Army Malaria Institute – its Evolution Third Decade (1st Half): 1985-1990Karl H. Rieckmann, Anthony W. Sweeney, Michael D. Edstein, Robert D. Cooper, Stephen P. Frances AbstractThe first half of the third decade (1985-1990) after the establishment of the Malaria Research Unit was characterised by a substantial increase in laboratory and field activities. Various new procedures were developed to discover and counter the increasing problem of parasite resistance to established antimalarial drug regimens. After early findings that the currently-used pyrimethamine/dapsone (Maloprim®) prophylaxis was no longer effective against Plasmodium falciparum malaria, in vitro, ex vivo and pharmacological investigations were undertaken with proguanil and a low dose of dapsone. This led to a field study in Papua New Guinea (PNG) which indicated that such a drug combination might be more effective than Maloprim®. Before the effectiveness of this combination could be determined further, it became obvious that Maloprim®/chloroquine had become inadequate for the protection of ADF soldiers on exercises in PNG. During one of these exercises, the landmark discovery that P. vivax had developed resistance to chloroquine was the first documented evidence that this previously successful standard medication had lost its efficacy against vivax malaria. These findings in 1988/1989 led to the evaluation of mefloquine and doxycycline as alternative prophylactic regimens for ADF personnel serving in malarious areas. In efforts to identify and assess other potential antimalarial drugs, in vitro studies were continued with various strains of P. falciparum. These studies were supplemented by investigations with P. falciparum and P. vivax in non-human primates and by malaria transmission studies with Anopheles farauti mosquitoes. The survey of anopheline mosquitoes in northern Australia indicated the widespread presence of three isomorphic species of An. farauti and the ever-present possibility of re-introducing malaria into northern Australia. Investigations relating to the biological control of mosquitoes were gradually phased out and emphasis was given to the assessment of novel mosquito repellent measures for improved personal protection against malaria. because they were affordable and largely effective The global malaria situation had generally not in suppressing malaria in populations with some improved during the previous decade. Following degree of background immunity to malaria. In a few the principles of primary health care outlined at the areas, though, travellers or residents with little or no Alma Ata Conference in 1978, increasing emphasis immunity to malaria were not responding adequately was given to applying strategies of malaria control to prophylaxis or treatment with standard drugs. tailored more specifically for different epidemiological This raised the possibility of using alternative drugs, such as the tetracyclines and mefloquine, for malaria sensible approach, lack of funding and decreased prioritisation of malaria activities in many malarious Research activities at the Army Malaria Research countries meant that there was generally little or no Unit (AMRU), commenced in the mid-1960s,5 were improvement in the malaria situation. The extension able to be increased following its relocation from of chloroquine-resistant falciparum malaria to other the University of Sydney to the Ingleburn military areas, including Africa and the Southwest Pacific facility in 1974.6 This was possible because improved region was a contributing factor hindering malaria housing facilities enabled the gradual acquisition of control activities. In some countries, a combination more suitable equipment for the laboratories, the of pyrimethamine and sulfadoxine (Fansidar®) had establishment of an appropriate mosquito insectary largely replaced chloroquine for malaria treatment.3 and animal quarters, and a gradual increase in staff However, since most infections still responded from 9 to 23 positions by the end of the decade. clinically to chloroquine and related drugs, they Significant progress was made in assessing in vitro continued to be used as first-line treatment drug resistance and screening potential antimalarial drugs by short-term and long-term culture of addition, office space was acquired from 2nd Military Plasmodium falciparum, in determining the Hospital to accommodate a refurbished library, staff synergistic activity of antifolate drug combinations offices and a small laboratory. In late 1985, Professor in rodent malaria, and in developing sensitive Karl Rieckmann was recruited as Director of Medical methods for estimating drug concentrations in body Research, following a 25-year absence from Australia fluids with a view to optimising drug doses used during which he conducted laboratory and field for malaria prophylaxis and treatment.6 Following investigations to control the increasing problem of the Unit’s documentation of chloroquine-resistant parasite resistance to antimalarial drugs. After the falciparum malaria in Papua New Guinea (PNG) at transfer of Lieutenant Colonel John Twartz to 2nd the start of the second decade, increasing attention Military Hospital in 1986, Major Andrew Taylor was was given to the investigation of various antifolate posted to the Unit as medical officer for 12 months. drug combinations. Although studies with dapsone/ Following Colonel Donaldson’s re-assignment to proguanil combinations continued for a while, the Army Aviation Centre at Oakey, Queensland, in greater attention was placed on dapsone combined August 1987, Lieutenant Colonel Sweeney became with another dihydrofolate reductase (DHFR) Commanding Officer and Acting Director of the inhibitor – pyrimethamine. This pyrimethamine/ Unit. In 1988, Ms Barbara Kotecka was appointed dapsone combination, widely known as Maloprim®, Parasitologist following the resignation of Dr Haydn was registered and approved for malaria prophylaxis Scott, and Lieutenant Colonel Donald Davis was posted to the Unit as Medical Officer for a short period of time.
By 1980, standard malaria prophylaxis for Australian military personnel consisted of weekly Maloprim® Figure 1: Army Malaria Research Unit Staff (1986). and chloroquine while on deployment overseas and Standing (L to R): Mr H. Scott, PTE A.Topping, SGT C. for 4 weeks after return to Australia to suppress Rogers, SGT J. Staley, CAPT R. Cooper, CPL M. Baker, Mr falciparum and vivax malaria. In addition, a 14-day T. Haddon, CPL M. Sellars, SGT K. Newman, CPL G. Owen, course of primaquine (7.5 mg base 3 times a day) was taken to eradicate any residual liver stages of vivax Sitting (L to R): MAJ M. Edstein, Prof K. Rieckmann, BRIG malaria. With good drug compliance, these regimens G. Maynard (DMS-A), COL E. Donaldson, MAJGEN W. appeared to be largely effective in preventing malaria Rodgers (DGAHS), LTCOL A. Sweeney, MAJ R. Veenendaal. in the rather limited number of military personnel Absent: MAJ J. O’Brien, CAPT W. Backhouse, CAPT A. deployed overseas during this period.
