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Cost Analysis of a Nucleic Acid Amplification Test in the Diagnosis of Pulmonary Tuberculosis at an Urban Hospital with a High Prevalence of TB/HIV  [PDF]
Max W. Adelman, Ekaterina Kurbatova, Yun F. Wang, Michael K. Leonard, Nancy White, Deborah A. McFarland, Henry M. Blumberg
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0100649
Abstract: Introduction The Centers for Disease Control and Prevention has recommended using a nucleic acid amplification test (NAAT) for diagnosing pulmonary tuberculosis (TB) but there is a lack of data on NAAT cost-effectiveness. Methods We conducted a prospective cohort study that included all patients with an AFB smear-positive respiratory specimen at Grady Memorial Hospital in Atlanta, GA, USA between January 2002 and June 2008. We determined the sensitivity, specificity, and positive and negative predictive value of a commercially available and FDA-approved NAAT (amplified MTD, Gen-Probe) compared to the gold standard of culture. A cost analysis was performed and included costs related to laboratory tests, hospital charges, anti-TB medications, and contact investigations. Average cost per patient was calculated under two conditions: (1) using a NAAT on all AFB smear-postive respiratory specimens and (2) not using a NAAT. One-way sensitivity analyses were conducted to determine sensitivity of cost difference to reasonable ranges of model inputs. Results During a 6 1/2 year study period, there were 1,009 patients with an AFB smear-positive respiratory specimen at our public urban hospital. We found the NAAT to be highly sensitive (99.6%) and specific (99.1%) on AFB smear-positive specimens compared to culture. Overall, the positive predictive value (PPV) of an AFB smear-positive respiratory specimen for culture-confirmed TB was 27%. The PPV of an AFB smear-positive respiratory specimen for culture-confirmed TB was significantly higher for HIV-uninfected persons compared to those who were HIV-seropositive (152/271 [56%] vs. 85/445 [19%]; RR = 2.94, 95% CI 2.36–3.65, p<0.001). The cost savings of using the NAAT was $2,003 per AFB smear-positive case. Conclusions Routine use of the NAAT on AFB smear-positive respiratory specimens was highly cost-saving in our setting at a U.S. urban public hospital with a high prevalence of TB and HIV because of the low PPV of an AFB smear for culture-confirmed TB.
Comparison of the Efficacies of Loop-Mediated Isothermal Amplification, Fluorescence Smear Microscopy and Culture for the Diagnosis of Tuberculosis  [PDF]
Geojith George, Prem Mony, John Kenneth
PLOS ONE , 2011, DOI: 10.1371/journal.pone.0021007
Abstract: Background Despite the advent of novel diagnostic techniques, smear microscopy remains as the most practical test available in resource-limited settings for tuberculosis (TB) diagnosis. Due to the low sensitivity of microscopy and the long time required for culture, feasible and accessible rapid diagnostic methods are urgently needed. Loop-mediated Isothermal Amplification (LAMP) is a promising nucleic-acid amplification assay, which could be accessible, cost-effective and more suited for use with unpurified samples. Methodology/Principal Findings In the current study, the objective was to assess the efficacy of a LAMP assay for tuberculosis compared with fluorescence smear microscopy as well as L?wenstein-Jensen (LJ) and Mycobacteria Growth Indicator Tube (MGIT) cultures for the diagnosis of pulmonary tuberculosis using sputum samples. Smear microscopy and culture were performed for decontaminated and concentrated sputum from TB suspects and the LAMP was also performed on these specimens. The LAMP and smear microscopy were compared, in series and in parallel, to culture. LAMP and smear microscopy showed sensitivities of 79.5% and 82.1% respectively and specificities of 93.8% and 96.9% respectively, compared to culture. LAMP and smear in series had sensitivity and specificity of 79.5% and 100.0% respectively. LAMP and smear in parallel had sensitivity and specificity of 82.1% and 90.6% respectively. Conclusions/Significance The overall efficacies of LAMP and fluorescence smear microscopy in the current study were high and broadly similar. LAMP and smear in series had high specificity (100.0%) and can be used as a rule-in test combination. However, the performance of LAMP in smear negative samples was found to be insufficient.
