Pablo Bifani

• PhD
• Yong Loo Lin School of Medicine, National University of Singapore

Testing Plasmodium vivax antimicrobial sensitivity is limited to ex vivo schizont maturation assays, which preclude determining the IC50s of delayed action antimalarials such as doxycycline. Using Plasmodium cynomolgi as a model for P. vivax, we determined the physiologically significant delayed death effect induced by doxycycline [IC50(96 h), 1,40...

Testing Plasmodium vivax antimicrobial sensitivity is limited to ex vivo schizont maturation assays, which precludes determining the IC50s of delayed action antimalarials such as doxycycline. Using Plasmodium cynomolgi as a model for P. vivax , we determined the physiologically significant delayed death effect induced by doxycycline (IC50(96h), 140...

The lack of a long-term in vitro culture method has severely restricted the study of Plasmodium vivax, in part because it limits genetic manipulation and reverse genetics. We used the recently optimized P. cynomolgi Berok in vitro culture model to investigate the putative P. vivax drug resistance marker MDR1 Y976F. Introduction of this mutation usi...

Improved control of Plasmodium vivax malaria can be achieved with the discovery of new antimalarials with radical cure efficacy, including prevention of relapse caused by hypnozoites residing in the liver of patients. We screened several compound libraries against P. vivax liver stages, including 1565 compounds against mature hypnozoites, resulting...

Improved control of malaria caused by Plasmodium vivax can be achieved with the discovery of new antimalarials with radical cure efficacy, including prevention of relapse caused by hypnozoites residing in the liver of vivax patients. We screened several compound libraries against P. vivax liver stages, including 1566 compounds against mature hypnoz...

A major obstacle impeding malaria research is the lack of an in vitro system capable of supporting infection through the entire liver stage cycle of the parasite, including that of the dormant forms known as hypnozoites. Primary hepatocytes lose their liver specific functions in long‐term in vitro culture. The malaria parasite Plasmodium initiates...

The ability to culture pathogenic organisms substantially enhances the quest for fundamental knowledge and the development of vaccines and drugs. Thus, the elaboration of a protocol for the in vitro cultivation of the erythrocytic stages of Plasmodium falciparum revolutionized research on this important parasite. However, for P. vivax, the most wid...

Plasmodium vivax hypnozoites persist in the liver, cause malaria relapse and represent a major challenge to malaria elimination. Our previous transcriptomic study provided a novel molecular framework to enhance our understanding of the hypnozoite biology (Voorberg-van der Wel A, et al., 2017). In this dataset, we identified and characterized the Li...

Hypnozoites are the liver stage non-dividing form of the malaria parasite that are responsible for relapse and acts as a natural reservoir for human malaria Plasmodium vivax and P. ovale as well as a phylogenetically related simian malaria P. cynomolgi. Our understanding of hypnozoite biology remains limited due to the technical challenge of requir...

Treating M. abscessus infection is challenging due to the potent β-lactamase BlaMab (Beta-lactamase of M. abscessus). Avibactam is a non-β-lactam, β-lactamase inhibitor shown to inhibit BlaMab. We tested whether avibactem can render piperacillin effective against M. Abscessus. In-vitro, avibactam enhanced the activity of piperacillin by 16–32 fold,...

Artemisinin (ART) resistance has spread through Southeast Asia, posing serious threat to the control and elimination of malaria. ART resistance has been associated with mutations in the Plasmodium falciparum kelch-13 ( Pfk13 ) propeller domain. Phenotypically, ART resistance is defined as delayed parasite clearance in patients' due to the reduced s...

Plasmodium liver hypnozoites, which cause disease relapse, are widely considered to be the last barrier towards malaria eradication. The biology of this quiescent form of the parasite is poorly understood which hinders drug discovery. We report a comparative transcriptomic dataset of replicating liver schizonts and dormant hypnozoites of the relaps...

Neocuproine treatment of liver schizonts and hypnozoites. Results of the three assays presented in Figure 4C.