The accidental discovery of a fungus killing mosquito larvae in the Unit insectary generated considerable interest in the possible use of Culicinomyces clavisporus as a potential fungal larvicide to control mosquito breeding, although its practical application in the field remained in doubt. In addition to preliminary studies with another mosquito pathogen, other entomological activities included speciation and malaria transmission studies with Anopheles farauti, the major malaria vector in the Southwest Pacific region, and the initiation of annual surveys to map the distribution of this vector in northern Australia.6 In 1988, Emeritus Professor (Colonel) Black died In January 1985, Colonel Eric Donaldson became after a distinguished career in tropical medicine Director of AMRU, following the promotion of Colonel during which he was also the motivating force in re- Graham Maynard to Brigadier and his transfer to establishing the Army’s active participation in malaria Army Headquarters in Canberra. Colonel Donaldson research activities. In the same year, Karl Rieckmann inherited a unit which had grown considerably since was appointed Professor of Medicine at the University its inception about 20 years earlier and which was of Sydney. After his appointment as Director of destined to contribute significantly to international AMRU in 1989, the long-standing association with efforts to control drug-resistant malaria. During the University of Sydney was strengthened when 1985 and 1986, there was a substantial extension AMRU was accorded departmental status within of primate, mosquito and parasitology facilities. In the Faculty of Medicine during the following year. In Journal of Military and Veterans’ Health 1989, Major Michael Edstein commenced a 3-year prophylaxis in PNG.10 Furthermore, adequate posting to the Armed Forces Research Institute plasma concentrations of pyrimethamine, dapsone of Medical Research (AFRIMS), Thailand, and and chloroquine were observed in 3 of the men who contributed to joint efforts by AMRU and US and Thai developed falciparum malaria while they were still Army scientists to counter the increasing problem of on weekly prophylaxis following the 1988/1989 SAS exercise in PNG.7 Six other men became ill with falciparum malaria within 4 weeks after completing prophylaxis, suggesting that parasites were only Upsurge in malaria during military exercises in PNG. partially suppressed by Maloprim®/chloroquine Current malaria prophylaxis appeared to control malaria in the ADF quite well until the deployment P. vivax resistance to chloroquine. Following the of 163 members of the Special Air Services (SAS) 26-day deployment of SAS soldiers to PNG during regiment to PNG for 3 to 4 weeks during 1988 1989, 2 soldiers developed acute attacks of vivax and 1989. Five soldiers developed malaria (3 P. malaria 3 to 15 days after return to Australia.8 Both falciparum, 2 P. vivax) while taking prophylaxis and men were still on weekly chloroquine and Maloprim® 36 of them had attacks of malaria (6 P. falciparum, 30 prophylaxis, and their plasma chloroquine levels P. vivax) after completing prophylaxis.7 The malaria were considerably higher than those generally attack rates were similar to those observed during regarded as therapeutically effective against P. vivax. the Pacific campaign of World War II and higher than About 30 years after the emergence of chloroquine- those observed during the Malayan emergency or the resistant P. falciparum, this was the first documented Vietnam conflict. This suggested very strongly that evidence that P. vivax was also capable of developing weekly Maloprim®/chloroquine prophylaxis was no resistance to chloroquine. Chloroquine-resistant P. longer able to protect soldiers adequately against vivax was also observed in a traveller returning to falciparum and vivax malaria and that the current Australia from PNG and the Solomon Islands.11 primaquine eradication regime was becoming less effective in preventing relapses of vivax malaria. P. vivax tolerance to primaquine. Relapses of vivax The gravity of the situation was heightened even malaria acquired in the southwest Pacific area further by the documentation that chloroquine was have traditionally been treated with a total daily unable to suppress vivax malaria in 2 soldiers who primaquine dose of 22.5 mg base, rather than 15 were treated with chloroquine after their return mg base, because parasites from this area were considered to be more tolerant to the drug than 8 There was obviously an urgent need for improved protection of soldiers against malaria.
in other parts of the world. The unacceptably high number of vivax infections observed in the SAS soldiers after their return to Australia suggested that the parasites were becoming even more tolerant to In vitro test for drug resistance. The increased likelihood of ADF personnel acquiring drug-resistant infections highlighted the importance of forwarding Malaria diagnosis. The increasing problem of parasitised blood samples to AMRU to assess the in drug resistance highlighted the importance of vitro sensitivity of parasites to chloroquine and other early microscopic diagnosis of malaria and correct drugs.9 By determining the presence and degree of identification of malaria species. The diagnostic drug resistance in patients with falciparum malaria, verification service provided by the Unit revealed that medical personnel could modify their treatment the wrong plasmodial species was being identified regimens, if necessary, for patients whose infections in up to one in 5 military personnel, raising the had been acquired in the same geographical area. likelihood that some military personnel had received These tests could also be helpful in formulating more inappropriate treatment. This emphasised the need effective prophylactic and therapeutic drug regimens to forward duplicate blood films to AMRU as quickly as possible to enable treatment to be modified if necessary. As species identification by microscopic P. falciparum resistance to pyrimethamine/ examination of blood films can sometimes be dapsone (Maloprim®). The first documented notoriously difficult, it was hoped that this problem evidence that P. falciparum was becoming resistant could be partly resolved by reviving 2-4 week training to Maloprim® was in 1987 when high performance courses for pathology technicians working at defence liquid chromatography (HPLC) analysis revealed that high plasma concentrations of pyrimethamine and dapsone did not prevent an Australian soldier from developing falciparum malaria while on Proguanil/Dapsone as a possible alternative to mg dapsone in 6 healthy male soldiers.17 After the seventh and last daily dose, mean plasma maximum (minimum) concentrations of proguanil, cycloguanil Earlier favourable field and laboratory findings with and dapsone were 151 (21), 56 (15) and 285 (125) proguanil/dapsonel5,6 led to a reconsideration of ng/mL, respectively. Respective elimination half- using proguanil, in combination with a low-dose of lives were 23, 15 and 18 hours. When serum samples dapsone, for malaria prophylaxis. Further studies collected between 4 and 24 hours were incubated were carried out with proguanil, chlorproguanil, with parasites of the K1 isolate, no parasite growth and their active triazine metabolites to gain a better understanding of the pharmacokinetics of these drugs.12,13 Addition of proguanil, dapsone The complete inhibition of parasite growth following or monoacetyl-dapsone (principal metabolite of daily co-administration of proguanil and dapsone dapsone) to the pyrimethamine-resistant K1 isolate was in marked contrast to observations in 6 soldiers of P. falciparum showed little or no antimalarial following weekly administration of pyrimethamine/ activity in vitro. On the other hand, cycloguanil dapsone (Maloprim®).18 Although sera collected from (active metabolite of proguanil) was several orders of the 6 volunteers inhibited parasite growth of the magnitude more active than its parent