Convenient detection of E. coli in Ringer's solution.

An easy and inexpensive detection method for DNA hybridization assays combining magnetic beads and enzymatically generated silver nanoparticles is introduced. The main advantage of this approach is the possibility to distinguish between positive and negative test results with the naked eye. In the case of complementary DNA sequences the sample will turn black within a few minutes, allowing readout without any hardware. In order to illustrate the applicability of the assay genomic DNA isolated from E. coli contaminated Ringer's solution was used for testing the sensitivity as well as specificity.

A rapid, cost-effective method of assembly and purification of synthetic DNA probes >100 bp.

Here we introduce a rapid, cost-effective method of generating molecular DNA probes in just under 15 minutes without the need for expensive, time-consuming gel-extraction steps. As an example, we enzymatically concatenated six variable strands (50 bp) with a common strand sequence (51 bp) in a single pool using Fast-Link DNA ligase to produce 101 bp targets (10 min). Unincorporated species were then filtered out by passing the crude reaction through a size-exclusion column (<5 min). We then compared full-length product yield of crude and purified samples using HPLC analysis; the results of which clearly show our method yields three-quarters that of the crude sample (50% higher than by gel-extraction). And while we substantially reduced the amount of unligated product with our filtration process, higher purity and yield, with an increase in number of stands per reaction (>12) could be achieved with further optimization. Moreover, for large-scale assays, we envision this method to be fully automated with the use of robotics such as the Biomek FX; here, potentially thousands of samples could be pooled, ligated and purified in either a 96, 384 or 1536-well platform in just minutes.

Use of the AMPLIFIED Mycobacterium tuberculosis direct test for the diagnosis of tuberculosis.

A comparative analysis was made of 3 conventional tests for tuberculosis (TB) versus a DNA probe technique among suspected TB patients at a reference centre in Greece. During 2004, we tested 2961 biological specimens from 2234 patients with the following methods: Ziehl-Neelsen staining, LöwensteinOJensen culture, BACTEC mycobacteria growth indicator tubes (MGIT) and the Gen-Probe AMPLIFIED Mycobacterium tuberculosis direct test (MTD). Of a total of 136 TB patients diagnosed and under anti-TB treatment, 133 of them (98%) were positive by amplified MTD. There were 112 TB (82%) detected by the MGIT method, 102 (75%) by Löwenstein-Jensen culture and 75 (55%) by Ziehl-Neelsen staining. Using MTD the positive result is ready within hours compared with days or weeks.

"Shotgun" versus sequential testing. Cost-effectiveness of diagnostic strategies for vaginitis.

BACKGROUND: Although vaginitis is a common outpatient problem, only 60% of patients can be diagnosed at the initial office visit of a primary care provider using the office procedures of pH testing, whiff tests, normal saline, and potassium hydroxide preps. OBJECTIVE: To determine the most cost-effective diagnostic and treatment approach for the medical management of vaginitis. DESIGN: Decision and cost-effectiveness analyses. PARTICIPANTS: Healthy women with symptoms of vaginitis undiagnosed after an initial pelvic exam, wet mount preparations, pH, and the four criteria to diagnose bacterial vaginosis. SETTING: General office practice. METHODS: We evaluated 28 diagnostic strategies comprised of combinations of pH testing, vaginal cultures for yeast and Trichomonas vaginalis, Gram's stain for bacterial vaginosis, and DNA probes for Neisseria gonorrhoeae and Chlamydia. Data sources for the study were confined to English language literature. MEASUREMENT: The outcome measures were symptom-days and costs. RESULTS: The least expensive strategy was to perform yeast culture, gonorrhoeae and Chlamydia probes at the initial visit, and Gram's stain and Trichomonas culture only when the vaginal pH exceeded 4.9 (330 dollars, 7.30 symptom days). Other strategies cost 8 dollars to 76 dollars more and increased duration of symptoms by up to 1.3 days. In probabilistic sensitivity analysis, this strategy was always the most effective strategy and was also least expensive 58% of the time. CONCLUSIONS: For patients with vaginitis symptoms undiagnosed by pelvic examination, wet mount preparations and related office tests, a comprehensive, pH-guided testing strategy at the initial office visit is less expensive and more effective than ordering tests sequentially.

Clinical management of chlamydia and gonorrhea infection in a county teaching emergency department--concerns in overtreatment, undertreatment, and follow-up treatment success.

To date, several studies have examined overtreatment or undertreatment of Neisseria gonorrheae, Chlamydia trachomatis, or both in women. However, no study has looked at both subpopulations together, along with eventual treatment of disease-positive patients who were not empirically treated. This study is unique, for it looks at all of these subpopulations to assess overall efficacy of management of these diseases in women. A 1-year prospective, descriptive study was performed in a teaching county hospital Emergency Department (ED). There were 1260 women receiving a pelvic examination and routine GEN-PROBE testing for gonorrhea and chlamydia who were studied. The main outcome measures were the proportion of women disease positive and initially not treated (undertreated), the proportion of women disease negative who were initially treated (overtreated), as well as the follow-up treatment rate for those undertreated. Finally, the subpopulation of women disease positive and not empirically treated was examined in detail. Of 1260 GEN-PROBE-tested women, 81 (6.4%, 95% CI 1.1-11.7%) were disease positive and 31/81 (38.3%, 95% CI 21.2-55.4%) of these women were undertreated. Furthermore, 20/31 (64.5%, 95% CI 43.5-85.5%) women did not return for follow-up treatment. The billable health care dollars of routine GENPROBE testing per woman (n = 11/1260, 0.9%) returning for treatment as a result of the test was $4762.80 US dollars. Four hundred twenty-six (33.8%) of the 1260 women were empirically treated on the initial visit. Of these 426 initially treated women, 376 (88.3%, 95% CI 85.1-91.5%) were GEN-PROBE negative for disease (overtreated). The billable health care dollars of this overtreatment was $12,449.51 US dollars. This study demonstrates that health care providers are substantially overtreating women who are gonorrhea and chlamydia negative. This generates moral, ethical, health care, and financial concerns. Additionally, one-third of disease-positive women are not treated on initial visit and the majority of undertreated patients are not returning for subsequent treatment. This study provides support for investigating improved methods in the management of chlamydia and gonorrhea in women.

