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1.  Detection of Toxigenic Clostridium difficile: Comparison of the Cell Culture Neutralization, Xpert C. difficile, Xpert C. difficile/Epi, and Illumigene C. difficile Assays 
Journal of Clinical Microbiology  2012;50(4):1331-1335.
Clostridium difficile is the most important cause of nosocomial diarrhea. Several laboratory techniques are available to detect C. difficile toxins or the genes that encode them in fecal samples. We evaluated the Xpert C. difficile and Xpert C. difficile/Epi (Cepheid, CA) that detect the toxin B gene (tcdB) and tcdB, cdt, and a deletion in tcdC associated with the 027/NAP1/BI strain, respectively, by real-time PCR, and the Illumigene C. difficile (Meridian Bioscience, Inc.) that detects the toxin A gene (tcdA) by loop-mediated isothermal amplification in stool specimens. Toxigenic culture was used as the reference method for discrepant stool specimens. Two hundred prospective and fifty retrospective diarrheal stool specimens were tested simultaneously by the cell cytotoxin neutralization assay (CCNA) and the Xpert C. difficile, Xpert C. difficile/Epi, and Illumigene C. difficile assays. Of the 200 prospective stools tested, 10.5% (n = 23) were determined to be positive by CCNA, 17.5% (n = 35) were determined to be positive by Illumigene C. difficile, and 21.5% (n = 43) were determined to be positive by Xpert C. difficile and Xpert C. difficile/Epi. Of the 50 retrospective stools, previously determined to be positive by CCNA, 94% (n = 47) were determined to be positive by Illumigene C. difficile and 100% (n = 50) were determined to be positive by Xpert C. difficile and Xpert C. difficile/Epi. Of the 11 discrepant results (i.e., negative by Illumigene C. difficile but positive by Xpert C. difficile and Xpert C. difficile/Epi), all were determined to be positive by the toxigenic culture. A total of 21% of the isolates were presumptively identified by the Xpert C. difficile/Epi as the 027/NAP1/BI strain. The Xpert C. difficile and Xpert C. difficile/Epi assays were the most sensitive, rapid, and easy-to use assays for the detection of toxigenic C. difficile in stool specimens.
doi:10.1128/JCM.06597-11
PMCID: PMC3318532  PMID: 22278839
2.  Evaluation of the Cepheid Xpert Clostridium difficile Epi Assay for Diagnosis of Clostridium difficile Infection and Typing of the NAP1 Strain at a Cancer Hospital ▿  
Journal of Clinical Microbiology  2010;48(12):4519-4524.
Clostridium difficile is the most common cause of health care-associated diarrhea. Accurate and rapid diagnosis is essential to improve patient outcome and prevent disease spread. We compared our two-step diagnostic algorithm, an enzyme immunoassay for glutamate dehydrogenase (GDH) followed by the cytotoxin neutralization test (CYT) with a turnaround time of 24 to 48 h, versus the Cepheid Xpert C. difficile Epi assay, a PCR-based assay with a turnaround time of <1 h. In the first phase of the study, only GDH-positive stool samples were tested by both CYT and Xpert PCR. Discordant results were resolved by toxigenic culture. In the second phase, all stool samples were tested by GDH and Xpert PCR. Only GDH-positive stools were further tested by CYT. Genotypic characterization of 45 Xpert PCR-positive stools was performed by sequencing of the tcdC gene and PCR ribotyping. In phase 1, the agreement between the GDH-CYT and the GDH-Xpert PCR was 72%. The sensitivities and specificities of GDH-CYT and GDH-Xpert PCR were 57% and 97% and 100% and 97%, respectively. In phase 2, the agreement between GDH-CYT and Xpert PCR alone was 95%. As in phase 1, sensitivity of the Xpert PCR was higher than that of the GDH-CYT. The correlation between PCR-ribotyping, sequencing, and Xpert PCR for detection of NAP1 strains was excellent (>90%). The excellent sensitivity and specificity and the rapid turnaround time of the Xpert PCR assay as well as its strain-typing capability make it an attractive option for diagnosis of C. difficile infection.
doi:10.1128/JCM.01648-10
PMCID: PMC3008447  PMID: 20943860
3.  Impact of Strain Type on Detection of Toxigenic Clostridium difficile: Comparison of Molecular Diagnostic and Enzyme Immunoassay Approaches ▿  
Journal of Clinical Microbiology  2010;48(10):3719-3724.
A multicenter clinical trial assessed the performance of the Cepheid Xpert C. difficile assay on stool specimens collected from patients suspected of having Clostridium difficile infection (CDI). A total of 2,296 unformed stool specimens, collected from seven study sites, were tested by Xpert C. difficile enrichment culture followed by cell culture cytotoxicity testing of the isolates (i.e., toxigenic culture with enrichment) and the study sites' standard C. difficile test methods. The methods included enzyme immunoassay (EIA), direct cytotoxin testing, and two- and three-step algorithms using glutamate dehydrogenase (GDH) screening followed by either EIA or EIA and an in-house PCR assay. All C. difficile strains were typed by PCR-ribotyping. Compared to results for toxigenic culture with enrichment, the sensitivity, specificity, and positive and negative predictive values of the Xpert assay were 93.5, 94.0, 73.0, and 98.8%, respectively. The overall sensitivity of the EIAs compared to that of enrichment culture was 60.0%, and the sensitivity of combined GDH algorithms was 72.9%; both were significantly lower than that of Xpert C. difficile (P < 0.001 and P = 0.03, respectively). The sensitivity of the EIA was significantly lower than that of the Xpert C. difficile assay for detection of ribotypes 002, 027, and 106 (P < 0.0001, P < 0.0001, and P = 0.004, respectively, Fisher's exact test), and the sensitivity of GDH algorithms for ribotypes other than 027 was lower than that for Xpert C. difficile (P < 0.001). The Xpert C. difficile assay is a simple, rapid, and accurate method for detection of toxigenic C. difficile in unformed stool specimens and is minimally affected by strain type compared to EIA and GDH-based methods.
doi:10.1128/JCM.00427-10
PMCID: PMC2953097  PMID: 20702676
4.  Comparison of Strain Typing Results for Clostridium difficile Isolates from North America▿ 
Journal of Clinical Microbiology  2011;49(5):1831-1837.
Accurate strain typing is critical for understanding the changing epidemiology of Clostridium difficile infections. We typed 350 isolates of toxigenic C. difficile from 2008 to 2009 from seven laboratories in the United States and Canada. Typing was performed by PCR-ribotyping, pulsed-field gel electrophoresis (PFGE), and restriction endonuclease analysis (REA) of whole-cell DNA. The Cepheid Xpert C. difficile test for presumptive identification of 027/NAP1/BI isolates was also tested directly on original stool samples. Of 350 isolates, 244 (70%) were known PCR ribotypes, 224 (68%) were 1 of 8 common REA groups, and 187 (54%) were known PFGE types. Eighty-four isolates typed as 027, NAP1, and BI, and 83 of these were identified as presumptive 027/NAP1/BI by Xpert C. difficile. Eight additional isolates were called presumptive 027/NAP1/BI by Xpert C. difficile, of which three were ribotype 027. Five PCR ribotypes contained multiple REA groups, and three North American pulsed-field (NAP) profiles contained both multiple REA groups and PCR ribotypes. There was modest concordance of results among the three methods for C. difficile strains, including the J strain (ribotype 001 and PFGE NAP2), the toxin A-negative 017 strain (PFGE NAP9 and REA type CF), the 078 animal strain (PFGE NAP7 and REA type BK), and type 106 (PFGE NAP11 and REA type DH). PCR-ribotyping, REA, and PFGE provide different but overlapping patterns of strain clustering. Unlike the other methods, the Xpert C. difficile 027/NAP1/BI assay gave results directly from stool specimens, required only 45 min to complete, but was limited to detection of a single strain type.
doi:10.1128/JCM.02446-10
PMCID: PMC3122689  PMID: 21389155
5.  Clostridium difficile Testing in the Clinical Laboratory by Use of Multiple Testing Algorithms ▿  
Journal of Clinical Microbiology  2010;48(3):889-893.