P. cynomolgi samples used for RNAseq, related to Figure 1

Gene expression values (FPKM after host normalization) generated by RNA-seq of hypnozoite (Hz) and liver schizont (Sz) samples, related to Figure 1

List of differentially expressed genes between hypnozoite and liver schizont samples passing the cut-off of >2 fold change absolute value and a 10% false discovery rate, related to Figure 2

Orthology groups obtained from the mapping of P. cynomolgi proteins to the proteomes of the following species: P. falciparum 3D7, P. berghei ANKA, P. knowlesi H, P. vivax Sal1, P. yoelii yoelii 17X, H. sapiens, D. melanogaster, M. musculus, R. norvegicus, and S. cerevisiae, related to Figure 3

Gene expression values (>1 FPKM after host normalization) for putative transporters generated by RNA-seq of hypnozoite (Hz) and schizont (Sz) samples, related to Figure 4

Reference genomes used in the analysis, related to Figure 1

Alignment statistics per sample, related to Figure 1

Enrichment for genes expressed in the schizont samples (Sz). Gene sets from the Gene Ontology (GO) and PlasmoDB were used and mapped to P. cynomologi identifiers. p is the adjusted p-value (Benjamini-Hochberg), related to Figure 3

Normalization of selected Plasmodium genes.

P. cynomolgi proteins linked to malaria pathways mapped from PlasmoDB, related to Figure 3

Enrichment for genes expressed in the hypnozoite samples (Hz). Gene sets from the Gene Ontology (GO) and PlasmoDB were used and mapped to P. cynomologi identifiers. p is the adjusted p-value (Benjamini-Hochberg), related to Figure 3

Plasmodium liver hypnozoites, which cause disease relapse, are widely considered to be the last barrier towards malaria eradication. The biology of this quiescent form of the parasite is poorly understood which hinders drug discovery. We report a comparative transcriptomic dataset of replicating liver schizonts and dormant hypnozoites of the relaps...

Key Points Zoonotic P cynomolgi switches red cell tropism for reticulocytes expressing Trf1 (CD71+) and DARC (CD234+). In the human host, P cynomolgi displays an almost identical rheopathobiology to P vivax.

Malaria control and elimination are threatened by the emergence and spread of resistance to artemisinin-based combination therapies (ACTs). Experimental evidence suggests that when an artemisinin (ART)-sensitive (K13 wild-type) Plasmodium falciparum strain is exposed to ART derivatives such as dihydroartemisinin (DHA), a small population of the ear...

Background Whole genome sequencing (WGS) has rapidly become an important research tool in tuberculosis epidemiology and is likely to replace many existing methods in public health microbiology in the near future. WGS-based methods may be particularly useful in areas with less diverse Mycobacterium tuberculosis populations, such as New York City, wh...

A molecular understanding of drug resistance mechanisms enables surveillance of the effectiveness of new antimicrobial therapies during development and deployment in the field. We used conventional drug resistance selection as well as a regime of limiting dilution at early stages of drug treatment to probe two antimalarial imidazolopiperazines, KAF...

Mutations in the Plasmodium falciparum cyclic amine resistance locus (PfCARL) are associated with parasite resistance to the imidazolopiperazines, a potent class of novel antimalarial compounds that display both prophylactic and transmission-blocking activity, in addition to activity against blood-stage parasites. Here, we show that pfcarl encodes...

SNVs and indels identified in GNF179-selected parasite lines. All SNVs and indels within three new (sample set no. 1) and four previously reported (from reference 16—sample set no. 2) IZP-selected parasite lines are listed. Mutation call data indicate whether a given sample is a WT sample (0/0), a mutant sample (1/1), or a sample with mixed read id...

SNPs present in pfcarl within 203 lines whose sequences are publically available (18). Mutations are separated in the categories of nonsynonymous coding, synonymous coding, and noncoding, and data represent the corresponding genomic position on chromosome 3, minor-allele frequency, and nucleotide change (and amino acid change if applicable).

Digital drop PCR for growth competition assay schematic for experiment performed as described for Fig. 3B. (a) Schematic diagram of competitive growth kinetics experiment. (b) Signals of WT (green) and S1076I mutant (blue) pfcarl probes. Download

IZP analogs tested as described for Fig. 5, with specific IC50s for each indicated parasite line and the structure of each compound listed.

Pyrazinamide (PZA) forms part of first line anti-TB treatment, and will continue to play a important role as it has been included in the latest shortened WHO approved multi-drug resistant (MDR)-TB treatment regimen. The pncA gene encodes for the pyrazinamidase enzyme required to convert PZA to the active form of pyrazinoic acid (POA). Mutations pre...

