Plasmodium spp. are common in low endemicity areas worldwide, but outside Africa, their contribution to malaria transmission is poorly understood. Community-based studies with highly sensitive molecular diagnostics are needed to quantify the asymptomatic reservoir of Plasmodium falciparum and P. vivax infections in Thai communities.Low-density asymptomatic infections of P. falciparum and P. vivax parasites was determined using 18S rRNA qPCR. Gametocytes were also detected by pfs25 / pvs25 qRT-PCRs.A cross-sectional survey of 4309 participants was conducted in three endemic areas in Kanchanaburi and Ratchaburi provinces of Thailand in 2012. The presence of P. vivax (3.09%), 37 with P. falciparum (0.86%), and 11 with mixed P. vivax/ P. falciparum (0.26%). The clear majority of both P. vivax (91.7%) and P. falciparum (89.8%) infections were not accompanied by any febrile symptoms. Infections with either species were most common in adolescent and adult males. Recent travel to Myanmar was highly associated with P. falciparum  but not P. vivax infections (P = 0.13). A large number of P. vivax (71.5%) and P. falciparum (72.0%) infections were gametocyte positive by pvs25/pfs25 qRT-PCR. Detection of gametocyte-specific pvs25 and pfs25 transcripts was strongly dependent on parasite density. pvs25 transcript numbers, a measure of gametocyte density, were also highly correlated with parasite density .A total of 133 individuals were found infected with Plasmodium spp. were common in western Thai communities in 2012. The high prevalence of gametocytes indicates that these infections may contribute substantially to the maintenance of local malaria transmission.Asymptomatic infections with The online version of this article (10.1186/s13071-017-2407-y) contains supplementary material, which is available to authorized users. P. falciparum than for P. vivax, which now accounts for most local cases in Thailand [The last 30 years have seen a great reduction in the burden of malaria in South East Asia in general, and particularly in Thailand . As a coThailand . The advThailand  and moreThailand , 6, has Thailand , 8. DespThailand , there iPlasmodium spp. are common in low endemicity areas worldwide [P. falciparum and P. vivax infections in Thai communities.Low density, asymptomatic infections with orldwide –14. Studorldwide , 16. Howorldwide –19 and, orldwide , were smP. falciparum and P. vivax infections could infect mosquitos [Little is known about the role of asymptomatic infections in sustaining local malaria transmission in Thailand. In a single study conducted in Mae Hong Son, it was found that blood from 15.4% and 60% of study participants with afebrile osquitos . In thisosquitos .P. vivax compared to P. falciparum patients [Anopheles dirus, P. falciparum gametocyte observation by LM was strongly linked to Anopheles dirus infectivity; only a few patient blood samples without LM-detectable gametocytaemia were found to infect mosquitoes [P. vivax appears to be more infectious, and a gametocyte density under LM limit of detection can readily lead to mosquito infection [Light microscopy (LM) is relatively insensitive for detecting gametocytes , particularly in asymptomatic individuals with low-density infections . In Thaipatients . In membsquitoes , 25. In nfection .P. falciparum and from 31.1 to 91.1% for P. vivax [Molecular techniques are much more sensitive at detecting gametocytes than LM . In a stP. vivax . To dateWe, therefore, conducted a cross-sectional survey of 4309 people living in three malaria endemic communities in Kanchanaburi and Ratchaburi provinces. We combined full population sampling with the sensitive molecular diagnosis of both asexual blood-stage parasites and gametocytes.Three sites on a 250 km central span of the Thai-Burmese border were selected for our cross-sectional survey: (i) Suan Phueng district of Ratchaburi Province; (ii) Ban Kong Mong Tha, Sangkhla Buri district of Kanchanaburi Province; and (iii) Ban Bong Ti, Sai Yok district of Kanchanaburi Province. All areas had stable populations (main ethnic groups Thai and Karen) with less than 2% reporting to have travelled in the previous month. Agriculture, farming and forest foraging were main occupations. The annual peak malaria season of these areas was April-July .Anopheles minimus and Anopheles maculatus, exophagic and nighttime feeders, were the primary malaria vectors in this area. A large-scale cross-sectional survey conducted in 2003–2004 reported monthly prevalence rates of 0.1–1.5% for P. falciparum and 0.2–0.6% for P. vivax by microscopy [The southernmost study site, Suan Phueng district, is located 163 km west of Bangkok and encompasses the mountainous regions of the Tanaosri Mountain range. This study site was composed of four connected small villages: Wangko, Huai Krawan, Pong Hang and Huai Phak. The combined population size was 2188. Most houses were clustered along a stream. croscopy .P. falciparum and 5.9% for P. vivax by LM and identified the predominant anophelines as A. minimus, A. sawadwongporni, A. maculatus, A. campestris and A. barbirostris [The central site, Bong Ti, was composed of three villages: Ban Bong Ti Bon, Ban Bong Ti Lang and Ban Thai Muang. The combined population size was 3176. The site was situated 65 km west of Kanchanaburi in hilly terrain. To date, no detailed malaria epidemiological and entomological study has been conducted in this area. Most houses were accessible by paved roads. The northernmost site Kong Mong Tha was a single village located in a remote valley surrounded by mountains. The population size was 959. Once accessible only by boats or foot for 10 months of the year, the village had become accessible by trucks throughout the year at the time of this study. Previous studies done in 2000 estimated malaria prevalence to be 5.2% for irostris .Participation in the study was household based. Convenience sampling of households was used, and all household members were invited to participate. The expected sample sizes, i.e. the total residents in the participating households, were 1858 (Suan Phueng), 3034 (Bong Ti), and 678 (Kong Mong Tha). Informed consent was obtained from each participant and, when needed, a legal guardian. Demographic characteristics and behavioural risk data were obtained by trained interviewers using a structured questionnaire. Before blood collection, body temperature was measured with an infrared thermometer. Participants who had body temperature > 37.5 °C received a rapid diagnosis test  and, if positive, were referred for treatment at the nearest malaria clinic. In this study, “fever” was defined as body temperature > 37.5 °C, and “unwell” as any feeling of illness reported by the participants including but not limited to chills, nausea, headache, muscle pain and abdominal pain.Capillary blood (250 μl) was collected by finger prick from each participant into an EDTA-containing microtainer. A 50 μl portion was immediately preserved for RNA extraction onsite by mixing with 250 μl RNAprotect  and transferred to -20 °C for long-term storage within 12 h. The remaining blood was separated into pellet and plasma on the same day in a temporary field laboratory and stored at -20 °C. If the finger prick blood was < 250 μl, the volume of the draw was recorded.P. falciparum NF54 strain (100 μl packed cells with 0.01% ring stage parasitemia) were used as DNA extraction controls for every 81 survey samples. RNA was extracted with RNeasy Plus Mini kit (Qiagen). In addition to the proprietary gDNA elimination step of the kit, on-column treatment with RNase-Free DNase set (Qiagen) was used on all samples to further remove trace genomic DNA contaminant.After sample shipment on dry ice to Faculty of Tropical Medicine, Mahidol University, Bangkok, DNA was purified from each blood pellet (≤ 100 μl) with FavorPrep 96-Well Genomic DNA Extraction Kit  at following manufacturer’s protocol. Purified DNA was eluted in 100 μl Elution Buffer and stored at -20 °C. Two samples of Taq Universal Probes Supermix (Bio-Rad Laboratories), and all qRT-PCRs used Superscript III One-Step Quantitative RT-PCR System . Copy number was estimated using in-plate standards generated from a serial dilution of a plasmid containing an appropriate DNA fragment. The performance of each assay can be found in Additional file All qPCR and qRT-PCR measurements were performed on a CFX96 Realtime PCR Detection System and analysed with CFX Manager Software Version 3.0 . All qPCRs used iP. falciparum and P. vivax as previously described [P. falciparum.Malaria parasite infection was first identified by the QMAL assay, a genus-specific probe-based qPCR targeting a conserved region in 18S rRNA genes of plasmodia , using 4escribed  except fpfs25 or pvs25 transcripts. Four microliters of purified RNA were used in a single-step qRT-PCR for pfs25 or pvs25 [Gametocyte carriage was inferred from the presence of or pvs25 . Two quahttps://CRAN.R-project.org).Standard methods for the analyses of epidemiological data were used. A chi-square test was used to determine the statistical significance of the difference between proportions. Multivariate analysis of factors associated with parasite carriage was conducted using logistic regression. Association between gametocyte and parasite densities was investigated by linear regression. All analyses were performed using Stata 13  or R version 2.14.0  had resided in the village for longer than 2 months. Children aged 1–6 and 7–12 years accounted for 17.6 and 19.7% of participants, respectively. 9.5% were adolescents (13–17 years), and 39.4% and 13.8% were adults aged 19–49 and 50+, respectively. Only 78 participants (1.8%) reported travel outside the village in the previous month, with 27 of these having visited Myanmar. 