%) or of CSM and the filter method (91%). Extending the incubation time from 48 to 72 h led to an increase in the isolation rate regardless of the medium used (P less than 0.001). CSM and CCDA were the most selective media. SKM and CampyBAP appeared to be the most inhibitory media for the isolation of C. coli.     

Comparison of CHROMagar Salmonella medium and xylose-lysine-desoxycholate and Salmonella-Shigella agars for isolation of Salmonella strains from stool samples

The growth and appearance of 115 stock Salmonella isolates on a new formulation of CHROMagar Salmonella (CAS) medium were compared to those on xylose-lysine-desoxycholate agar (XLD), Salmonella-Shigella agar (SS), and Hektoen enteric agar (HEA) media. CAS medium was then compared prospectively to XLD and SS for the detection and presumptive identification of Salmonella strains in 500 consecutive clinical stool samples. All stock Salmonella isolates produced typical mauve colonies on CAS medium. Nine Salmonella strains were isolated from clinical specimens. The sensitivities for the detection of salmonellae after primary plating on CAS medium and the combination of XLD and SS after enrichment were 100%. The specificity for the detection of salmonellae after primary plating on CAS medium (83%) was significantly (P < 0.0001) higher than that after primary plating on the combination of SS and XLD media (55%) (a 28% difference in rates; 95% confidence interval, 23.0 to 34%). Twenty-nine non-Salmonella organisms produced mauve colonies on CAS medium, including 17 Candida spp. (59%) and 8 Pseudomonas spp. (28%). These were easily excluded as salmonellae by colony morphology, microscopic examination of a wet preparation, or oxidase testing. One biochemically inert Escherichia coli isolate required further identification to differentiate it from Salmonella spp. The use of plating on CAS medium demonstrated high levels of sensitivity and specificity and reduced the time to final identification of Salmonella spp., resulting in substantial cost savings. It can be recommended for use for the primary isolation of Salmonella spp. from stool specimens. Other media (e.g., XLD) are required to detect Shigella spp. concurrently.     

Flow cytometric detection of Cryptosporidium oocysts in human stool samples.

Cryptosporidium parvum is an important pathogen that causes diarrhea in virtually all human populations. Improved diagnostic methods are needed to understand the risk factors, modes of transmission, and impact of cryptosporidiosis. In the present study, we fluorescently labeled and counted C. parvum oocysts by flow cytometry (FC) and developed a simple and efficient method of processing human stool samples for FC analysis. Formed stool (suspended in phosphate-buffered saline) from an asymptomatic, healthy individual was seeded with known concentrations of oocysts, and oocysts were labeled with a cell wall-specific monoclonal antibody and detected by FC. The method described herein resulted in a mean oocyst recovery rate of 45% +/- 16% (median, 42%), which consistently yielded a fourfold increase in sensitivity compared to direct fluorescent-antibody assay of seeded stool samples. However, in many instances, FC detected as few as 10(3) oocysts per ml. Thus, FC provides a reproducible and sensitive method for C. parvum oocyst detection.     

Comparison of Rambach agar, SM-ID medium, and Hektoen Enteric agar for primary isolation of non-typhi salmonellae from stool samples.

Stool samples (n = 504) were streaked simultaneously onto Rambach agar (R agar; E. Merck, Darmstadt, Germany), SM-ID medium (bioMérieux S.A., Montalieu-Vercieu, France), and Hektoen Enteric (HE) agar (BBL Becton-Dickinson, Baltimore, Md.) in order to evaluate the performances of the first two media in comparison with that of the well-established HE agar. Following overnight cultivation at 37 degrees C, 29 samples (5.8%) were positive for non-typhi salmonellae on at least one of the three media. Sensitivities and specificities were 69 and 98%, 79 and 85%, and 100 and 79% for R, SM-ID, and HE agars, respectively. On the basis of the poor sensitivities, R and SM-ID agars are not recommended as primary plating media when screening for non-typhi salmonellae. However, the high specificity of R agar may help to reduce the work load when this medium is used for plating after enrichment.     

