Enhancing TB Case Detection: Experience in Offering Upfront Xpert MTB/RIF Testing to Pediatric Presumptive TB and DR TB Cases for Early Rapid Diagnosis of Drug Sensitive and Drug Resistant TB

Background

Diagnosis of pulmonary tuberculosis (PTB) in children is challenging due to difficulties in obtaining good quality sputum specimens as well as the paucibacillary nature of disease. Globally a large proportion of pediatric tuberculosis (TB) cases are diagnosed based only on clinical findings. Xpert MTB/RIF, a highly sensitive and specific rapid tool, offers a promising solution in addressing these challenges. This study presents the results from pediatric groups taking part in a large demonstration study wherein Xpert MTB/RIF testing replaced smear microscopy for all presumptive PTB cases in public health facilities across India.

Methods

The study covered a population of 8.8 million across 18 programmatic sub-district level tuberculosis units (TU), with one Xpert MTB/RIF platform established at each study TU. Pediatric presumptive PTB cases (both TB and Drug Resistant TB (DR-TB)) accessing any public health facilities in study area were prospectively enrolled and tested on Xpert MTB/RIF following a standardized diagnostic algorithm.

Results

4,600 pediatric presumptive pulmonary TB cases were enrolled. 590 (12.8%, CI 11.8–13.8) pediatric PTB were diagnosed. Overall 10.4% (CI 9.5–11.2) of presumptive PTB cases had positive results by Xpert MTB/RIF, compared with 4.8% (CI 4.2–5.4) who had smear-positive results. Upfront Xpert MTB/RIF testing of presumptive PTB and presumptive DR-TB cases resulted in diagnosis of 79 and 12 rifampicin resistance cases, respectively. Positive predictive value (PPV) for rifampicin resistance detection was high (98%, CI 90.1–99.9), with no statistically significant variation with respect to past history of treatment.

Conclusion

Upfront access to Xpert MTB/RIF testing in pediatric presumptive PTB cases was associated with a two-fold increase in bacteriologically-confirmed PTB, and increased detection of rifampicin-resistant TB cases under routine operational conditions across India. These results suggest that routine Xpert MTB/RIF testing is a promising solution to present-day challenges in the diagnosis of PTB in pediatric patients.     

The deubiquitinating enzyme USP37 enhances CHK1 activity to promote the cellular response to replication stress

The deubiquitinating enzyme USP37 is known to contribute to timely onset of S phase and progression of mitosis. However, it is not clear if USP37 is required beyond S-phase entry despite expression and activity of USP37 peaking within S phase. We have utilized flow cytometry and microscopy to analyze populations of replicating cells labeled with thymidine analogs and monitored mitotic entry in synchronized cells to determine that USP37-depleted cells exhibited altered S-phase kinetics. Further analysis revealed that cells depleted of USP37 harbored increased levels of the replication stress and DNA damage markers γH2AX and 53BP1 in response to perturbed replication. Depletion of USP37 also reduced cellular proliferation and led to increased sensitivity to agents that induce replication stress. Underlying the increased sensitivity, we found that the checkpoint kinase 1 is destabilized in the absence of USP37, attenuating its function. We further demonstrated that USP37 deubiquitinates checkpoint kinase 1, promoting its stability. Together, our results establish that USP37 is required beyond S-phase entry to promote the efficiency and fidelity of replication. These data further define the role of USP37 in the regulation of cell proliferation and contribute to an evolving understanding of USP37 as a multifaceted regulator of genome stability.     

A neural network model for kindling of focal epilepsy: basic mechanism

A simple neural network model is proposed for kindling — the phenomenon of generating epilepsy by means of repeated electrical stimulation. The model satisfies Dale's hypothesis, incorporates a Hebb-like learning rule and has low periodic activity in absence of shocks. Many of the experimental observations are reproduced and some new experiments are suggested. It is proposed that the main reason for kindling is the formation of a large number of excitatory synaptic connections due to learning.     

