Wen Dai

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.

Human B cells have an active phagocytic capability and undergo immune activation upon phagocytosis of Mycobacterium tuberculosis.

The paradigm that B cells are nonphagocytic was taken for granted for a long time until phagocytic B cells were found in early vertebrate animals. Thereafter, limited evidence has shown that human B cells may also internalize bacteria. However, whether human B cells can actively phagocytose bacteria has been less extensively investigated; in particular, the mechanisms and significance of the phagocytosis require clarification. Here, we show that the human Raji B cell line can phagocytose both live and dead Mycobacterium tuberculosis (Mtb), and the phagocytosed Mtb in turn affects the immune functions of the B cells. After incubation of Raji cells with Mtb, our confocal microscopy, electron microscopy and flow cytometry data showed that Raji cells effectively engulfed Mtb as well as latex beads. The phagocytic rate was proportional to the incubation time and the amount of Mtb or beads added. Additionally, we found that normal human serum could enhance the ability of Raji cells to phagocytose Mtb, while heat-inactivated serum reversed this promoting effect. The phagocytic process of B cells could partially be inhibited by cytochalasin B, an actin inhibitor. Importantly, the phagocytosed Mtb could regulate B cell immune functions, such as stimulating IgM production and upregulating the expression of the antigen-presenting costimulatory molecules CD80 and CD86. Therefore, our results provide the first evidence that human B cells can phagocytose Mtb in an active manner that is independent of bacterial viability, and phagocytosed Mtb can in turn regulate the immune activation of B cells.

Cerebrospinal Fluid Cytology and Clinical Analysis of 34 Cases with Leptomeningeal Carcinomatosis

The clinical characteristics and cerebrospinal fluid (CSF) cytological features of 34 hospitalized patients with leptomeningeal carcinomatosis (LC) were studied. Most patients presented with signs of meningeal irritation (19 cases) and intra-cranial hypertension (23 cases). Computed tomography (CT) and/or magnetic resonance imaging (MRI) revealed brain parenchymal lesions, hydrocephalus and leptomeningeal enhancement (nine, six and eight cases, respectively). The CSF changes included high opening pressure (21 cases), increased white blood cell count (23 cases), elevated protein levels (25 cases) and low glucose levels (17 cases). Malignant cells were found in all CSF specimens and 32 cases had malignant cells in their initial CSF examinations. High serum levels of carcinoembryonic antigen (CEA) occurred in 11 patients. Signs of meningeal irritation and intra-cranial hypertension were common. It is concluded that serum CEA measurement along with CT and MRI scanning are helpful in the diagnosis of LC. Crucially, however, CSF cytology could be the most important technique for the diagnosis of LC.

High rate of drug resistance among tuberculous meningitis cases in Shaanxi province, China

The clinical and mycobacterial features of tuberculous meningitis (TBM) cases in China are not well described; especially in western provinces with poor tuberculosis control. We prospectively enrolled patients in whom TBM was considered in Shaanxi Province, northwestern China, over a 2-year period (September 2010 to December 2012). Cerebrospinal fluid specimens were cultured for Mycobacterium tuberculosis; with phenotypic and genotypic drug susceptibility testing (DST), as well as genotyping of all positive cultures. Among 350 patients included in the study, 27 (7.7%) had culture-confirmed TBM; 84 (24.0%) had probable and 239 (68.3%) had possible TBM. DST was performed on 25/27 (92.3%) culture positive specimens; 12/25 (48.0%) had “any resistance” detected and 3 (12.0%) were multi-drug resistant (MDR). Demographic and clinical features of drug resistant and drug susceptible TBM cases were similar. Beijing was the most common genotype (20/25; 80.0%) with 9/20 (45%) of the Beijing strains exhibiting drug resistance; including all 3 MDR strains. All (4/4) isoniazid resistant strains had mutations in the katG gene; 75% (3/4) of strains with phenotypic rifampicin resistance had mutations in the rpoB gene detected by Xpert MTB/RIF®. High rates of drug resistance were found among culture-confirmed TBM cases; most were Beijing strains.

Simultaneous Detection of Five Pathogens from Cerebrospinal Fluid Specimens Using Luminex Technology.

Early diagnosis and treatment are crucial for the outcome of central nervous system (CNS) infections. In this study, we developed a multiplex PCR-Luminex assay for the simultaneous detection of five major pathogens, including Mycobacterium tuberculosis, Cryptococcus neoformans, Streptococcus pneumoniae, and herpes simplex virus types 1 and 2, which frequently cause CNS infections. Through the hybridization reaction between multiplex PCR-amplified targets and oligonucleotide “anti-TAG” sequences, we found that the PCR-Luminex assay could detect as low as 101–102 copies of synthetic pathogen DNAs. Furthermore, 163 cerebrospinal fluid (CSF) specimens from patients with suspected CNS infections were used to evaluate the efficiency of this multiplex PCR-Luminex method. Compared with Ziehl-Neelsen stain, this assay showed a high diagnostic accuracy for tuberculosis meningitis (sensitivity, 90.7% and specificity, 99.1%). For cryptococcal meningitis, the sensitivity and specificity were 92% and 97.1%, respectively, compared with the May Grunwald Giemsa (MGG) stain. For herpes simplex virus types 1 and 2 encephalitis, the sensitivities were 80.8% and 100%, and the specificities were 94.2% and 99%, respectively, compared with Enzyme Linked Immunosorbent Assay (ELISA) assays. Taken together, this multiplex PCR-Luminex assay showed potential efficiency for the simultaneous detection of five pathogens and may be a promising supplement to conventional methods for diagnosing CNS infections.

