Strengthening capacity, collaboration and quality of clinical research in Africa: EDCTP Networks of Excellence.

Developing countries bear 90% of the global disease burden, but only access about 10% of globally available health research funding. Weak south-south networking hampers effective use of limited resources, production of critical mass of quality scientists, career opportunities and incentives to retain the few available scientists. The south must urgently act strategically to accelerate generation of talented scientists, create enabling environment and incentives to retain scientists and attract back those in diaspora. The creation of strong networks of excellence for clinical research among southern academic and research institutions is a novel strategic approach championed by European and Developing Countries Clinical Trials Partnership to achieve the aforementioned goals and mitigate the high disease burden. It will promote strong collaboration, resource sharing and cross-mentorship allowing each partner to grow with complementary capacities that support each other rather than compete negatively. It will enable the south and Africa in particular to participate actively and own the means for solving its own health problems and raise the professional quality and capacity of southern institutions to forge better and equal partnership with northern institutions.

A continuing high incidence of subacute sclerosing panencephalitis (SSPE) in the Eastern Highlands of Papua New Guinea.

The aims of this descriptive study were to confirm the high incidence of subacute sclerosing panencephalitis (SSPE) previously reported from Papua New Guinea (PNG) and to relate SSPE to previous measles vaccination and measles illness. From February 1997 to April 1999 we diagnosed a total of 55 patients with SSPE at Goroka Base General Hospital in Eastern Highlands Province (EHP) of PNG. The diagnosis was based on high cerebrospinal fluid and serum measles virus antibody titres with progressive neurological disorder and myoclonic jerks. Of these 55 patients 42 were from EHP, including 32 whose onset was in the 2-year period 1997-1998. The annual incidence of SSPE in EHP in these 2 years was 98 per million population under 20 years of age, the highest ever reported. This incidence was more than ten times higher than the highest incidence in the prevaccine era reported from elsewhere. The mean age of onset of SSPE was 7.7 years (range 2.8-14.8 years) and the interval between measles and the onset of SSPE, where known, had a mean of 5.9 years and a range of 2.5-11.1 years. Among the SSPE patients 19 had a documented history of measles vaccination. Eight of these 19 also had documentation of previous measles illness; of these, seven were vaccinated after the development of measles and one was vaccinated 20 days before measles illness. Two non-SSPE children received vaccination twice which was documented and subsequently developed measles which was also substantiated by documentation. Two patients with SSPE yielded amplified nucleotide sequences of measles virus that were different from any of the vaccine strains. We found no evidence to implicate measles vaccination in the development of SSPE.

The association of the glycophorin C exon 3 deletion with ovalocytosis and malaria susceptibility in the Wosera, Papua New Guinea.

Erythrocyte polymorphisms, including ovalocytosis, have been associated with protection against malaria. This study in the Wosera, a malaria holoendemic region of Papua New Guinea, examined the genetic basis of ovalocytosis and its influence on susceptibility to malaria infection. Whereas previous studies showed significant associations between Southeast Asian ovalocytosis (caused by a 27- base pair deletion in the anion exchanger 1 protein gene) and protection from cerebral malaria, this mutation was observed in only 1 of 1019 individuals in the Wosera. Polymerase chain reaction strategies were developed to genotype individuals for the glycophorin C exon 3 deletion associated with Melanesian Gerbich negativity (GPCDeltaex3). This polymorphism was commonly observed in the study population (GPCDeltaex3 frequency = 0.465, n = 742). Although GPCDeltaex3 was significantly associated with increased ovalocytosis, it was not associated with differences in either Plasmodium falciparum or P vivax infection measured over the 7-month study period. Future case-control studies will determine if GPCDeltaex3 reduces susceptibility to malaria morbidity.

New JC virus (JCV) genotypes from papua new guinea and micronesia (type 8 and type 2E) and evolutionary analysis of 32 complete JCV genomes.

The JC virus (JCV) is a ubiquitous human polyomavirus that frequently resides in the kidneys of healthy individuals and is excreted in the urine of a large percentage of the population. Geographic-specific JCV variants, isolated from urine and from brain of progressive multifocal leukoencephalopathy (PML) patients, have been grouped into seven distinct genotypes based on whole genome analysis and by individual polymorphic nucleotides (typing sites) in the VP1 coding region. Mutations in the archetypal regulatory region, sometimes consisting of deletions and/or duplications, are also useful taxonomic characters for further characterizing and subdividing genotypes. Investigation of JCV variation in Papua New Guinea (PNG) revealed three distinct variants called PNG- 1, PNG-2, and PNG-3. These variants exhibited consistent coding region and regulatory region mutations. Evolutionary analysis of 32 complete JCV genomes including six new viral genomes from the western Pacific suggests that the new PNG JCV variants are closely associated with the broad group of Type 2 strains of JCV found throughout Asia, forming a monophyletic group with the Northeast Asian strains (Type 2A). Within the Type 2 clade, however, the PNG JCV variants cluster as two distinct groups and are therefore described here as new JCV genotypes designated Type 2E and Type 8.

