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Introduction: Elderly people with haemophilia (PwH) develop haemophilic arthropathy, pain, and reduced health‐related quality of life (HR‐QoL). The condition of elderly mild haemophilia patients have rarely been evaluated. This study aimed to compare joint status, pain, and HR‐QoL between elderly with mild, moderate/severe haemophilia and healthy elderlies. Methods: Knee/ankle abnormalities were assessed by ultrasound (HEAD‐US) and physical examination (HJHS 2.1). Pain severity and pain interference were investigated using the Brief Pain Inventory. Pressure pain thresholds (PPTs) were obtained at knees/ankles and forehead. Functional limitations were evaluated using the 2‐Minute‐Walking‐Test, Timed‐Up‐and‐Go and HAL. The EQ‐5D‐5L questionnaire evaluated HR‐QoL. Healthy controls (HCs) and elderly individuals with moderate/severe and mild haemophilia were compared using Kruskal–Wallis and Mann–Whitney U tests. Results: From the 46 elderly PwH approached, 40 individuals (≥60 years) with haemophilia A/B (17 moderate/severe; 23 mild) and 20 age‐matched HCs were recruited. Moderate/severe PwH displayed worse joint status, lower PPTs, and poorer HR‐QoL than mild PwH and HCs (p‐value =.010–<.001). HEAD‐US abnormalities were observed in 100% of knees and 94% of ankles in moderate/severe PwH, versus 50% of knees and 61% of ankles in mild PwH. Pain was reported by 80% and 57% of moderate/severe and mild PwH, respectively. Low PPTs, functional limitations, and poor HR‐QoL scores were likewise observed in some mild PwH, yet without significantly differing from HCs. Conclusion: This study highlights poor joint/functional status, pain, and HR‐QoL outcomes in elderly with moderate/severe haemophilia. A few mild haemophilia subjects presented joint abnormalities, pain, functional limitations, and poor HR‐QoL, without significantly differing from HCs. Highlights: Elderly individuals with mild haemophilia have not yet been extensively studied, whereas moderate/severe haemophilia individuals have proven to suffer from haemophilic arthropathy, pain, and poor health‐related quality of life (HR‐QoL).Using a case‐control design, joint status, pain, and HR‐QoL outcomes were examined in elderly haemophilia individuals and compared with those of healthy controls (HCs).Elderly moderate/severe haemophilia individuals exhibited worse joint status, increased joint pain sensitivity, and reduced HR‐QoL compared with both mild haemophilia subjects and HCs. A subset of mild haemophilia subjects exhibited poor joint status, pain, and HR‐QoL outcomes, without any differences noted when compared with HCs. [ABSTRACT FROM AUTHOR]

In people with haemophilia (PwH), joint pain is a major comorbidity that is often overlooked and under-treated. It is believed that, to ensure the most successful outcome, pain management should be tailored to the predominant pain phenotype (i.e., nociceptive, neuropathic and nociplastic). The 2021 clinical criteria and grading system for nociplastic pain, established by the International Association for the Study of Pain (IASP), emphasize the necessity of early-stage identification and predominant pain type classification. Consistent with findings in other chronic musculoskeletal pain conditions, studies suggest that a subgroup of PwH suffers from nociplastic pain, i.e., pain arising from altered nociception rather than structural damage, but this has not yet been explored in PwH. This study aimed to identify PwH with "unlikely", "possible" and "probable" nociplastic pain and investigate differences in anthropometric, demographic and clinical characteristics and psychological factors between subgroups of PwH and healthy individuals.: The IASP clinical criteria and grading system were used to classify pain types in adult men with moderate or severe haemophilia recruited from two Belgian haemophilia treatment centres. Statistical analyses were applied to study between-subgroup differences. Of 94 PwH, 80 PwH (85%) were classified with "unlikely" and 14 (15%) with "at least possible" nociplastic pain (including 5 PwH (5%) with "possible" and 9 PwH (10%) with "probable" nociplastic pain). PwH in both the "unlikely" and "at least possible" nociplastic pain groups showed significantly higher levels of unhelpful psychological factors compared to healthy individuals. Additionally, age may partially account for the observed differences in body height and psychological factors. Larger sample sizes may be needed to detect more subtle between-group differences. study confirmed the presence of nociplastic pain in haemophilia, categorising a notable subgroup as individuals who experience at least possible nociplastic pain. These exploratory insights may provide a starting point for future studies and the development of more effective and tailored pain management. [ABSTRACT FROM AUTHOR]

Abstract Background The entomological inoculation rate (EIR) is an important indicator in estimating malaria transmission and the impact of vector control. To assess the EIR, the enzyme-linked immunosorbent assay (ELISA) to detect the circumsporozoite protein (CSP) is increasingly used. However, several studies have reported false positive results in this ELISA. The false positive results could lead to an overestimation of the EIR. The aim of present study was to estimate the level of false positivity among different anopheline species in Cambodia and Vietnam and to check for the presence of other parasites that might interact with the anti-CSP monoclonal antibodies. Methods Mosquitoes collected in Cambodia and Vietnam were identified and tested for the presence of sporozoites in head and thorax by using CSP-ELISA. ELISA positive samples were confirmed by a Plasmodium specific PCR. False positive mosquitoes were checked by PCR for the presence of parasites belonging to the Haemosporidia, Trypanosomatidae, Piroplasmida, and Haemogregarines. The heat-stability and the presence of the cross-reacting antigen in the abdomen of the mosquitoes were also checked. Results Specimens (N = 16,160) of seven anopheline species were tested by CSP-ELISA for Plasmodium falciparum and Plasmodium vivax (Pv210 and Pv247). Two new vector species were identified for the region: Anopheles pampanai (P. vivax) and Anopheles barbirostris (Plasmodium malariae). In 88% (155/176) of the mosquitoes found positive with the P. falciparum CSP-ELISA, the presence of Plasmodium sporozoites could not be confirmed by PCR. This percentage was much lower (28% or 5/18) for P. vivax CSP-ELISAs. False positive CSP-ELISA results were associated with zoophilic mosquito species. None of the targeted parasites could be detected in these CSP-ELISA false positive mosquitoes. The ELISA reacting antigen of P. falciparum was heat-stable in CSP-ELISA true positive specimens, but not in the false positives. The heat-unstable cross-reacting antigen is mainly present in head and thorax and almost absent in the abdomens (4 out of 147) of the false positive specimens. Conclusion The CSP-ELISA can considerably overestimate the EIR, particularly for P. falciparum and for zoophilic species. The heat-unstable cross-reacting antigen in false positives remains unknown. Therefore it is highly recommended to confirm all positive CSP-ELISA results, either by re-analysing the heated ELISA lysate (100°C, 10 min), or by performing Plasmodium specific PCR followed if possible by sequencing of the amplicons for Plasmodium species determination.

