FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1547343313> ?p ?o ?g. }

• W1547343313 endingPage "367" @default.
• W1547343313 startingPage "360" @default.
• W1547343313 abstract "Objective To compare the effectiveness and feasibility of an insertion sequence (IS6110)-based polymerase chain reaction (PCR) assay with conventional methods of detecting Mycobacterium tuberculosis and to analyse mutations present in the hot spot region of the RNA polymerase B subunit (rpoB) gene associated with rifampin resistance by DNA sequencing. Methods Ninety-five sputum samples from 84 clinically suspected cases of tuberculosis were tested for mycobacterial infections by Ziehl Neelsen smear examination, Lowenstein-Jensen culture and IS6110-based PCR assay. Results Sensitivity and specificity of the PCR were 94%; the sensitivity of culture was 65%, and of smear tests, 59%. Both smear microscopy and culture had 100% specificity. DNA sequencing data of the 305-bp fragment of the rpoB gene for nine clinical isolates revealed one point mutation at position I572F and double mutations at position S531F in two isolates obtained from two patients who did not respond to the anti-tuberculosis therapy. Conclusion IS6110-based PCR can be used routinely in clinical laboratories for rapid detection of Mycobacterium tuberculosis and thus allow early diagnosis and treatment of any contacts by the cheapest method currently available in the Palestinian Authority region. Rapid detection of rifampin resistance isolates will enable efficient treatment of patients and assist in eradication of the disease in the Palestinian territories. Objectif: Comparer l’efficacité et la faisabilité d’un test PCR (réaction en chaîne de la polymérase) basé sur une séquence d’insertion (IS6110) avec celle des méthodes classiques de détection de Mycobacterium tuberculosis et analyser par séquençage de l’ADN les mutations présentes dans la région critique du gène de la sous-unitéβ de l’ARN polymérase (rpoB), associéà la résistance à la rifampicine. Méthodes: 95 échantillons d’expectorations de 84 cas cliniquement suspects de tuberculose ont été testés pour des infections à mycobactéries par examen Ziehl Neelsen des frottis, la culture sur milieu Löwenstein Jensen et la PCR basée sur l’IS6110. Résultats: La sensibilité et la spécificité de la PCR étaient de 94%, la sensibilité de la culture était de 65% et celle des frottis 59%. Les frottis et la culture avaient une spécificité de 100%. Les données de séquençage de l’ADN du fragment de 305pb du gène rpoB de 9 isolats cliniques ont révélé une mutation ponctuelle en position I572F et une double mutation en position S531F dans 2 isolats obtenus de deux patients qui ne répondaient pas au traitement antituberculeux. Conclusion: La PCR basée sur l’IS6110 peut être utilisée en routine dans les laboratoires cliniques pour la détection rapide de Mycobacterium tuberculosis et donc permettre un diagnostic précoce et le traitement rapide des contacts par la méthode la moins chère actuellement disponible dans la région de l’Autorité Palestinienne. La détection rapide des isolats résistant à la rifampicine permettra un traitement efficace des patients et aidera à l’éradication de la maladie dans les territoires palestiniens. Objetivo: Comparar la efectividad y la viabilidad del ensayo de la reacción en cadena de la polimerasa (PCR) amplificando una secuencia de inserción (IS6110), con los métodos convencionales de detección del Mycobacterium tuberculosis, y analizar mediante secuenciación del ADN las mutaciones presentes en la región “caliente” del gen rpoB que codifica para la subunidad B de la polimerasa, asociada con la resistencia a Rifampin. Methods: Se testaron 95 muestras de esputo de 84 casos con sospecha clínica de tuberculosis, en busca de infecciones micobacterianas, utilizando la técnica de Ziehl Neelsen, cultivos en medio de Lowenstein Jensen y el ensayo de PCR IS6110. Resultados: La sensibilidad y especificidad de la PCR eran del 94%; la sensibilidad del cultivo era del 65%; y en frotis del 59%. Tanto la microscopía de frotis como el cultivo tenían una especificidad del 100%. Los datos de secuenciación del ADN del fragmento de 305 pb del gen rpoB de nueve aislados clínicos mostraron una mutación puntual en la posición