Strengthening the Diagnosis of Drug-Resistant Tuberculosis Using NGS-Based Approaches and Bioinformatics Pipelines for Data Analysis in India.

In India, drug-resistant tuberculosis (DR-TB) is a major public health issue and a significant challenge to stop TB program. An estimated 27% of new TB cases and 44% of previously treated TB cases are resistant to at least one anti-TB drug. The conventional methods for DR-TB diagnosis are time-consuming and have limitations, leading to delays in treatment initiation and the spread of the disease. Next-generation sequencing (NGS) based approaches have emerged as a promising tool for diagnosing DR-TB, simultaneously offering rapid and accurate detection of resistance mutations in multiple genes. NGS-based approaches generate a large amount of data, which requires efficient and reliable bioinformatics pipelines for data analysis. TBProfiler and Mykrobe are the bioinformatics pipelines that have been created to analyze NGS data for the diagnosis of DR-TB. These pipelines use reference-based and machine-learning approaches to detect resistance mutations and predict drug susceptibility, enabling clinicians to make informed treatment decisions. Implementing NGS-based approaches and bioinformatics pipelines for DR-TB diagnosis can potentially improve patient outcomes by facilitating early detection of drug resistance and guiding personalized treatment regimens. However, the widespread adoption of these approaches in India faces several challenges, including high costs, limited infrastructure, and a lack of trained personnel. Addressing these challenges requires concerted effort to ensure equitable access to and effective implementation of these innovative technologies.

First successful field evaluation of new, one-minute haemozoin-based malaria diagnostic device.

BACKGROUND: Early and accurate diagnosis of malaria is critical to the success of malaria elimination. However, the current mainstay of malaria diagnosis in the field, such as light microscopy and rapid diagnostic tests (RDTs), have limitations due to low parasite density or mutation in diagnostic markers. METHODS: We evaluated an inexpensive, robust, rapid, malaria diagnostic device, called Gazelle, that employs magneto-optical detection to identify haemozoin crystals (Hz) produced by all species of human malaria parasites in infected individuals. A beam of polarised light is passed through the lysed diluted blood sample under the influence of high (~.55T) and low magnetic fields. The difference in light transmission through the sample between the high and low magnetic fields indicates presence of Hz, suggesting possible malarial infection. A total of 300 febrile patients were screened at the malaria clinic of Indian Council of Medical Research-National Institute of Research in Tribal Health (ICMR-NIRTH), Jabalpur, India, from August 2018 to November 2018. Malaria diagnosis was done using four diagnostic methods: Gazelle, light microscopy, RDT, and malaria specific Polymerase Chain Reaction (PCR). Measures of diagnostic accuracy were compared. FINDINGS: Out of 300 febrile patients enroled and tested for the presence of malaria parasites, 262 patient samples were included in the final analysis. The sensitivity and specificity of Gazelle was 98% and 97% in comparison to light microscopy, 82% and 99% to PCR and 78% and 99% to RDT, respectively. The results of the four diagnostic methods were comparable and statistically no significant differences in sensitivity or specificity was observed between these methods. Enhanced diagnostic accuracy of Gazelle in malaria patients with no prior history of malaria treatment was observed in this study. INTERPRETATION: The diagnostic ability of Gazelle was comparable to light microscopy and better than RDTs even in low parasitemia and in presence of pfhrp2/3 deletion mutant parasites. Gazelle may be a novel valuable diagnostic tool in resource poor settings where (i) microscopy is not feasible and (ii) pfhrp2/3gene deleted parasite are present. Its speed, cost-efficiency, and alternative to lack of microscopists makes it an important adjunct in field settings. FUNDING: HemexDx, India.

Association of p53 codon72 Arg>Pro polymorphism with susceptibility to nasopharyngeal carcinoma: evidence from a case-control study and meta-analysis.

