Where malaria endemicity is low, control programmes need increasingly sensitive tools for monitoring malaria transmission intensity (MTI) and to better define health priorities. A cross-sectional survey was conducted in a low endemicity area of the Peruvian north-western coast to assess the MTI using both molecular and serological tools.Plasmodium vivax merozoite surface protein-119 (PvMSP119) and to Plasmodium falciparum glutamate-rich protein (PfGLURP) were detected by ELISA. Risk factors for exposure to malaria (seropositivity) were assessed by multivariate survey logistic regression models. Age-specific antibody prevalence of both P. falciparum and P. vivax were analysed using a previously published catalytic conversion model based on maximum likelihood for generating seroconversion rates (SCR).Epidemiological, parasitological and serological data were collected from 2,667 individuals in three settlements of Bellavista district, in May 2010. Parasite infection was detected using microscopy and polymerase chain reaction (PCR). Antibodies to P. vivax, and 0 and 0.04%, respectively for P. falciparum, while seroprevalence was much higher, 13.6% for P. vivax and 9.8% for P. falciparum. Settlement, age and occupation as moto-taxi driver during previous year were significantly associated with P. falciparum exposure, while age and distance to the water drain were associated with P. vivax exposure. Likelihood ratio tests supported age seroprevalence curves with two SCR for both P. vivax and P. falciparum indicating significant changes in the MTI over time. The SCR for PfGLURP was 19-fold lower after 2002 as compared to before (λ1 = 0.022 versus λ2 = 0.431), and the SCR for PvMSP119 was four-fold higher after 2006 as compared to before (λ1 = 0.024 versus λ2 = 0.006).The overall parasite prevalence by microscopy and PCR were extremely low: 0.3 and 0.9%, respectively for Combining molecular and serological tools considerably enhanced the capacity of detecting current and past exposure to malaria infections and related risks factors in this very low endemicity area. This allowed for an improved characterization of the current human reservoir of infections, largely hidden and heterogeneous, as well as providing insights into recent changes in species specific MTIs. This approach will be of key importance for evaluating and monitoring future malaria elimination strategies. Plasmodium falciparum strains to chloroquine (CQ) and sulphadoxine-pyrimethamine (SP) ) and pre-2006 λ2 = 0.006 (95% CI [0.002–0.017]). For PfGLURP, best estimates indicated a 19–fold drop in the SCR approximately eight years prior to the survey (year 2002): post-2002 λ1 = 0.022 (95% CI [0.014–0.033]), and pre-2002 λ2 = 0.431 (95% CI [0.041–4.54]).Age seroprevalence plots for PvMSP1P. falciparum cases were recorded (1998), P. falciparum incidence dropped and stabilized around 4,000–5,000 cases annually in 2000–2001. This was followed by a steady decrease, and since 2006, autochthonous P. falciparum reported cases have become scarce . Similarly to P. falciparum, P. vivax incidence decreased gradually after the ENSO  reaching very low levels during the four-year period of intense droughts, with only 700, 315, 168, and 606 reported cases, respectively from 2004 to 2007. However, since then, P. vivax cases have increased with 4,185, 2,735 and 2,153 cases in 2008, 2009 and 2010, respectively. During the same period, annual rainfall in Piura dramatically dropped from 1998  to minimum values (between 14.3 and 59.4 mm) during the drought period, but subsequently increased since 2008 (193.5 mm in 2008).The retrospective analysis of 20-year annual malaria incidence and rainfall from 1990 to 2010 in Piura department showed that after the El Niño-Southern Oscillation (ENSO) phenomenon in 1997–1998 Figure , when neP. vivax was predominant, species-specific seroprevalence showed that exposure to P. falciparum still occurs in the study area. Interestingly, over the past ten years, both species experienced significant but opposite changes in MTI: while P. falciparum MTI dropped after 2002, the one of P. vivax significantly increased after 2006.The combination of molecular and serological tools, in addition to standard microscopy, allowed for an in-depth characterization of the current malaria transmission pattern as well as recent changes in species-specific MTI in this peri-urban area of the Peruvian northern coast. Most of the malaria infections were submicroscopic (70%), as well as asymptomatic (89%) despite the low level of transmission. Though Despite having been conducted at the end of rainy season, when the health information system usually records the highest malaria incidence , few malIn areas of low malaria transmission with seasonal patterns affected by climatic conditions, such as the Peruvian northern coast region, parasitological indicators determined by microscopy or PCR can be insensitive. In these areas, serological measures can be used as a proxy for malaria transmission intensity ,23,25,36P. falciparum reported cases in Piura department have been extremely rare in the past five years, P. vivax and P. falciparum seroprevalence indicated ongoing exposure to both species. The low PfGLURP seropositivity in young children and the only one P. falciparum case detected by PCR suggest that a