Agar Plate Culture Method Research Articles

Diagnosis of Strongyloides Stercoralis infection: meta-analysis on evaluation of conventional parasitological methods (1980-2013)

Research on diagnostic methods have strongyloidiasis divergent validity and incomplete by not reporting data on safety, efficiency and performance diagnosis. To assess validity, performance, efficiency and safety of four diagnostic conventional parasitological methods for detection of Strongyloides stercoralis infection in the period 1980-2013. Systematic review with meta-analysis, exhaustive and reproducible literature search of six databases. Quality of the articles was assessed and meta-analysis was performed under the random effects model, calculating sensitivity, specificity, likelihood ratios, predictive values, proportion of false results, accuracy, odds ratio and Youden index J and ROC curve using Meta-DiSc(es) and Epidat 3.1. 11 studies with 9,025 individuals were included. Sensitivity of the Baermann method was 72%, positive likelihood ratio (LR+) 228 and negative likelihood ratio (LR-) 0.32. The agar plate culture (APC) had a sensitivity of 89%, LR+ 341 and LR- 0.11. Stool sensitivity was 21%, LR+ 67 and LR- 0.67. Sensitivity of the formol-ether concentration was 48%, LR+ 110 and LR- 0.59. Areas under the ROC curve were 0.999 in Baermann and APC, 0.977 in the stool and 0.829 in formalin-ether concentration; specificity was 100% in all tests. The four conventional parasitological methods tested in this study to detect S. stercoralis can be helpful; however, agar plate culture and Baermann method are best suited.

Membrane-assisted culture of fungal mycelium on agar plates for RNA extraction and pharmacological analyses

Fungal mycelium grown in liquid culture is easy to harvest for RNA extraction and gene expression analyses, but liquid cultures often develop rather heterogeneously. In contrast, growth of fungal mycelium on agar plates is highly reproducible. However, this biological material cannot be harvested easily for downstream analyses. This article describes a PVDF (polyvinylidene difluoride) membrane-assisted agar plate culture method that enables the harvest of mycelium grown on agar plates. This culture method leads to a strongly reduced variation in gene expression between biological replicates and requires less growth space as compared with liquid cultures.

Conventional PCR for molecular diagnosis of human strongyloidiasis

Strongyloidiasis is frequently asymptomatic and diagnosis of latent infection is difficult due to limitations of current parasitological and serological methods. This study aimed to verify the use of conventional polymerase chain reaction (PCR) assay for molecular diagnosis of Strongyloides stercoralis infection. Fresh stool samples were obtained from 103 individuals: 33 S. stercoralis positive, 30 positive for other parasites and 40 negative for parasitological methods. These samples were examined by the Lutz, Rugai and agar plate culture methods and conventional PCR assay. Two sets of primers (S. stercoralis species-specific and genus-specific sets), located in the 18S ribosomal RNA gene, were used for PCR. Of the 33 samples positive for S. stercoralis by parasitological methods, 28 (84.8%) were also detected by PCR assay using species-specific primers and 26 (78.8%) using genus-specific primers. Among the stool samples negative by parasitological methods, seven (17.5%) were positive by PCR using species-specific primers and two (5.0%) using genus-specific primers. In conclusion, the conventional PCR assay described in this study using a species-specific primer pair provided a molecular method for S. stercoralis diagnosis in human stool samples.

Modified Formalin-Ether Concentration Technique for Diagnosis of Human Strongyloidiasis

We compared the efficacy and applicability of a modified formalin-ether concentration technique (M-FECT) to the conventional FECT (C-FECT) and the agar plate culture (APC) method for the detection of Strongyloides stercoralis larvae. For this purpose, we used 600 human fecal specimens collected in an endemic area of southern Thailand. In the M-FECT, we used 2 layers of wire meshes, instead of gauze, to avoid the loss by absorption/adhesion of larvae to the gauze during filtration, and we reduced the exposure time of S. stercoralis larvae in stool samples to formalin. By such simple modifications, the efficacy of M-FECT has become comparable to APC and was much better than that of C-FECT for the diagnosis of strongyloidiasis.

