Rebecca Rockett

A pilot randomised trial of induced blood-stage Plasmodium falciparum infections in healthy volunteers for testing efficacy of new antimalarial drugs.

Background Critical to the development of new drugs for treatment of malaria is the capacity to safely evaluate their activity in human subjects. The approach that has been most commonly used is testing in subjects with natural malaria infection, a methodology that may expose symptomatic subjects to the risk of ineffective treatment. Here we describe the development and pilot testing of a system to undertake experimental infection using blood stage Plasmodium falciparum parasites (BSP). The objectives of the study were to assess the feasibility and safety of induced BSP infection as a method for assessment of efficacy of new drug candidates for the treatment of P. falciparum infection. Methods and Findings A prospective, unblinded, Phase IIa trial was undertaken in 19 healthy, malaria-naïve, male adult volunteers who were infected with BSP and followed with careful clinical and laboratory observation, including a sensitive, quantitative malaria PCR assay. Volunteers were randomly allocated to treatment with either of two licensed antimalarial drug combinations, artemether–lumefantrine (A/L) or atovaquone-proguanil (A/P). In the first cohort (n = 6) where volunteers received ∼360 BSP, none reached the target parasitemia of 1,000 before the day designated for antimalarial treatment (day 6). In the second and third cohorts, 13 volunteers received 1,800 BSP, with all reaching the target parasitemia before receiving treatment (A/L, n = 6; A/P, n = 7) The study demonstrated safety in the 19 volunteers tested, and a significant difference in the clearance kinetics of parasitemia between the drugs in the 13 evaluable subjects, with mean parasite reduction ratios of 759 for A/L and 17 for A/P (95% CI 120–4786 and 7–40 respectively; p<0.01). Conclusions This system offers a flexible and safe approach to testing the in vivo activity of novel antimalarials. Trial Registration: ClinicalTrials.gov NCT01055002

Different serologic behavior of MCPyV, TSPyV, HPyV6, HPyV7 and HPyV9 polyomaviruses found on the skin.

The polyomavirus family is rapidly expanding with twelve new human viruses identified since 2007. A significant number of the new human polyomaviruses (HPyV) has been found on the skin. Whether these viruses share biological properties and should be grouped together is unknown. Here we investigated the serological behavior of cutaneous HPyVs in a general population. 799 sera from immunocompetent Australian individuals aged between 0–87 were analyzed with a Luminex xMAP technology-based immunoassay for the presence of VP1-directed IgG antibodies against MCPyV, HPyV6, HPyV7, TSPyV, HPyV9, and BKPyV as a control. Except for HPyV9, overall seropositivity was high for the cutanous polyomaviruses (66–81% in adults), and gradually increased with age. Children below 6 months displayed seropositivity rates comparable to the adults, indicative of maternal antibodies. TSPyV seroreactivity levels strongly increased after age 2 and waned later in life comparable to BKPyV, whereas MCPyV, HPyV6 and HPyV7 seroreactivity remained rather stable throughout. Based on the identified serologic profiles, MCPyV seems to cluster with HPyV6 and HPyV7, and TSPyV and HPyV9 by themselves. These profiles indicate heterogeneity among cutaneous polyomaviruses and probably reflect differences in exposure and pathogenic behavior of these viruses.

Experimentally Induced Blood-Stage Plasmodium vivax Infection in Healthy Volunteers

BACKGROUND Major impediments to development of vaccines and drugs for Plasmodium vivax malaria are the inability to culture this species and the extreme difficulty in undertaking clinical research by experimental infection. METHODS A parasite bank was collected from a 49-year-old woman with P. vivax infection, characterized, and used in an experimental infection study. RESULTS The donor made a full recovery from malaria after collection of a parasite bank, which tested negative for agents screened for in blood donations. DNA sequence analysis of the isolate indicated that it was clonal. Two subjects inoculated with the isolate became polymerase chain reaction positive on days 8 and 9, with onset of symptoms and positive blood smears on day 14, when they were treated with artemether-lumefantrine, with rapid clinical and parasitologic response. Transcripts of the parasite gene pvs25 that is expressed in gametocytes, the life cycle stage infectious to mosquitoes, were first detected on days 11 and 12. CONCLUSIONS This experimental system results in in vivo parasite growth, probably infectious to mosquitoes. It offers the opportunity to undertake studies previously impossible in P. vivax that will facilitate a better understanding of the pathology of vivax malaria and development of antimalarial drugs and vaccines. Trial Registration. ANZCTR: 12612001096842.