Human polyomavirus JC variants in Papua New Guinea and Guam reflect ancient population settlement and viral evolution.

The peopling of the Pacific was a complex sequence of events that is best reconstructed by reconciling insights from various disciplines. Here we analyze the human polyomavirus JC (JCV) in Highlanders of Papua New Guinea (PNG), in Austronesian-speaking Tolai people on the island of New Britain, and in nearby non-Austronesian-speaking Baining people. We also characterize JCV from the Chamorro of Guam, a Micronesian population. All JCV strains from PNG and Guam fall within the broad Asian group previously defined in the VP1 gene as Type 2 or Type 7, but the PNG strains were distinct from both genotypes. Among the Chamorro JCV samples, 8 strains (Guam-1) were like the Type 7 strains found in Southeast Asia, while nine strains (Guam-2) were distinct from both the mainland strains and most PNG strains. We identified three JCV variants within Papua New Guinea (PNG-1, PNG-2 and PNG-3), but none of the Southeast Asian (Type 7) strains. PNG-1 strains were present in all three populations (Highlanders and the Baining and Tolai of New Britain), but PNG-2 strains were restricted to the Highlanders. Their relative lack of DNA sequence variation suggests that they arose comparatively recently. The single PNG-3 strain, identified in an Austronesian-speaking Tolai individual, was closely related to the Chamorro variants (Guam-2), consistent with a common Austronesian ancestor. In PNG-2 variants a complex regulatory region mutation inserts a duplication into a nearby deletion, a change reminiscent of those seen in the brains of progressive multifocal leukoencephalopathy patients. This is the first instance of a complex JCV rearrangement circulating in a human population.

Control measures and the outcome of the measles epidemic of 1999 in the Eastern Highlands Province.

In the Eastern Highlands Province (EHP) of Papua New Guinea (PNG) measles outbreaks have occurred regularly every 3 to 4 years since 1980. The latest was between September 1998 and March 2000. Between July 1999 and March 2000 314 children with measles were reviewed at Goroka Base Hospital. The majority of these children were very young: 55% were under 1 year and 27% under 6 months. The median age of the measles cases was 11 months (range 10 days to 13 years). 40% of the children had a verifiable history of having received at least one dose of measles vaccine. The majority were vaccinated during the epidemic and included many children who either were below 6 months of age or who developed measles within 2 weeks of vaccination. Measles complications occurred in 82% of the children, the most common being pneumonia. Serious complications, particularly severe pneumonia, were more common among the unvaccinated children than in those who had received at least a single dose of the measles vaccine. No deaths occurred among 82 children who had received measles vaccine more than 2 weeks before the onset of clinical measles, compared with 10 deaths in 206 children who had never been vaccinated against measles or were vaccinated in the 2 weeks before presentation (p=0.067). The overall case fatality was 4%: 14% among the hospital-acquired and 2.5% in community-acquired measles. Improvement in the measles vaccination coverage and supplementary vaccination campaigns are required to prevent measles outbreaks in PNG. Intensified measles vaccination campaigns, such as the one conducted in EHP in 1999, are recommended during epidemics to minimize deaths due to measles and to rapidly control outbreaks. The efficacy of measles vaccination can only be measured in total mortality, not in the prevention of clinical measles.

Emergence of FY*A(null) in a Plasmodium vivax-endemic region of Papua New Guinea.