Oligo-DNA custom macroarray for monitoring major pathogenic and non-pathogenic fungi and bacteria in the phyllosphere of apple trees.

BackgroundTo monitor the richness in microbial inhabitants in the phyllosphere of apple trees cultivated under various cultural and environmental conditions, we developed an oligo-DNA macroarray for major pathogenic and non-pathogenic fungi and bacteria inhabiting the phyllosphere of apple trees.Methods and FindingsFirst, we isolated culturable fungi and bacteria from apple orchards by an agar-plate culture method, and detected 32 fungal and 34 bacterial species. Alternaria, Aureobasidium, Cladosporium, Rhodotorula, Cystofilobasidium, and Epicoccum genera were predominant among the fungi, and Bacillus, Pseudomonas, Sphingomonas, Methylobacterium, and Pantoea genera were predominant among the bacteria. Based on the data, we selected 29 major non-pathogenic and 12 phytopathogenic fungi and bacteria as the targets of macroarray. Forty-one species-specific 40-base pair long oligo-DNA sequences were selected from the nucleotide sequences of rDNA-internal transcribed spacer region for fungi and 16S rDNA for bacteria. The oligo-DNAs were fixed on nylon membrane and hybridized with digoxigenin-labeled cRNA probes prepared for each species. All arrays except those for Alternaria, Bacillus, and their related species, were specifically hybridized. The array was sensitive enough to detect 103 CFU for Aureobasidium pullulans and Bacillus cereus. Nucleotide sequencing of 100 each of independent fungal rDNA-ITS and bacterial 16S-rDNA sequences from apple tree was in agreement with the macroarray data obtained using the same sample. Finally, we analyzed the richness in the microbial inhabitants in the samples collected from apple trees in four orchards. Major apple pathogens that cause scab, Alternaria blotch, and Marssonina blotch were detected along with several non-phytopathogenic fungal and bacterial inhabitants.ConclusionsThe macroarray technique presented here is a strong tool to monitor the major microbial species and the community structures in the phyllosphere of apple trees and identify key species antagonistic, supportive or co-operative to specific pathogens in the orchard managed under different environmental conditions.

Efficacy of parasitological methods for the diagnosis of Strongyloides stercoralis and hookworm in faecal specimens

To compare the efficacy of stool examination for the detection of Strongyloides stercoralis and hookworm, a total of 634 stool samples from the routine laboratory service of the Pharmacia Faculty, Federal University of Bahia, Brazil, were examined by agar plate culture (APC), Baermann-Moraes and spontaneous sedimentation. The sensitivity of agar plate culture, calculated by combining results of all 3 methods, was 95% for S. stercoralis and 77.6% for hookwoorm. Moreover, APC had superior accuracy than Baermann-Moraes and spontaneous sedimentation for S. stercoralis and hookworm diagnosis, respectively. The S. stercoralis and hookworm positive samples from the laboratory routine, obtained after the previous analysis, along with those initially selected, were used to evaluate the concordance between microscopic examination and both the type of furrows left by larvae and the time for culture positivity using the APC method. Of 115 stool samples positive for S. stercoralis and 92 positive for hookworm, 110 (95.7%) and 89 (96.7%), respectively, had concordant results for furrows and morphological characteristics. The cumulative percentage of positivity increased to 94% by the third day of observation; at this time, only 19.6% of hookworm-positive samples had positive culture plates. Analyses of 74 S. stercoralis-positive stool samples stored at 4°C for 24, 48 and 72h showed the presence of larvae in 48.6%, 28.4% and 23% of samples, respectively when re-examined by the APC. As a definitive diagnosis of strongyloidiasis depends on the microscopic demonstration of parasites, increasing the sensitivity of the detection requires the use of different parasitological methods, including APC.