90.5% of participants reported using a bednet last night with most (80.5%) owning a net for more than 2 years.P. vivax or P. falciparum : 133 P. vivax (Pv: 3.09%), 37 P. falciparum (Pf: 0.86%) and 11 mixed P. vivax/ P. falciparum (0.26%) infections.A total of 180 participants were found to be infected with either Pv: 3.82%; Pf: 1.36%) and Suan Phueng . However, in Kong Mong Tha only 6 P. vivax  and no P. falciparum infections (Fisher’s exact test: P = 0.012) were detected. Infections with either P. vivax or P. falciparum were significantly more common in male participants  and varied among age groups  peaking in adolescents for P. falciparum (2.20%) and adults for P. vivax  and not P. vivax . Remarkably, 4 of 27 people (14.8%) that travelled to Myanmar the previous month had Plasmodium spp. infections . The use of a bednet was, however, not associated with a reduction in risk of infection for either P. vivax  or P. falciparum . Five of the 19 participants  that reported having had a malaria episode in the last 2 weeks were infected with P. falciparum, none with P. vivax.Participants that reported recent travel were more likely to be infected but this only reached statistical significance for P. vivax (91.7%) and P. falciparum (89.8%) infections were not accompanied by any febrile symptoms. However, participants with either P. vivax and P. falciparum infections were nevertheless significantly more likely to report having febrile symptoms in the previous 2 days than uninfected individuals . Participants with P. vivax more commonly reported feeling ‘unwell’ at the time of the surveys .The majority of P. vivax infection  and 22.9/μl (95% CI: 8.9–59.5) for Plasmodium vivax parasite densities decreased significantly with age  = 4.56, P = 0.002) and were higher in people reporting feeling “unwell”: 37.3 (95% CI: 14.3–97.3) vs 7.0 (95% CI: 4.6–10.9) 18S copies/μl; ANOVA: F = 6.73, P = 0.011). The low number of P. falciparum infections precluded an analysis of factors associated with P. falciparum parasite densities.P. vivax (71.5%) and 36 of 50 P. falciparum  infections were positive respectively for pvs25 and pfs25 by qRT-PCR, resulting in a population prevalence of 2.40% P. vivax and 0.84% P. falciparum gametocytemic individuals. The presence of pvs25 and pfs25 transcripts in a sample was dependent on parasite density  for P. vivax and 1.83-fold  test: χ2 = 5.2, df = 1, P = 0.045) for P. falciparum, respectively. No other variables were significantly associated with the detection of P. vivax or P. falciparum gametocytes.Overall, 103 of 144 ity Fig. : with eapvs25 and 57.8 (95% CI: 16.0–208.3) pfs25 transcripts/μl were detected in P. vivax and P. falciparum gametocyte positive samples, respectively. pvs25 transcript numbers were highly correlated with parasite density as gauged by the 18S rDNA copy numbers . No significant correlation was found between pfs25 transcript- and 18S rDNA copy numbers .On average 26.3  Plasmodium spp. infections. Our study filled this gap in knowledge. Using sensitive molecular techniques, we measured the prevalence of P. falciparum and P. vivax together with their gametocyte prevalence in three populations on the western border of the country.As a result of the sustained Thai malaria control programs that is based on clinical case management and vector controls over the last decades, the burden of malaria in Thailand has progressively been reduced, restricting transmission mostly to the national borders with Myanmar and Cambodia . The ThaPlasmodium infections in Bong Ti and Suan Phueng had an overall prevalence rate of 4.2%. These two sites accounted for 90% of the participants in this study. Because both sites were part of larger communities and were located on the hilly terrain occupied primarily by agricultural Thai and Karen residents, they should be representative of many areas along Thailand western border. On the other hand, Kong Mong Tha was a small village geographically isolated from the nearest populous areas. It thus represented a unique setting where P. falciparum appeared to have been eliminated.We found that Plasmodium by approximately two-fold.Because a portion of very low-density infections inevitably has escaped detection, the prevalence likely was underestimated. Recently, Imwong and coworkers used an “high-volume” 18S rRNA qPCR to determine the distribution of parasite density in endemic populations of northwestern Thailand and western Cambodia . The disP. vivax parasitemia as age increased  where acquired immunity against the parasite likely took longer to develop.We observed a decline in sed Fig. . This trPlasmodium spp. infections by light microscopy to be 0.1–2.1% in 2004 and showed a higher prevalence of P. falciparum than P. vivax [P. vivax. It is difficult to directly compare the prevalence in both surveys because different diagnoses were used. Interestingly, we found that Hamlet 2, a village in Suan Phueng which previously had distinctly high prevalence, still had the highest prevalence in 2012, its value exceeding twice the average of the other villages (data not shown). This village, unlike others, also saw no decrease in parasite prevalence during the 1994–2004 control program. Thus, it appears that this village was a local transmission hotspot that needed special attention by the malaria control program.A previous study conducted in Suan Phueng estimated the prevalence of P. vivax . In the P. vivax and P. falciparum infections appears to have shifted towards adults, suggesting reduced risks of malaria transmission within the household and a relative increase in occupation-related outdoor inoculations. This may reflect the effectiveness of the local indoor residual spraying and bednet donation programmes. However, the shift may be explained by improved diagnosis, which allowed better detection of low parasitemia infections in adults [Comparing our current results to those from 2004 in Suan Phueng, the age structure of both n adults , 35.Plasmodium infections of 4.9% in Bong Ti is comparable to that observed (5.7% by PCR) in a small active case detection survey conducted in the same district during the high transmission peak in June 2012 [The overall prevalence of une 2012 .P. falciparum or mixed infections. Given with the overall low prevalence of P. falciparum infections (0.9%), this suggests that a large portion of the P. falciparum infections may be imported by people who travelled across the border.Interestingly, 14.8% (4/27) of people from Suan Phueng and Bong Ti who reported to have travelled to Myanmar during the previous month had infections. Of these, three had P. vivax and none had P. falciparum. The overall prevalence by PCR (0.6%) was much lower than the average LM prevalence of 10% in 2000–2001 [P. vivax appears to be more resilient than P. falciparum.We only identified six infected individuals in Kong Mong Tha, all of whom had 000–2001 . Thus, d000–2001 , P. vivaPlasmodium spp. infections. Because 10% (5/50) of all P. falciparum infections were detected within 2 weeks after a previous malaria episode, these infections may not truly represent asymptomatic infections, but rather, symptomatic infections after anti-malarial treatment. Thus, the proportion of non-febrile symptoms at the time of survey may have slightly underestimated the risk of symptomatic infection.Most infections were of low density, and only 10% of infections were accompanied by febrile symptoms. This suggests that, despite low transmission levels in recent years, residents still acquire and maintain a substantial level of clinical immunity to pvs25 and pfs25 transcripts were found in a large proportion of asymptomatic infections, indicating a potentially large contribution to the transmission reservoir. We found that both the probability of detecting pvs25 transcripts and density were strongly correlated with the 18S rDNA copy number, a proxy for circulating parasite density. While such a correlation had been reported previously for clinical cases [r2 = 0.82) between pvs25 transcript and 18S rDNA copy numbers will permit future estimation of the gametocyte reservoir in the population from blood DNA without costly qRT-PCR.al cases , 38, oural cases . The tigpfs25 transcript abundance and P. falciparum parasitemia. This may be a result of the slow development of P. falciparum gametocytes, which takes approximately 2 weeks, and their longevity [P. falciparum gametocyte density may be correlated with parasitemia during the preceding weeks but not at the time of blood sampling. In addition, because of cytoadherence and parasite sequestration, the total P. falciparum load may not be faithfully reflected in the peripheral blood sampled. The faster development of the P. vivax gametocyte, its shorter life-span, and the presence of all blood stages in the circulation may together permit the detection of a positive relationship between the copy numbers of pvs25 transcripts and 18S rDNA.In contrast, we found no correlation between ongevity . In otheAn. dirus, a major local vector of southeast Asia. These studies demonstrated that robust mosquito infection by P. vivax requires only a few gametocytes/μl blood [P. falciparum requires ~100 gametocytes/μl [P. vivax than to P. falciparum transmission in the population. Using the parasite density distribution in Thailand and Cambodia [P. vivax carriers contribute significantly to transmission [P. vivax compared to P. falciparum in the area.Two studies recently reported the relationship between gametocytemia and infection of μl blood  while incytes/μl , suggestCambodia , we prevsmission . BecauseP. vivax, should be considered transmission-competent. The lack of an association of bednet use with risk of either P. vivax or P. falciparum infection indicates that current vector control methods may have reached the limit of effectiveness. A novel approach to target the large reservoir of asymptomatic parasites may thus be required if Thailand’s goal of malaria elimination in 2025 is to be achieved.The high proportion of asymptomatic, gametocyte positive infection presents a significant challenge to the malaria control programme that is based exclusively on case management and vector control. Our data thus suggest that all infected individuals, particularly those with Asymptomatic infections were common in endemic areas of western Thailand in 2012. Most infections were associated with gametocytemia and thus may contribute to onward transmission. Targeting these silent transmission reservoirs will be an important task for Thailand’s malaria elimination program.