Acute leukemia presenting with blasts first found in the cerebrospinal fluid but not in the peripheral blood

Acute leukemia presenting with central nervous system (CNS) signs and symptoms is uncommon and prone to be misdiagnosed. Here, we report nine patients with acute leukemia, including five patients with acute lymphoblastic leukemia (ALL) and four patients with acute myeloid leukemia (AML). These patients presented with symptoms suggestive of involvement of multiple cranial nerves, the spinal cord, and meningeal involvement. Moreover, we found that all these patients unexpectedly showed the presence of blasts in the cerebrospinal fluid (CSF) but not in the peripheral blood despite repeated examinations. Bone marrow examination confirmed the presence of acute leukemia in these patients. Seven patients died within 18months of diagnosis and two patients developed stable disease. Our findings show a novel presenting feature of acute leukemia and highlight the importance of CSF cytology in the diagnosis of acute leukemia.

Simultaneous detection of 13 viruses involved in meningoencephalitis using a newly developed multiplex PCR Mag-array system

BACKGROUND The early detection and identification of pathogens in central nervous system viral infections associated with neurological disease increases the survival rate. However, the limitations of current diagnostic methods contribute to a lack of proper diagnosis in 62% of patients. Therefore, a robust method for detecting multiple viruses in a single reaction with high specificity, throughput, and speed is required. METHODS A multiplex PCR Mag-Array (MPMA) system was developed that integrates three strategies: chimeric primer design, temperature switch PCR, and MagPlex-TAG techniques. The MPMA was used to amplify 13 target viral sequences simultaneously, with plasmids containing specific viral sequences as standard samples. To evaluate its clinical performance, 177 cerebrospinal fluid (CSF) samples were tested. RESULTS The MPMA system presented high specificity and efficiency in detecting a control panel of 13 plasmids. Among 177 CSF samples, consistent results were achieved for 19 samples pre-tested using a commercial kit. Viral pathogens were found in 28/138 undiagnosed samples, with herpes simplex viruses (HSV-1 and HSV-2) being predominant. The 20 non-infectious samples revealed negative results. Compared to sequencing methods, sensitivity for detecting HSV-1 and HSV-2 was 100% and 98.78%, respectively, and specificity was 100% and 98.22%, respectively. CONCLUSIONS A robust MPMA system that can simultaneously and reliably detect 13 meningoencephalitis-associated viruses with high specificity, throughput, and speed has been developed.

Sub-optimal Specificity of Modified Ziehl-Neelsen Staining for Quick Identification of Tuberculous Meningitis.

Background: Microbiological confirmation of tuberculous meningitis (TBM) remains problematic. We assessed the diagnostic performance of a modified Ziehl-Neelsen (MZN) staining method that showed promise in earlier studies. Methods: Patients evaluated for TBM in Shaanxi province, China, were prospectively enrolled from May, 2011 to April, 2013. Cerebrospinal fluid (CSF) specimens were evaluated using the Xpert MTB/RIF® assay, MZN staining, and standard biochemical and microbiological tests, together with detailed clinical and radiological assessment. Results: Among 316 patients included in the study, 38 had definite TBM, 66 probable TBM, 163 possible TBM and 49 “no TBM,” using consensus uniform research case definition criteria. Comparing “definite or probable TBM” to “no TBM” MZN staining had higher sensitivity than Xpert MTB/RIF® (88.5 vs. 36.5%), but greatly reduced specificity (71.4 vs. 100.0%); 14/49 (28.6%) cases with “no TBM” tested positive on MZN. Mycobacterium tuberculosis culture was performed in 104/179 (58.1%) of MZN positive samples; 12.5% (13/104) were positive. Using Xpert MTB/RIF® as the reference standard, MZN had a sensitivity of 92.1% (95% CI 79.2–97.3) and specificity of 71.4% (95% CI 57.6–82.2). Conclusion: Xpert MTB/RIF® offered a rapid and specific TBM diagnosis, but sensitivity was poor. MZN was mainly hampered by false positives. Strategies to enhance the sensitivity of Xpert MTB/RIF® or improve the diagnostic accuracy of MZN should be explored.

Active phagocytosis of Mycobacterium tuberculosis (H37Ra) by T lymphocytes (Jurkat cells)

This study aimed to co-culture Jurkat T lymphocytes with inactivated Mycobacterium tuberculosis (Mtb H37Ra), explore whether T lymphocytes could phagocytose H37Ra cells, and determine the underlying mechanism. Jurkat T lymphocytes were co-cultured with H37Ra cells, and confocal laser scanning microscopy, electron microscopy, and flow cytometry techniques were used to identify phagocytosis and elucidate its mechanism. After Jurkat T lymphocytes phagocytosed H37Ra cells, the cell body became larger, with abundant cytoplasm, the portion of the nucleus closest to the bacterium deformed, long and short pseudopodia were extended, and the folds of the cell membrane formed depressions that created phagocytic vesicles surrounding the bacterium. The macropinocytosis inhibitor amiloride and the cytoskeletal inhibitor cytochalasin D were found to inhibit phagocytic efficacy; serum complements might enhance phagocytosis through opsonization. Jurkat T lymphocytes could actively phagocytose inactivated Mtb via the macropinocytotic mechanism. Actin remodeling played an important role in the macropinocytotic process. Serum complements may regulate phagocytosis.