Tag Archives: C diff testing

New Study Evaluated Rectal Swabs For Clostridium difficile Testing

Clostridium difficile (C. diff) is among one of the top 18-drug-resistant threats to the United States according to the Centers for Disease Control and Prevention, responsible for around 250,000 infections on an annual basis and 14,000 deaths.

When it comes to diagnosis, microbiological testing of stool samples is often used. However, a new study suggests that for simple PCR-based detection of C. diff, dry rectal swabs were an effective substitute for the use of stool samples.

To read this article in its entirely please click on the following link:

http://www.contagionlive.com/news/dry-rectal-swabs-prove-effective-alternative-to-stool-samples-for-c-diff-diagnosis

“With this study, we proved that rectal swabs for the diagnosis of C. diff infection by PCR can replace the actually used stool samples,” study author Nathalie Jazmati, MD, University Hospital of Cologne, told our sister publication MD Magazine. “That will be more convenient for both patients and health care workers. Nevertheless, this was only a small study and our results have to be confirmed in a bigger clinical trial.”

In an effort to examine methods other than the analysis of stool specimens for C. diff confirmation, a research team from Germany examined the way rectal swabs with liquid transport medium and nylon flocked dry swabs performed for the detection of C. diff; they also evaluated the impact of storage temperature on the swabs.

For their study, the researchers collected 60 clinical stool samples that tested positive for C. diff by PCR and used them to simulate rectal swabs. Then, researchers dipped both wet and dry swabs into the stool and tested by PCR 3 times.

The first test took place immediately after the simulation “swab,” then, after 1 month and 3 months storage at -80°C. When the researchers tested the frozen samples, they first thawed them at room temperature for 15 minutes and the liquid swabs were vortexed for 30 seconds.

Testing all of the dry swabs 100% successfully detected C. diff, an equal rate of the stool sample testing; this proved true for all 3 phases of testing, and the researchers learned that no significant differences were found on the samples after they were frozen and thawed.

The detection rate for the other 30 liquid swabs was lower, at 83.2% accuracy. However, the researchers determined the temperature and the freezing and thawing of these samples did not have any significant impact.

The authors added that their results fall in line with other studies that tested PCR from rectal swabs in the detection of C. diff. The idea of using rectal swabs instead of stool samples isn’t new—it dates back to 1987.

Liquid swabs are currently cleared by the US Food and Drug Administration (FDA) for transport and the culture of gastrointestinal pathogens, the study authors continued, but it is not FDA approved for use with any molecular gastrointestinal assays.

In the future, dry swabs would “be appropriate and can probably speed up and facilitate the diagnosis of C. diff infection,” the researchers wrote, but warned, “nevertheless, using single step PCR-based detection of C. diff may lead to overdiagnosis of C. diff infection due to the high sensitivity but lower specificity of PCR.”

That marks a heightened importance for the careful clinical evaluation of the patient: Are they an asymptomatic carrier? Is there another reason for the patient’s diarrhea? Do they truly have a C. diff infection? All important questions to continue to ask.

While liquid swabs cannot substitute for the two-step laboratory diagnosis of C. diff, the researchers believe that their study shows the dry swab is a suitable alternative to stool sample testing.

C diff Infection Compared Control in 6 United Kingdom Hospitals With Whole-Genome Sequencing (WGS)

David W. Eyre
Warren N. Fawley
Anu Rajgopal
Christopher Settle
Kalani Mortimer
Simon D. Goldenberg
Susan Dawson
Derrick W. Crook
Tim E. A. Peto
A. Sarah Walker

.

Clin Infect Dis cix338.
Published:
29 May 2017 

Abstract

Background Variation in Clostridium difficile infection (CDI) rates between healthcare institutions suggests overall incidence could be reduced if the lowest rates could be achieved more widely.
Methods.

We used whole-genome sequencing (WGS) of consecutive C. difficile isolates from 6 English hospitals over 1 year (2013–14) to compare infection control performance. Fecal samples with a positive initial screen for C. difficile were sequenced. Within each hospital, we estimated the proportion of cases plausibly acquired from previous cases.

Results.

Overall, 851/971 (87.6%) sequenced samples contained toxin genes, and 451 (46.4%) were fecal-toxin-positive. Of 652 potentially toxigenic isolates >90-days after the study started, 128 (20%, 95% confidence interval [CI] 17–23%) were genetically linked (within ≤2 single nucleotide polymorphisms) to a prior patient’s isolate from the previous 90 days. Hospital 2 had the fewest linked isolates, 7/105 (7%, 3–13%), hospital 1, 9/70 (13%, 6–23%), and hospitals 3–6 had similar proportions of linked isolates (22–26%) (P ≤ .002 comparing hospital-2 vs 3–6). Results were similar adjusting for locally circulating ribotypes. Adjusting for hospital, ribotype-027 had the highest proportion of linked isolates (57%, 95% CI 29–81%). Fecal-toxin-positive and toxin-negative patients were similarly likely to be a potential transmission donor, OR = 1.01 (0.68–1.49). There was no association between the estimated proportion of linked cases and testing rates.

Conclusions.

WGS can be used as a novel surveillance tool to identify varying rates of C. difficile transmission between institutions and therefore to allow targeted efforts to reduce CDI incidence.

To view the article in its entirety please click on the following link:

Preventing Clostridium difficile infection (CDI) is a priority for infection control teams, as it remains a major healthcare-associated infection; although the incidence of healthcare-associated CDI in the United Kingdom has fallen to 1.5 per 10000 inpatient bed-days [1], rates across Europe range from 0.7 to 28.7/10000 bed-days [2], and there were an estimated 293000 healthcare-associated cases in the United States in 2011 [3].