The emergence of resistant Mycobacterium tuberculosis (Mtb) infection and the dearth of drugs against tuberculosis have made it imperative to identify and validate novel targets and classes of drugs for treatment. The pyrimidine operon regulatory protein (PyrR), a regulator of de novo pyrimidine synthesis, is an essential enzyme and a probable 5-fl...

Tuberculosis (TB) remains the second leading cause of death from an infectious disease globally, despite the persistent efforts to control it. Research and development into new TB medicines is imperative for effective TB control; however, new strategies for the rational use of existing drugs, such as through the identification of new drug targets,...

Alleviating the burden of tuberculosis (TB) requires an understanding of the genetic basis that determines the emergence of drug resistant mutants. PA-824 (Pretonamid) is a bicyclic nitroimidazole class compound presently undergoing the phase III STAND clinical trial despite lacking identifiable genetic markers for drug-specific resistant Mycobacte...

This case report describes an early presentation (<6 hours) of Neisseria meningitidis serogroup A in a neonate born to a mother who was a carrier of the bacterium (nasopharynx, intracervical and whole blood). The neonate succumbed to the infection. N. meningitidis was recovered from cerebrospinal fluid. The isolates of the mother and infant were pr...

Mycobacterium tuberculosis is wrapped in complex waxes, impermeable to most antibiotics. Comparing M. bovis BCG and M. tuberculosis mutants, lacking phthiocerol dimycocerosates (PDIM) and/or phenolic glycolipids, with wild-type strains, we observed that glycopeptides strongly inhibited PDIM deprived mycobacteria. Vancomycin together with a drug tar...

Dengue fever is currently the most important mosquito-borne viral disease in Indonesia. In South Sulawesi province, most regions report dengue cases including the capital city, Makassar. Currently, no information is available on the serotypes and genotypes of the viruses circulating in the area. To understand the dynamic of dengue disease in Makass...

[This corrects the article on p. 451 in vol. 4.].

New chemotherapeutic compounds against multidrug-resistant Mycobacterium tuberculosis (Mtb) are urgently needed to combat drug resistance in tuberculosis (TB). We have identified and characterized the indolcarboxamides as a new class of antitubercular bactericidal agent. Genetic and lipid profiling studies identified the likely molecular target of...

para-Aminosalicylic acid (PAS) is one of the antimycobacterial drugs currently used for multidrug-resistant tuberculosis. Although it has been in clinical use for over 60 years, its mechanism(s) of action remains elusive. Here we report that PAS is a prodrug targeting dihydrofolate reductase (DHFR) through an unusual and novel mechanism of action....

Indole-2-carboxamides have been identified as a promising class of anti-tuberculosis agents from phenotypic screening against mycobacteria. One of the hits indole-2-carboxamide analog (1) had low µM potency against Mycobacterium tuberculosis (Mtb), high mouse liver microsomal clearance and low aqueous solubility. Structure activity relationship stu...

New therapeutic strategies are needed to combat the tuberculosis pandemic and the spread of multidrug-resistant (MDR) and extensively drug-resistant (XDR) forms of the disease, which remain a serious public health challenge worldwide. The most urgent clinical need is to discover potent agents capable of reducing the duration of MDR and XDR tubercul...

Most candidate anti-bacterials are identified on the basis of their whole cell anti-bacterial activity. A critical bottleneck in the early discovery of novel anti-bacterials is tracking the structure activity relationship (SAR) of the novel compounds synthesized during the hit to lead and lead optimization stage. It is often very difficult for medi...

Minimal number of biomarker genes for MoA deconvolution on a PCR array. Note the gene function were as depicted in <http://genolist.pasteur.fr/TubercuList/>. (DOCX)

A prediction correlation matrix using feature-reduced data to validate the classification of individual inhibitors. A heat map representation of gene expression similarity matrix of chemical inhibitors using selected features. The blue-red color scale shows the degree of correlation of drugs expression profiles ranging from −1 to 1 respectively. Al...