P. falciparum and P. vivax as prevalent species for Thailand, however, P. knowlesi infections have been occasionally reported in Thailand. In German travellers, only five cases of P. knowlesi infections have been reported to date.Thailand is a major destination for German travellers with more than 760,000 arrivals in 2015. At the same time, malaria is a concern in travel recommendations with regard to this destination. The World Malaria Report of 2016 mentions only A 45-year-old German male tourist travelled to Thailand from 25 December 2016 to 13 January 2017. On 14 January he developed fever with no other symptoms, and presented on 17 January at the Division for Tropical Medicine and Infectious Diseases in Munich, Germany.P. knowlesi infection.Malaria was diagnosed, primarily based on a single parasite in the thin smear microscopy, while commercial rapid diagnostic testing remained negative. Only the result of a differential PCR assay revealed P. knowlesi has to be considered in travellers returning from Thailand. Cases may present with an extremely low parasitaemia. This is in contrast to the assumption that P. knowlesi was likely to cause high parasitaemia due to its short replication cycle. Despite considerable reductions in morbidity, malaria remains to be a relevant public health concern for Southeast Asia. The WHO World Malaria Report 2016 estimates 52,000 malaria infections for the year 2015 for Thailand among a total population of 68.2 million . ThailanPlasmodium species in travellers to Thailand from 12 European countries and the USA has been reported by Behrens et al. at 0.60 cases per 100,000 visits for the year 2008 [P. falciparum and at 58.2% for P. vivax [Plasmodium knowlesi infections are not reported at all in some publications, including the WHO world malaria report of 2016 [The incidence of malaria across all  of 2016 –11.P. knowlesi has been known as the “fifth malaria species” that naturally infects humans . In contrast to P. falciparum, P. vivax and P. ovale spp. it is mostly a zoonotic parasite that shows a high prevalence in macaques, in particular in Malaysia. The species P. malariae is known as an anthropozoonotic parasite. Different Anopheles species have been described as potential vectors, with divergent feeding affinities towards humans and non-human primates. First infections in humans were reported from Malaysia in 1965, and human infections have been reported as rare events ever since. However, in a retrospective study in Malaysia on samples of patients that had been diagnosed with P. malariae infections, actually 82% of infections were revealed by species-specific molecular analysis as having been caused by P. knowlesi [P. knowlesi infections [P. knowlesi poses also challenges on malaria prevention efforts, as transmission appears to frequently occur outside homes, rendering bed net use futile [knowlesi  . Also pfections . P. knowe futile .P. knowlesi infections are analogously considered very rare events. In the available literature, there have been five cases reported in returning German travellers altogether, which have all been visitors to Thailand [In German travellers returning from Southeast Asia, Thailand –19.P. knowlesi infection in a returning German traveller from Thailand, and highlights the difficulties in accomplishing the correct diagnosis. In addition, possible consequences for current travel recommendations are considered.This article is presenting a further case of a The patient, a 45-year-old male Caucasian German, born and residing in the German county of Bavaria, travelled to Thailand for holiday purposes repeatedly over the past years. The most recent journey started on 25 December 2016 with a flight from Munich, Germany to Muscat, Oman, where he had a 2-h stopover, before continuing to Bangkok, Thailand, where he arrived on 26 December. He spent the time from 26 December 2016 to 01 January 2017 in Chiang Mai in the north of Thailand where he visited the Doi Inthanon National Park. On 01 January he travelled to Bangkok where he stayed until 03 January, followed by a stay in Ranong city from 03 to 05 January. He then spent the time from 05 to 11 January on the island of Little Koh Chang in the Andaman Sea, which is part of Ranong Province. Via the city of Ranong he continued back to Bangkok where on 13 January he boarded a flight to Munich, Germany via Muscat, Oman  of the Medical Centre of the University of Munich (LMU) for further investigation.Upon presentation, the patient’s general condition was only slightly reduced, temperature by tympanic measurement was 38.8 °C. The physical examination did not reveal any other remarkable findings. On abdominal ultrasound no pathological findings could be established, particularly the spleen appeared normal in size and ultrasound pattern.P. falciparum and/or P. vivax/P. ovale spp./P. malariae, Dengue Duo  for the detection of the non-structural protein 1 (NS1)/IgM/IgG and the Afinion C-reactive protein (CRP) test (Alere) were conducted. The Plasmodium genus-specific real-time PCR FTD Malaria  was carried out followed by the multiplex PCR FTD Malaria differentiation (Mikrogen) for the detection of P. falciparum, P. vivax, P. ovale spp. and P. malariae. In cases of positive amplification of Plasmodium genus-specific sequences and negative multiplex PCR FTD Malaria differentiation test results, the P. knowlesi nested PCR as first described by Imwong et al. based on a 410 base pair fragment of the species-specific P. knowlesi 18S rRNA gene is applied at DIDTM [P. falciparum and P. vivax antigens was conducted.Laboratory analyses were conducted in-house on whole blood and plasma samples upon attendance of the patient at DIDTM on day 4 after onset of symptoms. Automated full blood count was followed by visual differentiation of thin smears. As stipulated by the malaria emergency diagnostics protocol of DIDTM, screening for parasites was initiated by microscopical examination of thin smears on 100 fields at 400-fold magnification following Diff-Quik staining . Subsequently, Giemsa stained thick smears were examined. Furthermore, the rapid diagnostic tests BinaxNow Malaria  for the detection of antigens of at DIDTM . For immIn addition, a full blood sample was sent to the national reference laboratory for PCR-based malaria differentiation at the Bernhard-Nocht-Institute for Tropical Medicine (BNITM), Hamburg, Germany.As the automated blood count and the CRP rapid test results are conducted in-house at DIDTM, these results were available within the first half hour of presentation, and revealed a marked thrombocytopenia of 79/nl  and an elevated CRP . At the same time the Dengue rapid test and the malaria rapid diagnostic test were negative.Plasmodium infection at an extremely low parasitaemia. In the examination of 100 investigated fields at × 400 magnification only 1 parasite was detected. Consecutively the full thin smear slides were investigated, revealing a total of 3 parasites: one gametocyte and 2 trophozoites (parasite density 0.0002%) (Fig. The thin blood smears revealed a P. vivax. Examination of the thick smear again revealed only 1 structure suggesting a Plasmodium infection without allowing further differentiation. With this finding, treatment was initiated with a 3-day course of atovaquone 250 mg/ proguanil 100 mg, 4 tablets daily, as recommended by the national malaria guidelines of the Association of the Scientific Medical Societies in Germany [Plasmodium genus-specific PCR tested positive but the differential PCR revealed negative results for P. falciparum, P. vivax, P. ovale and P. malariae. Both the DIDTM and the national reference laboratory at BNITM returned a positive PCR result for P. knowlesi.The aspects of the identified gametocyte and the trophozoites rather resembled  Germany . Due to P. falciparum and P. vivax remained negative. The patient improved without any signs of adverse treatment effects (Table Fever and sweats settled the evening the first dose of atovaquone/ proguanil was given. The patient was seen again at DIDTM on 18 and 23 January, when repeated blood counts and smears were performed. Thrombocyte levels went down to 75/nl on 18 January, and returned to normal levels by 23 January. Blood smears on both follow-up days remained negative for parasites. The serology for P. knowlesi infection for Thailand, but only P. falciparum and P. vivax infections [P. knowlesi also in other Southeast Asian countries, such as Myanmar and Indonesia [In the currently available literature, knowlesi malaria remains a rather rare event outside Malaysia, and particularly rare in travellers returning from Southeast Asia. Of note, the World Malaria Report 2016 does not state any ndonesia , 23.P. knowlesi, further studies are needed to assess the prevalence of the parasite in the monkey population on this island, which is a hideaway recommendation for tourists that intend to avoid the main crowds on the Thai islands on the gulf side.Based on reported incubation periods for knowlesi malaria of about 9 to 12 days , it seemPlasmodium-species differentiation is hampered, it is recommended to do a differential PCR [P. knowlesi has to be suspected when the commercially available malaria PCR assays reveal a constellation of a positive genus specific sequence together with negative results for species specific sequences, as these usually do not comprise P. knowlesi.As has been described in other publications, knowlesi malaria has to be suspected in situations where commercial rapid diagnostic tests reveal negative results in samples with positive blood