The incidence of Clostridium difficile infection (CDI) has risen almost 3-fold in the United States over the past decade, emphasizing the need for rapid and accurate tests for CDI. The Cepheid Xpert C. difficile assay is an integrated, closed, nucleic acid amplification system that automates sample preparation and real-time PCR detection of the toxin B gene (tcdB). A total of 432 stool specimens from symptomatic patients were tested by a glutamate dehydrogenase (GDH) assay, a toxin A and B enzyme immunoassay (EIA), the Xpert C. difficile assay, and a cell culture cytotoxicity neutralization assay (CCCN). The results of these methods, used individually and in combination, were compared to those of toxigenic culture. Results for the Xpert C. difficile assay alone showed a sensitivity, specificity, positive predictive value, and negative predictive value (NPV) of 94.4, 96.3, 84.0, and 98.8%, while the EIA alone gave corresponding values of 58.3, 94.7, 68.9, and 91.9%, respectively. An algorithm using the GDH assay and the EIA (plus the CCCN if the EIA was negative) showed corresponding values of 83.1, 96.7, 83.1, and 96.1%. The Xpert C. difficile assay was statistically superior to the EIA (P, <0.001 by Fisher's exact test) and to the GDH-EIA-CCCN algorithm (P, 0.0363). Combining the GDH and Xpert C. difficile assays lowered both the sensitivity and the NPV of the Xpert assay. The GDH-EIA-CCCN procedure required, on average, 2 days to complete testing on GDH-positive results, while testing by the Xpert C. difficile assay was completed, on average, in less than 1 h. Xpert C. difficile testing yielded the highest sensitivity and NPV, in the least amount of time, of the individual- and multiple-test algorithms evaluated in this study.
doi:10.1128/JCM.01801-09
PMCID: PMC2832460  PMID: 20071552
6.  Indeterminate tcdB using a Clostridium difficile PCR assay: a retrospective cohort study 
BMC Infectious Diseases  2013;13:324.
Background
C. difficile (CD) real-time polymerase chain reaction (PCR) for toxin B gene (tcdB) is more sensitive, and reduces turnaround time when compared to toxin immunoassay. We noted typical amplification curves with high tcdB cycle thresholds (Ct) and low endpoints (Ept) that are labeled negative by the Xpert® C. difficile assay (Cepheid) and undertook this study to determine their significance.
Methods
We defined an indeterminate CD assay result as detection of a typical PCR amplification curve with an Ept >10 that was interpreted as negative by the Xpert® assay. Samples with indeterminate Xpert® result were collected for 5 months and retested by Xpert®, cultured for toxigenic CD, and isolates subjected to PCR ribotyping, detection of toxin genes and multilocus variable-number tandem repeat analysis (MLVA) typing. Chart reviews were completed to assess if patients met the Society of Healthcare Epidemiology of America and the Infectious Diseases Society of America CD infection (CDI) clinical case definition. Illness severity was compared with tcdB Ct and culture results.
Results
During the 5-month study period, 48/3620 (1%) of specimens were indeterminate and 387/3620 (11%) were positive. Of the 48 patients with indeterminate results, 39 (81%) met the clinical case definition of CDI, and 7 of these (18%) met criteria for severe CDI. Toxigenic stool cultures were positive for 86% (6/7) of patients with severe CDI, 19% (6/32) of patients with non-severe CDI, and 44% (4/9) of patients who did not meet the clinical case definition of CDI (p = 0.002). Lower tcdB Ct and higher Ept were associated with greater likelihood of toxigenic culture positivity (p = 0.03) and more severe symptoms (p = 0.06). Indeterminate results were not associated with a particular technologist or instrument module, or CD strain type.
Conclusions
A subset of specimens (1%) using the Xpert® C. difficile assay have typical amplification curves and are interpreted as negative. At least one-third of these results are associated with positive CD culture. The mechanism of these indeterminate results is not technique-related, equipment-related, or due to particular CD strains. Clinicians should be aware that even PCR testing has the potential to miss CDI cases and further highlights the importance of clinical context when interpreting results.
doi:10.1186/1471-2334-13-324
PMCID: PMC3718660  PMID: 23865713
Clostridium difficile; Polymerase chain reaction; Toxin B gene; False negative results
7.  Comparison of Xpert MTB/RIF with Other Nucleic Acid Technologies for Diagnosing Pulmonary Tuberculosis in a High HIV Prevalence Setting: A Prospective Study 
PLoS Medicine  2011;8(7):e1001061.
In this prospective, real-world cohort study nested within a national screening program for tuberculosis, Lesley Scott and colleagues compare the performance of Xpert MTB/RIF on a single sputum sample with different TB sputum detection technologies.
Background
The Xpert MTB/RIF (Cepheid) non-laboratory-based molecular assay has potential to improve the diagnosis of tuberculosis (TB), especially in HIV-infected populations, through increased sensitivity, reduced turnaround time (2 h), and immediate identification of rifampicin (RIF) resistance. In a prospective clinical validation study we compared the performance of Xpert MTB/RIF, MTBDRplus (Hain Lifescience), LightCycler Mycobacterium Detection (LCTB) (Roche), with acid fast bacilli (AFB) smear microscopy and liquid culture on a single sputum specimen.
Methods and Findings
Consecutive adults with suspected TB attending a primary health care clinic in Johannesburg, South Africa, were prospectively enrolled and evaluated for TB according to the guidelines of the National TB Control Programme, including assessment for smear-negative TB by chest X-ray, clinical evaluation, and HIV testing. A single sputum sample underwent routine decontamination, AFB smear microscopy, liquid culture, and phenotypic drug susceptibility testing. Residual sample was batched for molecular testing. For the 311 participants, the HIV prevalence was 70% (n = 215), with 120 (38.5%) culture-positive TB cases. Compared to liquid culture, the sensitivities of all the test methodologies, determined with a limited and potentially underpowered sample size (n = 177), were 59% (47%–71%) for smear microscopy, 76% (64%–85%) for MTBDRplus, 76% (64%–85%) for LCTB, and 86% (76%–93%) for Xpert MTB/RIF, with specificities all >97%. Among HIV+ individuals, the sensitivity of the Xpert MTB/RIF test was 84% (69%–93%), while the other molecular tests had sensitivities reduced by 6%. TB detection among smear-negative, culture-positive samples was 28% (5/18) for MTBDRplus, 22% (4/18) for LCTB, and 61% (11/18) for Xpert MTB/RIF. A few (n = 5) RIF-resistant cases were detected using the phenotypic drug susceptibility testing methodology. Xpert MTB/RIF detected four of these five cases (fifth case not tested) and two additional phenotypically sensitive cases.
Conclusions
The Xpert MTB/RIF test has superior performance for rapid diagnosis of Mycobacterium tuberculosis over existing AFB smear microscopy and other molecular methodologies in an HIV- and TB-endemic region. Its place in the clinical diagnostic algorithm in national health programs needs exploration.
Please see later in the article for the Editors' Summary
Editors' Summary
Background
Tuberculosis (TB)—a contagious bacterial infection that mainly affects the lungs—is a global public health problem. In 2009, 9.4 million people developed TB, and 1.7 million people died from the disease; a quarter of these deaths were in HIV-positive individuals. People who are infected with HIV, the virus that causes AIDS, are particularly susceptible to TB because of their weakened immune system. Consequently, TB is a leading cause of illness and death among people living with HIV. TB is caused by Mycobacterium tuberculosis, which is spread in airborne droplets when people with the disease cough or sneeze. Its characteristic symptoms are a persistent cough, night sweats, and weight loss. Diagnostic tests for TB include sputum smear analysis (the microscopic examination of mucus brought up from the lungs by coughing for the presence of M. tuberculosis) and mycobacterial liquid culture (in which bacteriologists try to grow M. tuberculosis from sputum samples and test its drug sensitivity). TB can usually be cured by taking several powerful drugs daily for at least six months.
Why Was This Study Done?
Mycobacterial culture is a sensitive but slow way to diagnose TB. To halt the disease's spread, it is essential that TB—particularly TB that is resistant to several treatment drugs (multidrug-resistant, or MDR, TB)—is diagnosed quickly. Recently, several nucleic acid amplification technology (NAAT) tests have been developed that rapidly detect M. tuberculosis DNA in patient samples and look for DNA changes that make M. tuberculosis drug-resistant. In December 2010, the World Health Organization (WHO) endorsed Xpert MTB/RIF—an automated DNA test that detects M. tuberculosis and rifampicin resistance (an indicator of MDR TB) within two hours—for the investigation of patients who might have TB, especially in regions where MDR TB and HIV infection are common. TB diagnosis in HIV-positive people can be difficult because they are more likely to have smear-negative TB than HIV-negative individuals. In this prospective study, the researchers compare the performance of Xpert MTB/RIF on a single sputum sample with that of smear microscopy, liquid culture, and two other NAAT tests (MTBDRplus and LightCycler Mycobacterium Detection) in adults who might have TB in Johannesburg (South Africa), a region where many adults are HIV-positive.
What Did the Researchers Do and Find?
The researchers evaluated adults with potential TB attending a primary health care clinic for TB according to national guidelines and determined their HIV status. A sputum sample from 311 participants underwent smear microscopy, liquid culture, and drug susceptibility testing; 177 samples were also tested for TB using NAAT tests. They found that 70% of the participants were HIV-positive and 38.5% had culture-positive TB. Compared to liquid culture, smear microscopy, MTBDRplus, LightCycler Mycobacterium Detection, and Xpert MTB/RIF had sensitivities of 59%, 76%, 76%, and 86%, respectively. That is, assuming that liquid culture detected everyone with TB, Xpert MTB/RIF detected 86% of the cases. The specificity of all the tests compared to liquid culture was greater than 97%. That is, they all had a low false-positive rate. Among people who were HIV-positive, the sensitivity of Xpert MTB/RIF was 84%; the sensitivities of the other NAAT tests were 70%. Moreover, Xpert MTB/RIF detected TB in 61% of smear-negative, culture-positive samples, whereas the other NAATs detected TB in only about a quarter of these samples. Finally, although some TB cases were identified as drug-resistant by one test but drug-sensitive by another, the small number of drug-resistant cases means no firm conclusions can be made about the accuracy of drug resistance determination by the various tests.