A real-time, quantitative PCR method using hydrolysis probes for the monitoring of Plasmodium falciparum load in experimentally infected human volunteers

BackgroundThe accurate quantification of Plasmodium falciparum parasite numbers by PCR is an important tool for monitoring growth kinetics in subjects infected and subsequently treated with anti-malarial agents.MethodsA real-time quantitative PCR (rt-qPCR) method using primers and a hydrolysis probe that targets the 18S rRNA gene was adapted and optimized to estimate parasite load in blood samples. Samples included laboratory prepared blood samples of varying parasite concentrations (6.4 × 105 to 6.4 parasites per 500 μl of packed red blood cells (500pRBC)) and blood samples collected from an experimentally infected human subject collected at 19 time points over 10 days. Sample preparation and extraction, detection chemistry, assay reproducibility, and limit of detection were compared to a previously published SYBR Green rt-qPCR used in a malaria vaccine clinical trial.ResultsBoth the rt-qPCR hydrolysis probe assay and SYBR Green rt-qPCR provided a limit of detection of 6.4 × 101 parasites per 500pRBC. However non-specific amplification in the SYBR Green rt-qPCR assay led to either inaccurate estimation of parasite load at levels below 6.4 × 102 parasites per 500pRBC and to false-positive detection of parasites in negative samples. The rt-qPCR hydrolysis probe assay was specific and provided reliable quantification of parasitaemia down to 6.4 × 101 parasites per 500pRBC. Notably, 12 of the 19 consecutive samples collected from the experimentally infected subject were at or below 6.4 × 102 copies per 500pRBC.ConclusionsThese results show that the hydrolysis probe rt-qPCR assay is superior to the SYBR Green rt-qPCR for the quantification of P. falciparum in human blood samples. The hydrolysis probe rt-qPCR is now in use in the Queensland paediatric infectious diseases laboratory (QPID) to monitor parasitaemia in experimentally-infected clinical trial subjects.

Evaluation of the cobas 4800 CT/NG test for detecting Chlamydia trachomatis and Neisseria gonorrhoeae

Objectives To investigate the performance of the fully automated cobas 4800 CT/NG test for detection of Chlamydia trachomatis and Neisseria gonorrhoeae. Methods The study was conducted using 900 clinical specimens (496 urine and 404 swab specimens) for C trachomatis testing, of which 498 specimens (318 urine and 180 swab specimens) were also tested for N gonorrhoeae. The results of the cobas 4800 CT/NG test were compared with those obtained from the Roche COBAS AMPLICOR CT/NG and COBAS TaqMan CT assays. N gonorrhoeae-positive specimens were further tested using in-house, real-time PCR assays. A panel of 223 Neisseria isolates was used to further investigate the performance of the cobas 4800 N gonorrhoeae assay. Results For urine specimens, the sensitivity, specificity and negative and positive predictive values of the cobas 4800 CT/NG test were 94.5%, 99.5%, 98.8% and 97.7%, respectively, for C trachomatis, and 92.9%, 100%, 99.7% and 100%, respectively, for N gonorrhoeae. For swab specimens, the sensitivity, specificity and negative and positive predictive values were 92.0%, 100%, 99.5% and 100%, respectively, for C trachomatis, and 100%, 99.4%, 100% and 90.0%, respectively, for N gonorrhoeae. All N gonorrhoeae isolates were positive and all non-gonococcal Neisseria strains were negative by the cobas 4800 N gonorrhoeae assay. Conclusions The cobas 4800 CT/NG test is suitable for high through-put identification of C trachomatis and N gonorrhoeae infections.

Detection of Novel Polyomaviruses, TSPyV, HPyV6, HPyV7, HPyV9 and MWPyV in Feces, Urine, Blood, Respiratory Swabs and Cerebrospinal Fluid

Eight novel human polyomaviruses have been discovered since 2007. Prevalence rates and tissue tropism for the most recent members HPyV 6, 7, 9, TSPyV and MWPyV are largely unknown. We used real-time PCR to determine the presence of HPyV 6, 7, 9, TSPyV and MWPyV in feces (n = 263), urine (n = 189), blood (n = 161), respiratory swabs (n = 1385) and cerebrospinal fluid (n = 171) from both healthy control children and children and adults undergoing diagnostic testing. Whole genome sequencing was able to be performed on 9 MWPyV positive specimens. Novel polyomaviruses were only detected in respiratory swabs and feces, with no detections of HPyV 9 in any sample type. MWPyV was found to be the most prevalent novel polyomavirus, being detected in 18 (1.5%) respiratory specimens from symptomatic patients, 16 (9.8%) respiratory sample from healthy control children, 11 (5.9%) fecal specimens from patient suffering gastrointestinal illness, and in 13 (15.3%) of feces from healthy control children. MWPyV was found only in respiratory and fecal specimens from children, the oldest being 9 years old. HPyV 6, 7, 9 and TSPyV were also detected in respiratory specimens and fecal specimens at low prevalence (<1.3%). The majority of these detections were found in immunocompromised patients. Our findings suggest that MWPyV can result in a subclinical infection, persistent or intermittent shedding, particularly in young children. The other novel polyomaviruses were also found in respiratory and fecal specimens, but at lower prevalence and most commonly in immunocompromised individuals.