In Papua New Guinea (PNG), numerous blood group polymorphisms and hemoglobinopathies characterize the human population. Human genetic polymorphisms of this nature are common in malarious regions, and all four human malaria parasites are holoendemic below 1500 meters in PNG. At this elevation, a prominent condition characterizing Melanesians is alpha(+)-thalassemia. Interestingly, recent epidemiological surveys have demonstrated that alpha(+)-thalassemia is associated with increased susceptibility to uncomplicated malaria among young children. It is further proposed that alpha(+)-thalassemia may facilitate so-called "benign" Plasmodium vivax infection to act later in life as a "natural vaccine" against severe Plasmodium falciparum malaria. Here, in a P. vivax-endemic region of PNG where the resident Abelam-speaking population is characterized by a frequency of alpha(+)-thalassemia >/=0.98, we have discovered the mutation responsible for erythrocyte Duffy antigen-negativity (Fy[a-b-]) on the FY*A allele. In this study population there were 23 heterozygous and no homozygous individuals bearing this new allele (allele frequency, 23/1062 = 0.022). Flow cytometric analysis illustrated a 2-fold difference in erythroid-specific Fy-antigen expression between heterozygous (FY*A/FY*A(null)) and homozygous (FY*A/FY*A) individuals, suggesting a gene-dosage effect. In further comparisons, we observed a higher prevalence of P. vivax infection in FY*A/FY*A (83/508 = 0.163) compared with FY*A/FY*A(null) (2/23 = 0.087) individuals (odds ratio = 2.05, 95% confidence interval = 0.47-8.91). Emergence of FY*A(null) in this population suggests that P. vivax is involved in selection of this erythroid polymorphism. This mutation would ultimately compromise alpha(+)-thalassemia/P. vivax-mediated protection against severe P. falciparum malaria.

Prevention of cerebral malaria in children in Papua New Guinea by southeast Asian ovalocytosis band 3.

Southeast Asian ovalocytosis (SAO) occurs at high frequency in malarious regions of the western Pacific and may afford a survival advantage against malaria. It is caused by a deletion of the erythrocyte membrane band 3 gene and the band 3 protein mediates the cytoadherence of parasitized erythrocytes in vitro. The SAO band 3 variant may prevent cerebral malaria but it exacerbates malaria anemia and may also increase acidosis, a major determinant of mortality in malaria. We undertook a case-control study of children admitted to hospital in a malarious region of Papua New Guinea. The SAO band 3, detected by the polymerase chain reaction, was present in 0 of 68 children with cerebral malaria compared with six (8.8%) of 68 matched community controls (odds ratio = 0, 95% confidence interval = 0-0.85). Median hemoglobin levels were 1.2 g/dl lower in malaria cases with SAO than in controls (P = 0.035) but acidosis was not affected. The remarkable protection that SAO band 3 affords against cerebral malaria may offer a valuable approach to a better understanding of the mechanisms of adherence of parasitized erythrocytes to vascular endothelium, and thus of the pathogenesis of cerebral malaria.

HLA-DQA1 genotyping by polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) and restriction endonuclease digestion in Papua New Guinea.

We have used PCR-SSCP, a technique based on the conformation of single-stranded DNA, to characterize the HLA-DQA1 gene in four geographically diverse population groups in Papua New Guinea. Among the 294 individuals that were studied from Goroka, north coast of Madang, Kimbe and Wanigela, we detected 5 of the 20 known variants of this gene locus. These included alleles 0101, 0102, 0103, 0301 and 0501. Furthermore, variable mobility shifts observed for alleles 0301 and 0501 from Madang suggested a further 3 variants. All 15 combinations of the 5 confirmed alleles were detected and their respective gene frequencies found to be consistent with the groups' ethnic and linguistic diversity. In respect to their frequencies and the observed overall allelic heterozygosity, the distribution in Kimbe showed some similarity to that in the north coast of Madang while Madang and Goroka were the most different. The distribution of alleles 0102 and 0501 was observed to be similar for Goroka and Wanigela as was 0301 for Madang and Wanigela. Our results, confirmed by endonuclease digestion, show PCR-SSCP to be a highly sensitive technique that can be used to characterize HLA-DQ antigens. In addition, the simplicity of the method provides an opportunity for large-scale typing of HLA antigens.

A novel JC virus variant found in the Highlands of Papua New Guinea has a 21-base pair deletion in the agnoprotein gene.

OBJECTIVES: This paper describes a unique JC virus (JCV) variant recovered from the Highlands of Papua New Guinea that contains an inframe 21-bp deletion in the agnoprotein gene. We characterize the mutation and suggest possible roles for the deletion in JCV evolution. STUDY DESIGN/METHODS: JCV DNA was extracted from urine and polymerase chain reaction (PCR) amplified using whole genome primers. PCR products were cloned, and multiple clones were sequenced. The JCV agnogene was PCR amplified to verify the presence of the agnogene deletion. RESULTS: This mutation creates a 21-bp deletion near the 3' end, which alters the predicted secondary structure of the messenger RNA and changes local codon usage at the 3' end of the agnogene. Protein secondary structure predictions suggest the deleted portion of the agnoprotein may be a flexible surface feature. CONCLUSIONS: We describe the first stable coding region deletion in JCV that presumably signifies a single evolutionary event that led to the split from other Highlands viral groups and occurred well after the human expansions that led to the peopling of the Southwest Pacific.