smears , 17, 18.tial PCR . In the P. knowlesi and P. falciparum infections, these should be considered as first line presumptive treatments where inconsistent laboratory results hamper an instant conclusive diagnosis. In our patient a rapid cessation of fever could be reached with atovaquone/ proguanil within 12 h of the first administered dose.As both artemisinine combination therapies  and atovP. knowlesi infection. Nevertheless, the authors are just able to present one case in this report, whereas recommendations for malaria prevention have to be based on risk modelling for geographic regions which are based on larger datasets. However, it has to be kept in mind that Thailand remains a favourite travel destination for German tourists, and diagnostic and treatment algorithms for febrile returning travellers have to take into consideration P. knowlesi infections. Diagnosis can be challenging due to ambiguous morphology of plasmodial parasites, very low levels of parasitaemia and negative results in commercially available rapid diagnostic tests. Treatment with atovaquone/ proguanil showed to be effective with rapid clinical improvement.It is noteworthy that malaria prevention guidelines regarding travels to Thailand do differ between Germany, Switzerland, the UK and the USA. Both the German and the Swiss guidelines do not recommend antimalarial chemoprophylaxis for any region of Thailand, but recommend provision of standby medication for stays in the border regions particularly when outside reach of health care facilities , 5. Cont


Malaria control interventions have led to a decline in transmission intensity in many endemic areas, and resulted in elimination in some areas. This decline, however, will lead to delayed acquisition of protective immunity and thus impact disease manifestation and outcomes. Therefore, the variation in clinical and haematological parameters in children with malaria was assessed across three areas in Ghana with varying transmission intensities.A total of 568 children between the ages of 2 and 14 years with confirmed malaria were recruited in hospitals in three areas with varying transmission intensities (Kintampo > Navrongo > Accra) and a comprehensive analysis of parasitological, clinical, haematological and socio-economic parameters was performed.P = 0.016), although this was not significant in Accra and Navrongo. Parasitaemia levels were not a significant predictor of haemoglobin level after controlling for age and gender. However, higher haemoglobin levels in children were associated with certain socioeconomic factors, such as having fathers who had any type of employment (P < 0.05) and mothers who were teachers (P < 0.05).Areas of lower malaria transmission tended to have lower disease severity in children with malaria, characterized by lower parasitaemias and higher haemoglobin levels. In addition, total white cell counts and percent lymphocytes decreased with decreasing transmission intensity. The heterozygous sickle haemoglobin genotype was protective against disease severity in Kintampo (The findings demonstrate significant differences in the haematological presentation and severity of malaria among areas with different transmission intensity in Ghana, indicating that these factors need to be considered in planning the management of the disease as the endemicity is expected to decline after control interventions. Plasmodium falciparum, which accounts for more than 90% of malaria deaths globally  are highest in Kintampo (>250 ib/p/y), followed by Navrongo (<200 ib/p/y), and lowest in Accra (<50 ib/p/y) –26. A deChildren between the ages of 2–14 years who had lived in the community for at least 6 months and presented with symptoms of malaria were screened for malaria with rapid diagnostic tests (RDTs) using a drop of blood from a finger prick. After obtaining informed consent from guardians of RDT-positive children, 0.5 mL of blood was collected into EDTA-coated vacutainer tubes (BD biosciences) for parasitological and haematological analysis.Haemoglobin levels, erythrocyte indices , leucocyte indices  were analysed using an automated haematology analyser. Thick blood smears were stained with Giemsa and examined for parasites using oil immersion microscopy. Parasite density per µL of blood was determined by counting the number of parasites per 200 WBCs and multiplying by the total WBC count obtained from an automated haematology analyser.Haemoglobin types were determined by Hb electrophoresis according to manufacturer’s instructions . Briefly, whole blood was haemolysed using haemolysing reagent containing 0.005 M EDTA with 0.01% potassium cyanide. Patient haemolysates and controls were then loaded on to cellulose acetate plates and electrophoresed at 350 V for 25 min in a Tris–EDTA/boric acid buffer . Hb electrophorotypes were identified by comparing to control bands.P. falciparum parasitaemia (of any density) without anaemia ; (2) mild malarial anaemia (MMA): children with a malaria-positive smear for P. falciparum parasitaemia (of any density) with 8.0 ≤ Hb < 11 g/dL; (3) Moderate-to-severe malarial anaemia (MSMA): children with a malaria-positive smear for P. falciparum parasitaemia (of any density) with Hb <8.0 g/dL. For some analyses, participants were classified according to parasite density, using ≥10,000 parasites/µL as definition for high-density parasitaemia.Studies have shown that the WHO definition of severe malarial anaemia of <5.0 g/dL with ≥10,000 parasites/µL is restrictive as it excludes children who do not meet the criteria of >10,000 parasite/µL, but still have potentially fatal malarial anaemia . In ordeP < 0.05 was considered statistically significant.Data were analysed using Minitab (version 17). Kolmogorov–Smirnov tests were used to determine normality of the data before application of further tests. One way ANOVA or Kruskal–Wallis tests were applied for across group comparisons for normally and non-normally distributed data respectively. Post-hoc tests of either student t test or Mann–Whitney U tests were applied for pairwise analysis where necessary. Chi square analysis was used to compare categorical variables and determine likelihood ratio for association. Univariate and multivariate regression analyses were carried out to determine the predictors of malaria severity. For all analyses, P. falciparum transmission (Kintampo > Navrongo > Accra) were enrolled. While there was no difference in the proportion of females recruited at each site . However, there was no significant difference in haemoglobin levels in children with HbC genotypes relative to those with the HbAA genotype , MMA  and MSMA (Hb < 8.0 g/dL n = 65). The relationship between malaria severity and various demographic, clinical and haematological parameters was then examined by comparing across the malaria groups. Children with UM were significantly older than children with MMA (P < 0.001) and MSMA  and anaemia severity. There was however, evidence of the protective effects of HbAS, such that children with this genotype had a significantly lower proportion of MSMA (P = 0.011) and a higher proportion of UM (P < 0.001) compared to the HbAA group Fig. d. There To determine indicators of disease severity, haematological indices were compared across the three categories. With the exception of RDW, all red cell indices examined were lower in the malarial anaemia groups compared to UM Table , which wMultiple regression analyses were performed to determine the socio-demographic predictors of haemoglobin level and parasitaemia, as these are the main clinical indicators of anaemia severity in this cohort. Although several factors were associated with haemoglobin levels in univariate analyses  and the self-reported use of bed nets. As expected, children in Navrongo and Kintampo were more likely to have higher parasitaemia, which is consistent with the reported EIR for these areas.As a result of concerted intervention strategies, malaria transmission in endemic areas has been declining in the last decade, resulting in a cumulative drop in the incidence of malaria cases worldwide . HoweverMalarial anaemia severity was significantly associated with erythropenia, lymphocytosis, monocytosis, agranulosis, reticulocytosis and thrombocytopenia. Thrombocytopaenia is commonly associated with malaria, and is a marker of malaria severity , 35. RedMultivariate analysis revealed that the educational status of the father as well as the economic status of the household are major predictors of the severity of anaemia. Typically in African households, decision-making is the responsibility of the father and hence his level of education and understanding of malaria, as well as the availability of funds would determine whether healthcare would be sought early. In addition, better education may influence nutritional choices, such as intake of vitamins and minerals, which have an impact on haemoglobin levels. Recent reports by Diiro and colleagues in Kenya have shown that households headed by males with a higher level of education were at lower risk of malaria . ChildreThe results presented in this study demonstrate significant changes in the clinical manifestations as well as the determinants of diseases severity of malaria as transmission intensity decreases. As transmission intensity decreases, the average age of children presenting to hospital with malaria increases while the risk of developing anaemia decreases. Children living in households where the head-of household had Middle school education or better were less likely to develop high density parasitaemia.