Rapid molecular diagnosis of tuberculosis and other mycobacteriosis in smear-negative clinical specimens  [cached]
Revista de Ciências Farmacêuticas Básica e Aplicada , 2009,
Abstract: Delay in diagnosis of pulmonary and other forms of tuberculosis (TB) can be fatal, particularly in HIV-infected patients. Hence, techniques based on nucleic acid amplification, which are both rapid and of high specificity and sensitivity, are now widely used and recommended for laboratories that diagnose TB. In the present study, diagnostic methods based on mycobacterial DNA amplification were evaluated in comparative trials alongside tradicional bacterial methods, using negative smear samples from patients with clinically-suspected TB (sputum samples from 25 patients with suspected pulmonary TB, urine samples from two patients with suspected renal TB and cerebrospinal fluid samples from one patient with suspected meningeal TB). A specificity of 100% was achieved with DNA amplification methods and tradicional culture/identification methods, in relation to clinical findings and treatment results. For the smear-negative sputa, conventional PCR for M. tuberculosis was positive in 62% of suspected lung TB case, showing the same sensitivity as bacterial identification. Both techniques failed in the detection of extra-pulmonary samples. Nested PCR showed, after species-specific amplification, a sensitivity of 100% for M. avium and 85% for M. tuberculosis. For extra-pulmonary smear-negative samples, only Nested PCR detected M. tuberculosis and all cases were confirmed clinically. Nested PCR, in which two-step amplification reactions are performed, can identify the two most important mycobacteria in human pathology quickly and directly from clinical spicimens. Keywords: tuberculosis; M. avium; Nested PCR; Smearnegative specimens
Association of BMI Category Change with TB Treatment Mortality in HIV-Positive Smear-Negative and Extrapulmonary TB Patients in Myanmar and Zimbabwe  [PDF]
Lenka Benova, Katherine Fielding, Jane Greig, Bern-Thomas Nyang'wa, Esther Carrillo Casas, Marcio Silveira da Fonseca, Philipp du Cros
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0035948
Abstract: Objective The HIV epidemic has increased the proportion of patients with smear-negative and extrapulmonary tuberculosis (TB) diagnoses, with related higher rates of poor TB treatment outcomes. Unlike in smear-positive pulmonary TB, no interim markers of TB treatment progress are systematically used to identify individuals most at risk of mortality. The objective of this study was to assess the association of body mass index (BMI) change at 1 month (±15 days) from TB treatment start with mortality among HIV-positive individuals with smear-negative and extrapulmonary TB. Methods and Findings A retrospective cohort study of adult HIV-positive new TB patients in Médecins Sans Frontières (MSF) treatment programmes in Myanmar and Zimbabwe was conducted using Cox proportional hazards regression to estimate the association between BMI category change and mortality. A cohort of 1090 TB patients (605 smear-negative and 485 extrapulmonary) was followed during TB treatment with mortality rate of 28.9 per 100 person-years. In multivariable analyses, remaining severely underweight or moving to a lower BMI category increased mortality (adjusted hazard ratio 4.05, 95% confidence interval 2.77–5.91, p<0.001) compared with remaining in the same or moving to a higher BMI category. Conclusions We found a strong association between BMI category change during the first month of TB treatment and mortality. BMI category change could be used to identify individuals most at risk of mortality during TB treatment among smear-negative and extrapulmonary patients.
