Richard G. Andre

Highly efficient dry season transmission of malaria in Thailand

Man-biting collections were made for 7 consecutive nights per month for 24 months at 2 sites in a Thai village regularly treated with DDT and fenitrothion yet hyperendemic for Plasmodium falciparum and P. vivax. Only Anopheles dirus was incriminated as a vector: 1.6% were infective and 2.4% were infected (median numbers of oocysts = 3.5). Transmission occurred within the village, which was located in groves of rubber and fruit trees, during the dry months of November to May only, when rates of parity (64%) and biting (2/man-night) were higher than during the monsoon (38% and 0.8%/man-night). Vectorial capacity and inoculation rates surged and then fell during 30 d at the end of the monsoon, quickly reinitiating transmission. Sporozoite species were identified using indirect fluorescent antibody tests or enzyme-linked immunosorbent assays: 76% were P. falciparum, compared to 78% of gametocytes; one mosquito was infected with both species. Vector survival and inoculation rates differed between similar sites 800 m apart. Dry season breeding occurred at the bottom of a deep, concrete-lined well. Much of the natural forest habitat of An. dirus in south-eastern Thailand that was once destroyed for farming is now being replaced with orchards; this ecological change may reintroduce malaria to a wide area.

A stable, oligosymptomatic malaria focus in Thailand

Blood from most of the 250 residents of a non-migratory farming village in south-eastern Thailand was visually examined for malaria parasites monthly for 2 years. Nearly 97% of the population had at least one (median = 5) patent Plasmodium falciparum infection per year; 72% had one due to P. vivax (median = 1). This contrasted with a slide positivity rate of 17% calculated from 12 months of passive case detection before the study began. Children 1-9 years old had the highest mean monthly prevalence (51%) and highest geometric mean density (10/500 white blood cells) of P. falciparum. Fewer than half the expected number of mixed infections were found but these were more common at high densities of P. falciparum. Individuals over 19 years old comprised 52% of the population but accounted for only 18% of P. vivax and 32% of P. falciparum gametocytaemias. Fever rates were marginally higher in those below 10 years old (8%) but occurred with equal frequency in those with patent infections or negative. The spleen rate (89% stage 1) was 24% in those under 15 years old and 7% in those older. No malaria mortality was seen P. falciparum cases treated for 10 d with quinine+tetracycline (QT) cleared the infection as often as those given one dose of mefloquine+sulfadoxine+pyrimethamine (MSP); both treatments reduced densities in cases not cured. Apparently unsupervised compliance was no better with MSP than with QT. The role played by hyperendemic, cryptic foci in Asian epidemics of malaria may have been underestimated.

Seasonal fluctuation of Plasmodium falciparum gametocytaemia

Two numerically minor components of Plasmodium falciparum prevalence--gametocytaemia and trophozoite densities greater than 99/500 white blood cells--displayed an annual cycle that reflected the seasonal abundance of infective Anopheles dirus at a hyperendemic focus in Thailand, even though the gross monthly prevalence for combined ages remained stable. Gametocyte prevalence rose more than 300% within 30 d after the capture of the dry seasons first infective mosquito, remained at about 8% until the beginning of the monsoon 7 months later, then fell within 60 d to about 2%. The number of cases with a high density of trophozoites behaved similarly. These periodic fluctuations represented changes in incidence, at least half of which appeared to be due to superinfection. Almost 49% of all gametocyte carriers were older than 14 years, but nearly all gametocyte densities greater than 20/500 white blood cells were in children. These observations, as well as the calculated efficiency of human infectivity, imply that superinfection of adults may contribute significantly to transmission in semi-immune populations.

In vivo and in vitro studies of chloroquine-resistant malaria in West Malaysia.