compound, drug-sensitive FC27 isolate completely at trough with 90% parasite growth of the drug-resistant K1 concentrations of both drugs, even maximum serum isolate being inhibited at a concentration of 80 concentrations failed to inhibit the growth of K1 ng/mL. However, only 20 ng/mL cycloguanil was parasites. Infections with less resistant parasites required to inhibit parasite growth when 40 ng/mL might still be able to be suppressed at peak dapsone was added to the culture. Parasite growth concntrations, but this would be unlikely for serum was also inhibited when 20 ng/mL cycloguanil was samples collected towards the end of the weekly added to sera collected from volunteers 24 hours dose of Maloprim® This is due to the ill-matched after administration of a low dose of 10 mg dapsone.14 elimination half-lives and clearance of pyrimethamine and dapsone, with mean half-lives in the volunteers These encouraging results led to the use of a bioassay being 105 hours and 23 hours, respectively.18 The (see below) to assess the activity of proguanil and considerably more rapid elimination of dapsone dapsone by incubating sera, collected at various implied that residual levels of pyrimethamine times after drug administration, with in vitro cultured would not be able to suppress parasites without the parasites. By determining serum antimalarial activity synergistic activity of dapsone. This very significant in vitro, preliminary information about the duration difference in the pharmacokinetics of the two drugs and synergy of drug activity could be obtained ex probably explained the decreasing effectiveness of vivo, before proceeding to in vivo assessment of Maloprim® in areas where parasites were becoming such activity in individuals exposed to or infected increasingly resistant to pyrimethamine. with malaria. Thus, 6 healthy Army volunteers, not serving in a malarious area, received 200 mg Field study with proguanil and low-dose dapsone.
proguanil daily for 7 days and, after an interval of at least a month, 10 mg dapsone daily for 7 days.14,15 The results of these findings were shared with the Serum specimens were collected at various intervals PNG Medical Research Advisory Committee and it up to 24 hours after the end of proguanil or dapsone approved a joint proposal by Ramu Sugar Limited administration. None of the dapsone sera showed (RSL) and AMRU to evaluate the effectiveness of any antimalarial activity against the K1 isolate and, proguanil/dapsone among seasonal workers at RSL, although sera from a few of the proguanil-treated PNG. Some consideration had been given by RSL volunteers (containing cycloguanil) showed partial to using proguanil alone for malaria prophylaxis inhibition of parasite growth for up to 12 hours, none because chloroquine prophylaxis was not protecting of them did so 24 hours after drug administration. their relatively non-immune workers from highland On the other hand, when dapsone- and cycloguanil- provinces against malaria. However, because in vitro containing sera were combined with each other, tests in 1987 had shown parasites in this malarious parasite inhibition varied between 77% and 99% in area to have a degree of resistance to cycloguanil the samples collected from volunteers 24 hours after similar to that observed during studies at AMRU, the sugar company was keen to evaluate the effectiveness of a 12-week supervised course of proguanil in The favourable ex vivo antimalarial activity of combination with a low dose of dapsone. As a result, proguanil combined with dapsone prompted a further 280 out of 1800 workers living in highly malarious study to determine the steady-state pharmacokinetics villages indicated their willingness to participate in of 200 mg proguanil co-administered daily with 10 a supervised 12-week course of proguanil/dapsone. Journal of Military and Veterans’ Health Three weeks after receiving a standard 3-day highly effective in curing drug-resistant falciparum course of amodiaquine to eliminate any circulating malaria.20 However, despite indications that the asexual parasites, they received 200 mg proguanil selective use of the tetracyclines might be beneficial once a day and 25 mg dapsone twice a week (10 mg for “personal prophylaxis by non-immune individuals tablets for daily administration were unavailable). visiting or working temporarily in areas with a high Finger-tip blood specimens were collected every transmission of multidrug resistant strains”,20 two weeks for examination of malaria parasites and the first field study to determine the prophylactic white cell counts. In addition, blood specimens were effectiveness of doxycycline had not been conducted examined for malaria parasites from any villagers (participants and non-participants) who developed During 1989, 184 Australian soldiers volunteered fever or reported to the RSL medical clinic with any to take 100 mg doxycycline daily during their illness. None of the 225 participants who completed deployments to PNG for 3 to 6 weeks. In addition the 12-week proguanil/dapsone course showed any to doxycycline prophylaxis, 69 men also took a parasites in their blood films during the period of daily dose of 7.5 mg primaquine base. Medication drug administration, but 16 of them developed was generally well-tolerated and, although about 1 symptomatic infections 2 to 12 weeks after the end of in 20 soldiers experienced some photosensitivity, drug administration. During the 12-week proguanil/ the incidence of gastrointestinal disturbances dapsone course, 72 symptomatic infections were was similar to that observed with mefloquine or detected in the 1,500 or so non-participants, Maloprim®/chloroquine prophylaxis.7 All of the men indicating continuous malaria transmission during were protected against P. falciparum and P. vivax while they were on medication. None of them developed These findings suggested that this prophylactic falciparum malaria after returning to Australia but, regimen was effective in preventing malaria in an despite a 14-day primaquine eradication course, area with proven resistance to chloroquine and 13% (15 out of 115) of the soldiers taking doxycyline proguanil. The cumulative weekly dose of dapsone alone experienced delayed primary attacks of vivax (50 mg) was one-half the weekly dose of dapsone malaria, usually within 2 to 3 weeks after returning to (100 mg) in Maloprim® and less than one-third the Australia. On the other hand, none of the 69 soldiers cumulative weekly dose given to Australian and taking doxycycline, in combination with a low-dose American soldiers during the Vietnam conflict (175 of primaquine, developed vivax malaria.7 Leaving mg).4 No decrease in white blood cells was observed aside these preliminary findings with primaquine, throughout the course of the study and, in reality, it seemed likely that short-term administration of agranulocytosis would be unlikely to manifest doxycycline might play a useful role in protecting itself after such low doses of dapsone. Due to the healthy non-pregnant adults against falciparum marked drop in malaria cases and no discernible malaria and in suppressing vivax malaria during drug toxicity, the company expressed its interest in promoting the wider use of this drug combination among its workers to enhance their health and to reduce the number of man-hours lost by malaria Mefloquine is a 4-quinolinemethanol drug which had been developed under the auspices of the antimalarial Although favourable results were obtained at Ramu, drug development program of the US Army. Initial further studies were obviously required before this clinical studies with this drug in 1974 had shown it drug combination could be considered for use as an to be very effective in the prophylaxis and treatment alternative to Maloprim®. Therefore, field studies were of multidrug-resistant falciparum malaria.23,24 After started in 1989 with two possible alternative drugs extensive field studies over the next decade, this – doxycycline (a tetracycline drug) and mefloquine.
