Evaluation of combined LED-fluorescence microscopy and bleach sedimentation for diagnosis of tuberculosis at peripheral health service level

Background

Sputum microscopy is the only diagnostic for tuberculosis (TB) available at peripheral levels of health service in resource-poor countries. Its sensitivity is reduced in high HIV-prevalence settings. Sodium hypochlorite (NaOCl) specimen sedimentation prior microscopy and light-emitting diode (LED)-fluorescence microscopy (FM) can individually improve performance of microscopy. This study aimed to evaluate the performance of combined LED-FM and NaOCl sputum sedimentation for TB detection at peripheral level of health services.

Methods

A prospective study was conducted in an urban health clinic in Nairobi, Kenya. Three sputum specimens were collected over 2 days from consecutive TB suspects. Smears were prepared and stained with auramine O and Ziehl-Neelsen (ZN) methods. Bleach (3.5%) was added to the remaining specimen before overnight sedimentation at room temperature. Auramine O staining was performed on smears of sediment. A 4^th specimen was collected for TB culture. Auramine smears were read under the same microscope as used for ZN smears, but equipped with the LED FluoLED™ fluorescence illuminator.

Results

497 patients were included, and 1394 specimens collected. The yield of positive specimen was significantly increased after NaOCl sedimentation (24.9%) compared to direct LED-FM (20.6%) and direct ZN (20.3%). In detecting smear-positive patients, sensitivity was 78.5% for LED-FM after NaOCl sedimentation compared to 73.2% and 72.0% for direct LED-FM (P = 0.06) and direct ZN (P = 0.06), respectively. Specificity was 87.8% for LED-FM after NaOCl sedimentation compared to 96.7% and 95.9% for direct LED-FM (P<0.01) and direct ZN (P<0.01), respectively. Inter-reading agreement (kappa = 0.7) and technicians'' acceptability were good.

Conclusion

NaOCl sedimentation did not improve the performance of LED-FM in the diagnosis of pulmonary TB at peripheral health service level.     

Comparative Yield of Different Diagnostic Tests for Tuberculosis among People Living with HIV in Western Kenya

Background

Diagnosis followed by effective treatment of tuberculosis (TB) reduces transmission and saves lives in persons living with HIV (PLHIV). Sputum smear microscopy is widely used for diagnosis, despite limited sensitivity in PLHIV. Evidence is needed to determine the optimal diagnostic approach for these patients.

Methods

From May 2011 through June 2012, we recruited PLHIV from 15 HIV treatment centers in western Kenya. We collected up to three sputum specimens for Ziehl-Neelsen (ZN) and fluorescence microscopy (FM), GeneXpert MTB/RIF (Xpert), and culture, regardless of symptoms. We calculated the incremental yield of each test, stratifying results by CD4 cell count and specimen type; data were analyzed to account for complex sampling.

Results

From 778 enrolled patients, we identified 88 (11.3%) laboratory-confirmed TB cases. Of the 74 cases who submitted 2 specimens for microscopy and Xpert testing, ZN microscopy identified 25 (33.6%); Xpert identified those plus an additional 18 (incremental yield = 24.4%). Xpert testing of spot specimens identified 48 (57.0%) of 84 cases; whereas Xpert testing of morning specimens identified 50 (66.0%) of 76 cases. Two Xpert tests detected 22/24 (92.0%) TB cases with CD4 counts <100 cells/μL and 30/45 (67.0%) of cases with CD4 counts ≥100 cells/μl.

Conclusions

In PLHIV, Xpert substantially increased diagnostic yield compared to smear microscopy and had the highest yield when used to test morning specimens and specimens from PLHIV with CD4 count <100 cells/μL. TB programs unable to replace smear microscopy with Xpert for all symptomatic PLHIV should consider targeted replacement and using morning specimens.     