What Do These Findings Mean?
Although these findings are likely to be affected by the study's small size, they suggest that Xpert MTB/RIF may provide a more accurate rapid diagnosis of TB than smear microscopy and other currently available NAAT tests in regions where HIV and TB are endemic (i.e., always present). Indeed, the reported accuracy of Xpert MTB/RIF for TB diagnosis—85% sensitivity and 97% specificity—has the potential to save more than 400,000 lives per year. Taken together with the results of other recent studies (including an accompanying article by Lawn et al. that investigates the use of Xpert MTB/RIF for screening for HIV-associated TB and rifampicin resistance), these findings support the WHO recommendation that Xpert MTB/RIF, rather than smear microscopy, should be the initial test in HIV-infected individuals who might have TB.
Additional Information
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001061.
This study is further discussed in a PLoS Medicine Perspective by Carlton Evans; a related PLoS Medicine Research Article by Lawn et al. is also available
WHO provides information (in several languages) on all aspects of tuberculosis, including general information on tuberculosis diagnostics and specific information on the Xpert MTB/RIF test; further information about WHO's endorsement of Xpert MTB/RIF is included in a recent Strategic and Technical Advisory Group for Tuberculosis report
WHO also provides information about tuberculosis and HIV
The US National Institute of Allergy and Infectious Diseases has detailed information on tuberculosis and HIV/AIDS
The US Centers for Disease Control and Prevention also has information about tuberculosis, including information on the diagnosis of and on tuberculosis and HIV co-infection
Information is available from Avert, an international AIDS charity on many aspects of HIV/AIDS, including information on HIV-related tuberculosis (in English and Spanish)
doi:10.1371/journal.pmed.1001061
PMCID: PMC3144192  PMID: 21814495
8.  Screening for HIV-Associated Tuberculosis and Rifampicin Resistance before Antiretroviral Therapy Using the Xpert MTB/RIF Assay: A Prospective Study 
PLoS Medicine  2011;8(7):e1001067.
In a prospective study, Stephen Lawn and colleagues find that pre-ART screening with Xpert MTB/RIF increased tuberculosis case detection by 45% compared to smear microscopy in HIV-positive patients at high risk of TB risk. AE competing interests must also pull through to the proof. “The Academic Editor, Madhukar Pai, declares that he consults for the Bill & Melinda Gates Foundation (BMGF). The BMGF supported FIND which was involved in the development of the Xpert MTB/RIF assay. He also co-chairs the Stop TB Partnership's New Diagnostics Working Group that was involved in the WHO endorsement of the Xpert assay.” Linked: Scott pmed.1001061; Evans pmed.1001064; Dowdy pmed.1001063
Background
The World Health Organization has endorsed the Xpert MTB/RIF assay for investigation of patients suspected of having tuberculosis (TB). However, its utility for routine TB screening and detection of rifampicin resistance among HIV-infected patients with advanced immunodeficiency enrolling in antiretroviral therapy (ART) services is unknown.
Methods and Findings
Consecutive adult HIV-infected patients with no current TB diagnosis enrolling in an ART clinic in a South African township were recruited regardless of symptoms. They were clinically characterised and invited to provide two sputum samples at a single visit. The accuracy of the Xpert MTB/RIF assay for diagnosing TB and drug resistance was assessed in comparison with other tests, including fluorescence smear microscopy and automated liquid culture (gold standard) and drug susceptibility testing. Of 515 patients enrolled, 468 patients (median CD4 cell count, 171 cells/µl; interquartile range, 102–236) produced at least one sputum sample, yielding complete sets of results from 839 samples. Mycobacterium tuberculosis was cultured from 81 patients (TB prevalence, 17.3%). The overall sensitivity of the Xpert MTB/RIF assay for culture-positive TB was 73.3% (specificity, 99.2%) compared to 28.0% (specificity, 100%) using smear microscopy. All smear-positive, culture-positive disease was detected by Xpert MTB/RIF from a single sample (sensitivity, 100%), whereas the sensitivity for smear-negative, culture-positive TB was 43.4% from one sputum sample and 62.3% from two samples. Xpert correctly identified rifampicin resistance in all four cases of multidrug-resistant TB but incorrectly identified resistance in three other patients whose disease was confirmed to be drug sensitive by gene sequencing (specificity, 94.1%; positive predictive value, 57%).
Conclusions
In this population of individuals at high risk of TB, intensive screening using the Xpert MTB/RIF assay increased case detection by 45% compared with smear microscopy, strongly supporting replacement of microscopy for this indication. However, despite the ability of the assay to rapidly detect rifampicin-resistant disease, the specificity for drug-resistant TB was sub-optimal.
Please see later in the article for the Editors' Summary
Editors' Summary
Background
Tuberculosis (TB)—a contagious bacterial infection that mainly affects the lungs—is a leading cause of illness and death among people who are infected with HIV, the virus that causes AIDS by destroying the immune system, which leaves infected individuals susceptible to other infections. TB is caused by Mycobacterium tuberculosis, which is spread in airborne droplets when people with the disease cough or sneeze. Its symptoms include a persistent cough, weight loss, and night sweats. Diagnostic tests for TB include chest X-rays, sputum smear analysis (microscopic examination of mucus coughed up from the lungs for M. tuberculosis bacilli), and mycobacterial liquid culture (the growth of M. tuberculosis from sputum and determination of its drug sensitivity). TB can be cured by taking several drugs daily for six months, although the recent emergence of multidrug-resistant TB (MDR-TB) is making the disease increasingly hard to treat.
Why Was This Study Done?
TB is a major problem in clinics that provide antiretroviral therapy (ART) for HIV-positive people in resource-limited settings. Not only is it a major cause of sickness and mortality in those affected by it, but TB (especially MDR-TB) can also spread to other patients attending the same clinic for health services. Rapid diagnosis and appropriate treatment are very important to reduce these risks. Unfortunately, sputum smear analysis—the mainstay of TB diagnosis in resource-limited settings—only detects about a fifth of TB cases when used as a screening tool before initiating ART. Chest X-rays are costly and don't always detect TB, and liquid culture—the gold standard method for TB diagnosis—is costly, technically difficult, and slow. Consequently, the World Health Organization (WHO) recently endorsed a new test for the investigation of patients suspected of having TB, especially in regions where HIV infection and MDR-TB are common. Xpert MTB/RIF is an automated DNA test that detects M. tuberculosis and DNA differences that make the bacteria resistant to the drug rifampicin (an indicator of MDR-TB) within 2 hours. In this study, the researchers investigate whether Xpert MTB/RIF could be used as a routine screening test to increase TB detection among HIV-positive people initiating ART.
What Did the Researchers Do and Find?
The researchers collected sputum from HIV-infected adults with no current TB diagnosis enrolling at an ART clinic in a South African township where HIV infection and TB are both common. They then compared the diagnostic accuracy of Xpert MTB/RIF (performed at a centralized laboratory) with that of several other tests, including liquid culture (the reference test). Nearly a fifth of the patients had culture-positive TB. Xpert MTB/RIF identified three-quarters of these patients (a sensitivity of 73.3%). By contrast, the sensitivity of smear microscopy was 28%. The new test's specificity (the proportion of patients with a negative Xpert MTB/RIF result among patients without TB) was 99.2%. That is, Xpert MTB/RIF had a low false-positive rate. Notably, Xpert MTB/RIF detected all cases of smear-positive, culture-positive TB but only 43.4% of smear-negative, culture-positive cases from a single sputum sample; it detected 62.3% of such cases when two sputum samples were analyzed. Finally, Xpert MTB/RIF correctly identified rifampicin resistance in all four patients who had MDR-TB but incorrectly identified resistance in three patients with drug-sensitive TB.
What Do These Findings Mean?
In this population of HIV-positive patients with a high TB risk, pre-ART screening with Xpert MTB/RIF increased case detection by 45% compared to smear microscopy, a finding that needs confirming in other settings. Importantly, Xpert MTB/RIF reduced the delay in diagnosis of TB from more than 20 days to two days. This delay would be reduced further by doing the assay at ART clinics rather than at a centralized testing facility, but the diagnostic accuracy of point-of-care testing needs evaluating. Overall, these findings (and those of an accompanying article by Scott et al. that examines the performance of Xpert MTB/RIF in an area where HIV infection is common) support the replacement of smear microscopy with Xpert MTB/RIF for pre-ART TB screening (provided misdiagnosis of rifampicin resistance can be reduced). These findings also suggest that routine screening with Xpert MTB/RIF could reduce the risk of MDR-TB outbreaks in HIV care and treatment settings and improve outcomes for HIV-positive patients with MDR-TB who currently often die before a diagnosis of TB can be made.
Additional Information
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001056.
