High Levels of Genetic Diversity of Plasmodium falciparum Populations in Papua New Guinea despite Variable Infection Prevalence

High levels of genetic diversity in Plasmodium falciparum populations are an obstacle to malaria control. Here, we investigate the relationship between local variation in malaria epidemiology and parasite genetic diversity in Papua New Guinea (PNG). Cross-sectional malaria surveys were performed in 14 villages spanning four distinct malaria-endemic areas on the north coast, including one area that was sampled during the dry season. High-resolution msp2 genotyping of 2,147 blood samples identified 761 P. falciparum infections containing a total of 1,392 clones whose genotypes were used to measure genetic diversity. Considerable variability in infection prevalence and mean multiplicity of infection was observed at all of the study sites, with the area sampled during the dry season showing particularly striking local variability. Genetic diversity was strongly associated with multiplicity of infection but not with infection prevalence. In highly endemic areas, differences in infection prevalence may not translate into a decrease in parasite population diversity.     

High complexity of Plasmodium vivax infections in Papua New Guinean children

Although genetically distinct malaria parasites have been shown to simultaneously infect an individual, the total number of unique parasites has not been systematically studied. We examined multiple clones (8-38) from individual blood samples collected from Papua New Guinean children for polymorphisms in the Plasmodium vivax Duffy binding protein (dbpII) and the merozoite surface protein 3alpha (msp3alpha). We found a median of 4 (range = 2-6) and 12 (range = 2-23) unique genotypes based on dbpII and msp3alpha, respectively, per person at one time point and at least 12-33 unique genotypes per person over a four-month period. Control polymerase chain reactions (PCRs) detected 0-31% of clones with haplotypes that arose from PCR artifacts, indicating that caution must be taken when using PCR-based analysis to examine complex infections. To reduce artifacts from clones, analysis was based on haplotypes unlikely to have been generated by PCR artifacts or had been previously identified. Plasmodium vivax infections can be highly complex in disease-endemic areas, suggesting continual genetic mixing that could have significant implications for the use of antimalarial drugs and malaria vaccines.     

Diversity of antigens expressed on the surface of erythrocytes infected with mature Plasmodium falciparum parasites in Papua New Guinea

Antigens were detected on the surface of erythrocytes from children with acute falciparum malaria in Madang, Papua New Guinea. These parasite-induced erythrocyte surface antigens (PIESA) were serotyped with convalescent sera from children and hyperimmune sera from adults in parasite infected cell agglutination assays (PICAs) and by inhibition of binding of infected cells to melanoma cells. Extensive serological diversity of PIESA was demonstrated. A significant correlation between serotypes defined by reactivity of immune sera in PICA and inhibition of melanoma cell binding (MCB) was observed. This suggests that both assays measure antibody responses to the same antigen(s). Increased recognition of different PIESA specificities with age is consistent with the hypothesis that repeated exposure to malaria confers immunity against a range of PIESA serotypes and parallels the development of clinical immunity to malaria in this area of Papua New Guinea.     

Low efficacy of amodiaquine or chloroquine plus sulfadoxine-pyrimethamine against Plasmodium falciparum and P. vivax malaria in Papua New Guinea

Because of increasing resistance to 4-aminoquinolines in Papua New Guinea, combination therapy of amodiaquine (AQ) or chloroquine (CQ) plus sulfadoxine-pyrimethamine (SP) was introduced as first-line treatment against uncomplicated malaria in 2000. The purpose of this study was to monitor in vivo efficacy of the current standard combination therapy against Plasmodium falciparum and P. vivax malaria. Studies were conducted between 2003 and 2005 in the Simbu, East Sepik, and Madang Provinces in Papua New Guinea according to the revised protocol of the World Health Organization (WHO) for assessment of antimalarial drug efficacy. Children between six months and seven years of age with clinically overt and parasitologically confirmed P. falciparum or P. vivax malaria were treated according to the new policy guidelines (i.e., AQ plus SP given to patients weighing < 14 kg and CQ plus SP given to patients weighing < 14 kg). Children were monitored up to day 28 and classified according to clinical and parasitological outcome as adequate clinical and parasitological response (ACPR), early treatment failure (ETF), late clinical failure (LCF), or late parasitological failure (LPF). For P. falciparum malaria, polymerase chain reaction (PCR)-corrected treatment failure rates up to day 28 ranged between 10.3% and 28.8% for AQ plus SP and between 5.6% and 28.6% for CQ plus SP, depending on the region and the year of assessment. Overall treatment failure rate with AQ or CQ plus SP for P. vivax malaria was 12%. Our results suggest that the current first-line treatment in Papua New Guinea is not sufficiently effective. According to the new WHO guidelines for the treatment of malaria, a rate of parasitological resistance greater than 10% in the two dominant malaria species in the country justifies a change in treatment policy.     

