Bert Mulder

Mosquito Feeding Assays to Determine the Infectiousness of Naturally Infected Plasmodium falciparum Gametocyte Carriers

Introduction In the era of malaria elimination and eradication, drug-based and vaccine-based approaches to reduce malaria transmission are receiving greater attention. Such interventions require assays that reliably measure the transmission of Plasmodium from humans to Anopheles mosquitoes. Methods We compared two commonly used mosquito feeding assay procedures: direct skin feeding assays and membrane feeding assays. Three conditions under which membrane feeding assays are performed were examined: assays with i) whole blood, ii) blood pellets resuspended with autologous plasma of the gametocyte carrier, and iii) blood pellets resuspended with heterologous control serum. Results 930 transmission experiments from Cameroon, The Gambia, Mali and Senegal were included in the analyses. Direct skin feeding assays resulted in higher mosquito infection rates compared to membrane feeding assays (odds ratio 2.39, 95% confidence interval 1.94–2.95) with evident heterogeneity between studies. Mosquito infection rates in membrane feeding assays and direct skin feeding assays were strongly correlated (p<0.0001). Replacing the plasma of the gametocyte donor with malaria naïve control serum resulted in higher mosquito infection rates compared to own plasma (OR 1.92, 95% CI 1.68–2.19) while the infectiousness of gametocytes may be reduced during the replacement procedure (OR 0.60, 95% CI 0.52–0.70). Conclusions Despite a higher efficiency of direct skin feeding assays, membrane feeding assays appear suitable tools to compare the infectiousness between individuals and to evaluate transmission-reducing interventions. Several aspects of membrane feeding procedures currently lack standardization; this variability makes comparisons between laboratories challenging and should be addressed to facilitate future testing of transmission-reducing interventions.

Effect of gametocyte sex ratio on infectivity of Plasmodium falciparum to Anopheles gambiae

Insectary-reared Anopheles gambiae were experimentally fed with the blood of 90 naturally infected human volunteers carrying gametocytes of Plasmodium falciparum. At least one mosquito was successfully infected in 74% of experiments. The probability that a gametocyte carrier was infective, the probability that a mosquito became infected, and the number of oocysts harboured were related to gametocyte density. The mean proportion of male gametocytes was 0.217 (i.e., 3.6 females for every male). Sex ratios differed significantly between gametocyte carriers. Variation in sex ratio was not related to the probability that a gametocyte carrier was infective. Among infective people whose sex ratio estimates were based on a reasonable number of gametocytes, sex ratio significantly predicted the proportion of infected mosquitoes and mean oocyst load, with infectivity rising as the proportion of the male gametocytes increased towards 50%. There was no indication that infectivity reached a peak at some intermediate sex ratio, as would be expected if random mating of gametes was the primary determinant of fertilization success. These results raise 2 interesting questions: why should higher sex ratios be more infective, and why is the observed population sex ratio lower than that which produces the greatest infectivity?

The early sporogonic cycle of Plasmodium falciparum in laboratory‐infected Anopheles gambiae: an estimation of parasite efficacy

This study investigated the successive losses in the parasite densities of Plasmodium falciparum stages during the early sporogony in laboratory‐reared Anopheles gambiae infected by membrane feeding with blood from naturally infected gametocyte carriers (>50 gametocytes/mm3). The developmental stages of P. falciparum in the mosquito were studied from zygote to oocyst, by immunofluorescent method using monoclonal antibodies against the Pfs25 protein present on the surface of newly formed gametes. This method allows for assessment of the various sporogonic stages before, during and after passage of the midgut wall. Parasite densities were determined within the entire blood meal at 3 h (zygotes and macrogametes) and 24 h (ookinetes) post‐infection. At 48 h after the mosquito blood meal, midguts were checked for the presence of early oocysts. For the mid‐size oocysts count, classic microscopy examination was used at day 7 post‐infection. The parasite efficacy was estimated by following successive losses in parasite densities between different early stages of the sporogonic cycle in A. gambiae. Thirty‐seven experimental infections were realized with high gametocyte densities, ranging from 64 to 2392 gametocytes/mm3. All gametocyte carriers showed infection with round forms 100%; ookinetes were found in 91.9%. The prevalences of infections with oocysts were 48.6% at day 2 (young oocyst) and 37.8% at day 7 (mid‐size oocyst). The mean densities per mosquito for each parasite stage were 12.6 round forms, 5.5 ookinetes, 1.8 young oocyst and 2 mid‐size oocysts. Significant correlations were found between two consecutive parasite stages (round forms/ookinetes, ookinetes/young oocysts, young oocysts/mid‐size oocysts) and between round forms and mid‐size oocysts. The mean parasite density significantly decreased between round forms and ookinetes (yield Y1 = 41.6%) and between ookinetes and young oocysts (Y2 = 61.4%). By contrast, no significant decrease was observed between young oocysts and mid‐size oocysts (Y3 = 91.2%). The overall yield of the early sporogonic cycle (from round form to oocyst at day 7) was equal to 25.7%, indicating that almost 3/4 of the total parasites were lost during the early step of the sporogonic cycle, from 3 h post‐infection to day 7.