Performance of LED-based fluorescence microscopy to diagnose tuberculosis in a peripheral health centre in Nairobi

Background

Sputum microscopy is the only tuberculosis (TB) diagnostic available at peripheral levels of care in resource limited countries. Its sensitivity is low, particularly in high HIV prevalence settings. Fluorescence microscopy (FM) can improve performance of microscopy and with the new light emitting diode (LED) technologies could be appropriate for peripheral settings. The study aimed to compare the performance of LED-FM versus Ziehl-Neelsen (ZN) microscopy and to assess feasibility of LED-FM at a low level of care in a high HIV prevalence country.

Methods

A prospective study was conducted in an urban health clinic in Nairobi, Kenya. Three sputum specimens were collected over 2 days from suspected TB patients. Each sample was processed with Auramine O and ZN methods and a 4^th specimen was collected for TB culture reference standard. Auramine smears were read using the same microscope, equipped with the FluoLED™ fluorescence illuminator. Inter-reader agreement, reading time and technicians'' acceptability assessed feasibility.

Results

497 patients were included and 1394 specimens were collected. The detection yields of LED-FM and ZN microscopy were 20.3% and 20.6% (p = 0.64), respectively. Sensitivity was 73.2% for LED-FM and 72% for ZN microscopy, p = 0.32. It was 96.7% and 95.9% for specificity, p = 0.53. Inter-reader agreement was high (kappa = 0.9). Mean reading time was three times faster than ZN microscopy with very good acceptance by technicians.

Conclusions

Although it did not increase sensitivity, the faster reading time combined with very good acceptance and ease of use supports the introduction of LED-FM at the peripheral laboratory level of high TB and HIV burden countries.     

Performance of three LED-based fluorescence microscopy systems for detection of tuberculosis in Uganda

Background

Direct smear microscopy using Ziehl-Neelsen (ZN) staining is the mainstay of tuberculosis (TB) diagnosis in most high burden countries, but is limited by low sensitivity in routine practice, particularly in high human immunodeficiency virus (HIV) prevalence settings.

Methods

We compared the performance of three commercial light emitting diode (LED)-based microscopy systems (Primostar™ iLED, Lumin™ and AFTER®) for fluorescent detection of Mycobacterium tuberculosis with ZN microscopy on slides prepared from sputum of TB suspects. Examination time for LED-based fluorescent microscopy (LED FM) and ZN slides was also compared, and a qualitative user appraisal of the LED FM systems was carried out.

Results

LED FM was between 5.6 and 9.4% more sensitive than ZN microscopy, although the difference was not statistically significant. There was no significant difference in the sensitivity or specificity of the three LED FM systems, although the specificity of Fraen AFTER was somewhat lower than the other LED FM methods. Examination time for LED FM was 2 and 4 times less than for ZN microscopy. LED FM was highly acceptable to Ugandan technologists, although differences in operational performance of the three systems were reported.

Conclusions

LED FM compares favourably with ZN microscopy, with equivalent specificity and a modest increase in sensitivity. Screening of slides was substantially quicker using LED FM than ZN, and LED FM was rated highly by laboratory technologists. Available commercial systems have different operational characteristics which should be considered prior to programmatic implementation.     

Role of modified bleach method in staining of acid-fast bacilli in lymph node aspirates

OBJECTIVE: To correlate acid-fast bacilli (AFB) positivity with cytomorphologic patterns of tuberculous lymphadenitis and evaluate bleach concentration method in diagnosing lymph node tuberculosis compared to Ziehl-Neelsen (ZN) method. STUDY DESIGN: One hundred cases of tuberculous lymphadenitis diagnosed by fine needle aspiration cytology (FNAC) were analyzed and classified into 6 cytomorphologic patterns and correlated with bacillary load using routine and modified bleach methods of ZN staining. Smears were graded for AFB positivity. Sensitivity of routine ZN and modified bleach concentration was compared. RESULTS: The classic cytomorphologic pattern of tuberculosis showing epithelioid granulomas, Langerhans giant cells and caseous necrosis was seen in 23% of cases. Routine ZN staining detected AFB in 27% of cases and the modified bleach method in 72%. In 58 cases the modified bleach method had a higher grade of AFB positivity than the routine method. The modified bleach method did not miss any AFB positivity detected on routine ZN staining. CONCLUSION: The modified bleach method demonstrated AFB positivity in 72% of cases. AFB positivity grade was much higher than with routine ZN staining, making bacilli easily visible, with shorter screening time. The modified bleach method is inexpensive, easily performed and more sensitive and safe than routine ZN staining.     