Treatment of Tuberculosis in a Region with High Drug Resistance: Outcomes, Drug Resistance Amplification and Re-Infection  [PDF]
Maryline Bonnet, Manuela Pardini, Francesca Meacci, Germano Orrù, Hasan Yesilkaya, Thierry Jarosz, Peter W. Andrew, Mike Barer, Francesco Checchi, Heinz Rinder, Graziella Orefici, Sabine Rüsch-Gerdes, Lanfranco Fattorini, Marco Rinaldo Oggioni, Juliet Melzer, Stefan Niemann, Francis Varaine
PLOS ONE , 2011, DOI: 10.1371/journal.pone.0023081
Abstract: Introduction Emerging antituberculosis drug resistance is a serious threat for tuberculosis (TB) control, especially in Eastern European countries. Methods We combined drug susceptibility results and molecular strain typing data with treatment outcome reports to assess the influence of drug resistance on TB treatment outcomes in a prospective cohort of patients from Abkhazia (Georgia). Patients received individualized treatment regimens based on drug susceptibility testing (DST) results. Definitions for antituberculosis drug resistance and treatment outcomes were in line with current WHO recommendations. First and second line DST, and molecular typing were performed in a supranational laboratory for Mycobacterium tuberculosis (MTB) strains from consecutive sputum smear-positive TB patients at baseline and during treatment. Results At baseline, MTB strains were fully drug-susceptible in 189/326 (58.0%) of patients. Resistance to at least H or R (PDR-TB) and multidrug-resistance (MDR-TB) were found in 69/326 (21.2%) and 68/326 (20.9%) of strains, respectively. Three MDR-TB strains were also extensively resistant (XDR-TB). During treatment, 3/189 (1.6%) fully susceptible patients at baseline were re-infected with a MDR-TB strain and 2/58 (3.4%) PDR-TB patients became MDR-TB due to resistance amplification. 5/47 (10.6%) MDR- patients became XDR-TB during treatment. Treatment success was observed in 161/189 (85.2%), 54/69 (78.3%) and 22/68 (32.3%) of patients with fully drug susceptible, PDR- and MDR-TB, respectively. Development of ofloxacin resistance was significantly associated with a negative treatment outcome. Conclusion In Abkhazia, a region with high prevalence of drug resistant TB, the use of individualized MDR-TB treatment regimens resulted in poor treatment outcomes and XDR-TB amplification. Nosocomial transmission of MDR-TB emphasizes the importance of infection control in hospitals.
Age and Sex Differences in Sputum Smear Microscopy Results for Acid Fast Bacilli in a Tertiary Care Centre, South India  [PDF]
Palanivel Chinnakali,Kalaiselvi Selvaraj,Pruthu Thekkur,Gomathi Ramasamy,Mahalakshmy Thulasingam,Kavita Vasudevan
Journal of Respiratory Medicine , 2014, DOI: 10.1155/2014/674942
Abstract: Background and Objectives. Low counts are more difficult to find in microscopic sputum examination and thus are more likely to be missed. In this study, we aimed to estimate the proportion of low-count grading and assessing any age and gender differences in sputum smear grading in a low HIV prevalence setting. Materials and Methods. From the tuberculosis laboratory register information on sputum positivity including the grading of smears, age and gender were extracted for January 2011–December 2011. Smears were examined using Ziehl-Neelsen technique and graded as per the Program Guidelines. Positive smears were classified into low grade positive smears (scanty and 1+) and high grade positive smears (2+ and 3+). Differences in grading of smear based on gender and age were analysed using chi square test. Results. Of 9000 smears examined, 8210 (91.2%) were collected as diagnostic smears from tuberculosis suspects. Low grade positivity was 37.5% among diagnostic smears and 69.6% among follow-up smears. Sputum smears from female examinees had higher proportions of low grade positive smears . Stratification of age and sex within TB suspects had clearly demonstrated the observance of higher low grade positivity among female TB suspects at extremes of age. 1. Introduction Sputum smear microscopy remains the mainstay in the diagnosis of pulmonary tuberculosis in many developing countries including India. Though newer techniques like real time cartridge based nucleic acid amplification test (CBNAAT) with higher sensitivity and specificity have been introduced in fewer centres in India; good quality sputum microscopy remains the backbone in the diagnosis of pulmonary tuberculosis under Revised National Tuberculosis Control Program (RNTCP). Sensitivity (probability of finding the acid fast bacilli if present) of the sputum smear microscopy depends on many factors like quality of sputum obtained from patients, concentration of bacilli in the sputum, skill of the laboratory personnel in staining procedure, and duration between sputum sample collection and smear examination [1]. Grading of sputum smear microscopy in a patient population may differ due to difference in patient characteristics like the severity of disease, HIV status, and previous or current medication [2, 3]. Grading of sputum smear can be used as crude indicator of transmissibility of tuberculosis from patients to household and other contacts [4, 5]. It also predicts the speed of bacteriologic conversion once patients were initiated on chemotherapy [4–6]. Low counts are more difficult to find in