Variation in CDI incidence across countries and between healthcare institutions [4] suggests overall incidence could be reduced if the lowest rates could be achieved more widely. Surveillance programs [5] and penalties for healthcare institutions [6] have been implemented to promote reductions. However, robustly identifying the best performing institutions is challenging.

Variations in true incidence can arise from differences in patient risk factors or locally circulating strains. However, testing strategy also influences reported incidence; reported CDI incidence is associated with testing rates [2]. With low testing rates, CDI ascertainment is likely to be suboptimal. Conversely, high testing rates may lead to overdiagnosis, for example, from testing C. difficile colonized patients, who do not have CDI but may have diarrhea of another cause.

The lack of a universally accepted objective CDI case definition means that robust comparisons of infection rates between institutions should ideally also consider independent measures of which patients are being tested to assess the comparability of differing testing strategies [7].

Additionally, assessing potential sources of healthcare- attributed CDI cases [8] is complex, requiring differentiation between lapses in infection control around symptomatic cases or more generally, deviation from optimal antimicrobial stewardship, and external factors, for example, the food chain. Healthcare exposure increases the risk of C. difficile acquisition; both CDI and colonization increase during hospital stay [9]. However, despite this strong association, studies using whole-genome sequencing (WGS) [10–12] and other genotyping schemes [13–15] have shown that, in endemic settings with standard infection control, only the minority of infections are likely to have been acquired from other hospitalized CDI cases. However, the extent to which this proportion of linked cases varies between hospitals is unknown. Furthermore, such potential variance in linkage rates could identify a potentially preventable group of CDIs.

We investigated variation in the proportion of linked cases using WGS of consecutive C. difficile isolates from 6 hospitals in England and explored whether this could be used to assess their infection control effectiveness, by assessing the proportion of cases plausibly acquired from (linked to) previous cases.

METHODS

Samples and Settings

Hospitals in England are recommended to store frozen aliquots of C. difficile–positive fecal samples for 12 months [16]. Stored consecutive hospital and community diarrheal samples submitted for routine C. difficile testing at 6 hospital laboratories were studied, including a tertiary referral center and teaching hospital, and 5 district general hospitals serving a mix of urban and rural populations (see Supplement). Samples were obtained for a one-year period at each hospital between January 2013 and October 2014. Results were anonymized by assigning a computer-generated random identifier, hospital 1 to hospital 6.

Each hospital used the United Kingdom-recommended 2-stage C. difficile testing algorithm [17]. Hospital 1 used toxin gene polymerase chain reaction (PCR) as a screening test, hospital 2 both glutamate dehydrogenase (GDH) enzyme immunoassay (EIA) and toxin gene PCR as a combined screening test, and hospitals 3–6 a GDH screen. Screen-positive samples underwent confirmatory fecal-toxin EIA testing. Screen-positive, fecal-toxin-positive patients were regarded as having CDI. Toxin gene PCR was also performed as a third-line test on all GDH-positive samples at hospitals 3 and 6, and on samples from inpatients at hospital 5. PCR-positive, fecal-toxin-negative patients, with a clinical syndrome in keeping with CDI, were regarded as potential cases for treatment and infection control purposes.

All screen-positive fecal samples were sent to Leeds General Infirmary microbiology laboratory, United Kingdom (except hospital 2, which submitted isolates and excluded toxin EIA-negative/PCR-negative samples), where they underwent selective culture for C. difficile [18] and capillary electrophoresis ribotyping [19]. Individual patient consent for use of anonymized bacterial isolates was not required.

Sequencing

DNA was extracted from subculture of a single colony from each culture-positive sample and sequenced using Illumina HiSeq2500. Sequence data were processed as previously (see Supplement) [10, 20], mapping sequenced reads to the C. difficile 630 reference genome [21]. Sequences were compared using single-nucleotide polymorphisms (SNPs) between sequences obtained from maximum-likelihood phylogenies [22], corrected for recombination [23]. Potentially toxigenic strains were identified as those containing toxin genes using BLAST searches of de novo [24] assemblies.

Analysis

For each sample, only the hospital, collection date, and fecal-toxin EIA result were known; no further epidemiological data were available. Within each hospital, sequences were compared with all sequences from samples obtained in the prior 90 days. Samples from the community and hospital were included to increase the chance of identifying transmission events occurring in hospital but leading to CDI onset after discharge. From previous estimates of C. difficile evolution and within-host diversity [10, 25, 26], ≤2 SNPs are expected between isolates linked by transmission within 90 days. Therefore, where ≥1 prior sequences within ≤2 SNPs were identified, a case was considered to have been potentially acquired from another case. A 90 day threshold for linking cases was chosen assuming that cases were rapidly treated and infectiousness declined, and that subsequent cases related by direct transmission occurred within incubation periods implied by surveillance definitions [8] and previous studies [13]. As the sources of cases occurring at the start of the study may themselves have been sampled before the study started, the proportion of cases linked to a prior case was only calculated for cases occurring after the first 90 days, with cases in the first 90 days included only as potential sources for subsequent cases.

Two differing case definitions were considered. Initially, all patients with culture-positive potentially toxigenic C. difficile were considered “cases” to capture possible transmission events involving potentially toxigenic C. difficile irrespective of fecal-toxin status. The analysis was then repeated restricted only to fecal-toxin-positive CDI cases. For comparisons with previously published data, the same definition and analysis approach was applied to fecal-toxin-positive CDI cases occurring within 90 days in Oxford (September 2007 to December 2010, split by calendar year) [10] and Leeds (August 2010 to April 2012) [11].