I572F y mutaciones dobles en la posición S531F en dos aislados obtenidos de dos pacientes que no respondían a la terapia antituberculosa. Conclusión: La PCR IS6110 puede utilizarse de forma rutinaria en laboratorios clínicos para la detección rápida de Mycobacterium tuberculosis y por lo tanto permitir el diagnóstico y tratamiento tempranos de cualquier contacto, siendo el método más barato actualmente disponible en la región de Palestina. La detección rápida de aislados resistentes a Rifampin permitirá realizar un tratamiento eficiente a los pacientes y ayudará en la erradicación de la enfermedad en los territorios Palestinos. Pulmonary tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), continues to have a high fatality rate worldwide. One-third of the world’s population is infected with MTB. However, only 10% of people infected with TB will show symptoms or develop active TB (WHO 2009). The common methods used to diagnose TB are microscopic analysis of smears and culture of the bacteria. As microscopic examination requires a relatively high number of bacteria in the sample, it is considered to have low sensitivity (Srisuwanvilai et al. 2008; Daley et al. 2009). To determine the presence of the bacteria in a culture, the sample has to be incubated for 2 to 8 weeks prior to analysis, leading to significant delays in diagnosis (Hashimoto et al. 1995). Thus, there is a need for a rapid, sensitive and specific diagnostic method for TB bacteria. Various PCR methods with different DNA targets are specific and sensitive for the rapid detection of MTB in sputum and other body fluids (Eisenach et al. 1991; Salian et al. 1998; Haldar et al. 2007; Gopinath & Singh 2009). As the insertion sequence IS6110 is normally present in multiple copies (1–25 copies) in the MTB genome and as it is specific for the MTB complex (Hellyer et al. 1996), we considered it an excellent target for the detection of MTB. According to the Palestinian Ministry of Health reports, the estimated incidence in 2006 for all TB cases was 21 per 100 000 population/year. In 2007, the case detection rate was 4.2%. These low detection rates can be explained by misdiagnosis because of the low sensitivity of the diagnostic methods used or by overestimated incidence. Sputum smear-positive cases account for 88% of pulmonary TB cases and 41% of all TB cases. The age range of the majority of positive diagnosed patients was 15–44 years; the incidence was higher among males (ratio 1.8:1). Early diagnosis of the disease and rapid identification of resistance to primary anti-tuberculosis agents are essential for efficient treatment and control of multidrug-resistant (MDR) strains. Resistance to rifampin (RIF), a highly potent anti-tuberculosis drug, often results in high rates of clinical relapse (Soudani et al. 2007). As 90% of RIF-resistant strains are also resistant to isoniazid (INH) (Drobniewski & Wilson 1998), RIF-resistance is a valuable surrogate marker for MDR TB. DNA sequencing studies demonstrate that 95% of RIF-resistant strains have a single point mutation at the 81-bp stretch (hot spot region) of the RNA polymerase B subunit gene (rpoB) (Mani et al. 2001; Patnaik et al. 2001; Hoek et al. 2008; Pietzka et al. 2009). Other studies have revealed that mutations can also occur outside this region within the rpoB gene (Heep et al. 2001; Cavusoglu et al. 2002). The TB incidence rate in Palestinian territories is low but MDR cases have been reported, and it is recognized that the deterioration in the economical, nutritional and health situations caused by regional conflict increases the likelihood of an increase in MDR TB cases. The TB detection method must be affordable and relatively cheap in order to be used in Palestinian hospitals and clinics; therefore, it was not practicable in this study to use expensive commercial kits, as opposed to an in-house method based on PCR primers, which are readily available and affordable. This study is part of a Palestinian TB eradication programme that aims to create a more rapid and efficient system for detecting and combating TB in the West Bank (WB). To our knowledge, PCR assay for direct diagnosis of TB from clinical specimens has not been applied in the WB. Therefore, the goal of our study was to determine the sensitivity