Clinical profiling and use of loop-mediated isothermal amplification assay for rapid detection of Mycobacterium tuberculosis from sputum  [PDF]
A Poudel,BD Pandey,B Lekhak,B Rijal,BR Sapkota,Y Suzuki
Kathmandu University Medical Journal , 2009, DOI: 10.3126/kumj.v7i2.2701
Abstract: Background: Tuberculosis is a global health problem and the situation is worsening with newer incidences of drug resistance and HIV association. Diagnosis of tuberculosis can be done by many methods and test, culture of sputum being the ideal one. Nucleic acid amplification (NAA) assay are more time efficient one, that amplify and detect specific nucleic acid sequences allows rapid, sensitive and specific detection of M. tuberculosis in sputum samples. Objectives : The present study intends to compile the clinical presentations of the pulmonary tuberculosis (PTB) patients and to evaluate the efficacy of in-house loop-mediated isothermal amplification (LAMP) in detecting Mycobacterium tuberculosis in sputum samples by comparing with microscopy and culture. Materials and methods : Two hundred two sputum samples were collected from 202 patients at National Tuberculosis Center, Bhaktapur, Nepal. Complete clinical profiling, epidemiological data and record on BCG vaccination were noted and the samples were subjected for microscopy, culture and in-house LAMP with six primers specific for 16S RNA gene of Mycobacterium tuberculosis . Result : Of the 176 cases of clinical profiling, productive cough was most common symptom in 147 (83.52%), followed by chest pain 136 (77.27%), fever 133 (75.56%) and haemoptysis 61 (34.66%). There was a statistically significant association between BCG vaccination and development of TB (Χ 2 =5.33, P=0.02). Of 202 cases, 115 (56.93%) were chest X-ray positive, 101(50%) were direct smear-positive and 100 (49.51%) were culture positive. LAMP had a sensitivity of 97% and specificity of 94.12% while comparing with culture. In addition, its sensitivity and specificity were 91.09% and 89.11% respectively with reference to microscopy. Conclusion : As in our previous study, overall, the result of present study further confirms that the in-house LAMP is a simple, rapid, sensitive and specific DNA amplification technique for PTB diagnosis. Because of rapidity of microscopy and specificity of culture, in-house LAMP assay can be used as a very powerful and useful supplementary tool with complete clinical profiling of the patients for rapid diagnosis of TB in both AFB-positive and negative cases who are suspected as PTB in disease endemic country like Nepal. Key words: clinical profiling; Sputum; DNA; LAMP; M. tuberculosis; Nepal DOI: 10.3126/kumj.v7i2.2701 Kathmandu University Medical Journal (2009) Vol.7, No.2 Issue 26, 109-114
Considerations on the use of nucleic acid-based amplification for malaria parasite detection
Stéphane Proux, Rossarin Suwanarusk, Marion Barends, Julien Zwang, Ric N Price, Mara Leimanis, Lily Kiricharoen, Natthapon Laochan, Bruce Russell, Fran?ois Nosten, Georges Snounou
Malaria Journal , 2011, DOI: 10.1186/1475-2875-10-323
Abstract: Blood samples were collected from patients attending malaria clinics on the Thai-Myanmar border. Frozen aliquots from 413 samples were tested independently in two laboratories by nested PCR assay. Dried blood spots on filter papers from the same patients were also tested by the nested PCR assay in one laboratory and by a multiplex PCR assay in another. The aim was to determine which protocol best detected parasites below the sensitivity level of microscopic examination.As expected PCR-based assays detected a substantial number of infected samples, or mixed infections, missed by microscopy (27 and 42 for the most sensitive assay, respectively). The protocol that was most effective at detecting these, in particular mixed infections, was a nested PCR assay with individual secondary reactions for each of the species initiated with a template directly purified from the blood sample. However, a lesser sensitivity in detection was observed when the same protocol was conducted in another laboratory, and this significantly altered