Successful freeze storage and lyophilisation for preservation of Helicobacter pylori.

Long term storage techniques for the preservation of Helicobacter pylori were developed. The cells survived at -75 degrees C in the presence of glycerol and at +4 degrees C after freeze-drying. Both techniques are suitable for routine use.     

Detection of clarithromycin-resistant Helicobacter pylori in stool samples

The recognition of the role of Helicobacter pylori in gastric diseases has led to the widespread use of antibiotics in the eradication of this pathogen. The most advocated therapy, triple therapy, often includes clarithromycin. It is well known that clarithromycin resistance is one of the major causes of eradication failure. The development of a rapid noninvasive technique that could easily be performed on fecal samples and that could also provide information about the antibiotic resistance of this microorganism is therefore advisable. Previous findings have demonstrated that clarithromycin resistance is due to a single point mutation in the 23S rRNA. All the mutations described have been associated with specific restriction sites, namely BsaI (A2143G), MboII (A2142C/G), and HhaI (T2717C). On this basis we have developed a new method, a seminested PCR, allowing screening for clarithromycin resistance of H. pylori directly on stool samples. This method furnished a 783-bp fragment of the 23S rRNA, which was subsequently digested by MboII, BsaI, and HhaI, in order to identify single point mutations associated with clarithromycin resistance. Of a total of 283 stool samples examined, 125 were H. pylori positive and two of them were shown to contain clarithromycin-resistant strains due to the presence of a mutation at position 2717, whereas no PCR products contained mutations at position 2142 or 2143. In order to evaluate the reliability of the new system, we compared the results of restriction analysis of the PCR products with the MICs shown by the H. pylori isolates by culturing gastric biopsies from the same patients.     

Comparison of Sabouraud dextrose and Pagano-Levin agar media for detection and isolation of yeasts from oral samples.

The sensitivities of Sabouraud dextrose agar and modified Pagano-Levin agar for the primary isolation of yeasts and the recovery of multiple yeast species from single clinical samples were compared by using oral-rinse samples. Although there was a highly significant positive correlation between the numbers of yeasts recovered from both media, modified Pagano-Levin agar was far superior in detecting multiple yeast species in a single sample. Of 150 oral samples containing yeasts, 23 (15.3%) contained more than one yeast species. The most frequent combination of different yeasts was Candida albicans and Torulopsis glabrata.     

Direct detection of verotoxin-producing Escherichia coli in stool samples by PCR.

A method for the rapid detection of verotoxin-producing Escherichia coli in stool samples by PCR was evaluated. Verotoxin-1 and verotoxin-2 genes in DNA extracted directly from stool samples were amplified with oligonucleotide primers. Stools spiked with control organisms, E. coli C600 (H19B) (verotoxin-1) or E. coli C600 (933W) (verotoxin-2), demonstrated that verotoxin-1-containing organisms could be detected at 10(2) CFU per 0.1 g of stool and verotoxin-2-containing organisms could be detected at 10(7) CFU per 0.1 g of stool. Testing of stool samples from patients with diarrhea showed a high concordance between PCR positivity and the presence of verotoxin-producing E. coli, determined by isolation of serotype O157:H7 on sorbitol-MacConkey medium (34 of 35 stool samples) or by colony blots with gene probes (19 of 21 stool samples). Conversely, only 1 of 20 (5.0%) stool samples that were O157:H7 culture negative and colony blot negative and that contained free verotoxin only was positive by PCR. As well, only 4 of 145 (2.8%) stool samples that were negative for serotype O157:H7 or free verotoxin were PCR positive. PCR of DNA extracted directly from stool samples provides a rapid method for the detection of stool samples containing verotoxin-producing E. coli compared with colony blot testing.     