Plasmodium falciparum antigens, as a tool for assessing malaria transmission intensity along the border area of China-Myanmar.Following the decline of malaria transmission in many countries and regions, serological parameters have become particularly useful for estimating malaria transmission in low-intensity areas. This study evaluated a novel serological marker, Malaria Random Constructed Antigen-1 (M.RCAg-1), which contains 11 epitopes from eight Plasmodium falciparum and P. vivax patients was used to detect the properties of M.RCAg-1 and antibody responses. Cross-sectional surveys were conducted at the China-Myanmar border and in Hainan province in 2012 and 2013 using cluster sampling. Filter blood spot papers were collected from all participants. Antibodies against M.RCAg-1 were detected using indirect ELISA. The Mann–Whitney test and Spearman’s rank correlation test were performed to analyze antibody data. P. falciparum malaria transmission intensity was estimated using a catalytic conversion model based on the maximum likelihood of generating a community seroconversion rate (SCR).Serum from P. falciparum patients and had very limited cross-reactivity with P. vivax infection. The total amount of IgG antibodies was decreased with the decrease in parasitemia after taking medication and lasted several weeks. In a population survey, the antibody levels were higher in residents living close to the China-Myanmar border than those living in non-epidemic areas (P < 0.0001), but no significant difference was observed between residents from Hainan and non-epidemic areas. The calculated SCR was 0.0128 for Jieyangka, 0.004 for Susuzhai, 0.0047 for Qiushan, and 0.043 for Kayahe. The estimated exposure rate obtained from the anti-M.RCAg-1 antibody level correlated with traditional measures of transmission intensity derived from altitude.M.RCAg-1 was well-recognized by the naturally acquired anti-malaria antibodies in P. falciparum malaria, especially for malaria surveillance in low transmission areas.Our study demonstrates that M.RCAg-1 is potentially useful as a serological indicator of exposure to The online version of this article (doi:10.1186/s40249-016-0194-x) contains supplementary material, which is available to authorized users. Please see Additional file Plasmodium falciparum (Pf) [Pf transmission intensity can help us understand the disease burden and the risk of being infected, provide guidance for control and prevention strategies and confirm an area is Pf malaria-free with reliable evidence. Until now, the industry standard for malaria transmission intensity has been entomological inoculation rate (EIR), which is time-consuming, expensive, and imprecise in low-transmission districts [Pf transmission intensity. However, these methods are not sensitive enough in communities and are inaccurate, especially in areas of low transmission.Approximately 3.3 billion people are still at risk for malaria globally, most of them infected by rum (Pf) , even thrum (Pf) . Unlike istricts . Other m19 (MSP-119). Antibodies elicited by these well-characterized Pf antigens have been tested at relatively stable rates in some communities. However, each of the individual antigens has its own limitations. For example, MSP-2 with a high rate of polymorphism [19 has been used to estimate Pf malaria transmission in many African areas [Pf transmission in the short-term. Considering of great individual variations in antibody responses and multiple malaria antigens expressed during the process of Pf infection [To efficiently assess malaria transmission and endemicity, many researchers have suggested that serological parameters offer more advantages than other approaches, such as EIR –8, becau19 MSP-9. Antibomorphism  will undmorphism ; saturatmorphism , even atmorphism ; and MSPan areas , but thenfection , antibodnfection . A multinfection . HoweverPf exposure for not only communities [Pf in different populations is still difficult to avoid. Our lab has successfully constructed a multi-epitope chimeric protein, Malaria Random Constructed Antigen-1 (M.RCAg-1) [Pf antigens. This chimeric antigen was selected from a DNA library containing thousands of diverse multi-epitope chimeric antigen genes constructed using epitope shuffling and an isocaudamer technique due to its high specific immunogenicity and anti-parasite efficacy [More novel serological biomarkers are being found that can accurately estimate recent munities , but als.RCAg-1) , which cefficacy .Pf malaria transmission dynamics. Using a simple indirect ELISA, we detected anti-M.RCAg-1 antibodies in the serum of Pf-infected patients from Pf malaria endemic areas or non-endemic areas. The serological parameter obtained in our study demonstrated that this chimeric antigen can be used as an indicator to estimate malaria transmission dynamics.The objective of this study was to estimate whether M.RCAg-1 can be used as an indicator of Pf patients and 38 Plasmodium vivax (Pv) patients were enrolled in the study. The population details have been described elsewhere [Serum samples were collected from malaria patients in Laza, Myanmar, between September and December 2008 to detect antibody responses against M.RCAg-1. The patients were diagnosed by microscopic examination of Giemsa-stained blood smears and treated promptly with the appropriate antimalarial and supportive therapy. Four plasma samples were collected from each patient: prior to drug therapy (D0), the first day of treatment (D1), the third day after treatment (D3), and the seventh day after treatment (D7). A Giemsa-stained blood smear was prepared at the same time plasma samples were collected. Plasma was used for the detection of antibody against M.RCAg-1 and epitopes. Giemsa-stained blood smears were used to determine parasitemia levels and the Plasmodium species. A total of 67 lsewhere .Pf malaria, has decreased significantly in recent years because of the implementation of malaria control strategies. Only 15 indigenous Pf malaria cases were reported in Yunnan province in 2012–2013 [Pf malaria cases was much greater in Laza, Myanmar, than Yunnan province in China; 415 Pf malaria cases were reported in 2012–2013 [This study comprised two cross-sectional surveys conducted in the border area of China-Myanmar and Hainan province of China. The border area in Yunnan province with Myanmar , 28 was 012–2013 ; thus, t012–2013 . One vilPf malaria case has been reported since 2010 [Pf epidemic area.Hainan province used to be a malaria endemic area with the highest disease burden, but no locally acquired nce 2010 , 32. In Cluster sampling was used in this study, and residents in all age groups were included from each village. Pregnant women were excluded from this study. All participants were requested to provide finger blood for immediate microscopy (thick and thin blood smears) and dried blood spots on filter paper (Whatman 3MM Chr) for subsequent serological tests. Two professional microscopists diagnosed all of the participants independently after reading the blood films. All of the blood filter papers were put in sealed plastic bags and stored at −80 °C after being dried at room temperature with abundant self-indicating silica desiccants. Information on gender and age was recorded.A total of 108 healthy volunteer donors from the General Hospital of Chinese People’s Liberation, Beijing, China, who had never been exposed to malaria were chosen as non-epidemic controls. Blood plasma and dried blood spots were prepared from each control. The plasma was used as a control for the plasma from malaria patients and the blood spots as a control for the dried blood spots collected from study sites.Ethical approval was received from the Institutional Review Board (IRB) of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences. Informed consent was obtained from all participants, and from guardians for children <15 years of age.Escherichia coli and prepared and purified by the Institute of Process Engineering, Chinese Academy of Science. The purity of the protein was verified through HP-SEC and RP-HPLC analysis and the stabilities examined by SDS-PAGE at regular intervals. All batches of proteins we obtained had good stability with purity >95 %. All epitopes were synthesized by China Peptides Co., Ltd, and the quality confirmed through mass spectrometry and HPLC analysis. The purity of all of the epitopes (from Epitope-1 to Epitope-11) included in the M.RCAg-1 protein was >95 %. All proteins and epitopes were split charged and stored at −80 °C.M.RCAg-1 proteins were produced in Antibodies were recovered from the blood spots following a propositional method . Plastic2CO3 (pH 9.2) at 1 μg/ml (or 5 μg/ml for epitopes), coated on high absorbance plates (Corning), and kept at 4 °C overnight. After washing five times with PBST, the plates were blocked with 3 % (vol/vol) sheep serum in PBS following incubation for 2 h at 37 °C. Plasma diluted 1∶200 with blocking buffer or 100 μl of eluate from blood spots was added in duplicate and incubated and washed as described above. Next, the plates were incubated with peroxidase-conjugated goat anti-human IgG antibodies (Sigma) at a dilution of 1∶20 000. H2O2 with tetramethylbenzidine  was chosen as the chromogenic substrate and the reaction terminated with 1 M H2SO4 after 10 min. The optical density (OD) was determined at both 450 and 630 nm using a microplate reader . Each plate contained three negative control serum samples from Beijing donors who had never been exposed to malaria and two positive control serum samples from malaria patients with high antimalarial antibody concentrations. All antibody values were expressed in arbitrary units (AU) calculated by dividing the mean OD by the mean OD + 3 standard deviations (SDs) for the three negative controls tested simultaneously. These relative OD values were referred to as OD %.IgG antibodies to M.RCAg-1 protein and its epitopes were measured by indirect ELISA. Briefly, M.RCAg-1 was dissolved in 0.1 M NaDifferences between the antibody concentrations of two groups were analyzed using the Mann-Whitney test. Spearman’s rank correlation test was used to test the relationship between antibody levels and different factors, including altitude, age, and parasitemia. The mean titer of the non-epidemic controls + 2 SDs was set as the cutoff and the antibody data dichotomized as seronegative or seropositive. A simple reversible catalytic model was fitted to the dichotomized data using maximum likelihood methods . The modPv infections were detected in Jieyangka and 5 in Qiushan, Yunnan. No clinical symptom, such as fever, occurred. As no Pf infection was detected in the participants, it confirmed that the Pf malaria transmission intensity was low in all of these areas and the parasite prevalence not suitable for estimating the Pf malaria exposure level in these areas.Details about the study population are given in Table Pf patients than in Pv patients (P < 0.0001) and negative controls (P < 0.0001), and there was very limited cross-reactivity of M.RCAg-1 with Pv infection  in Pf patient serum   Fig. . The lon01) Fig. , suggestPf malaria, especially in low transmission areas, we tested the antibody levels in eluates from blood spots collected from residents living in Hainan province and along the China-Myanmar border. We found no significant differences in the antibody levels of residents from Hainan and participants from Beijing (P = 0.176). However, on the China-Myanmar border, the antibody levels of residents from the four villages were significantly higher than the levels in participants from Beijing (P < 0.0001). In the three villages in Yunnan province, antibody levels decreased with increasing elevation .To determine whether M.RCAg-1 can be used to estimate exposure to Pf infection was more serious in this area. The parameter ρ was set to 0 in this study [Pf malaria transmission in Jieyangka was underestimated here, especially in children under 14 years of age. Importantly, we identified a semi-logarithmic relationship between the village-specific rate of seroconversion (λ) and altitude, and the log (λ) linearly correlated with log (EIR), which was estimated from altitude [To obtain the parameter λ for each village, we calculated the seroprevalence data for each settlement stratified by age Fig. , and theis study . Parametaltitude  Fig. 7)Pf infectPf antigens that have been shown to play essential roles in malaria immune responses [Even though serological biomarker detection is theoretically the best tool for malaria surveillance, the properties of selected antigens are an important point of the technique. Only sensitive and specific antibodies can fulfill the requirements for estimating the malaria exposure level in a population precisely, monitor the changes over time and the impact of intervention on transmission, and confirm the elimination of malaria. The chimeric antigen M.RCAg-1 was identified using malaria parasite-immunized animal serum from a library containing thousands of chimeric antigen genes . This anesponses , 37–39. esponses , and theesponses , 41.Pf-infected patients from Myanmar but not the serum of healthy individuals from several locations in China, which is in line with previous data from Brazil and Cameroon [Pf-infected patients, even if at different levels, suggesting that M.RCAg-1 is a well-recognized chimeric antigen to the naturally acquired anti-malaria antibodies and has the advantage of few polymorphisms. In addition, we found that anti-M.RCAg-1 antibodies rapidly appeared in the sera of patients when the infection started, confirming the sensitivity of M.RCAg-1 to Pf infection.Our data demonstrate that M.RCAg-1 can be recognized by serum from Cameroon . Our datPf-infected