Risk Factor Analysis

Univariate logistic regression was used to determine whether a case’s toxin status affected the risk of it being genetically related to a prior case, that is, potentially acquired from another case. Similarly, logistic regression was used to determine whether a case’s fecal-toxin status affected the risk of it being genetically linked to a subsequent case, that is, to assess the relative infectiousness of fecal-toxin-positive and toxin-negative patients.

To assess whether the locally circulating strain mix affected transmission estimates, hospital-specific estimates were adjusted for ribotype using multivariate logistic regression (see Supplement).

Simulations

To estimate the impact of missing data (as not all sampled cases were sequenced at some hospitals), we simulated transmission at a theoretical hospital. We subsampled simulated cases and calculated the change in the percentage of cases linked to a prior case as the proportion of missing samples increases (details in Supplement).

METHODS

Samples and Settings

Hospitals in England are recommended to store frozen aliquots of C. difficile–positive fecal samples for 12 months [16]. Stored consecutive hospital and community diarrheal samples submitted for routine C. difficile testing at 6 hospital laboratories were studied, including a tertiary referral center and teaching hospital, and 5 district general hospitals serving a mix of urban and rural populations (see Supplement).

Samples were obtained for a one-year period at each hospital between January 2013 and October 2014. Results were anonymized by assigning a computer-generated random identifier, hospital 1 to hospital 6.

Each hospital used the United Kingdom-recommended 2-stage C. difficile testing algorithm [17].

Hospital 1 used toxin gene polymerase chain reaction (PCR) as a screening test,

Hospital 2 both glutamate dehydrogenase (GDH) enzyme immunoassay (EIA) and toxin gene PCR as a combined screening test, and hospitals 3–6 a GDH screen.

Screen-positive samples underwent confirmatory fecal-toxin EIA testing. Screen-positive, fecal-toxin-positive patients were regarded as having CDI. Toxin gene PCR was also performed as a third-line test on all GDH-positive samples at hospitals 3 and 6, and on samples from inpatients at hospital 5. PCR-positive, fecal-toxin-negative patients, with a clinical syndrome in keeping with CDI, were regarded as potential cases for treatment and infection control purposes.

All screen-positive fecal samples were sent to Leeds General Infirmary microbiology laboratory, United Kingdom (except hospital 2, which submitted isolates and excluded toxin EIA-negative/PCR-negative samples), where they underwent selective culture for C. difficile [18] and capillary electrophoresis ribotyping [19]. Individual patient consent for use of anonymized bacterial isolates was not required.

Sequencing

DNA was extracted from subculture of a single colony from each culture-positive sample and sequenced using Illumina HiSeq2500. Sequence data were processed as previously (see Supplement) [10, 20], mapping sequenced reads to the C. difficile 630 reference genome [21]. Sequences were compared using single-nucleotide polymorphisms (SNPs) between sequences obtained from maximum-likelihood phylogenies [22], corrected for recombination [23]. Potentially toxigenic strains were identified as those containing toxin genes using BLAST searches of de novo [24] assemblies.

Analysis

For each sample, only the hospital, collection date, and fecal-toxin EIA result were known; no further epidemiological data were available. Within each hospital, sequences were compared with all sequences from samples obtained in the prior 90 days. Samples from the community and hospital were included to increase the chance of identifying transmission events occurring in hospital but leading to CDI onset after discharge. From previous estimates of C. difficile evolution and within-host diversity [10, 25, 26], ≤2 SNPs are expected between isolates linked by transmission within 90 days. Therefore, where ≥1 prior sequences within ≤2 SNPs were identified, a case was considered to have been potentially acquired from another case. A 90 day threshold for linking cases was chosen assuming that cases were rapidly treated and infectiousness declined, and that subsequent cases related by direct transmission occurred within incubation periods implied by surveillance definitions [8] and previous studies [13]. As the sources of cases occurring at the start of the study may themselves have been sampled before the study started, the proportion of cases linked to a prior case was only calculated for cases occurring after the first 90 days, with cases in the first 90 days included only as potential sources for subsequent cases.

Two differing case definitions were considered. Initially, all patients with culture-positive potentially toxigenic C. difficile were considered “cases” to capture possible transmission events involving potentially toxigenic C. difficile irrespective of fecal-toxin status. The analysis was then repeated restricted only to fecal-toxin-positive CDI cases. For comparisons with previously published data, the same definition and analysis approach was applied to fecal-toxin-positive CDI cases occurring within 90 days in Oxford (September 2007 to December 2010, split by calendar year) [10] and Leeds (August 2010 to April 2012) [11].

Risk Factor Analysis

Univariate logistic regression was used to determine whether a case’s toxin status affected the risk of it being genetically related to a prior case, that is, potentially acquired from another case. Similarly, logistic regression was used to determine whether a case’s fecal-toxin status affected the risk of it being genetically linked to a subsequent case, that is, to assess the relative infectiousness of fecal-toxin-positive and toxin-negative patients.

To assess whether the locally circulating strain mix affected transmission estimates, hospital-specific estimates were adjusted for ribotype using multivariate logistic regression (see Supplement).

Simulations

To estimate the impact of missing data (as not all sampled cases were sequenced at some hospitals), we simulated transmission at a theoretical hospital. We sub-sampled simulated cases and calculated the change in the percentage of cases linked to a prior case as the proportion of missing samples increases (details in Supplement).

