Screening Criteria for Ophthalmic Manifestations of Congenital Zika Virus Infection.

Importance: Current guidelines recommend screening eye examinations for infants with microcephaly or laboratory-confirmed Zika virus infection but not for all infants potentially exposed to Zika virus in utero. Objective: To evaluate eye findings in a cohort of infants whose mothers had polymerase chain reaction-confirmed Zika virus infection during pregnancy. Design, Setting, and Participants: In this descriptive case series performed from January 2 through October 30, 2016, infants were examined from birth to 1 year of age by a multidisciplinary medical team, including a pediatric ophthalmologist, from Fernandes Figueira Institute, a Ministry of Health referral center for high-risk pregnancies and infectious diseases in children in Rio de Janeiro, Brazil. Participants: Mother-infant pairs from Rio de Janeiro, Brazil, who presented with suspected Zika virus infection during pregnancy were referred to our institution and had serum, urine, amniotic fluid, or placenta samples tested by real-time polymerase chain reaction for Zika virus. Main Outcomes and Measures: Description of eye findings, presence of microcephaly or other central nervous system abnormalities, and timing of infection in infants with confirmed Zika virus during pregnancy. Eye abnormalities were correlated with central nervous system findings, microcephaly, and the timing of maternal infection. Results: Of the 112 with polymerase chain reaction-confirmed Zika virus infection in maternal specimens, 24 infants (21.4%) examined had eye abnormalities (median age at first eye examination, 31 days; range, 0-305 days). Ten infants (41.7%) with eye abnormalities did not have microcephaly, and 8 (33.3%) did not have any central nervous system findings. Fourteen infants with eye abnormalities (58.3%) were born to women infected in the first trimester, 8 (33.3%) in the second trimester, and 2 (8.3%) in the third trimester. Optic nerve and retinal abnormalities were the most frequent findings. Eye abnormalities were statistically associated with microcephaly (odds ratio [OR], 19.1; 95% CI, 6.0-61.0), other central nervous system abnormalities (OR, 4.3; 95% CI, 1.6-11.2), arthrogryposis (OR, 29.0; 95% CI, 3.3-255.8), and maternal trimester of infection (first trimester OR, 5.1; 95% CI, 1.9-13.2; second trimester OR, 0.5; 95% CI, 0.2-1.2; and third trimester OR, 0.3; 95% CI, 0.1-1.2). Conclusions and Relevance: Eye abnormalities may be the only initial finding in congenital Zika virus infection. All infants with potential maternal Zika virus exposure at any time during pregnancy should undergo screening eye examinations regardless of the presence or absence of central nervous system abnormalities.

Validation of a Prediction Model for Vaginal Birth after Cesarean Delivery Reveals Unexpected Success in a Diverse American Population.

Objective To investigate the validity of a prediction model for success of vaginal birth after cesarean delivery (VBAC) in an ethnically diverse population. Methods We performed a retrospective cohort study of women admitted at a single academic institution for a trial of labor after cesarean from May 2007 to January 2015. Individual predicted success rates were calculated using the Maternal-Fetal Medicine Units Network prediction model. Participants were stratified into three probability-of-success groups: low (<35%), moderate (35-65%), and high (>65%). The actual versus predicted success rates were compared. Results In total, 568 women met inclusion criteria. Successful VBAC occurred in 402 (71%), compared with a predicted success rate of 66% (p = 0.016). Actual VBAC success rates were higher than predicted by the model in the low (57 vs. 29%; p < 0.001) and moderate (61 vs. 52%; p = 0.003) groups. In the high probability group, the observed and predicted VBAC rates were the same (79%). Conclusion When the predicted success rate was above 65%, the model was highly accurate. In contrast, for women with predicted success rates <35%, actual VBAC rates were nearly twofold higher in our population, suggesting that they should not be discouraged by a low prediction score.

The value of the first trimester ultrasound in the era of cell free DNA screening.

OBJECTIVE: To describe the clinically relevant findings detected by the first trimester ultrasound (FTU) and to determine the additional value of the FTU compared to cell free DNA (cfDNA) alone. METHOD: Retrospective cohort study of patients undergoing a FTU at a maternal-fetal medicine referral practice. Fetal, gynecologic, and placental findings detected by ultrasound were analyzed with available cfDNA and diagnostic testing results. A subgroup analysis of positive ultrasound findings and cfDNA results was performed to assess the additional benefit of ultrasound evaluation in FT prenatal screening. RESULTS: There were 1906 FTU between 1 October 2013 and 1 October 2014. CfDNA results were available for 959 (50%) patients. FTU detected: 42 fetal (2.2%), 286 gynecologic (15.0%), and 317 placental (16.6%) findings. CfDNA results were discordant with invasive testing results in 8/61 cases (13%) and with ultrasound findings in 18/42 (42%) cases. There were six false positive and two false negative cfDNA results confirmed by diagnostic testing. Subgroup analysis revealed that cfDNA as the sole method of prenatal screening in the FT would miss 95% of the fetal findings detected with ultrasound. CONCLUSION: The comprehensive FTU provides valuable clinical information about fetal and maternal anatomy that cannot be detected with cfDNA alone. © 2016 John Wiley & Sons, Ltd.