Malaria transmission-blocking activity in experimental infections of Anopheles gambiae from naturally infected Plasmodium falciparum gametocyte carriers.

Experimental infections of anopheline mosquitoes were carried out with Plasmodium falciparum gametocytes from 65 naturally infected patients in Cameroon. A comparison was made between infections with blood containing autologous plasma and blood in which the plasma was replaced with plasma from a donor without previous malaria exposure. A lower infection rate was observed in 50 of 65 autologous plasma samples. Transmission was significantly blocked in 3 infections. This indicates that, in a population living in an area endemic for malaria, blood plasma factor(s) can reduce the transmission capacity of gametocyte carriers to mosquitoes.

Human immune responses that reduce the transmission of Plasmodium falciparum in African populations

Graphical abstract Research highlights ► Gametocyte density is positively associated with mosquito infection rates. ► Autologous plasma reduces the transmission of malaria parasites. ► Transmission reducing activity is associated with indicators of recent exposure to gametocytes.

Laboratory diagnosis of pulmonary tuberculosis in TB and HIV endemic settings and the contribution of real time PCR for M. tuberculosis in bronchoalveolar lavage fluid

Background  Tuberculosis (TB) in Africa is increasing because of the human immunodeficiency virus (HIV) epidemic, and in HIV/AIDS patients it presents atypically. Pulmonary tuberculosis (PTB) in Africa is mainly diagnosed clinically, by chest radiograph or by sputum smear for acid fast bacilli (AFB).

Plasmodium Falciparum Transmission Blocking Immunity Under Conditions Of Low And High Endemicity In Cameroon

Transmission Blocking Immunity (Tbi) Was Studied In Relation To Age, Gametocyte Density And Transmission Intensity. Subjects With High Gametocytaemias Were Selected In A Hypo‐endemic Urban District And A Hyper‐endemic Rural Area In South Cameroon. Tbi Was Determined In Blood From Gametocyte Carriers In A Bioassay (Direct Membrane Feeding Assay), With Either Autologous Plasma (Own) Or Control Serum (Ab). Mosquito Infection Rates (Ir) Were Compared. Infection Rates Correlated Positively With Gametocyte And Oocyst Densities. Three Tbi Indicators Were Analysed: The Proportion Of Transmission Reducers (Irab > Irown, P < 0·01), The Mean Intensity Of Tbi (Irab – Irown), And The Contribution Of Tbi To Total Inhibition [(Irab – Irown)/(100 −irown)]. We Could Not Discriminate Between Areas With Regard To Either The Proportion Of Transmission Reducers (Urban 15% And Rural 29%) Or The Mean Levels Of Tbi (Urban 10% And Rural 9%), Or Contribution Of Tbi To Total Inhibition (Urban 10% And Rural 13%). There Was No Relationship Between Tbi Indicators And Age, But A Trend Of Increasing Values Was Observed With Rising Gametocytaemia, Which Was Considered As A Confusing Factor. A Multivariable Analysis Showed That The Probability Of Being A Reducer Was 4·6 Fold Higher In The Rural Area Than In The Urban District.

Xpert MTB/RIF(R), a novel automated polymerase chain reaction-based tool for the diagnosis of tuberculosis

There is an urgent need for new point of care tests for tuberculosis (TB). Xpert MTB/RIF® is a real-time polymerase chain reaction-based system that detects Mycobacterium tuberculosis DNA and rifampicin (RMP) resistance modulating mutations directly from clinical samples in 2 h. The sensitivity for detecting M. tuberculosis in culture-positive samples was 93.8% (60/64) and exceeded smear microscopy (40/64, 62.5%). The specificity for detecting M. tuberculosis was 92.0% (23/25) and for RMP resistance it was 100% (8/8). The test is simple to conduct and requires basic sputum handling facilities only. These characteristics render it a promising close-to-patient test for TB in various settings.

Stabilization of chloroquine resistance in vivo of Plasmodium falciparum in Edea, south Cameroon

Chloroquine resistant falciparum malaria has rapidly spread over central and west Africa (WERNSDORFER & PAYNE, 1991). In southern Cameroon chloroquine resistance was first reported by SANSONNETTI et al. (1985). We measured chloroquine sensitivity in vivo of Plasmodium falciparum in May 1989 and 1991 in schoolchildren in the town of Edea, southern Cameroon, and observed considerable increase over 2 years (GAZIN et al., 1990; MULDER et al., 1992). Parallel studies in vivo in

Mycobacterium bovis infection in a young Dutch adult : transmission from an elderly human source?

A young female health professional was diagnosed with pulmonary tuberculosis caused by Mycobacterium bovis. Source finding and contact tracing was initiated by the regional municipal health service using both tuberculin skin test and QuantiFERON®-TB Gold (QFT-GIT (IGRA). The strain appeared near-identical to that of an elderly Dutch patient.