RESULTS

Consecutive samples sent for C. difficile testing at 6 hospitals were studied for 12 months (Table 1). In total, 1052/1098 (96%) of GDH/toxin-PCR screen-positive samples were available: 95/98 (97%) at hospital 1, 144/178 (81%) at hospital 2, 118/127 (93%) at hospital 5 and otherwise 100%. 974/1052 (93%) available samples were confirmed as C. difficile on culture. For the 5 hospitals with available testing data, 887/21539 (4.1%) of samples submitted for testing were culture-positive (Table 1); 971/974 (99.7%) culture-positive samples were successfully sequenced. Of sequenced culture-positive samples, 451/971 (46.4%) were EIA fecal-toxin-positive, 35–71% by hospital. By contrast, 851/971 (87.6%) were potentially toxigenic, that is, had toxin genes detected via sequence data. Hence, 400/851 (47.0%) samples containing potentially toxigenic C. difficile did not have fecal-toxin detected. In the 971 sequenced isolates, the most common ribotypes identified were 014, 015, 005, 002, 020, and 078 (Table 2). Ribotype-027(NAP1/ST-1) only accounted for 16 (2%) cases.

To view graphs and tables, please click the following link:

https://academic.oup.com/cid/article/doi/10.1093/cid/cix338/3857742/Comparison-of-Control-of-Clostridium-difficile

Relatedness to Prior Cases

The proportion of cases plausibly linked to a prior case by recent transmission varied by hospital. Of 851 sequenced potentially toxigenic cases, all were considered as potential sources of infection, but only the 652 obtained after the first 90 days of sampling at each hospital were assessed for linkage to a previous case. Across the 6 hospitals, 128/652 (20%, 95% confidence interval [CI] 17–23%) potentially toxigenic cases were genetically linked to a prior case from the previous 90 days. Hospital 2 had the fewest cases linked to a prior case, 7/105 (7%, 3–13%), hospital 1 had an intermediate number, 9/70 (13%, 6–23%), and hospitals 3–6 had similar numbers of linked cases, 37/153 (24%, 18–32%), 32/134 (24%, 17–32%), 18/76 (24%, 15–35%), and 25/113 (22%, 15–31%), respectively. Hospital 2 had significantly fewer linked cases than hospitals 3–6 (P ≤ .002), with weaker evidence for lower rates in hospital 1 than hospitals 3, 4, and 5 (P = .05, .07, .09, respectively). Overall, 48/128 (38%) of potential transmission recipients were fecal-toxin-negative (11–68% across hospitals, Figure 1A). Fecal-toxin detection in a recipient was associated with increased odds of having a potential transmission donor, odds ratio 1.67 (95% CI 1.12–2.48, P = .01).

 

In total, 59/128 (46%) putative transmission recipients were only linked to ≥1 fecal-toxin-positive potential donors, 50 (39%) to only fecal-toxin-negative donors, and 19 (15%) to both toxin-positive and toxin-negative donors. Considering the 667 cases occurring in the first 270 days at each hospital, that is, the cases with an opportunity to transmit to a sampled case within the next 90 days, 120 (18%) were potential donors. Fecal-toxin-positive and -negative cases were similarly infectious: the odds ratio for a fecal-toxin- positive case, compared to a fecal-toxin-negative case, being a potential transmission donor was 1.01 (95% CI 0.68–1.49, P = .97).

When only considering transmission to and from fecal- toxin-positive cases, fewer cases were genetically linked to a previous case within 90 days, 51/335 (15%, 95% CI 12–20%). We observed a different “ranking” of hospitals compared with the above analysis of linkage rates based on potentially toxigenic isolate-positive patients: hospital 3 had the greatest proportion of fecal-toxin-positive cases genetically related to a prior fecal-toxin-positive case, 31% (22–41%), and hospital 6 the lowest, 0% (0–9%) (Figure 1B).

Results were similar to those for all potentially toxigenic C. difficile (Figure 1A) if all C. difficile sequences, nontoxigenic as well as potentially toxigenic, were considered (Figure 1C). Considering only nontoxigenic isolates, very similarly to potentially toxigenic isolates, 19/96 (20%, 95% CI 12–29%) were genetically linked to a prior patient isolate from the previous 90 days.

There was no evidence that the number of linked cases varied during the study at any hospital (Figure 1D). Because different numbers of sequences were obtained from the different hospitals, we investigated how this affected the estimated proportions of cases linked to a prior case. Estimated proportions of linked cases were relatively stable once approximately 50 cases had been sequenced (Figure 2).

Impact of Testing Frequency

The proportion of originally tested samples that were stored and then culture-positive was similar across the 5 hospitals with testing data, 3.8%–4.3% (P = .89, Table 1). In contrast, testing rates ranged from 98 to 239 samples per 10000 bed-days. There was no association between the estimated proportion of cases linked to a previous case within 90 days and testing rates (P = .19 for all potentially toxigenic cases, Figure 3A, and P = .60 for fecal-toxin-positive cases only, Figure 3B). For comparison, Figure 3B also displays rates of linked cases for previously published data from Oxford and Leeds.

Figure 2:  Proportion of potentially toxigenic cases linked to a previous potentially toxigenic case by hospital and number of sequences obtained. Abbreviation: SNP, single-nucleotide polymorphism.

Adjustment for Ribotype

After adjustment for locally circulating ribotypes, estimates of the proportion of potentially toxigenic cases related to a previous potentially toxigenic case within ≤2 SNPs and ≤90 days remained largely unchanged (Figure 4A). Using the same model, per-ribotype estimates for the proportion of related cases, adjusted for differences across hospitals, showed more variation (Figure 4B, Table 2 for unadjusted proportions). Ribotype-027 had significantly more related cases (adjusted proportion, 57%, 95% CI 29–81%, n = 12) than the comparison group of all other ribotypes (11%, 7–18%, P = .002, n = 124), as did ribotype-002 (25%, 15–38%, P = .04, n = 53), 012 (50%, 29–71%, P = .001, n = 22), and 087 (44%, 23–67%, P = .005, n = 18).