Antibodies to Plasmodium falciparum gamete surface antigens in Papua New Guinea sera

Sera from individuals living in malaria endemic areas of Papua New Guinea were tested for their effect on infectivity of Plasmodium falciparum gametocytes grown in culture to Anopheles freeborni mosquitoes. Consistent reduction of infectivity to less than 5% of control was observed with nine out of the 41 sera from the endemic area tested and also with three out of seven sera tested from individuals rarely exposed to malaria infection. Gamete surface antigens recognized by the sera were investigated by immunoprecipitation from 125I surface-labelled gametes extracted in SDS and Triton X-100. The main antigens recognized were of the same mol. wt (230, 48 and 45 kD) as those known to be targets of transmission-blocking monoclonal antibodies. A significant negative correlation was observed between the total ct/min immunoprecipitated from surface-labelled gametes by the sera and the average number of oocysts per gut observed in membrane feeding experiments with these sera. Spearmann's rank correlation coefficient indicated that suppression of infectivity correlated strongly with the presence of antibodies against the 230 kD protein; there was no significant correlation between suppression and antibodies to the 48/45 kD proteins. The antibody response to the different gamete surface antigens varied greatly in sera from the endemic areas suggesting that individuals respond differently to each gamete antigen.     

Insight into the early spread of chloroquine-resistant Plasmodium falciparum infections in Papua New Guinea

The first report of Plasmodium falciparum chloroquine (CQ) resistance (CQR) in Papua New Guinea (PNG) appeared in 1974. Although the current prevalence of CQR-associated parasite gene polymorphisms has been documented for some regions, the spatial and temporal relationships that characterize CQ-resistant parasites in PNG are unknown. Insight into the evolution of CQ-resistant parasites could be provided by evaluating genetic markers in parasite populations. We compared pfcrt and pfmdr1 polymorphisms and flanking microsatellite (MS) polymorphisms between P. falciparum-infected placental tissue (early 1980s) and blood (late 1990s) samples collected throughout PNG. Consistent with the results of recent studies, pfcrt-SVMNT and pfmdr1-86Y were the only CQR-associated alleles observed in the placental tissue samples, and they were observed together in 79% of the samples. Results of analysis of MS flanking pfcrt (approximately 40 kb) suggested that there was less diversity in the samples collected during the 1980s than in those collected during the 1990s and that the 1990s parasites were significantly differentiated from the 1980s parasites. On the other hand, for MS flanking pfmdr1 (approximately 5 kb) and for 1 putatively neutral locus, diversity levels were similar, and the 2 parasite populations were not significantly differentiated. These results suggest that selection for CQR was operating on the pfcrt-SVMNT allele during the early 1980s. Thus, archival samples can provide novel insight into the dynamics of CQR.     

Diversity in the immunodominant determinants of the circumsporozoite protein of Plasmodium falciparum parasites from malaria-endemic regions of Papua New Guinea and Brazil.