Cell-free DNA screening in clinical practice: abnormal autosomal aneuploidy and microdeletion results.

BACKGROUND: Since its commercial release in 2011 cell-free DNA screening has been rapidly adopted as a routine prenatal genetic test. However, little is known about its performance in actual clinical practice. OBJECTIVE: We sought to investigate factors associated with the accuracy of abnormal autosomal cell-free DNA results. STUDY DESIGN: We conducted a retrospective cohort study of 121 patients with abnormal cell-free DNA results from a referral maternal-fetal medicine practice from March 2013 through July 2015. Patients were included if cell-free DNA results for trisomy 21, trisomy 18, trisomy 13, or microdeletions (if reported by the laboratory) were positive or nonreportable. The primary outcome was confirmed aneuploidy or microarray abnormality on either prenatal or postnatal karyotype or microarray. Secondary outcomes were identifiable associations with in vitro fertilization, twins, ultrasound findings, testing platform, and testing laboratory. Kruskal-Wallis or Fisher exact tests were used as appropriate. RESULTS: A total of 121 patients had abnormal cell-free DNA results for trisomy 21, trisomy 18, trisomy 13, and/or microdeletions. In all, 105 patients had abnormal cell-free DNA results for trisomy 21, trisomy 18, and trisomy 13. Of these, 92 (87.6%) were positive and 13 (12.4%) were nonreportable. The results of the 92 positive cell-free DNA were for trisomy 21 (48, 52.2%), trisomy 18 (22, 23.9%), trisomy 13 (17, 18.5%), triploidy (2, 2.2%), and positive for >1 parameter (3, 3.3%). Overall, the positive predictive value of cell-free DNA was 73.5% (61/83; 95% confidence interval, 63-82%) for all trisomies (by chromosome: trisomy 21, 83.0% [39/47; 95% confidence interval, 69-92%], trisomy 18, 65.0% [13/20; 95% confidence interval, 41-84%], and trisomy 13, 43.8% [7/16; 95% confidence interval, 21-70%]). Abnormal cell-free DNA results were associated with positive serum screening (by group: trisomy 21 [17/48, 70.8%]; trisomy 18 [7/22, 77.8%]; trisomy 13 [3/17, 37.5%]; nonreportable [2/13, 16.7%]; P = .004), and abnormal first-trimester ultrasound (trisomy 21 [25/45, 55.6%]; trisomy 18 [13/20, 65%]; trisomy 13 [6/14, 42.9%]; nonreportable [1/13, 7.7%]; P = .003). There was no association between false-positive rates and testing platform, but there was a difference between the 4 laboratories (P = .018). In all, 26 patients had positive (n = 9) or nonreportable (n = 17) microdeletion results. Seven of 9 screens positive for microdeletions underwent confirmatory testing; all were false positives. CONCLUSION: The positive predictive value of 73.5% for cell-free DNA screening for autosomal aneuploidy is lower than reported. The positive predictive value for microdeletion testing was 0%. Diagnostic testing is needed to confirm abnormal cell-free DNA results for aneuploidy and microdeletions.

From Mosquitos to Humans: Genetic Evolution of Zika Virus.

Initially isolated in 1947, Zika virus (ZIKV) has recently emerged as a significant public health concern. Sequence analysis of all 41 known ZIKV RNA open reading frames to date indicates that ZIKV has undergone significant changes in both protein and nucleotide sequences during the past half century.

Zika Virus Infection in Pregnant Women in Rio de Janeiro.