This study is further discussed in a PLoS Medicine Perspective by Carlton Evans; a related PLoS Medicine Research Article by Scott et al. is also available
WHO provides information (in several languages) on all aspects of tuberculosis, including general information on tuberculosis diagnostics and specific information on the Xpert MTB/RIF test; further information about WHO's endorsement of Xpert MTB/RIF is included in a recent Strategic and Technical Advisory Group for Tuberculosis report
WHO also provides information about tuberculosis and HIV
The US National Institute of Allergy and Infectious Diseases has detailed information on tuberculosis and HIV/AIDS
The US Centers for Disease Control and Prevention also has information about tuberculosis, including information on the diagnosis of and on tuberculosis and HIV co-infection
Information is available from Avert, an international AIDS charity, on many aspects of HIV/AIDS, including information on HIV-related tuberculosis (in English and Spanish)
doi:10.1371/journal.pmed.1001067
PMCID: PMC3144215  PMID: 21818180
9.  Evaluation of Four Different Diagnostic Tests To Detect Clostridium difficile in Piglets▿ 
Journal of Clinical Microbiology  2011;49(5):1816-1821.
Clostridium difficile is emerging as pathogen in both humans and animals. In 2000 it was described as one of the causes of neonatal enteritis in piglets, and it is now the most common cause of neonatal diarrhea in the United States. In Europe, C. difficile infection (CDI) in both neonatal piglets and adult sows has also been reported. Diagnosis of this infection is based on detection of the bacterium C. difficile or its toxins A and B. Most detection methods, however, are only validated for diagnosing human infections. In this study three commercially available enzyme immunoassays (EIAs) and a commercial real-time-PCR (Becton, Dickinson, and Company) were evaluated by testing 172 pig fecal specimens (139 diarrheic and 33 nondiarrheic piglets). The results of each test were compared to those of cytotoxicity assays (CTAs) and toxigenic culture as the “gold standards.” Compared to CTAs, the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were, respectively, as follows: for real-time PCR, 91.6, 37.1, 57.6, and 82.5%; for Premier Toxins A&B (Meridian), 83.1, 31.5, 53.1, and 66.7%; for ImmunoCard Toxins A&B kit (ICTAB; Meridian), 86.6, 56.8, 66.9, and 80.7%; and for VIDAS (bioMérieux), 54.8, 92.6, 85.0, and 72.8%. Compared to toxigenic culture, the sensitivity, specificity, PPV, and NPV were, respectively, as follows: for real-time PCR, 93.0, 34.7, 50.0, and 87.5%; for Premier Toxins A&B, 80.3, 27.7, 43.8, and 66.7%; and for ICTAB, 80.0, 46.2, 52.8, and 75.4%; and for VIDAS, 56.4, 89.8, 77.5, and 76.7%. We conclude that all tests had an unacceptably low performance as a single test for the detection of C. difficile in pig herds and that a two-step algorithm is necessary, similar to that in cases of human CDI. Of all of the assays, the real-time PCR had the highest NPV compared to both reference methods and is therefore the most appropriate test to screen for the absence of C. difficile in pigs as a first step in the algorithm. The second step would be a confirmation of the positive results by toxigenic culture.
doi:10.1128/JCM.00242-11
PMCID: PMC3122649  PMID: 21411571
10.  Evaluation of a New Automated Homogeneous PCR Assay, GenomEra C. difficile, for Rapid Detection of Toxigenic Clostridium difficile in Fecal Specimens 
Journal of Clinical Microbiology  2013;51(9):2908-2912.
We evaluated a new automated homogeneous PCR assay to detect toxigenic Clostridium difficile, the GenomEra C. difficile assay (Abacus Diagnostica, Finland), with 310 diarrheal stool specimens and with a collection of 33 known clostridial and nonclostridial isolates. Results were compared with toxigenic culture results, with discrepancies being resolved by the GeneXpert C. difficile PCR assay (Cepheid). Among the 80 toxigenic culture-positive or GeneXpert C. difficile assay-positive fecal specimens, 79 were also positive with the GenomEra C. difficile assay. Additionally, one specimen was positive with the GenomEra assay but negative with the confirmatory methods. Thus, the sensitivity and specificity were 98.8% and 99.6%, respectively. With the culture collection, no false-positive or -negative results were observed. The analytical sensitivity of the GenomEra C. difficile assay was approximately 5 CFU per PCR test. The short hands-on (<5 min for 1 to 4 samples) and total turnaround (<1 h) times, together with the high positive and negative predictive values (98.8% and 99.6%, respectively), make the GenomEra C. difficile assay an excellent option for toxigenic C. difficile detection in fecal specimens.
doi:10.1128/JCM.01083-13
PMCID: PMC3754623  PMID: 23804386
11.  Evaluation of a Rapid Membrane Enzyme Immunoassay for the Simultaneous Detection of Glutamate Dehydrogenase and Toxin for the Diagnosis of Clostridium difficile Infection 
Annals of Laboratory Medicine  2014;34(3):235-239.
We evaluated the new C. DIFF QUIK CHEK COMPLETE (CD COMPLETE; TechLab, USA), which is a rapid membrane enzyme immunoassay that uses a combination of glutamate dehydrogenase (GDH) antigen and toxin A and B detection. A total of 608 consecutive loose stool specimens collected from the patients with suspected Clostridium difficile infection (CDI) from August to December 2012 were subjected to the CD COMPLETE and VIDAS Clostridium difficile A & B (VIDAS CDAB; bioMérieux, France). Their performances were compared with a toxigenic culture as a reference. Stool specimens that were culture-negative and CD COMPLETE- or VIDAS CDAB-positive were analyzed by using an enrichment procedure. In comparison to the toxigenic cultures, sensitivity, specificity, positive predictive values (PPV), and negative predictive values (NPV) were 63.6%, 98.0%, 76.1%, and 96.4%, respectively, for the CD COMPLETE-toxin and 75.5%, 97.4%, 72.5%, and 97.8%, respectively, for the VIDAS CDAB. In comparison to the enriched C. difficile cultures, the sensitivity, specificity, PPV, and NPV for the CD COMPLETE-GDH were 91.0%, 92.4%, 70.5%, and 98.1%, respectively. The CD COMPLETE is a reliable method for the diagnosis of CDI and provides greater sensitivity than toxin enzyme immunoassay alone. Furthermore, the CD COMPLETE-GDH has advantages over direct culture in detecting C. difficile.
doi:10.3343/alm.2014.34.3.235
PMCID: PMC3999323  PMID: 24790912
Glutamate dehydrogenase; Toxin A; Toxin B; Clostridium difficile; Enzyme immunoassays; Culture
12.  Role of Fecal Clostridium difficile Load in Discrepancies Between Toxin Tests and PCR: Is Quantitation the Next Step in C. difficile Testing? 
Purpose
Direct tests for Clostridium difficile are 30–50% more sensitive than tests for C. difficile toxins but the reasons for this discrepancy are incompletely understood. In addition to toxin degradation and strain differences, we hypothesized that C. difficile concentration could be important in determining whether toxins are detected in fecal samples.
Methods
We performed standard curves on an FDA-approved real-time PCR test for the C. difficile tcdB gene (Xpert C. difficile/Epi, Cepheid) during a prospective comparison of a toxin immunoassay (Meridian Premier), PCR and toxigenic culture. Immunoassay-negative, PCR-positive samples were retested with a cell cytotoxin assay (TechLab).
Results
Among 107 PCR-positive samples, 46 (43.0%) had toxins detected by immunoassay and an additional 18 (16.8%) had toxin detected by the cytotoxin assay yielding 64 (59.8%) toxin-positive and 43 (40.2%) toxin-negative samples. Overall, toxin-negative samples with C. difficile had 101–104 fewer DNA copies than toxin-positive samples and most discrepancies between toxin tests and PCR were associated with a significant difference in C. difficile quantity. 95% of toxin-positive samples had ≥4.1 log10 C. difficile tcdB DNA copies/mL. 52% of immunoassay-negative samples and 70% of immunoassay and cytotoxin negative samples had <4.1 log10 C. difficile tcdB DNA copies/mL.
Conclusions
These findings suggest that fecal C. difficile concentration is a major determinant of toxin detection and C. difficile quantitation may add to the diagnostic value of existing test methods. Future studies are needed to validate the utility of quantitation and determine the significance of low concentrations of C. difficile in the absence of detectable toxin.
doi:10.1007/s10096-012-1695-6
PMCID: PMC3753214  PMID: 22814877
Clostridium difficile; toxin; PCR; quantitation; diarrhea
13.  Comparison of a Rapid Molecular Method, the BD GeneOhm Cdiff Assay, to the Most Frequently Used Laboratory Tests for Detection of Toxin-Producing Clostridium difficile in Diarrheal Feces ▿  
Journal of Clinical Microbiology  2009;47(11):3478-3481.