To determine the nature and extent of variation in the T cell sites of the Plasmodium falciparum circumsporozoite (CS) protein, a candidate antigen in the development of a malaria vaccine, we cloned and sequenced 69 recombinant clones of the CS protein gene representing 18 and 17 P. falciparum isolates from infected individuals from Madang, Papua New Guinea (PNG), a holoendemic malaria region, and Paragaminos and Jacunda, Brazil, relatively low endemic regions, respectively. As previously known, the amino acid sequence polymorphism was restricted to the three immunodominant regions of the protein, Th1R-N1, Th2R, and Th3R. While some of the observed nonsilent mutations in the T cell determinants of the CS protein were similar to those previously identified, we have found new amino acid changes in each of the polymorphic sequences in parasites from PNG and Brazil. A comparison of the CS epitope sequences of parasites from PNG and Brazil with the previously known CS epitope sequences of parasites from Brazil and The Gambia showed the following: 1) polymorphism was found in the Th1R-N1, Th2R, and Th3R region; however, while amino acid substitutions in the Th1R-N1 and Th2R region tended to be conservative, the substitutions found in the Th3R region were not, suggesting that the Th3R epitope may be rapidly evolving to allow parasites to escape host antiparasite cytotoxic T cell-enforced immune responses, and 2) the CS proteins of P. falciparum from high malaria-transmission regions (PNG and The Gambia) appear more polymorphic than the CS proteins of parasites from relatively low malaria-endemic regions in Brazil, where P. falciparum infection has been recently established.     

Molecular analysis of Plasmodium falciparum from drug treatment failure patients in Papua New Guinea

A study was conducted in Papua New Guinea to analyze Plasmodium falciparum drug resistance polymorphisms in patients presenting with resistant malaria. One hundred ninety-nine P. falciparum-positive patients were recruited at two sites, Madang and Maprik. Exposure to the 4-aminoquinolines chloroquine and amodiaquine was uniformly high, at 84% overall. However, 59% of these were taken in various combinations of sulfadoxine/pyrimethamine and/or primaquine and/or quinine. Two markers for 4-aminoquinoline resistance, P. falciparum chloroquine resistance transporter 76T and P. falciparum multidrug resistance 1, were fixed in the population and two markers for pyrimethamine resistance, dihydrofolate reductase (dhps) 59R and 108N, were found at moderate to high levels, overall 60% and 75%, respectively. No polymorphisms in dhps associated with sulfadoxine resistance were present. Differences between the two sites are analyzed. The study period encompasses a change in standard malaria treatment policy. These findings stress the need for regular monitoring of the effects of standard drug treatment of uncomplicated malaria in Papua New Guinea.     

High frequency of antibody response to Plasmodium falciparum gametocyte antigens during acute malaria infections in Papua New Guinea highlanders.

Sera from 62 adult Papua New Guinea highlanders with suspected acute malaria were tested by competitive ELISA for the presence of antibodies capable of inhibiting binding of 8 monoclonal antibodies (Mabs) directed against epitopes on gametocytes of Plasmodium falciparum. Between 33% and 72% of the malaria cases were inhibitory, depending on the Mab. There was no difference between the proportion of persons with P. falciparum (asexuals or gametocytes) and P. vivax whose sera inhibited Mab binding, but all 3 categories had a significantly higher proportion of inhibitors than persons who were malaria negative. The amount of gametocyte antibody recognizing epitopes on Pfs 48/45 and Pfs 230 increased with increasing numbers of previous malaria episodes. The proportion of sera from these relatively nonimmune adults which had gamete antibodies was similar to the proportion seen in sera from a highly endemic area, suggesting that antibody responses to these epitopes are a part of the initial response observed after a limited number of malaria episodes.     

Genetic variations of the Plasmodium vivax dihydropteroate synthase gene

Dihydropteroate synthase gene of Plasmodium vivax was recently identified. In the present study, the sequences of the dyhydropteroate synthase gene of 68 P. vivax isolates from various geographic areas were compared. Sequencing revealed limited polymorphism at codons 383 and 553 in all analyzed samples. Interstrain analysis showed several genotypic variations in the tandem repeats domain which produce length polymorphism between different parasite isolates.     

In vitro sensitivity of Plasmodium falciparum to conventional and novel antimalarial drugs in Papua New Guinea

