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Carriage and density of gametocytes, the transmission stages of malaria parasites, are determined for predicting the infectiousness of humans to mosquitoes. This measure is used for evaluating interventions that aim at reducing malaria transmission. Gametocytes need to be detected by amplification of stage-specific transcripts, which requires RNA-preserving blood sampling. For simultaneous, highly sensitive quantification of both, blood stages and gametocytes, we have compared and optimized different strategies for field and laboratory procedures in a cross sectional survey in 315 5-9 yr old children from Papua New Guinea. qRT-PCR was performed for gametocyte markers pfs25 and pvs25, Plasmodium species prevalence was determined by targeting both, 18S rRNA genes and transcripts. RNA-based parasite detection resulted in a P. falciparum positivity of 24.1%; of these 40.8% carried gametocytes. P. vivax positivity was 38.4%, with 38.0% of these carrying gametocytes. Sensitivity of DNA-based parasite detection was substantially lower with 14.1% for P. falciparum and 19.6% for P. vivax. Using the lower DNA-based prevalence of asexual stages as a denominator increased the percentage of gametocyte-positive infections to 59.1% for P. falciparum and 52.4% for P. vivax. For studies requiring highly sensitive and simultaneous quantification of sexual and asexual parasite stages, 18S rRNA transcript-based detection saves efforts and costs. RNA-based positivity is considerably higher than other methods. On the other hand, DNA-based parasite quantification is robust and permits comparison with other globally generated molecular prevalence data. Molecular monitoring of low density asexual and sexual parasitaemia will support the evaluation of effects of up-scaled antimalarial intervention programs and can also inform about small scale spatial variability in transmission intensity.

Background Diagnosis of soil-transmitted helminths (STHs) in developing countries is commonly based on microscopic detection of eggs in stool samples, using the Kato-Katz (KK) method, which has a poor sensitivity for detecting light intensity infections. We compared the performance of the KK method and real-time PCR in the framework of a randomized trial, which evaluated four novel treatments against Trichuris trichiura and concomitant STH infections. Results Two stool samples obtained from 320 participants were examined at baseline and follow-up with quadruplicate KK and PCR analyses of one of the two samples using “bead-beating” for DNA extraction. At follow-up, 80 samples were negative according to both PCR and KK and 173 were positive with both methods for any of the STHs. Relative to PCR, the calculated sensitivity of KK at follow-up was 83.6%, 43.0% and 53.8% for T. trichiura , for hookworm and for Ascaris lumbricoides , respectively. The sensitivity of PCR compared with KK at this time point was 89.1% for T. trichiura , 72.7% for hookworm and 87.5% for A. lumbricoides. Cure rates (CRs) for T. trichiura and A. lumbricoides were slightly lower with the PCR method. For hookworm CRs with KK were mostly significantly lower, namely 36.7%, 91.1%, 72.2% and 77.8% for moxidectin, moxidectin in combination with tribendimidine, moxidectin in combination with albendazole and albendazole in combination with oxantel pamoate, respectively, whereas with PCR the CRs were 8.3%, 82.6%, 37.1% and 57.1%, respectively. Conclusions In conclusion, a single real-time PCR is as sensitive as quadruplicate KK for T. trichiura and A. lumbricoides detection but more sensitive for hookworm , which has an influence on the estimated treatment efficacy. PCR method with DNA extraction using the “bead-beating protocol” should be further promoted in endemic areas and laboratories that can afford the needed equipment. The study is registered at ISRCTN (no. 20398469).

A better understanding of human-to-mosquito transmission is crucial to control malaria. In order to assess factors associated with gametocyte carriage, 2083 samples were collected in a cross-sectional survey in Papua New Guinea. Plasmodium species were detected by light microscopy and qPCR and gametocytes by detection of pfs25 and pvs25 mRNA transcripts by reverse-transcriptase PCR (qRT-PCR). The parasite prevalence by PCR was 18.5% for Plasmodium falciparum and 13.0% for P. vivax. 52.5% of all infections were submicroscopic. Gametocytes were detected in 60% of P. falciparum-positive and 51% of P. vivax-positive samples. Each 10-fold increase in parasite density led to a 1.8-fold and 3.3-fold increase in the odds of carrying P. falciparum and P. vivax gametocytes. Thus the proportion of gametocyte positive and gametocyte densities was highest in young children carrying high asexual parasite densities and in symptomatic individuals. Dilution series of gametocytes allowed absolute quantification of gametocyte densities by qRT-PCR and showed that pvs25 expression is 10-20 fold lower than pfs25 expression. Between 2006 and 2010 parasite prevalence in the study site has decreased by half. 90% of the remaining infections were asymptomatic and likely constitute an important reservoir of transmission. However, mean gametocyte densities were low (approx. 1-2 gametocyte/μL) and it remains to be determined to what extent low-density gametocyte positive individuals are infective to mosquitos.