Adjustment for Completeness of Testing

As only 144/178 (81%) of GDH-positive samples at hospital 2 were retrievable for culture we assessed the likely impact of these missing samples on the estimated proportion of linked cases by simulating transmission and sampling at a theoretical hospital (Figure S1). As sampling becomes increasingly less complete, the estimated proportion of linked cases declines proportional to the probability of a case being sampled. Applying our simulation to hospital 2 provides a revised estimate of 8% of cases being linked to a prior case (see Supplement for details).

……………………….

DISCUSSION

Here, we demonstrate the value of WGS as a tool to estimate different rates of C. difficile transmission across institutions. Sequencing consecutive C. difficile isolates from routine testing over one year, we found transmission rates varied between 6 hospitals. Considering all patients with potentially toxigenic C. difficile, irrespective of fecal-toxin status, in the best performing hospital only 7% of patients’ isolates were sufficiently genetically related to a previous isolate from another patient to support transmission (8% adjusting for incomplete sampling). By contrast, approximately 3–4-fold more isolates (22–26%) were related in 4 of the other hospitals. These results remained similar after adjusting for the locally circulating strains.

Restricting to only patients with fecal-toxin-positive CDI, we confirmed previous findings that only a minority of CDI cases arise from contact with another symptomatic case: 35% in Oxford [10], 35% in Leeds [11], and 37% of ribotype-027 cases in Liverpool [12], were genetically linked to a previous case, with only a subset of these cases sharing time and space on the same hospital ward.

Applying the criteria for linking cases used in the present study to the Oxford and Leeds data sets, 38% of cases in Oxford were linked to a previous case in 2008 falling to 19% in 2010, and 30% of cases were similarly linked in Leeds. Across the 6 study hospitals, serving a range of populations, toxin-positive CDI linkage rates were all <15% with the exception of hospital 3, where 31% of cases were linked. It is likely the lower linkage rates in the current study in part reflect the falling incidence of ribotype-027 [11], associated with more onward transmission in this study, likely as a result of national fluoroquinolone restriction [27] but may also represent changes in infection prevention and control practice.

Our findings also support the recently reported role in transmission of GDH-positive patients with toxigenic C. difficile, but no detected fecal-toxin [28]. By sequencing all GDH-positive cases, we were able to compare the probability of fecal-toxin-positive and toxin-negative patients being potential sources of transmission, that is, having C. difficile genetically linked to a subsequent C. difficile isolate in another patient. Fecal-toxin-negative patients were similarly infectious to fecal-toxin-positive patients: fecal-toxin status did not affect the odds of being a potential transmission source. Strategies to identify and institute infection control measures around patients with potentially toxigenic C. difficile without detected fecal-toxin are therefore likely to reduce overall CDI incidence, although may be more costly, for example if toxin gene PCR is used as an initial screen rather than GDH EIA. Toxin-positive patients, that is, CDI cases, were more likely to have an identified potential transmission donor, than toxin-negative patients. This is in keeping with previous observations that recent C. difficile acquisition is associated with increased risk of disease, whereas long-term carriage is relatively protective [29].

It is likely that differing clinical CDI testing thresholds applied across the study hospitals, despite each being guided by national recommendations; notably, testing rates varied more than 2-fold between hospitals (98–239 tests/10000 bed-days). However, despite this variation, the overall proportion of samples tested that were C. difficile culture-positive was very similar across hospitals (~4%). These 2 findings combined resulted in varying rates of potentially toxigenic C. difficile isolation, 4.2–8.2/10000 bed-days, and varying (fecal-toxin-positive) CDI rates, 1.8–5.7/10000 bed-days. As the proportion of samples that were C. difficile culture-positive was close to reported community asymptomatic C. difficile colonization rates (~4%), and lower than reported colonization rates in asymptomatic hospital inpatients, (~10%) [30], it is possible that the higher reported CDI rates in some study hospitals may reflect overascertainment; independent assessment of which symptomatic patients are tested for CDI would be required to resolve this with certainty [7]. As designed, the study did not measure the extent of transmission involving asymptomatic patients, and therefore it is likely that not all hospital-associated transmission is captured. However, as this was the case for all hospitals, comparisons can still be made between hospitals and with previous studies investigating symptomatic patients.

Interestingly, we did not find any evidence of a relationship between rates of C. difficile testing and proportions of cases that could be linked to a previous case. Differing sampling/testing will likely mean the study populations at each hospital varied, for example with some institutions potentially more likely to include milder CDI cases than others. It should also be noted that differences in the population sampled by a particular testing strategy may affect the proportion of cases linked differently to incomplete sampling of a given population. We quantified the impact of the latter through simulation. Unfortunately, incomplete sampling could appear very similar to the impact of good infection control, as both results in low proportions of linked cases. One study limitation is that we only sequenced 81% GDH-positive samples at hospital 2. However, we demonstrate it may be possible to adjust for incomplete sampling, providing missed cases as assumed missing at random, and the number of onward transmissions from each case was random.

Both a limitation and a strength of our approach is that it relies only on sequencing laboratory samples and sampling dates. We demonstrate this allows comparative hospital surveillance with very limited, and no personal, sensitive or confidential, data. However, without ward admission and patient contact data, it is possible some genetically linked cases do not represent direct transmission from other cases. Genetic links might also arise through indirect healthcare-associated transmission via unsampled hosts or the hospital environment. Additionally, a minority of cases, without healthcare exposure in the last 90 days, may still have been genetically linked. However, there is no obvious reason why genetically related community C. difficile exposures, and therefore the proportion of such cases linked, should vary across England at a population level, even if other CDI risk factors do vary geographically, for example, antimicrobial use. Therefore, although we analyze transmission within the populations served by each hospital, as most CDI cases have recent healthcare exposure, the overall proportion of linked cases is still likely to be a reasonable combined indicator of infection control performance around cases and more generally. Without patient-level identifiers some repeat tests from the same patient may have been wrongly assigned as transmission events; however, we anticipate this was uncommon; repeat testing within 28 days is discouraged in national guidelines [17], and such samples are frequently not routinely processed.