Evaluation of seven immunoassays for detection of rotavirus in pediatric stool samples

The performance of seven commercially manufactured rotavirus assays was evaluated with 144 pediatric stool specimens and compared with electron microscopy (EM) findings. The four enzyme-linked immunosorbent assays used were Rotazyme II, Pathfinder, IDL rotavirus immunoassay, and Enzygnost (Behring) rotavirus assay. The three latex tests were Meritec rotavirus detection test, Virogen Rotatest, and Bartels rotavirus latex test. Test outcomes were compared with EM on the basis of sensitivity, specificity, positive-negative predictive value, and the kappa statistic. Relative to EM, Meritec had the highest specificity (97%), followed by Virogen (95%), IDL (91%), Pathfinder (85%), Behring (81%), Bartels (72%), and Rotazyme (71%). The sensitivities were as follows: Rotazyme (92%), Pathfinder (89%), Bartels (86%), Virogen (86%), Behring (82%), Meritec (71%), and IDL (75%). Patient age and sex did not influence test results. Owing to the absence of a true standard, the tests were also compared with each other on the basis of the kappa statistic, the frequency of positive test results, and the frequency of samples in which a test differed from all other tests. Using these measures, the assays could be classified into three groups with progressively decreasing utility: group 1 (Virogen, Meritec, IDL, and EM), group 2 (Pathfinder and Behring), and group 3 (Rotazyme and Bartels). Laboratory criteria were also compared. Latex tests were faster and required less equipment than enzyme-linked immunosorbent assays. The Virogen latex assay showed the best overall performance, which made it our choice for rapid and accurate rotavirus diagnosis. However, in children who have gastrointestinal symptoms with negative rotavirus test results, EM will be useful until such time as immunological tests for other enteric viruses are available.     

Latex agglutination test for detection of Clostridium difficile toxin in stool samples

A total of 163 stool specimens were tested for detection of Clostridium difficile and its toxin by cytotoxicity assay with tissue culture, latex agglutination test, and isolation of the organism. From 33 specimens which were positive for toxin by cytotoxicity, 30 were positive by the latex agglutination test; the organism was isolated from 21. The total number of samples which were positive with the latex agglutination test was 44. The predictive value of a positive latex agglutination result relative to the cytotoxicity test was 68%, and the predictive value of a negative result was 97.5%. The specificity and sensitivity of the latex agglutination test relative to the cytotoxicity assay and the low cost and simple facilities required indicate that the latex agglutination test is a useful procedure for screening for C. difficile toxins, provided that positive latex results are confirmed by cytotoxicity assay.     

Detection of astrovirus in pediatric stool samples by immunoassay and RNA probe.

Two new astrovirus assays, a rapid biotin-avidin enzyme immunoassay (EIA) and RNA probe hybridization, were developed and compared with an established astrovirus assay, an indirect EIA, and immune electron microscopy. Sensitivity and specificity were evaluated by using a screening panel of 22 astrovirus-positive and 305 astrovirus-negative fecal specimens. The biotin-avidin assay was equivalent in performance to the reference indirect assay, and both could detect about 10 ng of viral protein. Although the probe was more sensitive than either EIA and could detect higher dilutions of virus in tissue culture and stool specimens, it did not detect more astrovirus-positive fecal specimens. Of the 22 astrovirus-positive specimens detected by the EIAs, 20 were confirmed by immune electron microscopy with hyperimmune rabbit antiserum. To determine the usefulness of EIAs for large epidemiologic studies, EIAs were used to screen 1,289 stool specimens from three studies of children with and without diarrhea. Astrovirus was detected in 3.5% of specimens from children with diarrhea and 1.9% of specimens from those without diarrhea. Our results indicate that the biotin-avidin EIA is an efficient, sensitive, and specific method for routinely screening large numbers of fecal samples and that its application in epidemiologic studies may yield higher rates of astrovirus infection than have been found previously by other methods.