LED Fluorescence Microscopy for the Diagnosis of Pulmonary Tuberculosis: A Multi-Country Cross-Sectional Evaluation

Background

The diagnosis of tuberculosis (TB) in resource-limited settings relies on Ziehl-Neelsen (ZN) smear microscopy. LED fluorescence microscopy (LED-FM) has many potential advantages over ZN smear microscopy, but requires evaluation in the field. The aim of this study was to assess the sensitivity/specificity of LED-FM for the diagnosis of pulmonary TB and whether its performance varies with the timing of specimen collection.

Methods and Findings

Adults with cough ≥2 wk were enrolled consecutively in Ethiopia, Nepal, Nigeria, and Yemen. Sputum specimens were examined by ZN smear microscopy and LED-FM and compared with culture as the reference standard. Specimens were collected using a spot-morning-spot (SMS) or spot-spot-morning (SSM) scheme to explore whether the collection of the first two smears at the health care facility (i.e., “on the spot”) the first day of consultation followed by a morning sample the next day (SSM) would identify similar numbers of smear-positive patients as smears collected via the SMS scheme (i.e., one on-the-spot-smear the first day, followed by a morning specimen collected at home and a second on-the-spot sample the second day). In total, 529 (21.6%) culture-positive and 1,826 (74.6%) culture-negative patients were enrolled, of which 1,156 (49%) submitted SSM specimens and 1,199 (51%) submitted SMS specimens. Single LED-FM smears had higher sensitivity but lower specificity than single ZN smears. Using two LED-FM or two ZN smears per patient was 72.8% (385/529, 95% CI 68.8%–76.5%) and 65.8% (348/529, 95% CI 61.6%–69.8%) sensitive (p<0.001) and 90.9% (1,660/1,826, 95% CI 89.5%–92.2%) and 98% (1,790/1,826, 95% CI 97.3%–98.6%) specific (p<0.001). Using three LED-FM or three ZN smears per patient was 77% (408/529, 95% CI 73.3%–80.6%) and 70.5% (373/529, 95% CI 66.4%–74.4%, p<0.001) sensitive and 88.1% (95% CI 86.5%–89.6%) and 96.5% (95% CI 96.8%–98.2%, p<0.001) specific. The sensitivity/specificity of ZN smear microscopy and LED-FM did not vary between SMS and SSM.

Conclusions

LED-FM had higher sensitivity but, in this study, lower specificity than ZN smear microscopy for diagnosis of pulmonary TB. Performance was independent of the scheme used for collecting specimens. The introduction of LED-FM needs to be accompanied by appropriate training, quality management, and monitoring of performance in the field.

Trial Registration

Current Controlled Trials ISRCTN53339491 Please see later in the article for the Editors'' Summary     

Implementation of LED Fluorescence Microscopy for Diagnosis of Pulmonary and HIV-Associated Tuberculosis in a Hospital Setting in Indonesia

Background

Fluorescence microscopy (FM) has not been implemented widely in TB endemic settings and little evaluation has been done in HIV-infected patients. We evaluated diagnostic performance, time and costs of FM with light-emitting diodes technology (LED-FM), compared with conventional (Zieh-Neelsen) microscopy in a hospital in Indonesia which acts as referral centre for HIV-infected patients.

Method

We included pulmonary tuberculosis suspects from the outpatient and HIV clinic. Direct and concentrated sputum smears were examined using LED-FM and ZN microscopy by two technicians who were blinded for the HIV-status and the result of the comparative test. Mean reading time per slide was recorded and cost of each slide was calculated. Mycobacteria culture served as the reference standard.