A distinctive feature of Plasmodium vivax infections is the overall low parasite density in peripheral blood. Thus, identifying asymptomatic infected individuals in endemic communities requires diagnostic tests with high sensitivity. The detection limits of molecular diagnostic tests are primarily defined by the volume of blood analysed and by the copy number of the amplified molecular marker serving as the template for amplification. By using mitochondrial DNA as the multi-copy template, the detection limit can be improved more than tenfold, compared to standard 18S rRNA targets, thereby allowing detection of lower parasite densities. In a very low transmission area in Brazil, application of a mitochondrial DNA-based assay increased prevalence from 4.9 to 6.5%. The usefulness of molecular tests in malaria epidemiological studies is widely recognized, especially when precise prevalence rates are desired. Of concern, however, is the challenge of demonstrating test accuracy and quality control for samples with very low parasite densities. In this case, chance effects in template distribution around the detection limit constrain reproducibility. Rigorous assessment of false positive and false negative test results is, therefore, required to prevent over- or under-estimation of parasite prevalence in epidemiological studies or when monitoring interventions.

Background Co-infection of the four major species of human malaria parasite Plasmodium falciparum (Pf), P. vivax (Pv), P. malariae (Pm), and P. ovale sp. (Po) is regularly observed, but there is limited understanding of between-species interactions. In particular, little is known about the effects of multiple Plasmodium species co-infections on gametocyte production. Methods We developed molecular assays for detecting asexual and gametocyte stages of Pf, Pv, Pm, and Po. This is the first description of molecular diagnostics for Pm and Po gametocytes. These assays were implemented in a unique epidemiological setting in Papua New Guinea with sympatric transmission of all four Plasmodium species permitting a comprehensive investigation of species interactions. Findings The observed frequency of Pf-Pv co-infection for asexual parasites (14.7%) was higher than expected from individual prevalence rates (23.8%Pf x 47.4%Pv = 11.3%). The observed frequency of co-infection with Pf and Pv gametocytes (4.6%) was higher than expected from individual prevalence rates (13.1%Pf x 28.2%Pv = 3.7%). The excess risk of co-infection was 1.38 (95% confidence interval (CI): 1.09, 1.67) for all parasites and 1.37 (95% CI: 0.95, 1.79) for gametocytes. This excess co-infection risk was partially attributable to malaria infections clustering in some villages. Pf-Pv-Pm triple infections were four times more frequent than expected by chance alone, which could not be fully explained by infections clustering in highly exposed individuals. The effect of co-infection on parasite density was analyzed by systematic comparison of all pairwise interactions. This revealed a significant 6.57-fold increase of Pm density when co-infected with Pf. Pm gametocytemia also increased with Pf co-infection. Conclusions Heterogeneity in exposure to mosquitoes is a key epidemiological driver of Plasmodium co-infection. Among the four co-circulating parasites, Pm benefitted most from co-infection with other species. Beyond this, no general prevailing pattern of suppression or facilitation was identified in pairwise analysis of gametocytemia and parasitemia of the four species. Trial registration This trial is registered with ClinicalTrials.gov, Trial ID: NCT02143934.