BACKGROUND: Zika virus (ZIKV) has been linked to central nervous system malformations in fetuses. To characterize the spectrum of ZIKV disease in pregnant women and infants, we followed patients in Rio de Janeiro to describe clinical manifestations in mothers and repercussions of acute ZIKV infection in infants. METHODS: We enrolled pregnant women in whom a rash had developed within the previous 5 days and tested blood and urine specimens for ZIKV by reverse-transcriptase-polymerase-chain-reaction assays. We followed women prospectively to obtain data on pregnancy and infant outcomes. RESULTS: A total of 345 women were enrolled from September 2015 through May 2016; of these, 182 women (53%) tested positive for ZIKV in blood, urine, or both. The timing of acute ZIKV infection ranged from 6 to 39 weeks of gestation. Predominant maternal clinical features included a pruritic descending macular or maculopapular rash, arthralgias, conjunctival injection, and headache; 27% had fever (short-term and low-grade). By July 2016, a total of 134 ZIKV-affected pregnancies and 73 ZIKV-unaffected pregnancies had reached completion, with outcomes known for 125 ZIKV-affected and 61 ZIKV-unaffected pregnancies. Infection with chikungunya virus was identified in 42% of women without ZIKV infection versus 3% of women with ZIKV infection (P<0.001). Rates of fetal death were 7% in both groups; overall adverse outcomes were 46% among offspring of ZIKV-positive women versus 11.5% among offspring of ZIKV-negative women (P<0.001). Among 117 live infants born to 116 ZIKV-positive women, 42% were found to have grossly abnormal clinical or brain imaging findings or both, including 4 infants with microcephaly. Adverse outcomes were noted regardless of the trimester during which the women were infected with ZIKV (55% of pregnancies had adverse outcomes after maternal infection in the first trimester, 52% after infection in the second trimester, and 29% after infection in the third trimester). CONCLUSIONS: Despite mild clinical symptoms in the mother, ZIKV infection during pregnancy is deleterious to the fetus and is associated with fetal death, fetal growth restriction, and a spectrum of central nervous system abnormalities. (Funded by Ministério da Saúde do Brasil and others.).

Differential drug efflux or accumulation does not explain variation in the chloroquine response of Plasmodium falciparum strains expressing the same isoform of mutant PfCRT.

Mutant forms of the Plasmodium falciparum chloroquine resistance transporter (PfCRT) mediate chloroquine resistance by effluxing the drug from the parasite's digestive vacuole, the acidic organelle in which chloroquine exerts its parasiticidal effect. However, different parasites bearing the same mutant form of PfCRT can vary substantially in their chloroquine susceptibility. Here, we have investigated the biochemical basis for the difference in chloroquine response among transfectant parasite lines having different genetic backgrounds but bearing the same mutant form of PfCRT. Despite showing significant differences in their chloroquine susceptibility, all lines with the mutant PfCRT showed a similar chloroquine-induced H+ leak from the digestive vacuole, indicative of similar rates of PfCRT-mediated chloroquine efflux. Furthermore, all lines showed similarly reduced levels of drug accumulation. Factors other than chloroquine efflux and accumulation therefore influence the susceptibility to this drug in parasites expressing mutant PfCRT. Furthermore, in some but not all strains bearing mutant PfCRT, the 50% inhibitory concentration (IC50) for chloroquine and the degree of resistance compared to that of recombinant control parasites varied with the length of the parasite growth assays. In these parasites, the 50% inhibitory concentration for chloroquine measured in 72- or 96-h assays was significantly lower than that measured in 48-h assays. This highlights the importance of considering the first- and second-cycle activities of chloroquine in future studies of parasite susceptibility to this drug.

Investigations into the role of the Plasmodium falciparum SERCA (PfATP6) L263E mutation in artemisinin action and resistance.

Artemisinin-based combination therapies (ACTs) are highly effective for the treatment of Plasmodium falciparum malaria, yet their sustained efficacy is threatened by the potential spread of parasite resistance. Recent studies have provided evidence that artemisinins can inhibit the function of PfATP6, the P. falciparum ortholog of the ER calcium pump SERCA, when expressed in Xenopus laevis oocytes. Inhibition was significantly reduced in an L263E variant, which introduced the mammalian residue into a putative drug-binding pocket. To test the hypothesis that this single mutation could decrease P. falciparum susceptibility to artemisinins, we implemented an allelic-exchange strategy to replace the wild-type pfatp6 allele by a variant allele encoding L263E. Transfected P. falciparum clones were screened by PCR analysis for disruption of the endogenous locus and introduction of the mutant L263E allele under the transcriptional control of a calmodulin promoter. Expression of the mutant allele was demonstrated by reverse transcriptase (RT) PCR and verified by sequence analysis. Parasite clones expressing wild-type or L263E variant PfATP6 showed no significant difference in 50% inhibitory concentrations (IC(50)s) for artemisinin or its derivatives dihydroartemisinin and artesunate. Nonetheless, hierarchical clustering analysis revealed a trend toward reduced susceptibility that neared significance (artemisinin, P approximately = 0.1; dihydroartemisinin, P = 0.053 and P = 0.085; and artesunate, P = 0.082 and P = 0.162 for the D10 and 7G8 lines, respectively). Notable differences in the distribution of normalized IC(50)s provided evidence of decreased responsiveness to artemisinin and dihydroartemisinin (P = 0.02 for the D10 and 7G8 lines), but not to artesunate in parasites expressing mutant PfATP6.

