Increased case finding of tuberculosis from sputum and sputum deposits after magnetic bead concentration of mycobacteria

BackgroundMulti drug resistant tuberculosis (MDR-TB) poses formidable challenges to TB control due to its complex diagnostic and treatment challenges and often associated with a high rate of mortality. Accurate and rapid detection of MDR-TB is critical for timely initiation of treatment. Line Probe Assay (LPA) is a qualitative in vitro diagnostic test based on DNA-STRIP technology for the identification of the M. tuberculosis complex and its resistance to rifampicin (RMP) and/or isoniazid (INH). Hain Lifescience, GmbH, Germany has improved the sensitivity of Genotype MTBDRplus VER 2.0 LPA for the detection of MDR-TB; with the possibility of applying the tool in smear negative sputum samples.MethodA cross sectional study was conducted on 274 presumptive MDR-TB patients referred to the National TB Reference Laboratory (NTRL), Ethiopian Public Health Institute (EPHI) who submitted sputum samples for laboratory diagnosis of drug resistant-TB testing. Seventy-two smear and culture positive samples processed in smear positive direct LPA category and 197 smear negative sputum samples were processed for direct LPA. Among the smear negative samples 145 (73.6%) were culture negative and 26 (13.2%) were culture positive. All specimens were processed using NALC-NaOH method and ZN smear microscopy done from sediments. Genotype MTBDRplus VER 2.0 done from processed sputum sediments and the result was compared against the reference, BACTEC MGIT 960 culture and DST. Sensitivity, specificity, PPV and NPV of Genotype MTBDRplus VER 2.0 assay was determined and P-value <0.05 was considered as statistically significant.ResultsThe sensitivity, specificity, PPV and NPV of Genotype MTBDRplus VER 2.0 LPA were 96.4, 100, 100 and 96.9%, respectively for the detection of MDR-TB from direct smear positive sputum samples. The sensitivity, specificity, PPV and NPV of Genotype MTBDR plus VER 2.0 LPA were 77.8, 97.2, 82.4 and 97.2%, respectively, for the detection of M. tuberculosis from direct smear negative sputum samples. Fourteen (53.8%) samples had valid results with LPA among the 26 smear negative culture positive samples. The remaining 8 (30.8%) and 4 (15.4%) were invalid and negative with LPA, respectively. The sensitivity and specificity of Genotype MTBDRplus VER 2.0 LPA were 100% for the detection of MDR-TB among 14 direct smear negative and culture positive sputum samples.The most common mutations associated with RMP and INH resistance were S531L and S315TL, respectively. A single rare mutation (C15T/A16G) was detected for INH resistance.ConclusionThe diagnostic performance of Genotype MTBDRplus VER 2.0 LPA in direct smear positive sputum sample was highly sensitive and specific for early detection of MDR-TB. However, the diagnostic performance of this molecular assay in direct smear negative sputum sample was low and showed a high level of invalid results for detection of M. tuberculosis and its resistance to RMP and/or INH so it is unlikely to implement Genotype MTBDRplus VER 2.0 for the detection of MDR-TB in direct smear negative sample in our routine settings. The sensitivity of the assay should be improved for detection of MDR-TB in direct smear negative sputum specimens.

ObjectiveDirect sputum smear microscopy (DSSM) has a low detection rate. This study investigated whether an alternative method called Mono-Prep smear microscopy (MPSM) can enhance the diagnosis of tuberculosis in tuberculosis laboratories that perform direct smear microscopy in China.MethodsA total of 117 sputum samples were collected from outpatients who attended Beijing Chest Hospital. DSSM, MPSM, solid culture, and Xpert MTB/RIF were performed on the samples.ResultsThe positive rates of DSSM, MPSM, solid culture, and Xpert MTB/RIF were 27.4% (32/117), 40.2% (47/117), 35.9% (42/117), and 52.1% (61/117), respectively. MPSM could detect 15 more cases of tuberculosis compared with DSSM (47 vs 32) among 117 sputum samples. This represented a significantly higher positive rate and sensitivity of MPSM compared with DSSM. However, MPSM appeared to have a lower specificity (81.3%) compared with DSSM (90.7%) with the solid culture used as a standard.ConclusionUse of MPSM can increase the number of positive sputum samples, but it still needs improvement.