The molecular force of blood-stage infection (molFOB) is a quantitative surrogate metric for malaria transmission at population level and for exposure at individual level. Relationships between molFOB, parasite prevalence and clinical incidence were assessed in a treatment-to-reinfection cohort, where P.vivax (Pv) hypnozoites were eliminated in half the children by primaquine (PQ). Discounting relapses, children acquired equal numbers of new P. falciparum (Pf) and Pv blood-stage infections/year (Pf-molFOB = 0–18, Pv-molFOB = 0–23) resulting in comparable spatial and temporal patterns in incidence and prevalence of infections. Including relapses, Pv-molFOB increased >3 fold (relative to PQ-treated children) showing greater heterogeneity at individual (Pv-molFOB = 0–36) and village levels. Pf- and Pv-molFOB were strongly associated with clinical episode risk. Yearly Pf clinical incidence rate (IR = 0.28) was higher than for Pv (IR = 0.12) despite lower Pf-molFOB. These relationships between molFOB, clinical incidence and parasite prevalence reveal a comparable decline in Pf and Pv transmission that is normally hidden by the high burden of Pv relapses. Clinical trial registration: ClinicalTrials.gov NCT02143934 Malaria is caused by five different species of parasites that are transmitted to humans by bites from parasite-carrying mosquitos. Once in human blood, the parasites rapidly multiply. People who live in countries where malaria is common may become infected and never show any symptoms because their immune systems are able to keep parasite numbers low. Repeated infections, or infection with more than one species of malaria parasite also are common. Some species of malaria, including Plasmodium vivax, can hibernate in the liver for weeks or months after the infection and only become active later. Asymptomatic infections, multi-parasite infections, and reactivating parasites make it hard to measure how often new malaria infections occur. One way scientists can determine if a new infection has occurred is by genotyping the parasites in a person’s blood. Genotyping involves looking for small differences in the parasite DNA. For example, a study in Papua New Guinea, where P. vivax is very common, showed that reactivations of hibernating parasites were more common than new infections. Now, Hofmann et al. use the same study in Papua New Guinea to compare the frequency and consequences of new infections with P. vivax and another malaria parasite, Plasmodium falciparum. In the study, 466 children from 6 villages were followed for 8 months with tests every 2 to 4 weeks to genotype the parasites in their blood. Some of the children were treated with antimalarial drugs to help wipe out any existing parasites including hibernating ones. While P. vivax was about twice as common in blood samples—likely due to reactivation—genotyping showed that new infections with the two parasites occur at equal rates and often at the same times and locations. Hofmann et al. also showed that some villages and some children had much higher rates of infection than others. This difference could not fully be explained by use of bednets or other preventive measures. Children were more likely to become ill from P. falciparum than P. vivax even though P. vivax was more common. But children with more frequent infections with P. falciparum seemed better able to manage the parasites and were less likely to develop symptoms that those with infrequent infections. The experiments show that genotyping may help scientists better track new malaria infections and develop better strategies to prevent or treat malaria.

Background In malaria endemic populations, complex patterns of Plasmodium vivax and Plasmodium falciparum blood-stage infection dynamics may be observed. Genotyping samples from longitudinal cohort studies for merozoite surface protein ( msp ) variants increases the information available in the data, allowing multiple infecting parasite clones in a single individual to be identified. msp genotyped samples from two longitudinal cohorts in Papua New Guinea (PNG) and Thailand were analysed using a statistical model where the times of acquisition and clearance of each clone in every individual were estimated using a process of data augmentation. Results For the populations analysed, the duration of blood-stage P. falciparum infection was estimated as 36 (95% Credible Interval (CrI): 29, 44) days in PNG, and 135 (95% CrI 94, 191) days in Thailand. Experiments on simulated data indicated that it was not possible to accurately estimate the duration of blood-stage P. vivax infections due to the lack of identifiability between a single blood-stage infection and multiple, sequential blood-stage infections caused by relapses. Despite this limitation, the method and data point towards short duration of blood-stage P. vivax infection with a lower bound of 24 days in PNG, and 29 days in Thailand. On an individual level, P. vivax recurrences cannot be definitively classified into re-infections, recrudescences or relapses, but a probabilistic relapse phenotype can be assigned to each P. vivax sample, allowing investigation of the association between epidemiological covariates and the incidence of relapses. Conclusion The statistical model developed here provides a useful new tool for in-depth analysis of malaria data from longitudinal cohort studies, and future application to data sets with multi-locus genotyping will allow more detailed investigation of infection dynamics.

Background Despite the high prevalence of strongyloidiasis in the Laotian population, Laotian hospitals still lack diagnostic capacity to appropriately diagnose Strongyloides stercoralis infections. This cross-sectional hospital-based study was conducted to assess the prevalence of Strongyloides stercoralis infection among hospitalized patients treated at Mahosot Hospital, the primary reference hospital of Lao People’s Democratic Republic (Lao PDR), and to validate feasible methods for diagnosing S. stercoralis infection at hospital’s laboratory. Methods Between September and December 2018, stool samples of 104 inpatients were investigated for S. stercoralis infection by wet smear, Baermann technique, Koga Agar plate culture (KAPC), and real-time detection polymerase chain reaction (RTD-PCR) at the Infectious Diseases Ward of the Mahosot Hospital in Vientiane. The sensitivity, the specificity, the negative predictive value (NPV) of each diagnostic test, as well as their combination(s) was calculated using a composite reference standard (CRS). The correlation of the different test methods was assessed by chi-square or Fisher’s exact test. Cohen’s kappa coefficient was used to assess the diagnostic agreement of the different test methods. Results The overall prevalence of S. stercoralis infections among the study population was 33.4%. The cumulative infection prevalence statistically significantly increased from the lowest age group of 40 years and below (22.4%), to the medium (40.0%) and to the oldest age group of 61 year and above (72.7%)( P  = 0.003). The cumulative infection prevalence of CRS was considerably higher in male (40.4%) compared to female patients (28.1%), but not statistically different ( P  = 0.184). The diagnostic sensitivity of Baermann technique, KAPC, RTD-PCR, and the combination of Baermann technique and KAPC were 60.0, 60.0, 74.3, and 77.1%, respectively. Only 13 patients (37.1%) of the total 35  S. stercoralis patients diagnosed with any technique had a simultaneously positive diagnostic test with Baermann, KAPC and RTD-PCR. Conclusions We identified Baermann technique and KAPC to be currently the most feasible and implementable standard methods for diagnosing S. stercoralis at a hospital setting such as Mahosot Hospital and provincial and district hospitals in Lao PDR and other low- and middle income countries in Southeast Asia. Trial registration This study was approved by the National Ethics Committee for Health Research in Lao PDR (reference no. 083/NECHR) and by the Ethics Committee Northwest and Central Switzerland (reference no. 2018–00594).