patients than Pv-infected patients, we conclude that it is a Pf-specific sero-biomarker when used in malaria sero-surveillance.The specificity of sero-surveillance tests could be influenced by the potential cross-reactivity of antibodies to antigens from different malaria species –44. Ther19), a serological assay may not distinguish significant recent deviations from the historic pattern of transmission [One of the potential disadvantages of the serological approach is that, if antibody responses are very long-lived (such as MSP-1smission . Some stsmission , 46. In In endemic areas, a decrease in malaria transmission will always wane the population immunity, which makes the local residents vulnerable to outbreaks of this disease. Therefore, immune monitoring is necessary for identifying the susceptible population and helping provide additional protective interventions, as demonstrated by Richards, who showed that sero-surveillance has the potential to indicate population immunity except exposure . In thisPf malaria transmission have been reported to be dramatically decreased [Pf infection has been reported in Hainan province since 2010, we conclude that M.RCAg-1 can be used to confirm the elimination of Pf malaria. Moreover, the anti-M.RCAg-1 antibody levels increased with the increasing age, which can be explained by the cumulative exposure to malaria parasites over time [In our study, all of the areas selected for evaluating whether M.RCAg-1 can be used as a serological marker were areas where ecreased  and cannver time , 52.Altitude has long been assumed to represent a proxy for malaria transmission  because Pf malaria exposure in Laza is much more serious than in Yunnan. Our data are in line with a report on the malaria situation in the same areas [From our studies, seropositivity is mainly due to adults in Yunnan province in China, whereas the seroprevalence is high in children under 14 years of age in Laza, Myanmar, meaning that the recent or on-going me areas  that menme areas , 54.E. coli expression system using a routine process. In addition, to test the serum antibodies with an indirect ELISA assay, all of these specialties make the task not only simple, but also inexpensive.Several reports have shown that using multiple antigens or epitopes in sero-surveillance assays has advantages over the use of one antigen , 47, 55.Pf malaria transmission. However, more investigations in different populations are needed to confirm its usefulness and ensure the generalizability of results.Many challenges still exist for malaria elimination as the focus shifts from the detection of symptomatic patients to the detection and clearance of all infections. Effective tests capable of estimating and monitoring transmission intensity with high sensitivity and accuracy in the field will play an important role. This study indicates that multi-epitope chimeric antigens may have a great advantage as serological markers to estimate Pf malaria transmission intensity, especially at a fairly low level, to monitor its recent trends, and to confirm the elimination of Pf malaria infection in malaria surveillance systems.Our study demonstrates that the chimeric multi-epitope antigen M.RCAg-1 has the potential as a sero-biomarker to estimate the


Studies designed to determine the effects of mass administration of azithromycin on trachoma have suggested that mass azithromycin distributions may also reduce the prevalence of malaria. These studies have typically examined the impact of a small number of treatments over short durations. In this prespecified substudy of a cluster-randomized trial for trachoma, we compared malaria parasitemia prevalence in 24 communities in Niger randomized to receive either annual or biannual mass azithromycin distributions over 3 years. The 12 communities randomized to annual azithromycin received three treatments during the high-transmission season, and the 12 communities randomized to biannual azithromycin received a total of six treatments: three during the high-transmission season and three during the low-transmission season. Blood samples were taken to assess malariometric indices among children in all study communities at a single time point during the high-transmission season after 3 years of the intervention. No significant differences were identified in malaria parasitemia, parasite density, or hemoglobin concentration between the annual and biannual treatment arms. When compared with annual mass azithromycin alone, additional mass azithromycin distributions given during the low-transmission season did not significantly reduce the subsequent prevalence of malaria parasitemia or parasite density after 3 years, as measured during the high-transmission season. Studies have also identified potential secondary benefits of mass azithromycin given for trachoma among children, including reductions in the prevalence of malaria and its sequelae.–Mass distributions of oral azithromycin are currently used to control ocular strains of ,A previously published substudy of a large, cluster-randomized trial for trachoma conducted in Niger found that malaria parasitemia among children was significantly lower in communities that received two mass distributions of azithromycin compared with communities that received one distribution.The Partnership for the Rapid Elimination of Trachoma was a group of cluster-randomized trials conducted in The Gambia, Niger, and Tanzania .A 2 × 2 factorial design was used to evaluate both varying treatment frequencies and targeted treatment coverage levels. Communities were randomized into four arms with 12 communities each: 1) annual treatment at standard (80%) coverage, 2) annual treatment at enhanced (90%) coverage, 3) biannual treatment at standard (80%) coverage, and 4) biannual treatment at enhanced (90%) coverage. In this report, the two enhanced coverage arms comparing annual to biannual treatment at a target coverage of 90% are examined (2 and 4 in the preceding list). Within each CSI, stratified blocked randomization of communities was performed based on clinical trachoma prevalence in children as described previously.www.r-project.org) was used to generate the random allocation sequence of clusters (Travis C. Porco).The statistical package R .Community residents were advised to alert village health workers within 2 weeks after mass treatment if they and/or their children experienced an adverse event, defined as diarrhea, nausea, and vomiting for more than 2 days, hospitalization for any cause, or death. Residents were referred to the nearest health-care center as needed.Blood samples were collected from children in both the annual and biannual treatment arms at 36 months after study initiation. The study aimed to collect thick blood smears and hemoglobin concentration from 50 children in each study community. Sixty-two children 6–60 months of age from each community were randomly selected from the most recent census prior to field data collection. If a community had fewer than 50 children, blood specimens were taken from all children. After obtaining verbal consent from a parent or guardian for each study participant, thick blood smears and hemoglobin concentration were collected at a centralized exam station in each community according to methods previously described.Plasmodium parasites on the slides. The microscopists were masked to treatment arm. If both microscopists observed parasites, then the smear was considered positive. Discordant slides were considered negative. To assess parasite density, the microscopists counted the number of asexual parasites per 200 white blood cells .Thick blood smears were collected on glass slides, air-dried, and stored at room temperature. Two experienced microscopists at the Zinder Regional Hospital in Niger stained the thick blood smears with 3% Giemsa and used a light microscope to determine the presence or absence of We estimated that 24 communities (12 communities per arm) would provide greater than 80% power to detect a 3% absolute difference in malaria parasitemia. The sample size calculation assumed a baseline prevalence of 10% and an intraclass correlation coefficient (ICC) of 0.075.t test. To ensure results were not dependent on assumptions, sensitivity analyses were conducted with nonparametric tests (Wilcoxon signed rank) at the community level. A mixed-effects logistic regression model was used to examine effects at the individual level while clustering for community. Secondary outcomes, parasite density, gametocytemia, and hemoglobin concentration, were assessed similarly. All analyses were performed as intent-to-treat using Stata 13 .The primary analysis accounted for clustering at the level of the randomization unit (community) by comparing the community prevalence of malaria parasitemia in the annual and biannual treatments at 36 months after study initiation arms using a paired Ethical approval was obtained from the University of California, San Francisco Committee for Human Research and the Comité d’Ethique du Niger . This study is registered at clinicaltrials.gov (NCT00792922) and was implemented in accordance with the Declaration of Helsinki. Given the high rates of illiteracy in the study area, the institutional review boards approved verbal informed consent, both from the local chiefs of each community before randomization and each child participant’s parent or guardian before data collection.From May 2010 to September 2013, 24 communities in the enhanced (90%) coverage arm of the main Niger trial were followed. At baseline, the 12 communities randomized to annual mass azithromycin had a mean of 138 children  and the 12 biannually treated communities had a mean of 125 children  6–60 months of age . As showAll communities received three mass antibiotic distributions during the high-transmission season . The 12 communities randomized to biannual mass azithromycin were treated an additional three times during the low-transmission season . Treatment coverage is shown in Children from each study community were randomly selected for this substudy from the most recent census in May 2013. Thick blood smears were collected from a total of 1,032 children in 24 communities  3 years after enrollment . Hemoglobin concentration was collected from 1,033 children . Specimen collection occurred more than 1 year after the last treatment in the annually treated communities and approximately 8 months after the last treatment in the biannually treated communities.P = 0.995). A mixed-effects logistic regression model with treatment arm as a fixed effect and community as a random effect showed similar results . No significant differences were seen in community-level parasite density, gametocytemia, or hemoglobin concentration when comparing annually treated communities to biannually treated communities. Analyses conducted with Wilcoxon signed rank tests and mixed-effects regression models for each of these three assessments showed similar results.Grades for the presence or absence of malaria parasites were concordant between the two microscopists in 99.4% of samples with an ICC of 0.994 . Comparing the two assessments of parasite density resulted in an ICC of 0.957 (95% CI = 0.951–0.962). In this cluster-randomized trial, we were unable to demonstrate a difference in malaria parasitemia, parasite density, or hemoglobin concentration between children who received annual or biannual treatment with azithromycin after 3 years of the intervention. A previous study in Niger found a significant reduction in malaria parasitemia and parasite density in communities with an additional treatment during the low-transmission season compared with communities with a single mass treatment after 1 year.Plasmodium species resistance after mass azithromycin for trachoma in the area.There are several possible explanations for the discrepancy in results between annual and biannual azithromycin after 1 year compared with 3 years. Azithromycin may lose effectiveness after multiple mass treatments due to increasing resistance. A previous study suggested that azithromcyin–artesunate treatment failure may have resulted from Another explanation for the difference in study results could be that the previous study assessed malaria outcomes in the low-transmission season and the present study assessed outcomes in the high-transmission season. Single mass treatments with azithromycin have shown reductions in malaria prevalence immediately after treatment, but these reductions are not maintained over time without additional treatment.,This study has several important limitations. First, no baseline assessment of the prevalence of malarial parasitemia was conducted, which may have revealed whether parasitemia decreased in both arms. Baseline prevalence could have offered a more powerful study design, though randomized posttest analysis does permit valid inference,After 3 years of multiple distributions of mass azithromycin, we were unable to show a significant reduction in community-level prevalence of malaria parasitemia during the high-transmission season in communities randomized to biannual versus annual treatment. In this setting, additional mass azithromycin distributions during the low-transmission seasons did not reduce community-level prevalence of malaria parasitemia or parasite density. Mass azithromycin may have a modest impact on malaria prevalence during the low-transmission season, but there may be little protection from reinfection after a single treatment during the high-transmission season. Additional studies could examine malaria risk over time to better compare the impacts of different azithromycin distributions on malaria prevalence during both the low- and high-transmission seasons.