A study was conducted to detect environmental pathogen, Mycobacterium ulcerans which is the causative agent of Buruli ulcer (BU) isolated from small mammals collected in areas in Benin. Isolated samples from mammals which did not show any M. ulcerans recommended more research on M. ulcerans in African (small) mammals.

A study is conducted to determine the nature and distribution of free-living amoebae in areas of Benin with low and high endemicities for Buruli ulcer (BU). The differences observed in socio-economic status between the areas and high and of low BU endemicity fail to explain the actual mechanism linking BU endemicity and the occurrence of free-living amoebae.

The prevalence of mycobacteria in rodents and insectivores are studied in Morogoro, Tanzania, by using direct smear examination (DSE) after acid-fast staining technique. The findings have shown that potentially pathogenic mycobacteria have posed a risk for other animals and humans, especially HIV/AIDS patients, having a weakened immune system.

Background: Malaria "hotspots" have been proposed as potential intervention units for targeted malaria elimination. Little is known about hotspot formation and stability in settings outside sub-Saharan Africa.Methods: Clustering of Plasmodium infections at the household and hotspot level was assessed over 2 years in 3 villages in eastern Cambodia. Social and spatial autocorrelation statistics were calculated to assess clustering of malaria risk, and logistic regression was used to assess the effect of living in a malaria hotspot compared to living in a malaria-positive household in the first year of the study on risk of malaria infection in the second year.Results: The crude prevalence of Plasmodium infection was 8.4% in 2016 and 3.6% in 2017. Living in a hotspot in 2016 did not predict Plasmodium risk at the individual or household level in 2017 overall, but living in a Plasmodium-positive household in 2016 strongly predicted living in a Plasmodium-positive household in 2017 (Risk Ratio, 5.00 [95% confidence interval, 2.09-11.96], P < .0001). There was no consistent evidence that malaria risk clustered in groups of socially connected individuals from different households.Conclusions: Malaria risk clustered more clearly in households than in hotspots over 2 years. Household-based strategies should be prioritized in malaria elimination programs in this region. [ABSTRACT FROM AUTHOR]

Aim: This study aims to reconstruct the evolutionary history of the Crocidura poensis species complex and to identify factors driving diversification within it. We tested whether: (a) there is a pattern of allopatric differentiation coincident with the location of hypothesized Pleistocene forest refugia, (b) sister taxa are separated by broad rivers, (c) sister taxa occupy adjacent but distinct habitat. Location: Sub‐Saharan African forests and adjacent savanna. Taxon: Shrews. Methods: Analyses were based on 247 specimens collected from across the distribution of the species complex. We reconstructed the phylogeny (Bayesian and maximum likelihood methods) and assessed historical biogeography of this taxonomic group using a combination of mitochondrial and nuclear markers. We mapped the genetic diversity and estimated the divergence times by a relaxed clock model. Informed by multilocus species delimitation methods, we discussed possible taxonomic implications. Results: This complex is composed of nine major genetic lineages (proposed species). The earliest split within this complex occurred after 2.0–2.4 Ma, which corresponds to a period of increased aridity and/or extreme environmental variability. Most other divergence events occurred after the Early‐Middle Pleistocene Transition (1.2–0.8 Ma). Divergent selection across ecological gradients could explain diversification within the West African lineage. In Central Africa, the observed phylogeographic pattern fits the Pleistocene refuge hypothesis and supports the existence of multiple small rather than a few large forest refugia during glacial maxima. Large rivers, like the Congo and Sanaga Rivers, are important barriers to gene flow for several lineages but probably were not the primary cause of differentiation. Main conclusions: Both geographic isolation in distinct forest refugia and divergent selection along ecological gradients could explain Pleistocene diversification within this complex. [ABSTRACT FROM AUTHOR]

Background: Malaria elimination needs a concentration of activities towards identification of residual transmission foci and intensification of efforts to eliminate the last few infections, located in so-called 'malaria hotspots'. Previous work on characterizing malaria transmission hotspots has mainly focused on falciparum malaria and especially on symptomatic cases, while the malaria reservoir is expected to be mainly concentrated in the asymptomatic human population when transmission is low. For Plasmodium vivax, there has been less effort in identifying transmission hotspots. The main aim of this study was to uncover micro-epidemiological mechanisms of clustering of malaria infections at a sub-village level, based on geographical or behavioural features. Methods: A cross-sectional survey was performed in three villages within the highest malaria endemic province of Cambodia. The survey took place in the dry season, when the malaria reservoir is expected to be low and residing in the asymptomatic part of the population. Village and field locations of households were georeferenced, blood samples were taken from as many residents as possible and a short questionnaire probing for individual risk factors was taken. Asymptomatic malaria carriers were detected by PCR, and geographical clustering analysis (SaTScan) as well as risk factor analysis were performed. Results: A total of 1540 out of 1792 (86%) individuals were sampled. Plasmodial DNA was detected in 129 individuals (8.4%). P. vivax was most prevalent (5.5%) followed by Plasmodium malariae (2.1%) and Plasmodium falciparum (1.6%). Mixed infection occurred in 12 individuals. In two out of three villages geographical clustering of high and low malaria infection risk was clearly present. Cluster location and risk factors associated with the infection differed between the parasite species. Age was an important risk factor for the combined Plasmodium infections, while watching television at evenings was associated with increased odds of P. vivax infections [OR (CI): 1.86 (0.95-3.64)] and bed net use was associated with reduced odds of P. falciparum infections [OR (CI): 0.25 (0.077-0.80)]. Conclusions: Clusters of malaria carriers were malaria species specific and often located remotely, outside village centres. As such, at micro-epidemiological level, malaria is not a single disease. Further unravelling the micro-epidemiology of malaria can enable programme managers to define the interventions likely to contribute to halt transmission in a particular hotspot location. [ABSTRACT FROM AUTHOR]