Our method of comparing infection control performance depends on culturing C. difficile, which is not routinely undertaken, and on sequencing at least 6 months of samples, at around US$100 per sample. However, if samples are stored, as recommended in England, C. difficile could be cultured and sequenced retrospectively if increased incidence was noted and then continued prospectively to monitor the impact of any interventions. The cost-effectiveness of such an approach needs further evaluation.

In summary, here we present a novel method that enables assessment of the extent of hospital-acquired infection transmission within healthcare institutions. This approach revealed differences in CDI transmission rates across 6 English hospitals. It demonstrates the potential of whole-genome sequencing as a nationwide tool to identify institutions with excellent and also suboptimal infection control and therefore has the potential to allow targeted efforts to reduce CDI incidence.

Resources:  https://academic.oup.com/cid/article/doi/10.1093/cid/cix338/3857742/Comparison-of-Control-of-Clostridium-difficile

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

Clostridium difficile Diagnostic Testing Ranging From Most Sensitive to Lower End Sensitivity

labatorymicroscopeimage

Clostridium difficile Diagnostic Testing and C.diff. Background Information

 

 

Background:

According to the CDC statistic reporting 2015, there are 453,000 CDI cases diagnosed each year. 2/3 of the cases are Inpatient HAI only 24% have hospital onset, 23% Long-term care, 18% post discharge.

Rate of Colectomies have increased as high as 6.2% in epidemic periods.

CDI extends inpatient hospital stays by 2.3 to 12 days increasing financial burden by $2,454 to $27,160 EACH CASE.

More than 82% or 8 in 10 individuals are diagnosed with Community-associated CDI and had a recent healthcare exposure such as a Doctor’s office or Dentist office visit within 12 weeks time.

Each Year – 29k patients newly diagnosed with a CDI die within 30 days of a CDI (mostly senior population) 14k patients die each year from a CDI involvement.

Each Year – 83k patients are being treated for Recurrent CDI within 8 weeks of initial onset.

The most prevalent Clostridium strain today is the B1/NAP1/027 – the Hypervirulent Strain vs the 078 strain which was the typical strain.

027 Ribotype is the largest recorded outbreak and fatalities .  Mode of transmission remains the same – Fecal to Oral route transmission and lives on inanimate objects and surfaces longer than 6 months.

According to researchers Merrigan and colleagues (https://www.ncbi.nlm.nih.gov/pubmed/20675495)

Examined the accumulation of spores over the bacterial growth cycle and demonstrated that hypervirulent strains sporulated earlier and accumulated significantly more spores per total volume of culture than non-hypervirulent strains (078).  This increased rate of sporulation may explain the observation of unusually high relapse rates associated with hyupervirulent strains because patients are more likely to contaminate their local environment and subsequently re-infect themselves.  More research needs to be done to confirm this theory and it remains contentious.

Diagnostic Testing – Clostridium difficile (C.diff.):

There are a number of diagnostics and studies continue to create debate and discussions about CDI testing and diagnosis and the connection between testing methods and clinical outcomes.

Per studies and research by Dr.’s Dale Gerding, MD, Dr. M. Thomas, Jr.MD, Dr. Clifford McDonald in January 2016 the Diagnosis and Treatment of Clostridium difficile Infection   Gerding, Dale N. MD*†; File, Thomas M. Jr MD, MSc‡§; McDonald, L. Clifford MD

Infectious Diseases in Clinical Practice: January 2016 – Volume 24 – Issue 1 – p 3–10 doi: 10.1097/IPC.0000000000000350 NFID Clinical Updates

A 2006 survey found that the most common lab test for CDI diagnosis was EIA (Enzyme Immunoassay) for toxins A and B.  48-96 Hours turn around time. 

Found to generate false positives and false negative results.

Today PCR is by far the most common test. It became available for laboratory diagnosis C.diff. associated diarrhea (CDAD) and colitis in 2010.

>PCR = Polymerase Chain Reaction with a 1 day turn around.

>Proven sensitivity of 100%,,  96.9% A/B specific accuracy and superior to A/B EIA testing.

  • The MOST Sensitive test in use today is Culture plus Toxin Confirmation, but it is too SLOW to be of practical use.
  • Nucleic Acid Amplification Test –This test may lead to over diagnosing by detecting colonized patient with diarrhea from another cause (viral or other ).
  • Glutamate dehydrogenase EIA is very sensitive but not specific and cell cytotoxin is also too slow for practical use today.
  • At the lower end of sensitivity are toxins A and B EIA, toxin A  EIA,  GDH latex test, and endoscopy, which is approximately 50% sensitive.

Summary:

If Labs have no clinical input and accept any unformed stool for testing, it may be most appropriate to use a test that better identifies CDI such as a relatively sensitive test for toxin in the stool (eg., cell cytotoxin or GDH = Glutamate dehydrogenase,  coupled with EIA for toxin).

If patients are screened carefully for clinical symptoms associated with a CDI (at least 3 unformed stools within 24 hours plus a history of antibiotic therapy) then a highly sensitive test such as the NAAT or a toxigenic culture, or GDH plus toxin detection may be best.