Comparison of the BD™ GeneOhm MRSA assay, broth enrichment culture and chromID™ MRSA for detection of methicillin-resistant Staphylococcus aureus from inter-hospital intensive care transfer patients

The objective of the present study was to compare the current chromogenic methicillin-resistant Staphylococcus aureus (MRSA) agar plate culture method with broth enrichment and the BD GeneOhm MRSA assay. Another key objective was to determine whether two admission surveillance swabs collected 1 h apart would reliably detect MRSA colonisation in patients transferred to a tertiary referral intensive care unit from other hospitals compared with the current routine method of two samples collected 24 h apart. In total, 593 swabs from 102 consecutive patients transferred from another hospital to the Princess Alexandra Hospital ICU were screened for the MRSA using nose and groin swabs collected on admission, 1 h and 24 h after admission. The non-selective broth enrichment step produced results in complete concordance with the existing method without an increase in sensitivity. The GeneOhm MRSA assay demonstrated 100% sensitivity, 97% specificity and 100% negative predictive values. The initial positive predictive value of this assay, however, was only 28%, largely due to the low prevalence of MRSA. Despite this low positive predictive value and because of the demonstrated 100% negative predictive value, the use of this assay could significantly improve turn-around times of surveillance screens in our laboratory by obviating the need for culture of over 90% of MRSA screening swabs. Positive polymerase chain reaction results require confirmation by culture, given that phenotypical characterisation of isolates is required for infection prevention and control. Comparison of the two screening collection timings in determining MRSA carriage could not be answered, due to insufficient positive results in the study group.

Increased Detection Rate of Strongyloides stercoralis by Repeated Stool Examinations Using the Agar Plate Culture Method

To clarify the efficacy of repeated stool examinations by the agar plate culture method for the detection of Strongyloides stercoralis infection, 4,071 stool samples collected from 2,406 patients > 50 years of age in Ryukyu University Hospital were examined. The cumulative detection rate of S. stercoralis infection was 4.7% (112/2,406). At the first, second, third, and beyond fourth examinations, the detection rates were 3.6% (86/2,406), 1.5% (12/786), 2.6% (10/392), and 2.0% (4/198), respectively. From these results, the cumulative detection rate was estimated to be 7.4% when three stool samples were examined for all patients. Our study showed that repeated stool examinations increase the sensitivity of detection of S. stercoralis infection.

Prevalence of Blastocystis hominis and Strongyloides stercoralis infection in Okinawa, Japan

This study was conducted to clarify the prevalence of Blastocystis hominis and Strongyloides stercoralis infection in Ryukyu University Hospital, Okinawa, Japan, between January 2004 and November 2006. Stool samples collected from 3,292 patients were examined by the direct smear method, formalin-ether sedimentation method, and agar plate culture method. The prevalence rate of B. hominis and S. stercoralis infection was 1.0 and 3.4%, respectively. The prevalence rate of B. hominis infection in patients aged >80 years old was significantly higher than that in patients <80 years old (P < 0.001). The prevalence rate of S. stercoralis infection was significantly higher in patients with B. hominis infection compared with those without (P < 0.001). This study demonstrated a prevalence rate for B. hominis and S. stercoralis infection and an association between B. hominis and S. stercoralis infection in Okinawa, Japan.

Evaluation of biofilm production and prevalence of theicaDgene in methicillin-resistant and methicillin-susceptibleStaphylococcus aureusstrains isolated from patients with nosocomial infections and carriers

Staphylococcus aureus is one of the most important etiological agents responsible for healthcare-associated infections and is capable of producing many virulence factors including biofilm. The aim of the present study was to analyze the correlation between the presence of the icaD and icaA genes and the ability to produce biofilm in vitro in 302 methicillin-resistant (MRSA) and 268 methicillin-sensitive S. aureus (MSSA) strains isolated in the Provincial Hospital in Gdansk. Presence of the icaD and icaA genes was detected by PCR and the ability to produce biofilm in vitro was measured both spectrophotometrically and via Congo Red Agar plate culture methods. We found that 91% of MRSA strains harbored the icaD gene. Moreover, all icaD-negative strains were icaA-positive. Of MRSA and MSSA strains, 47% and 69%, respectively, produced biofilm in vitro. The level of consistency between the two applied phenotypic methods was 96%. Additionally, we found that strains with the same biofilm status may be present in asymptomatic carriers and cause infections.