the data obtained on the parasite species distribution.The sensitivity of a given PCR assay varies between laboratories. Although, the variations are relatively minor, they primarily diminish the ability to detect low-level and mixed infections and are sufficient to obviate the main rationale to use PCR assays rather than microscopy or rapid diagnostic tests. The optimal approach to standardise methodologies is to provide PCR template standards. These will help researchers in different settings to ensure that the nucleic acid amplification protocols they wish to use provide the requisite level of sensitivity, and will permit comparison between sites.Microscopic examination of Giemsa-stained blood smears remains the most reliable method for routine clinical diagnosis of malaria. The technique is robust and except for minor variations, it has remained unmodified since the invention of the thick smear more than 108 years ago [1]. At present, reliable
Locked Nucleic Acid Pentamers as Universal PCR Primers for Genomic DNA Amplification  [PDF]
Zhen Sun, Zhi Chen, Xiaoli Hou, Shuping Li, Haihong Zhu, Ji Qian, Daru Lu, Wei Liu
PLOS ONE , 2008, DOI: 10.1371/journal.pone.0003701
Abstract: Background Multiplexing technologies, which allow for simultaneous detection of multiple nucleic acid sequences in a single reaction, can save a lot of time, cost and labor compared to traditional single reaction detection methods. However, the multiplexing method currently used requires precise handiwork and many complicated steps, making a new, simpler technique desirable. Oligonucleotides containing locked nucleic acid residues are an attractive tool because they have strong affinities for their complementary targets, they have been used to avoid dimer formation and mismatch hybridization and to enhance efficient priming. In this study, we aimed to investigate the use of locked nucleic acid pentamers for genomic DNA amplification and multiplex genotyping. Results We designed locked nucleic acid pentamers as universal PCR primers for genomic DNA amplification. The locked nucleic acid pentamers were able to prime amplification of the selected sequences within the investigated genomes, and the resulting products were similar in length to those obtained by restriction digest. In Real Time PCR of genomic DNA from three bacterial species, locked nucleic acid pentamers showed high priming efficiencies. Data from bias tests demonstrated that locked nucleic acid pentamers have equal affinities for each of the six genes tested from the Klebsiella pneumoniae genome. Combined with suspension array genotyping, locked nucleic acid pentamer-based PCR amplification was able to identify a total of 15 strains, including 3 species of bacteria, by gene- and species-specific probes. Among the 32 species used in the assay, 28 species and 50 different genes were clearly identified using this method. Conclusion As a novel genomic DNA amplification, the use of locked nucleic acid pentamers as universal primer pairs in conjunction with suspension array genotyping, allows for the identification of multiple distinct genes or species with a single amplification procedure. This demonstrates that locked nucleic acid pentamer-based PCR can be utilized extensively in pathogen identification.
Isothermal Amplification Methods for the Detection of Nucleic Acids in Microfluidic Devices  [PDF]
Laura Maria Zanoli,Giuseppe Spoto
Biosensors , 2013, DOI: 10.3390/bios3010018
Abstract: Diagnostic tools for biomolecular detection need to fulfill specific requirements in terms of sensitivity, selectivity and high-throughput in order to widen their applicability and to minimize the cost of the assay. The nucleic acid amplification is a key step in DNA detection assays. It contributes to improving the assay sensitivity by enabling the detection of a limited number of target molecules. The use of microfluidic devices to miniaturize amplification protocols reduces the required sample volume and the analysis times and offers new possibilities for the process automation and integration in one single device. The vast majority of miniaturized systems for nucleic acid analysis exploit the polymerase chain reaction (PCR) amplification method, which requires repeated cycles of three or two temperature-dependent steps during the amplification of the nucleic acid target sequence. In contrast, low temperature isothermal amplification methods have no need for thermal cycling thus requiring simplified microfluidic device features. Here, the use of miniaturized analysis systems using isothermal amplification reactions for the nucleic acid amplification will be discussed.
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