How to manage adult patients with malaria in the non-endemic setting.

BACKGROUND: The diagnosis and management of malaria in non-endemic countries presents a continuing challenge. Plasmodium falciparum, which is capable of rapidly inducing severe and life-threatening multiorgan disease, is the species most frequently diagnosed in Europe and North America. OBJECTIVES: To summarise the more relevant diagnostic findings and clinical features of malaria observed in non-endemic settings and to provide an update of the key management decision points using three illustrative clinical scenarios of uncomplicated and severe malaria. SOURCES: The discussion is based on a relevant literature search spanning the last 20 years. Recommendations are based on available clinical guidelines including those of the WHO, observational studies conducted in non-endemic settings, and, when available, extrapolation from randomised studies from malaria-endemic settings. CONTENT: The following topics are covered: diagnosis, including the use of molecular biology; clinical characteristics; management with a specific focus on complicated (severe) and uncomplicated malaria; and areas of resistance to available antimalarial drugs. IMPLICATIONS: Malaria imported to non-endemic settings, especially P. falciparum malaria, is sometimes initially overlooked and the delayed diagnosis is responsible for every year of preventable deaths. This review aims to raise awareness of malaria outside endemic countries and to provide clinicians with a practical guide for efficient diagnosis and targeted therapy for the different species involved.

Real-time fluorescence loop-mediated isothermal amplification assays for detection of zoonotic malaria Plasmodium parasites.

BACKGROUND: Natural human infections by Plasmodium cynomolgi and P. inui have been reported recently and gain the substantial attention from Southeast Asian countries. Zoonotic transmission of non-human malaria parasites to humans from macaque monkeys occurred through the bites of the infected mosquitoes. The objective of this study is to establish real-time fluorescence loop-mediated isothermal amplification (LAMP) assays for the detection of zoonotic malaria parasites by combining real-time fluorescent technology with the isothermal amplification technique. METHODS: By using 18S rRNA as the target gene, the primers for P. cynomolgi, P. coatneyi and P. inui were newly designed in the present study. Four novel real-time fluorescence LAMP assays were developed for the detection of P. cynomolgi, P. coatneyi, P. inui and P. knowlesi. The entire amplification process was completed in 60 min, with the assays performed at 65 °C. By using SYTO-9 as the nucleic acid intercalating dye, the reaction was monitored via real-time fluorescence signal. RESULTS: There was no observed cross-reactivity among the primers from different species. All 70 field-collected monkey samples were successfully amplified by real-time fluorescence LAMP assays. The detection limit for P. cynomolgi, P. coatneyi and P. knowlesi was 5 × 109 copies/µL. Meanwhile, the detection limit of P. inui was 5 × 1010 copies/µL. CONCLUSION: This is the first report of the detection of four zoonotic malaria parasites by real-time fluorescence LAMP approaches. It is an effective, rapid and simple-to-use technique. This presented platform exhibits considerable potential as an alternative detection for zoonotic malaria parasites.

Improved limit of detection for zoonotic Plasmodium knowlesi and P. cynomolgi surveillance using reverse transcription for total nucleic acid preserved samples or dried blood spots.

Background: Zoonotic P. knowlesi and P. cynomolgi symptomatic and asymptomatic infections occur across endemic areas of Southeast Asia. Most infections are low-parasitemia, with an unknown proportion below routine microscopy detection thresholds. Molecular surveillance tools optimizing the limit of detection (LOD) would allow more accurate estimates of zoonotic malaria prevalence. Methods: An established ultra-sensitive Plasmodium genus quantitative-PCR (qPCR) assay targeting the 18S rRNA gene underwent LOD evaluation with and without reverse transcription (RT) for P. knowlesi, P. cynomolgi and P. vivax using total nucleic acid preserved (DNA/RNA Shield™) isolates and archived dried blood spots (DBS). LODs for selected P. knowlesi-specific assays, and reference P. vivax- and P. cynomolgi-specific assays were determined with RT. Assay specificities were assessed using clinical malaria samples and malaria-negative controls. Results: The use of reverse transcription improved Plasmodium species detection by up to 10,000-fold (Plasmodium genus), 2759-fold (P. knowlesi), 1000-fold (P. vivax) and 10-fold (P. cynomolgi). The median LOD with RT for the Kamau et al. Plasmodium genus RT-qPCR assay was ≤0.0002 parasites/µL for P. knowlesi and 0.002 parasites/µL for both P. cynomolgi and P. vivax. The LODs with RT for P. knowlesi-specific PCRs were: Imwong et al. 18S rRNA (0.0007 parasites/µL); Divis et al. real-time 18S rRNA (0.0002 parasites/µL); Lubis et al. hemi-nested SICAvar (1.1 parasites/µL) and Lee et al. nested 18S rRNA (11 parasites/µL). The LOD for P. vivax- and P. cynomolgi-specific assays with RT were 0.02 and 0.20 parasites/µL respectively. For DBS P. knowlesi samples the median LOD for the Plasmodium genus qPCR with RT was 0.08, and without RT was 19.89 parasites/uL (249-fold change); no LOD improvement was demonstrated in DBS archived beyond 6 years. The Plasmodium genus and P. knowlesi-assays were 100% specific for Plasmodium species and P. knowlesi detection, respectively, from 190 clinical infections and 48 healthy controls. Reference P. vivax-specific primers demonstrated known cross-reactivity with P. cynomolgi. Conclusion: Our findings support the use of an 18S rRNA Plasmodium genus qPCR and species-specific nested PCR protocol with RT for highly-sensitive surveillance of zoonotic and human Plasmodium species infections.