residual P. falciparum transmission is still present in the study area. This residual transmission may be due to asymptomatic P. falciparum infections that remain undetected, unless a population-wide survey (such as the present study) is carried out. However, the implementation of mass screening as a public health strategy in low transmission settings remains a matter of debate [Even though f debate ,40,41, af debate .P. falciparum seropositivity could be the presence of antibodies cross-reacting with P. vivax or other infectious agents’ antigens. Limited information is available about the cross-reactivity between P. falciparum and P. vivax antigens [P. vivax infections by PCR and PfGLURP seropositivity, and the absence of correlation between antibody responses to the two species suggest that cross-reactivity is probably minimal. A few studies have suggested the cross-reactive potential of PfGLURP with Schistosoma haematobium[One might argue that an alternative explanation for the observed antigens ,45, and ematobium, which iP. vivax and P. falciparum exposure with increasing age. As antibody responses will persist for prolonged periods, the rate of acquisition in young age groups can be used to determine current malaria transmission intensity [Serologic markers can also be used to determine risk factors for malaria. This is particularly important in low endemicity areas as the one reported here, where the low malaria prevalence jeopardizes the risk factor analysis. In the study area, age was the only independent risk factor that was associated to both species. The age-dependent PvMSP119 and PfGLURP seropositivity indicates cumulative ntensity -25 as weP. vivax malaria episodes also confirmed that PvMSP119 antibody detection measures cumulative exposure. However, the history of P. vivax malaria in the past year was not included in the multivariate risk factor analysis for P. vivax exposure, since it is not a risk factor for P. vivax infections per se but rather a consequence of previous exposure to a given risk factor. Plasmodium vivax infections are on the pathway between risk factors and the presence of P. vivax antibodies.The higher seroprevalence in individuals with several P. vivax and P. falciparum seropositivity reflect the heterogeneity of malaria transmission intensity between species. Age and distance to the water drain were the only two independent risk factors significantly associated with P. vivax exposure. As mentioned above, the effect of age on seroprevalence usually reflects the cumulative exposure to malaria infections occurring with age rather than a higher risk of exposure in adults as compared to children. However, the significant interaction found between age and house distance to the drain, beside the cumulative effect of age on seroprevalence, indicates a significant lower exposure in children aged < ten years. Indeed, the effect of distance (<100 m versus ≥100 m) on P. vivax seropositivity was only significant in adults, indicating that children living close to the drain were not exposed any more to infectious bites compared to children living farther. Water accumulation in the drain provides breeding habitats for Anopheles malaria vectors after rains [P. vivax infection. However, since adults are more likely to have outdoor evening activities, this could explain why children below ten, were no more at risk of malaria while living close to the drain.Differences in the risk factors for er rains ,39, resuP. falciparum seropositivity. Historically, the three study settlements were important foci of P. falciparum transmission during the epidemic of 1998 and the early 2000s [P. falciparum exposure support the hypothesis that transmission occurs not only in the immediate house environment as previously described by Guthmann et al. [An. albimanus bites increases. Occupational risk for malaria has also been found in the Peruvian Amazon Region, where malaria morbidity has been clearly associated with forest-related activities such as farming, land clearing and wood extraction [Besides age, settlement and prior work as moto-taxi driver in the previous year, there were two additional independent risk factors for ly 2000s ,48. Thern et al. , but outtraction ,50.P. falciparum transmission occurred in the study area approximately eight years before the current study (around 2002). This supports the idea that the wide scale implementation of ACT since 2001 has significantly contributed to the reduction of P. falciparum MTI. Indeed, following the 1998 epidemic and the subsequent intensified control interventions implemented [P. falciparum incidence returned to the pre-epidemic levels (1997) by 2000–2001 , and since 2002 it steadily decreased to reach almost undetected levels from 2007 onwards (Figure P. falciparum surge after the drought period in 2007, as observed with P. vivax, advocates for the additional significant effect of the ACT deployment on P falciparum MTI in the northern coast region [The catalytic model used to generate PfGLURP seroprevalence curves suggested that a significant decline in s Figure . In addiP. vivax incidence substantially increased [P. vivax MTI after 2006. Plasmodium vivax transmission in this area seems to be more sensitive than P. falciparum to changes in climatological and environmental conditions, especially