Phylogenetic analysis and evolution of morphological characters in the genus <Emphasis Type="Italic">Jasminum</Emphasis> L. (Oleaceae) in India

Jasminum L. (Oleaceae) consists of \(\sim 200\) species that are distributed in tropical, subtropical and temperate regions of the world. In India, this genus is represented by ca 47 species of which 16 are endemic. Based on the nuclear (internal-transcribed spacer (ITS) region of nrDNA and chloroplast markers (matK, trnL-F and trnH-psbA), phylogenetic relationships in 22 species including one variety of Jasminum in India have been assessed. Maximum likelihood and Bayesian analyses from individual markers, as well as from combined dataset, reveal that the group is monophyletic if Menodora spp. are excluded from the analyses. Our analyses recovered three strongly supported clades. Ancestral character state reconstruction of taxonomically useful characters (leaf forms, leaf arrangement and flower colour) which were used to demarcate sections within the genus reveals homoplasy. Our study suggests that after split from the last common ancestor, there have been at least four reversals to unifoliolate condition. Pinnately compound leaf form evolved at least twice and trifoliolate condition evolved one time only. Alternate leaf form evolved at least twice, once in clade 1 and once in clade 3 and all the time from ancestors having opposite leaf forms. Flower colour evolution clearly depicts that clade 1 is yellow-flowered and clades 2 and 3 have admixture of white and yellow-flowered Jasminum species. Our study suggests that yellow-flowered condition evolved from the white-flowered ancestor. The present study is first to estimate the evolutionary history of Indian Jasmines.     

The sheep CD1 gene family contains at least four CD1B homologues

We identified four cDNA sequences encoding sheep homologues of the CD1 molecule. The sheep sequences were selected from λgt11 thymocyte cDNA libraries by hybridization with a humanCD1C probe and a homologous sheep probe. TheSCD1B-42 andSCD1A25 sequences encode complete CD1 molecules. The third sequence,SCD1B-52, which is closely related toSCD1B-42 and may be an allele, has the sequence encoding the α3 region precisely deleted. The fourth sequence,SCD1T10, is truncated at the 5′ end. All four sequences are related to the humanCD1B and domestic rabbitCD1B-like sequences at both nucleotide and amino acid level. Comparison of the derivedCD1 amino acid sequences with the sequence of major histocompatibility complex class I molecules showed that the sheep CD1 molecules, like human CD1 molecules, lack most of the conserved class I residues known to be involved in interaction with 132-microglobulin and the CD8 molecule. They do not contain the peptide docking residues involved in anchoring peptides in the peptide binding groove of class I molecules. Southern hybridization of sheep DNA with a sheepCD1 exon 4/ga3 probe showed that the sheep genome encodes at least sevenCD1 genes. The implications of these analyses for CD1 function are discussed. The nucleotide sequence data reported in this paper have been submitted to the EMBL/GenBank nucleotide sequence databases and have been assigned the accession numbers Z36890 (SCD1A25), Z36891 (SCD1B-42), Z36892 (SCD1B-52), and X90567 (SCDIT10)     

Mutualistic acacia ants exhibit reduced aggression and more frequent off‐tree movements near termite mounds

In many ant–plant mutualisms, ants establish colonies in hollow thorns, leaf pouches, or other specialized structures on their host plants, which they then defend from herbivores. Resource heterogeneity could affect the maintenance of these mutualisms if it leads to one or both partners altering their investment in the interaction. Such a phenomenon may be especially pertinent to the Acacia–ant mutualism found in East African savannas, where termite mounds have a profound effect on the spatial structuring of resources used by both plants and ants. Here, we examined whether the proximity to termite mounds of Acacia drepanolobium trees is associated with variation in the behavior of one of their ant associates, Crematogaster nigriceps. We found that ant colonies near termite mounds had decreased aggressive responses to simulated herbivory as well as increased off‐tree movement. We hypothesize that these changes are the result of resident ant colonies near termite mounds shifting investment from defense of their host plant to foraging for nearby resources.     