Abstract Studies to detect the presence of resistant Plasmodium falciparum strains in West Malaysia were conducted in 4 States. Investigations utilized the in vivo technique for determining the susceptibility of falciparum asexual parasites to chloroquine. A total of 6,880 individuals were examined for malaria, with an overall prevalence of 20%. Of 864 P. falciparum cases, 395 met all the criteria of the 7-day study. The equivalent of a single adult dose of chloroquine (600 mg. base) was given to 217 of the participants; of these, 36% were positive on the seventh day following treatment. 178 people were treated with 25 mg./kg. chloroquine base over a 3-day period, with 3% being found positive on Day 7. It can be seen from these results and those of similar surveys in Thailand that a gradient between a low amount of resistance in West Malaysia and a high amount of resistance in Thailand exists. Limited studies utilizing the newly developed in vitro cultivation of P. falciparum parasites in the presence of drugs, further support the in vivo findings that both resistant and sensitive strains are present in West Malaysia.

Species-and infective stage-specific monoclonal antibodies to Leishmania major produced by an in vitro immunization method

Monoclonal antibodies specific to the infective-stage promastigotes of Leishmania major are needed for developing rapid diagnostic assays of infected sand flies. An in vitro immunization protocol was applied for the production of monoclonal antibodies using small amounts of L. major. Infective-stage promastigotes were isolated from sand flies (Phlebotomus papatasi) 7-10 days after infection and used as antigen for immunization. Two weeks after a primary immunization, murine splenocytes were removed and immunized in vitro with antigen in murine EL-4 thymoma cell conditioned medium. Three fusions were performed using X63-Ag.653 myeloma cells as fusion partners and two fusions were performed using FOX-NY cells. Antibodies specific to promastigotes were detected using an indirect enzyme-linked immunosorbent assay (ELISA). Initially 56 monoclonal antibodies were selected, and their species and stage specificity were determined using both an ELISA and an indirect fluorescent antibody assay (IFA). Twelve monoclonal antibodies showed species specificity to L. major when tested against four sympatric species of Leishmania. Four other monoclonal antibodies showed species and infective-stage specificity to L. major promastigotes. When tested in immunoblots, all four species- and stage-specific monoclonal antibodies bound to five protein bands that were unique to the infective-stage promastigotes.

Antagonism of diisopropyl fluorophosphate (DFP) poisoning by carbamate pretreatments in house flies (musca domestica L.)

Abstract 1. Pretreatments with the carbamates mobam and pyridostigmine, but not neostigmine, “protected” house flies against the lethality of diisopropyl fluorophosphate (DFP). 2. Histochemical evidence demonstrated that DFP, when administered to flies by injection of a dose of 50 ng/fly (spld 95 ), inactivated cholinesterase throughout the fused lobes of the compound thoracic ganglia. 3. Flies pretreated with pyridostigmine and challenged with 50 ng of DFP ( ld 95 ) recovered from poisoning by 1 hr post-treatment. 4. The thoracic ganglia removed at 1 hr from recovered flies had a cholinesterase distribution similar to control (saline-treated) flies. 5. In contrast, flies pretreated with neostigmine before DFP challenge did not fully recover from poisoning by 1 hr and did not survive to 24 hr. 6. The thoracic ganglia removed from these flies contained active cholinesterase sites only in the mesothoracic lobe of the fused ganglion.

In vivo and in vitro assays for organophosphate poisoning therapeutic chemicals using the house fly, Musca domestica L.

Abstract 1. 1. Thoracic injection of 31 μg of N-methyl pyridinium-2-aldoxime chloride (2-PAM) per fly (1.55 g/kg) 1 hr before or after diisopropylfluorophosphate (DFP) elevated the baseline LD50, of DFP (14 ng/fly; 0.7 mg/kg) by 50 and 162 times, respectively. 2. 2. The net increase in the acetylcholinesterase (AChE) activity (“regeneration”) by the addition of 2-PAM (1.55 × 10−4 M) 3 min before or after the addition of DFP (8.43 × 10−7 M) to the fly homogenate ranged from 48.5 to 50.5%. Toxogonin was comparable to 2-PAM. HI-6 and HS-4 were less effective than 2-PAM. 3. 3. The toxic syndrome casued by injection of acetylcholine chloride, edrophonium chloride, atropine sulfate and gallamine triethiodide showed that the cholinergic nervous system was readily accessible. 4. 4. Brain AChE showed typical substrate-inhibition and a Km of 141 μM with acetylthiocholine. Fly brain AChE was comparable electrophoretically with mammalian AChE (fetal bovine serum) but was immunochemically distinct from several mammalian AChE.