Identification of Mycobacterium tuberculosis in the cerebrospinal fluid of patients with meningitis using nested PCR

Abstract

Abstract. Tuberculous meningitis (TBM) is the most severe form of tuberculosis. It is caused by Mycobacterium tubercu losis (M. tuberculosis; MT) and it is very difficult to diagnose. The symptoms are similar to other infectious neurological diseases, such as neurocysticercosis, neuroborreliosis, or herpes viral infection. The aim of this study was to identify tuberculosis (TB) in cases of meningitis with clinical and laboratory evidence suggestive of TBM, and to confirm our findings with molecular tests for TB infection. We recruited patients with neurological symptoms who were examined at the neurology services of Hospitals of Instituto Mexicano del Seguro Social (IMSS) in Mexico City. A total of 144 consecutive patients with suggestive infectious meningitis were initially included; 94 cases of meningitis with clinical and laboratory evidence suggestive of TBM were included, but only 50 of these cases fulfilled the criteria for probable TBM. As the controls, we included 50 cases of meningitis with clinical and laboratory evidence suggestive of non-TBM. Cerebrospinal fluid (CSF) was collected from all 100 patients (cases and controls) and tested for TB by multiplex and nested PCR analyses. Nested PCR detected 0.1 fg of M. tuberculosis DNA. TB infection was confirmed with molecular tests in 49 patients from the 50 cases suggestive of TBM and in 1 of the 50 non-TBM cases. The analysis exhibited a sensitivity of 98.0%, a specificity of 92.0%, a positive predictive value of 88.0% and a negative predictive value of 98.0%. The use CSF for the analyses proved to be effective for the rapid diagnosis of TBM using a developed system of multiplex and nested PCR analyses in patients presenting neurological symptoms.

Diagnosis of urogenital tuberculosis by multiplex-nested PCR targeting mpt64 (Rv1980c) and IS6110: comparison with multiplex PCR and GeneXpert® MTB/RIF

“…Likewise, we obtained similar LODs of 100 fg and 10 ng with the menstrual blood of healthy females spiked with the purified Mtb DNA by M-nested PCR and M-PCR respectively (data not shown). Our finding is comparable with a previous report on monoplex-nested PCR targeting mtp40 (Rv2351c), wherein an LOD of 1 fg was obtained for the purified Mtb DNA, which was 10 6 -fold more sensitive than M-PCR (IS6110 + mtp40 + fbpB) (Rios-Sarabia et al 2016). In a similar manner, Sun et al (2017) demonstrated an LOD of 1 fg using a single-tube IS6110 nested PCR, where outer primers were bound at high annealing temperature (≥10°C), compared to inner primers and the final amplified product was evaluated by a lateral flow strip assay.…”

“…Our finding is comparable with a previous report on monoplex‐nested PCR targeting mtp40 (Rv2351c), wherein an LOD of 1 fg was obtained for the purified Mtb DNA, which was 10 6 ‐fold more sensitive than M‐PCR (IS 6110 + mtp40 + fbpB ) (Rios‐Sarabia et al . 2016). In a similar manner, Sun et al .…”

“…Our findings on M‐nested PCR/M‐PCR are well in consensus with the previous report, wherein mtp40 nested PCR revealed superiority over M‐PCR (IS 6110 + mtp40 + fbpB ) to diagnose TB meningitis (Rios‐Sarabia et al . 2016). While evaluating EPTB specimens including UGTB ( n = 20), mtp40 nested PCR exhibited a high sensitivity of 100% but moderate specificity (82%) within urine samples of UGTB cases, against culture (García‐Elorriaga et al .…”

Novel approach based on one-tube nested PCR and a lateral flow strip for highly sensitive diagnosis of tuberculous meningitis

“…Recently, nested PCR methods have been developed for detection and amplification of M . tuberculosis DNA with high sensitivity and specificity [ 10 – 12 ]. Nested PCR is a two-step procedure that is performed by first amplifying the target sequence with the outer primers and then using the product as the DNA template for the inner primers for amplification.…”

Herpes Simplex Virus and Tuberculosis-Associated Encephalitis in Northeast of Iran

“…In a large study in Taiwan between 1997 and 2001, it was reported that 1.5% of tuberculosis deaths were related to CNS involvement (17). A recent study has shown that tuberculosis meningitis should be detected by more accurate techniques with high sensitivity and specificity such as PCR method (18).…”