since the implementation of ACT. The recent changes in species specific MTIs are also in line with the results of the multivariate risk factor analysis. While children under-five were significantly at lower risk of P. falciparum exposure compared to older children age 6-10, this was not the case for the P. vivax exposure where the under five were at similar risk of exposure compared to the 6-10 years old.Since the end of the drought period and the resuming of the rains, ncreased  (Figure 19 are highly suitable for the detection of antibodies against P. falciparum and P. vivax, respectively [. Multiplexing the serological tests as well as standardizing the antigens used is of crucial importance for monitoring elimination efforts and comparing trends across countries and regions.The number of antigens used in the two serological tests could be a limitation of the study. Due to the degree of sequence variability, it is recommended to use multiple antigens for each species in order to optimize the sensitivity of the antibody detection and to improve the identification of changes in malaria transmission over time in low transmission settings ,38. Howeectively ,52. MultP. vivax with a majority of asymptomatic and sub-microscopic infections. Additionally, serological markers reflected exposure to malaria and allowed for species-specific risk factor analysis, as well as showing recent changes in respective MTIs. In conclusion, the combination of both serological and molecular tools improved detection sensitivity, and provided new insights into recent changes in transmission intensity for both species. This invaluable information can help decision-makers to adjust malaria control strategies in order to maintain current achievements, avoid outbreaks, and possibly move towards the elimination of malaria.Monitoring MTI in areas of low endemicity and seasonal patterns, such as the north-western coast of Peru, poses a major challenge to national malaria control programmes. In this setting, molecular tools confirmed the current malaria transmission pattern characterized by low parasite rates, mainly due to The authors declare that they have no competing interests.AR contributed to the study design, analysed the data and wrote the paper; ALL and VS contributed to the study design and paper review; NS and JC contributed to the data analysis and paper review; JCM contributed to the fieldwork supervision and processed the blood samples with ELISA and PCR; EP and OJP carried out and coordinated the fieldwork; DG contributed to the supervision of the sample processing and paper review; MOP, ISS and MT contributed antigen to the project and reviewed the article; UDA contributed to the study design and reviewed the manuscript; AE contributed to the study design, the data analysis and reviewed the paper. All authors read and approved the final manuscript.


Plasmodium knowlesi and HIV co-infection is reported in a German traveller returning from Thailand. The 54 year-old patient presented to the Institute of Tropical Medicine in Tübingen with a 11-day history of daily fever and chills. Initial microscopic evaluation of Giemsa-stained thin blood smears was suggestive of a mixed infection with Plasmodium falciparum and Plasmodium malariae. However, PCR amplification of small subunit ribosomal RNA gene revealed a P. knowlesi infection. Parasitaemia was 473 parasites/μl and the platelet count was within the normal range. Oral treatment with Malarone® was initiated and resulted in a fast recovery without any complications.A case of P. knowlesi malaria-induced temporary elevation of viral load similar to that reported for P. falciparum and HIV co-infection.As part of routine screening the patient also underwent HIV testing and was found to be HIV positive with a CD4 cell count of 115/μl and a viral load of 34,799 copies/ml. A follow-up measurement of the viral load seven days after the first quantification revealed an increase to 102,000 copies/ml. Three months after the first quantification the viral load had dropped to 10,000 copies/ml without the initiation of antiretroviral treatment. This suggests the possibility of a  Plasmodium capable of causing human malaria. The fifth species is Plasmodium knowlesi, a malaria parasite naturally circulating in long-tailed (Macaca fascicularis) and pig-tailed (Macaca nemestrina) macaques in Southeast Asia. In 2004, Singh and colleagues described the first large focus of naturally acquired P. knowlesi infections in humans in the Kapit division of Sarawak  [P. knowlesi infections throughout Malaysian Borneo [P. knowlesi in patients with microscopically diagnosed malaria ranges from less than 1%  [P. knowlesi malaria to non-endemic countries have been published [P. knowlesi malaria have been described in Southeast Asia. Case-fatality rates of 2%  [P. knowlesi cases were treated with oral artesunate combination therapy or intravenous artesunate [In the last eight years a growing body of literature confirmed the existence of a fifth species of  Borneo) . Thereafn Borneo  and Penin Borneo , as welln Borneo , Vietnamn Borneo , Philippn Borneo , Thailann Borneo , Cambodin Borneo , Singapon Borneo ). The frhailand)  to up tohailand) . Since 2ublished -24. Elevublished . Howeverospital)  and 11% ospital)  have beetesunate .Plasmodium