Background: While community distribution of topical repellents has been proposed as an additional malaria control intervention, the safety of this intervention at the population level remains poorly evaluated. We describe the safety of mass distribution of the picaridin repellent during a cluster-randomised trial in rural Cambodia in 2012–2013. Methods: The repellent was distributed among 57 intervention villages with around 25,000 inhabitants by a team of village distributors. Information on individual adverse events, reported by phone by the village distributors, was obtained through home visits. Information on perceived side effects, reported at the family level, was obtained during two-weekly bottle exchange. Adverse events were classified as adverse reactions (events likely linked to the repellent), cases of repellent abuse and events not related to the repellent use, and classified as per Common Terminology Criteria for Adverse Events. Findings: Of the 41 adverse events notified by phone by the village distributors, there were 22 adverse reactions, 11 cases of repellent abuse (6 accidental, 5 suicide attempts) and 8 non-related events. All adverse reactions were mild, occurred in the first few months of use, and mainly manifested as skin conditions. Of the 11 cases of abuse, 2 were moderate and 2 life-threatening. All cases with adverse reactions and repellent abuse recovered completely. 20% of families reported perceived side effects, mainly itching, headache, dizziness and bad smell, but few discontinued repellent use. Conclusions: Adverse reactions and abuse during mass use of picaridin were uncommon and generally mild, supporting the safety of the picaridin repellent for malaria control. [ABSTRACT FROM AUTHOR]

Background: Cambodia reduced malaria incidence by more than 75% between 2000 and 2015, a target of the Millennium Development Goal 6. The Cambodian Government aims to eliminate all forms of malaria by 2025. The country's malaria incidence is highly variable at provincial level, but less is known at village level. This study used passive case detection (PCD) data at village level in Ratanakiri Province from 2010 to 2014 to describe incidence trends and identify high-risk areas of malaria to be primarily targeted towards malaria elimination. Methods: In 2010, the Cambodian malaria programme created a Malaria Information System (MIS) to capture malaria information at village level through PCD by village malaria workers and health facilities. The MIS data of Ratanakiri Province 2010-2014 were used to calculate annual incidence rates by Plasmodium species at province and commune levels. For estimating the trend at provincial level only villages reporting each year were selected. The communal incidences and the number of cases per village were visualized on a map per Plasmodium species and per year. Analysis of spatial clustering of village malaria cases by Plasmodium species was performed by year. Results: Overall, malaria annual incidence rates per 1000 inhabitants decreased from 86 (2010) to 30 (2014). Falciparum incidence decreased (by 79% in 2014 compared to 2010; CI 95% 76-82%) more rapidly than vivax incidence (by 19% in 2014 compared to 2010; CI 95% 5-32%). There were ten to 16 significant spatial clusters each year. Big clusters tended to extend along the Cambodian-Vietnamese border and along the Sesan River. Three clusters appeared throughout all years (2010-2014): one with 21 villages appeared each year, the second shrunk progressively from 2012 to 2014 and the third was split into two smaller clusters in 2013 and 2014. Conclusion: The decline of malaria burden can be attributed to intensive malaria control activities implemented in the areas: distribution of a long-lasting insecticidal net per person and early diagnosis and prompt treatment. Dihydro-artemisinin piperaquine was the only first-line treatment for all malaria cases. No radical treatment with primaquine was provided for Plasmodium vivax cases, which could explain the slow decrease of P. vivax due to relapses. To achieve malaria elimination by 2025, priority should be given to the control of stable malaria clusters appearing over time. [ABSTRACT FROM AUTHOR]

Background: Serological markers for exposure to different Plasmodium species have recently been used in multiplex immunoassays based on the Luminex technology. However, interpretation of the assay results requires consideration of the half-life of specific antibodies against these markers. Therefore, the aim of the present study was to document the half-life of malaria specific serological makers, as well as assessing the sensitivity of these markers to pick up recent changes in malaria exposure. Methods: A recently developed multiplex immunoassay was used to measure the intensity of antibody (Ab) responses against 19 different Plasmodium specific antigens, covering different human malaria parasites and two vector saliva antigens. Therefore, 8439 blood samples from five cross-sectional surveys in Ratanakiri, Cambodia, were analysed. These involve a random selection from two selected surveys, and an additional set of blood samples of individuals that were randomly re-sampled three, four or five times. A generalized estimating equation model and linear regression models were fitted on log transformed antibody intensity data. Results: Results showed that most (17/21) Ab-responses are higher in PCR positive than PCR negative individuals. Furthermore, these antibody-responses follow the same upward trend within each age group. Estimation of the half-lives showed differences between serological markers that reflect short-(seasonal) and long-term (year round) transmission trends. Ab levels declined significantly together with a decrease of PCR prevalence in a group of malaria endemic villages. Conclusion: For Plasmodium falciparum, antibodies against LSA3.RE, GLURP and Pf.GLURP.R2 are most likely to be a reflexion of recent (range from 6 to 8 months) exposure in the Mekong Subregion. PvEBP is the only Plasmodium vivax Ag responding reasonably well, in spite of an estimated Ab half-life of more than 1 year. The use of Ab intensity data rather dichotomizing the continuous Ab-titre data (positive vs negative) will lead to an improved approach for serological surveillance. [ABSTRACT FROM AUTHOR]

Background: Malaria transmission is highly heterogeneous, especially in low endemic countries, such as Cambodia. This results in geographical clusters of residual transmission in the dry, low transmission season, which can fuel the transmission to wider areas or populations during the wet season. A better understanding of spatial clustering of malaria can lead to a more efficient, targeted strategy to reduce malaria transmission. This study aims to evaluate the potential of the use of serological markers to define spatial patterns in malaria exposure. Methods: Blood samples collected in a community-based randomized trial performed in 98 high endemic communities in Ratanakiri province, north-eastern Cambodia, were screened with a multiplex serological assay for five serological markers (three Plasmodium falciparum and two Plasmodium vivax). The antibody half-lives range from approximately six months until more than two years. Geographical heterogeneity in malaria transmission was examined using a spatial scan statistic on serology, PCR prevalence and malaria incidence rate data. Furthermore, to identify behavioural patterns or intrinsic factors associated with malaria exposure (antibody levels), risk factor analyses were performed by using multivariable random effect logistic regression models. The serological outcomes were then compared to PCR prevalence and malaria incidence data. Results: A total of 6502 samples from two surveys were screened in an area where the average parasite prevalence estimated by PCR among the selected villages is 3.4 %. High-risk malaria pockets were observed adjacent to the 'Tonle San River' and neighbouring Vietnam for all three sets of data (serology, PCR prevalence and malaria incidence rates). The main risk factors for all P. falciparum antigens and P. vivax MSP1.19 are age, ethnicity and staying overnight at the plot hut. Conclusion: It is possible to identify similar malaria pockets of higher malaria transmission together with the potential risk factors by using serology instead of PCR prevalence or malaria incidence data. In north-eastern Cambodia, the serological markers show that malaria transmission occurs mainly in adults staying overnight in plot huts in the field. Pf.GLURP.R2 showed a shrinking pocket of malaria transmission over time, and Pf.MSP1.19, CSP, PvAMA1 were also informative for current infection to a lesser extent. Therefore, serology could contribute in future research. However, further in-depth research in selecting the best combination of antigens is required. [ABSTRACT FROM AUTHOR]

