Comparison of the Xpert MTB/RIF and Cobas TaqMan MTB Assays for Detection of Mycobacterium tuberculosis in Respiratory Specimens

The Xpert MTB/RIF assay (Cepheid, Sunnyvale, CA) is a fully automated, cartridge-based real-time PCR assay designed to detect Mycobacterium tuberculosis and rifampin resistance within 2 h. The performance of the Xpert assay has been evaluated in various clinical settings. However, there are few data comparing the Xpert assay to the Cobas TaqMan MTB test (Roche Diagnostics, Basel, Switzerland), one of the most widely utilized molecular assays for M. tuberculosis detection. In this prospective study, 320 consecutive respiratory specimens were processed simultaneously using acid-fast bacillus (AFB) staining, mycobacterial cultures with both solid and liquid media, and the Cobas and Xpert assays. The Xpert assay was performed with direct respiratory specimens, while the Cobas assay was done with decontaminated concentrated specimens. Based on the culture as a reference method, the overall sensitivities of the Cobas and Xpert assays were 71.4% and 67.9%, respectively. When AFB smear results were taken into consideration, the sensitivities of the Cobas assay for smear-positive and -negative specimens were 87% and 54%, while those of the Xpert assay were 67% and 69%, respectively. The Cobas assay showed 100% specificity and 100% positive predictive value (PPV) regardless of smear results, while the Xpert assay showed 100% specificity and 100% PPV for smear-positive specimens but 98% specificity and 60% PPV for smear-negative specimens. In conclusion, the Xpert assay showed performance that was slightly inferior to that of the Cobas assay but seems useful for the rapid detection of M. tuberculosis, considering that it was performed without laborious and time-consuming decontamination and concentration procedures.     

Intestinal parasitic infections and swamp development in Sierra Leone

The prevalence of Entamoeba histolytica, Giardia lamblia and other intestinal and urogenital parasites were assessed in five Inland Valley Swamp (IVS) development faming communities in the Moyamba District, South-central Sierra Leone. Stool and urine samples were submitted by 1106 individuals and examined by the iron-haematoxylin staining and the formalin-ether concentration techniques for faecal sample and centrifugation method for the urine samples. The overall parasitic infection rate was 61.7% while 5.9% of the population had multiple infections. E. histolytica infection rate was 12.3 % and most of the infected individuals were passing cysts. Giardia lamblia and Trichomonas vaginalis infection rates were 10.0% and 0.4% respectively. Among the helminth infections, Ascaris lumbricoides was the most commonly observed (13.7%), followed by hookworms (12.1 %), Trichuris trichiura (9.3%), Strongyloides stercoralis (7.7%) and tapeworms (2.6%). The high parasitic infection rate (61.7%) and the frequency of multiple infections indicate an interrelationship of environmental factors which support transmission rather than a single factor.     

Evaluation of the Biofire FilmArray BioThreat-E Test (v2.5) for Rapid Identification of Ebola Virus Disease in Heat-Treated Blood Samples Obtained in Sierra Leone and the United Kingdom

Rapid Ebola virus (EBOV) detection is crucial for appropriate patient management and care. The performance of the FilmArray BioThreat-E test (v2.5) using whole-blood samples was evaluated in Sierra Leone and the United Kingdom and was compared with results generated by a real-time Ebola Zaire PCR reference method. Samples were tested in diagnostic laboratories upon availability, included successive samples from individual patients, and were heat treated to facilitate EBOV inactivation prior to PCR. The BioThreat-E test had a sensitivity of 84% (confidence interval [CI], 64% to 95%) and a specificity of 89% (CI, 73% to 97%) in Sierra Leone (n = 60; 44 patients) and a sensitivity of 75% (CI, 19% to 99%) and a specificity of 100% (CI, 97% to 100%) in the United Kingdom (n = 108; 70 patients) compared to the reference real-time PCR. Statistical analysis (Fisher''s exact test) indicated there was no significant difference between the methods at the 99% confidence level in either country. In 9 discrepant results (5 real-time PCR positives and BioThreat-E test negatives and 4 real-time PCR negatives and BioThreat-E test positives), the majority (n = 8) were obtained from samples with an observed or probable low viral load. The FilmArray BioThreat-E test (v2.5) therefore provides an attractive option for laboratories (either in austere field settings or in countries with an advanced technological infrastructure) which do not routinely offer an EBOV diagnostic capability.     