Results

Among 404 tuberculosis suspects from the outpatient clinic and 256 from the HIV clinic, mycobacteria culture was positive in 12.6% and 27%, respectively. The optimal sensitivity of LED-FM was achieved by using a threshold of ≥2 AFB/length. LED-FM had a higher sensitivity (75.5% vs. 54.9%, P<0.01) but lower specificity (90.0% vs 96.6%, P<0.01) compared to ZN microscopy. HIV was associated with a lower sensitivity but similar specificity. The average reading time using LED-FM was significantly shorter (2.23±0.78 vs 5.82±1.60 minutes, P<0.01), while costs per slide were similar.

Conclusion

High sensitivity of LED-FM combined with shorter reading time of sputum smear slides make this method a potential alternative to ZN microscopy. Additional data on specificity are needed for effective implementation of this technique in high burden TB laboratories.     

Tuberculosis of the breast. A cytomorphologic study

OBJECTIVE: Extrapulmonary tuberculosis occurring in the breast is rare despite the fact that 1-2 billion people worldwide suffer from tuberculosis. The aim of this study was to examine the cytomorphology of breast tuberculosis (breast TB) and to review the literature. STUDY DESIGN: Old records from the Cytopathology Laboratory, All India Institute of Medical Sciences, were reviewed from January 1980 to December 1998. Cases of breast TB where a cytologic diagnosis was rendered or a histologic diagnosis with prior fine needle aspiration cytology (FNAC) was available were selected. These slides were reviewed for determining the cytologic findings. RESULTS: One hundred sixty cases of breast TB were included in the study. Six males and 154 females with a clinical suspicion of carcinoma had undergone FNA that was reported as TB. The majority of the patients (111) were in the reproductive age group, 21-40 years. Of the 160 cases, 118 (73.75%) had cytomorphology diagnostic of tuberculosis--epithelioid cell granulomas with caseous necrosis. Eleven of the remaining 42 cases were positive for acid-fast bacilli (AFB) on Ziehl-Neelsen (ZN) staining, while 31 cases were confirmed to be tubercular on histology. ZN staining was done in 44 cases, and AFB were demonstrated in only 38.6% of cases. CONCLUSION: Up to 73% of breast TB can be confidently diagnosed when both epithelioid cell granulomas and necrosis are present. Also, the possibility that a woman in the reproductive age group who presents with a palpable lump in the breast may have tuberculosis must be kept in mind, especially as the incidence of breast TB may increase in the future with the global spread of AIDS.     

Epidemiology of Enterovirus D68 in Ontario

In August 2014, children’s hospitals in Kansas City, Missouri and Chicago, Illinois notified the Centers for Disease Control and Prevention (CDC) about increased numbers of pediatric patients hospitalized with severe respiratory illness (SRI). In response to CDC reports, Public Health Ontario Laboratories (PHOL) launched an investigation of patients being tested for enterovirus D-68 (EV-D68) in Ontario, Canada. The purpose of this investigation was to enhance our understanding of EV-D68 epidemiology and clinical features. Data for this study included specimens submitted for EV-D68 testing at PHOL from September 1, 2014 to October 31, 2014. Comparisons were made between patients who tested positive for the virus (cases) and those testing negative (controls). EV-D68 was identified in 153/907 (16.8%) of patients tested. In the logistic regression model adjusting for age, sex, setting and time to specimen collection, individuals younger than 20 years of age were more likely to be diagnosed with EV-D68 compared to those 20 and over, with peak positivity at ages 5–9 years. Cases were not more likely to be hospitalized than controls. Cases were more likely to be identified in September than October (OR 8.07; 95% CI 5.15 to 12.64). Routine viral culture and multiplex PCR were inadequate methods to identify EV-D68 due to poor sensitivity and inability to differentiate EV-D68 from other enterovirus serotypes or rhinovirus. Testing for EV-D68 in Ontario from July to December, 2014 detected the presence of EV-D68 virus among young children during September-October, 2014, with most cases detected in September. There was no difference in hospitalization status between cases and controls. In order to better understand the epidemiology of this virus, surveillance for EV-D68 should include testing of symptomatic individuals from all treatment settings and patient age groups, with collection and analysis of comprehensive clinical and epidemiological data.     

