Fungal endocarditis in neonate

Neonatal endocarditis is a rare but usually fatal disease. Fungal endocarditis is an uncommon complication of invasive fungal infections and is associated with a high burden of morbidity and mortality. It frequently occurs in premature infants. The majority of these infections are caused by Candida (60-70%) and Aspergillus species (20-25%). The diagnosis is difficult because the criteria that have suggested and used in adults are not readily applicable for neonates. The incidence of fungal endocarditis in a neonate is on the rise, reported in the last decade secondary to use of central venous lines, frequent use of broad-spectrum antibiotics and neonatal surgical interventions.

Role of procalcitonin in diagnosis of neonatal sepsis and procalcitonin guided duration of antibiotic therapy

Background: Neonatal sepsis is one of the important causes of neonatal morbidity and mortality particularly in the developing countries. Accurate and quick diagnosis is difficult because clinical presentation is non-specific, bacterial cultures are time-consuming and other laboratory tests lack sensitivity and specificity. Procalcitonin (PCT) is often reported to be more superior to C-reactive protein (CRP), being more sensitive and specific, starts to rise earlier and returns to normal concentration more rapidly than CRP.Methods: It is a hospital based prospective observational study. Blood samples were obtained and analyzed for blood culture, septic screen including serum CRP and PCT. Neonates were categorized into proven sepsis (n=39), probable sepsis (n=21) and clinical sepsis (n=40) groups on the basis of laboratory findings and risk factors. Data was analyzed by using standard statistical tests using SPSS 16.Results: Out of 100 cases, elevated PCT level >0.5 ng/dl was detected in 75 and >2 ng/dl was detected in 51 whereas CRP was positive only in 61 cases. Among the 39 culture positive cases, elevated serum PCT level was noticed in 39 (100%) cases whereas CRP level was noticed in 30 (76.9%) cases. Mean PCT levels were significantly high according to infection severity (P<0.01). Procalcitonin (sensitivity 87.2%, specificity 72.13%, positive predictive value 66.7% and negative predictive value 89.8% and with p value of <0.001) is more superior than CRP to predict sepsis in neonate. The mean duration of antibiotic therapy was 12.46±4.62 days in definite sepsis, 4.53±1.78 days in probable sepsis group and in clinical sepsis group 3.75±1.33 days by serial PCT measurement.Conclusions: Serum PCT levels >2 ng/dl has got a better sensitivity and NPV, which help us not only in the early diagnosis but also in the prognosis and duration of antibiotic therapy.

Childhood Moyamoya Disease: A Clinical and Angiographic Study from Eastern India.

Moyamoya disease (MMD) is a rare cerebrovascular disease of childhood with majority of cases from Japan. This is a case series of 14 children diagnosed on the basis of characteristic angiographic findings. Various clinical features and chief angiographic findings were analyzed.

Molecular typing of Mycobacterium leprae strains from northern India using short tandem repeats.

Background & objectives: Due to the inability to cultivate Mycobacterium leprae in vitro and most cases being paucibacillary, it has been difficult to apply classical genotyping methods to this organism. The objective of this study was therefore, to analyze the diversity among M. leprae strains from Uttar Pradesh, north India, by targeting ten short tandem repeats (STRs) as molecular markers. Methods: Ninety specimens including 20 biopsies and 70 slit scrappings were collected in TE buffer from leprosy patients, who attended the OPD of National JALMA Institute for Leprosy and Other Mycobacterial Diseases, Tajganj, Agra, and from villages of Model Rural Health Research Unit (MRHRU) at Ghatampur, Kanpur, Uttar Pradesh. DNA was extracted from these specimens and ten STRs loci were amplified by using published and in-house designed primers. The copy numbers were determined by electrophoretic mobility as well as sequence analysis. Phylogenetic analysis was done on variable number of tandem repeats (VNTRs) data sets using start software. Results: Diversity was observed in the cross-sectional survey of isolates obtained from 90 patients. Allelic index for different loci was found to vary from 0.7 to 0.8 except for rpoT for which allelic index was 0.186. Similarity in fingerprinting profiles observed in specimens from the cases from same house or nearby locations indicated a possible common source of infection. Such analysis was also found to be useful in discriminating the relapse from possible reinfection. Interpretation & conclusions: This study led to identification of STRs eliciting polymorphism in north Indian strains of M. leprae. The data suggest that these STRs can be used to study the sources and transmission chain in leprosy, which could be very important in monitoring of the disease dynamics in high endemic foci.