A first report on the characterization of rotavirus strains in Sierra Leone

In an effort to reduce the high mortalities associated with rotavirus infections, a number of African countries are considering introducing human rotavirus vaccines. The demonstrated safety and efficacy of the live‐attenuate human rotavirus vaccines in several clinical trials worldwide has accelerated such initiatives. Although the percentage‐mortality rates for Sierra Leone are top of the list for rotavirus‐associated deaths in Africa, no study has reported the prevalent strains circulating within this country. In this study, stool specimens were collected from 128 Sierra Leonean children presenting with diarrhea in 2005. Almost 37.5% (48/128) were rotavirus positive by EIA, of which 89.6% (43/48) revealed a short electropherotype, and a further 6.98% (3/48) could not be assigned a PAGE pattern. Genotyping analysis revealed G2P[4] (30.23%), G2P[6] (13.95%), G8P[6] (11.63%), G2P[8] (4.65%), G8P[4] (4.65%), and G8P[8] (2%) strains. About 11% were only assigned VP7 genotypes (G2), while 20.9% had mixed G and P types. The frequent detection of G2 rotaviruses could be of concern considering data generated from some studies that suggests lower efficacy of Rotarix® vaccine against G2 rotaviruses. This underscores the need for extensive and continuous regional strain surveillance to support rotavirus vaccines introduction and guide future vaccine development efforts. Such information will be useful before considering administration of specific rotavirus vaccine candidates in countries like Sierra Leone where little is known about circulating rotavirus strains. J. Med. Virol. 83:540–550, 2011. © 2011 Wiley‐Liss, Inc.     

Emergency conflict-related psychosocial interventions in Sierra Leone and Uganda: lessons from Medecins Sans Frontieres

Médecins Sans Frontières has been involved in emergency mental health or psychosocial programmes since 1990. In this article the intervention model developed for emergency settings is shared. Psychosocial programmes distinguish two elements. The 'psycho'-component facilitates the reconnection of the affected individual to his environment. The 'socio'-element aims to create an environment that facilitates the individual to re-integrate. The nature of mental health and psychosocial programmes requires a multidisciplinary approach. Emotional support can also be provided by regular medical staff and does not always require a specialist. The years ahead of us are important for the development of psychosocial interventions. Fundamental issues such as programme evaluation need systematic research.     

Serological and entomological study on yellow fever in Sierra Leone]

In a serological and entomological survey on yellow fever carried out in Sierra-Leone in 1972, altogether 899 sera from children 0 to 14 years were tested with 12 antigens by haemagglutination-inhibition and complement fixation tests. Mouse neutralization test with yellow fever, West-Nile and Zika viruses were also performed on selected sera. Generally speaking, the incidence of arboviruses is low but the prevalence of antibodies for some viruses was found to vary considerably between different areas. As regards yellow fever, the virus has recently been in circulation in only two areas: Bafodia and Lalehun-Labour Camp and there is no risk for a yellow fever outbreak to occur in the near future. Due to the shortness of the survey, entomological prospections were confined to a search for Ae. aegypti larvae in and around dwellings: no breeding places are found in houses and Breteau indices are usually low, especially in forest villages. On the other hand, in urban settlements in the mining areas, breeding places around houses are numerous and are bound to increase in number. All the conditions necessary for the outbreak of an epidemic would be present within few years: such a situation would appear in Labour Camp where yellow fever virus has been circulating, where most of the population has no immunity and where Breteau indice goes as high as 34.4. As regards the other arboviruses, Zika virus is active in most areas and Chikungunya virus is particularly active in the plateau and savanna zones, in the North-East.     

Stability of Cervical Specimens in SurePath Medium for Human Papillomavirus Testing with the Roche cobas 4800 Assay

The stability of cervical specimens in SurePath preservative fluid for human papillomavirus (HPV) testing with Roche cobas 4800 was determined using a panel of 308 pooled specimens from a colposcopy referral population. The SurePath specimens appeared to be stable for up to 10 weeks at ambient temperature for HPV testing with cobas 4800.     