and specificity of the PCR assay compared to smear microscopy and bacterial culture. An additional aim was to detect and identify mutations present in the hot spot region of the rpoB gene, which is associated with RIF-resistance. The results of the study may improve the diagnosis of patients with TB in the Palestinian territories WB. A total of 95 sputum samples were included in this study, all samples of insufficient volume were excluded. The samples were from 84 Palestinian patients suspected of having pulmonary TB, based on their clinical symptoms. The patients originated from various parts of the West Bank and were referred to the Palestinian Ministry of Health central laboratory for microbiological testing. The sputum samples were processed using the N-acetyl-Cysteine-NaOH method (Suresh et al. 2007a), and aliquots were collected for ZN-staining and inoculation onto a slant of Lowenstein-Jensen medium. All cultures were incubated at 37 °C for up to 8 weeks (Srisuwanvilai et al. 2008). Basic demographic information about patients, including full names, age, sex and geographic location were obtained from their medical records. All participants gave their written informed consent to participate. The protocol for the study was approved by both the Ministry of Health and the Al-Quds University Ethics Committees. The ZN-stained material was scraped off from the microscopic slides (Suresh et al. 2007b) followed by DNA extraction according to the manufacture’s instructions (High pure PCR template preparation, Roche Diagnostics GmbH, Mannheim, Germany). MTB DNA complex was amplified using a published primer pair (Eisenach et al. 1990) targeting a 123-bp segment of the insertion sequence IS6110. A DNA extraction control and a negative control (ddH2O) were included in each PCR to detect any contamination. The PCR was carried out in a final volume of 25 μl using PCR-Ready SupremeTM mix (Syntezza Bioscience, Jerusalem, Israel) including primers at 1 μm final concentration and 5 μl of the extracted DNA. The PCR conditions were as follows: 5 min at 95 °C followed by 38 cycles of 94 °C for 1 min, 68 °C for 20 s, 72 °C for 20 s with a final extension of 5 min at 72 °C. The PCR product was separated on a 2% agarose gel by electrophoresis in TAE buffer. The pre- and post-PCR procedures were performed in separate rooms to prevent contamination. To determine the analytical sensitivity of the IS6110-based PCR, 10-fold serial dilutions of 4 ng of pure DNA of M. tuberculosis (H37Rv) reference strain were used. DNA concentration was measured by a Nanodrop ND-1000 UV-Vis spectrophotometer (Thermo Fisher Scientific Inc, Waltham MA, USA). For each PCR, 1 μl of the extracted DNA was used as a template. The PCR detection limits were determined by the presence of a clearly detectable 123-bp band on 2% agarose gel. The specificity of the amplified target (123 bp) was confirmed either by restriction fragment length polymorphism analysis (RFLP) using HaeIII enzyme (Zink et al. 2001) or by direct sequencing. For the digestion reaction, 15 μl of the PCR product was incubated with 10 U of HaeIII (Promega, Madison, WI, USA) for 2 h at 37 °C. The digestion product was electrophoresed in a 4% agarose gel in TAE buffer. The expected 94- and 29-bp fragments were determined after ethidium bromide staining and visualized by UV light. DNA molecular weight ladder (100 bp) was used as a molecular size marker (Promega, Madison, WI, USA). For direct sequencing, the samples were purified using PCR product purification kit (Roche Diagnostics GmbH, Mannheim, Germany). The eluted DNA sequenced at the Center for Genomic Technologies, The Hebrew University of Jerusalem. Nine clinical isolates were obtained from nine different patients (three of them not responding to anti-TB therapy) and analysed for the presence of mutations associated with RIF-resistance. A 305-bp amplicon of the rpoB gene covering cluster I (507 to 533 codons; numbering according to the Escherichia coli rpoB sequence) and cluster II (codons 571 and 572) was sequenced after amplification by PCR using previously reported primer sets (Williams et al. 1994). The positive amplifications were purified using PCR product purification kit (Roche Diagnostics GmbH, Mannheim, Germany) and directly sequenced