Primaquine (PQ) is the only currently licensed antimalarial that prevents Plasmodium vivax (Pv) relapses. It also clears mature P. falciparum (Pf) gametocytes, thereby reducing post-treatment transmission. Randomized PQ treatment in a treatment-to-reinfection cohort in Papua New Guinean children permitted the study of Pv and Pf gametocyte carriage after radical cure and to investigate the contribution of Pv relapses. Children received radical cure with Chloroquine, Artemether-Lumefantrine plus either PQ or placebo. Blood samples were subsequently collected in 2-to 4-weekly intervals over 8 months. Gametocytes were detected by quantitative reverse transcription-PCR targeting pvs25 and pfs25. PQ treatment reduced the incidence of Pv gametocytes by 73%, which was comparable to the effect of PQ on incidence of blood-stage infections. 92% of Pv and 79% of Pf gametocyte-positive infections were asymptomatic. Pv and to a lesser extent Pf gametocyte positivity and density were associated with high blood-stage parasite densities. Multivariate analysis revealed that the odds of gametocytes were significantly reduced in mixed-species infections compared to single-species infections for both species (ORPv = 0.39 [95% CI 0.25-0.62], ORPf = 0.33 [95% CI 0.18-0.60], p<0.001). No difference between the PQ and placebo treatment arms was observed in density of Pv gametocytes or in the proportion of Pv infections that carried gametocytes. First infections after blood-stage and placebo treatment, likely caused by a relapsing hypnozoite, were equally likely to carry gametocytes than first infections after PQ treatment, likely caused by an infective mosquito bite. Pv relapses and new infections are associated with similar levels of gametocytaemia. Relapses thus contribute considerably to the Pv reservoir highlighting the importance of effective anti-hypnozoite treatment for efficient control of Pv. ClinicalTrials.gov NCT02143934.

Background Researching a water-borne disease in the middle of the Sahara desert might not seem the most relevant concern. However, nomadic Sahelian pastoralists health concerns regarding their livestock and anecdotal reports about trematode infections of Fasciola spp. and Schistosoma spp. in desert-raised animals justified an exploratory study focusing on the lakes of Ounianga in Northern Chad. The aim was to test whether trematode parasites such as Schistosoma spp. occur in human populations living around the Sahara desert lakes of Ounianga Kebir and Ounianga Serir in northern Chad. Methods The study was carried out in January 2019 and comprised of three components. First, a cross sectional survey based on a random sample drawn from the population to detect infections with S. haematobium and S. mansoni ; second, focus group discussions exploring disease priorities, access to health and health seeking behaviour; and third, surveying water contact sites for intermediate host snails. Samples of trematode parasites and snails were confirmed on species level by molecular genetic methods. For parasitological and malacological surveys descriptive statistics were performed. Qualitative data analysis included the full review of all transcripts, followed by a descriptive and explorative thematic analysis. Results Among 258 participants, the overall S. haematobium prevalence using urine filtration was 39.2% [95% confidence interval ( CI ): 33.5–45.1%], with 51.5% of the infected suffering from heavy infection. The intermediate host snail of S. haematobium ( Bulinus truncatus ) occurred at water contact sites near both study villages, revealing the potential for local transmission. Although a positive S. mansoni point-of-care circulating cathodic antigen (POC-CCA) test result was obtained from 8.6% (95% CI 5.7–12.8%) of the samples, no intermediate host snails of S. mansoni were found, and the relevance of S. mansoni remains uncertain. Qualitative findings underline the importance of morbidity caused by urinary schistosomiasis, and the lack of access to diagnostics and treatment as a major health concern. Conclusions This research revealed a high prevalence of urinary schistosomiasis in the population living around the lakes of Ounianga in the Sahara, a United Nations Educational, Scientific and Cultural Organization (UNESCO) world heritage site in Chad. Despite the high public health importance of the associated morbidity expressed by the population, there is no access to diagnostics and treatment. Further work is needed to develop and test a context-adapted intervention. Graphical Abstract