LED-Fluorescence Microscopy for Diagnosis of Pulmonary Tuberculosis under Programmatic Conditions in India

Background

Light-emitting diode fluorescence microscopy (LED-FM) has been shown to be more sensitive than conventional bright field microscopy using Ziehl-Neelsen (ZN) stain in detecting sputum smear positive tuberculosis in controlled laboratory conditions. In 2012, Auramine O staining based LED-FM replaced conventional ZN microscopy in 200 designated microscopy centres (DMC) of medical colleges operating in collaboration with India’s Revised National Tuberculosis Control Programme. We aimed to assess the impact of introduction of LED-FM services on sputum smear positive case detection under program conditions.

Methods

This was a before and after comparison study. In 15 randomly selected medical college DMCs, all presumptive TB patients who underwent sputum smear examination in the years 2011 (before LED-FM) and 2012 (after LED-FM) were compared. An additional 15 comparable DMCs that implemented conventional ZN sputum smear microscopy were also selected for comparison between 2011 and 2012.

Results

The proportion of presumptive TB patients (PTP)found sputum smear positive increased by 30%- from 13.6% (3432/25159) in 2011 to 17.8% (4706/26426) in 2012 (P value <0.01) in the sites that implemented LED-FM microscopy, whereas in DMCs where the ZN staining procedure is followed the proportion of sputum smear positive had remained unchanged (13.0%versus 12.6%;P value0.31).

Conclusion

Use of LED-FM significantly increased the proportion of smear positive cases among presumptive TB patients under routine program conditions in high workload laboratories. The study provides operational evidence needed to scale-up the use of LED-FM in similar settings in India and beyond.     

A sensitive and specific ES-31 antigen detection based fluorometric assay for confirmation of Mycobacterium tuberculosis in cell culture

Confirmation of presence of M. tuberculosis bacilli on microscopic examination is very important in diagnosis of tuberculosis. The present study was undertaken to find the usefulness of mycobacterial ES-31 serine protease as a marker to detect tuberculosis bacilli using fluorescein isothiocyanate conjugated anti-ES-31 serine protease antibody. This immunofluorescence method was compared with Ziehl-Neelsen and auramine-O staining methods for detection of tuberculosis bacilli. Slides were prepared for each serially diluted tuberculosis H37Ra bacilli (1 x 10(7) bacilli/ml to 5 bacilli/ml). Slides for each dilution group were stained by ZN method, auramine-O and immunostaining methods using fluorescein isothiocyanate conjugated anti-ES-31 serine protease antibody. ZN staining method showed efficacy for detection of M. tuberculosis H37Ra upto 1 x 10(4) bacilli/ml while auramine-O method showed upto 1 x 10(2) bacilli/ml. The presence of bacilli was indicated by green fluorescence on immunostaining using anti-ES-31 antibody conjugate and this method was effective upto 10 bacilli/ml. The slides which were negative for ZN (1 x 10(3) cells/ml) and auramine-O (100 cells/ml) method showed positivity on restaining with immunofluorescent staining method. The results of this preliminary study showed that immunofluorescent staining method using specific anti-ES-31 antibody conjugate was more sensitive for detection of tuberculosis bacilli than ZN and auramine-O methods in samples of laboratory strain. The utility of this method will be studied further in clinical specimens.     

Yield of Two Consecutive Sputum Specimens for the Effective Diagnosis of Pulmonary Tuberculosis

Background

From long instances, it is debatable whether three sputum specimens are required for the diagnosis of pulmonary tuberculosis (TB) or TB can be diagnosed effectively using two consecutive sputum specimens. This study was set out to evaluate the significance of examining multiple sputum specimens in diagnosis of TB.