for both sense and antisense strands. The obtained sequences were aligned against the reference sequence of H37Rv for the detection of mutations, indicating RIF-resistance. For statistical analysis: one sputum sample per patient was included, and thirteen samples were excluded (11 follow-up samples from three patients and two samples from two household contacts with a patient with TB). The overall sensitivity and specificity for each diagnostic assay was calculated using online calculator (http://faculty.vassar.edu/lowry/clin1.html) based on the assumption that, the patient was confirmed positive when, one assay was positive for MTB (either by culture or by smear examination) or had a previous history of TB. These confirmed values were used as a “gold standard” against which each diagnostic assay performance was determined. The level of agreement between each diagnostic test and the “gold standard” was tested by kappa coefficient (K, 95% CI) (http://www.graphpad.com/quickcalcs/kappa1.cfm). Of the 84 pulmonary TB suspected patients, 49% were females and 51% were males. The average age of the patients was 46.4 (±2.5) years. The common clinical symptoms of all these patients were fever, weight loss and cough with expectoration for more than 2 weeks. The patients came from cities located in different geographical areas in the Palestinian Authority with varied population densities: 32 patients were from Nablus, 21 from Hebron, 12 from Ramallah, nine from Jenin, five from Tulkarm, two from Jericho and one patient each from Jerusalem, Qalqilya and Tubas. Among the samples, two suspected multidrug resistance cases from the Nablus district were reported (cases 354 and 178). Case 354, a 37-year-old woman, was diagnosed and treated as an extrapulmonary case in 2003/2004. She developed a bronchopulmonary fistula, an empyema abscess, which was treated surgically by a lobotomy on the right side. Subsequently, she was reported as having pulmonary TB in November 2004 and treated according to the DOTS programme. In 2008, she was considered as a pulmonary TB relapse case and re-treated with first-line drugs, but remained sputum smear positive. Now she is clinically considered as a MDR case in need of advanced treatment, and she is still in need of supervision. Case 178, a 41-year-old diabetic man, first reported as a pulmonary TB case in July 2005 and was treated according to the DOTS programme. He responded to treatment and was MTB negative by both smear and culture. In June 2006, he was assessed as a TB relapse case, as he was MTB positive by smear and culture showing no response to the treatment, which continued until May 2007. At that time, he was considered clinically as a MDR case but he abandoned treatment. The 95 sputum samples were studied for the presence of M. tuberculosis using microscopy, bacterial culture and PCR methods. The molecular part of the study was conducted at Al-Quds nutrition and health research institute, Al-Quds University. The microscopy and bacterial culture tests were carried out at the Palestinian Ministry of Health central laboratory. Comparison of the results from the two laboratories indicated differences in identification of positive cases (Table 1). Using the bacterial culture method, 11 samples (12%) were found positive for TB; using the ZN-microscopic method, 13 samples (14%) were positive for TB (Figure 1); using the PCR assay, 30 samples (32%) were positive for TB. The PCR assay was performed on all samples (Figure 2) after determination of its sensitivity and specificity. The lowest detectable band was clearly observed using 40 fg of M. tuberculosis DNA (Figure 3). Among the positive PCR-samples, seven were confirmed by RFLP (Figure 4) and nine samples were confirmed by direct sequencing. Only four samples were confirmed by both techniques. ZN-stained sputum smear of Case 354 The arrow indicates the Acid Fast Bacilli(×1000). Amplification of a 123-bp fragment from the M. tuberculosis IS6110 gene. DNA was amplified using P1P2 primers pair targeting a 123-bp segment of the IS6110 gene. Samples 1, 2, 9, 10 and 11 were negative for MTB. Samples 3, 4, 5, 6, 7, 8 and 12 were positive for MTB using this assay. P was the positive control and represents the reference strain M. tuberculosis (H37Rv). N