Characterization of Transferrable Mechanisms of Quinolone Resistance (TMQR) among Quinolone-resistant Escherichia coli and Klebsiella pneumoniae causing Urinary Tract Infection in Nepalese Children.

BACKGROUND: Transferrable mechanisms of quinolone resistance (TMQR) can lead to fluoroquinolone non-susceptibility in addition to chromosomal mechanisms. Some evidence suggests that fluoroquinolone resistance is increasing among the pediatric population. We sought to determine the occurrence of TMQR genes among quinolone-resistant E. coli and K. pneumoniae causing urinary tract infections among Nepalese outpatient children (< 18 years) and identify molecular characteristics of TMQR-harboring isolates. METHODS: We performed antimicrobial susceptibility testing, phenotypic extended-spectrum ß-lactamase (ESBL) and modified carbapenem inactivation method tests, and investigated the presence of six TMQR genes (qnrA, qnrB, qnrS, aac(6')-Ib-cr, oqxAB, qepA), three ESBL genes (blaCTX-M, blaTEM, blaSHV), and five carbapenemase genes (blaNDM, blaOXA-48, blaKPC, blaIMP, blaVIM). The quinolone resistance-determining region (QRDR) of gyrA and parC were sequenced for 35 TMQR-positive isolates. RESULTS: A total of 74/147 (50.3%) isolates were TMQR positive by multiplex PCR [aac(6')-Ib-cr in 48 (32.7%), qnrB in 23 (15.7%), qnrS in 18 (12.3%), qnrA in 1 (0.7%), and oqxAB in 1 (0.7%) isolate]. The median ciprofloxacin minimum inhibitory concentration of TMQR-positive isolates (64 µg/mL) was two-fold higher than those without TMQR (32 µg/mL) (p = 0.004). Ser-83→Leu and Asp-87→Asn in GyrA and Ser-80→Ile in ParC were the most common QRDR mutations (23 of 35). In addition, there was a statistically significant association between TMQR and two ß-lactamase genes; blaCTX-M (p = 0.037) and blaTEM (p = 0.000). CONCLUSION: This study suggests a high prevalence of TMQR among the quinolone-resistant E. coli and K. pneumoniae isolates causing urinary tract infection in children in this area of Nepal and an association with the carriage of ESBL gene. This is a challenge for the management of urinary infections in children. Comprehensive prospective surveillance of antimicrobial resistance in these common pathogens will be necessary to devise strategies to mitigate the emergence of further resistance.

Characteristics of Pregnant Women and Risk Factors of Their Newborn for Admission to a Neonatal Intensive Care Unit in Nepal.

BACKGROUND: Nepal has progressed gradually in reducing under-5 mortality and neonatal mortality; however, they are still high at 30.8 and 19.8 per 1000 live births, respectively. Neonatal mortality constituted about 64% of the under-5 mortality in 2019, higher than the global average of 47%. METHODS: This is a prospective study among pregnant women and their newborn babies in Siddhi Memorial Hospital, Bhaktapur, Nepal, from October 2017 to April 2018. Demographic and clinical data, high vaginal swabs of pregnant mothers, and umbilical cord blood were collected. High vaginal swabs were cultured, and umbilical cord blood samples were cultured and tested for inflammatory markers. After discharge to home, neonates were followed for 28 days of life by weekly phone calls. RESULTS: Total number of pregnant mothers enrolled was 151. The median age was 26 years (IQR: 18-40), and the proportion of adolescent mothers was 4.7%. Half of the deliveries were done by the caesarian section, and 8.6% had gestational age <37 weeks. High vaginal swab cultures were positive in 8.2% of the samples (n=135), and Escherichia coli was the most common bacteria. Out of 153 newborn babies (2 were twins), 8 (5.2%) were admitted to the neonatal intensive care unit. The proportion of low birth weight was 13%, and it was independently associated with neonatal intensive care unit admission (adjusted OR=9.4, 95%CI 1.8-50.1; P value= 0.009). CONCLUSIONS: Adolescent pregnancies and Low Birth Weight babies were commonly observed. Both of these issues need to be addressed by effective measures that would improve the current situation of maternal and child health in Nepal.

Assessment of Biofilm Formation by Candida albicans Strains Isolated from Hemocultures and Their Role in Pathogenesis in the Zebrafish Model.

Candida albicans, an opportunistic pathogen, has the ability to form biofilms in the host or within medical devices in the body. Biofilms have been associated with disseminated/invasive disease with increased severity of infection by disrupting the host immune response and prolonging antifungal treatment. In this study, the in vivo virulence of three strains with different biofilm formation strengths, that is, non-, weak-, and strong biofilm formers, was evaluated using the zebrafish model. The survival assay and fungal tissue burden were measured. Biofilm-related gene expressions were also investigated. The survival of zebrafish, inoculated with strong biofilms forming C. albicans,, was significantly shorter than strains without biofilms forming C. albicans. However, there were no statistical differences in the burden of viable colonogenic cell number between the groups of the three strains tested. We observed that the stronger the biofilm formation, the higher up-regulation of biofilm-associated genes. The biofilm-forming strain (140 and 57), injected into zebrafish larvae, possessed a higher level of expression of genes associated with adhesion, attachment, filamentation, and cell proliferation, including eap1, als3, hwp1, bcr1, and mkc1 at 8 h. The results suggested that, despite the difference in genetic background, biofilm formation is an important virulence factor for the pathogenesis of C. albicans. However, the association between biofilm formation strength and in vivo virulence is controversial and needs to be further studied.

Prevalence of vitamin D deficiency in pregnant women and their babies in Bhaktapur, Nepal.

BACKGROUND: Vitamin D deficiency has been observed worldwide in pregnant women and their newborns. Maternal vitamin D deficiency can lead to deficiency in their newborn baby and has been linked with various complications during pregnancy and delivery. There is risk of premature delivery and it is associated with high neonatal mortality. METHODS: Seventy-nine pregnant women who were admitted to the Siddhi Memorial Hospital for delivery and their newborn babies were enrolled in the study. Maternal blood samples were taken before delivery while umbilical cord blood samples of their babies were taken after delivery. Serum vitamin D level and calcium level were assessed by fluorescence immunoassay using Ichromax vitamin D kit and endpoint method, respectively in the Siddhi Memorial Hospital laboratory. RESULTS: Mean +/- SD serum vitamin D and calcium levels in pregnant mother before delivery were 14.6 +/- 8.5 ng/ml and 8.0 +/- 0.5 mg/dl, respectively, and in the cord blood were 25.7 +/- 11.2 ng/ml and 8.6 +/- 0.9 mg/dl, respectively. Eighty-one percent of the mothers and 35.8% of their babies were found to have vitamin D deficiency. Although 97.5% of the pregnant women were taking calcium supplementation, serum calcium was found lower than the normal reference value in 67% of the pregnant women and 64.2% of their babies. There were a linear relationship between the maternal and baby's serum vitamin D (P < 0.001) and calcium (P < 0.001) levels. CONCLUSION: There is high prevalence of vitamin D and calcium deficiency in pregnant mothers and newborn babies in Bhaktapur, Nepal. Pregnant women need to be supplemented with adequate amounts of these nutrients.