Evaluation of the Analytical Performance of the Xpert MTB/RIF Assay

We performed the first studies of analytic sensitivity, analytic specificity, and dynamic range for the new Xpert MTB/RIF assay, a nucleic acid amplification-based diagnostic system that detects Mycobacterium tuberculosis and rifampin (RIF) resistance in under 2 h. The sensitivity of the assay was tested with 79 phylogenetically and geographically diverse M. tuberculosis isolates, including 42 drug-susceptible isolates and 37 RIF-resistant isolates containing 13 different rpoB mutations or mutation combinations. The specificity of the assay was tested with 89 nontuberculosis bacteria, fungi, and viruses. The Xpert MTB/RIF assay correctly identified all 79 M. tuberculosis isolates and correctly excluded all 89 nontuberculosis isolates. RIF resistance was correctly identified in all 37 resistant isolates and in none of the 42 susceptible isolates. Dynamic range was assessed by adding 10² to 10⁷ CFU of M. tuberculosis into M. tuberculosis-negative sputum samples. The assay showed a log-linear relationship between cycle threshold and input CFU over the entire concentration range. Resistance detection in the presence of different mixtures of RIF-resistant and RIF-susceptible DNA was assessed. Resistance detection was dependent on the particular mutation and required between 65% and 100% mutant DNA to be present in the sample for 95% certainty of resistance detection. Finally, we studied whether assay specificity could be affected by cross-contaminating amplicons generated by the GenoType MTBDRplus assay. M. tuberculosis was not detected until at least 10⁸ copies of an MTBDRplus amplicon were spiked into 1 ml of sputum, suggesting that false-positive results would be unlikely to occur.Conventional diagnostic methods for Mycobacterium tuberculosis are slow and/or lack sensitivity. A number of new diagnostic approaches have brought incremental improvements to detection and drug susceptibility testing; however, the technical complexity of these assays and their dependence on dedicated laboratory infrastructure have limited their adoption, especially in low-resource, high-burden settings (1, 11, 12, 21). The recently introduced Xpert MTB/RIF (manufactured and marketed by Cepheid, Sunnyvale, CA) assay simultaneously detects the presence of M. tuberculosis and its susceptibility to the important first-line drug rifampin (RIF) (7). A sample processing system and an automated heminested real-time PCR assay are integrated into a single disposable cartridge. The assay can be performed directly from a clinical sputum sample or from a decontaminated sputum pellet and can generally be completed in less than 2 h (7).The Xpert MTB/RIF assay detects M. tuberculosis and RIF resistance by PCR amplification of the rifampin resistance-determining region (RRDR) of the M. tuberculosis rpoB gene and subsequent probing of this region for mutations that are associated with RIF resistance. Approximately 95% of RIF-resistant tuberculosis cases contain mutations in this 81-bp region (16). Our previous work has established that the Xpert MTB/RIF assay has a limit of detection (LOD), defined as the minimum number of bacilli that can be detected with 95% confidence) of 131 CFU per ml of clinical sputum (7). The assay was also able to identify RIF resistance in samples containing 23 common clinically occurring rpoB mutations. None of the 20 nontuberculosis mycobacteria (NTM) species tested, including the NTM species commonly described as causing human disease were falsely identified as M. tuberculosis (7), suggesting high specificity. Several small studies using clinical samples demonstrated 98% to 100% sensitivity overall, 72% sensitivity in smear-negative patients, and a specificity of 100% (7).In the present study, we expand upon our previous work and report on several critical analytical assay performance characteristics, including dynamic range, sensitivity, specificity, RIF resistance detection in heterogeneous samples, and resiliency against cross-contamination by other nucleic acid amplification techniques (NAATs).     

Case report and brief commentary: Wuchereria bancrofti and Onchocerca volvulus co-infection in a refugee from Sierra Leone

Filarial infection is endemic in the tropics and is a public health problem in Africa, Asia, South and Central America, and the Pacific Islands. Co-infection with filarial nematodes, if unrecognized, can result in untoward therapeutic consequences. We report a case of co-infection of Wuchereria bancrofti and Onchocerca volvulus that was diagnosed by direct blood smear (W. bancrofti ) and serology (O. volvulus) in a native of Sierra Leone. We comment briefly on the therapeutic implications of the co-infection.     