A visualized hybrid PCR-LAMP assay for the detection of human Plasmodium species.

Combining the advantages of PCR and LAMP, we described a new technique, namely PCR-LAMP, for malaria diagnosis. The whole process of DNA amplification can be completed in 35 min. This hybrid amplification technique markedly improved the sensitivity of detection compared to the classic single PCR or LAMP assay alone. PCR-LAMP assay had a detection limit of 1 copy/µL for P. knowlesi and P. ovale, 0.1 copy/µL for P. vivax, P. falciparum and P. malariae, respectively. To facilitate the endpoint detection, xylenol orange was added. Positive samples were indicated in orange while negative reactions were violet. The inclusion of xylenol orange into the LAMP reaction mix significantly reduces the post-amplification workload. Without relying on the use of specific instruments, the color changes of the amplicons could be visualized directly through the naked eye. In conclusion, PCR-LAMP poses the potential to be developed as a new malaria molecular diagnosis tool.

Disentangling the intersection of inequities with health and malaria exposure: key lessons from rural communities in Northern Borneo.

BACKGROUND: The increasing incidence of Plasmodium knowlesi malaria poses a significant challenge to efforts to eliminate malaria from Malaysia. Macaque reservoirs, outdoors-biting mosquitoes, human activities, and agricultural work are key factors associated with the transmission of this zoonotic pathogen. However, gaps in knowledge regarding reasons that drive malaria persistence in rural Kudat, Sabah, Northern Borneo remain. This study was conducted to address this knowledge gap, to better understand the complexities of these entangled problems, and to initiate discussion regarding new countermeasures to address them. This study aims to highlight rural community members' perspectives regarding inequities to health relating to P. knowlesi malaria exposure. METHODS: From January to October 2022, a study using qualitative methods was conducted in four rural villages in Kudat district of Sabah, Malaysia. A total of nine in-depth interviews were conducted with community and faith leaders, after the completion of twelve focus group discussions with 26 photovoice participants. The interviews were conducted using the Sabah Malay dialect, audio-recorded, transcribed, and translated into English. The research team led the discussion and analysis, which was approved by participants through member checking at the community level. RESULTS: Participants identified disparity in health as a key issue affecting their health and livelihoods. Injustice in the social environment was also identified as a significant challenge, including the importance of listening to the voices of affected communities in disentangling the social and economic phenomena that can impact malaria control. Specific concerns included inadequate access to health-related resources and degradation of the environment. Participants recommended improving access to water and other necessities, increasing the availability of malaria control commodities in healthcare facilities, and developing sustainable programs to reduce socioeconomic disparities. CONCLUSION: Inequities to health emerged as a key concern for malaria control in rural Kudat, Sabah. A locally targeted malaria programme cantered on improving the social and economic disparities associated with health outcomes, could be a potential strategy for malaria prevention in such areas. Community-level perspectives gathered from this study can be used as a foundation for future discussions and dialogues among policymakers and community members for achieving greater transparency, improving social equity, and interoperability in addressing P. knowlesi malaria control.

Evaluation of A Simple DNA Extraction Method and Its Combination with Loop-Mediated Isothermal Amplification Assays for Rapid Plasmodium knowlesi Diagnosis.