long-acting drug had progressively been approved for use as an antimalarial by various countries, and was registered in Australia in 1988. The following year, 40 soldiers agreed to take a weekly dose of 250 In 1970, the tetracyclines had been found to be mg mefloquine for 4 weeks during their deployment effective against both the pre-erythrocytic and to PNG; all of them remained well and the medication asexual erythrocytic stages of chloroquine-resistant was tolerated as well as Maloprim®/chloroquine P. falciparum malaria.19 Following that early prophylaxis.7 After return to Australia, despite taking discovery, clinical and field studies with various the primaquine eradication course, 10% (4 out of tetracyclines, including doxycycline, had confirmed 40) of the soldiers suffered acute attacks of vivax that this group of drugs, when given in combination malaria. As expected, mefloquine had acted against with a rapidly-acting blood schizontocide, was the asexual erythrocytic stages of P. falciparum and after drug administration to either uninfected non- P. vivax but not against the liver stages of P. vivax. human primates or human volunteers. Thus, vital These results provided further evidence that 22.5 mg information about the potential value of various primaquine a day for 2 weeks was not always able to drug combinations could be obtained before eliminate the latent tissue stages (hypnozoites) of P. formulating drug regimens for malaria prophylaxis Effectiveness of amodiaquine against chloroquine- Assessment of other potential antimalarial drugs resistant P. vivax. Historically, chloroquine and In vitro assessment of drug activity against drug- amodiaquine had been used interchangeably for resistant isolates of P. falciparum. Additional malaria prophylaxis and treatment because it was studies were performed with various pyrimidine considered that malaria parasites were equally and purine antimetabolites in collaboration with the susceptible to both these 4-aminoquinoline drugs. Department of Biochemistry, University of New South However, in 1969, amodiaquine was shown to Wales. Some synergistic activity against P. falciparum be more effective than chloroquine in treating was observed between pyrazofurin and tubercidin, multidrug-resistant infections of P. falciparum.30 but none was observed in most combinations of Would chloroquine-resistant P. vivax respond in these compounds or when they were combined the same way? The findings in 2 Aotus monkeys with dihydrofolate reductase (DHFR) inhibitors inoculated with the AMRU 1 isolate indicated that such as pyrimethamine or cycloguanil (active this isolate was also more susceptible to amodiaquine metabolite of proguanil).25 These investigations and that this 4-aminoquinoline drug could play a were followed by collaborative studies with the useful role in the treatment of chloroquine-resistant Department of Biochemistry at the University of Sydney to determine the activity of TDHO-Me, BW566C80 (a new antimalarial undergoing clinical trials), and pyrazofurin, which inhibit the third, Malaria transmission via mosquitoes using cultured fourth and sixth step of the de novo pyrimidine pathway, respectively. Unfortunately, no synergism was observed between any of these compounds nor As part of AMRU’s collaboration with the Walter and between them and dihydrofolate reductase (DHFR) Eliza Hall Institute (WEHI) and the Australian Malaria inhibitors, such as cycloguanil. This was in marked Vaccine Joint Venture (Saramane Pty Ltd), colony contrast to the intense synergistic activity observed specimens of An. farauti 1 needed to be infected between DHFR inhibitors and dapsone.25 Because with in vitro cultured P. falciparum gametocytes. of these findings, further studies with pyrimidine This would enable immunisation studies to assess and purine antimetabolites were discontinued. By potential vaccine candidates as well as future trials contrast, collaborative studies with the Australian to evaluate the efficacy of antimalarial drugs. The An. National University, started in 1984, continued farauti 1 colony was chosen for this work as, unlike to show that Mannich bases exerted significant the An. farauti 2 and An. farauti 3 colonies, it was antimalarial activity against drug-resistant isolates self-mating and sufficient numbers to support the work could be readily maintained. The procedure of producing cultured gametocytes and infecting Bioassay for assessing drug activity. After anophelines was being routinely carried out by Dr establishing the continuous culture of several Imogene Schneider’s team at Walter Reed Army different strains of P. falciparum, it became possible Institute of Research (WRAIR) using the NF54 (WR) to use a bioassay method28 to complement HPLC strain of P. falciparum and An. stephensi. During drug analysis of serum specimens. In addition to February and March 1987 Captain Bob Cooper estimating the concentration of some drugs, such as learnt the technique at WRAIR and then transferred the active metabolite of proguanil (cycloguanil),29 the it to AMRU. Despite being able to induce P. falciparum bioassay could provide comparative data of biological cultures to produce healthy gametocytes, there was activity against parasite strains with defined drug a persistent failure to infect An. farauti 1 past the susceptibilities. The bioassay could also be used ookinete stage. To determine whether An. farauti to detect the presence of unrecognised active drug 1 was indeed refractory to cultured P. falciparum metabolites not yet detectable by HPLC analysis. gametocytes, colony An. farauti 1 material was sent But perhaps the most important contribution of the to Dr Schneider who ran parallel infection studies bioassay was that now the synergistic antimalarial with the same batch of gametocytes fed to both An. activity of various serum concentrations could be farauti and An. stephensi. The results showed that assessed against different strains of P. falciparum while 70% of An. stephensi became infected to the Journal of Military and Veterans’ Health sporozoite stage, only 2.1% of An. farauti 1 were transmit infections via the bites of these infective infected and this only to the oocyst stage.32 The mosquitoes were achieved in 9 out of 15 monkeys, importation of An. stephensi into Australia was not with an average prepatent period of 32 days (range possible due to quarantine regulations and, in the 15-79 days).34 The ability to use sporozoites to absence of a competent vector, these studies were initiate infections in monkeys offered the potential abandoned pending changes in the quarantine to develop a cyclical model for the evaluation of antimalarial drugs against the liver stages of P. vivax. Malaria transmission in Saimiri and Aotus monkeys Distribution of Anopheline mosquitoes in northern Aotus monkeys, first received at the Unit