Evaluation of Xpert MTB/RIF for Detection of Tuberculosis from Blood Samples of HIV-Infected Adults Confirms Mycobacterium tuberculosis Bacteremia as an Indicator of Poor Prognosis

Tuberculosis (TB) remains a leading cause of death among HIV-infected adults, in part because of delayed diagnosis and therefore delayed initiation of treatment. Recently, the Gene-Xpert platform, a rapid, PCR-based diagnostic platform, has been validated for the diagnosis of TB with sputum. We have evaluated the Xpert MTB/RIF assay for the diagnosis of Mycobacterium tuberculosis bacteremia and investigated its impact on clinical outcomes. Consecutive HIV-infected adults with fever and cough presenting to Queen Elizabeth Central Hospital, Blantyre, Malawi, were recruited and followed up for 2 months. At presentation, three sputum samples were examined by smear, culture, and Xpert MTB/RIF assay for the presence of M. tuberculosis and blood was drawn for PCR with Xpert, for mycobacterial culture (Myco/F Lytic), and for aerobic culture. One hundred four patients were recruited, and 44 (43%) were sputum culture positive for M. tuberculosis. Ten were Xpert blood positive, for a sensitivity of 21% and a specificity of 100%. The 2-week mortality rate was significantly higher among patients who were Xpert blood positive than among those who were negative (40% versus 3%; multivariate odds ratio [OR] for death if positive, 44; 95% confidence interval [CI], 3 to 662). This effect persisted on assessment of the mortality rate at 2 months (40% versus 11%; OR, 5.6; 95% CI, 1.3 to 24.6). When screening uncomplicated patients presenting with a productive cough for pulmonary TB, Xpert blood offers no diagnostic advantage over sputum testing. Despite this, Xpert blood positivity is highly predictive of early death and this test rapidly identifies a group of patients in urgent need of initiation of treatment.     

Supplementary Testing Is Not Required in the cobas 4800 CT/NG Test for Neisseria gonorrhoeae Weak-Positive Urogenital Samples

Weak-positive Neisseria gonorrhoeae nucleic acid amplification test results are difficult to interpret. We show that the frequency of unconfirmed N. gonorrhoeae results from the cobas 4800 test rises exponentially after 38.0 cycles, where the likelihood of an unconfirmed result exceeds 29%. Supplementary testing of such samples should be avoided; instead, treatment should be based on clinical pretest probability.     

Evaluation of GeneXpert MTB/RIF for Diagnosis of Tuberculous Meningitis

Tuberculous meningitis (TBM) is the most severe form of tuberculosis. Microbiological confirmation is rare, and treatment is often delayed, increasing mortality and morbidity. The GeneXpert MTB/RIF test was evaluated in a large cohort of patients with suspected tuberculous meningitis. Three hundred seventy-nine patients presenting with suspected tuberculous meningitis to the Hospital for Tropical Diseases, Ho Chi Minh City, Vietnam, between 17 April 2011 and 31 December 2012 were included in the study. Cerebrospinal fluid samples were tested by Ziehl-Neelsen smear, mycobacterial growth indicator tube (MGIT) culture, and Xpert MTB/RIF. Rifampin (RIF) resistance results by Xpert were confirmed by an MTBDR-Plus line probe assay and all positive cultures were tested by phenotypic MGIT drug susceptibility testing. Overall, 182/379 included patients (48.0%) were diagnosed with tuberculous meningitis. Sensitivities of Xpert, smear, and MGIT culture among patients diagnosed with TBM were 59.3% (108/182 [95% confidence interval {CI}, 51.8 to 66.5%]), 78.6% (143/182 [95% CI, 71.9 to 84.3%]) and 66.5% (121/182 [95% CI, 59.1 to 73.3%]), respectively. There was one false-positive Xpert MTB/RIF test (99.5% specificity). Four cases of RIF resistance (4/109; 3.7%) were identified by Xpert, of which 3 were confirmed to be multidrug-resistant (MDR) TBM and one was culture negative. Xpert MTB/RIF is a rapid and specific test for the diagnosis of tuberculous meningitis. The addition of a vortexing step to sample processing increased sensitivity for confirmed TBM by 20% (P = 0.04). Meticulous examination of a smear from a large volume of cerebrospinal fluid (CSF) remains the most sensitive technique but is not practical in most laboratories. The Xpert MTB/RIF represents a significant advance in the early diagnosis of this devastating condition.     

HIV-1, HCV and HBV seronegative window reduction by the new Roche cobas TaqScreen MPX test in seroconverting donors.