NR1I2 polymorphisms are related to tacrolimus dose-adjusted exposure and BK viremia in adult kidney transplantation

Background Pregnane X, encoded by the gene NR112, is a nuclear receptor whose primary role is to promote the detoxification and clearance of drugs and other foreign compounds from the body. Aim The aim of this study was to analyze associations between NR1I2 polymorphisms, immunosuppressant drug exposure, and clinical outcomes in adult kidney transplant recipients. Methods Exposures to tacrolimus, mycophenolic acid, and total and free prednisolone were estimated at month 1 posttransplant using validated multiple regression-derived limited sampling strategies. Results In the 158 subjects studied, median (interquartile range) dose-adjusted exposure to tacrolimus was significantly higher in individuals carrying the NR1I2 8055T variant allele, when compared with exposure in wild-type individuals [20 (14, 22) &mgr;g·h/L/mg versus 15 (9, 24) &mgr;g·h/L/mg; P =0.0007]. Using multivariable logistic regression, NR1I2 8055T carrier status was independently predictive of higher dose-adjusted tacrolimus exposure (P=0.0005). Moreover, BK viremia was seen significantly more frequently in NR1I2 8055T allele carriers compared with wild-type individuals (38% vs 18%, P=0.005) and possession of the NR1I2 8055T allele imposed significantly higher odds of BK viremia (adjusted odds ratio, 2.76 [95% confidence interval, 1.33–7.73]; P=0.006). No significant difference in geometric mean peak BK viral replication titer was observed between 8055T carriers and noncarriers. No NR1I2 SNP or haplotype was significantly, independently associated with total or free prednisolone or MPA exposure. Conclusions These data demonstrate an impact of pregnane X receptor polymorphisms on tacrolimus pharmacokinetics. Association of the 8055T allele with BK viremia suggests clinically significant “overimmunosuppression” in individuals with this genotype.

Exploring the prevalence of ten polyomaviruses and two herpes viruses in breast cancer.

Several different viruses have been proposed to play a role in breast carcinogenesis. The aim of this study was to investigate the prevalence of a subset of viruses in breast cancer tissue. We investigated the prevalence of 12 DNA viruses: EBV and CMV from the Herpesviridae family and SV40, BKV, JCV, MCV, WUV, KIV, LPV, HPyV6, HPyV7, and TSV from the Polyomaviridae family in 54 fresh frozen breast tumour specimens. Relevant clinical data and basic lifestyle data were available for all patients. The tissue samples were DNA extracted and real-time PCR assays were used for viral detection. The highest prevalence, 10% (5/54), was found for EBV. MCV, HPyV6, and HPyV7 were detected in single patient samples (2% each), while WUV, KIV, JCV, BKV, LPV, SV40, TSV and CMV were not detected in the 54 breast cancer specimens analysed here. Further investigations are needed to elucidate the potential role of viruses, and particularly EBV, in breast carcinogenesis.

Association of micropapillary urothelial carcinoma of the bladder and BK viruria in kidney transplant recipients.

BK virus (BKV) is an ubiquitous human polyomavirus that establishes latency in urothelium. BKV is known to re‐activate in immunosuppressed individuals, and is an increasingly important cause of nephropathy and graft loss in kidney transplant recipients. Animal studies have demonstrated BKV has a potential role as a tumor virus. However, its role in precipitating or facilitating oncogenesis in humans is still debated.

Piperaquine Monotherapy of Drug-Susceptible Plasmodium falciparum Infection Results in Rapid Clearance of Parasitemia but Is Followed by the Appearance of Gametocytemia

Background. Piperaquine, coformulated with dihydroartemisinin, is a component of a widely used artemisinin combination therapy. There is a paucity of data on its antimalarial activity as a single agent. Such data, if available, would inform selection of new coformulations. Methods. We undertook a study in healthy subjects, using the induced blood stage malaria (IBSM) model to test the antimalarial activity of single doses of piperaquine (960, 640, and 480 mg) in 3 cohorts. In a pilot study in the third cohort, gametocyte clearance following administration of 15 mg, or 45 mg or no primaquine was investigated. Results. Parasite clearance over the 48-hour period after piperaquine administration was more rapid in the 960 mg cohort, compared with the 640 mg cohort (parasite reduction ratio, 2951 [95% confidence interval {CI}, 1520–5728] vs 586 [95% CI, 351–978]; P < .001). All 24 subjects developed gametocytemia as determined by pfs25 transcripts. Clearance of pfs25 was significantly faster in those receiving primaquine than in those not receiving primaquine (P < .001). Conclusions. Piperaquine possesses rapid parasite-clearing activity, but monotherapy is followed by the appearance of gametocytemia, which could facilitate the spread of malaria. This new information should be taken into account when developing future antimalarial coformulations. Clinical Trials Registration ACTRN12613000565741.