Targeted Next Generation Sequencing (tNGS) for detection of drug- resistant tuberculous meningitis: is this sequencing technology ready for prime time?

PURPOSE: Tuberculous meningitis (TBM) is the most severe form of tuberculosis (TB). Difficulty in diagnosing the condition along with other factors, increases its potential for high morbidity and mortality. Targeted Next Generation Sequencing (tNGS) generates high quality sequence read depths, enabling the identification of low-frequency alleles linked to Drug resistance (DR). The paucibacillary nature of tuberculous meningitis is a challenge for making a definitive diagnosis. METHODS: tNGS was performed on 20 cerebrospinal fluid (CSF) samples where, MGIT has shown Positive MTB Cultures. We simultaneously performed pyrosequencing (PSQ) and phenotypic Drug susceptibility testing (pDST) for these 20 samples. RESULTS: Sequencing results (from tNGS and PSQ) were compared with reference standards i.e. pDST. tNGS detected MTB in 7/20 (35%) CSF samples whereas, PSQ detected MTB in 17/20 (85%). CONCLUSION: Although tNGS has ability to detect minority variants along with detection of additional targets than PSQ, PSQ remains the diagnostic choice in our tertiary lab.

Ceftazidime-resistant Burkholderia pseudomallei masquerading as isolated atypical neuromelioidosis.

A middle-aged recently diagnosed uncontrolled diabetic from Mumbai, India presented with fever and headache for 8 weeks. Two weeks prior to his symptoms, he travelled to Lonavala during the monsoons and gave history of indoor swimming in a chlorinated pool. Investigations showed isolated neurological involvement with multiple brain abscesses. Abscess cultures grew a non-lactose fermenter. Automated systems failed to identify it. After assessment of his background history and detailed microbiological analysis of the organism, specific investigations were requested which confirmed the suspected diagnosis of melioidosis. We report an unusual presentation of ceftazidime-resistant Burkholderia pseumodmallei as isolated neuromelioidosis with brain abscesses.

Laboratory Diagnosis of Tropical Infections.

HIGHLIGHTS: (1) Blood culture is the gold standard for the diagnosis of bacterial infections. (2) Bone marrow culture is more sensitive than blood culture even in patients with enteric fever receiving antibiotics. (3) Microscopic agglutination test is considered the gold standard for diagnosing leptospirosis; however, now IgM ELISA and polymerase chain reaction (PCR) are more frequently used for diagnosis. (4) Tuberculosis is diagnosed with the help of nucleic acid amplification tests like Xpert MTB/RIF Ultra which also detects rifampicin resistance. Other tests include microscopy, Lowenstein-Jensen and mycobacteria growth indicator tube culture, line probe assay. (5) Tropical rickettsial infections are diagnosed by serological reactions (Weil-Felix, ELISA for antibodies) and PCR. (6) For Brucellosis culture from blood, bone marrow or tissue specimens remain the mainstay in diagnosis. (7) Dengue, Zika, Crimean-Congo hemorrhagic fever, Ebola, hantavirus, rabies are diagnosed with reverse transcriptase-polymerase chain reaction. Serological tests like IgM ELISA or paired sera samples for IgG are also used for diagnosis. HOW TO CITE THIS ARTICLE: Basu S, Shetty A. Laboratory Diagnosis of Tropical Infections. Indian J Crit Care Med 2021;25(Suppl 2):S122-S126.