Human malaria diagnosis using a single-step direct-PCR based on the Plasmodium cytochrome oxidase III gene

Background: Nested PCRs based on the Plasmodium 18s-rRNA gene have been extensively used for human malaria diagnosis. However, they are not practical when large quantities of samples need to be processed, further there have been challenges in the performance and when interpreting results, especially when submicroscopic infections are analysed. Here the use of "direct PCR" was investigated with the aim of improving diagnosis in the malaria elimination era.\ud \ud Methods: The performance of the Plasmodium cytochrome oxidase III gene (COX-III) based novel malaria detection strategies (direct nested PCR and direct single PCR) were compared using a 18s-rRNA direct nested PCR as a reference tool. Evaluations were based on sensitivity, specificity and the ability to detect mixed infections using control blood spot samples and field collected blood samples with final species diagnosis confirmation by sequencing.\ud \ud Results: The COX-III direct PCR (limit of detection: 0.6–2 parasites/μL) was more sensitive than the 18s-rRNA direct nested PCR (limit of detection: 2–10 parasites/μL). The COX-III direct PCR identified all 21 positive controls (no mixed infections detected) while the 18s-rRNA direct nested PCR identified 18/21 (including four mixed infections). Different concentrations of simulated mixed infections (Plasmodium vivax and Plasmodium falciparum) suggest that the COX-III direct PCR detects only the predominant species. When the 18s-rRNA direct nested PCR was used to detect Plasmodium in field collected bloods spots (n = 3833), there was discrepancy in the results from the genus PCR (16 % positive) and the species-specific PCR (5 % positive). Further, a large portion of a subset of these positive samples (93 % for genus and 60 % for P. vivax), did not align with Plasmodium sequences. In contrast, the COX-III direct PCR clearly identified (single bands confirmed with sequencing) 2 % positive Plasmodium samples including P. vivax, P. falciparum, Plasmodium malariae and Plasmodium ovale wallikeri.\ud \ud Conclusions: The COX-III single direct PCR is an alternative method for accurate detection of Plasmodium microscopic and submicroscopic infections in humans, especially when a large number of samples require screening. This PCR does not require DNA isolation, is sensitive, quick, produces confident/clear results, identifies all the Plasmodium species infecting humans, and is cost-effective.\u

Post-exposure prophylaxis with doxycycline to prevent sexually transmitted infections in men who have sex with men: an open-label randomised substudy of the ANRS IPERGAY trial

International audienceBACKGROUND:Increased rates of sexually transmitted infections (STIs) have been reported among men who have sex with men. We aimed to assess whether post-exposure prophylaxis (PEP) with doxycycline could reduce the incidence of STIs.METHODS:All participants attending their scheduled visit in the open-label extension of the ANRS IPERGAY trial in France (men aged 18 years or older having condomless sex with men and using pre-exposure prophylaxis for HIV with tenofovir disoproxil fumarate plus emtricitabine) were eligible for inclusion in this open-label randomised study. Participants were randomly assigned (1:1) at a central site to take a single oral dose of 200 mg doxycycline PEP within 24 h after sex or no prophylaxis. The primary endpoint was the occurrence of a first STI (gonorrhoea, chlamydia, or syphilis) during the 10-month follow-up. The cumulative probability of occurrence of the primary endpoint was estimated in each group with the Kaplan-Meier method and compared with the log-rank test. The primary efficacy analysis was done on the intention-to-treat population, comprising all randomised participants. All participants received risk-reduction counselling and condoms, and were tested regularly for HIV. This trial is registered with ClinicalTrials.gov number, NCT01473472.FINDINGS:Between July 20, 2015, and Jan 21, 2016, we randomly assigned 232 participants (n=116 in the doxycycline PEP group and n=116 in the no-PEP group) who were followed up for a median of 8·7 months (IQR 7·8-9·7). Participants in the PEP group used a median of 680 mg doxycycline per month (IQR 280-1450). 73 participants presented with a new STI during follow-up, 28 in the PEP group (9-month probability 22%, 95% CI 15-32) and 45 in the no-PEP group (42%, 33-53; log-rank test p=0·007). The occurrence of a first STI in participants taking PEP was lower than in those not taking PEP (hazard ratio [HR] 0·53; 95% CI 0·33-0·85; p=0·008). Similar results were observed for the occurrence of a first episode of chlamydia (HR 0·30; 95% CI 0·13-0·70; p=0·006) and of syphilis (0·27; 0·07-0·98; p=0·047); for a first episode of gonorrhoea the results did not differ significantly (HR 0·83; 0·47-1·47; p=0·52). No HIV seroconversion was observed, and 72 (71%) of all 102 STIs were asymptomatic. Rates of serious adverse events were similar in the two study groups. Gastrointestinal adverse events were reported in 62 (53%) participants in the PEP group and 47 (41%) in the no-PEP group (p=0·05).INTERPRETATION:Doxycycline PEP reduced the occurrence of a first episode of bacterial STI in high-risk men who have sex with men.FUNDING:France Recherche Nord & Sud Sida-HIV Hépatites (ANRS) and Bill & Melinda Gates Foundation

Fast and robust single PCR for Plasmodium sporozoite detection in mosquitoes using the cytochrome oxidase I gene

Abstract Background Molecular tools for detecting malaria-infected mosquitoes with improved practicality, sensitivity and specificity, and high-throughput are required. A common PCR technique used to detect mosquitoes infected with Plasmodium spp. is a nested PCR assay based on the 18s-rRNA gene. However, this technique has several technical limitations, is laborious and time consuming. Methods In this study, a PCR-based on the Plasmodium cytochrome oxidase I (COX-I) gene was compared with the 18s-rRNA nested PCR using serial dilutions (330–0.0012 pg) of DNA from Plasmodium vivax, Plasmodium falciparum and Plasmodium knowlesi and with DNA from 48 positive and negative Kenyan mosquitoes (previously detected by using both ELISA and PCR). This assay for Plasmodium spp. DNA detection using the fast COX-I PCR assay was then performed individually on 2122 field collected mosquitoes (from the Solomon Islands) in which DNA was extracted from head and thorax. Results The fast COX-I PCR assay took 1 h to run and consistently detected as low as to 0.043 pg of parasite DNA (equivalent to two parasites) in a single PCR, while analyses with the 18s-rRNA nested PCR required 4 h to complete with a consistent detection threshold of 1.5 pg of DNA. Both assays produced concordant results when applied to the 48 Kenyan control samples with known Plasmodium spp. infection status. The fast COX-I PCR identified 23/2122 Plasmodium-infected mosquitoes from the Solomon Islands. Conclusions This new COX-I PCR adapted for a single PCR reaction is a faster, simpler, cheaper, more sensitive technique amenable to high-throughput analyses for Plasmodium DNA detection in mosquitoes and is comparable to the 18s-rRNA nested PCR. The improved sensitivity seen with the fast COX-I PCR will improve the accuracy of mosquito infection rate determination