BACKGROUND: By shortening the pre-seroconversion window in the viral screening of donated blood, nucleic acid amplification testing greatly improves safety and efficiency, particularly when combined with multiple target detection and maximal automation. OBJECTIVES: Evaluation of seronegative window reduction during HIV-1, HCV and HBV infection by the novel cobas TaqScreen MPX test for simultaneous nucleic acid detection of HIV-1 (groups M and O), HIV-2, HCV and HBV using the cobas s 201 system. STUDY DESIGN: Testing of HIV-1, HCV, and HBV seroconversion panels (20 each) using the cobas TaqScreen MPX test versus reference immuno- and nucleic acid technology assays. RESULTS: The cobas TaqScreen MPX test detected HIV-1 and HCV infection earlier than immunoassays in 20/20 and 19/20 panels, and HBV DNA earlier than or on the same day as HBsAg in 19/20 and 18/20 panels, and later in 1 and 2 panels on neat samples and 1:6 dilutions. Pre-seroconversion sensitivity exceeded that of COBAS AmpliScreen testing in pools of 24. CONCLUSION: The cobas TaqScreen MPX test shortens the pre-seroconversion window in minipools of six, evidencing high sensitivity, and significantly enhances blood-screening efficiency by the simultaneous automated detection of multiple viruses in a single test.     

Evaluation of the COBAS AMPLICOR MTB system.

A panel of 1,012 respiratory sediments was retrospectively tested by PCR amplification to detect Mycobacterium tuberculosis with the COBAS AMPLICOR MTB system. The sensitivities and specificities of COBAS and fluorescence microscopy compared to culture were 92.6 versus 95.6% and 99.6 versus 95.3%, respectively. Inhibition occurred in 48 (4.7%) specimens.     

Evaluation of Cobas TaqMan MTB for Direct Detection of the Mycobacterium tuberculosis Complex in Comparison with Cobas Amplicor MTB

The Roche Cobas Amplicor MTB assay, recently replaced by the Roche Cobas TaqMan MTB assay, was one of the first commercially available assays for detection of the Mycobacterium tuberculosis complex based on nucleic acid amplification. We reported previously on the limited specificity of the Cobas Amplicor MTB assay, in particular for positive samples with an optical density at 660 nm (OD₆₆₀) of <2.0. Using a selected set of respiratory samples, which were scored as false positive by the Cobas Amplicor test, we demonstrate here that the specificity of the Cobas TaqMan assay is significantly improved. In addition, our study of a set of 133 clinical samples revealed that the Cobas TaqMan MTB assay showed significantly less PCR inhibition than the Cobas Amplicor test. An overall concordance of 98.2% was observed between the two assays. In a subsequent prospective study, we evaluated the performance of the Roche Cobas TaqMan MTB assay on 1,143 clinical specimens, including respiratory (n = 838) and nonrespiratory (n = 305) specimens. Using culture as the gold standard, we found a sensitivity of 88.4% and a specificity of 98.8% for the 838 respiratory specimens, compared to a sensitivity of 63.6% and a specificity of 94.6% for the 305 nonrespiratory specimens. We conclude that the Cobas TaqMan MTB assay is a significantly improved tool for the direct detection of M. tuberculosis DNA in clinical specimens.     

Evaluation of Xpert MTB/RIF for Detection of Mycobacterium tuberculosis in Cerebrospinal Fluid

Studies investigating Xpert MTB/RIF diagnostic performance on cerebrospinal fluid (CSF) samples are lacking in resource-rich settings. Xpert MTB/RIF results for 740 CSF samples from 698 patients across England were retrospectively compared with the results of culture of the same and contemporary samples. The overall sensitivity was calculated at 55%.     

MDR-TB诊断试剂盒鉴定广东结核分枝杆菌复合群及RIF和INH药敏效果分析

目的评价耐多药结核病（MDR-TB）快速诊断试剂盒对广东地区结核分枝杆菌复合群的鉴定效果及利福平（RIF）和异烟肼（INH）的药敏检测效果。方法选取151份痰标本和150份细菌培养物,采用MDR-TB快速诊断试剂盒对其进行菌群鉴定及RIF和INH药敏检测,结果分别与常规法进行比较。结果与常规生化菌种鉴定结果相比,两者鉴定结果一致率为96.7%（291/301）;与常规比例法药敏试验结果相比,诊断试剂盒检测单耐RIF的灵敏度为94.1%（64/68）,检测单耐INH的灵敏度为87.7%（57/65）,检测RIF和INH皆耐药株的灵敏度为79.3%（46/58）,检测RIF和INH皆敏感株的特异性为100%。结论该试剂盒能简单快速地鉴定结核分枝杆菌复合群,检测RIF和INF的敏感性高、特异性好,在广东地区应用前景较好。