A correlation matrix based on Pearson correlations of compounds from the same SAR library. Note high levels of correlation as evident by the SAR clustering. NCE1 SAR compounds and NCE2 SAR compounds form tight clusters while NCE3 SAR compounds form two clusters. Red blocks indicate correlations greater than 0.98, blue blocks indicate correlations b...

para-Aminosalicylic acid (PAS) is one of the antimycobacterial drugs currently used for multidrug-resistant tuberculosis. Although it has been in clinical use for over 60 years, its mechanism(s) of action remains elusive. Here we report that PAS is a prodrug targeting dihydrofolate reductase (DHFR) through an unusual and novel mechanism of action....

Tetrahydropyrazolo[1,5-a]pyrimidine scaffold was identified as a hit series from a Mycobacterium tuberculosis (Mtb) whole cell high through-put screening (HTS) campaign. A series of derivatives of this class were synthesized to evaluate their structure–activity relationship (SAR) and structure–property relationship (SPR). Compound 9 had a promising...

This book includes 19 chapters and shows how the challenge of tuberculosis is currently being met, providing insights into the evidence base underlying developments in laboratory diagnosis and techniques, drug development and treatment, as well as measurement of drug resistance and development of vaccines.

Patients with suspected pulmonary tuberculosis (TB) visiting government TB diagnostic and treatment centres in Makassar City, South Sulawesi Province, Indonesia, from February to October 2008 were included in the study. To determine the distribution of Mycobacterium tuberculosis genotypes in Makassar. Cross-sectional study. Spoligotyping, mycobacte...

Pyrazinamide (PZA) is a first-line antitubercular drug known for its activity against persistent Mycobacterium tuberculosis bacilli. We set out to systematically determine the PZA susceptibility profiles and mutations in the pyrazinamidase (pncA) gene of a collection of multidrug-resistant tuberculosis (MDR-TB) clinical isolates and PZA-resistant (...

Recent reports highlight the incursion of community-associated MRSA within healthcare settings. However, knowledge of this phenomenon remains limited in Latin America. The aim of this study was to evaluate the molecular epidemiology of MRSA in three tertiary-care hospitals in Medellín, Colombia. An observational cross-sectional study was conducted...

Results of MLST in representative strains of major spa types. (XLS)

Growing evidence suggests that the presence of a subpopulation of hypoxic non-replicating, phenotypically drug-tolerant mycobacteria is responsible for the prolonged duration of tuberculosis treatment. The discovery of new antitubercular agents active against this subpopulation may help in developing new strategies to shorten the time of tuberculos...

Evidence from genotype-phenotype studies suggests that genetic diversity in pathogens have clinically relevant manifestations that can impact outcome of infection and epidemiologic success. We studied 5 closely related Mycobacterium tuberculosis strains that collectively caused extensive disease (n = 862), particularly among US-born tuberculosis pa...

Influx of T cells in the lungs at 60 days post-challenge. Wistar rats were vaccinated with BCG and after 6 weeks vaccinated and age-matched unvaccinated rats were challenged with ∼100 CFU of Mtb W4. At 60 days post-challenge number of total T cells, total CD3+CD4+ and total CD3+CD8+ cells was assessed in the lung. Bars show mean ± SM for cells from...

Specific T cell responses in BCG vaccinated rats. Wistar rats were sub-cutaneously vaccinated by 106 BCG. After 6 weeks rats were sacrificed and lung cells were isolated. Lung cells from BCG vaccinated and age matched unvaccinated controls were restimulated with heat killed BCG overnight and cells were analyzed for IFN-γ+ T cells as described in Ma...

Cluster diagram of 20 genes, fold difference of which was statistically significant (P<0.05) irrespective of their fold change (look Table S1), N = 5. (PDF)

Our understanding of the correlation of Mycobacterium bovis Bacille Calmette-Guerin (BCG)-mediated immune responses and protection against Mycobacterium tuberculosis (Mtb) infection is still limited. We have recently characterized a Wistar rat model of experimental tuberculosis (TB). In the present study, we evaluated the efficacy of BCG vaccinatio...

Mycobacterium tuberculosis is the causative agent of a pulmonary epidemic that is estimated to infect one-third of the world's population and that has an increased incidence of multidrug resistance. The evaluation of new chemical entities against M. tuberculosis is hampered by the lack of biological tools to help predict efficacy, from early drug d...