Methods

We retrospectively reviewed the acid-fast bacillus (AFB) smear and culture results of three consecutive days’ sputum specimens from 413 confirmed TB patients which were detected as part of a larger active case finding study in Dhaka Central Jail, the largest correctional facility in Bangladesh.

Results

AFB was detected from 81% (n = 334) patients, of which 89% (n = 297) were diagnosed from the first and additional 9% (n = 30) were from the second sputum specimen. M. tuberculosis growth was observed for 406 patients and 85% (n = 343) were obtained from the first sputum and additional 10% (n = 42) were from the second one. The third specimen didn’t show significant additional diagnostic value for the detection of AFB by microscopy or growth of the M. tuberculosis.

Conclusions

We concluded from our study results that examining two consecutive sputum specimens is sufficient enough for the effective diagnosis of TB. It can also decrease the laboratory workload and hence improve the quality of work in settings with high TB burden like Bangladesh.     

Species-specific immunocytochemical localization of Mycobacterium tuberculosis complex in fine needle aspirates of tuberculous lymphadenitis using antibody to 38 kDa immunodominant protein antigen

OBJECTIVE: To examine immunocytochemical localization of Mycobacterium tuberculosis (MTB) complex antigen in fine needle aspiration (FNA) smears of tuberculous lymphadenitis (TBLN) using species-specific monoclonal antibody MTSS to 38-kDa immnunodominant protein antigen as a diagnostic adjunct to conventional cytomorphology and its advantage over Ziehl-Neelsen (ZN) microscopy. Study Design FNA smears from 340 cases-174 TBLN; 34 negative controls from nontuberculous, positive controls of 13 known acid-fast bacilli (AFB)-positive sputum smears; 50 blind controls; and 69 other controls (smears from stock cultures of bacterial, atypical mycobacteria and fungal species) were subjected to ZN and immunocytochemical staining using MTSS by the streptavidin-biotin method. RESULTS: Immunocytochemical staining was positive in 59 of 61 (96.7%) archival and 110 of 113 (97.3%) fresh FNA smears; ZN positivity for AFB was observed in 27 of 61 (44.2%) archival and 48 of 113 (42.4%) fresh FNA smears of TBLN. CONCLUSION: The immunostaining using MTSS showed a definite advantage over conventional ZN staining for detection and specific diagnosis of TBLN in FNA smears with 0% false positive results. Immunostaining of cytosmears with species specific antibody to MTB would prove to be a good diagnostic adjunct to morphologic diagnosis.     

Comparison of two methods of storing breast fine needle aspirates (FNAs) using oestrogen receptor immunocytochemical assay as a method of evaluating the storage methods

Two methods of storing fine needle aspirates were compared in 14 patients with breast cancer. the methods of storage were: (1) as a Cytospin slide prepared immediately from the aspirated material and stored at −80°C; (2) as a suspension of cells in tissue culture medium, stored at −80°C. the effect of storage on the cells was assessed by means of an oestrogen receptor immunocytochemical assay (ER-ICA). an ER positivity of 100% was obtained by ER-ICA staining of cells after storage method 1, whilst all of the specimens stored by method 2 were ER-negative. the data demonstrate that cells stored in tissue culture medium at −80°C are not suitable for ER measurement. the storage method of choice for specimens intended for ERICA is as a Cytospin slide. the ER status of cells deposited on Cytospin slides prepared immediately and stored at −80°C for 2 years could be demonstrated despite the delay in processing the specimen.     

Optimizing Mycobacterial Culture in Smear-Negative,Human Immunodeficiency Virus-Infected Tuberculosis Cases

Introduction

Tuberculosis (TB) is a significant public health problem and the diagnosis in human immunodeficiency virus (HIV)—infected individuals is challenging. The use of mycobacterial culture remains an important complementary tool and optimizing it has important benefits. We sought to determine the effect of an increase in the number of specimens evaluated, addition of nutritional supplementation to the culture medium, sputum appearance and volume on diagnostic yield and time to detection of pulmonary TB among smear-negative, HIV-infected adults.