A rapid DNA probe test compared to culture methods for identification of subgingival plaque bacteria.

There has been a significant amount of interest in developing a more rapid and cost-effective test to identify bacterial pathogens in plaque. DNA probe technology may meet both these objectives, it is more rapid and cost-effective when compared to culture methods. The purpose of this study was to compare an automated DNA probe test with classical culture methods for identifying Bacteroides forsythus and Porphyromonas gingivalis in subgingival plaque of patients with adult periodontitis. Subgingival plaque samples were collected from sites with moderate to severe periodontitis and divided into two aliquots for analysis by either DNA probe or culture methods. When the DNA probe method was compared with the culture method (gold standard), the sensitivity and specificity for B. forsythus were 92.0% (SE = 3.4%) and 50.5% (SE = 7.8%), respectively; for P. gingivalis they were 52.2% (SE = 8.7%) and 74.7% (SE = 5.9%), respectively. Detection of B. forsythus and P. gingivalis by DNA probe correlated with probing depth (P = 0.01 for B. forsythus and P = 0.03 for P. gingivalis). It was concluded the DNA probe test was comparable to culture methods in detecting B. forsythus. In addition, when compared to the culture method, a better correlation was obtained with DNA probe detection of B. forsythus or P. gingivalis and clinical parameters.

Evaluation of nonisotopic DNA hybridization methods for detection of the tdh gene of vibrio parahaemolyticus.

Production of the thermostable direct hemolysin (TDH) by Vibrio parahaemolyticus is associated with pathogenicity of the organism and is encoded by the tdh gene. The timely resolution of seafood-associated outbreaks requires rapid and accurate detection of pathogenic V. parahaemolyticus. The specificity of alkaline phosphatase- and digoxigenin-labeled tdh gene probes was evaluated against 61 strains of V. parahaemolyticus (including isolates from recent outbreaks involving oysters from the Pacific Northwest, Texas, and New York), 85 strains of other vibrios, and 7 strains of non-vibrio species from clinical and environmental sources. The probes were specific for detection of the V. parahaemolyticus tdh gene.

Comparison of Gram stain with DNA probe for detection of Neisseria gonorrhoeae in urethras of symptomatic males.

The comparison of Gram-stained urethral smears with Gen-Probe for the detection of Neisseria Gonorrhoeae in the urethras of males with symptomatic urethritis revealed a 99.6% correlation between the two methods. A simple Gram stain would appear to be the method of choice for the detection of gonorrhea in symptomatic males, because it is much less expensive and much more rapid than the Gen-Probe method.

Impact of technological developments and organizational strategies on clinical laboratory cost reduction.

Health care reform efforts, largely under the aegis of managed health care initiatives, have prompted clinical laboratories to increase efficiency and reduce both expenditures and test turnaround times. The adoption of newer technologies is viewed as a mechanism of achieving the latter objectives, but direct and indirect costs and outcomes are often difficult to project. Issues explored in this article include the impact on a large university hospital-based clinical microbiology laboratory following the application of various technological approaches to organism recognition and susceptibility testing and the consolidation of certain testing services. Included are applications of an automated blood culture system; radiometric detection, identification, and susceptibility testing of mycobacteria; and the use of molecular probes to identify various microorganisms. Assessment was made through retrospective review of direct costs, estimates of average test report turnaround times, work flow changes, and real or perceived outcomes. Both the application of technology per se and consolidation of an independent virology service into the general microbiology laboratory enabled improvement in test report times and led to direct or indirect cost reduction. Managerial strategies to bring about organization changes throughout all clinical laboratories in response to a major hospital-wide cost reduction program are also presented together with financial outcomes achieved.

DNA probes as epidemiological tools for surveillance of Plasmodium falciparum malaria in Thailand.

We have previously reported on development of a DNA probe-based method for diagnosing Plasmodium falciparum infection directly from patient blood samples. In the present studies, we sought to examine applicability of the method to large epidemiological surveys, comparing sensitivity, specificity, time required to obtain results, and costs with those of conventional microscopic examination. Results of DNA probe hybridization were also compared between laboratories in the US and Thailand, to assess transferability of the DNA probe technology. Five separate surveys of approximately 5000 villagers each were performed between December 1987 and June 1989 (26,176 samples total). Sensitivity ranged from 61% to 92% for both US and Thai laboratories, while specificity ranged from 98.2% to 99.9%. Agreement between the US and Thai laboratories was good, with kappa coefficients between 0.62 and 0.78 for different surveys. Between 4 and 8 person-days were required to obtain results from each set of 5000 samples by DNA hybridization, whereas microscopic examination required 150 person-days. Approximate costs were US 0.17 per sample for DNA probe analysis, and US$0.36 for microscopic examination. We conclude that the DNA probe method offers significant advantages when large numbers of samples must be surveyed for P. falciparum.