The initial and vital stage in the diagnosis of malaria involves extracting DNA. The efficiency of malaria testing is restricted by the multiple steps involved in commercial DNA extraction kits. We attempted to improve an existing loop-mediated isothermal amplification (LAMP) for the detection of Plasmodium knowlesi by using a simple DNA extraction approach, making it a feasible option for mass screening. We utilized a simple nucleic acid extraction method directly from whole blood for the detection of P. knowlesi, taking only 5 min to complete. The extracted DNA was evaluated by two fluorescent-based LAMP and one colorimetric-based LAMP assay. The detection limit for both SYTO-LAMP and SYBR green-LAMP was 0.00001% and 0.0001% parasitemia, respectively. Meanwhile, neutral red-LAMP had a detection limit of 0.01% parasitemia. Combining this simple and inexpensive DNA extraction method, SYTO-LAMP could serve as an alternative molecular diagnosis for the detection of P. knowlesi and other human Plasmodium spp.

Plasmodium knowlesi circumsporozoite protein: genetic characterisation and predicted antigenicity of the central repeat region

@#Circumsporozoite protein (CSP) central repeat region is one of the main target regions of the RTS,S/AS01 vaccine for falciparum infection as it consists of immunodominant B cell epitopes. However, there is a lack of study for P. knowlesi CSP central repeat region. This study aims to characterise the CSP repeat motifs of P. knowlesi isolates in Peninsular Malaysia. CSP repeat motifs of 64 P. knowlesi isolates were identified using Rapid Automatic Detection and Alignment of Repeats (RADAR). Antigenicity of the repeat motifs and linear B cell epitopes were predicted using VaxiJen 2.0, BepiPred-2.0 and BCPred, respectively. A total of 35 dominant repeat motifs were identified. The repeat motif “AGQPQAQGDGANAGQPQAQGDGAN” has the highest repeat frequency (n=15) and antigenicity index of 1.7986. All the repeat regions were predicted as B cell epitopes. In silico approaches revealed that all repeat motifs were antigenic and consisted of B cell epitopes which could be designed as knowlesi malaria vaccine.

The presence of Plasmodium malariae and Plasmodium knowlesi in near malaria elimination setting in western Indonesia.

BACKGROUND: Indonesia is progressing towards malaria elimination. To achieve this goal, intervention measures must be addressed to cover all Plasmodium species. Comprehensive control measures and surveillance programmes must be intensified. This study aims to determine the prevalence of microscopic and submicroscopic malaria in Langkat district, North Sumatera Province, Indonesia. METHODS: A cross-sectional survey was conducted in six villages in Langkat district, North Sumatera Province in June 2019. Data were recorded using a standardized questionnaire. Finger pricked blood samples were obtained for malaria examination using rapid diagnostic test, thick and thin blood smears, and polymerase chain reaction. RESULTS: A total of 342 individuals were included in the study. Of them, one (0.3%) had a microscopic Plasmodium malariae infection, no positive RDT examination, and three (0.9%) were positive for P. malariae (n = 1) and Plasmodium knowlesi (n = 2). The distribution of bed net ownership was owned by 40% of the study participants. The participants had a house within a radius of 100-500 m from the forest (86.3%) and had the housing material of cement floor (56.1%), a tin roof (82.2%), wooden wall (35.7%), bamboo wall (28.1%), and brick wall (21.6%). CONCLUSION: Malaria incidence has substantially decreased in Langkat, North Sumatera, Indonesia. However, submicroscopic infection remains in the population and may contribute to further transmission. Surveillance should include the detection of microscopic undetected parasites, to enable the achievement of malaria elimination.

Optimizing Plasmodium vivax serological surveillance within a coendemic epidemiological landscape.

Serological surveillance is a useful tool for revealing hotspots of transmission intensity or cryptic asymptomatic reservoirs, especially as malaria transmission declines. Such approaches can help us to understand malaria epidemiology, but also to guide interventions. Recently, Longley et al. refined a panel for Plasmodium vivax serological surveillance to aid in malaria elimination.

Comparison of two malaria multiplex immunoassays that enable quantification of malaria antigens.