Methods

In this prospective study conducted at the Tshwane District Hospital and Academic TB Laboratory, Pretoria, South Africa we collected three sputum specimens an hour apart from presumptive TB cases at an antiretroviral treatment site. We analysed specimens from 236 patients. Specimen appearance and volume were recorded. All specimens were processed for culture using both standard and supplemented media.

Results

A single specimen identified 79% of PTB cases using standard media; the second and third specimens added 12.5% and 8.3% respectively. Media supplementation, sputum appearance and specimen volume had no effect on culture yield or contamination rates. The mean time to detection was reduced from 19.8 days in standard cultures to 11.8 days in nutrient supplemented cultures (p = 0.002). For every 1 ml increase in sputum volume, time to detection was decreased by a factor of 0.797 (p = 0.011).

Conclusion

Use of an inexpensive culture supplement substantially reduced time to detection and could contribute to reducing treatment delay among HIV-infected cases.     

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

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.     

Microscopic-Observation Drug-Susceptibility Assay for the Diagnosis of Drug-Resistant Tuberculosis in Harare,Zimbabwe

Introduction

Limited data exist on use of the microscopic-observation drug-susceptibility (MODS) assay among persons suspected of MDR-TB living in high HIV-prevalence settings.

Methods

We retrospectively reviewed available clinical and drug susceptibility data for drug-resistant TB suspects referred for culture and drug-susceptibility testing between April 1, 2011 and March 1, 2012. The diagnostic accuracy of MODS was estimated against a reference standard including Löwenstein-Jensen (LJ) media and manual liquid (BACTEC MGIT) culture. The accuracy of MODS drug-susceptibility testing (DST) was assessed against a reference standard absolute concentration method.

Results

One hundred thirty-eight sputum samples were collected from 99 drug-resistant TB suspects; in addition, six previously cultured MDR isolates were included for assessment of DST accuracy. Among persons with known HIV infection status, 39/59 (66%) were HIV-infected. Eighty-six percent of patients had a history of prior TB treatment, and 80% of individuals were on antituberculous treatment at the time of sample collection. M. tuberculosis was identified by reference standard culture among 34/98 (35%) MDR-TB suspects. Overall MODS sensitivity for M. tuberculosis detection was 85% (95% CI, 69–95%) and specificity was 93% (95% CI, 84–98%); diagnostic accuracy did not significantly differ by HIV infection status. Median time to positivity was significantly shorter for MODS (7 days; IQR 7–15 days) than MGIT (12 days; IQR 6–16 days) or LJ (28 days; IQR 21–35 days; p<0.001). Of 33 specimens with concurrent DST results, sensitivity of the MODS assay for detection of resistance to isoniazid, rifampin, and MDR-TB was 88% (95% CI, 68–97%), 96% (95% CI, 79–100%), and 91% (95% CI, 72–99%), respectively; specificity was 89% (95% CI, 52–100%), 89% (95% CI, 52–100%), and 90% (95% CI, 56–100%), respectively.

Conclusion

In a high HIV-prevalence setting, MODS diagnosed TB and drug-resistant TB with high sensitivity and shorter turnaround time compared with standard culture and DST methods.     

A Pilot Study of Short-Duration Sputum Pretreatment Procedures for Optimizing Smear Microscopy for Tuberculosis

Background

Direct sputum smear microscopy for tuberculosis (TB) lacks sensitivity for the detection of acid fast bacilli. Sputum pretreatment procedures may enhance sensitivity. We did a pilot study to compare the diagnostic accuracy and incremental yield of two short-duration (<1 hour) sputum pretreatment procedures to optimize direct smears among patients with suspected TB at a referral hospital in India.