BackgroundSputum concentration increases the sensitivity of smear microscopy for the diagnosis of tuberculosis (TB), but few studies have investigated this method in human immunodeficiency virus (HIV)-infected individuals.MethodsWe performed a prospective, blinded evaluation of direct and concentrated Ziehl-Neelsen smear microscopy on a single early-morning sputum sample in HIV-infected patients with > 2 weeks of cough hospitalized in Kampala, Uganda. Direct and concentrated smear results were compared with results of Lowenstein-Jensen culture.ResultsOf 279 participants, 170 (61%) had culture-confirmed TB. The sensitivity of direct and concentrated smear microscopy was not significantly different (51% vs. 52%, difference 1%, 95% confidence interval (CI): [-7%, 10%], p = 0.88). However, when results of both direct and concentrated smears were considered together, sensitivity was significantly increased compared with either method alone (64%, 95% CI: [56%, 72%], p < 0.01 for both comparisons) and was similar to that of direct smear results from consecutive (spot and early-morning) specimens (64% vs. 63%, difference 1%, 95% CI: [-6%, 8%], p = 0.85). Among 109 patients with negative cultures, one had a positive direct smear and 12 had positive concentrated smears (specificity 99% vs. 89%, difference 10%, 95% CI: [2%, 18%], p = 0.003). Of these 13 patients, 5 (38%) had improved on TB therapy after two months.ConclusionSputum concentration did not increase the sensitivity of light microscopy for TB diagnosis in this HIV-infected population. Given the resource requirements for sputum concentration, additional studies using maximal blinding, high-quality direct microscopy, and a rigorous gold standard should be conducted before universally recommending this technique.

Direct sputum smear is still the first-choice tool for screening of tuberculosis worldwide. Variants of this technique, to improve the sensitivity are desired. Two microbiological variants of the standard sputum smear ("pellet" and "diluted-pellet") for both Ziehl-Neelsen (ZN) and auramine fluorescence (AF) staining were evaluated. In addition, two methods for concentration of mycobacteria in sputum, using positive and negative pressure filtration, were tested and compared. The evaluation of the microbiological variants was performed on 98 culture positive sputum samples from different TB patients. The diagnostics sensitivity and the level of detritus in the processed sputum were determined. Bacilli load in the smear variants was determined by microscopic observation and by manual inspection of microscopic digital images. The comparison of the mycobacteria filtration methods was performed on 76 smear positive sputum samples. Filters retaining the concentrated mycobacteria were stained with AF and compared with the direct smear. Bacilli load, detritus level, filtered volume, filtration time and background noise level, were determined. The sensitivity of microscopy with the microbiological variants was 7.1% and 2% higher in ZN and AF respectively, compared to direct smear. The sensitivity of AF in diluted pellet was significantly higher than all ZN variants (P < 0.05). Detritus level observed in slides was significantly lower in the diluted pellet than the pellet and direct smear in ZN and AF (P < 0.001). A significant increase in the bacilli load in microscopic observation and digital images analysis was observed in pellet and diluted pellet than the direct method (P <0.0001). The concentration of mycobacteria using positive-pressure filtration showed a trend to produce a higher bacilli load compared to the negative-pressure filtration and direct smear, although it was not significant. Detritus levels were significantly higher in both variants of filtration (P < 0.0001). Filtered volumes were higher in positive-pressure compared to negative-pressure filtration. Filtration times were significantly higher in negative-pressure compared to positive-pressure filtration (P < 0.0001). The proposed variants improved the performance of the standard sputum smear, making it an important test for settings with high rates of smear-negative TB cases.

An alternative bio-friendly sputum processing method is the need of the hour to augment the rate of detection of TB cases and to improve the sensitivity of rapid growth based diagnostic methods. Chitin, mucolytic in nature and present ubiquitously in animal kingdom, was found to have decontaminating activity when used for processing sputum specimens. The aim of the present study is to develop an alternative bio friendly sputum processing method using chitin. Smear microscopy was done on direct sputum samples and on the deposits obtained after processing with modified Petroff’s method as well as Chitin method. Two direct smears were made from each of the sputum samples and stained by Ziehl Neelsen and Auramine phenol (AP) method. The samples were divided in to two aliquots and processed by chitin and modified Petroff’s method. Smears were made from each of the deposits and stained by both methods. The deposits were inoculated on to two Lowenstein Jensen slopes. AP method showed a sensitivity of 95% in direct smear. Samples processed by chitin and the deposit smears stained by AP method showed a sensitivity of 80% and a specificity of 89% compared to that of modified Petroff’s method. The sensitivity of chitin culture is 87% and the specificity is 85%. Chitin–H2So4 solution took less time compared to 4% NaOH to homogenize the mucopurulent sputum specimens. Chitin–H2So4 can be used as an alternative method of sputum processing for the detection of M. tuberculosis.