in 1982, continued to be regarded as the best experimental host for human malaria parasites. Although Operation Anopheles, initiated in 1984 to determine the distribution of the An. farauti sibling species in live births, older monkeys were dying from old age, northern Queensland, was extended during 1985- so that the colony was not increasing in size. As 1990 to cover all of northern Australia where malaria outbreaks had previously occurred and to include Aotus monkeys could not be obtained from all species of anophelines. Cape York Peninsula was Saimiri sciureus monkeys were acquired from the Commonwealth Serum Laboratories (CSL), surveyed in 1985 and 1986, the Gulf of Carpentaria Melbourne, to determine whether they could be used in 1987 and 1988, and the Northern Territory in 1989 as an alternative host for human malaria parasites, and 1990. The work was supported by members of the 1st Preventive Medicine Company. Kiowa light observation helicopters from 162 Reconnaissance Commencing in 1989 attempts were made to infect Squadron, Army Aviation, provided access to the Saimiri and Aotus monkeys with various strains of P. remote and isolated areas of the region. Adult vivax (Chesson and Salvador 1). Following isolation anophelines, collected by using CO baited light of the chloroquine resistant strain - designated as traps, were identified morphologically. This was also AMRU 1 - from an ADF soldier exercising in Papua done with adult mosquitoes which were reared from New Guinea, studies using this strain became larvae that had been collected from breeding sites the main focus of the work. The aim was to adapt and characterise the natural course of infection of this strain in Specimens identified as An. farauti were Saimiri and Aotus monkeys so that a monkey malaria model could be used to evaluate the cryopreserved and transported back to AMRU where the isomorphic species were separated using isoenzyme electrophoresis or P32 DNA labelled probes. Studies with Saimiri monkeys showed that these The isoenzyme electrophoresis method, developed animals were refractory to the chloroquine resistant by Dr Rod Mahon (Division of Entomology, CSIRO, AMRU 1 strain of P. vivax, though they were infected Canberra), worked well for specimens collected in with the AMRU 2 strain which was a chloroquine northern Queensland but failed to identify An. farauti sensitive strain isolated from another ADF soldier specimens collected in the Northern Territory. The latter specimens were examined using species specific Aotus monkeys, on the other hand, could be DNA probes designed to separate the isomorphic infected quite readily with the AMRU 1 strain. Over species - An. farauti 1, An. farauti 2, and An. farauti a period of a year 12 monkeys were inoculated 3.35 This method, developed in collaboration with Dr with AMRU 1 parasites, either by blood passage or Tom Burkot and Leanne Cooper of the Queensland using ccryopreserved parasites. Parasites appeared Institute of Medical Research, does not require field in the peripheral blood of all monkeys within 3-18 specimens to be preserved as stringently as those days (mean 7.5 days) following inoculation, with collected for analysis by isoenzyme electrophoresis. maximum parasitaemias (range 1200 - 68800/ This is due to the DNA molecule being quite robust µL; mean 20587) being achieved within 19 days of and less susceptible to degradation than enzymes. inoculation. However, within 13 days, parasitaemias An. farauti 1 could also be identified by a simple in all monkeys fell to less than 500/µL. Gametocytes field technique not requiring the sophisticated were produced during the course of the infection equipment used for isoenzyme electrophoresis and and appeared to be most infective 3-4 days prior to DNA hybridisation. It relied on the fact that larvae of peak parasitaemia. Infection rates in An. farauti 1, this species breed in brackish water and can tolerate An. farauti 2, and An. farauti 3 were 16.8%, 8.9%, higher levels of salinity than either An. farauti 2 or 3. and 16.9%, respectively, though rates as high as Thus, exposure of first instar larvae to sea water for 96% were observed in some batches. Attempts to 1 hour killed 0.5% of An. farauti 1 larvae but >99.1% of An. farauti 2 and 3 larvae.36 Of the 12 species of anophelines collected from 620 (derived from dead infected mosquito larvae) to sites, the 9 most common were An. farauti 1 (128 sites), An. farauti 2 (67 sites), An. farauti 3 (93 sites), Subsequent studies with another microsporidium - An. annulipes (335 sites), An. bancroftii (181 sites), Amblyospora indicola – infecting Culex sitiens (with An. meraukensis (162 sites), An. hilli (88 sites), An. Apocyclops as the copepod intermediate host) showed amictus (93 sites), and An. novaguinensis (70 sites). that its life cycle was similar to that of Amblyospora All species were found in areas with rainfall above dyxenoides. Furthermore, the developmental stages the 1200 mm p.a. isohyet, but only An. annulipes of these two microsporidia looked to be the same and An. amictus were found below the 1000 mm in both mosquito hosts and also in the different p.a. isohyet. While the breeding of most species was dependent on adequate rainfall, An. farauti 1 and An. hilli also required brackish water. Geographically, the The possibility that these two microsporidia were distribution of the three isomorphic species of An. the same species was investigated by host specificity farauti varied from one area to another. An. farauti experiments in which spores produced in both 1 was common along all coastal areas of northern mosquitoes were exposed separately to the two Australia. An. farauti 2 was the most common and copepods. The results indicated that spores from widespread of the three isomorphic species in Cape Cx. annulirostris were not able to infect Apocyclops York Peninsula, while An. farauti 3 was the most sp. copepods and that spores from Cx. sitiens were abundant species in inland areas of the Northern not infectious to M. albicans. Further experiments showed that copepod spores produced in M. albicans were infectious to Cx. annulirostris larvae but not to Operation Anopheles indicated the wide distribution Cx. sitiens larvae. Similarly, spores from Apocyclops of various species of the An. farauti taxon, the main copepods were infectious to Cx. sitiens but not to malaria vectors in northern Australia. As vast tracts Cx. annulirostris larvae. These findings suggested of the Northern Territory and northern Queensland that there was a high level of specificity of these within the range of these species are frequently used microsporidia in their invertebrate hosts.42 by the ADF for training exercises, they remain a potential threat to ADF personnel if malaria is re- A computer simulation model of the dynamics of introduced into Australia. In 1989-1990, about 1000 A. dyxenoides in its mosquito and copepod hosts malaria cases were imported annually into Australia, showed that the biological complexity of this host- of which about 1 in 20 were in Army personnel parasite system made it difficult to adequately assess returning from overseas exercises. A small malaria its potential for control of mosquito populations.43 outbreak did, in fact, occur in northern Queensland. Moreover, there were technical problems in the Although it was not related to any movement of production of inocula for field release that would military personnel, it illustrated the importance of have to be solved before this approach could be prompt malaria diagnosis and treatment to decrease implemented, including the artificial culture of the likelihood of gametocytes infecting local vector microsporidia and the mass laboratory rearing of spores in copepods or mosquito larvae.44 Even though the specificity experiments were based Potential of Microsporidia for mosquito control.