Six hundred diarrheal stool specimens were collected from inpatients and outpatients at local university hospitals for the detection of toxigenic Clostridium difficile using three parallel methods, the BD GeneOhm Cdiff assay, the tissue culture cytotoxicity assay, and a commercially available enzyme-linked fluorescence immunoassay (ELFA) (Vidas C. difficile toxin A and B assay; bioMérieux). Toxigenic C. difficile culture was also performed to further clarify discordant results. During a 3-month study period, 58 (9.7%) of the 600 diarrheal samples examined were positive by the BD GeneOhm Cdiff assay, while the Vidas C. difficile toxin A and B assay and the cytotoxicity assay performed directly on stool samples gave 4.7% and 6.3% positivity rates, respectively. In the case of four samples, BD GeneOhm Cdiff assay results were not evaluable at first because of the presence of PCR inhibitors, but upon repeat testing from the frozen lysates, all of these samples proved to be negative. After resolution with toxigenic culture, the cytotoxicity assay proved to be positive in 55 samples (9.2%), while the ELFA was positive in 37 samples (6.2%). Results of culture and repeated cytotoxicity assays emphasized the importance of the culture method, because the use of ELFA or enzyme immunoassay without a culture method may lead to a substantial portion of toxigenic C. difficile strains being missed.
doi:10.1128/JCM.01133-09
PMCID: PMC2772624  PMID: 19794052
14.  Comparison of a Commercial Multiplex Real-Time PCR to the Cell Cytotoxicity Neutralization Assay for Diagnosis of Clostridium difficile Infections▿  
Journal of Clinical Microbiology  2009;47(11):3729-3731.
A commercial multiplex real-time PCR assay (Cepheid Xpert C. difficile assay) for the diagnosis of Clostridium difficile infection was evaluated. The sensitivity and specificity of the Cepheid assay were 97.1% and 93.0% for fresh stools, using the cell cytotoxicity neutralization assay as the reference. Using PCR ribotyping as the reference for ribotype 027 strains, the corresponding figures were 100% and 98.1%, respectively.
doi:10.1128/JCM.01280-09
PMCID: PMC2772590  PMID: 19741082
15.  Population Health Impact and Cost-Effectiveness of Tuberculosis Diagnosis with Xpert MTB/RIF: A Dynamic Simulation and Economic Evaluation 
PLoS Medicine  2012;9(11):e1001347.
Nicolas Menzies and colleagues investigate the potential impact and cost-effectiveness of implementing Xpert MTB/RIF for diagnosing tuberculosis in five southern African countries.
Background
The Xpert MTB/RIF test enables rapid detection of tuberculosis (TB) and rifampicin resistance. The World Health Organization recommends Xpert for initial diagnosis in individuals suspected of having multidrug-resistant TB (MDR-TB) or HIV-associated TB, and many countries are moving quickly toward adopting Xpert. As roll-out proceeds, it is essential to understand the potential health impact and cost-effectiveness of diagnostic strategies based on Xpert.
Methods and Findings
We evaluated potential health and economic consequences of implementing Xpert in five southern African countries—Botswana, Lesotho, Namibia, South Africa, and Swaziland—where drug resistance and TB-HIV coinfection are prevalent. Using a calibrated, dynamic mathematical model, we compared the status quo diagnostic algorithm, emphasizing sputum smear, against an algorithm incorporating Xpert for initial diagnosis. Results were projected over 10- and 20-y time periods starting from 2012. Compared to status quo, implementation of Xpert would avert 132,000 (95% CI: 55,000–284,000) TB cases and 182,000 (97,000–302,000) TB deaths in southern Africa over the 10 y following introduction, and would reduce prevalence by 28% (14%–40%) by 2022, with more modest reductions in incidence. Health system costs are projected to increase substantially with Xpert, by US$460 million (294–699 million) over 10 y. Antiretroviral therapy for HIV represents a substantial fraction of these additional costs, because of improved survival in TB/HIV-infected populations through better TB case-finding and treatment. Costs for treating MDR-TB are also expected to rise significantly with Xpert scale-up. Relative to status quo, Xpert has an estimated cost-effectiveness of US$959 (633–1,485) per disability-adjusted life-year averted over 10 y. Across countries, cost-effectiveness ratios ranged from US$792 (482–1,785) in Swaziland to US$1,257 (767–2,276) in Botswana. Assessing outcomes over a 10-y period focuses on the near-term consequences of Xpert adoption, but the cost-effectiveness results are conservative, with cost-effectiveness ratios assessed over a 20-y time horizon approximately 20% lower than the 10-y values.
Conclusions
Introduction of Xpert could substantially change TB morbidity and mortality through improved case-finding and treatment, with more limited impact on long-term transmission dynamics. Despite extant uncertainty about TB natural history and intervention impact in southern Africa, adoption of Xpert evidently offers reasonable value for its cost, based on conventional benchmarks for cost-effectiveness. However, the additional financial burden would be substantial, including significant increases in costs for treating HIV and MDR-TB. Given the fundamental influence of HIV on TB dynamics and intervention costs, care should be taken when interpreting the results of this analysis outside of settings with high HIV prevalence.
Please see later in the article for the Editors' Summary
Editors' Summary
Background
In 2010, about 9 million people developed tuberculosis (TB)—a contagious bacterial disease that usually infects the lungs—and about 1.5 million people died from the disease. Most of these deaths were in low- and middle-income countries, and a quarter were in HIV-positive individuals, who are particularly susceptible to TB. Mycobacterium tuberculosis, the bacterium that causes TB, is spread in airborne droplets when people with active disease cough or sneeze. The characteristic symptoms of TB are a persistent cough, weight loss, fever, and night sweats. Diagnostic tests for TB include sputum smear analysis (microscopic examination of mucus coughed up from the lungs for the presence of M. tuberculosis) and mycobacterial liquid culture (growth of M. tuberculosis from sputum and determination of its drug sensitivity). TB can be cured by taking several antibiotics daily for at least six months, although the recent emergence of multidrug-resistant TB (MDR-TB) is making the disease increasingly hard to treat.
Why Was This Study Done?
To reduce the global TB burden, active disease must be diagnosed quickly and accurately. In most high-burden settings, however, TB diagnosis relies on sputum smear analysis, which fails to identify some people (especially HIV-infected individuals) who have TB. Mycobacterial culture correctly identifies more infected people but is slow and costly, and many high-burden settings lack the infrastructure for high-volume culture diagnosis of TB. Faced with these diagnostic inadequacies, the World Health Organization (WHO) recently recommended the use of Xpert MTB/RIF for initial diagnosis in patients suspected of having MDR-TB or HIV-associated TB. This new, automated DNA test detects M. tuberculosis and DNA differences that make the bacteria resistant to the drug rifampicin (an indicator of MDR-TB) within two hours. Many countries are moving toward adopting Xpert for TB diagnosis, so it is essential to understand the population health impact and cost-effectiveness of diagnostic strategies based on this test. Here, the researchers use a calibrated, dynamic mathematical model of TB to investigate the consequences of Xpert MTB/RIF implementation in five southern African countries where both TB-HIV coinfection and MDR-TB are common.
What Did the Researchers Do and Find?
The researchers used their mathematical model, which simulates the movement of individuals through different stages of TB infection, to investigate the potential health and economic consequences of implementing Xpert for initial TB diagnosis in Botswana, Lesotho, Namibia, South Africa, and Swaziland. In the modeled scenarios, compared to an diagnostic approach based on sputum smear (the “status quo”), implementation of Xpert averted an estimated 132,000 TB cases and 182,000 TB deaths in southern Africa over the ten years following its introduction, reduced the proportion of the population with TB by 28%, and increased health service costs by US$460 million. Much of this cost increase reflected increased antiretroviral therapy costs for TB/HIV-infected individuals who survived TB infection because of better case-finding and treatment. Finally, relative to the status quo, over ten years, Xpert implementation in southern Africa cost US$959 for every DALY (disability-adjusted life-year) averted. Cost-effectiveness ratios in individual countries ranged from US$792 per DALY averted in Swaziland to US$1,257 per DALY averted in Botswana.
What Do These Findings Mean?
These findings suggest that Xpert implementation in southern Africa could substantially reduce TB illness and death through improved case-finding and treatment, but that the impact of Xpert on long-term transmission dynamics may be more limited. Although the additional financial burden associated with Xpert roll-out is likely to be substantial, these findings suggest that using Xpert for TB diagnosis offers reasonable value given its cost. WHO considers any intervention with a cost-effectiveness ratio less than the per-capita gross domestic product (GDP) highly cost-effective—in 2010, the per-capita GDP ranged from US$7,000 in South Africa and Botswana to US$982 in Lesotho.
These findings may not be generalizable to regions with different HIV infection rates, and their accuracy is likely to be affected by the quality of the data fed into the mathematical model and by the structure of the model. Thus, it is essential that the impact of Xpert-based TB diagnosis be carefully evaluated as the approach is rolled out, and that the information generated by these evaluations be used to improve the accuracy of model-based estimates of the long-term effects of this new strategy for TB diagnosis.
Additional Information
Please access these websites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001347.