jefA (Rv2459), a drug efflux gene in Mycobacterium tuberculosis confers resistance to isoniazid & ethambutol.

Background & objectives: Drug efflux pumps have been contributing factor(s) in the development of multidrug resistance in various clinically relevant bacteria. During efflux pump gene expression studies on mycobacteria, we have found a previously uncharacterized open reading frame (ORF) Rv2459 to be overexpressed in drug stressed conditions. The objective of the present study was to investigate the role of this ORF as a drug efflux pump, which might add new information in our understanding about the alternative mechanisms of drug resistance in mycobacteria. Methods: The open reading frame Rv2459 of Mycobacterium tuberculosis encoding a probable drug efflux protein has been cloned using pSD5 E.coli-Mycobacterium shuttle vector and overexpressed in M. tuberculosis H37Rv. This ORF was named as jefA. Overexpression of this gene in clones has been verified by real-time reverse transcription PCR. Minimum inhibitory concentrations (MICs) of recombinant as well as non-recombinant clones were determined by resazurin microtitre assay plate method (REMA) with and without efflux pump inhibitors carbonyl cyanide m-chlorophenylhydrazone (CCCP) and verapamil. Results: In recombinant strains of M. tuberculosis, the overexpression of this gene led to an increase in MIC of anti-tubercular drugs isoniazid and ethambutol when tested by REMA. In the presence of CCCP and verapamil, the recombinant strains showed decrease in MIC for these drugs. Bioinformatic analysis has shown a close relation of JefA protein with drug efflux pumps of other clinically relevant bacteria. In homology derived structure prepared from nearest available model, it was observed that amino acids forming TMH 1, 8 and 11 participated in ethambutol specificity and those forming TMH 2, 7 and 10 participated in isoniazid specificity in JefA. Interpretation & conclusion: The increased transcription of jefA leads to increased resistance to ethambutol and isoniazid in M. tuberculosis via efflux pump like mechanism and contributes in the development of resistance to these drugs. JefA amino acid sequence is well conserved among clinically important bacterial genera, which further provides evidence of being a potent drug efflux pump. The involvement in drug resistance and very little homology with any of the human proteins makes JefA important to be included in the list of potential drug targets.

Estimation of efflux mediated multi-drug resistance and its correlation with expression levels of two major efflux pumps in mycobacteria.

Multidrug resistance has been posing an increasing problem in the treatment of tuberculosis. Mutations in the genomic targets of drugs have been identified as the major mechanism behind this resistance. However, high degree of resistance in some isolates towards major drugs like rifampicin, isoniazid, ethambutol and streptomycin can not be explained solely on the basis of mutations. Besides this, certain other mechanisms like efflux pumps have also been considered as alternative mechanisms in the drug resistant isolates where there is no mutation and these mechanisms are specially important for drug resistance in non-tuberculous mycobacteria (NTM). In this study, we have estimated efflux pump mediated drug resistance in different mycobacterial species with the help of efflux pump inhibitors. All major anti-tuberculous drugs have been shown to be extruded by efflux pumps and the degree to which these drugs are extruded, vary in different mycobacterial species and isolates. The correlation of this resistance with functional activity of two major efflux pump genes pstB and Rv1258c was also assessed by reverse transcription PCR. Besides the significant role of these pumps observed, other efflux pumps, present in mycobacteria, may also be involved in drug resistance and need to be investigated.