on only two mosquito/copepod combinations, Earlier field observations at Mildura conducted each Amblyospora species probably only infects a by AMRU had discovered a microsporidian – single mosquito host species, thereby preventing Amblyospora - parasitising Culex annulirostris its biocontrol potential against a range of disease mosquitoes in Mildura, Victoria.5 With the support vectors. For these reasons it was concluded that, on of research grants from the National Health and the basis of current knowledge, such parasites could Medical Research Council and the World Health not be considered as promising biological control Organization, the complete life cycle of Amblyospora in mosquitoes and the copepod intermediate host No further investigations with any other potential was established. This provided the opportunity to biological agents were carried out, including some evaluate the potential value of these parasites as that were discovered accidentally during Operation biological control agents of mosquitoes.
Anopheles in northern Queensland. They included The particular Amblyospora species infecting Culicinomyces bisporalis, Crypticola clavulifera and Cx. annulirostris mosquitoes and Mesocyclops Lagenidium giganteum, observed in mosquito and albicans copepods was described as Amblyospora biting midge larvae living in water contained in plant dyxenoides.40 Its infection rates in copepods were directly proportional to the concentration of spores Journal of Military and Veterans’ Health Figure 2: Entomology staff at Cowley Beach Training monitored by placing swatches of fabric in the pockets of Disruptive Pattern Combat Uniforms (DPCU) and (L to R) LTCOL A. W. Sweeney, CAPT R. D. Cooper, SGT C. by attaching them to ADF bednets before treatment. Samples returned to AMRU by the health officer for chemical analysis showed that the treatment was carried out successfully. Furthermore, bioassay of the samples showed a marked knockdown and mortality of An. farauti mosquitoes. However, due to operational limitations, it was not possible to determine how long the permethrin treatment persisted in DPCU.
Also in 1989, the persistence of permethrin in bednets was investigated in collaboration with the World Health Organization entomologist in Honiara, Solomon Islands. Net samples used in the field evaluation of impregnated nets on Guadalcanal were sent to AMRU before treatment and 2, 5, and 8 months after treatment. Chemical analysis and bioassays showed that permethrin persisted in treated nets for 8 months, but was lost more readily Deet – applied to skin. The topical application from the bottom of nets, probably due to abrasion of mosquito repellents was (and continues to be) an important means of protecting oneself against Deet combined with permethrin. In a collaborative malaria. The ADF mosquito repellent, a liquid project with the Letterman Army Institute of Research, formulation containing 95% diethylmethylbenzamide US Army, the efficacy of 2 new ‘controlled release’ (also known as diethyltoluamide, commonly called formulations of deet and the US Army’s standard deet) in ethanol, felt oily when applied to skin and repellent (75% deet in ethanol) were compared with was not particularly well accepted by soldiers in the each other at the Cowley Beach Training Area.50 In field. As there was also some concern about the safety addition, mosquito biting activity was determined of using such high concentrations of deet, studies when each of these topical skin applications were were undertaken by AMRU to evaluate alternate used in combination with permethrin-impregnated approaches to reducing vector-human contact. uniforms. This was the first US Army/ Australian A novel approach, developed by a chemist in Army entomological research undertaken since Melbourne, was to incorporate 20% deet and 0.5% World War II. The 3 deet formulations and the permethrin in a bar of soap, and using it as a impregnated uniforms had similar repellent activity. repellent by rubbing moistened soap onto the surface However, when any of the deet repellents were used of the skin. When field studies were undertaken in combination with the impregnated uniforms, there at Homebush Bay, Sydney and the Cowley Beach was a marked decrease in mosquito biting activity.50 Training Area in Queensland. the soap formulation Repellents against “Chiggers”. Studies were provided protection against mosquitoes for only also carried out to evaluate the activity of various one hour, much shorter than the ADF mosquito repellents against the chigger mite, Eutrombicula hirsti. Trombiculid mites can transmit scrub typhus, Permethrin – impregnated in uniforms and a potential public health problem for soldiers in bednets. Another field study at Cowley Beach northern Australia. They are also responsible for was conducted using an alternative approach for ‘Scrub Itch’, a painful condition which is often repelling mosquitoes – the impregnation of clothing encountered by soldiers in the wet tropics. After with permethrin, a synthetic pyrethroid compound. establishing a colony of these mites and studying The results showed that impregnated jungle green their biology,51 repellent studies showed that low uniforms prevented more mosquitoes from biting concentrations of deet, dimethylphthlate, benyl benzoate and permethrin were highly toxic to chiggers.52 A subsequent field study conducted In 1989, permethrin-impregnated uniforms and in 1990 at Cowley Beach Training Area showed bednets were first used by ADF personnel during that Disruptive Pattern Combat Uniforms treated their service with the UNTAG Mission to Namibia. with permethrin and dibutylphthalate provided The effectiveness of permethrin impregnation was excellent protection against E. hirtsi chiggers.53 These investigations represented the first Australian Mannich base compounds (up to 1990).
