Paul de Kruif, American science writer on malaria: a case study.

From 1926 onward, science writer Paul de Kruif has popularized the history of great discoveries in the science of diseases, malaria among them. During the thirties he focused on abuses and neglects of medical care and pointed to new developments, e.g., the treatment of syphilis with heat, rather than with malaria therapy. He conveyed his messages in popular magazines, using a romantic style and explicit language that was unheard of. He promoted new drugs, like Atabrine for endemic malaria and for the military engaged in (sub-) tropical war zones. Medical authorities were unhappy that his message about Atabrine was so dramatic, raising unwarranted hope of eradication. By the end of the Second World War, however, the usefulness of Atabrine was recognized. In my research I consulted original sources: letters, articles and books of De Kruif, as well as contemporary scientific publications; recent books on malaria history helped to design the historiography. Paul de Kruif's dealing with various malaria topics is presented as a case study of science writing. Modern malaria research requires skilled journalists: they can excite public interest about problems in distant parts of the tropical world and work to raise funds. Journalists should also push for the translation of (bio-) medical progress to applicable tools in malaria control.

Role of heat-labile serum factor or host complement in the inhibition of Plasmodium falciparum sporogonic stages in Anopheles stephensi by gametocyte carriers' serological factors.

This study investigated the significance of serum complement on transmission-reducing activity (TRA) of field sera from 24 infected Plasmodium falciparum gametocyte carriers (from Cameroon) against cultured NF54 P. falciparum. Laboratory-reared Anopheles stephensi were given infectious blood meals prepared either with sera from naïve Dutch donor (AB type) or pair-matched field serum samples, both with and without active complement. TRA of serum factors and host complement on mosquito infection rate and oocyst intensity were divided into the various components involved in the early stages of sporogony. The majority (>80%) of sera tested showed positive antibody titres to Pfs230, the relevant complement-dependent target of transmission-reducing mechanisms. Regardless of the presence of active complement, bloodmeals with field sera exhibited significantly lower infection rates and oocyst intensity than the control group. Serological reactivity in Capture-ELISA against Pfs230 was significantly correlated with the reduction of parasite infectivity. Contrary to our expectation, the presence of active complement in the mosquito bloodmeal did not increase parasite losses and therefore the magnitude of transmission reduction by individual immune sera. Our findings on P. falciparum are consistent with previous studies on animal hosts of Plasmodium, indicating that early P. falciparum sporogonic stages may be insensitive to the antibody-dependent pathways of complement in human serum.

Stage-specific effects of host plasma factors on the early sporogony of autologous Plasmodium falciparum isolates within Anopheles gambiae.

Summary Quantitatively assessing the impact of naturally occurring transmission-blocking (TB) immunity on malaria parasite sporogonic development may provide a useful interpretation of the underlying mechanisms. Here, we compare the effects of plasma derived from 23 naturally infected gametocyte carriers (OWN) with plasma from donors without previous malaria exposure (AB) on the early sporogonic development of Plasmodium falciparum in Anopheles gambiae. Reduced parasite development efficiency was associated with mosquitoes taking a blood meal mixed with the gametocyte carriers' own plasma, whereas replacing autologous plasma with non-immune resulted in the highest level of parasite survival. Seven days after an infective blood meal, 39.1% of the gametocyte carriers' plasma tested showed TB activity as only a few macrogametocytes ingested along with immune plasma ended up as ookinetes but subsequent development was blocked in the presence of immune plasma. In other experiments (60.9%), the effective number of parasites declined dramatically from one developmental stage to the next, and resulted in an infection rate that was two-fold lower in OWN than in AB infection group. These findings are in agreement with those in other reports and go further by quantitatively examining at which transition stages TB immunity exerts its action. The transitions from macrogametocytes to gamete/zygote and from gamete/zygote to ookinete were identified as main targets. However, the net contribution of host plasma factors to these interstage parasite reductions was low (5-20%), suggesting that irrespective of the host plasma factors, mosquito factors might also lower the survival level of parasites during the early sporogonic development.

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.

Underreporting of malaria incidence in The Netherlands: results from a capture-recapture study.

The aim of this study was to estimate the completeness of notification of malaria by physicians and laboratories in the Netherlands in 1996. We used a capture-recapture (CRC) analysis of three incomplete, partially overlapping registers of malaria cases: a laboratory survey, the Notification Office and the hospital admission registration. The response of the laboratories was 83.2%. In 1996 the laboratories microscopically identified 535 cases of malaria, 330 patients with malaria were admitted to hospital and physicians notified 311 malaria cases. 667 malaria cases were recorded in at least one register. CRC analysis estimated the total number of malaria cases at 774 (95 % CI of 740-821). This implies a completeness of notification of 40.2% for physicians and 69.1% for the laboratories. It can be concluded that laboratory-based notification can considerably increase the number of officially reported malaria cases as compared to notification by physicians. However, possibly one-third of the cases may still go unreported.