indicates the negative control (ddH2O). Determination of the IS6110-based PCR sensitivity. Amplification of the purified DNA titration of MTB DNA from 4 ng down to 0.004 fg using the P1P2 primers. The PCR detection limit was shown at the DNA concentration of 40 fg. NC is the negative control representing the extraction buffer, NC2 representing the negative control of the PCR assay. Confirmation of M. tuberculosis present by restriction enzyme HaeIII. Positive PCR products were confirmed as M. tuberculosis using the HaeIII restriction enzyme, which generated low molecular weight fragments of 94 and 29 bp long. Samples 1, 2, 3, 4, 5, and 6 were digested indicating the presence of MBT, UC: undigested sample, MW represents DNA molecular weight marker. The sequences obtained representing the 123 bp of the IS6110 region were identical to each other and to the MTB repetitive IS6110 sequence in the National Center for Biotechnology Information Database (http://www.ncbi.nih.gov/BLAST/). One representative sequence was deposited in the GenBank (Accession no. GU968453). The PCR assay detected six cases negative by bacterial cultures but smear positive: five samples were from patients who had previous positive bacterial culture and subsequently had received anti-tuberculosis drugs; the sixth sample was from a patient who was diagnosed with pulmonary TB based on his clinical symptoms and microscopy, but attempts to isolate TB by culture failed (Table 1). Among the 78 samples considered negative to MTB by bacterial cultures and smears, the PCR assay identified 14 samples as positive for MTB: four samples were from patients who had previous history of pulmonary TB, five samples were from patients who had positive culture and were treated with anti-tuberculosis drugs. Of the remaining five samples, two were household contacts with a patient with TB and had a positive skin test with clinical signs of the disease, the other three samples were from patients who had no history of TB (Table 1). The PCR diagnostic method was found to be sensitive and specific. It detected all samples with positive results by bacterial culture and smears, in samples diagnosed negative with smear and positive by bacterial culture the PCR sensitivity was only 67%. The overall sensitivity and specificity of the PCR according to our gold standard were 94%. The positive and negative predictive values and the kappa coefficient of the used methods are presented in Table 2. DNA was extracted from nine clinical isolates: six isolates from six patients responding to treatment and three isolates from three patients not responding to anti-tuberculosis therapy. The DNA of the nine isolates was amplified using the primer sets for the rpoB gene covering (cluster I and II). The positive amplifications were directly sequenced for both sense and antisense, and a consensuses sequence was determined. Among the six clinical isolates obtained from patient responding to treatment, no mutations along the 305-bp fragment of the rpoB gene were detected in comparison with the reference sequence of H37Rv. Two sequences were deposited to the GenBank (Accession no. HM048901 and HM048902). Among the three isolates obtained from three patients that did not respond to the anti-tuberculosis therapy, one isolate showed a single point mutation from ATC (I; isoleucine) to TTC (F; phenylalanine) at codon 572 (cluster II), (Accession no. HM048904). The other isolate showed a double base-pair mutation in a single codon TCG (S; serine) to TTT (F; phenylalanine) (cluster I), (Accession no. HM048903). The third isolate revealed no mutation in the sequence obtained from the same region (Figure 5). Alignment of deduced amino acid sequences from the 305-bp fragments of the rpoB genes of mycobacterial isolates, H37Rv (accession number: BX842574.1) was used as a reference sequence for drug susceptible isolate. CaseYA66 showed S to F transition at the sequence position 42, while Case 178 showed I to F transition at the sequence position 83. CaseN98, CaseD190, CaseR99, CaseS78, Case 354 and CaseA11 showed no mutations in the sequenced region. Several studies show that DNA extracts from stained microscopic preparations can be used for rapid molecular diagnosis and resistance detection of M. tuberculosis (Patnaik