Neither approaches have been established today – appropriate testing strategy remains a dilemma.

Clinicians should be aware of the test being used in their laboratories. 

 >If a NAAT = Nucleic Acid Amplification Test  (PCR=Polymerase Chain Reaction or LAMP = Loop Mediated Isothermal Amplification) is being used, then they should recognize the potential for over diagnosis, especially if the specimens are sent from patients with minimal diarrhea.

>If EIA toxin testing is being used, it is more likely that a positive test represents CDI, but EIA testing yields FALSE negatives in patients with CDI due to the lack of sensitivity.

A less complicated breakdown of diagnostic testing:

Cultures:  Stool culture for C. diff.  most sensitive test available. 48-96 hours turn around.

Molecular tests:  FDA approved PCR assays, test for the gene encoding toxin B, are highly sensitive and specific for the presence of a toxin-producing Clostridium difficile organism.

Antigen detection:  rapid tests – less than 1 hour – detect presence of C. diff. antigen by latex agglutination or Immunochromatographic assays.  (used often in ER).

Toxin testing – tissue culture cytotoxicity assay detects toxin B only. Costly and requires 24-48 hours for final result. Historical gold standard for diagnosing clinical significant disease caused by C. diff. it is recognized as less sensitive than PCR or culture for detecting the organism in patients with CDI symptoms.

Enzyme immunoassay detects toxin A , toxin B  or both A and B.  Due to concerns overtoxin A-negative, B-positive strains causing disease, most laboratories employ a toxin B only or A and B assay.  Because these are same day assays that are relatively inexpensive and easy to perform, they are popular with clinical labs. There are increasing concerns about their relative insensitivity – less than tissue culture cytotoxicity and much less than the PCR or toxigenic culture.

C. diff. toxin is very unstable. The toxin degrades at room temperature and may be undetectable within 2 hours after collection of a specimen.

False-negative results occur when specimens are not properly tested or kept refrigerated until the testing can be done.

To learn more about how to collect and transport stool specimens to the laboratory click on the link below:

https://cdifffoundation.org/2015/06/17/c-difficile-laboratory-test-information-for-patients-and-healthcare-providers/

Great Basin Scientific — A Molecular Diagnostics Company

About Great Basin Scientific

Great Basin Scientific is a molecular diagnostics company that commercializes breakthrough chip based technologies. The Company is dedicated to the development of simple, yet powerful, sample to – result technology and products that provide fast, multiple pathogen
diagnoses of infectious diseases. The Company’s vision is to make molecular diagnostic testing so simple and cost -­ effective that every patient will be tested for every serious infection, reducing misdiagnoses and significantly limiting the spread of infectious disease. More information can be found on the company’s website at www.gbscience.com

PRODUCT:

Toxigenic Clostridium difficile (C. diff)

The Toxigenic Clostridium difficile (C. diff) assay is a DNA test that detects pathogens from a patient’s raw stool sample. The sample-to-result molecular test detects Toxigenic C. diff by targeting the Toxin B gene (tcdB) with fewer hands-on steps than competitors.

Need for Testing

Toxigenic C. diff causes 15-25% of antibiotic associated diarrhea. Toxigenic C. diff intestinal colonization can cause Clostridium difficile infection (CDI) when antibiotic use disrupts normal intestinal flora. According to the Association for Professionals in Infection Control and Epidemiology, the prevalence and mortality rate of CDI has risen markedly since 2003. Early recognition of CDI with accurate and timely diagnosis is essential to aid vital patient management decisions.

Advantage of Great Basin’s Toxigenic Clostridium difficile Assay over conventional culture methodology

  • Conventional tests have laborious methodology, poor sensitivity and long turnaround times (<96 hours)
  • Great Basin’s diff Assay has easy-to-use molecular methodology, higher sensitivity and a faster turnaround time (<3hours)

FOR MORE PRODUCT INFORMATION :    http://gbscience.com/products/test/c-diff/

Great Basin Scientific, Inc.  announced that it has initiated a clinical trial for its Stool Bacterial Pathogens Panel, a multi-plex molecular assay detecting common bacterial agents, including food-borne pathogens, present in individuals suspected of acute gastroenteritis. The Stool Bacterial Pathogens Panel is the Company’s second multiplex panel.

The Stool Bacterial Pathogens Panel will target nucleic acids of Salmonella species, Shigella species, Shiga toxin-producing E. coli (stx1 and stx2 genes), the E. coli O157 serotype (reported if stx1 and/or stx2 are positive), and Campylobacter species (combined C. jejuni and C. coli).

This is the second start of five clinical trials Great Basin has planned in 2016. The Company began the clinical trial on its Bordetella Direct Test in May of this year. Both assays are on-track with the guidance for trial starts in 2016 that the Company provided in its business update call in April of this year.

“Our second on-time clinical trial start this year highlights the commitment, organization and talent in our R&D group,” said Ryan Ashton, co-founder and Chief Executive Officer of Great Basin Scientific. “We are proud of their accomplishments so far in 2016 and have confidence in their ability to bring our aggressive product development plans to fruition.

Great Basin is particularly excited to begin this trial on another sample-to-result multi-plex panel, which will provide a great value to our customers, and provides significant opportunity to increase our revenue per customer and further demonstrates the unique and powerful versatility of our platform to the market.”