BACKGROUND: Immunoassay platforms that simultaneously detect malaria antigens including histidine-rich protein 2 (HRP2)/HRP3 and Plasmodium lactate dehydrogenase (pLDH), are useful epidemiological tools for rapid diagnostic test evaluation. This study presents the comparative evaluation of two multiplex platforms in identifying Plasmodium falciparum with presence or absence of HRP2/HRP3 expression as being indicative of hrp2/hrp3 deletions and other Plasmodium species. Moreover, correlation between the malaria antigen measurements performed at these platforms is assessed after calibrating with either assay standards or international standards and the cross-reactivity among Plasmodium species is examined. METHODS: A 77-member panel of specimens composed of the World Health Organization (WHO) international Plasmodium antigen standards, cultured parasites for P. falciparum and Plasmodium knowlesi, and clinical specimens with mono-infections for P. falciparum, Plasmodium vivax, and Plasmodium malariae was generated as both whole blood and dried blood spot (DBS) specimens. Assays for HRP2, P. falciparum-specific pLDH (PfLDH), P. vivax-specific pLDH (PvLDH), and all human Plasmodium species Pan malaria pLDH (PanLDH) on the Human Malaria Array Q-Plex and the xMAP platforms were evaluated with these panels. RESULTS: The xMAP showed a higher percent positive agreement for identification of hrp2-deleted P. falciparum and Plasmodium species in whole blood and DBS than the Q-Plex. For whole blood samples, there was a highly positive correlation between the two platforms for PfLDH (Pearson r = 0.9926) and PvLDH (r = 0. 9792), moderate positive correlation for HRP2 (r = 0.7432), and poor correlation for PanLDH (r = 0.6139). In Pearson correlation analysis between the two platforms on the DBS, the same assays were r = 0.9828, r = 0.7679, r = 0.6432, and r = 0.8957, respectively. The xMAP HRP2 assay appeared to cross-react with HRP3, while the Q-Plex did not. The Q-Plex PfLDH assay cross-reacted with P. malariae, while the xMAP did not. For both platforms, P. knowlesi was detected on the PvLDH assay. The WHO international standards allowed normalization across both platforms on their HRP2, PfLDH, and PvLDH assays in whole blood and DBS. CONCLUSIONS: Q-Plex and xMAP show good agreement for identification of P. falciparum mutants with hrp2/hrp3 deletions, and other Plasmodium species. Quantitative results from both platforms, normalized into international units for HRP2, PfLDH, and PvLDH, showed good agreement and should allow comparison and analysis of results generated by either platform.

Quantification of the misidentification of Plasmodium knowlesi as Plasmodium malariae by microscopy: an analysis of 1569 P. knowlesi cases.

BACKGROUND: Plasmodium knowlesi is recognized as the fifth Plasmodium species causing malaria in humans. It is morphologically similar to the human malaria parasite Plasmodium malariae, so molecular detection should be used to clearly discriminate between these Plasmodium species. This study aimed to quantify the rate at which P. knowlesi is misidentified as P. malariae by microscopy in endemic and non-endemic areas. METHODS: The protocol of this systematic review was registered in the PROSPERO International Prospective Register of Systematic Reviews (ID = CRD42020204770). Studies reporting the misidentification of P. knowlesi as P. malariae by microscopy and confirmation of this by molecular methods in MEDLINE, Web of Science and Scopus were reviewed. The risk of bias in the included studies was assessed using the Quality Assessment of Diagnostic Accuracy Studies (QUADAS). The pooled prevalence and 95% confidence interval (CI) of the misidentification of P. knowlesi as P. malariae by microscopy were estimated using a random effects model. Subgroup analysis of the study sites was performed to demonstrate any differences in the misidentification rates in different areas. Heterogeneity across the included studies was assessed and quantified using Cochran's Q and I2 statistics, respectively. Publication bias in the included studies was assessed using the funnel plot, Egger's test and contour-enhanced funnel plot. RESULTS: Among 375 reviewed studies, 11 studies with a total of 1569 confirmed P. knowlesi cases in humans were included. Overall, the pooled prevalence of the misidentification of P. knowlesi as P. malariae by microscopy was estimated at 57% (95% CI 37-77%, I2: 99.3%). Subgroup analysis demonstrated the highest rate of misidentification in Sawarak, Malaysia (87%, 95% CI 83-90%, I2: 95%), followed by Sabah, Malaysia (85%, 95% CI 79-92%, I2: 85.1%), Indonesia (16%, 95% CI 6-38%), and then Thailand (4%, 95% CI 2-9%, I2: 95%). CONCLUSION: Although the World Health Organization (WHO) recommends that all P. malariae-positive diagnoses made by microscopy in P. knowlesi endemic areas be reported as P. malariae/P. knowlesi malaria, the possibility of microscopists misidentifying P. knowlesi as P. malariae is a diagnostic challenge. The use of molecular techniques in cases with malariae-like Plasmodium with high parasite density as determined by microscopy could help identify human P. knowlesi cases in non-endemic countries.