Objective Recent clinical studies have shown high rates of malaria treatment failure in endemic areas of Papua New Guinea (PNG), necessitating a change of treatment from chloroquine (CQ) or amodiaquine (AQ) plus sulphadoxine‐pyrimethamine to the artemisinin combination therapy (ACT) artemether plus lumefantrine (LM). To facilitate the monitoring of antimalarial drug resistance in this setting, we assessed the in vitro sensitivity of Plasmodium falciparum isolates from Madang Province. Methods A validated colorimetric lactate dehydrogenase assay was used to assess growth inhibition of 64 P. falciparum isolates in the presence of nine conventional or novel antimalarial drugs [CQ, AQ, monodesethyl‐amodiaquine (DAQ), piperaquine (PQ), naphthoquine (NQ), mefloquine (MQ), LM, dihydroartemisinin and azithromycin (AZ)]. Results The geometric mean (95% confidence interval) concentration required to inhibit parasite growth by 50% (IC₅₀) was 167 (141–197) nm for CQ, and 82% of strains were resistant (threshold 100 nm ), consistent with near‐fixation of the CQ resistance‐associated pfcrt allele in PNG. Except for AZ [8.351 (5.418–12.871) nm ], the geometric mean IC₅₀ for the other drugs was <20 nm . There were strong associations between the IC₅₀s of 4‐aminoquinoline (CQ, AQ, DAQ and NQ), bisquinoline (PQ) and aryl aminoalcohol (MQ) compounds suggesting cross‐resistance, but LM IC₅₀ only correlated with that of MQ. Conclusions Most PNG isolates are resistant to CQ in vitro but not to other ACT partner drugs. The non‐isotopic semi‐automated high‐throughput nature of the Plasmodium lactate dehydrogenase assay facilitates the convenient serial assessment of local parasite sensitivity, so that emerging resistance can be identified with relative confidence at an early stage.     

Epidemic malaria in the highlands of Papua New Guinea

As part of a larger study into the epidemiology of malaria in the highlands of Papua New Guinea, outbreak investigations were carried out at the end of the 2002 rainy season in 11 villages situated between 1,400 and 1,700 meters above sea level that had reported epidemics. Locations and timing of these epidemics corresponded largely to those reported in the pre-control era of the 1960s and 1970s. On average, 28.8% (range = 10.3-63.2%) of people in each of the 11 villages were found to be infected with malaria. Plasmodium falciparum accounted for 59% of all identified infections and P. vivax for 34%. The majority (53%) of infections were symptomatic. Although symptomatic infections were most common in children 2-9 years of age (36%), even in adults a prevalence of 20% was observed. A comparison with earlier non-epidemic data in three of the villages without easy access to health care showed markedly increased levels of morbidity, with 6-10-fold increases in parasite prevalence, a 3-fold increase in both measured and reported fevers, and a 12-fold increase in enlarged spleens. The average hemoglobin levels were reduced by 2.3-3.5 g/dL, with a concurrent increase in moderate to severe anemia (hemoglobin level < 7.5 g/dL) from 0.0-3.3% to 3.8-18.4%. These massive increases in morbidity have devastating impact on the affected communities and highlight that malaria epidemics are a serious and increasing public health problem in the highlands of Papua New Guinea.     

Arthritis in the highlands of Papua New Guinea.

Acute polyarthritis is an important cause of morbidity in many tropical countries. Classification has often been difficult, with the term tropical polyarthritis used for those in whom a diagnosis could not be made. The implication that this is a distinct entity is probably incorrect, with likely causes being septic arthritis or post-infective reactive arthritis. This study aimed to determine the types of arthritis found in 43 patients (30 men) presenting consecutively to the Goroka Base Hospital in the Eastern Highlands of Papua New Guinea. Gonococcal arthritis was diagnosed in eight patients (six men) on the basis of isolation of Neisseria gonorrhoeae from the joint aspirate. In all cases the N gonorrhoeae was identified by the closed culture system on chocolate agar, but not always by routine plating. There were no specific clinical features that identified patients with a gonococcal septic arthritis. The remaining 34 patients had an undifferentiated oligoarthritis. The pattern of arthritis in men and women was of a lower limb pauciarticular arthritis with a predilection for the knee and ankle joints. A total of 30% of male patients had a history of urethral discharge and 44% of all patients had preceding diarrhoea. Arthritis was the only feature in 59% of patients and in 32% there was an associated enthesitis. In this study most patients had an oligoarthritis consistent with a reactive arthritis or a septic arthritis due to N gonorrhoeae. Broth inoculation of synovial fluid was the best method to isolate N gonorrhoeae, with standard methods for gonococcal isolation failing in some patients. It is recommended that the term 'tropical polyarthritis' is no longer used as it does not refer to a specific entity but consists of several known arthritides.