research in this field since World War II.
• Annual surveys and characterisation of anopheline mosquitoes in northern Australia (up to 1990).
• Experiments with microsporidia (Amblyospora) for The first half of the third decade (1985-1990) was biocontrol of mosquitoes (up to 1990).
characterised by greater emphasis on practical problems facing ADF personnel in malarious areas. In vitro, in vivo and ex vivo tests and procedures • Ex vivo and pharmacological studies with were used successfully to investigate the increasing prevalence of P. falciparum and P. vivax to standard • Trials with topical mosquito repellents and antimalarial drugs. These investigations revealed, for insecticide-impregnated clothing and bed nets (up the first time, that P. vivax could develop resistance to chloroquine, the standard drug used world-wide for the prevention and treatment of vivax malaria. Laboratory and field studies were also able to identify • First documented evidence of P. falciparum potentially useful drugs and drug combinations for preventing drug-resistant malaria infections. Although wider biological control of mosquitoes Attempts at transmission of cultured P. falciparum proved difficult, significant advances were made in identifying repellent formulations and procedures for providing better personal protection against • Field study with proguanil and low-dose dapsone.
mosquitoes and other arthropods. As northern Australia remains receptive to malaria, the Unit • Upsurge in malaria during ADF exercises in PNG.
conducted the most extensive survey of anopheline mosquitoes and characterisation of malaria vectors ever performed in that region. • Professor Karl Rieckmann appointed fifth Director • Discovery that P. vivax is able to develop resistance The opinions expressed are those of the authors and do not necessarily reflect those of the Joint Health • Course of chloroquine-resistant P. vivax infections Command or any extant Australian Defence Force • Evaluation of doxycycline and mefloquine for Authors’ affiliation: Australian Army Malaria Institute Corresponding author: Karl Rieckmann • Colonel Eric Donaldson appointed fourth Director • Professor Karl Rieckmann appointed Director of Plasmodium falciparum in vitro studies with
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4. Rieckmann KH. Falciparum malaria: The urgent need for safe and effective drugs. Ann Rev Med 1983; 5. Rieckmann KH, Sweeney AW. Army Malaria Institute: its evolution and achievements. First decade: 1965- Journal of Military and Veterans’ Health 6. Rieckmann KH, Edstein MD, Cooper RD, Sweeney AW. Army Malaria Institute: its evolution and achievements. Second decade: 1975-1985. JMVH 2012; 20 (3): 9-20.
7. Rieckmann KH, Yeo AET, Davis DR, Hutton DC, Wheatley PF, Simpson R. Recent military experience with malaria prophylaxis. Med J Aust 1993; 158: 446-449.
8. Rieckmann KH, Davis DR, Hutton DC. Plasmodium vivax resistance to chloroquine. Lancet 1989; 2:1183- 9. Rieckmann KH, Sax LJ, Campbell GH, Mrema JE. Drug sensitivity of Plasmodium falciparum. An in-vitro microtechnique. Lancet 1978; 1:22-23.
10. Edstein MD, Veenendaal JR, Rieckmann KH, O’Donoghue M. Failure of dapsone/pyrimethamine plus chloroquine against falciparum malaria in Papua New Guinea. Lancet 1988; 1:237 11. Whitby M, Wood G, Veenendaal JR, Rieckmann K. Chloroquine-resistant Plasmodium vivax. Lancet 1989; 12. Edstein MD, Veenendaal JR, Scott HV, Rieckmann. Steady-state kinetics of proguanil and its active metabolite, cycloguanil, in man. Chemotherapy 1988; 34:385-392.
13. Veenendaal JR, Edstein MD, Rieckmann KH. Pharmacokinetics of chlorproguanil in man after a single oral dose of Lapudrine®. Chemotherapy 1988; 34:277-283.
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16. Army Malaria Research Unit. Report on activities for year 1987/1988. Australian Government Publishing 17. Edstein MD, Veenendaal JR, Rieckmann KH. Multiple-dose kinetics in healthy volunteers and in vitro antimalarial activity of proguanil plus dapsone. Chemotherapy 1990; 36:169-176.
18. Edstein MD, Rieckmann KH, Veenendaal JR. Multiple-dose pharmacokinetics and in vitro antimalarial activity of dapsone and pyrimethamine. Br J Clin Pharmacol 1990; 30:259-265.
19. Rieckmann KH, Powell RD, McNamara JV, Willerson D, Kass L, Frischer H, Carson PE. Effects of tetracycline against chloroquine-resistant and chloroquine-sensitive strains of Plasmodium falciparum. Am J Trop Med Hyg 1971; 20:811-815.
20. Rieckmann KH. Antibiotics. In: Handbook of Experimental Pharmacology: Antimalarial Drugs 1984; Volume 68/II, Chapter 14, pp. 443-470. Springer-Verlag. Berlin, Heidelberg, New York.
21. Pang LW, Limsomwong N, Boudreau EF et al. Doxycycline prophylaxis for falciparum malaria. Lancet 22. Rieckmann KH. Tetracycline prophylaxis for malaria. Lancet 1987; 2:507-508.