et al. 2001; Van Der Zanden et al. 2003; Suresh et al. 2007b). In this study, we evaluated the utility of IS6110-based PCR as a rapid diagnostic tool for pulmonary tuberculosis using DNA directly eluted from ZN-stained sputum smears. Based on our results, the IS6110-based PCR had a detection limit of 40 fg corresponding to five bacterial cells, which is consistent with other studies (Hashimoto et al. 1995; Sun et al. 2009). The possibility of detecting the bacteria in low concentrations encouraged us to apply the assay directly to clinical samples, using the bacterial culture on Lowenstein-Jensen slant and ZN-microscopy tests as controls. Of the three methods used in this study, the overall sensitivity of the PCR assay was the highest, agreeing with other studies, which found an accordance sensitivity of 74–100% (Beige et al. 1995; Lima et al. 2008; Ani et al. 2009). In samples that were negative by sputum smear and positive by cell culture, the sensitivity of the PCR assay was statistically lower (67%). This phenomenon is not unique as it has been reported in several studies using similar methods (Clarridge et al. 1993; Patnaik et al. 2001; Lima et al. 2008). To overcome the low sensitivity from sputum smear-negative samples, a nested PCR amplification has been used to enhance the DNA amplicon and found to be sufficient (Garcia-Quintanilla et al. 2000). Detection of the bacteria among patients with a history of TB and patients having treatment because of active tuberculosis has been described previously (Rajalahti et al. 1998). Therefore, the detection of MTB DNA by PCR in sputum smears of three patients 3–13 months after infection and treatment is consistent with previous findings, which showed that non-viable bacteria could be detected by PCR (Rajalahti et al. 1998; Lima et al. 2008). Thus, it is important to carefully evaluate the medical history of the patient, during the diagnosis procedure especially using the sensitive PCR assay. The drawback of the high sensitivity PCR assay is the possibility of contamination and/or false positive diagnosis of the DNA samples, which is crucial in diagnosis and treatment of patients. In our study, we carried out all precautions, including separate laboratories for different stages of the PCR, use of sterile reagents and disposables. However, we identified three patients, with no history of TB who had never been treated with anti-tuberculosis drugs, to be positive for TB based on the PCR assay. These patients were negative for TB by sputum smear and bacterial culture methods. In such cases, follow-up specimens are needed to demonstrate whether or not PCR has the ability to detect MTB DNA earlier than conventional diagnostic methods. We believe that the discrepant positive diagnosis is an outcome of the high sensitivity of the assay and we recommend confirming the result by another diagnostic method or by further PCR analysis of subsequent clinical samples. One sample found to be positive by culture and negative by sputum smear did not amplify in the PCR. In order to rule out the possibility of inhibitory factors in the PCR, the sample was spiked with purified MTB DNA and amplified along with positive and negative controls. No evidence for inhibition was observed during the test. The other possible explanation for failing to amplify the IS6110 gene is the lack of homogeneity in small volume samples used in the PCR assay, which may contribute to this false negative result (Palomino 2005; Lima et al. 2008). Overall, the PCR assay presented in this study was found to be a practical and reliable method to diagnose TB despite the drawbacks of its high sensitivity because reduction of the time required for TB diagnosis is crucial in controlling the spread of the disease and allowing adequate treatment. Moreover, the ability to detect MDR mutation using a PCR assay has significant implication in identification of MDR patients at early stage. The two cases from Nablus (354 and 178) were identified as MDR after five and three years, respectively. The late diagnosis influences the patient and their surroundings. For case 354, because of the health insurance policy and the low economic income of the family, the patient did not get adequate treatment and the only option was to transfer