In the present review, we aim to provide a general introduction to different facets of the arms race between pathogens and their hosts/environment, emphasizing its evolutionary aspects. We focus on vector-borne parasitic protozoa, which have to adapt to both invertebrate and vertebrate hosts. Using Leishmania, Trypanosoma and Plasmodium as main models, we review successively (i) the adaptations and counter-adaptations of parasites and their invertebrate host, (ii) the adaptations and counter-adaptations of parasites and their vertebrate host and (iii) the impact of human interventions (chemotherapy, vaccination, vector control and environmental changes) on these adaptations. We conclude by discussing the practical impact this knowledge can have on translational research and public health. [ABSTRACT FROM AUTHOR]

The burden of leptospirosis in humans and animals in Africa is higher than that reported from other parts of the world. However, the disease is not routinely diagnosed in the continent. One of major factors limiting diagnosis is the poor availability of live isolates of locally circulating Leptospira serovars for inclusion in the antigen panel of the gold standard microscopic agglutination test (MAT) for detecting antibodies against leptospirosis. To gain insight in Leptospira serovars and their natural hosts occurring in Tanzania, concomitantly enabling the improvement of the MAT by inclusion of fresh local isolates, a total of 52 Leptospira isolates were obtained from fresh urine and kidney homogenates, collected between 1996 and 2006 from small mammals, cattle and pigs. Isolates were identified by serogrouping, cross agglutination absorption test (CAAT), and molecular typing. Common Leptospira serovars with their respective animal hosts were: Sokoine (cattle and rodents); Kenya (rodents and shrews); Mwogolo (rodents); Lora (rodents); Qunjian (rodent); serogroup Grippotyphosa (cattle); and an unknown serogroup from pigs. Inclusion of local serovars particularly serovar Sokoine in MAT revealed a 10-fold increase in leptospirosis prevalence in Tanzania from 1.9% to 16.9%in rodents and 0.26% to 10.75% in humans. This indicates that local serovars are useful for diagnosis of human and animal leptospirosis in Tanzania and other African countries. [ABSTRACT FROM AUTHOR]

Background: The public health value of a vector control tool depends on its epidemiological efficacy, but also on its ease of implementation. This study describes an intensive distribution scheme of a topical repellent implemented in 2012 and 2013 for the purpose of a cluster-randomized trial using the existing public health system. The trial aimed to assess the effectiveness of repellents in addition to long-lasting insecticidal nets (LLIN) and occurred in a province of Cambodia. Determinants for accessibility and consumption of this tool were explored. Methods: 135 individuals were appointed to be repellent distributors in 57 villages. A 2-weekly bottle exchange programme was organized. Distributors recorded information regarding the amount of bottles exchanged, repellent leftover, and reasons for not complying in household data sheets. Distributor-household contact rates and average 2-weekly consumption of repellent were calculated. Household and distributors characteristics were obtained using questionnaires, surveying 50 households per cluster and all distributors. Regression models were used to explore associations between contact and consumption rates and determinants such as socio-economic status. Operational costs for repellent and net distribution were obtained from the MalaResT project and the provincial health department. Results: A fourfold increase in distributor-household contact rates was observed in 2013 compared to 2012 (median2012 = 20 %, median2013 = 88.9 %). Consumption rate tripled over the 2-year study period (median2012 = 20 %, median2013 = 57.89 %). Contact rates were found to associate with district, commune and knowing the distributor, while consumption was associated with district and household head occupation. The annual operational cost per capita for repellent distribution was 31 times more expensive than LLIN distribution (USD 4.33 versus USD 0.14). Discussion: After the existing public health system was reinforced with programmatic and logistic support, an intense 2-weekly distribution scheme of a vector control tool over a 2-year period was operated successfully in the field. Lack of associations with socio-economic status suggested that the free distribution strategy resulted in equitable access to repellents. The operational costs for the repellent distribution and exchange programme were much higher than LLIN distribution. Such effort could only be justified in the context of malaria elimination where these interventions are expected to be limited in time [ABSTRACT FROM AUTHOR]

Background: Epidemiological surveillance is a key activity in malaria control and elimination in low-transmission and pre-elimination settings. Hence, sensitive tools for estimating malaria force of infection are crucial. Serological markers might provide additional information in estimating force of infection in low-endemic areas along with classical parasite detection methods. Serological markers can be used to estimate recent, past or present malaria exposure, depending on the used markers and their half-life. Methods: An assay based on 14 Plasmodium-specific peptides, one peptide specific for Anopheles gambiae saliva protein and five Plasmodium-specific recombinant proteins was developed for the MAGPIX system, assessed for its performance, and applied on blood spots from 2000 individuals collected in the Ratanakiri Province, Cambodia. Results: A significant correlation for the use of 1000 and 2000 beads/antigen/well as well as for the monoplex versus multiplex assay was observed for all antigens (p < 0.05). For the majority of antigens, antigen-coupled beads were stable for at least 2 months. The assay was very reproducible with limited intercoupling, interplate and intraplate variability (mean RSD <15 %). Estimating seroconversion and seroreversion per antigen using reversible catalytic models and models allowing two seroconversion rates showed higher seroconversion rates in adults. Conclusion: The multiplex bead-based immunoassay was successfully implemented and analysis of field blood samples shows that changes detected in force of malaria infection vary according to the serological markers used. Multivariate analysis of the antibody responses and insights into the half-life of antibodies are crucial for improving the interpretation of these results and for identifying the most useful serological markers of past and recent malaria infection. [ABSTRACT FROM AUTHOR]