Sulphadoxine/pyrimethamine: an appropriate first-line alternative for the treatment of uncomplicated falciparum malaria in Ghanaian children under 5 years of age.

OBJECTIVE: To assess whether chloroquine (CQ) still is an appropriate first-line drug for the treatment of uncomplicated falciparum malaria in Ghana and whether sulphadoxine/pyrimethamine (SP) could be a good alternative. METHOD: The parasitological, clinical and haematological responses to CQ and SP were studied in children < 5 years of age according to a modified WHO 28-day in vivo protocol. A total of 142 children attending the outpatients department meeting the inclusion criteria were randomly assigned to the CQ (n=72) or SP (n=70) group. RESULTS: In the CQ group, 15 children (20.8%) exhibited early clinical failure (within 3 days) compared with only 1 (1.4%) in the SP group (P < 0.01). The clinical failure rate before day 14 (early treatment failure plus late treatment failure before day 14) also showed a marked advantage in favour of the SP group (1.4 against 29.2%). The median time to clinical failure was 11.5 days in the CQ group and 26 days in the SP group (P < 0.01). Of the 72 children treated with CQ, 9 (12.5%) had RIII resistance and 19 (26.4%) had RII resistance. A total of 36 (50.0%) were sensitive to CQ. From the 70 children treated with SP, none had RIII or RII resistance. There was no difference in haematological response between the two treatment groups. CONCLUSION: Although there is little concordance on when to change treatment policy, the high resistance to CQ in this study supports the change to another first-line drug for children under 5 years of age. SP seems to be a good alternative, although a high RII and RIII resistance against this drug has already been reported in the coastal zones of Ghana.

[Considerable underreporting of malaria in the Netherlands; a capture-recapture analysis]. / Sterke onderrapportage van malaria in Nederland; een vangst-hervangstanalyse.

OBJECTIVE: To estimate the completeness of notification of malaria by physicians and laboratories in the Netherlands. METHOD: Capture-recapture analysis was applied to three incomplete, partially overlapping registers of malaria cases in 1995 and 1996: a laboratory survey, the Notification Office and the hospital admission registration. RESULTS: The average response of the 107 laboratories approached was 83.6% over both years. In 1995 and 1996 581 and 535 malaria cases respectively were microscopically diagnosed. In each year physicians officially notified 311 patients. 350 and 330 patients respectively were admitted to hospital. Capture-recapture analysis estimated the total number of new malaria cases at 933 (95% confidence interval: 849-1072) in 1995 and at 774 cases (740-821) in 1996. The estimated completeness of notification in 1995 and 1996 was therefore 33.3% and 40.2% for physicians and 62.3% and 69.1% for the laboratories. CONCLUSION: Laboratory-based notification, introduced in the Infectious Diseases Act, can considerably increase the number of officially reported malaria cases as compared with notification by physicians. However, approximately one-third of the estimated number of cases may still go unreported.

Anti-NANP antibody and treatment efficacy in patients with acute uncomplicated falciparum malaria attacks.

African patients originating from the hypoendemic, urban area of Greater Dakar (Senegal, West Africa) who presented with an acute Plasmodium falciparum infection were studied using an in-vivo chloroquine sensitivity assay for 28 days. Forty-seven patients with acute malaria infections were treated with 25 mg/body weight of chloroquine. Adequate responses to treatment were observed in 24 patients (51%), whereas 23 (49%) were resistant. On the day of admission, these two groups of patients were comparable with respect to age, level of parasitemia and delay before initiation of treatment, but not with respect to gametocyte prevalence which was higher in patients resistant to therapy (48%) than in those who responded to treatment (17%). In order to evaluate whether the therapeutic response was associated with any given specific immune response, antibody activities against different stages of the parasite cycle were evaluated: anti-NANP repeats (i.e. antisporozoite stage antigen), anti-Pfs 45 kDa (i.e. antigametocyte stage antigen), and anti-MSP3 (i.e. antimerozoite stage antigen) antibodies were measured by ELISA at day 0 (i.e. on the day of admission and before initiation of treatment), day 7 and day 28. No significant differences between treatment-sensitive and treatment-resistant infections were observed for antibody prevalences and optical densities, except at day 0, when the prevalence of antibodies against NANP repeats was 2.4 times more frequent in the group of patients with a propitious response to treatment: 62.5% of the patients with an infection sensitive to chloroquine had anti-NANP antibodies, whereas only 26.1% of the patients resistant to chloroquine treatment had such a humoral response. These observations are discussed in relation to (1) the finding that gametocyte prevalence was markedly increased at a time when resistance to antimalarial treatment was observed; (2) the possibility that the efficacy of the therapeutic response could be the result of the combined effects of treatment and the individual immune status of the patients at the time of drug cure; and (3) the presence of detectable anti-NANP activity as potential indicator of the level of premunition acquired in an area of low and seasonal malaria transmission.