Food preservative based on propolis: Bacteriostatic activity of propolis polyphenols and flavonoids upon Escherichia coli

Propolis was tested as food preserver, due its recognized bactericidal and bacteriostatic properties. Furthermore, most propolis components are natural constituents of food and recognized as safe substances. Fifteen propolis from Santa Fe, Argentine in 20% w/w ethanolic extracts, were tested upon Escherichia coli ATCC 25922 by agar diffusion and plate culture methods. Considering propolis physicochemical characteristics and inhibitory effects, tested samples were classified in three groups. A minimum inhibitory concentration mean value of 14.3 ± 6 mg soluble compounds/ml of the most active propolis was capable of inhibiting 10 5 cfu/ml cellular concentration. Such extract had 32.31% total soluble compounds (2.1% coumaric + siringic acids, 5.16% quercetin, 0.47 apigenine, 8.15 galangine, 7.2 caffeic acid + crisine and 9.23% no-identified phenolics compounds). By relating the zone of growth inhibition with extracts concentration, a linear response was obtained. On the propolis samples tested, a single value of the minimum inhibitory concentration could not be established. Those values were strongly dependent on propolis composition and botanical origin. The propolis extracts tested, may successfully inhibit the E. coli development in vitro, and consequently may be useful as natural food preserver.

In vitro antiparasitic activity of extracts of Cardiospermum halicacabum against third-stage larvae of Strongyloides stercoralis

Extracts of Cardiospermum halicacabum, medicinal plant, were tested in vitro for their effectiveness against third-stage larvae of Strongyloidesstercoralis. Third-stage larvae of S. stercoralis were isolated from cultures of dog's feces using agar plate culture method. The larvae (1,000 larvae/ml), suspended in phosphate buffer saline solution, pH 7.4, were exposed to aqueous and alcohol extracts (2,000 microg/ml) of C. halicacabum at 37 degrees C with 5% CO2. Ivermectin (250 microg/ml) and piperazine (2,000 microg/ml) were also used as the reference drugs. The survival of Strongyloides larvae based on its motility was determined daily for 7 days. Strongyloides larvae were viable after contact with ivermectin, piperazine and C. halicacabum (aqueous and alcohol) solutions, but most of them were immobilized, after exposure to aqueous and alcohol extracts of C. halicacabum within 72 and 48 h, respectively, while ivermectin took from 72 to 144 h, and piperazine more than 7 days, to achieve the same rate of nonmotility. Clearly, the viability of S. stercoralis larvae was significantly reduced when exposed to extracts of C. halicacabum. Further study is needed on the antiparasitic activity of aqueous and alcohol extracts of C. halicacabum against S. stercoralis.

Strongyloidiasis--progress in diagnosis and treatment.

Strongyloidiasis is an intestinal parasitic disease caused by Strongyloides stercoralis. Basically, detecting larvae of S. stercoralis in feces makes definitive diagnosis. The ordinary agar plate culture method developed at our department is much simpler to handle and much more sensitive than the conventional filter paper culture method. It is considered to be the most useful method in the diagnosis of strongyloidiasis and in evaluation of the eradicating effect. Among chemotherapeutic agents, thiabendazole representing the benzimidazole compounds is most effective. However, it has a problem in safety, since its adverse effects and liver dysfunction occur with a high incidence, and it can be severe. Regarding the effects of mebendazole, albendazole and ivermectin, a study was conducted which included many patients. A high incidence of liver dysfunction was observed with mebendazole, and eradicating effect was not sufficient with albendazole. Ivermectin is different from benzimidazole compounds in a pharmacokinetic profile. However, ivermectin showed a strong anthelmintic effect with the least toxicity. We therefore consider ivermectin is the most useful drug for the treatment of strongyloidiasis.