Methodology/Findings

Blinded laboratory comparison of bleach and universal sediment processing (USP) pretreated centrifuged auramine smears to direct Ziehl-Neelsen (ZN) and direct auramine smears and to solid (Loweinstein-Jensen (LJ)) and liquid (BACTEC 460) culture. 178 pulmonary and extrapulmonary TB suspects were prospectively recruited during a one year period. Thirty six (20.2%) were positive by either solid or liquid culture. Direct ZN smear detected 22 of 36 cases and direct auramine smears detected 26 of 36 cases. Bleach and USP centrifugation detected 24 cases each, providing no incremental yield beyond direct smears. When compared to combined culture, pretreated smears were not more sensitive than direct smears (66.6% vs 61.1 (ZN) or 72.2 (auramine)), and were not more specific (92.3% vs 93.0 (ZN) or 97.2 (auramine).

Conclusions/Significance

Short duration sputum pretreatment with bleach and USP centrifugation did not increase yield as compared to direct sputum smears. Further work is needed to confirm this in a larger study and also determine if longer duration pre-treatment might be effective in optimizing smear microscopy for TB.     

Evaluation of Cobas Amplicor MTB test to detect Mycobacterium tuberculosis in pulmonary and extrapulmonary specimens

Tuberculosis (TB) is one of the major public health problems in the world. Effective control of TB depends on rapid and correct diagnosis and appropriate treatment. The aim of this study was to evaluate the performance of Cobas Amplicor MTB (CA-MTB) test for pulmonary and extrapulmonary specimens isolated in our laboratory. A total of 424 specimens obtained from the suspected TB patients from January 2003 to August 2004 were included in this study. All specimens (173 pulmonary and 251 extrapulmonary specimens) were processed, stained, cultured and assayed using the CA-MTB test for identification of Mycobacterium tuberculosis. The CA-MTB test results were compared to culture and acid-fast staining as gold standard. The sensitivity, specificity, positive and negative predictive values of CA-MTB were determined as 73%, 100%, 100%, and 97% for pulmonary specimens, and 45%, 100%, 100% and 96% for extrapulmonary specimens respectively. The sensitivity of the test for acid-fast bacilli (AFB) smear positive pulmonary and extrapulmonary specimens was 92% and 75%. These results indicate that the CA-MTB is a rapid test for detection of tuberculosis in pulmonary specimens, but does not perform well enough in extrapulmonary specimens.     

Classification of lesions and comparison of immunohistochemical and acid fast staining in diagnosis of naturally occurring paratuberculosis in goats

Lesions associated with paratuberculosis and degrees of positivity by Ziehl-Neelsen (ZN) and Avidin–Biotin Complex peroxidase (ABC) techniques in 26 adult goats grossly or clinically suspected to paratuberculosis were studied. Of these, 17 (65.4%) had microscopic lesions associated with paratuberculosis which were classified into three categories. Diffuse multibacillary lesions (30.8%) characterized by a diffuse granulomatous enteritis with epithelioid macrophages infiltration as main inflammatory cells into lamina propria that arranged mosaic-like appearance, and filled with numerous acid fast bacilli in ZN and highly positive in ABC. Diffuse lymphocytic lesions (11.5%) composed of lymphocytes as main inflammatory infiltrate, with some epithelioid macrophages or giant cells containing few if any mycobacteria. Diffuse mixed lesions (23%) characterized by a diffuse granulomatous enteritis with infiltration of mixture of large numbers of lymphocytes and epithelioid macrophages with varying degrees of positivity in ZN and ABC. In all types of lesions, other lesions such as granulomatous lymphangitis and arteritis, lymphangiectasis, thrombi composed of degenerated epithelioid macrophages were observed. Caseous necrosis, calcification and fibrosis in mesenteric lymph nodes commonly, especially in diffuse lymphocytic form, were observed. In conclusion, the present study showed three categories of lesions in which diffuse multibacillary form was the commonest type. Both ABC and ZN methods can detect (ABC with more positivity) almost all the goats with diffuse multibacillary and mixed lesions; more cases with lymphocytic forms were positive by ABC than by ZN. ABC method seems to be an efficient diagnostic adjunct to conventional ZN staining for diagnosis of paratuberculosis in goats. The importance of sampling the ileum (especially ileocecal valve), jejunum and mesenteric lymph nodes to find microscopic lesions of paratuberculosis in goats is emphasized.