High incidence of Plasmodium knowlesi malaria compared to other human malaria species in several hospitals in Malaysia

@# Through the regional control programme, Malaysia has been successfully reducing the incidence of Plasmodium falciparum and Plasmodium vivax infections. However, the incidence of zoonotic malaria Plasmodium knowlesi infection is increasing and now has been the major cause of malaria in Malaysia especially Malaysian Borneo. The emergence of knowlesi infection has threatened the malaria elimination programme which the government aims to reduce the overall malaria infections by 2020. Unlike other benign human Plasmodium spp., P. knowlesi can cause fatal infections. The aim of this study was to determine the incidence and distribution of five human malaria parasites including P. knowlesi in Peninsular Malaysia and Malaysian Borneo. A total of 112 blood samples were collected from seven states and district hospitals in Peninsular Malaysia and Malaysian Borneo from year 2015 to 2016. The samples were examined by microscopy and further confirmed by nested PCR assay targeting 18S rRNA gene of Plasmodium spp. Following the nested PCR assays, a total of 54 (48.2%) samples were positive for P. knowlesi infections, 12 (10.7%) cases were positive for P. vivax infections, followed by 7 (6.3%) cases of P. falciparum and 4 (3.5%) cases of P. malariae. There were 3 cases (2.7%) of mixed infections (P. knowlesi/P. vivax). However, no cases were identified as P. ovale. A total of 32 (28.6%) cases were found as negative infections. LoopMediated Isothermal Amplification Assay (LAMP) was performed to confirm inconclusive results produced by microscopy and nested PCR. P. knowlesi showed the highest prevalence in Sarawak (n= 30), Sabah (n=13), Pulau Pinang (n=5) and Pahang (n=6). PCR and LAMP was not able to detect a large number of microscopy positive samples due to DNA degradation during storage and shipping. Among all the states involved in this study, the highest prevalence of P. knowlesi infection was found in Sabah and Sarawak.

Trends of neglected Plasmodium species infection in humans over the past century in India.

BACKGROUND: Efforts for malaria elimination in India focus solely on the more prevalent human malaria parasites of Plasmodium falciparum (Pf) and Plasmodium vivax (Pv). The three non-Pf/Pv species - Plasmodium malariae (Pm), Plasmodium ovale (Po) and Plasmodium knowlesi (Pk) are seldom studied though they are often present as mixed infections with Pf/Pv and thus may be misdiagnosed. This study provides a comprehensive landscape of Pm, Po, and Pk infections from 1930 to 2020. METHODOLOGY: We systematically searched for published literature on Pm, Po, and Pk in India from PubMed database and collated data from 35 studies. The data, starting from 1930, were mapped decade-wise across India. The prevalence of the three neglected Plasmodium species and their proportional contribution to reported Plasmodium mixed-infection were also calculated and analysed. PRINCIPAL FINDINGS: Amongst the three non-Pf/Pv species, Pm infections have been reported in greater numbers across India and were mostly mono-infections till 1980. From 1983 onwards, reports of Pm mixed infections with Pf/Pv started to emerge. In contrast, reports on occurrence of Po are still rare barring few mixed infection studies. Further, Pk mono- and mixed cases were first reported in 2004 in India and Pk now has been found reported from four Indian states. CONCLUSION: This is the first account of country-wide assimilation of reported malaria parasite species data that covers Pm, Po, and Pk infection profiles from 1930 to 2020. This study illustrates the need to survey all 5 human malaria parasite species in India and to target them collectively during the malaria elimination phase.

Quantification of Plasmodium knowlesi versus Plasmodium falciparum in the rhesus liver: implications for malaria vaccine studies in rhesus models.