Identification of a mutant PfCRT-mediated chloroquine tolerance phenotype in Plasmodium falciparum.

Mutant forms of the Plasmodium falciparum transporter PfCRT constitute the key determinant of parasite resistance to chloroquine (CQ), the former first-line antimalarial, and are ubiquitous to infections that fail CQ treatment. However, treatment can often be successful in individuals harboring mutant pfcrt alleles, raising questions about the role of host immunity or pharmacokinetics vs. the parasite genetic background in contributing to treatment outcomes. To examine whether the parasite genetic background dictates the degree of mutant pfcrt-mediated CQ resistance, we replaced the wild type pfcrt allele in three CQ-sensitive strains with mutant pfcrt of the 7G8 allelic type prevalent in South America, the Oceanic region and India. Recombinant clones exhibited strain-dependent CQ responses that ranged from high-level resistance to an incremental shift that did not meet CQ resistance criteria. Nonetheless, even in the most susceptible clones, 7G8 mutant pfcrt enabled parasites to tolerate CQ pressure and recrudesce in vitro after treatment with high concentrations of CQ. 7G8 mutant pfcrt was found to significantly impact parasite responses to other antimalarials used in artemisinin-based combination therapies, in a strain-dependent manner. We also report clinical isolates from French Guiana that harbor mutant pfcrt, identical or related to the 7G8 haplotype, and manifest a CQ tolerance phenotype. One isolate, H209, harbored a novel PfCRT C350R mutation and demonstrated reduced quinine and artemisinin susceptibility. Our data: 1) suggest that high-level CQR is a complex biological process dependent on the presence of mutant pfcrt; 2) implicate a role for variant pfcrt alleles in modulating parasite susceptibility to other clinically important antimalarials; and 3) uncover the existence of a phenotype of CQ tolerance in some strains harboring mutant pfcrt.

Gene encoding a deubiquitinating enzyme is mutated in artesunate- and chloroquine-resistant rodent malaria parasites.

Artemisinin- and artesunate-resistant Plasmodium chabaudi mutants, AS-ART and AS-ATN, were previously selected from chloroquine-resistant clones AS-30CQ and AS-15CQ respectively. Now, a genetic cross between AS-ART and the artemisinin-sensitive clone AJ has been analysed by Linkage Group Selection. A genetic linkage group on chromosome 2 was selected under artemisinin treatment. Within this locus, we identified two different mutations in a gene encoding a deubiquitinating enzyme. A distinct mutation occurred in each of the clones AS-30CQ and AS-ATN, relative to their respective progenitors in the AS lineage. The mutations occurred independently in different clones under drug selection with chloroquine (high concentration) or artesunate. Each mutation maps to a critical residue in a homologous human deubiquitinating protein structure. Although one mutation could theoretically account for the resistance of AS-ATN to artemisinin derivates, the other cannot account solely for the resistance of AS-ART, relative to the responses of its sensitive progenitor AS-30CQ. Two lines of Plasmodium falciparum with decreased susceptibility to artemisinin were also selected. Their drug-response phenotype was not genetically stable. No mutations in the UBP-1 gene encoding the P. falciparum orthologue of the deubiquitinating enzyme were observed. The possible significance of these mutations in parasite responses to chloroquine or artemisinin is discussed.

X-ray structural analysis of Plasmodium falciparum enoyl acyl carrier protein reductase as a pathway toward the optimization of triclosan antimalarial efficacy.

The x-ray crystal structures of five triclosan analogs, in addition to that of the isoniazid-NAD adduct, are described in relation to their integral role in the design of potent inhibitors of the malarial enzyme Plasmodium falciparum enoyl acyl carrier protein reductase (PfENR). Many of the novel 5-substituted analogs exhibit low micromolar potency against in vitro cultures of drug-resistant and drug-sensitive strains of the P. falciparum parasite and inhibit purified PfENR enzyme with IC50 values of <200 nM. This study has significantly expanded the knowledge base with regard to the structure-activity relationship of triclosan while affording gains against cultured parasites and purified PfENR enzyme. In contrast to a recent report in the literature, these results demonstrate the ability to improve the in vitro potency of triclosan significantly by replacing the suboptimal 5-chloro group with larger hydrophobic moieties. The biological and x-ray crystallographic data thus demonstrate the flexibility of the active site and point to future rounds of optimization to improve compound potency against purified enzyme and intracellular Plasmodium parasites.