BackgroundSputum smear microscopy is fast and inexpensive technique for detecting tuberculosis (TB) in high incidence areas but has low sensitivity. Physical and chemical sputum processing along with centrifugation have been found to show promise in overcoming this limitation. Our objective was to compare the sensitivity of smear microscopy obtained with smears made directly from respiratory specimens to those from concentrated specimens.MethodsBy active screening, 915 TB suspects were identified from Dhaka Central Jail and sputum specimens were aseptically collected. Direct smears were prepared by taking a small portion of the purulent part of the sputum with a sterile loop. The specimens were then processed by a standard N-acetyl-L-cysteine-NaOH digestion-decontamination method to prepare concentrated specimens. Both smears were then air dried, heat fixed, and stained by the Ziehl-Neelsen staining technique. The stained slides were examined under oil immersion and were graded following International Union Against Tuberculosis and Lung Diseases guidelines. All the specimens were inoculated into Lowenstein-Jensen (L-J) media and culture results were considered as gold standard to calculate sensitivity.ResultsOf 915 specimens, 73 (8%) specimens were positive both on direct and concentrated methods, one sample was positive on direct microscopy but was negative on concentrated method. An extra 14 (1.5%) samples were positive on concentrated method which were negative on direct smear. In L-J media 105 specimens were found positive for TB bacilli and of them, 74 (70.5%) and 87 (82.9%) were positive in direct and concentrated smear, respectively. The sensitivity of direct and concentrated smear microscopy was different when using positive culture as the gold standard (71% vs. 83%).ConclusionsThe results showed that concentrated technique increases the sensitivity of microscopy up to 12%. Therefore, the national programs in high TB burden countries may consider incorporating the technique into their guidelines at least in the district and higher level laboratories to improve case finding strategy.

BackgroundDiagnostic options for pulmonary tuberculosis in resource-poor settings are commonly limited to smear microscopy. We investigated whether bleach concentration by sedimentation and sputum cytology analysis (SCA) increased the positivity rate of smear microscopy for smear-positive tuberculosis.MethodsWe did a prospective diagnostic study in a Médecins Sans Frontières-supported hospital in Mindouli, Republic of Congo. Three sputum samples were obtained from 280 consecutive pulmonary tuberculosis suspects, and were processed according to WHO guidelines for direct smear microscopy. The remainder of each sputum sample was homogenised with 2.6% bleach, sedimented overnight, smeared, and examined blinded to the direct smear result for acid-fast bacilli (AFB). All direct smears were assessed for quality by SCA. If a patient produced fewer than three good-quality sputum samples, further samples were requested. Sediment smear examination was performed independently of SCA result on the corresponding direct smear. Positivity rates were compared using McNemar's test.ResultsExcluding SCA, 43.2% of all patients were diagnosed as positive on direct microscopy of up to three samples. 47.9% were diagnosed on sediment microscopy, with 48.2% being diagnosed on direct microscopy, sediment microscopy, or both. The positivity rate increased from 43.2% to 47.9% with a case definition of one positive smear (≥1 AFB/100 high power fields) of three, and from 42.1% to 43.9% with two positive smears. SCA resulted in 87.9% of patients producing at least two good-quality sputum samples, with 75.7% producing three or more. Using a case definition of one positive smear, the incremental yield of bleach sedimentation was 14/121, or 11.6% (95% CI 6.5-18.6, p = 0.001) and in combination with SCA was 15/121, or 12.4% (95% CI 7.1-19.6, p = 0.002). Incremental yields with two positive smears were 5/118, or 4.2% (95% CI 1.4-9.6, p = 0.062) and 7/118, or 5.9% (95% CI 2.4-11.8, p = 0.016), respectively.ConclusionsThe combination of bleach sedimentation and SCA resulted in significantly increased microscopy positivity rates with a case definition of either one or two positive smears. Implementation of bleach sedimentation led to a significant increase in the diagnosis of smear-positive patients. Implementation of SCA did not result in significantly increased diagnosis of tuberculosis, but did result in improved sample quality. Requesting extra sputum samples based on SCA results, combined with bleach sedimentation, could significantly increase the detection of smear-positive patients if routinely implemented in resource-limited settings where gold standard techniques are not available. We recommend that a pilot phase is undertaken before routine implementation to determine the impact in a particular context.

Comparative study between routine and molecular diagnosis of Mycobacterium species isolated from humans and bovines

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Background: Rose Bengal is a common test for serologic diagnosis of brucellosis. The aim of this study was to evaluate the agreement rate between modified rose Bengal, rose Bengal and rapid Wright tests for detection of antibody positive serum samples. Methods: C ross section study was designed. 941 serum sample of patients referred to the central medical laboratory of Kermanshah, were evaluated by rose Bengal, modified rose Bengal and rapid Wright, using convenient sampling method. Positive samples were confirmed and their titers were defined by Wright agglutination test. Results: The number of positive serum samples with modified rose Bengal was two fold higher than conventional rose Bengal test (P<0.001), which included all positive samples with rose Bengal. On the other hand, although the number of positive diagnosed serum samples with modified rose Bengal were nearly two times more than Wright test (P<0.001), but it did not include all positive samples detected by Wright test. All three tests showed acceptable agreement rate (k≥0.583). Conclusion: Results of this study confirmed that modified rose Bengal able to detect more positive serum sample than conventional rose Bengal and it would be substituted rose Bengal. However, the rapid Wright test should be used aside with modified rose Bengal, because it could not cover all positive samples detected by rapid Wright test.