Strongyloidiasis Detected by the Agar Plate Culture Method among Patients Infected by HIV

To evaluate the rate of strongyloidiasis among HIV/AIDS patients, stools and duodenal juice were examined using the agar plate culture method. From January to June 1993, a total of 60 HIV/AIDS patients were required for duodenal aspirate and three serial samples of freshly passed stools. Stools and duodenal aspirate were dispensed on an agar plate culture; after incubation at 28 degrees C during 48 h, screening of plates was made at 10 x. The presence of furrows and worms of short buccal chamber and prominent genital primordium were positive for Strongyloides stercoralis. Most patients were men (91.7%); their mean age, of 33.9 years (SD +/- 10.6); their median CD4(+) T-cells count, of 105/microL (range of 12 to 646). S. stercoralis was detected in three patients (5%). In duodenal juice, the three patients showed the parasite, but in feces, only two (3.3%). In these two individuals, the worms were found in feces by agar culture and Faust's concentration method. The rate of S. stercoralis in feces of HIV/AIDS individuals (3.3%) by agar culture method was similar to that formerly reported from the general Mexican population (2.9%) using standard concentration procedures. Hence, in this immunocompromised population of a low prevalence city, there was no advantage to using an agar plate culture for strongyloidiasis.

Efficacy of stool examination for detection of Strongyloides infection.

To determine the efficacy of stool examination for detection of Strongyloides infection, 1,350 stool samples collected in Japan, Brazil, and Thailand were examined by four different methods (direct fecal smear, formalin-ether concentration. Harada-Mori filter paper culture, and agar plate culture). The newly developed agar plate culture method was highly effective; more than 96% of the positive cases were diagnosed by this method. The coprologic examination, however, was not sensitive enough for detecting chronic infections because more than 40% of the positive cases were overlooked even when persons with proven Strongyloides infection were re-examined several months later without intervening treatment. Therefore, it is essential to examine stool samples repeatedly to achieve a correct diagnosis, and even so, it is important to note that a negative result does not necessarily indicate the unequivocal absence of the infection.

Prevalence of intestinal parasitic infection in five farms in Holambra, São Paulo, Brazil.

A parasitological survey was carried out on 222 inhabitants of five farms in Holambra, located 30 km north of Campinas, São Paulo, Brazil, on October 1992. Approximately 70% of the inhabitants were found to be infected with at least one species of intestinal parasite. The positive rates of 6 helminths and 7 protozoan species detected are as follows: 5.4% Ascaris lumbricoides; 8.6% Trichuris trichiura; 19.8% Necator americanus; 10.4% Strongyloides stercoralis; 1.4% Enterobius vermicularis; 0.9% Hymenolepis nana; 3.2% Entamoeba histolytica; 2.7% E. hartmanni; 9.9% E. coli; 14.0% Endolimax nana; 2.3% Iodamoeba butschlii; 10.4% Giardia lamblia; 37.8% Blastocystis hominis. The positive rates of helminth infection were generaly higher in the younger-group under 16 years-old than those in the elder group aged 16 or more, whereas the infection rates of protozoan species were higher in the elder group. The infection rate of Strongyloides was found to be 10.4% by a newly developed sensitive method (an agarplate culture methods).

Evaluation of Three Methods for Laboratory Diagnosis of Strongyloides stercoralis Infection

The direct smear, a modified Baermann technique, and the agar plate culture method were compared for cost and efficiency in recovering Strongyloides stercoralis larvae in a hospital setting in Honduras. Of 427 stool samples, 9 were positive by the direct smear and 33 additional ones were detected by the Baermann technique; the agar plate culture method disclosed 28 more cases for a total of 70 S. stercoralis infections. The modified Baermann method was 3.6 times more efficient than the direct smear, and the agar plate culture increased the modified Baermann efficiency by 0.8 times. Cost for materials alone was cheapest for the direct smear; it increased 4 times for the modified Baermann method and 15 times for the agar plate culture method. This last technique required a better equipped laboratory, more time to perform, and the best trained, most skillful laboratory personnel. In field studies, prevalence of infection should determine the cost-effectiveness of the agar plate culture method; in a clinical setting, when S. stercoralis infection must be ruled out, the agar plate culture method should be made available to the medical community.