Background: In the Indian subcontinent, Visceral leishmaniasis is endemic in a geographical area coinciding with the Lower Gangetic Plain, at low altitude. VL occurring in residents of hill districts is therefore often considered the result of Leishmania donovani infection during travel. Early 2014 we conducted an outbreak investigation in Okhaldhunga and Bhojpur districts in the Nepal hills where increasing number of VL cases have been reported. Methodology/Principal Findings: A house-to-house survey in six villages documented retrospectively 35 cases of Visceral Leishmaniasis (VL). Anti-Leishmania antibodies were found in 22/23 past-VL cases, in 40/416 (9.6%) persons without VL and in 12/155 (7.7%) domestic animals. An age- and sex- matched case-control study showed that exposure to known VL-endemic regions was no risk factor for VL, but having a VL case in the neighbourhood was. SSU-rDNA PCR for Leishmania sp. was positive in 24 (5%) of the human, in 18 (12%) of the animal samples and in 16 (14%) bloodfed female Phlebotomus argentipes sand flies. L. donovani was confirmed in two asymptomatic individuals and in one sand fly through hsp70-based sequencing. Conclusions/Significance: This is epidemiological and entomological evidence for ongoing local transmission of L. donovani in villages at an altitude above 600 meters in Nepal, in districts considered hitherto non-endemic for VL. The VL Elimination Initiative in Nepal should therefore consider extending its surveillance and control activities in order to assure VL elimination, and the risk map for VL should be redesigned. [ABSTRACT FROM AUTHOR]

Background: In certain regions in Southeast Asia, where malaria is reduced to forested regions populated by ethnic minorities dependent on slash-and-burn agriculture, malaria vector populations have developed a propensity to feed early and outdoors, limiting the effectiveness of long-lasting insecticide-treated nets (LLIN) and indoor residual spraying (IRS). The interplay between heterogeneous human, as well as mosquito behaviour, radically challenges malaria control in such residual transmission contexts. This study examines human behavioural patterns in relation to the vector behaviour. Methods: The anthropological research used a sequential mixed-methods study design in which quantitative survey research methods were used to complement findings from qualitative ethnographic research. The qualitative research existed of in-depth interviews and participant observation. For the entomological research, indoor and outdoor human landing collections were performed. All research was conducted in selected villages in Ratanakiri province, Cambodia. Results: Variability in human behaviour resulted in variable exposure to outdoor and early biting vectors: (i) indigenous people were found to commute between farms in the forest, where malaria exposure is higher, and village homes; (ii) the indoor/outdoor biting distinction was less clear in forest housing often completely or partly open to the outside; (iii) reported sleeping times varied according to the context of economic activities, impacting on the proportion of infections that could be accounted for by early or nighttime biting; (iv) protection by LLINs may not be as high as self-reported survey data indicate, as observations showed around 40% (non-treated) market net use while (v) unprotected evening resting and deep forest activities impacted further on the suboptimal use of LLINs. Conclusions: The heterogeneity of human behaviour and the variation of vector densities and biting behaviours may lead to a considerable proportion of exposure occurring during times that people are assumed to be protected by the distributed LLINs. Additional efforts in improving LLIN use during times when people are resting in the evening and during the night might still have an impact on further reducing malaria transmission in Cambodia. [ABSTRACT FROM AUTHOR]

Efforts to control the spread of Buruli ulcer – an emerging ulcerative skin infection caused by Mycobacterium ulcerans - have been hampered by our poor understanding of reservoirs and transmission. To help address this issue, we compared whole genomes from 18 clinical M. ulcerans isolates from a 30km2 region within the Asante Akim North District, Ashanti region, Ghana, with 15 other M. ulcerans isolates from elsewhere in Ghana and the surrounding countries of Ivory Coast, Togo, Benin and Nigeria. Contrary to our expectations of finding minor DNA sequence variations among isolates representing a single M. ulcerans circulating genotype, we found instead two distinct genotypes. One genotype was closely related to isolates from neighbouring regions of Amansie West and Densu, consistent with the predicted local endemic clone, but the second genotype (separated by 138 single nucleotide polymorphisms [SNPs] from other Ghanaian strains) most closely matched M. ulcerans from Nigeria, suggesting another introduction of M. ulcerans to Ghana, perhaps from that country. Both the exotic genotype and the local Ghanaian genotype displayed highly restricted intra-strain genetic variation, with less than 50 SNP differences across a 5.2Mbp core genome within each genotype. Interestingly, there was no discernible spatial clustering of genotypes at the local village scale. Interviews revealed no obvious epidemiological links among BU patients who had been infected with identical M. ulcerans genotypes but lived in geographically separate villages. We conclude that M. ulcerans is spread widely across the region, with multiple genotypes present in any one area. These data give us new perspectives on the behaviour of possible reservoirs and subsequent transmission mechanisms of M. ulcerans. These observations also show for the first time that M. ulcerans can be mobilized, introduced to a new area and then spread within a population. Potential reservoirs of M. ulcerans thus might include humans, or perhaps M. ulcerans-infected animals such as livestock that move regularly between countries. Author Summary: In this study we use the power of whole genome sequence comparisons to track the spread of Mycobacterium ulcerans, the causative agent of Buruli ulcer, through several villages in the Ashanti region of Ghana, providing new insights on the behaviour of this enigmatic and emerging pathogen. [ABSTRACT FROM AUTHOR]

Scaling up of insecticide treated nets has contributed to a substantial malaria decline. However, some malaria vectors, and most arbovirus vectors, bite outdoors and in the early evening. Therefore, topically applied insect repellents may provide crucial additional protection against mosquito-borne pathogens. Among topical repellents, DEET is the most commonly used, followed by others such as picaridin. The protective efficacy of two formulated picaridin repellents against mosquito bites, including arbovirus and malaria vectors, was evaluated in a field study in Cambodia. Over a period of two years, human landing collections were performed on repellent treated persons, with rotation to account for the effect of collection place, time and individual collector. Based on a total of 4996 mosquitoes collected on negative control persons, the overall five hour protection rate was 97.4% [95%CI: 97.1–97.8%], not decreasing over time. Picaridin 20% performed equally well as DEET 20% and better than picaridin 10%. Repellents performed better against Mansonia and Culex spp. as compared to aedines and anophelines. A lower performance was observed against Aedes albopictus as compared to Aedes aegypti, and against Anopheles barbirostris as compared to several vector species. Parity rates were higher in vectors collected on repellent treated person as compared to control persons. As such, field evaluation shows that repellents can provide additional personal protection against early and outdoor biting malaria and arbovirus vectors, with excellent protection up to five hours after application. The heterogeneity in repellent sensitivity between mosquito genera and vector species could however impact the efficacy of repellents in public health programs. Considering its excellent performance and potential to protect against early and outdoor biting vectors, as well as its higher acceptability as compared to DEET, picaridin is an appropriate product to evaluate the epidemiological impact of large scale use of topical repellents on arthropod borne diseases. Author Summary: Malaria and arboviruses are transmitted by several mosquitoes. Targeting these mosquitoes instead of the pathogens can contribute to prevention of these diseases. For mosquitoes biting throughout the night, mosquito nets (preferably impregnated with insecticides) are very effective for mosquito control. However, bites of day-, evening- and outdoor-biting mosquitoes have to be prevented in different ways, for example by applying repellents on the skin which contain DEET or other active ingredients such as picaridin. Here we report on the evaluation of the performance of two formulated picaridin repellents (lotion 10% and spray 20%) against mosquito bites, including vectors of arboviruses and malaria in the field in Cambodia. These repellent formulations were compared to a DEET solution (20%). In general, all repellents performed very well, providing more than 97% protection against mosquito bites when used for five consecutive hours. At the highest concentration, the picaridin repellent performed similarly to DEET. However, different mosquito species reacted differently to the repellents. As such, repellents can provide an additional protection against bites of malaria and arbovirus vectors. [ABSTRACT FROM AUTHOR]