him to a neighbouring country for antimicrobial therapy. The other example, case 178, the patient’s occupation required him to have direct contact with customers. Unfortunately, after his situation was revealed, he lost his customers, was stigmatized, and then fled to a neighbouring country where we lost all contact with him. These two MDR TB cases and many others show that the social and economic situation of the patients has a direct influence in their treatment and in eradicating the disease from the Palestinian territories. Unfortunately, although diagnosis of MDR TB requires phenotypic drug susceptibility testing this is not yet available in the WB. Thus, rapid detection of RIF-resistant isolates is essential to ensure an effective treatment. Among the three isolates that were obtained from three patients with no response to treatment, two of them had I572F and S531F mutations, which are associated with RIF-resistance by other studies (Van Der Zanden et al. 2003; Evans et al. 2009). No mutations were detected in the third isolate. The treatment failure could be because of either the presence of a mutation elsewhere in this gene or host immunological factors. In conclusion, this study demonstrates the feasibility of using IS6110-based PCR for detection of MTB DNA eluted directly from ZN-sputum smears in the WB of the Palestinian Authority. The PCR assay as a diagnostic method was found to be more rapid and more sensitive than smear microscopy and bacterial culture. We recommend that IS6110-based PCR be used routinely for the detection of MTB in the WB clinical laboratories supported by smear microscopy and bacterial culture. The interpretation of the results should be in the context of clinical presentation and medical history, especially in low-incidence areas such as the WB. Furthermore, the discovery and rapid detection of RIF-resistance strains among our population demonstrates the need to test for RIF-resistance routinely. Identification of MDR TB patients and early treatment is important in limiting transmission of drug-resistant strains to close contacts and enhance the overall control of tuberculosis in the region. The authors gratefully acknowledge Dr. Amira Hindi, Director of Public Health Affairs (Nablus, Palestinian Territories) and Ms. Siham Bushkar who provided necessary facilities to carry out this work. This study is supported by the German National Science Foundation (Deutsche Forschungsgemeinschaft (DFG) grant number NE575/4-1. The study material and supplement costs were funded by the DFG, Trilateral Project between German, Israeli, and Palestinian Researchers. This study is submitted in partial fulfillment of the requirements for the PhD program being pursued by Suheir Ereqat (DFG funds) at Hebrew University under the supervision of Dr. Gila Kahila Bar-Gal." @default.
• W1547343313 created "2016-06-24" @default.
• W1547343313 creator A5011592724 @default.
• W1547343313 creator A5019762716 @default.
• W1547343313 creator A5030461088 @default.
• W1547343313 creator A5033801177 @default.
• W1547343313 creator A5033913323 @default.
• W1547343313 creator A5067343642 @default.
• W1547343313 creator A5070320536 @default.
• W1547343313 creator A5071672903 @default.
• W1547343313 creator A5079088807 @default.
• W1547343313 creator A5090679995 @default.
• W1547343313 date "2010-12-16" @default.
• W1547343313 modified "2023-10-18" @default.
• W1547343313 title "Pulmonary tuberculosis in the West Bank, Palestinian Authority: molecular diagnostic approach" @default.
• W1547343313 cites W1628835506 @default.
• W1547343313 cites W1637046530 @default.
• W1547343313 cites W1864579407 @default.
• W1547343313 cites W1929979473 @default.
• W1547343313 cites W1968649252 @default.
• W1547343313 cites W1984460455 @default.
• W1547343313 cites W1984486728 @default.
• W1547343313 cites W1988917640 @default.
• W1547343313 cites W2005797570 @default.
• W1547343313 cites W2015257594 @default.
• W1547343313 cites W2017545490 @default.
• W1547343313 cites W2028836478 @default.
• W1547343313 cites W2053789574 @default.
• W1547343313 cites W2068148879 @default.
• W1547343313 cites W2078433429 @default.
• W1547343313 cites W2093529925 @default.
• W1547343313 cites W2101357517 @default.
• W1547343313 cites W2104697395 @default.
