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

Abstract

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.T uberculous meningitis (TBM) is the most severe form of tuberculosis and causes substantial morbidity and mortality (18). The early diagnosis of and prompt initiation of chemotherapy for TBM are crucial to a successful outcome. However, the early and accurate detection of Mycobacterium tuberculosis in the cerebrospinal fluid (CSF) of TBM patients still remains a challenge for clinicians, mainly due to the lack of rapid, efficient, and practical detection methods (30).Currently, mycobacterial culture is the gold standard for detecting M. tuberculosis, but it is time-consuming and requires specialized safety procedures in laboratories (19,26). Serological methods are convenient but lack sensitivity and specificity (4, 7). Although the PCR technique is rapid, it is costly for routine use in developing countries where most tuberculosis cases occur (5,17,21,24). Conventional smear microscopy with the Ziehl-Neelsen (ZN) stain is a rapid and practical method for detecting acid-fast bacilli (AFB), especially in low-income countries, due to its rapidity, low cost, and high positive predictive value for tuberculosis (14). However, the Ziehl-Neelsen method is severely handicapped by its low detection rate, ranging from 0 to 20% for CSF specimens (31-33). One of the main reasons behind this is that M. tuberculosis can hardly be stained by acid-fast dyes once it enters the cells. Another important reason is that the Ziehl-Neelsen method requires a large volume of CSF for TBM diagnosis, as it is incapable of detecting bacilli that are fewer than 10,000 in number per slide or per ml of specimen (32, 33). Therefore, it is important to develop an alternative, cost-effective method for detecting intracellular M. tuberculosis. Additionally, knowing which cell type is infected by M. tuberculosis in the CSF of TBM patients could help us to unravel new antituberculotic candidates (10).To reve...

Diagnostic Accuracy of Intracellular Mycobacterium tuberculosis Detection for Tuberculous Meningitis

“…The MZN stain was performed on all CSF specimens as described previously (9). In brief, 0.5 ml of CSF was loaded into a cytospin chamber with poly-Llysine-coated slides and centrifuged at 100 3 g for 5 minutes.…”

“…The first is based on the hypothesis that intracellular M. tuberculosis may be missed by conventional ZN stain and their detection may be enhanced by improving the presentation of cells on the slide and increasing the permeability of the cells to the stain (modified ZN [MZN] stain). A proof-of-concept study found it revealed AFB by microscopy in 48 of 48 CSF specimens taken from 29 patients treated for TBM as opposed to 8 of 48 positive by conventional ZN stain (9). The second test is based on the premise that the CSF white cells, particularly professional phagocytic cells, of patients with TBM may contain concentrations of M. tuberculosis-specific antigens, in particular, early secreted antigen target (ESAT)-6.…”

Efektivitas Variasi Garam Salmiak (NH4Cl) Dan Sentrifugasi Pada Pemeriksaan Basil Tahan Asam Penderita Tuberculosis

“…Prinsip dari Pewarnaan ZN adalah dinding bakteri yang tahan asam mempunyai lapisan lilin dan lemak yang sukar ditembus pengecatan, oleh karena pengaruh fenol dan pemanasan maka lapisan lilin dan lemak itu dapat ditembus cat karbol fuchsin. [4] Pada waktu pencucian dengan asam alkohol warna fuchsin tidak dilepas, Sedangkan pada bakteri tidak tahan asam akan luntur dan mengambil warna biru dari Metilen blue. Dalam metode pewarnaan ZN terdapat beberapa kekurangan yang akan mempengaruhi kesalahan pembacaan (negatif palsu), adanya artefak (sediaan kotor,endapan/Kristal reagen), sehingga dapat mempengaruhi pembacaan mikroskop.…”

“…Dalam metode pewarnaan ZN terdapat beberapa kekurangan yang akan mempengaruhi kesalahan pembacaan (negatif palsu), adanya artefak (sediaan kotor,endapan/Kristal reagen), sehingga dapat mempengaruhi pembacaan mikroskop. [4,5] Penggunaan garam salmiak 10% pada sputum yang disentrifugasi 3000 rpm selama 15 menit, hasil penelitian menunjukkan bahwa penggunaan garam salmiak 10% dengan sentrifugasi dapat meningkatkan penemuan jumlah BTA, Sediaan yang dibuat secara langsung dengan jumlah BTA yang sedikit akan menyebabkan penyebaran tidak merata, hal ini dapat menyebabkan negatif palsu, sentrifugasi dilakukan untuk mengumpulkan BTA pada endapan, sehingga apabila endapan diambil dan dibuat sediaan akan diperoleh jumlah BTA yang lebih banyak [6]. Terdapat pada penelitian lain pada pemeriksaan BTA dengan mikroskopis menggunakan sentrifugasi lebih baik dengan hasil yang signifikan dari pada metode langsung.…”

Pitfalls in clinical diagnosis and treatment of infectious meningitis in China

“…[3] Using the new acid-fast stain method of The Fourth Military Medical University, the positivity rate of CSF smear can be above 90%, enabling early diagnosis of tubercular meningitis. [4] Identifying the pathogens underlying cases of viral meningitis, weakly pathogenic bacterial meningitis, and parasitic meningitis is also difficult. For these reasons, the misdiagnosis rate in early meningitis is very high.…”