Background: The reservoir and mode of transmission of Mycobacterium ulcerans, the causative agent of Buruli ulcer, still remain a mystery. It has been suggested that M. ulcerans persists with difficulty as a free-living organism due to its natural fragility and inability to withstand exposure to direct sunlight, and thus probably persists within a protective host environment. Methodology/Principal Findings: We investigated the role of free-living amoebae as a reservoir of M. ulcerans by screening the bacterium in free-living amoebae (FLA) cultures isolated from environmental specimens using real-time PCR. We also followed the survival of M. ulcerans expressing green fluorescence protein (GFP) in Acanthameoba castellanii by flow cytometry and observed the infected cells using confocal and transmission electron microscopy for four weeks in vitro. IS2404 was detected by quantitative PCR in 4.64% of FLA cultures isolated from water, biofilms, detritus and aerosols. While we could not isolate M. ulcerans, 23 other species of mycobacteria were cultivated from inside FLA and/or other phagocytic microorganisms. Laboratory experiments with GFP-expressing M. ulcerans in A. castellani trophozoites for 28 days indicated the bacteria did not replicate inside amoebae, but they could remain viable at low levels in cysts. Transmission electron microscopy of infected A. castellani confirmed the presence of bacteria within both trophozoite vacuoles and cysts. There was no correlation of BU notification rate with detection of the IS2404 in FLA (r = 0.07, n = 539, p = 0.127). Conclusion/Significance: This study shows that FLA in the environment are positive for the M. ulcerans insertion sequence IS2404. However, the detection frequency and signal strength of IS2404 positive amoabae was low and no link with the occurrence of BU was observed. We conclude that FLA may host M. ulcerans at low levels in the environment without being directly involved in the transmission to humans. Author Summary: Mycobacterium ulcerans, the causative agent of Buruli ulcer (BU) is an environmental pathogen known to reside in aquatic habitat. However, the reservoir and modes of transmission to humans still remain unknown. M. ulcerans can probably not live freely due to its natural fragility and inability to withstand exposure to direct sunlight. This study investigated the hypothesis that free-living amoebae (FLA) can serve as a reservoir of M. ulcerans by testing for its presence in amoebae isolated from water bodies in BU endemic and non-endemic communities and whether the pathogen can remain viable when experimentally infected in amoebae in the laboratory. We detected only one (IS2404) of the three (IS2606 and KRB) targets for the presence of M. ulcerans in amoebae cultures and found no correlation between its presence in the environment and BU notification rate. M. ulcerans remained viable at low levels in amoebae for 28 days in vitro. We therefore conclude that FLA may host M. ulcerans at low levels in the environment without being directly involved in the transmission to humans. [ABSTRACT FROM AUTHOR]

Background: Malaria incidence worldwide has steadily declined over the past decades. Consequently, increasingly more countries will proceed from control to elimination. The malaria distribution in low incidence settings appears patchy, and local transmission hotspots are a continuous source of infection. In this study, species-specific clusters and associated risk factors were identified based on malaria prevalence data collected in the north-east of Cambodia. In addition, Plasmodium falciparum genetic diversity, population structure and gene flows were studied. Method: In 2012, blood samples from 5793 randomly selected individuals living in 117 villages were collected from Ratanakiri province, Cambodia. Malariometric data of each participant were simultaneously accumulated using a standard questionnaire. A two-step PCR allowed for species-specific detection of malaria parasites, and SNPgenotyping of P. falciparum was performed. SaTScan was used to determine species-specific areas of elevated risk to infection, and univariate and multivariate risk analyses were carried out. Result: PCR diagnosis found 368 positive individuals (6.4%) for malaria parasites, of which 22% contained mixed species infections. The occurrence of these co-infections was more frequent than expected. Specific areas with elevated risk of infection were detected for all Plasmodium species. The clusters for Falciparum, Vivax and Ovale malaria appeared in the north of the province along the main river, while the cluster for Malariae malaria was situated elsewhere. The relative risk to be a malaria parasite carrier within clusters along the river was twice that outside the area. The main risk factor associated with three out of four malaria species was overnight stay in the plot hut, a human behaviour associated with indigenous farming. Haplotypes did not show clear geographical population structure, but pairwise Fst value comparison indicated higher parasite flow along the river. Discussion: Spatial aggregation of malaria parasite carriers, and the identification of malaria species-specific risk factors provide key insights in malaria epidemiology in low transmission settings, which can guide targeted supplementary interventions. Consequently, future malaria programmes in the province should implement additional specific policies targeting households staying overnight at their farms outside the village, in addition to migrants and forest workers. [ABSTRACT FROM AUTHOR]