Tumor suppressor p53 is a critical player in the fight against cancer as it controls the cell cycle check point, apoptotic pathways and genomic stability. It is known to be the most frequently mutated gene in a wide variety of human cancers. Single-nucleotide polymorphism of p53 at codon72 leading to substitution of proline (Pro) in place of arginine (Arg) has been identified as a risk factor for development of many cancers, including nasopharyngeal carcinoma (NPC). However, the association of this polymorphism with NPC across the published literature has shown conflicting results. We aimed to conduct a case-control study for a possible relation of p53 codon72 Arg>Pro polymorphism with NPC risk in underdeveloped states of India, combine the result with previously available records from different databases and perform a meta-analysis to draw a more definitive conclusion. A total of 70 NPC patients and 70 healthy controls were enrolled from different hospitals of north-eastern India. The p53 codon72 Arg>Pro polymorphism was typed by polymerase chain reaction, which showed an association with NPC risk. In the meta-analysis consisting of 1842 cases and 2330 controls, it was found that individuals carrying the Pro allele and the ProPro genotype were at a significantly higher risk for NPC as compared with those with the Arg allele and the ArgArg genotype, respectively. Individuals with a ProPro genotype and a combined Pro genotype (ProPro+ArgPro) also showed a significantly higher risk for NPC over a wild homozygote ArgArg genotype. Additionally, the strength of each study was tested by power analysis and genotype distribution by Hardy-Weinberg equilibrium. The outcome of the study indicated that both allele frequency and genotype distribution of p53 codon72 Arg>Pro polymorphism were significantly associated with NPC risk. Stratified analyses based on ethnicity and source of samples supported the above result.

Delay in virus accumulation and low virus transmission from transgenic rice plants expressing Rice tungro spherical virus RNA.

Rice tungro, a devastating viral disease of rice in South and Southeast Asia, is caused by the joint infection of a DNA virus, Rice tungro bacilliform virus (RTBV) and an RNA virus Rice tungro spherical virus (RTSV). RTBV and RTSV are transmitted exclusively by the insect vector Green leafhopper (GLH). RTSV is necessary for the transmission of RTBV. To obtain transgenic resistance against RTSV, indica rice plants were transformed using DNA constructs designed to express an untranslatable sense or anti-sense RTSV RNA. Progeny of primary transformants showing low copies of the integrated transgenes and accumulating the corresponding transcripts at low levels were challenged with viruliferous GLH. Three out of four transgenic plant lines expressing untranslatable RTSV RNA in the sense orientation and two out of the four lines expressing an RTSV gene in the anti-sense orientation showed delayed buildup of RTSV RNA over time. Transmission of RTBV from the above lines was reduced significantly.

Both Indian cassava mosaic virus and Sri Lankan cassava mosaic virus are found in India and exhibit high variability as assessed by PCR-RFLP.

The biodiversity of geminiviruses associated with the Cassava Mosaic Disease (CMD) in India was investigated using PCR to specifically amplify the DNA of Indian cassava mosaic virus (ICMV) or Sri Lankan cassava mosaic virus (SLCMV) and also by using PCR to amplify specific viral genes, followed by digestion with different restriction endonucleases to obtain polymorphic patterns (PCR-RFLP). Results showed that both ICMV and SLCMV were present in mosaic-affected cassava; ICMV was geographically restricted to certain regions, whereas SLCMV was widespread. PCR-RFLP analysis showed that, in addition to ICMV-type and SLCMV-type patterns, a high proportion (40%) of the samples displayed novel patterns, some of which were localized in certain areas, whereas others were widely distributed.

Organic nitrogen stimulates caulogenesis from hypocotyl callus of Phyllanthus fraternus.

Factors involved in promoting caulogenesis from hypocotyl explants of Phyllanthus fraternus were studied. Hypocotyl explants were cultured on B5 medium supplemented with 2,4-D or NAA in the presence and absence of BAP (at concentrations 0, 10(-7), 10(-6) and 10(-5)M). Adventitious shoots differentiated from callus developed from the cut ends of 12.5% of the hypocotyl segments cultured on medium supplemented with 10(-6)M BAP in combination with 10(-6)M 2,4-D or 10(-6)M NAA. Profuse rooting occurred from the hypocotyl explants on medium supplemented with 10(-6)M BAP + 10(-6)M NAA. Incorporation of casein hydrolysate in B5 medium along with 10(-6)M BAP + 10(-7)M 2,4-D enhanced the frequency of cultures with adventitious shoots upto 68.0%. Glutamine, glutamic acid or proline could partially substitute for the effect of casein hydrolysate. Amongst the hypocotyls from 3-14 d old seedlings, the best caulogenesis was obtained with hypocotyls from 7 d old seedlings both in presence or absence of casein hydrolysate. Best rooting of shoots was achieved on half-strength B5 medium supplemented with 10(-6)M IBA. After hardening, plantlets were successfully transferred to the soil.