Fever remains a major public health problem. In Burkina Faso, more than half of febrile children are considered not to be infected by malaria. This study prospectively assessed probable (treatable) causes of fever in Burkinabe children.A prospective study was conducted among febrile children (≥37.5 °C) under 5 years of age presenting at four health facilities and one referral hospital in rural Burkina Faso. From each participant, blood was collected for malaria microscopy and culture, urine for dipstick testing and culturing if tested positive for leucocytes and nitrite, stool for rotavirus/adenovirus testing, culture and parasitology, and a nasopharyngeal swab for culture.Plasmodium falciparum malaria was found in 49.7% (340/684) of the participants and non-malaria infections in 49.1% (336/684) of children. The non-nalaria infections included gastro-intestinal infections (37.0%), common bacterial pathogens of nasopharynx (24.3%), bacterial bloodstream infections (6.0%) and urinary tract infections (1.8%). Nearly 45% (154/340) of the malaria infected children were co-infected with non-nalaria infections, but only 3.2% (11/340) of these co-infections could be considered as a possible alternative cause of fever. In contrast, in the malaria microscopy negative children 18.0% (62/344) of the infections could be the probable cause of the fever. Pathogens were not isolated from 23.7% (162/684) of the febrile cases.In total 684 febrile children were included in the study. Malaria remains the most common pathogen found in febrile children in Burkina Faso. However, a relative high number of febrile children had non-malaria infections. The correct diagnosis of these non-malaria fevers is a major concern, and there is an urgent need to develop more point-of-care diagnostic tests and capacities to identify and treat the causes of these fevers.The online version of this article (10.1186/s40249-018-0442-3) contains supplementary material, which is available to authorized users. Please Additional file Febrile illnesses remain a major public health problem in Sub-Saharan Africa (SSA). Fever is the most common clinical symptom found in children under five years of age presenting at health facilities , 2. NowaThe various possible aetiologies of a febrile illness are difficult to distinguish when only information from medical history and physical examination are available , 15. LabThe present prospective study was designed and conducted to identify the pathogens that could cause fever in children seeking care at five health centres in rural Burkina Faso. Febrile patients were evaluated for a wide range of infections, with a focus on treatable bacterial and parasitic infections, using conventional diagnostic. This study contributes to a better understanding of possible causes of fever in children under 5 years of age living in a malaria endemic region.This study was conducted in the rural district of Nanoro, Burkina Faso. Four peripheral health facilities  and the referral hospital Centre Medical Saint Camille of Nanoro were involved in the data and clinical specimen collection. The choice of the peripheral health facilities was based on their accessibility for patients and their distance to the research laboratory (maximum of 20 km). The district hospital was included as recruitment centre in order to be able to capture also the more severely ill children who were referred from peripheral health facilities.Haemophilus influenzae type b was introduced into the extended program of immunization (EPI) in January 2006  and agaA prospective study was conducted from January–December 2015. The study was approved by the National Ethical Committee in Health Research, Burkina Faso (Deliberation N°2014–11-130). All children under 5 years of age with an axillary temperature ≥ 37.5 °C presenting at one of the participating health facilities or the referral hospital were asked to participate in the study. Written informed consent was obtained from parent or legal guardian prior to enrolment. The medical history and findings from clinical examination of the child were recorded on a standard Case Report Form (CRF) by trained study nurses. The following clinical specimens were collected from each participant: blood, urine, stool and a nasopharyngeal swab. Parents or adult guardians were asked to complete sample collection within 48 h following inclusion when diagnostic specimen collection (stool and/or urine) could not be realized during the enrolment procedure. They were provided with instructions and sterile containers for that purpose. The parents or guardians were told to return the samples immediately after collection.The participants were managed according to the national guidelines. If culture results or other laboratory tests justified additional or alternative treatment, the results were forwarded to the health facility. The parents or guardians were contacted as soon as possible to arrange that the necessary treatment would be given to the participant.Nasopharyngeal swab and blood samples were also collected in Skim Milk-Tryptone-Glucose-Glycerol (STGG) and Ethylene Diamine Tetra-Acetic Acid (EDTA) tube respectively. All samples were transported to the central microbiology laboratory of the Clinical Research Unit of Nanoro (CRUN) where the various laboratory tests were conducted.Plasmodium species, difference in parasite density > Log10 or ratio > 2 in case of parasite density ≤ 400/μl or > 400/μl) the respective slide was read by a third independent reader to make a final decision. The final parasite counts were the geometric means of the two reader’s results or the geometric means of the two geometrically closest reading in case of third reading. These results were expressed as asexual parasites per microliter by using the patient’s white blood cell (WBC) count.Giemsa stained thick and thin blood smears were prepared and independently read by two experienced microscopists. In case of discordance  according to manufacturer’s instructions.From each child, 1–3 ml of venous blood was directly collected in a paediatric blood culture bottle  and incubated in a BACTEC 9050 instrument (Becton Dickinson) for a total of 5 days. If flagged for bacterial growth, the cultures were subsequently Gram stained, sub-cultured on Eosin-Methylene Blue (EMB) agar, 5% Sheep Blood agar  and chocolate + isovitalex agar, and incubated at 35–37 °C for 24 h. Isolates were identified by standard microbiological methods.Salmonella and Shigella agar (SS agar). To assess the prevalence of rotavirus and adenovirus after the introduction of vaccine against rotavirus in 2013, stool samples were also analysed for group A rotavirus using one step rotavirus and adenovirus serotype 40/41 in human faeces .Fresh stool smears were prepared in normal saline and examined by direct microscopy for the presence of intestinal parasites. Parasite elements looked for were: cysts, eggs, and vegetative forms of protozoa. Furthermore, stools were plated on EMB, Hektoen agar and inoculated in selenite of sodium broth and incubated at 35–37 °C. After 4 h of incubation, selenite of sodium broth was sub-cultured on 5 colonies forming units (CFU)/ml were considered significant bacteriuria.Dipstick testing  was done for each urine sample and the parameters collected were: presence or not of blood, protein, leucocytes and nitrite in the sample. A culture of the sample was done in case testing for leucocytes and nitrate was positive. Urine was plated on Cystine Lactose Electrolyte Deficient and EMB agar when dipstick testing for leucocytes and nitrite was positive. The cultures were incubated at 35–37 °C for 24 h. Only samples that yielded pure bacterial growth of more than 10Streptococcus and/or Staphylococcus species, respectively.A flexible flocked swab was introduced into the nasal passage and two rotations of swabbing were done. The swab was placed into STGG broth for storage and transportation. For analysis, nasopharyngeal swab was vortexed and 200 μl of this broth was inoculated in 10 ml of Todd-Hewitt (TH) broth for enrichment and incubated at 35–37 °C during 18 to 24 h. The TH broth was subsequently plated out and incubated at 35–37 °C for 24 h on sheep blood agar and chocolate + isovitalex agar and chapman  agar to detect For the purpose of this study, the infections found, except malaria, were considered to be non-malaria infections. The non-malaria infections were further classified as either “probable causes of fever” or “non-probable causes of fever”. The infections that could be considered as a “probable cause of fever” in the recruited cases were subsequently further regrouped in: bacterial bloodstream infections (bBSI), viral gastro-intestinal (vGII) caused by rotavirus or adenovirus, and urinary tract infections (UTI) –30. The t-test. Binomial logistic regression was used to compare the relation between the presence of infection and demographic, clinical and laboratory data. The P value < 0.05 was considered as significant.Data management involved double data entry using Open Clinica software. Data analysis was done by using R software version 3.3.1 . Categorical variables were summarized as proportions and Pearson’s Chi-square test or Fisher’s exact test were performed. Continuous variables were described by mean or median and compared by using the Student’s SD] = 14.1) and 29.4% were under 12 months of age. Mean axillary body temperature was 38.7 °C (37.5–41.0 °C)  children were febrile (axillary temperature ≥37.5 °C) and 684 febrile children (85.9%) were included in the study. The study flow chart is presented in Fig. Clinical specimens for blood culture and malaria microscopy were obtained from all study cases. The proportion of stool and urine samples collected was 76.5% 523/684) and 74.1% (507/684), respectively. A small proportion  of nasopharynx cultures could not be performed, because the training of study nurses to collect nasopharyngeal swabs was completed after the start of inclusion . The geometric mean parasite density was 24 003 parasites/μl (range: 72–2 272 500). Malaria was more prevalent in children