Background and objectives: With 2.2 million new cases every year, Tuberculosis (TB) continues to be an epidemic of large proportions in India. Conventional direct sputum smear microscopy, though limited in its sensitivity, is still the most common method of testing for TB. Newer techniques such as concentrated sputum microscopy, have shown some promise in improving this limited sensitivity. We have compared the efficacy of concentrated sputum versus the direct smear technique in 1000 sputum samples of patients suspected to be suffering from TB. Methods: A total of 1000 sputum specimens were collected for direct acid-fast bacilli (AFB) smear, concentrated AFB smear and culture from St. John’s Medical College and Hospital. 39 contaminated samples were (3.9%) omitted during the final analysis. Mycobacterial culture was used as the reference standard method for the detection of TB. Results: 184 and 198 of the 961 samples were found to AFB positive by direct smear microscopy and concentrated smear technique respectively. The measured sensitivity and specificity of direct smear microscopy were 69.86% and 95.82%, while that of concentrated smear microscopy was 76.71% and 95.96 % respectively. 33 samples found to be negative by the direct smear method turned out to be positive by the concentrated smear technique. Conclusions: Though our study suggests no significant statistical difference between the two techniques of detecting pulmonary tuberculosis, we recommend the use of the concentrated technique in centres such as ours, where facilities are already in place. In this way, the number of cases of TB that remain untreated may significantly come down.

Introduction and ObjectivesThe Gene Xpert® MTB/RIF test has been validated in sputum samples, facilitating rapid mycobacterium tuberculosis (MTB) diagnosis with improved sensitivity compared to smear alone. Its utility in bronchoalveolar...

Introduction: The examination of three sputum samples per suspect has been severely criticized from a public health viewpoint and several recent trials have documented the relative inefficiency of the third smear and the necessity for confirmation of a positive smear has also been contested. Aim: This study, undertaken in Qena, Egypt, aimed to determine the usefulness of examining the second and third direct smear microscopy (DSM) specimen in the diagnosis of pulmonary TB. Patients and methods: A retrospective study using record review at TB outpatient clinic; Qena Chest Hospital, Egypt, was done from 2010-2013. Direct smear results were collected as one of the following combinations PNN, PPP, PPN, PNP, NNP, NPP, and NPN, NNN, where N is a negative and P a positive smear. The proportion of positive, first, second and third specimen were calculated. Cases were considered positive having at least one positive smear confirmed by another positive one in the absence of sputum culture. Results: Out of 9420 recorded suspects, 719 of them were positive, so smear positivity was 7.6%. The majority of them were diagnosed from the first sample (96.4%). For only 3.6% (26 of 719), the second smear was positive and a third specimen was required (NPP) to make a definitive diagnosis of TB. No recorded isolated positive or negative smears in the third sample (NNP or PPN). Conclusions: These data indicated that, in our locality with limited financial resources, the incremental yield of a second sputum direct smear examination was low, and the third one was negligible indicating that examination of two sputum samples is enough among pulmonary TB patients. A third sample is required only as confirmatory if the second sample was positive. Smear microscopy can be substantially simplified with favourable resource implications.

SARS-CoV-2-Positive Sputum and Feces After Conversion of Pharyngeal Samples in Patients With COVID-19.

The present study deals to diagnose Toxoplasma gondii cysts in the soil of Mosul city, with a total of 50 samples were collected from each side of the city and three replications using flotation methods and microscopic methods. The study aims to detect contamination Toxoplasmosis oocysts, in the soil with making a comparison between the right and left side of the city and determine the effect of environmental factors such as humidity on the presence of the parasite through making a comparison between wet and dry soil samples. Molecular methods confirmed the presence of infection where the microscopic examination explained the presence of 20 positive samples out of 100 samples for both sides of the city where 11 positive cases found in the right side and 9 positive samples in the left side. The difference was clear between the wet samples with a rate of (14/48) positive samples, while the number of positive dry samples was (6/52) that confirmed infected 8 of the positive samples were subjected to a molecular examination using the polymerase chain reaction (PCR). Furthermore, the results showed the presence of 3 positive samples for B1 gene of the Toxoplasma parasite. The study proved the presence of soil contamination with parasite oocysts, which gives a measure of the high incidence of toxoplasmosis in Mosul city and the possibility of transmitting the infection to the rest of the intermediate hosts including humans.