Great Basin’s menu of FDA-cleared sample-to-result assays includes low-plex molecular tests for toxigenic Clostridium difficile (C.diff), Group B Streptococcus (GBS), Shiga toxin-producing E. coli (STEC) detection, and a multi-plex Staph ID/R Blood Culture Panel that identifies Staphylococcus aureus and Staphylococcus species and detects the mecA gene directly from positive blood cultures in about two hours. All assays run on the Great Basin Analyzer

PositiveID Corporation Expands Firefly Dx Testing Capabilities for Hospital Acquired Infection (HAI) Market

PositiveID Corporation (“PositiveID” or “Company”), a developer of biological detection and diagnostics solutions, announced today that it has successfully detected Clostridium difficile (“C. diff”) on its Firefly Dx polymerase chain reaction (“PCR”) breadboard prototype pathogen detection system (“prototype system”) in less than 20 minutes.

The C. diff assay, provided to the Company for testing by partner GenArraytion, Inc., is a more comprehensive and specific test than many other C. diff assays on the market as it tests for the C. diff chromosome as well as both Toxin A and Toxin B.

Clostridium difficile (C. diff.)  is a bacterium that most often affects older patients in hospitals or long-term care facilities after antibiotic use, and causes symptoms ranging from diarrhea to lethal inflammation of the colon.

In addition to C. diff, the Company recently announced its successful detection of

methicillin-resistant Staphylococcus aureus (“MRSA”), another common hospital-acquired infection, on its Firefly Dx prototype system. It has also successfully identified methicillin-susceptible Staphylococcus aureus (“MSSA”).

To read this news article in its entirety click on the following link

https://ca.finance.yahoo.com/news/positiveid-expands-firefly-dx-testing-123000203.html

ROCHE cobas® C. diff. Test approved by US Food and Drug Administration (FDA)

laboratorystillUS Food and Drug Administration (FDA) has provided 510(k) clearance for the cobas® Cdiff Test to detect Clostridium difficile (C. difficile) in stool specimens.

The cobas® Cdiff Test targets the toxin B gene found in toxigenic C. difficile strains directly in specimens from symptomatic patients. The test provides accurate information which assists clinicians in making timely treatment decisions and aids in the prevention of further infection in healthcare settings.

“Having the ability to provide a result quickly is important when supporting infection control for Clostridium difficile,” said Dr. Steve Young, Professor of Pathology, Department of Pathology UNMHSC and Tricore Reference Lab. “The cobas® 4800 System has the capability to allow for mixed batch testing of the cobas® Cdiff Test alongside testing for Methicillin-resistant Staphylococcus aureus, Staphylococcus aureus, and herpes simplex virus 1 and 2*, all on one platform. We can run these assays together at least once in each shift rather than once a day, which can greatly improve laboratory efficiency, ultimately leading to better infection control and patient care.”

In a clinical trial program conducted at sites throughout the United States, the cobas® Cdiff Test demonstrated excellent performance compared to direct and enrichment toxigenic culture. The test combines high assay sensitivity with rapid turnaround time and a minimum number of pre-analytic steps, to facilitate earlier intervention of patients suffering from

C. difficile-associated disease. Earlier intervention can also lead to more effective implementation of infection control measures, which can prevent further transmission to additional patients.

About the cobas® 4800 System
The cobas® 4800 System offers true walk-away automation of nucleic acid purification, PCR set-up and real-time PCR amplification and detection to help laboratories achieve maximum efficiency. The expanding system menu in the U.S. currently includes the cobas® MRSA/SA Test, cobas® CT/NG Test (Chlamydia trachomatis/Neisseria gonorrhoeae), cobas® HPV Test, cobas® BRAF V600 Mutation Test, cobas® EGFR Mutation Test and cobas® KRAS Mutation Test.

“With the addition of the cobas® Cdiff Test to the cobas® 4800 System menu, Roche is able to expand the tools available to assist clinicians in the management of healthcare associated infections,” said Paul Brown, head of Roche Molecular Diagnostics. “The cobas® Cdiff Test requires less sample handling and provides laboratories with a simplified workflow, when compared to other molecular methods. It also delivers a lower inhibition rate, which means fewer repeat samples and chances for error, enabling better patient care.”

 

To access the news article:

http://finance.yahoo.com/news/roche-receives-fda-clearance-cobas-050000123.html

 

Clostridium difficile (C.diff.) Testing Development and Validation of an Internationally-Standardized, High-Resolution Capillary Gel-Based Electrophoresis PCR-Ribotyping Protocol

c-diff

Authors:

Abstract

PCR-ribotyping has been adopted in many laboratories as the method of choice for C. difficile typing and surveillance. However, issues with the conventional agarose gel-based technique, including inter-laboratory variation and interpretation of banding patterns have impeded progress.

The method has recently been adapted to incorporate high-resolution capillary gel-based electrophoresis (CE-ribotyping), so improving discrimination, accuracy and reproducibility.

However, reports to date have all represented single-center studies and inter-laboratory variability has not been formally measured or assessed.

Here, we achieved in a multi-center setting a high level of reproducibility, accuracy and portability associated with a consensus CE-ribotyping protocol. Local databases were built at four participating laboratories using a distributed set of 70 known PCR-ribotypes.

A panel of 50 isolates and 60 electronic profiles (blinded and randomized) were distributed to each testing center for PCR-ribotype identification based on local databases generated using the standard set of 70 PCR-ribotypes, and the performance of the consensus protocol assessed.

A maximum standard deviation of only ±3.8bp was recorded in individual fragment sizes, and PCR-ribotypes from 98.2% of anonymised strains were successfully discriminated across four ribotyping centers spanning Europe and North America (98.8% after analyzing discrepancies). Consensus CE-ribotyping increases comparability of typing data between centers and thereby facilitates the rapid and accurate transfer of standardized typing data to support future national and international C. difficile surveillance programs.

 

For article/abstract in its entirety please click on the following link:

http://www.ncbi.nlm.nih.gov/pubmed/25679978?dopt=Abstract