The correlation between microscopical examination and erythrocyte band 3 (AE1) gene deletion in South-east Asian ovalocytosis.

South-east Asian ovalocytosis status was determined by microscopical examination of peripheral blood samples collected from 137 individuals in Papua New Guinea. The examination was performed separately by 2 microscopists, one of whom was very experienced in examining peripheral blood films for the diagnosis of south-east Asian ovalycytosis and the other was recently trained. The samples were also analysed by polymerase chain reaction (PCR) to determine ovalocytosis status by demonstrating a 27 base pair deletion in erythrocyte band 3 protein of the affected individuals. The microscopists were unaware of each other's results and of those obtained by PCR. Generally, there was very good agreement between the results obtained by both microscopists and the PCR. Although there was considerable inter-observer variation in the final ovalocyte count between the 2 microscopists, this did not affect their ability to discriminate between ovalocytic and normocytic individuals. Taking the PCR results as the standard, for the first, more experienced observer, the most efficient ovalocyte count cut-off point was around 50%. At this ovalocyte count the sensitivity and specificity of microscopical examination were 93.6% and 92.2%, and the positive and negative predictive values 86.3% and 96.5%, respectively. The second microscopist generally underscored the ovalocyte counts and his most efficient cut-off point was 20%, with sensitivity and specificity of 85.1% and 93.3% and positive and negative predictive values of 87.0% and 92.3%, respectively.

Screening for sexually transmitted diseases in rural women in Papua New Guinea: are WHO therapeutic algorithms appropriate for case detection?

The presence of a large reservoir of untreated sexually transmitted diseases (STDs) in developing countries has prompted a number of suggestions for improving case detection, including the use of clinical algorithms and risk assessments to identify women likely to be infected when they present to clinics for other reasons. We used data from a community-based study of STDs to develop and evaluate algorithms for detection of cervical infection with Chlamydia trachomatis or Neisseria gonorrhoeae, and for detection of vaginal infection with Trichomonas vaginalis or bacterial vaginosis. The algorithms were derived using data from 192 randomly selected women, then evaluated on 200 self-selected women. We evaluated the WHO algorithm for vaginal discharge in both groups. The prevalences of cervical and vaginal infection in the randomly selected group were 27% and 50%, respectively, and 23% and 52%, respectively, in the self-selected group. The derived algorithms had high sensitivities in both groups, but poor specificities in the self-selected women, and the positive predictive values were unacceptably low. The WHO algorithms had extremely low sensitivity for detecting either vaginal or cervical infection because relatively few women reported vaginal discharge. Simple algorithms and risk assessments are not valid for case detection in this population.

Red cell morphology and malaria anaemia in children with Southeast-Asian ovalocytosis band 3 in Papua New Guinea.

Southeast-Asian ovalocytosis (SAO) was diagnosed in children from Madang, Papua New Guinea, by detection of the SAO band 3 gene variant using the polymerase chain reaction. SAO band 3 was present in 16/241 (6.6%) children living in the community and 32/389 (8.2%) children with acute Plasmodium falciparum malaria (P=0.42). SAO band 3 was detected in 8.2% (23/281) of alpha+-thalassaemia homozygotes, 9.4% (20/214) of heterozygotes and 2.4% (2/85) of children with a normal alpha-globin genotype (P=0.12). The most consistent feature of SAO band 3 on microscopy of thin blood films was red cells with two or more linear or irregularly-shaped pale regions. In children living in the community, these were present in 15 with SAO band 3 (sensitivity 93.8%) and only two normals (specificity 99.1%). The presence of > or = 20% ovalocytosis was a poorer indicator of SAO band 3 (sensitivity 68.8% and specificity 100%). Haematological data were similar in SAO band 3 and normal children. However, in children with acute malaria, haemoglobin levels and red cell counts were significantly lower in SAO band 3 than normal children. The degree of ovalocytosis was lower in children with SAO band 3 during acute malaria, suggesting that a selective loss of ovalocytes may contribute to malaria anaemia in Southeast-Asian ovalocytosis.

Community based study of sexually transmitted diseases in rural women in the highlands of Papua New Guinea: prevalence and risk factors.