The disappearance of Dutch malaria and the Rockefeller Foundation.

Sixty years ago Professor Nico Swellengrebel wrote his famous book 'Malaria in the Netherlands' (Swellengrebel and de Buck, 1938). At that time tertian malaria was still endemic, with its epidemic ups and downs. Malaria disappeared as recently as 1960 and the Rockefeller Foundation (RF) contributed substantially to this effect. The Rockefeller Archives proved a valuable source of anecdotal information, which puts the scientific publications of the Dutch malariologists in a more vivid perspective. Following the course of history, first the already existing links with the RF are explained along with some peculiarities of tertian malaria in the Dutch temperate climate. The emergence of a new epidemic during the war years and the implication of new tools and principles for control as advocated by the RF are described. The subsequent shriveling of the vector population and the disappearance of malaria are presented, along with some details about the reluctance of WHO to declare the Netherlands malaria-free. Finally, recent unrest about possible return of malaria is put into perspective.

The use of anti-Pfs 25 monoclonal antibody for early determination of Plasmodium falciparum oocyst infections in Anopheles gambiae: comparison with the current technique of direct microscopic diagnosis.

Experimental infections of laboratory-reared anopheline mosquitoes were carried out with 57 Plasmodium falciparum gametocyte carriers from Cameroon. Prevalence of infected mosquitoes and oocyst intensity were determined by two independent methods. Young P. falciparum oocysts were detected on day 2 after feeding using an immunofluorescent assay, and the results were compared with direct microscopic examination of midgut oocysts on day 7 postinfection. The immunofluorescent assay was based on a FITC-labeled anti-25-kDa monoclonal antibody, while the direct microscopy was performed on midguts stained with 2% mercurochrome. Young oocysts were easily detected by their typical and bright green-fluorescing Pfs25 positive coat and their characteristic pattern of pigment granules under transmitted white light examination. The agreement between the results of the two methods was assessed using the Kappa coefficient on prevalences of positive infections and the interclass correlation coefficient on arithmetic mean oocyst load per infected midgut. The results indicated a low agreement between the two methods for the comparison of prevalences of infected mosquitoes. However, this agreement was near perfect for the comparison of mean oocyst intensities. Prevalences of positive infections and the overall number of parasites per positive gut were significantly correlated for both methods. Thus, the immunofluorescent test could be an appropriate tool for early determination of malaria infection in mosquitoes, particularly under laboratory conditions. The possible applications of this immuno-fluorescent technique are discussed.

Plasmodium falciparum: membrane feeding assays and competition ELISAs for the measurement of transmission reduction in sera from Cameroon.

The effect of natural malaria transmission-blocking factors in the blood of Plasmodium falciparum gametocyte carriers was assessed in two types of functional bioassays. In the direct membrane feeding assay (DMFA), a comparison is made between the infectivity of gametocytes from a naturally infected gametocyte carrier in the presence of autologous plasma and the infectivity in the presence of replacement plasma from nonimmune donors. In the standard membrane feeder assay (SMFA), cultured NF54 gametocytes are used to measure the capacity of endemic sera to block transmission. In the DMFA, 18 out of 48 sera (37.5%) from Cameroonian gametocyte carriers reduced transmission significantly, while in the SMFA 22 out of 48 sera (45.8%) produced transmission reduction. There was a positive correlation between both assays (r + 0.41, P < 0.05). Antibodies against epitopes of transmission-blocking target antigens Pfs48/45 and Pfs230 were measured in competition ELISAs and compared with the results of DMFA and SMFA. Serological reactivity in competition ELISAs against three epitopes of Pfs48/45 was significantly higher in the group of transmission-reducing sera in both the DMFA and the SMFA, especially for epitope III. No significant difference was found for Pfs230 antibodies (epitope I). Sensitivity of the serological assays was approximately 60%, with a specificity of around 70%. Serological tests cannot replace the functional bioassay in field situations as yet, but can contribute in the selection of sera for SMFA evaluation.

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 postinfection. 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 anticircumsporozoite antibodies in Dutch soldiers returning from sub-Saharan Africa.