Autologous Fibrin Gel

Fibrin glue is an effective hemostatic agent in a variety of clinical situations; its utility is limited by potential transmission of viral infection. We studied the bactericidal properties of fibrin gel (FG) in a murine contaminated hepatic injury model and in vitro by agar plate culture method. Intra-abdominal abscess formation and adhesion rate were assessed following controlled liver injury in association with abdominal contamination with 10(7) Bacteroides fragilis and hepatorrhaphy (H, n = 15) or FG (n = 12). Animals treated by hepatorrhaphy had a significantly greater intra-abdominal abscess rate (15/15 vs. 4/12, p less than 0.05) and adhesion rate (14/15 vs. 6/12, p less than 0.05) than animals treated with FG. Fibrin gel is bactericidal to Bacteroides fragilis, Enterobacter faecium, Escherichia coli, and Staphylococcus aureus but has no effect against Klebsiella pneumoniae or Pseudomonas aeruginosa; the plasma component appears active. Fibrin gel demonstrates significant improvement in adhesion formation and intra-abdominal abscess rate when compared with suture hepatorrhaphy. Fibrin gel appears protective in contaminated hepatic injury.

Automated methods for detection of bacteriuria

Urine specimens represent a large portion of cultures received by the clinical microbiology laboratory. Much time and effort are spent screening these specimens and approximately 80 percent show no growth. The methods employed for detection of bacteriuria include microscopic, chemical, culture, and automated. The most widely used procedure has been an agar plate culture method. However, this method requires overnight incubation and therefore results are delayed. Within the last decade many automated methods have been introduced for detecting bacteriuria, and results are available more rapidly than with the agar plate culture method. In addition to decreased detection time, these systems are accurate and costeffective. The purpose of this report is to review these automated bacteriuria screening systems, which include bioluminescence, colorimetry, electrochemical screening, electrical impedance, microcalorimetry, photometry, and radiometry. Accuracy, detection time, and cost are also discussed in this review.

Methods of producing perithecia of Gaeumannomyces graminis and their application to related fungi

Fifty-seven isolates of fungi of the Gaeumannomyces-Phialophora complex that had been maintained in stock culture were tested by host-infection (in vivo) and agar plate, filterpaper and flask culture (in vitro) methods for production of perithecia. Most isolates of each of the three varieties of G. graminis produced perithecia in one test or another ( avenae (Gga) , 100%; tritici (Ggt) , 75%; graminis (Ggg) , 71%), but stock cultures of the two varieties of Phialophora radicicola did not form perithecia by any method. For G. graminis , in vivo methods (89, 56–75, 50% of isolates producing perithecia) were generally rather more effective than in vitro methods ( 33 , 69 and 43%). No one in vitro method was the best for all varieties of G. graminis : full-strength potato-dextrose agar was the least effective medium for perithecia, but at quarter-strength and supplemented with yeast extract it ranked with malt extract broth agar and a filter paper method as the most effective in vitro methods for Ggt , but not Gga or Ggg . In host-infection tests using stock isolates those host-parasite combinations that produced most disease or most colonized roots tended also to be those in which the fungus produced perithecia. Perithecia that formed on roots excised from wheat seedlings used to bioassay field soil with little infestation by Ggt were mostly on roots without take-all lesions. In soils with more infestation there was a positive association between lesioned roots and perithecia. G. cylin- drosporus perithecia also occurred on these excised roots, but not on the same plants as Ggt and concurrence of Ggt and P. radicicola var. radicicola was ≤£ 30% of isolates. These fungi seemed to have different seasonal occurrences. Some factors that influence the production of perithecia in vivo are discussed.