WHO provides information (in several languages) on all aspects of tuberculosis, including general information on tuberculosis diagnostics and specific information on the roll-out of the Xpert MTB/RIF test; further information about WHO's endorsement of Xpert MTB/RIF is included in a recent Strategic and Technical Advisory Group for Tuberculosis report; WHO also provides information about tuberculosis and HIV
The Stop TB Partnership is working towards tuberculosis elimination; patient stories about TB-HIV coinfection are available
The US Centers for Disease Control and Prevention has information about tuberculosis, and about TB diagnosis
The US National Institute of Allergy and Infectious Diseases also has detailed information on all aspects of tuberculosis
The Tuberculosis Survival Project, which aims to raise awareness of tuberculosis and provide support for people with tuberculosis, provides personal stories about treatment for tuberculosis; the Tuberculosis Vaccine Initiative also provides personal stories about dealing with tuberculosis
MedlinePlus has links to further information about tuberculosis (in English and Spanish)
doi:10.1371/journal.pmed.1001347
PMCID: PMC3502465  PMID: 23185139
16.  Multicenter Evaluation of the Verigene Clostridium difficile Nucleic Acid Assay 
Journal of Clinical Microbiology  2013;51(12):4120-4125.
The Verigene Clostridium difficile Nucleic Acid test (Verigene CDF test) (Nanosphere, Northbrook, IL) is a multiplex qualitative PCR assay that utilizes a nanoparticle-based array hybridization method to detect C. difficile tcdA and tcdB in fecal specimens. In addition, the assay detects binary toxin gene sequences and the single base pair deletion at nucleotide 117 (Δ 117) in tcdC to provide a presumptive identification of the epidemic strain 027/NAP1/BI (referred to here as ribotype 027). This study compared the Verigene CDF test with anaerobic direct and enriched toxigenic culture on stool specimens from symptomatic patients among five geographically diverse laboratories within the United States. The Verigene CDF test was performed according to the manufacturer's instructions, and the reference methods performed by a central laboratory included direct culture onto cycloserine cefoxitin fructose agar (CCFA) and enriched culture using cycloserine cefoxitin mannitol broth with taurocholate and lysozyme. Recovered isolates were identified as C. difficile using gas liquid chromatography and were tested for toxin using a cell culture cytotoxicity neutralization assay. Strains belonging to ribotype 027 were determined by PCR ribotyping and bidirectional sequencing for Δ 117 in tcdC. A total of 1,875 specimens were evaluable. Of these, 275 specimens (14.7%) were culture positive by either direct or enriched culture methods. Compared to direct culture alone, the overall sensitivity, specificity, positive predictive value, and negative predictive value for the Verigene CDF test were 98.7%, 87.5%, 42%, and 99.9%, respectively. Compared to combined direct and enriched culture results, the sensitivity, specificity, positive predictive value, and negative predictive values of the Verigene CDF test were 90.9%, 92.5%, 67.6%, and 98.3%, respectively. Of the 250 concordantly culture-positive specimens, 59 (23.6%) were flagged as “hypervirulent”; 53 were confirmed as ribotype 027, and all 59 possessed Δ 117 in tcdC. Time to results was approximately 2.5 h per specimen. The Verigene CDF test is a novel nucleic acid microarray that reliably detects both C. difficile toxins A and B in unformed stool specimens and appears to adequately identify ribotype 027 isolates.
doi:10.1128/JCM.01690-13
PMCID: PMC3838056  PMID: 24088862
17.  Impact of Clinical Symptoms on Interpretation of Diagnostic Assays for Clostridium difficile Infections▿  
Journal of Clinical Microbiology  2011;49(8):2887-2893.
Asymptomatic Clostridium difficile colonization is common in hospitalized patients. Existing C. difficile assay comparisons lack data on severity of diarrhea or patient outcomes, limiting the ability to interpret their results in regard to the diagnosis of C. difficile infection (CDI). The objective of this study was to measure how including patient presentation with the C. difficile assay result impacted assay performance to diagnose CDI. Stool specimens from 150 patients that met inclusion and exclusion criteria were selected. Nine methods to detect C. difficile in stool were evaluated. All patients were interviewed prospectively to assess diarrhea severity. We then assessed how different reference standards, with and without the inclusion of patient presentation, impact the sensitivity, specificity, and positive and negative predictive values of the assays to diagnose CDI. There were minimal changes in sensitivity; however, specificity was significantly lower for the assays Tox A/B II, C. diff Chek-60, BD GeneOhm Cdiff, Xpert C. difficile, and Illumigene C. difficile and for toxigenic culture (P was <0.01 for all except Tox A/B II from fresh stool, for which the P value was 0.016) when the reference standard was recovery of toxigenic C. difficile from stool plus the presence of clinically significant diarrhea compared to when the reference standard was having at least four assays positive while ignoring diarrhea severity. There were 15 patients whose assay result was reported as negative but subsequently found to be positive by at least four assays in the comparison. None suffered from any CDI-related adverse events. In conclusion, clinical presentation is important when interpreting C. difficile diagnostic assays.
doi:10.1128/JCM.00891-11
PMCID: PMC3147743  PMID: 21697328
18.  Algorithm Combining Toxin Immunoassay and Stool Culture for Diagnosis of Clostridium difficile Infection▿  
Journal of Clinical Microbiology  2009;47(9):2952-2956.
Enzyme immunoassays (EIA) to detect glutamate dehydrogenase or toxins A (TcdA) and B (TcdB), a cytotoxicity assay, and bacteriologic culture have disadvantages when applied individually to diagnosis of Clostridium difficile infections. Stool specimens (n = 1,596) were subjected to toxin detection via an enzyme-linked fluorescent immunoassay (ELFA; Vidas CDAB assay) and bacteriologic culture for toxigenic C. difficile in a three-step algorithm with additional toxigenic culture. Isolates (n = 163) from ELFA-negative stool specimens were examined via ELFA for toxin production. We amplified tcdA and tcdB from C. difficile isolates and tcdB from stool specimens that were ELFA positive or equivocal and culture negative, and we compared the results to those obtained with the three-step algorithm. More than 26% of stool specimens (419/1,596) were culture positive, yielding 248 isolates (59.2%) with both toxin genes (tcdA- and tcdB-positive isolates), 88 isolates (21.0%) with either tcdA or tcdB, and 83 (19.8%) that had no toxin genes (tcdA- and tcdB-negative isolates). Among 49 (culture-negative/ELFA-positive or -equivocal) stool specimens, 53.1% (26/49) represented tcdB-positive isolates. Therefore, the total number of PCR-positive cases was 362, and 27.1% (98/362) of these were detected through toxigenic culture. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were 63.3%, 96.7%, 90.5%, and 92.4% (ELFA alone); 92.8%, 93.3%, 80.2%, and 97.8% (culture); and 70.7%, 91.4%, 95.5%, and 100% (three-step algorithm ELFA and bacterial culture with toxigenic culture), respectively, with culture and PCR for tcdA and tcdB as the standards. Thus, sensitivity and specificity were highest using culture and ELFA, respectively, but we recommend the three-step algorithm comprising EIA to detect both toxins and toxigenic culture for C. difficile as a practical method for achieving better PPV and NPV.
doi:10.1128/JCM.00609-09
PMCID: PMC2738110  PMID: 19625481
19.  Evaluation of a New Commercial TaqMan PCR Assay for Direct Detection of the Clostridium difficile Toxin B Gene in Clinical Stool Specimens▿  
Journal of Clinical Microbiology  2009;47(12):3846-3850.
The ProGastro Cd assay (Prodesse, Inc., Waukesha, WI) is a new commercial TaqMan PCR assay that detects tcdB. The ProGastro Cd assay was compared to the Wampole Clostridium difficile toxin B test (TOX-B test; TechLab, Blacksburg, VA), a cell culture cytotoxicity neutralization assay (CCCNA), and to anaerobic toxigenic bacterial culture, as the “gold standard,” for 285 clinical stool specimens. Assays were independently performed according to manufacturers' directions. A 1.0-ml sample was removed from the stool specimen, of which 20 μl was used for extraction on the NucliSENS easyMAG platform (bioMérieux, Inc., Durham, NC) for the Prodesse ProGastro Cd assay and 200 μl of the stool filtrate was used for the TOX-B CCCNA. Anaerobic toxigenic culture was done by heating an additional 1.0 ml of the stool sample to 80°C for 10 min before inoculation onto modified cycloserine, cefoxitin, and fructose agar with horse blood (Remel, Lenexa, KS) and into a prereduced chopped meat glucose broth (BBL, BD Diagnostics, Sparks, MD). The prevalence of toxin-producing strains of C. difficile was 15.7% (n = 44) as determined by anaerobic toxigenic culture. The sensitivity, specificity, and positive and negative predictive values of the Prodesse ProGastro Cd assay compared to the TOX-B test were 83.3%, 95.6%, 69.4%, and 98%, respectively. Compared to toxigenic culture, the sensitivity, specificity, and positive and negative predictive values of the Prodesse ProGastro Cd assay were 77.3%, 99.2%, 94.4%, and 95.9%, respectively, and those of the TOX-B test were 63.6%, 99.2%, 93.3%, and 93.6%, respectively. Although no statistical difference (Fisher's exact test) was detected (P = 0.242) between the sensitivities of the Prodesse ProGastro Cd assay and a standard CCCNA compared to anaerobic culture for the detection of toxigenic C. difficile, the Prodesse ProGastro Cd assay did detect more toxigenic C. difficile isolates than the CCCNA.
doi:10.1128/JCM.01490-09
PMCID: PMC2786685  PMID: 19846637
20.  Field Evaluation of the Cepheid GeneXpert Chlamydia trachomatis Assay for Detection of Infection in a Trachoma Endemic Community in Tanzania 
Purpose
To determine the sensitivity, specificity, and field utility of the Cepheid GeneXpert Chlamydia trachomatis (CT) Assay (GeneXpert) for ocular chlamydia infection compared to Roche Amplicor CT assay (Amplicor).