BACKGROUND: Rhesus macaques are valuable pre-clinical models for malaria vaccine development. The Plasmodium knowlesi/rhesus and Plasmodium falciparum/rhesus models are two established platforms for malaria vaccine testing, and both have previously been used to assess live-attenuated sporozoite vaccines. However, there is evidence that the susceptibility of the rhesus liver to P. knowlesi versus P. falciparum sporozoites likely differs, potentially complicating comparisons between these two platforms. METHODS: To quantify the differing susceptibility of rhesus to P. knowlesi and P. falciparum sporozoites, animals were infected by direct venous inoculation of purified, cryopreserved wild-type P. knowlesi sporozoites (PkSPZ) or P. falciparum sporozoites (PfSPZ). The entire liver was collected 5 days post-infection, and parasite burden in each liver lobe was quantified using an ultrasensitive Plasmodium 18S rRNA RT-PCR biomarker assay. The potential of using 18S rRNA copy number in the rhesus liver to directly measure the efficacy of vaccines targeting P. falciparum sporozoites and liver stages was also theoretically evaluated. RESULTS: Infection of rhesus with a high dose of PkSPZ led to consistently high burden liver stage infections (range 9.5-10.1 log10 copies 18S rRNA/g of liver), with similar amounts of parasite 18S rRNA detected in every liver lobe. Inoculation of rhesus with high doses of PfSPZ led to more variable, lower liver burdens (range 4.9-6.6 log10 copies 18S rRNA/g of liver in infected lobes), with parasite 18S rRNA below the limit of detection in some liver lobes. The low signal and heterogeneity of liver burden in the PfSPZ-infected animals indicates that even this extremely sensitive molecular assay cannot be used to assess reliably vaccine efficacy in the P. falciparum/rhesus platform. CONCLUSIONS: Detection of 18S rRNA in the liver following high dose intravenous PfSPZ confirmed that rhesus are modestly susceptible to wild-type P. falciparum sporozoites. However, comparison of 18S rRNA RT-PCR biomarker signal indicates that the P. falciparum liver burden was 3-5 logs lower than in PkSPZ-infected animals. Quantification of this difference in liver stage burden will help guide and interpret data from pre-clinical studies of live-attenuated sporozoite vaccines in rhesus models.

Detection of Plasmodium knowlesi using recombinase polymerase amplification (RPA) combined with SYBR Green I.

In this study, recombinase polymerase amplification (RPA) combined with SYBR Green I was developed for the detection of Plasmodium knowlesi. Positive samples were indicated with a green color while negative samples were orange. To increase the efficiency of amplification, an interval mixing step of samples after 3 to 6 min incubation was recommended. Different sets of reaction volumes from 6.25 to 50 µL were tested and the results indicated no differences in detection. RPA's combination with SYBR Green I is fast and easy to perform, hence this method is suitable for use in resource-limited settings.

Cardiac Tamponade: A Rare Complication of Plasmodium Knowlesi Malaria

Abstract@#Plasmodium knowlesi has been discovered as the fifth species causing malaria in humans. It is a major public health problem in South East Asia especially in Borneo. We report a case of pericardial effusion that rapidly progressing to cardiac tamponade, an atypical presentation of P. knowlesi malaria. Our patient had no underlying known medical illness, presented with high grade fever with chills and rigors, epigastric pain, nausea, vomiting and with poor oral intake. Initial bedside cardiac ultrasound showed minimal pericardial effusion. Within a few hours, she became hypotensive, deteriorated rapidly despite fluid resuscitation requiring mechanical ventilation and inotropic support. Bedside cardiac ultrasound showed cardiac tamponade and pericardiocentesis was done. We highlight the importance of having high level of suspicion for this atypical presentation of cardiac tamponade when a patient is hypotensive in P. knowlesi infection. Prompt diagnosis and management may prevent potentially fatal complication.

Changes in malaria epidemiology in Germany, 2001-2016: a time series analysis.