• W1547343313 cites W2119211279 @default.
• W1547343313 cites W2124148692 @default.
• W1547343313 cites W2127661592 @default.
• W1547343313 cites W2128110388 @default.
• W1547343313 cites W2129904819 @default.
• W1547343313 cites W2130716925 @default.
• W1547343313 cites W2135751095 @default.
• W1547343313 cites W2139158838 @default.
• W1547343313 cites W2142275507 @default.
• W1547343313 cites W2152489884 @default.
• W1547343313 cites W2164341034 @default.
• W1547343313 cites W2167047714 @default.
• W1547343313 cites W2093785970 @default.
• W1547343313 doi "https://doi.org/10.1111/j.1365-3156.2010.02697.x" @default.
• W1547343313 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/21159079" @default.
• W1547343313 hasPublicationYear "2010" @default.
• W1547343313 type Work @default.
• W1547343313 sameAs 1547343313 @default.
• W1547343313 citedByCount "9" @default.
• W1547343313 countsByYear W15473433132012 @default.
• W1547343313 countsByYear W15473433132013 @default.
• W1547343313 countsByYear W15473433132014 @default.
• W1547343313 countsByYear W15473433132017 @default.
• W1547343313 countsByYear W15473433132023 @default.
• W1547343313 crossrefType "journal-article" @default.
• W1547343313 hasAuthorship W1547343313A5011592724 @default.
• W1547343313 hasAuthorship W1547343313A5019762716 @default.
• W1547343313 hasAuthorship W1547343313A5030461088 @default.
• W1547343313 hasAuthorship W1547343313A5033801177 @default.
• W1547343313 hasAuthorship W1547343313A5033913323 @default.
• W1547343313 hasAuthorship W1547343313A5067343642 @default.
• W1547343313 hasAuthorship W1547343313A5070320536 @default.
• W1547343313 hasAuthorship W1547343313A5071672903 @default.
• W1547343313 hasAuthorship W1547343313A5079088807 @default.
• W1547343313 hasAuthorship W1547343313A5090679995 @default.
• W1547343313 hasBestOaLocation W15473433131 @default.
• W1547343313 hasConcept C114362828 @default.
• W1547343313 hasConcept C142724271 @default.
• W1547343313 hasConcept C195244886 @default.
• W1547343313 hasConcept C205649164 @default.
• W1547343313 hasConcept C2781069245 @default.
• W1547343313 hasConcept C2908628106 @default.
• W1547343313 hasConcept C3018316026 @default.
• W1547343313 hasConcept C71924100 @default.
• W1547343313 hasConcept C95457728 @default.
• W1547343313 hasConceptScore W1547343313C114362828 @default.
• W1547343313 hasConceptScore W1547343313C142724271 @default.
• W1547343313 hasConceptScore W1547343313C195244886 @default.
• W1547343313 hasConceptScore W1547343313C205649164 @default.
• W1547343313 hasConceptScore W1547343313C2781069245 @default.
• W1547343313 hasConceptScore W1547343313C2908628106 @default.
• W1547343313 hasConceptScore W1547343313C3018316026 @default.
• W1547343313 hasConceptScore W1547343313C71924100 @default.
• W1547343313 hasConceptScore W1547343313C95457728 @default.
• W1547343313 hasIssue "3" @default.
• W1547343313 hasLocation W15473433131 @default.
• W1547343313 hasLocation W15473433132 @default.
• W1547343313 hasOpenAccess W1547343313 @default.
• W1547343313 hasPrimaryLocation W15473433131 @default.
• W1547343313 hasRelatedWork W2073311344 @default.
• W1547343313 hasRelatedWork W2366490125 @default.
• W1547343313 hasRelatedWork W2367214875 @default.
• W1547343313 hasRelatedWork W2415759662 @default.
• W1547343313 hasRelatedWork W2431515385 @default.
• W1547343313 hasRelatedWork W2748952813 @default.

• next