Tuberculous meningitis leads to a devastating outcome, and early diagnosis and rapid chemotherapy are vital to reduce morbidity and mortality. Since Mycobacterium tuberculosis is a kind of cytozoic pathogen and its numbers are very few in cerebrospinal fluid, detecting M. tuberculosis in cerebrospinal fluid from tuberculous meningitis patients is still a challenge for clinicians. Ziehl-Neelsen stain, the current feasible microbiological method for the diagnosis of tuberculosis, often needs a large amount of cerebrospinal fluid specimen but shows a low detection rate of M. tuberculosis. Here, we developed a modified Ziehl-Neelsen stain, involving cytospin slides with Triton processing, in which only 0.5 ml of cerebrospinal fluid specimens was required. This method not only improved the detection rate of extracellular M. tuberculosis significantly but also identified intracellular M. tuberculosis in the neutrophils, monocytes, and lymphocytes clearly. Thus, our modified method is more effective and sensitive than the conventional Ziehl-Neelsen stain, providing clinicians a convenient yet powerful tool for rapidly diagnosing tuberculous meningitis.

A Highly Efficient Ziehl-Neelsen Stain: Identifying De Novo Intracellular Mycobacterium tuberculosis and Improving Detection of Extracellular M. tuberculosis in Cerebrospinal Fluid

Tuberculosis control program of Rwanda is currently phasing in light emitting diode-fluorescent microscopy (LED-FM) as an alternative to Ziehl-Neelsen (ZN) smear microscopy. This, alongside the newly introduced Xpert (Cepheid, Sunnyvale, CA, USA) is expected to improve diagnosis of tuberculosis and detection of rifampicin resistance in patients at health facilities. We assessed the accuracy of smear microscopy and the incremental sensitivity of Xpert at tuberculosis laboratories in Rwanda. This was a cross-sectional study involving four laboratories performing ZN and four laboratories performing LED-FM microscopy. The laboratories include four intermediate (ILs) and four peripheral (PLs) laboratories. After smear microscopy, the left-over of samples, of a single early-morning sputum from 648 participants, were tested using Xpert and mycobacterial culture as a reference standard. Sensitivity of each test was compared and the incremental sensitivity of Xpert after a negative smear was assessed. A total of 96 presumptive pulmonary tuberculosis participants were culture positive for M. tuberculosis. The overall sensitivity in PL of ZN was 55.1 % (40.2–69.3 %), LED-FM was 37 % (19.4–57.6 %) and Xpert was 77.6 % (66.6–86.4 %) whereas in ILs the same value for ZN was 58.3 % (27.7–84.8 %), LED-FM was 62.5 % (24.5–91.5 %) and Xpert was 90 (68.3–98.8 %). The sensitivity for all tests was significantly higher among HIV-negative individuals (all test p <0.05). The overall incremental sensitivity of Xpert over smear microscopy was 32.3 %; p < 0.0001. The incremental sensitivity of Xpert was statistically significant for both smear methods at PL (32.9 %; p = 0.001) but not at the ILs (30 %; p = 0.125) for both smear methods. Our study findings of the early implementation of the LED-FM did not reveal significant increment in sensitivity compared to the method being phased out (ZN). This study showed a significant incremental sensitivity for Xpert from both smear methods at peripheral centers where majority of TB patients are diagnosed. Overall our findings support the recommendation for Xpert as an initial diagnostic test in adults and children presumed to have TB.

/pdf/diagnostic-performance-of-smear-microscopy-and-incremental-3sr8b8kuhg.pdf

Diagnostic performance of smear microscopy and incremental yield of Xpert in detection of pulmonary tuberculosis in Rwanda

Introduction: Light emitting diode fluorescence microscopes have many practical advantages over conventional mercury vapour fluorescence microscopes, which would make them the preferred choice for laboratories in both low- and highresource settings, provided performance is equivalent. Methods: In a nested case-control study, we compared diagnostic accuracy and time required to read slides with the Zeiss PrimoStar iLED, LW Scientific Lumin, and a conventional fluorescence microscope (Leica DMLS). Mycobacterial culture was used as the reference standard, and subgroup analysis by specimen source and organism isolated were performed. Results: There was no difference in sensitivity or specificity between the three microscopes, and agreement was high for all comparisons and subgroups. The Lumin and the conventional fluorescence microscope were equivalent with respect to time required to read smears, but the Zeiss iLED was significantly time saving compared to both. Conclusions: Light emitting diode microscopy should be considered by all tuberculosis diagnostic laboratories, including those in high income countries, as a replacement for conventional fluorescence microscopes. Our findings provide support to the recent World Health Organization policy recommending that conventional fluorescence microscopy be replaced by light emitting diode microscopy using auramine staining in all settings where fluorescence microscopy is currently used.