DNA barcoding,microarrays and next generation sequencing: recent tools for genetic diversity estimation and authentication of medicinal plants

DNA barcoding, microarray technology and next generation sequencing have emerged as promising tools for the elucidation of plant genetic diversity and its conservation. They are proving to be immensely helpful in authenticating the useful medicinal plants for herbal drug preparations. These newer versions of molecular markers utilize short genetic markers in the genome to characterize the organism to a particular species. This has the potential not only to classify the known and yet unknown species but also has a promising future to link the medicinally important plants according to their properties. The newer trends being followed in DNA chips and barcoding pave the way for a future with many different possibilities. Several of these possibilities might be: characterization of unknown species in a considerably less time than usual, identification of newer medicinal properties possessed by the species and also updating the data of the already existing but unnoticed properties. This can assist us to cure many different diseases and will also generate novel opportunities in medicinal drug delivery and targeting.     

Human Exonuclease 5 Is a Novel Sliding Exonuclease Required for Genome Stability

Previously, we characterized Saccharomyces cerevisiae exonuclease 5 (EXO5), which is required for mitochondrial genome maintenance. Here, we identify the human homolog (C1orf176; EXO5) that functions in the repair of nuclear DNA damage. Human EXO5 (hEXO5) contains an iron-sulfur cluster. It is a single-stranded DNA (ssDNA)-specific bidirectional exonuclease with a strong preference for 5′-ends. After loading at an ssDNA end, hEXO5 slides extensively along the ssDNA prior to cutting, hence the designation sliding exonuclease. However, the single-stranded binding protein human replication protein A (hRPA) restricts sliding and enforces a unique, species-specific 5′-directionality onto hEXO5. This specificity is lost with a mutant form of hRPA (hRPA-t11) that fails to interact with hEXO5. hEXO5 localizes to nuclear repair foci in response to DNA damage, and its depletion in human cells leads to an increased sensitivity to DNA-damaging agents, in particular interstrand cross-linking-inducing agents. Depletion of hEXO5 also results in an increase in spontaneous and damage-induced chromosome abnormalities including the frequency of triradial chromosomes, suggesting an additional defect in the resolution of stalled DNA replication forks in hEXO5-depleted cells.     

Regulation of prolactin in mice with altered hypothalamic melanocortin activity

This study used two mouse models with genetic manipulation of the melanocortin system to investigate prolactin regulation. Mice with overexpression of the melanocortin receptor (MC-R) agonist, α-melanocyte-stimulating hormone (Tg-MSH) or deletion of the MC-R antagonist agouti-related protein (AgRP KO) were studied. Male Tg-MSH mice had lower blood prolactin levels at baseline (2.9±0.3 vs. 4.7±0.7ng/ml) and after restraint stress (68±6.5 vs. 117±22ng/ml) vs. WT (p<0.05); however, pituitary prolactin content was not different. Blood prolactin was also decreased in male AgRP KO mice at baseline (4.2±0.5 vs. 7.6±1.3ng/ml) and after stress (60±4.5 vs. 86.1±5.7ng/ml) vs. WT (p<0.001). Pituitary prolactin content was lower in male AgRP KO mice (4.3±0.3 vs. 6.7±0.5μg/pituitary, p<0.001) vs. WT. No differences in blood or pituitary prolactin levels were observed in female AgRP KO mice vs. WT. Hypothalamic dopamine activity was assessed as the potential mechanism responsible for changes in prolactin levels. Hypothalamic tyrosine hydroxylase mRNA was measured in both genetic models vs. WT mice and hypothalamic dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) content were measured in male AgRP KO and WT mice but neither were significantly different. However, these results do not preclude changes in dopamine activity as dopamine turnover was not directly investigated. This is the first study to show that baseline and stress-induced prolactin release and pituitary prolactin content are reduced in mice with genetic alterations of the melanocortin system and suggests that changes in hypothalamic melanocortin activity may be reflected in measurements of serum prolactin levels.