Transporters involved in resistance to antimalarial drugs.

The ability to treat and control Plasmodium falciparum infection through chemotherapy has been compromised by the advent and spread of resistance to antimalarial drugs. Research in this area has identified the P. falciparum chloroquine resistance transporter (PfCRT) and the multidrug resistance-1 (PfMDR1) transporter as key determinants of decreased in vitro susceptibility to several principal antimalarial drugs. Transfection-based in vitro studies are consistent with clinical findings of an association between mutations in the pfcrt gene and failure of chloroquine treatment, and between amplification of the pfmdr1 gene and failure of mefloquine treatment. Many countries are now switching to artemisinin-based combination therapies. These incorporate partner drugs of which some have an in vitro efficacy that can be modulated by changes in pfcrt or pfmdr1. Here, we summarize investigations of these and other recently identified P. falciparum transporters in the context of antimalarial mode of action and mechanisms of resistance.

Decreasing pfmdr1 copy number in plasmodium falciparum malaria heightens susceptibility to mefloquine, lumefantrine, halofantrine, quinine, and artemisinin.

The global dissemination of drug-resistant Plasmodium falciparum is spurring intense efforts to implement artemisinin (ART)-based combination therapies for malaria, including mefloquine (MFQ)-artesunate and lumefantrine (LUM)-artemether. Clinical studies have identified an association between an increased risk of MFQ, MFQ-artesunate, and LUM-artemether treatment failures and pfmdr1 gene amplification. To directly address the contribution that pfmdr1 copy number makes to drug resistance, we genetically disrupted 1 of the 2 pfmdr1 copies in the drug-resistant FCB line, which resulted in reduced pfmdr1 mRNA and protein expression. These knockdown clones manifested a 3-fold decrease in MFQ IC(50) values, compared with that for the FCB line, verifying the role played by pfmdr1 expression levels in mediating resistance to MFQ. These clones also showed increased susceptibility to LUM, halofantrine, quinine, and ART. No change was observed for chloroquine. These results highlight the importance of pfmdr1 copy number in determining P. falciparum susceptibility to multiple agents currently being used to combat malaria caused by multidrug-resistant parasites.

First evidence of pfcrt mutant Plasmodium falciparum in Madagascar.

The island of Madagascar, lying in the Indian Ocean approximately 250 miles from the African coast, has so far remained one of the few areas in the world without noticeable Plasmodium falciparum high-grade chloroquine (CQ) resistance. Here we report genotyping data on pfcrt in Madagascar. The pfcrt K76T mutation, which is critical for resistance to CQ, was detected in six (3.3%) of 183 P. falciparum isolates screened, within the mutant haplotypes CVIET and CVIDT. This is the first observation of pfcrt mutant parasites on the island. The current massive distribution of CQ for in-home management of fever in children will promote the dissemination of these mutant CQ-resistant parasites. In this context, genotyping of pfcrt remains a useful tool for CQ resistance surveillance as the prevalence of pfcrt mutations is far from saturation in Madagascar.

pfmdr1 mutations contribute to quinine resistance and enhance mefloquine and artemisinin sensitivity in Plasmodium falciparum.

The emergence and spread of multidrug resistant Plasmodium falciparum has severely limited the therapeutic options for the treatment of malaria. With ever-increasing failure rates associated with chloroquine or sulphadoxine-pyrimethamine treatment, attention has turned to the few alternatives, which include quinine and mefloquine. Here, we have investigated the role of pfmdr1 3' coding region point mutations in antimalarial drug susceptibility by allelic exchange in the GC03 and 3BA6 parasite lines. Results with pfmdr1-recombinant clones indicate a significant role for the N1042D mutation in contributing to resistance to quinine and its diastereomer quinidine. The triple mutations S1034C/N1042D/D1246Y, highly prevalent in South America, were also found to enhance parasite susceptibility to mefloquine, halofantrine and artemisinin. pfmdr1 3' mutations showed minimal effect on P. falciparum resistance to chloroquine or its metabolite mono-desethylchloroquine in these parasite lines, in contrast to previously published results obtained with 7G8 parasites. This study supports the hypothesis that pfmdr1 3' point mutations can significantly affect parasite susceptibility to a wide range of antimalarials in a strain-specific manner that depends on the parasite genetic background.