One hundred and twenty-five Dutch servicemen returning from central Africa after a short deployment were enrolled in a study aimed at assessing the effectiveness of malaria prevention measures. None of the persons developed an episode of clinically overt malaria during or after deployment, and no antibodies against blood stages of Plasmodium falciparum could be found. However, antibodies against the circumsporozoite protein (CS) of P. falciparum were demonstrable in 14 persons (11.2% of the study population) by an ELISA test using the recombinant CS-antigen R32tet32, while one person only was positive in an IFA test based on schizonts of P. fieldi as antigen. We concluded that the anti-CS-positive servicemen were probably bitten by mosquitoes carrying P. falciparum parasites while the IFA-positive person was possibly infected by P. vivax, P. ovale or P. malariae parasites. There was no significant association between the different antimalaria preventive measures and the development of anti-CS antibodies. Therefore mefloquine prophylaxis as the single most widely used preventive measure in this group of servicemen was possibly a major contributing factor in averting development of overt malaria.

Testing for anti-circumsporozoite and anti-blood-stage antibodies for epidemiologic assessment of Plasmodium falciparum infection in travelers.

The purpose of this investigation was to assess the role of serology for establishing incidences of Plasmodium falciparum malaria and of exposure to P. falciparum in epidemiologic studies of travelers using chemoprophylaxis. The design was a prospective cohort study involving 548 short-term Dutch travelers to areas endemic for P. falciparum malaria. Sera were collected before departure and, together with the medical history, 2-6 weeks after return. All sera were tested for anti-circumsporozoite (CS) antibodies by an R32tet32-ELISA; sera of subjects reporting febrile illness during travel or after return or with anti-CS responses were tested for anti-blood-stage antibodies by an indirect fluorescence antibody test (IFAT). Five subjects (0.9%) reported P. falciparum malaria confirmed by thick blood smear examination (documented cases) and six (1.0%) reported treatment for malaria without a documented diagnosis (presumptive cases). Conversions in the IFAT were detected in six subjects, including all five documented cases and one presumptive case. Anti-CS antibodies were detected in seven subjects (1.3%), including three documented cases and four of 442 subjects with no history of fever or malaria treatment (0.9%). Incidence rates per 1,000 person-months of travel (95% confidence interval) of infection with P. falciparum, whether or not suppressed by chemoprophylaxis, were 16.9 (8-31) for all destinations and 91.6 (33-200) for West Africa. In epidemiologic studies of P. falciparum malaria in travelers, testing for antibodies to blood stages can increase the sensitivity and specificity of case detection; testing for antibodies to sporozoites may be useful for the assessment of exposure to P. falciparum in travelers using chemoprophylaxis, but the sensitivity is limited.

Identification of human liver carboxylesterase as one of the proteins involved in Plasmodium falciparum malaria sporozoite invasion in primary cultures of human hepatocytes.

BACKGROUND/AIMS: In a previous study, we have demonstrated that primary human hepatocytes in culture are susceptible for Plasmodium falciparum sporozoite invasion and for development of parasites into exo-erythrocytic forms. In a separate study we demonstrated the involvement of two human liver plasma membrane proteins (55 kD and 20 kD) in the invasion of P. falciparum sporozoites in vitro. In this study, we have unravelled the nature of the 55 kD protein. METHODS: For the identification of this protein, a 53-58 kD membrane protein fraction from human liver was isolated, radioactively labelled, incubated with sporozoites and cross-linked. After reduction of the cross-linker, the released proteins were mixed with unlabelled 53-58 kD protein fraction and separated on two-dimensional SDS-PAGE. Autoradiography showed a single spot corresponding to a protein of 55 kD and pI of 5.7-5.8. RESULTS: Amino acid sequencing revealed the 55 kD protein as carboxylesterase. The biological activity of purified human liver carboxylesterase and of an antiserum against carboxylesterase on sporozoite invasion in vitro was evaluated. Human carboxylesterase as well as a rabbit antiserum against carboxylesterase inhibited the invasion of P. falciparum sporozoites into primary human hepatocytes in culture. A number of carboxylesterase cDNA clones were isolated from a human liver cDNA library. Sequence analysis revealed two different iso-types. Immunoaffinity purified recombinant human carboxylesterase was shown also to inhibit the invasion of sporozoites into primary human hepatocytes. Immunocytochemical analysis of the localisation of carboxylesterase in primary cultures of human hepatocytes using specific antibodies, showed its presence inside the hepatocytes and on the membrane. CONCLUSIONS: Carboxylesterase plays a role in the invasion process of P. falciparum sporozoites into human hepatocytes in vitro. The implications of these findings are further discussed.

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?