The MTBDR plus line probe assay (LPA) and Xpert MTB/RIF have been endorsed by the World Health Organization for the rapid diagnosis of drug-resistant tuberculosis. However, there is no clarity regarding the superiority of one over the other. In a double-blinded prospective study, we evaluated the efficacy of the Xpert MTB/RIF on samples that were first tested by LPA under the revised national tuberculosis control program of India. A total of 405 sputum samples from suspected drug-resistant tuberculosis patients were included. Of these, 285 smear-positive samples were subjected to LPA. Seventy-two (25.8%) samples showed multidrug resistance, 62 (22.2%) showed rifampin monoresistance, 29 (10.3%) showed isoniazid monoresistance, and 116 (41.5%) were pan-susceptible. Six (2.1%) of the samples gave invalid results. Of the 62 rifampin-monoresistant samples by LPA, 38 (61.4%) showed rifampin resistance, while 21 (33.8%) were found susceptible to rifampin by Xpert MTB/RIF using cartridge version G4. Three (4.8%) samples gave an error. Of the 116 pan-susceptible samples, only 83 were available for Xpert MTB/RIF testing; 4 (5.1%) were rifampin resistant, 74 (94.8%) were susceptible, and 5 (6.0%) showed an error. The 25 discrepant samples were further subjected to MGIT960 drug susceptibility testing. The MGIT960 results showed 100% agreement with LPA results but only 64.4% agreement with Xpert MTB/RIF results. Sequencing analysis of discrepant samples showed 91.3% concordance with LPA but only 8.7% concordance with the Xpert MTB/RIF assay. These findings indicate that by using Xpert MTB/RIF testing we might be underestimating the burden of drug-resistant tuberculosis and indicate that country-specific probes need to be designed to increase the sensitivity of the Xpert MTB/RIF.

Miltefosine unresponsive and relapse cases of visceral leishmaniasis (VL) are increasingly being reported. However, there has been no laboratory confirmed reports of miltefosine resistance in VL. Here, we report two laboratory confirmed cases of VL from India. Two patients with VL were referred to us with suspected VL. The first patient was a native of the VL endemic state of Bihar, but residing in Delhi, a VL non-endemic area. He was treated with broad-spectrum antibiotics and antipyretics but was unresponsive to treatment. The second patient was from Jharkhand state in eastern India (adjoining Bihar), another endemic state for VL. He was refractory to anti-leishmanial treatment, which included administration of miltefosine. Following investigation, both patients were serologically positive for VL, and blood buffy coat from both patients grew Leishmania donovani. The isolates derived from both cases were characterized for their drug susceptibility, genetically characterised, and SNPs typed for LdMT and LdROS gene expression. Both patients were successfully treated with amphotericin B. The in vitro drug susceptibility assays carried out on both isolates showed good IC50 values to amphotericin B (0.1 ± 0.0004 μg/ml and 0.07 ± 0.0019 μg/ml). One isolate was refractory to SbIII with an IC50 of > 200 μM while the second isolate was sensitive to SbIII with an IC50 of 36.70 ± 3.2 μM. However, in both the isolates, IC50 against miltefosine was more than 10-fold higher (> 100 μM) than the standard strain DD8 (6.8 ± 0.1181 μM). Furthermore, genetic analyses demonstrated single nucleotide polymorphisms (SNPs) (354Tyr↔Phe and 1078Phe↔Tyr) in the LdMT gene of the parasites. Here, we document two laboratory confirmed cases of miltefosine resistant VL from India. Our finding highlights the urgent need to establish control measures to prevent the spread of these strains. We also propose that LdMT gene mutation analysis could be used as a molecular marker of miltefosine resistance in L. donovani.

Colistin resistance is a globalized sensible issue because it has been considered a drug of the last-line resort to treat drug-resistant bacterial infections. The product of the mobilized colistin resistance (mcr) gene and its variants are the significant causes of colistin resistance, which is emerging due to the frequent colistin use in veterinary, and these genes circulate among the bacterial community. Apart from mcr genes, some other intrinsic genes and proteins are also involved in colistin resistance. Researchers focus on the most advanced genomics (whole genome sequencing), proteomics, and bioinformatics approaches to explore the question of colistin resistance. To combat colistin resistance, researchers developed various strategies such as the development of newer drugs, the repurposing of existing drugs, combinatorial treatment by colistin with other drugs, a nano-based approach, photodynamic therapy, a CRISPRi-based strategy, and a phage-based strategy. In this timeline review, we have discussed the development of colistin resistance and its management in developing countries.

Tuberculosis (TB) in children is neglected, mainly due to lack of sensitive diagnostic tools. Recently Xpert MTB/RIF assay has revolutionized the diagnostic field, but its usefulness in pediatric TB has not been reported from India and no report is available on its use on long term archived samples. We recruited 130 pediatric patients with probable intrathoracic tuberculosis and their gastric aspirate (GA) and induced sputum (IS) samples on 2 consecutive days were collected between January 2009 and December 2012. All samples (n = 520) were subjected to smear examination, BACTEC-MGIT culture and in-house multiplex PCR. An aliquot of each sample was stored at −80 °C and tested in Xpert MTB/RIF assay in 2013. Sample wise and patient wise detection rate of smear microscopy was 4.4 % and 10 %, while for BACTEC-MGIT culture this rate was 24.4 % and 46.9 %, respectively. Of the 130 day 1 GA samples, 31.5 % and 27.7 % day 2 GA samples were culture positive. Only 17.7 % GA samples were positive on both days. Of the 130 IS samples collected on day 1 and day 2, 15.4 % and 23.1 % samples were culture positive. A combination of GA and IS yielded best results. Combining both GA and IS, the overall sensitivity of Xpert MTB/RIF on smear and culture positive samples was 95.6 %. In smear negative and culture positive samples its sensitivity was 62.5 %. The duration of sample storage impacted the Xpert MTB/RIF test performance (p = 0.0001). In smear positive samples stored for 650–849 days, its sensitivity was 85.7 % and 77.1 % for IS and GA samples which dropped to 33.3 % and 50 %, respectively, if stored for more than 1050 days. Confirmatory diagnosis of tuberculosis particularly in children is a medical challenge. No laboratory or radiological test can reach to a satisfactory level of diagnostic sensitivity. However, in this study we foundthat combination of multiple samples and multiple diagnostic tests can give much better yield, though notoptimum. In present study, combination of 2 gastric aspirates (GA) and 2 induced sputum (IS) samples collected on two consecutive days, and tested on three diagnostic methods yielded a significantly high detection rate. Despite long term storage, the overall sensitivity of Xpert MTB/RIF on smear and -culture positive samples remained very high. But after storing these samples under subfreezing conditions thesensitivity of Xpert MTB/RIF decreased significantly. This is expected because even if the sample is smear and culture positive, the count of surviving mycobacteria goes down, after several years this count can reach to a undetectable level. This report shows that smear and culture positive samples stored at subfreezing conditions for several years can be used in the Xpert MTB/RIF assay, while maintaining appreciable diagnostic test sensitivity and specificity.

Prospectively, 162 pleural fluid samples from patients with probable tuberculous pleural effusion were tested by the Xpert MTB/RIF assay and the Bactec MGIT-960 culture system. Of these, 43 (26.5%) were positive in the MGIT-960 culture, and 23 (14.2%), in the Xpert MTB/RIF assay. The sensitivity and specificity of the Xpert MTB/RIF compared with the MGIT-960 culture were 54.8% and 100%, respectively.