Background: The evaluation of new long-lasting insecticidal bed nets (LLINs) is coordinated by the WHO Pesticide Evaluation Scheme (WHOPES). In 2007, Netprotect® was granted WHOPES interim recommendation after Phase I and II evaluations. Present study evaluates Netprotect® in a Phase III trial in rural Cambodia. Methods: A randomized, prospective longitudinal study design was used to assess the performance of Netprotect® over a period of three years, using conventionally-treated nets and a WHOPES recommended LLIN (PermaNet® 2.0) as positive controls. The primary outcomes were the physical integrity, insecticide content and cone bioassay performance using. Results: The baseline deltamethrin concentration of 43% of Netprotect® nets were below the tolerance limit while 27% of PermaNet® 2.0 nets were above the target dose limits. By 36 months Netprotect® retained 35% while PermaNet® 2.0 retained 49% of baseline insecticide dose. Moreover the proportion of the inactive deltamethrin R-alpha isomer in the Netprotect® nets was 33% at the baseline and increased to 69% after three years while it was low and almost constant for PermaNet® 2.0 (3-7%). Only 71% of Netprotect® met the WHO criteria for bio-efficacy after three years while at least 80% is required. Moreover Netprotect® nets failed for the WHOPES criteria after 12 and 24 months. The reference LLIN met the WHOPES criteria throughout the study. Over the entire three years the reference LLIN did obtain significant higher mosquito mortality than Netprotect®. The physical integrity was based on the proportionate hole index and after three years, 25% of Netprotect® and 30% of PermaNet® 2.0 were in a mediocre or poor state. Conclusion: Netprotect® did not meet the minimum WHO criteria for bio-efficacy after 12, 24 and 36 months. The use of a reference LLIN as positive control was helpful for data interpretation. However, for future three-year studies, it is essential that before initiating any study nets should be checked for their specifications and this for both the candidate LLIN as well as for the reference LLIN. Moreover, to improve the accuracy of the success rate of the candidate LLIN more nets should be tested for their bio-efficacy at the end of the trial. [ABSTRACT FROM AUTHOR]

Background: Despite progress in malaria control, malaria remains an important public health concern in Cambodia, mostly linked to forested areas. Large-scale vector control interventions in Cambodia are based on the free distribution of long-lasting insecticidal nets (LLINs), targeting indoor- and late-biting malaria vectors only. The present study evaluated the vector density, early biting activity and malaria transmission of outdoor-biting malaria vectors in two forested regions in Cambodia. Methods: In 2005 two entomological surveys were conducted in 12 villages and their related forest plots in the east and west of Cambodia. Mosquitoes were collected outdoors by human landing collections and subjected to enzyme-linked immunosorbent assay (ELISA) to detect Plasmodium sporozoites after morphological identification. Blood samples were collected in the same villages for serological analyses. Collected data were analysed by the classification and regression tree (CART) method and linear regression analysis. Results: A total of 11,826 anophelines were recorded landing in 787 man-night collections. The majority (82.9%) were the known primary and secondary vectors. Most of the variability in vector densities and early biting rates was explained by geographical factors, mainly at village level. Vector densities were similar between forest and village sites. Based on ELISA results, 29% out of 17 Plasmodium-positive bites occurred before sleeping time, and 65% in the forest plots. The entomological inoculation rates of survey 1 were important predictors of the respective seroconversion rates in survey 2, whereas the mosquito densities were not. Discussion: In Cambodia, outdoor malaria transmission in villages and forest plots is important. In this context, deforestation might result in lower densities of the primary vectors, but also in higher densities of secondary vectors invading deforested areas. Moreover, higher accessibility of the forest could result in a higher manvector contact. Therefore, additional vector control measures should be developed to target outdoor- and earlybiting vectors. [ABSTRACT FROM AUTHOR]

Background: The reservoir and mode of transmission of Mycobacterium ulcerans, the causative agent of Buruli ulcer, remain unknown. Ecological, genetic and epidemiological information nonetheless suggests that M. ulcerans may reside in aquatic protozoa. Methodology/Principal Findings: We experimentally infected Acanthamoeba polyphaga with M. ulcerans and found that the bacilli were phagocytised, not digested and remained viable for the duration of the experiment. Furthermore, we collected 13 water, 90 biofilm and 45 detritus samples in both Buruli ulcer endemic and non-endemic communities in Ghana, from which we cultivated amoeboid protozoa and mycobacteria. M. ulcerans was not isolated, but other mycobacteria were as frequently isolated from intracellular as from extracellular sources, suggesting that they commonly infect amoebae in nature. We screened the samples as well as the amoeba cultures for the M. ulcerans markers IS2404, IS2606 and KR-B. IS2404 was detected in 2% of the environmental samples and in 4% of the amoeba cultures. The IS2404 positive amoeba cultures included up to 5 different protozoan species, and originated both from Buruli ulcer endemic and non-endemic communities. Conclusions/Significance: This is the first report of experimental infection of amoebae with M. ulcerans and of the detection of the marker IS2404 in amoeba cultures isolated from the environment. We conclude that amoeba are potential natural hosts for M. ulcerans, yet remain sceptical about their implication in the transmission of M. ulcerans to humans and their importance in the epidemiology of Buruli ulcer. Author Summary: Buruli ulcer (BU) is a devastating skin disease caused by Mycobacterium ulcerans, an environmental bacterium that is probably linked to slow-running water. It is unlikely to occur free-living, but even though M. ulcerans DNA has been detected in quite a few different organisms (with most studies focusing on insects), it is still not clear what its real reservoir is. Amoeboid protozoa, inhabitants of biofilms in slow flowing water, are good candidates since all previously tested mycobacteria are resistant to the digestion by these macrophage-like organisms. In this paper we demonstrate that M. ulcerans can indeed infect Acanthamoeba polyphaga in the lab, and remain viable intracellularly. We also collected water, biofilm and detritus samples in BU endemic and non-endemic regions in Ghana. We found that several mycobacteria species commonly occur intracellularly in protozoa in these environments. Amoebae were isolated from almost all samples, and an M. ulcerans marker (IS2404) was detected in 4% of the amoeba cultures. We conclude that amoebae are potential hosts for M. ulcerans. However, because these IS2404 positive amoebae originated from both BU endemic and non-endemic areas, we remain sceptical about their implication in the transmission of M. ulcerans to humans. [ABSTRACT FROM AUTHOR]

This study reports the first successful application of real-time PCR for the detection of Mycobacterium ulcerans, the causative agent of Buruli ulcer (BU), in Ghana, a BU-endemic country. Environmental samples and organs of small mammals were analyzed. The real-time PCR assays confirmed the presence of M. ulcerans in a water sample collected in a BU-endemic village in the Ashanti Region. [ABSTRACT FROM AUTHOR]