Methods
In a trachoma-endemic community in Kongwa Tanzania, 144 children ages 0 to 9 were surveyed to assess clinical trachoma and had two ocular swabs taken. One swab was processed at Johns Hopkins University, Baltimore MD, using Amplicor, (Roche Molecular Diagnostics) and the other swab was processed at a field station in Kongwa using the GeneXpert Chlamydia trachomatis/Neisseria gonorrhoeae assay (Cepheid). The sensitivity and specificity of GeneXpert was compared to the Amplicor assay.
Results
Of the 144 swabs taken the prevalence of follicular trachoma by clinical exam was 43.7%, and by evidence of infection according to Amplicor was 28.5%. A total of 17 specimens (11.8%) could not be processed by GeneXpert in the field due to lack of sample volume, other specimen issues or electricity failure. The sensitivity of GeneXpert when compared to Amplicor was 100% and the specificity was 95%. The GeneXpert test identified more positives in individuals with clinical trachoma than Amplicor, 55% versus 52%.
Conclusion
The GeneXpert test for C. trachomatis performed with high sensitivity and specificity and demonstrated excellent promise as a field test for trachoma control.
Author Summary
Trachoma, an eye infection caused by C. trachomatis, is the leading cause of infectious blindness worldwide, affecting the developing world. The current standard for trachoma treatment involves mass drug administration (MDA) of an antibiotic that is given to a community to reduce transmission. A field test for the presence of infection would be a useful adjunct in measuring MDA impact. However, the current standard for measuring infection involves expensive, delicate instrumentation that is often only in laboratories in developed countries or capital cities, and eye swab specimens are mostly shipped to the developed world for analysis.
This study compared a standard method for infection analysis, Roche Amplicor, in the United States, with a new test, the Cepheid GeneXpert, in the field in Tanzania. We collected 144 duplicate eye swabs in children ages 0–9 years. 12% of specimens could not be analyzed by GeneXpert due to correctable technical difficulties. Of those analyzed, 100% of samples negative by Amplicor were also negative by GeneXpert, and 95% of samples positive by GeneXpert were also positive by Amplicor. The GeneXpert was easy to use with minimal opportunities for contamination, and is a promising new test for field-testing infection in trachoma control efforts.
doi:10.1371/journal.pntd.0002265
PMCID: PMC3701699  PMID: 23861986
21.  Characterisation and Carriage Ratio of Clostridium difficile Strains Isolated from a Community-Dwelling Elderly Population in the United Kingdom 
PLoS ONE  2011;6(8):e22804.
Background
Community-associated Clostridium difficile infection (CDI) appears to be an increasing problem. Reported carriage rates by C.difficile are debatable with suggestions that primary asymptomatic carriage is associated with decreased risk of subsequent diarrhoea. However, knowledge of potential reservoirs and intestinal carriage rates in the community, particularly in the elderly, the most susceptible group, is limited. We have determined the presence of C.difficile in the faeces of a healthy elderly cohort living outside of long-term care facilities (LCFs) in the United Kingdom.
Methods
Faecal samples from 149 community-based healthy elderly volunteers (median age 81 years) were screened for C.difficile using direct (Brazier's CCEY) and enrichment (Cooked Meat broth) culture methods and a glutamate dehydrogenase (GDH) immunoassay. Isolates were PCR-ribotyped and analysed for toxin production and the presence of toxin genes.
Results
Of 149 faecal samples submitted, six (4%) were found to contain C.difficile. One particular sample was positive by both the GDH immunoassay and direct culture, and concurrently produced two distinct strain types: one toxigenic and the other non-toxigenic. The other five samples were only positive by enrichment culture method. Overall, four C.difficile isolates were non-toxigenic (PCR-ribotypes 009, 026 (n = 2) and 039), while three were toxigenic (PCR-ribotypes 003, 005 and 106). All individuals who had a positive culture were symptom-free and none of them had a history of CDI and/or antibiotics use in the 3 month period preceding recruitment.
Conclusions
To our knowledge, this is the first study of the presence of C.difficile in healthy elderly community-dwelling individuals residing outside of LCFs. The observed carriage rate is lower than that reported for individuals in LCFs and interestingly no individual carried the common epidemic strain PCR-ribotype 027 (NAP1/BI). Further follow-up of asymptomatic carriers in the community, is required to evaluate host susceptibility to CDI and identify dynamic changes in the host and microbial environment that are associated with pathogenicity.
doi:10.1371/journal.pone.0022804
PMCID: PMC3160286  PMID: 21886769
22.  Clostridium difficile Isolates Resistant to Fluoroquinolones in Italy: Emergence of PCR Ribotype 018 ▿  
Journal of Clinical Microbiology  2010;48(8):2892-2896.
Recent evidence strongly suggests an association between the use of fluoroquinolones and Clostridium difficile infection (CDI). Resistance to fluoroquinolones has been described not only in the hypervirulent strain 027, but also in other important PCR ribotypes circulating in hospital settings. In a European prospective study conducted in 2005, strains resistant to moxifloxacin represented 37.5% of C. difficile clinical isolates. In this study, we investigated a sample of 147 toxigenic C. difficile isolates, collected in Italy from 1985 to 2008, for the presence of mutations in gyr genes that conferred resistance to fluoroquinolones based on a LightCycler assay. Results were confirmed by the determination of MICs for moxifloxacin. Strains resistant to moxifloxacin were also investigated for resistance to three other fluoroquinolones and for a possible association between fluoroquinolone and macrolide-lincosamide-streptogramin B resistance. C. difficile isolates were typed by PCR ribotyping. In total, 50 clinical isolates showed substitutions in gyr genes and were resistant to fluoroquinolones. Ninety-six percent of the C. difficile resistant isolates showed the substitution Thr82-to-Ile in GyrA, as already observed in the majority of resistant strains worldwide. A significant increase of resistance (P < 0.001) was observed in the period 2002 to 2008 (56% resistant) compared to the period 1985 to 2001 (10% resistant). Coresistance with erythromycin and/or clindamycin was found in 96% (48/50) of the isolates analyzed and, interestingly, 84% of resistant strains were erm(B) negative. The majority of the fluoroquinolone-resistant isolates belonged to PCR ribotype 126 or 018. PCR ribotype 126 was the most frequently found from 2002 to 2005, whereas PCR ribotype 018 was predominant in 2007 and 2008 and still represents the majority of strains typed in our laboratory. Overall, the results demonstrate an increasing number of C. difficile strains resistant to fluoroquinolones in Italy and changes in the prevalence and type of C. difficile isolates resistant to fluoroquinolones circulating over time.
doi:10.1128/JCM.02482-09
PMCID: PMC2916588  PMID: 20554809
23.  Clostridium difficile 027 infection in Central Italy 
BMC Infectious Diseases  2012;12:370.
Background
Clostridium difficile (CD) has increasingly become recognised as a significant international health burden, often associated with the healthcare environment. The upsurge in incidence of CD coincided with the emergence of a hypervirulent strain of CD characterized as 027.
In 2010, 8 cases of CD 027 infections were identified in Italy. Since then, no further reports have been published. We describe 10 new cases of CD 027 infection occurring in Italy.
Methods
Since December 2010, stool samples of patients with severe diarrhea and clinical suspicion of the presence of a hypervirulent strain, were tested for CD 027 by the Xpert C. difficile PCR assay (Cepheid, Sunnyvale, CA). Clinical, epidemiological and laboratory data were collected.
Results
From December 2010 to April 2012, 24 faecal samples from 19 patients who fit the above criteria were submitted to our laboratory. Samples were collected from 7 different hospitals.
Of these, 17 had a positive PCR for CD and 10 were the epidemic 027 strain (59%). All PCR positive samples had a positive EIA toxin A/B test. Nine of 10 patients were recently exposed to antimicrobials and were healthcare-associated, including 4 with a history of long term care facility (LTCF) admission; the remaining case was community-associated, namely the wife of a patient with hospital-acquired CD 027 infection. Five patients experienced at least one recurrence of CD associated diarrhea (CDAD) with a total of 12 relapsing episodes. Of these, two patients had 5 and 6 relapses respectively.
We compared the 10 patients with 027 CDAD versus the 7 patients with non-027 CDAD. None of the 7 patients with non-027 CDAD had a recent history of LTCF admission and no subsequent relapses were observed (p = 0.04).