BACKGROUND: German surveillance data showed a sharp rise of malaria cases in 2014 and 2015 due to the increased arrival of refugees from malaria endemic countries. A time series analysis of data from 2001 to 2016 was performed in order to describe the epidemiology of imported malaria in Germany in general and of the recent increase in particular. RESULTS: In total, 11,678 malaria cases were notified between 2001 and 2016 (range 526-1063 cases/year). Newly arriving refugees averaged 10 cases/year (1.5%) in 2001-13 and 292.5 cases/year (28.3%) in 2014-15. Plasmodium (P.) falciparum was the most frequently reported species (range 57.2-85.8%), followed by P. vivax (range during 2001-2013: 7.6-18.1%; during 2014-2015, mean 31.3%). In 2014-15, 22.3% of all P. vivax cases were refugees from Eritrea and 3.3% from other countries of the Horn of Africa; in 2015 and 2016, 19.5% were refugees from Afghanistan and Pakistan. Five P. knowlesi malaria infections were reportedly acquired in Thailand between 2012 and 2016. Total numbers of malaria notifications among native Germans and residents with migration background showed an increasing trend since 2007. Chemoprophylaxis use was reported for 24.3% (1695/6984) of cases and showed a declining trend. Native German cases took significantly more frequently chemoprophylaxis than cases with migration background (32.6% vs. 17.9%; p < 0.001). DISCUSSION/CONCLUSIONS: The steep rise in vivax malaria notifications in 2014 and 2015 was mainly due to newly arriving refugees from Eritrea but also from other countries of the Horn of Africa and South Asia. Clinicians should include malaria in their differential diagnosis in case of a febrile illness in the respective population and consider vivax malaria even if arrival to Germany dates back several months. Over the past 10 years, malaria notifications among native Germans and residents with migration background showed an increasing trend. Use of chemoprophylaxis was insufficient in both groups and deteriorating. New strategies need to be found to increase compliance to chemoprophylaxis recommendations. The surveillance provides valuable data for epidemiological assessment of imported malaria in Germany.

Clinical implications of a gradual dormancy concept in malaria.

Malaria recurrences after an initially successful therapy and malarial fever occurring a long time after infection are well-known problems in malariology. Currently, two distinct types of malaria recurrences are defined: recrudescence and relapse. A recrudescence is thought to originate from circulating Plasmodium blood stages which do not cause fever before a certain level of a microscopically detectable parasitemia is reached. Contrary, a relapse is thought to originate from quiescent intracellular hepatic parasite stages called hypnozoites. Recrudescences would typically occur in infections due to Plasmodium falciparum. Plasmodium knowlesi, and Plasmodium malariae, whereas relapses would be caused exclusively by Plasmodium vivax and Plasmodium ovale. This schematic view is, however, insufficiently supported by experimental evidence. For instance, hypnozoites of P. ovale have never been experimentally documented. On the other hand, the nonfinding of P. malariae hypnozoites turned into the proof for the nonexistence of P. malariae hypnozoites. Clinical relapse-type recurrences have been observed in both P. ovale and P. malariae infections, and decade-long incubation times have also been reported in P. falciparum infections. We propose a gradual hypothesis in accordance with the continuity concept of biological evolution: both, relapse and recrudescence may be potentially caused by all Plasmodium spp. We hypothesize that the difference between the various Plasmodium spp. is quantitative rather than qualitative: there are Plasmodium spp. which frequently cause relapses such as P. vivax, particularly the P.v. Chesson strain, species which cause relapses less frequently, such as P. ovale and sometimes P. malariae, and species which may exceptionally cause relapses such as P. falciparum. All species may cause recrudescences. As clinical consequences, we propose that 8-aminquinolines may be considered in a relapse-type recrudescence regardless of the causal Plasmodium sp., whereas primaquine relapse prevention might not be routinely indicated in malaria due to P. ovale.

Emerging vector-borne zoonoses: eco-epidemiology and public health implications in India.

The diseases originating from animals or associated with man and animals are remerging and have resulted in considerable morbidity and mortality. The present review highlights the re-emergence of emerging mainly zoonotic diseases like chikungunya, scrub typhus, and extension of spatial distribution of cutaneous leishmaniasis from western Rajasthan to Himachal Pradesh, Kerala, and Haryana states; West Nile virus to Assam, and non-endemic areas of Japanese encephalitis (JE) like Maharashtra and JE to Delhi; Crimean-Congo hemorrhagic fever making inroads in Ahmedabad; and reporting fifth parasite of human malaria with possibility of zoonosis have been highlighted, which necessitates further studies for prevention and control. Emphasis has been given on understanding the ecology of reservoir hosts of pathogen, micro niche of vector species, climatic, socioeconomic risk factors, etc. Development of facilities for diagnosis of virus from insects, reservoirs, and human beings (like BSL4, which has been established in NIV, Pune), awareness about symptoms of new emerging viral and other zoonotic diseases, differential diagnosis, risk factors (climatic, ecological, and socioeconomic) and mapping of disease-specific vulnerable areas, and mathematical modeling for projecting epidemiological scenario is needed for preparedness of public health institutes. It is high time to understand the ecological link of zoonotic or anthroponotic diseases for updated risk maps and epidemiological knowledge for effective preventive and control measures. The public health stakeholders in India as well as in Southeast Asia should emphasize on understanding the eco-epidemiology of the discussed zoonotic diseases for taking preventive actions.