OBJECTIVE: To estimate the prevalence of sexually transmitted diseases (STDs) and determine their risk factors/markers among a rural population of women in the highlands of Papua New Guinea. METHODS: Community based random cluster sample of women of reproductive age were interviewed and examined and had specimens collected for laboratory confirmation of chlamydial and trichomonal infection, gonorrhoea, syphilis, and bacterial vaginosis. RESULTS: Chlamydia trachomatis was detected in 26%, Trichomonas vaginalis in 46%, Neisseria gonorrhoeae in 1%, syphilis in 4%, pelvic inflammatory disease (PID) (diagnosed clinically) in 14%, and bacterial vaginosis in 9% of 201 women. 59% of the women had at least one STD. In a multivariate logistic regression analysis taking the clustered sampling into account, independent risk factors for chlamydial infection were age < or = 25 years, < four living children, visualization of yellow mucopurulent endocervical secretions on a white swab, and bacterial vaginosis. Being married to a man who did not have other wives was protective. For trichomonal infection, independent risk factors were having no formal education, infertility, more than one sexual partner in the previous 12 months, treatment for genital complaints in the previous 3 months, abnormal vaginal discharge detected on examination, and chlamydial infection. Similar levels of trichomonal infection were found in all age groups. Among married women, rates of infection correlated with their perception of their husband having had other sexual partners in the previous 3 months, and this relationship was significant for chlamydial infection among women over 25. CONCLUSION: STDs are a major problem in this population, with the risk factors varying by outcome. Current treatment regimens are inappropriate given the high prevalence of trichomonal infection, and the available services are inadequate. Effective interventions are required urgently to reduce this burden and to prevent the rapid transmission of HIV.

Sequence diversity and molecular evolution of the merozoite surface antigen 2 of Plasmodium falciparum.

Eleven new alleles of the Plasmodium falciparum merozoite surface antigen 2 (MSA2) from Papua New Guinea were analyzed by direct sequencing of polymerase chain reaction (PCR) products. We have used the sequence information to trace the molecular evolution of MSA2. The repeats of ten alleles belonging to the 3D7 allelic family differed considerably in size, nucleotide sequence, and repeat copy number. In the repeat region of these new alleles, codon usage was extremely biased with an exclusive use of NNT codons. Another new allele sequenced belonged to the FC27 family and confirmed the family-specific conserved structure of 96 and 36 bp repeats. In order to assess sequence microheterogeneity within samples defined as the same genotype by restriction fragment length polymorphism (RFLP), we have analyzed single-strand conformation polymorphism (SSCP) of different samples of the most frequent allele (D10 of the FC27 family) in the study population. No sequence heterogeneity could be detected within the repeat region. Based on analysis of the repeat regions in both allelic families, we discuss the hypothesis of a different evolutionary strategy being represented by each of the allelic families. Kew words: Merozoite surface antigen 2 - Nucleotide sequence comparisons - Molecular evolution

Occurrence of the erythrocyte band 3 (AE1) gene deletion in relation to malaria endemicity in Papua New Guinea.

South-east Asian ovalocytosis status was determined in 1629 individuals originating from 12 different geographical areas of Papua New Guinea, representing different ethnic groups and degrees of malaria endemicity. This was achieved by using polymerase chain reaction amplification to demonstrate a 27 base pair deletion in the erythrocyte band 3 (AE1) gene. By using this method, the prevalence of erythrocyte band 3 gene deletion was determined to range from zero in both the lowland inland area of Wosera, East Sepik Province and the highland region of Goroka, Eastern Highlands Province to 35% on the north coast of Madang Province. In general, the prevalence correlated well with altitude, being highest on the coast where malaria transmission is high, intermediate in the lowlands, and lowest in the non-malarious highlands. However, Wosera, a lowland area in the Sepik River Plains, which is hyperendemic for malaria, was an exception in that no ovalocytosis was detected. These results largely confirm the prevalence rates that have been reported in the past using microscopy. In keeping with the autosomal dominant mode of inheritance, the male:female ratio was 1.02 and no homozygote was detected, indicating that homozygosity for the ovalocytosis band 3 gene deletion is lethal.

Detection of four mutations in six unrelated South African patients with acute intermittent porphyria.

We have screened the hydroxymethylbilane synthase cDNA from six South African patients with acute intermittent porphyria, using a combination of chemical cleavage mismatch analysis and direct sequencing of asymmetrically amplified PCR products. Four mutations were detected, a novel T insertion (771insT) and three missense mutations (R26H, R116W and R173Q). The 771insT mutation produces a stop codon, thirty-three codons downstream and a loss of approximately 20% of the protein is predicted. The R116W mutation, which was found to have a high prevalence in the Dutch population, was detected in three unrelated South African patients.