/pdf/comparison-of-led-and-conventional-fluorescence-microscopy-398ke766lq.pdf

Comparison of LED and conventional fluorescence microscopy for detection of acid fast bacilli in a low-incidence setting.

During the last decade there has been a dramatic change in the laboratory approach to tuberculosis (TB) diagnosis in the developing world. This change began with the realization that acid-fast bacillus smear microscopy alone was totally inadequate to deal with the dual problems of human immunodeficiency virus (HIV)-associated TB and drug-resistant TB that threaten to undermine global progress in TB control. Subsequently, increased financial resources for TB laboratory services and the establishment of a systematic process for endorsement of new TB diagnostic tools and approaches by the World Health Organization (WHO) have led to rapid expansion of TB laboratory services and the availability of several new diagnostic tests that have been introduced. These include both commercial automated and noncommercial systems for phenotypic mycobacterial liquid culture and drug susceptibility testing, a simple and inexpensive test for mycobacterial species identification in culture isolates, light-emitting diode fluorescence microscopy, and rapid molecular methods for TB case detection and the diagnosis of drug-resistant TB. The latter methodologies that include line probe assays and an automated cartridge-based real-time polymerase chain reaction (PCR)-based test are being scaled up at an unprecedented pace and are truly revolutionizing the diagnosis of drug-resistant TB. On the other hand, little progress has been made in the quest for a true point-of-care test for TB. Fortunately, this is being addressed in several discovery initiatives that hopefully will provide impetus for the development of rapid, accurate TB diagnostics for the lowest level of the health system.

The changing landscape of diagnostic services for tuberculosis.

Light-emitting diode (LED) microscopy has recently been endorsed by the World Health Organization (WHO). However, it is unclear whether LED is as accurate and cost-effective as Ziehl–Neelsen (ZN) microscopy or mercury vapour fluorescence microscopy (MVFM) in tuberculosis (TB)–HIV-co-infected subjects. Direct and concentrated sputum smears from TB suspects were evaluated using combinations of LED microscopy, ZN microscopy and MVFM. Median reading time per slide was recorded and a cost analysis performed. Mycobacterial culture served as the reference standard. 647 sputum samples were obtained from 354 patients (88 (29.8%) were HIV-infected and 161 (26%) were culture-positive for Mycobacterium tuberculosis ) . Although overall sensitivity of LED compared with ZN microscopy or MVFM was similar, sensitivity of all three modalities was lower in HIV-infected patients. In the HIV-infected group, the sensitivity of LED microscopy was higher than ZN microscopy using samples that were not concentrated (46 versus 39%; p=0.25), and better than MVFM using concentrated samples (56 versus 44; p=0.5). A similar trend was seen in the CD4 count −1 subgroup. Median (interquartile range) reading time was quicker with LED compared with ZN microscopy (1.8 (1.7–1.9) versus 2.5 (2.2–2.7) min; p≤0.001). Average cost per slide read was less for LED microscopy (US$1.63) compared with ZN microscopy (US$2.10). Among HIV–TB-co-infected patients, LED microscopy was cheaper and performed as well as ZN microscopy or MVFM independent of the staining (ZN or auramine O) or processing methods used.

https://erj.ersjournals.com/content/erj/38/6/1393.full.pdf

Comparative cost and performance of light-emitting diode microscopy in HIV-tuberculosis-co-infected patients.

OBJECTIVES To evaluate fluorescence microscopy (FM) using light emitting diode (LED) technology for the detection of acid-fast bacilli at a tertiary referral centre in Mumbai, India, a tuberculosis-endemic country. DESIGN LED FM was introduced into a laboratory experienced with Ziehl-Neelsen (ZN) microscopy but unfamiliar with FM. It was evaluated in parallel with routine ZN microscopy services and compared with mycobacterial culture as a reference standard. RESULTS A total of 1357 pulmonary and 917 extra-pulmonary specimens were examined during the study. LED FM had 78.3% sensitivity and 92.0% specificity against mycobacterial culture when using pulmonary specimens, and 34.0% sensitivity and 88.8% specificity for extra-pulmonary specimens. The mean time per smear examination was 2.48 min for ZN vs. 1.41 min for LED FM. Several biases in study design and operation identified during analysis, which are likely to lead to underestimates of LED FM accuracy, are discussed in the context of future LED FM evaluations. CONCLUSIONS Although LED FM has significant benefits over both ZN microscopy and conventional FM, its implementation and validation may be prone to difficulties which could hamper evaluation of its performance. Adequate training and detailed standard operating procedures are important to maximise accuracy.

Evaluation of light emitting diode-based fluorescence microscopy for the detection of mycobacteria in a tuberculosis-endemic region.

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Evaluation of light emitting diode-based fluorescence microscopy for the detection of mycobacteria in a tuberculosis-endemic region.