Abstract: Mycobacterium ulcerans is the causative agent of Buruli ulcer, the third most common mycobacterial disease in humans after tuberculosis and leprosy. Although the disease is associated with aquatic ecosystems, cultivation of the bacillus from the environment is difficult to achieve. Therefore, at the moment, research is based on the detection by PCR of the insertion sequence IS2404 present in M. ulcerans and some closely related mycobacteria. In the present study, we compared four DNA extraction methods for detection of M. ulcerans DNA, namely the one tube cell lysis and DNA extraction procedure (OT), the FastPrep procedure (FP), the modified Boom procedure (MB), and the Maxwell® 16 Procedure (M16). The methods were performed on serial dilutions of M. ulcerans, followed by PCR analysis with different PCR targets in M. ulcerans to determine the detection limit (DL) of each method. The purity of the extracted DNA and the time and effort needed were compared as well. All methods were performed on environmental specimens and the two best methods (MB and M16) were tested on clinical specimens for detection of M. ulcerans DNA. When comparing the DLs of the DNA extraction methods, the MB and M16 had a significantly lower DL than the OT and FP. For the different PCR targets, IS2404 showed a significantly lower DL than mlsA, MIRU1, MIRU5 and VNTR6. The FP and M16 were considerably faster than the MB and OT, while the purity of the DNA extracted with the MB was significantly higher than the DNA extracted with the other methods. The MB performed best on the environmental and clinical specimens. This comparative study shows that the modified Boom procedure, although lengthy, provides a better method of DNA extraction than the other methods tested for detection and identification of M. ulcerans in both clinical and environmental specimens. [Copyright &y& Elsevier]

Abstract: In recent years, numerous IS2404 positive mycobacteria have been identified, compromising the detection of Mycobacterium ulcerans. In this study, variable number of tandem repeats (VNTR) analysis was successfully applied on cultures and tissue specimens to differentiate all currently known IS2404 positive mycobacteria from M. ulcerans. [Copyright &y& Elsevier]

Human population movements currently challenge malaria elimination in low transmission foci in the Greater Mekong Subregion. Using a mixed-methods design, combining ethnography (n = 410 interviews), malariometric data (n = 4996) and population surveys (n = 824 indigenous populations; n = 704 Khmer migrants) malaria vulnerability among different types of mobile populations was researched in the remote province of Ratanakiri, Cambodia. Different structural types of human mobility were identified, showing differential risk and vulnerability. Among local indigenous populations, access to malaria testing and treatment through the VMW-system and LLIN coverage was high but control strategies failed to account for forest farmers' prolonged stays at forest farms/fields (61% during rainy season), increasing their exposure (p = 0.002). The Khmer migrants, with low acquired immunity, active on plantations and mines, represented a fundamentally different group not reached by LLIN-distribution campaigns since they were largely unregistered (79%) and unaware of the local VMW-system (95%) due to poor social integration. Khmer migrants therefore require control strategies including active detection, registration and immediate access to malaria prevention and control tools from which they are currently excluded. In conclusion, different types of mobility require different malaria elimination strategies. Targeting mobility without an in-depth understanding of malaria risk in each group challenges further progress towards elimination. [ABSTRACT FROM AUTHOR]

In Cambodia, despite an impressive decline in prevalence over the last 10 years, malaria is still a public health problem in some parts of the country. This is partly due to vectors that bite early and outdoors reducing the effectiveness of measures such as Long-Lasting Insecticidal Nets. Repellents have been suggested as an additional control measure in such settings. As part of a cluster-randomized trial on the effectiveness of topical repellents in controlling malaria infections at community level, a mixed-methods study assessed user rates and determinants of use. Repellents were made widely available and Picaridin repellent reduced 97% of mosquito bites. However, despite high acceptability, daily use was observed to be low (8%) and did not correspond to the reported use in surveys (around 70%). The levels of use aimed for by the trial were never reached as the population used it variably across place (forest, farms and villages) and time (seasons), or in alternative applications (spraying on insects, on bed nets, etc.). These findings show the key role of human behavior in the effectiveness of malaria preventive measures, questioning whether malaria in low endemic settings can be reduced substantially by introducing measures without researching and optimizing community involvement strategies. [ABSTRACT FROM AUTHOR]

Background:Adherence to effective malaria medication is extremely important in the context of Cambodia’s elimination targets and drug resistance containment. Although the public sector health facilities are accessible to the local ethnic minorities of Ratanakiri province (Northeast Cambodia), their illness itineraries often lead them to private pharmacies selling “cocktails” and artemether injections, or to local diviners prescribing animal sacrifices to appease the spirits. Methods:The research design consisted of a mixed methods study, combining qualitative (in-depth interviews and participant observation) and quantitative methods (household and cross-sectional survey). Results:Three broad options for malaria treatment were identified: i) the public sector; ii) the private sector; iii) traditional treatment based on divination and ceremonial sacrifice. Treatment choice was influenced by the availability of treatment and provider, perceived side effects and efficacy of treatments, perceived etiology of symptoms, and patient-health provider encounters. Moreover, treatment paths proved to be highly flexible, changing mostly in relation to the perceived efficacy of a chosen treatment. Conclusions:Despite good availability of anti-malarial treatment in the public health sector, attendance remained low due to both structural and human behavioral factors. The common use and under-dosage of anti-malaria monotherapy in the private sector (single-dose injections, single-day drug cocktails) represents a threat not only for individual case management, but also for the regional plan of drug resistance containment and malaria elimination. [ABSTRACT FROM AUTHOR]

Buruli ulcer is an indolent, slowly progressing necrotizing disease of the skin caused by infection with Mycobacterium ulcerans. In the present study, we applied a redesigned technique to a vast panel of M. ulcerans disease isolates and clinical samples originating from multiple African disease foci in order to (i) gain fundamental insights into the population structure and evolutionary history of the pathogen and (ii) disentangle the phylogeographic relationships within the genetically conserved cluster of African M. ulcerans. Our analyses identified 23 different African insertion sequence element single nucleotide polymorphism (ISE-SNP) types that dominate in different areas where Buruli ulcer is endemic. These ISE-SNP types appear to be the initial stages of clonal diversification from a common, possibly ancestral ISE-SNP type. ISE-SNP types were found unevenly distributed over the greater West African hydrological drainage basins. Our findings suggest that geographical barriers bordering the basins to some extent prevented bacterial gene flow between basins and that this resulted in independent focal transmission clusters associated with the hydrological drainage areas. Different phylogenetic methods yielded two well-supported sister clades within the African ISE-SNP types. The ISE-SNP types from the "pan-African clade" were found to be widespread throughout Africa, while the ISE-SNP types of the "Gabonese/Cameroonian clade" were much rarer and found in a more restricted area, which suggested that the latter clade evolved more recently. Additionally, the Gabonese/Cameroonian clade was found to form a strongly supported monophyletic group with Papua New Guinean ISE-SNP type 8, which is unrelated to other Southeast Asian ISE-SNP types. [ABSTRACT FROM AUTHOR]