presenting at the health facilities than at the referral hospital  was found in 49.7% (340/684) of the cases. Malaria occurred whole year round Fig. . All 340Bacteria could be identified in 6.0% (41/684) of blood cultures, 2.0% (10/507) urine cultures, 7.7% (40/523) stools and 24.3% (154/623) nasopharyngeal cultures. Parasites were found in 27.3% (143/523) of the collected stool specimen and 4.8% (25/523) of these samples were positive for rotavirus/adenovirus. Pathogens could not be detected in 162 children (23.7% of the study population)  and all ten positive urine cultures contained Escherichia coli (10/10). Rotavirus was the main virus detected in positive stools tested with rotavirus/adenovirus tests . Pathogens that could be “a probable cause of fever”  were more often found in clinical specimen from children presenting at the referral hospital than in those presenting at the peripheral health facilities  was identified in 10.7% (73/684) of the febrile children that could be “a probable cause of fever” Table . Of thes 60%, 15/5. PathogGiardia intestinalis was the most frequently found parasite in stool samples 46.2% (66/143). Pathogenic bacteria were isolated from 7.7% (40/523) of the stool samples (Table Staphylococcus aureus was the most frequently found species 96.7% (148/153) amongst the common bacterial pathogens of nasopharynx (CBPN). The prevalence of pathogens that were probably not the cause of fever was more or less similar in clinical specimen from children presenting at the referral hospital and in those presenting at the peripheral health facilities  of the febrile children one or more pathogens were identified that could be considered as “non-probable cause of fever”  of the clinical samples obtained from children who were malaria microscopy positive (Table P < 0.001).Additional pathogens, next to OR = 2.85, 95% CI: 2.0–4.1, P < 0.001) or temperature > 39.5 °C . Furthermore, malaria was associated with white blood cell count > 12 × 103 cells/mm3, and haemoglobin concentration < 11 g/dl . Acorrelation between UTI or CBPN and the risk factors studied was not found.Only age > 12 months was significantly associated with GII  [Non-malaria infections that probably contributed to fever in this study were bBSI, UTI and vGII. The prevalence of bBSI was, despite differences in study designs, comparable to those reported in other studies in SSA (4 to 10%) , 38–40. al trend . UTI fous study) , 42–44. s study) . The lowThis study also identified the presence of pathogens that can be considered as “non-probable cause of fever”, such as gastro-intestinal parasites, common bacterial pathogens of the nasopharynx and bacteria in stool. Still these pathogens require attention in patient management as they may affect health, for example in case the protective mucosal barrier of the intestine or nasopharynx is broken and they enter the blood stream , 45. A lThis study showed that malaria remains strongly associated with paediatric fever episodes in Nanoro. A relatively high number of non-malaria infections was found in malaria microscopy positive as well as malaria microscopy negative febrile children. Among these non-malaria infections, a “probable cause of fever” was mainly found in the malaria-negative febrile children. As treatment of several of these non-malaria infections is possible, there is a need for a practical tool to help clinician to screen for treatable causes of the febrile episodes. In the absence of such a diagnostic tool, malaria diagnostic test can be of help for the clinician to decide who should receive additional treatment, but over-or under-treatment will remain likely and puts febrile children at significant risk of morbidity and mortality.Additional file 1:Multilingual abstract in the five official working languages of the United Nations. (PDF 320 kb)


Use of malaria rapid diagnostic tests (RDTs) has improved the management of this disease. We evaluated the validity of the SD-Bioline Malaria-Ag-Pf/Pan™ (Batch 60952) RDT supplied by the Malaria Control Program of the DRCongo.cChildren (n = 460) aged below 5 years seen in curative care (CC) for suspected malaria and in pre-school consultation (PSC) in two rural centers underwent clinical evaluation and capillary blood collection for microscopic reading of thick smear (TS) and thin film (BF), and for RDT. Sensitivity (Se), specificity (Sp), positive (PPV) and negative (NPV) predictive values of the RDT, and the corresponding accuracy and Youden indices were determined using microscopic data as reference. Results were compared using the Chi-square test.Microscopy showed malaria infection in 53.8% of CC and in 10.8% of PSC children. Similar results were obtained using the RDT . Se of the RDT was 82.1%, Sp 92.0%, PPV 88.5% and NPV 87.4%. RDT positivity was significantly (p < 0.01) associated with some symptoms  and with a recent history of malaria attack. In addition, Se of the RDT depended on parasitemia and decreased at low parasite denstity.SD-Bioline Malaria-Ag-Pf/Pan™ RDT has a relatively good sensitivity and specificity but seems useful only for high parasitemia. Negative SD Bioline Malaria Ag Pf/Pan™ RDT should be complemented with microscopy when clinical signs suggest malaria. Malaria remains a major public health concern. It is estimated that in 2013, malaria affected about 200 million people and caused 600 000 deaths [P. falciparum and with mouse monoclonal antibodies specific to lactate dehydrogenase (pLDH) of four Plasmodium species . The antibodies are mixed with colloid gold conjugate and react with the malaria antigen in the patients’ samples. The aim of this study is to contribute to improving the management of malaria cases by ensuring a proper diagnosis using valid and efficient tests.Early diagnosis of the disease remains the basis for improved case management. Laboratory diagnosis of malaria, classically based on microscopy, is faced with problems related to its technical implementation in resource-poor areas . The curThis is a cross-sectional study carried out in two Health Centers, Nuru and Ciranga, in the Katana Health Zone, from January to April 2014, in the middle of the rainy season. Our study population consisted of 460 children aged 6-59 months, including 340 seen during consultations for treatment and 120 seen during pre-school consultations, which in DRC are mandatory for health status suveillance and disease prevention (eg by vaccination) before 5 years of age [Data were analyzed using Epi Info and XL STAT 2014 . The chi-square test was used for comparison of results. The threshold for statistical significance was set at p < 0.05. Parasite density was calculated using the following formula , 7:  and Sp of 85.5% (95%-CI: 81.8 - 88.6) (accuracy 0.86 and Youden index of 0.76) for each species. We noticed a large statistically significant dependence (P < 0.0001) of the positivity of the RDT on the parasite density. Se of the RDT was 100% for parasite densities above 200/μm2 but decreased at lower parastiemias  in the populations studied. It has been noted that P. vivax is absent in Duffy gene-negative populations such as those inhabiting most of Central Africa  but preever see . Our resever see , but simnt women .P. falciparum in our population.The performance for P. falciparum is relatively lower than that found by WHO in 2012 for suspected malaria in Madagascar (Se and Sp of 92.9% and 98.9%) [P. falciparum was the only parasite species. The the SD Bioline RDT is highly sensitive to parasite densities above 200 parasites par μl of blood but loses specificity to distinguish between species, as high parasite density is frequently interpreted as poly parasitism. Because the SD Bioline RDT considers many cases of high parasite density as indicating poly-parasitism and because any polyparasitism case is considered as severe malaria, there is a clear risk to disorient the prescription of ACT. In both health centers (Nuru and Ciranga) several patients were hospitalized and treated with quinine perfusion based on the diagnosis made using the SD Bioline TDR. This carries the risk of promoting the emergence of strains resistant to quinine due to bad indications. Our results are also consistent with those found in the Philippines in 2002, in which a recent history of malaria history correlated with a positive RDT or a detectable parasite density [In the present study, SD Bioline RDT sensitivity (82.1%) and specificity (92.0%) did not reach the values indicated by the manufacturer (99.7%). The false positive rate (11.5%) is high compared with WHO standards that require a false positive rate of less than 10% , 14, 15.d 98.9%) . Similard 98.9%) ). Part oThis study shows that the use of SD Bioline Malaria Ag Pf/Pan ™ RDT to detect malaria parasites is more effective when the parasite density is high. This RDT loses its validity for low parasitemia. These results confirm that RDTs are valid in the diagnosis of malaria if they are complemented by microscopy. The SD Bioline remains a screening test only and is useful for large population screening, with diagnosis in individual cases suspected on infection based only on clinical evaluation requiring confirmation by microscopy. It is recommended that the NMCP should ensure the assessment and monitoring of the various RDTs offered by the different support organizations before adopting them in the health system.Rapid diagnostic tests (RDTs) are widely used for point-of-care diagnosis of Malaria in resource-limited settings;Malaria rapid diagnostic tests (RDTs) has improved the management of this disease;The existence of several RDTs on the market, the possibility of forgery, and the uncertainty about test stability during transport and storage, etc. are all reasons to reinforce quality control.Our Study shows the performance of Malaria RDTs in Eastern Democratic Republic of the Congo (DRC);Negative Malaria RDT should be complemented with microscopy when clinical signs suggest malaria;Positive RDT is correlated with the presence of sweating, recent history of malaria, and presence of chills.The authors declare no competing interest.