23. Rieckmann KH, Trenholme GM, Williams RL, Carson PE, Frischer H, Desjardins RE. Prophylactic activity of mefloquine hydrochloride (WR 142490) in drug-resistant malaria. Bull Wld Hlth Org 1974; 51:375-377.
24. Trenholme GM, Williams RL, Desjardins RE, Frischer H, Carson PE, Rieckmann KH. Mefloquine (WR 142490) in the treatment of human malaria. Science 1975; 190:792-794.
25. Scott HV, Rieckmann KH, O’Sullivan WJ. Synergistic antimalarial activity of dapsone/dihydrofolate reductase inhibitors and the interaction of antifol, antipyrimidine and antipurine combinations against Plasmodium falciparum in vitro. Trans R Soc Trop Med Hyg 1987; 81:715-721.
26. Scott HV, Tan WL, Barlin GB. Antimalarial activity of Mannich bases derived from 4-(7’-bromo-1’, 5’-naphthiridin-4’-ylamino) phenol and 4- (7’-trifluoromethylquinolin-4’-ylamino) phenol against Plasmodium falciparum in vitro. Ann Trop Med Parasitol 1987; 81:85-93.
27. Scott HV, Tan WL, Barlin GB. The in vitro and in vivo antimalarial activity of some Mannich bases derived from 4-(7’-bromo (and chloro) – 1’, 5’-naphthiridin – 4’ ylamino) phenol. Ann Trop Med Parasitol 1988; 82:127-134.
28. Mrema JEK, Rieckmann KH. A rabbit-in vitro system to evaluate drug action against Plasmodium falciparum. Trans R Soc Trop Med Hyg 1983; 77:130-135.
29. Scott HV, Edstein MD, Veenendaal JR, Rieckmann KH. A sensitive bioassay for serum cycloguanil using Plasmodium falciparum in vitro. Int J Parasit 1988; 18 (5):605-609.
30. Rieckmann KH. Determination of the drug sensitivity of Plasmodium falciparum. J Am Med Assoc 1971; 31. Cooper RD, Rieckmann KH. Efficacy of amodiaquine against a chloroquine-resistant strain of Plasmodium vivax. Trans R Soc Trop Med Hyg 1990; 84:473.
32. Cooper RD, Meza A, Schneider I. Anopheles farauti refractoriness to malaria infection with cultured gametocytes of Plasmodium falciparum. Med Vet Entomol 1994; 8:389-390 33. Cooper RD. Susceptibility of Guyanan Saimiri monkeys to a chloroquine-sensitive and a chloroquine- resistant strain of Plasmodium vivax from Papua New Guinea. J Parasitol 1995; 81:640-641.
34. Cooper RD. Studies of a chloroquine-resistant strain of Plasmodium vivax from Papua New Guinea in Aotus and Anopheles farauti s.l. J Parasitol 1994; 80:789-795.
35. Cooper L, Cooper RD, Burkot TR. The Anopheles punctulatus complex: DNA probes for identifying the Australian species using isotopic, chromogenic, and chemiluminescence detection systems. Exp Parasitol 1991; 73:27-35.
36. Sweeney AW. Larval salinity tolerances of the sibling species of Anopheles farauti. J Am Mosq Control Assoc 37. Sweeney AW, Cooper RD, Frances SP. Distribution of the sibling species of Anopheles farauti in the Cape York Peninsula, northern Queensland, Australia. J Am Mosq Control Assoc 1990; 6:425-429.
38. Cooper RD, Frances SP, Sweeney AW. Distribution of members of the Anopheles farauti complex in the Northern Territory of Australia. J Am Mosq Control Assoc1995; 11:66-71.
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41. Sweeney AW, Doggett SL, Gullick G. Laboratory experiments on infection rates of Amblyospora dyxenoides in the mosquito Culex annulirostris. J Invertebr Pathol 1989; 53:85-92.
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43. Larkin TS, Sweeney AW, Carruthers RI. Simulation of the dynamics of a microsporidian pathogen of mosquitoes. Ecological Modelling 1995; 77:143-165.
44. Sweeney AW, Becnel JJ. Potential of microsporidia for biological control of disease vectors. Parasitology 45. Sigler L, Frances SP, Panter C. Culicinomyces bisporalis, a new entomopathogenic hyphomycete from the larvae of the mosquito Aedes kochi. Mycologia 1987; 79 : 493-500.
46. Frances SP, Sweeney AW, Humber RA. Crypticola clavulifera gen. et. sp. nov. and Lagenidium giganteum: Oomycetes pathogenic for Dipterans infesting leaf axils in an Australian rainforest. J Invertebr Pathol 1989; 54 : 103-111.
47. Frances SP. Status of the Deuteromycete fungi, Tolypocladium and Culicinomyces as control agents for medically important Diptera. Proceedings of the Vth International Congress of Invertebrate Pathology. 1990; 103-106.
48. Frances SP. Pathogenicity, host range and temperature tolerance of Crypticola clavulifera (Oomycetes : Lagenidiales) in the laboratory. J Am Mosq Control Assoc 1991; 7: 504-506.
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50. Gupta RK, Sweeney AW, Rutledge LC, Cooper RD, Frances SP, Westrom DR. Effectiveness of Controlled- Release Personal-Use arthropod repellents and permethrin-impregnated clothing in the field. J Am Mosq Control Assoc 1987; 3:556-560.
51. Southcott RV, Frances SP. The life history stages of Odontacarus (Leogonius) barrienensis (Womersley) (Acari : Trombiculidae: Leewenhoekiinae). Internat J Acarol 1991; 17:275-287.
52. Frances SP. Response of a chigger, Eutrombicula hirsti (Acari : Trombiculidae) to repellent and toxicant compounds in the laboratory. J Med Ent 1994; 31:628-630. 53. Frances SP, Yeo AET, Brooke EW, Sweeney AW. Clothing impregnations of dibutylphthalate and permethrin as protectants against a chigger mite, Eutrombicula hirsti (Acari: Trombiculidae). J Med Ent 1992; 29:907-910.
Journal of Military and Veterans’ Health


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