Experimental Induction of Paromomycin Resistance in Antimony-Resistant Strains of L. donovani: Outcome Dependent on In Vitro Selection Protocol

Paromomycin (PMM) has recently been introduced for treatment of visceral leishmaniasis in India. Although no clinical resistance has yet been reported, proactive vigilance should be warranted. The present in vitro study compared the outcome and stability of experimental PMM-resistance induction on promastigotes and intracellular amastigotes. Cloned antimony-resistant L. donovani field isolates from India and Nepal were exposed to stepwise increasing concentrations of PMM (up to 500 µM), either as promastigotes or intracellular amastigotes. One resulting resistant strain was cloned and checked for stability of resistance by drug-free in vitro passage as promastigotes for 20 weeks or a single in vivo passage in the golden hamster. Resistance selection in promastigotes took about 25 weeks to reach the maximal 97 µM inclusion level that did not affect normal growth. Comparison of the IC50 values between the parent and the selected strains revealed a 9 to 11-fold resistance for the Indian and 3 to 5-fold for the Nepalese strains whereby the resistant phenotype was also maintained at the level of the amastigote. Applying PMM pressure to intracellular amastigotes produced resistance after just two selection cycles (IC50 = 199 µM) compared to the parent strain (IC50 = 45 µM). In the amastigote-induced strains/clones, lower PMM susceptibilities were seen only in amastigotes and not at all in promastigotes. This resistance phenotype remained stable after serial in vitro passage as promastigote for 20 weeks and after a single in vivo passage in the hamster. This study clearly demonstrates that a different PMM-resistance phenotype is obtained whether drug selection is applied to promastigotes or intracellular amastigotes. These findings may have important relevance to resistance mechanism investigations and the likelihood of resistance development and detection in the field

The effect of a face mask for respiratory support on breathing in preterm infants at birth

Development and application of statistical models for medical scientific researc

Effect of clinical chorioamnionitis on breathing effort in premature infants at birth: A retrospective case-control study

Rationale: Antenatal inflammation, usually associated with chorioamnionitis, is a major cause of premature birth. As inflammation could depress respiratory drive, we have examined the effect of clinical chorioamnionitis (CCA) on spontaneous breathing in premature infants at birth. Methods: Infants with CCA born 80% (3:37 (2:10-4:29) vs 2:25 (1:06-3:52) min, p=0.016) and had a lower oxygen saturation at 5 min (77 (66-92) vs 91 (68-94) %, p=0.028), despite receiving more oxygen (62 (48-76) vs 54 (43-73) %, p=0.036). Conclusion: CCA is associated with reduced breathing effort and oxygenation in premature infants at birth

Resuscitators' opinions on using a respiratory function monitor during neonatal resuscitation.

AIM: The aim of this study was to assess the resuscitators' opinions of the usefulness and clinical value of using a respiratory function monitor (RFM) when resuscitating extremely preterm infants with positive pressure ventilation. METHODS: The link to an online survey was sent to 106 resuscitators from six countries who were involved in a multicentre trial that compared the percentage of inflations within a predefined target range with and without the RFM. The resuscitators were asked to assess the usefulness and clinical value of the RFM. The survey was online for 4 months after the trial ended in May 2019. RESULTS: The survey was completed by 74 (70%) resuscitators of which 99% considered the RFM to be helpful during neonatal resuscitation and 92% indicated that it influenced their decision-making. The majority (76%) indicated that using the RFM improved their practice and made resuscitation more effective, even when the RFM was not available. Inadequate training was the key issue that limited the effectiveness of the RFM: 45% felt insufficiently trained, and 78% felt more training in using and interpreting the RFM would have been beneficial. CONCLUSION: Resuscitators considered the RFM to be helpful to guide neonatal resuscitation, but sufficient training was required to achieve the maximum benefit

Selection procedure for induction of PMM-resistance using intracellular <i>L. donovani</i> amastigotes.

<p>Late stationary-phase promastigotes were used to infect primary mouse macrophages exposed to 2-fold PMM dilutions starting from 500 µM. After 5 days, surviving intracellular amastigotes at the highest PMM concentration (checked after Giemsa staining on a duplicate plate) were allowed to transform back into promastigotes by replacing the RPMI cell culture medium by MEM-based promastigote medium and incubation at room temperature for 1 week. Next, the recovered promastigotes were expanded in 25 ml tissue culture bottles without PMM pressure and used to infect a new batch of primary mouse macrophages for another cycle of selection.</p

PMM resistance selection on intracellular amastigotes: <i>in vitro</i> susceptibility (IC₅₀) of the parent strain and the selected clones as promastigote and as intracellular amastigote to PMM, Sb^III, Sb^V and MIL.

<p>Intracellular amastigotes were transformed back to the extracellular (without drug exposure) promastigote stage after each selection cycle. After selection cycle-2, fourteen clones were obtained from the induced PMM-resistant promastigote population.</p><p>nd: not done.</p

PMM resistance selection in promastigotes: <i>in vitro</i> PMM susceptibility (IC₅₀) of <i>L. donovani</i> parasites cultured as promastigotes under increasing PMM drug pressure.

<p>To determine amastigote susceptibility of the induced promastigotes, stationary-phase stages were used to infect mouse primary macrophages and J774 macrophages.</p><p> <i>P = parent non selected strain/R = selected resistant strain</i></p><p> <i>nd = not done.</i></p>*<p> <i>BPK087/0 cl-11: susceptible to Sb^III (IC₅₀<15 µg/ml eq.) and resistant to Sb^V (IC₅₀>77 µg/ml eq.)</i></p