Conclusions
Our study shows that CD 027 is emerging in healthcare facilities in Italy. Whilst nosocomial acquisition accounted for the majority of such cases, 4 patients had history of a recent stay in a LTCF. We highlight the substantial risks of this highly transmissible organism in such environments. Moreover, 50% of our patients with CDAD from the 027 strain had high relapse rates which may serve to further establish this strain within the Italian health and social care systems.
doi:10.1186/1471-2334-12-370
PMCID: PMC3549759  PMID: 23259814
Clostridium difficile; CDAD; NAP1; 027; Italy
24.  Rapid Diagnosis of Tuberculosis with the Xpert MTB/RIF Assay in High Burden Countries: A Cost-Effectiveness Analysis 
PLoS Medicine  2011;8(11):e1001120.
A cost-effectiveness study by Frank Cobelens and colleagues reveals that Xpert MTB/RIF is a cost-effective method of tuberculosis diagnosis that is suitable for use in low- and middle-income settings.
Background
Xpert MTB/RIF (Xpert) is a promising new rapid diagnostic technology for tuberculosis (TB) that has characteristics that suggest large-scale roll-out. However, because the test is expensive, there are concerns among TB program managers and policy makers regarding its affordability for low- and middle-income settings.
Methods and Findings
We estimate the impact of the introduction of Xpert on the costs and cost-effectiveness of TB care using decision analytic modelling, comparing the introduction of Xpert to a base case of smear microscopy and clinical diagnosis in India, South Africa, and Uganda. The introduction of Xpert increases TB case finding in all three settings; from 72%–85% to 95%–99% of the cohort of individuals with suspected TB, compared to the base case. Diagnostic costs (including the costs of testing all individuals with suspected TB) also increase: from US$28–US$49 to US$133–US$146 and US$137–US$151 per TB case detected when Xpert is used “in addition to” and “as a replacement of” smear microscopy, respectively. The incremental cost effectiveness ratios (ICERs) for using Xpert “in addition to” smear microscopy, compared to the base case, range from US$41–$110 per disability adjusted life year (DALY) averted. Likewise the ICERS for using Xpert “as a replacement of” smear microscopy range from US$52–$138 per DALY averted. These ICERs are below the World Health Organization (WHO) willingness to pay threshold.
Conclusions
Our results suggest that Xpert is a cost-effective method of TB diagnosis, compared to a base case of smear microscopy and clinical diagnosis of smear-negative TB in low- and middle-income settings where, with its ability to substantially increase case finding, it has important potential for improving TB diagnosis and control. The extent of cost-effectiveness gain to TB programmes from deploying Xpert is primarily dependent on current TB diagnostic practices. Further work is required during scale-up to validate these findings.
Please see later in the article for the Editors' Summary
Editors' Summary
Background
Tuberculosis (TB) is a bacterial disease that infects one-third of the world's population. The disease is caused by Mycobacterium tuberculosis, a bacterium that most commonly infects the lungs (known as pulmonary TB) and is transmitted from person to person when an infected individual coughs, sneezes, or talks. The symptoms of TB include chest pain, weight loss, fever, and a persistent cough that sometimes contains blood. Only 5%–10% of people who are infected with TB become sick or infectious, but people with weakened immune systems, such as individuals who are HIV-positive, are more likely to develop the disease. TB is estimated to have killed 1.7 million people in 2009 and is currently the leading cause of death among people infected with HIV.
Why Was This Study Done?
Although TB can be treated with a six-month course of antibiotics, effectively diagnosing TB is not always straightforward and drug resistance is becoming an increasing problem. One of the most common and simple methods to diagnose TB is a technique called sputum smear microscopy, which involves examining matter from the lungs under a microscope for the presence of TB-causing bacteria. However, despite being cheap and relatively simple, the test does not always detect active TB (smear-negative) and cannot determine whether the TB-causing bacteria are resistant to antibiotics. The World Health Organization has recently endorsed a new rapid test, called Xpert MTB/RIF (referred to as Xpert), for the initial diagnosis of TB. The test uses DNA amplification methods to reliably and quickly detect TB and whether infecting bacteria are resistant to the antibiotic rifampicin. The new test is expensive so there are concerns that the test might not be cost-effective in low- and middle-income countries.
What Did the Researchers Do and Find?
The researchers used a technique called modeling to simulate the outcome of 10,000 individuals with suspected TB as they went through a hypothetical diagnostic and treatment pathway. The model compared the costs associated with the introduction of Xpert to a base case for two different scenarios. In the base case all individuals with suspected TB had two sputum smear microscopy examinations followed by clinical diagnosis if they were smear-negative. For the different scenarios Xpert was either used in addition to the two sputum smear microscopy examinations (if the patient was smear-negative) or Xpert was used as a replacement for sputum smear microscopy for all patients. Different input parameters, based on country-specific estimates, were applied so that the model reflected the implementation of Xpert in India, South Africa, and Uganda.
In the researcher's model the introduction of Xpert increased the proportion of TB-infected patients who were correctly diagnosed with TB in any of the settings. However, the cost per TB case detected increased by approximately US$100 in both scenarios. Although the cost of detection increased significantly, the cost of treatment increased only moderately because the number of false-positive cases was reduced. For example, the percentage of treatment costs spent on false-positive diagnoses in India was predicted to fall from 22% to 4% when Xpert was used to replace sputum smear microscopy. The model was used to calculate incremental cost effectiveness ratios (ICERs—the additional cost of each disability-adjusted life year [DALY] averted) for the different scenarios of Xpert implementation in the different settings. In comparison to the base case, introducing Xpert in addition to sputum smear microscopy produced ICERs ranging from US$41 to US$110 per DALY averted, while introducing Xpert instead of sputum smear microscopy yielded ICERs ranging from US$52 to US$138 per DALY averted.
What Do These Findings Mean?
The findings suggest that the implementation of Xpert in addition to, or instead of, sputum smear microscopy will be cost-effective in low- and middle-income countries. The calculated ICERs are below the World Health Organization's “willingness to pay threshold” for all settings. That is the incremental cost of each DALY averted by introduction of Xpert is below the gross domestic product per capita for each country ($1,134 for India, $5,786 South Africa, and $490 for Uganda in 2010). However, the authors note that achieving ICERs below the “willingness to pay threshold” does not necessarily mean that countries have the resources to implement the test. The researchers also note that there are limitations to their study; additional unknown costs associated with the scale-up of Xpert and some parameters, such as patient costs, were not included in the model. Although the model strongly suggests that Xpert will be cost-effective, the researchers caution that initial roll-out of Xpert should be carefully monitored and evaluated before full scale-up.
Additional Information
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001120.
The World Health Organization provides information on all aspects of tuberculosis, including tuberculosis diagnostics and the Stop TB Partnership (some information is in several languages)
The US Centers for Disease Control and Prevention has information about tuberculosis, including information on the diagnosis of tuberculosis disease
MedlinePlus has links to further information about tuberculosis (in English and Spanish)
doi:10.1371/journal.pmed.1001120
PMCID: PMC3210757  PMID: 22087078
25.  Prevalence of Clostridium difficile in raw beef, cow, sheep, goat, camel and buffalo meat in Iran 
BMC Public Health  2014;14:119.
Background
Clostridium difficile has been shown to be a nosocomial pathogen associated with diarrhoea and pseudomembranous colitis in hospitalised patients and the infection is believed to be acquired nosocomially. Recent studies have shown the occurrence of C. difficile in food animals which may act as a source of infection to humans.The aim of this study was to determine the occurrence of C. difficile in retail raw beef, cow, sheep, goat, camel and buffalo meat in Iran.
Method
From April to October 2012, a total of 660 raw meat samples from beef, cow, sheep, goat, camel and buffalo were purchased from 49 butcheries in Isfahan and Khuzestan provinces, Iran, and were evaluated for the presence of C. difficile using a method including selective enrichment in C. difficile broth, subsequent alcohol shock-treatment and plating onto C. difficile selective medium. C. difficile isolates were tested for the presence of toxin genes and were typed using PCR ribotyping.
Results
In this study, 13 of 660 meat samples (2%) were contaminated with C. difficile. The highest prevalence of C. difficile was found in buffalo meat (9%), followed by goat meat (3.3%), beef meat (1.7%), cow (0.94%) and sheep meat (0.9%). Seven of the 13C. difficile strains (53.9%) were positive for tcdA, tcdB and cdtB toxin genes and were classified as ribotype 078. Four strains (30.8%) were positive tcdA, and tcdB, and one strain (7.7%) was possessed only tcdB. The remaining isolate was non-toxigenic. Susceptibilities of 13C. difficile isolates were determined for 11 antimicrobial drugs using the disk diffusion assay. Resistance to clindamycin, gentamycin, and nalidixic acid was the most common finding.
Conclusions
To our knowledge, the present study is the first report of the isolation of C. difficile from raw buffalo meat. This study indicates the potential importance of food, including buffalo meat, as a source of transmission of C. difficile to humans.
doi:10.1186/1471-2458-14-119
PMCID: PMC3918140  PMID: 24499381
Clostridium difficile; Raw meat; Camel; Buffalo; Beef; Antimicrobial resistance

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