Hypoxia is believed to influence the metabolic state of Mycobacterium tuberculosis and cause phenotypic drug resistance. Using pimonidazole adduct staining, we show that lung lesions of infected rats contain regions of low oxygen tension. Our results support the use of the rat model for evaluating anaerobic drug activity in vivo.

Comparison of TB pathology in Wistar rats with other animal models and human. (DOC)

Despite the availability of many animal models for tuberculosis (TB) research, there still exists a need for better understanding of the quiescent stage of disease observed in many humans. Here, we explored the use of the Wistar rat model for the study of protective immunity and control of Mycobacterium tuberculosis (Mtb) infection. The kinetics of...

Drug resistance and molecular epidemiology of tuberculosis (TB) in the Murmansk region was investigated in a 2-year, population-based surveillance of the civilian population. During 2003 and 2004, isolates from all culture-positive cases were collected (n = 1,226). Prevalence of multi-drug resistance (MDR) was extremely high, as 114 out of 439 new...

Full list of 48 SNPs identified in this study. The first line indicates the gene and the second line indicates the position on that gene where polymorphisms were identified in relation to M. tuberculosis H37Rv strain (bottom). Polymorphisms that characterize and allowed discrimination of the 26 sequence types (Figure 2 and Table 1) are marked in re...

Description of M. tuberculosis Beijing strains belonging to each node found in Fig. 1 and 2 , and respective country of isolation. (DOC)

The Beijing family is a successful group of M. tuberculosis strains, often associated with drug resistance and widely distributed throughout the world. Polymorphic genetic markers have been used to type particular M. tuberculosis strains. We recently identified a group of polymorphic DNA repair replication and recombination (3R) genes. It was shown...

Pathogenic mycobacteria possess two homologous chaperones encoded by cpn60.1 and cpn60.2. Cpn60.2 is essential for survival, providing the basic chaperone function, while Cpn60.1 is not. In the present study, we show that inactivation of the Mycobacterium bovis BCG cpn60.1 (Mb3451c) gene does not significantly affect bacterial growth in 7H9 broth,...

Supplementary Tables S1–S2

Candidate antibacterials are usually identified on the basis of their in vitro activity. However, the apparent inhibitory activity of new leads can be misleading because most culture media do not reproduce an environment relevant to infection in vivo. In this study, while screening for novel anti-tuberculars, we uncovered how carbon metabolism can...

Variable-number tandem repeat (VNTRs) occur throughout the chromosome of Mycobacterium tuberculosis. Although these polymorphic VNTRs, also known as mycobacterial interspersed repetitive units (MIRUs), have proved to be useful tools in molecular epidemiology, their biological significance is less well understood. This study investigated the polymor...

Developing improved tuberculosis (TB) diagnostics is one of the international research priorities, as TB remains globally a major health threat. Loop-mediated isothermal amplification (LAMP) is a new nucleic acid detection method that can be used in low-resource settings, because it does not require expensive or complex instruments. Using the repet...

The side effects associated with tuberculosis therapy bring with them the risk of noncompliance and subsequent drug resistance. Increasing the therapeutic index of antituberculosis drugs should thus improve treatment effectiveness. Several antituberculosis compounds require in situ metabolic activation to become inhibitory. Various thiocarbamide-co...

A number of phylogenetic studies of Mycobacterium tuberculosis have suggested a highly clonal population structure. Despite the extreme homogeneity of M. tuberculosis strains, the genome is punctuated by a number of polymorphic regions that give rise to sufficient diversity, thus forming the basis for molecular epidemiologic studies of tuberculosis...

The emergence of Mycobacterium tuberculosis resistant to first-line antibiotics has renewed interest in second-line antitubercular agents. Here, we aimed to extend our understanding of the mechanisms underlying para-aminosalicylic acid (PAS) resistance by analysis of six genes of the folate metabolic pathway and biosynthesis of thymine nucleotides...

Fourteen strains of S. Typhi (n=13) and S. Paratyphi A (n=1) resistant to ciprofloxacin were compared with 30 ciprofloxacin decreased-susceptibility strains on the basis of qnr plasmid analysis, and nucleotide substitutions at gyrA, gyrB, parC and parE. In ciprofloxacin-resistant strains, five S. Typhi and a single S. Paratyphi A showed triple muta...