falciparum malaria [P. falciparum malaria causes a transient elevation of viral load in HIV-positive individuals [P. falciparum-infected patients. There is currently no published information about P. knowlesi malaria and HIV co-infection available. This is the first description of P. knowlesi malaria in an HIV-positive adult with advanced immunosuppression.It has been reported that HIV infection increases the susceptibility to malaria ,29 and t malaria -33. At tividuals -36. The P. falciparum ring stage parasites. However, several late trophozoite stage parasites with haemozoin pigment as well as gametocytes with a size of an uninfected red blood cell were identified. Based on these morphologic features the presumptive diagnosis of a co-infection with P. falciparum and Plasmodium malariae was made. Treatment with atovaquone/proguanil 250 mg/100 mg (four tablets/day for three days) was initiated and the patient was admitted to the hospital. During anti-malarial therapy the parasitaemia cleared rapidly. Serial blood smears revealed a parasitaemia of 70/μl after 24 hours and 11/μl after 48 hours of therapy. The platelet count after 24 hours and 48 hours of therapy was 249.000/μl and 257.000 μl respectively.A 54 year-old German traveller presented to the Institute of Tropical Medicine in Tübingen with fever, chills and severe headaches. Twenty days prior to presentation the patient had returned from a four-week trip to southern Thailand where he visited with his Thai wife. He stayed nearly all the time in Phuket and never took malaria chemoprophylaxis (please see below for a more detailed description of the trip). Eleven days prior to presentation the symptoms started with a sudden onset of fever  and chills. Thereafter fever decreased gradually, but the patient continued to experience low grade fevers around 38.5°C until the day of presentation. Eight days prior to presentation the patient noticed a temporary rash on the lower limbs that he related to the intermittent use of Paracetamol. On examination, the patient was in no acute distress but appeared fatigued. The temperature was 37.4°C and vital signs were stable . With the exception of tenderness in the left upper abdominal quadrant the physical examination was unremarkable. Abdominal ultrasound revealed mild splenomegaly (14.1 × 6.2 cm). Laboratory tests showed mild anaemia , elevation of lactate dehydrogenase  and C-reactive protein . Values were normal for platelet count , white cell count , total bilirubin  and plasma creatinine . Rapid diagnostic tests for dengue were negative . Giemsa-stained thick and thin blood smears showed the presence of malaria parasites  showed a 96%-match with P. knowlesi sequences. No other human plasmodial species were identified by the BLAST search. The patient was discharged after three days in improved conditions.Due to the uncertain blood smear identification of In addition, at day of presentation HIV testing was performed as part of routine screening. The patient’s last testing dated four years back and had been negative. Now HIV-1-antibodies were detected by ELISA and confirmed by Western blot. The CD4 cell count was 115/μl and viral load was 34,799 copies/ml. On repeated questioning the patient reported that the relationship with his Thai wife started four years ago. At that point both partners underwent HIV testing and were found to be HIV negative. During the patient’s yearly visits to Thailand the couple had unprotected intercourse. The patient stated that except for his wife he had not had any other sexual partners for the last four years. An illness suggestive of acute HIV could not be recalled.P. knowlesi infection the viral load follow-up revealed a drop to 17,000 copies/ml without antiretroviral therapy. Three months after the P. knowlesi infection, viral load was 10,000 copies/ml still without antiretroviral therapy. The CD4 cell count was 204/μl and 264/μl respectively . He noted that while walking in the local forests and fishing in the Andaman Sea he suffered a lot of mosquito bites. There was no contact with monkeys reported. For long-term HIV care the patient was referred to a specialized HIV centre. Five weeks after the According to official guidelines , the patPlasmodium knowlesi cases have been detected in several regions of Thailand mainly in forestal areas bordering Cambodia in the east, Malaysia in the south and Myanmar in the north-west [P. knowlesi in Ranong Province (southern part of the border to Myanmar) [P. knowlesi malaria in Thailand is low. Among 1874 febrile patients attending malaria clinics in different areas of Thailand the overall prevalence of P. knowlesi was 0.57% [P. knowlesi infection acquired in Thailand [P. knowlesi infections and that due to increasing travel activities to Southeast Asia, imported P. knowlesi malaria might become more important in non-endemic countries.rth-west . A recenMyanmar) , where tas 0.57% . NeverthThailand . This inPlasmodium species transmission is minimal [P. knowlesi infections have been reported. Only on repeat questioning during the follow-up visit did the patient recall a short visit to Ranong Province. This serves as a reminder that malaria has to be excluded in returning travellers from Thailand, even if they report primarily visiting areas with very low incidence of malaria.During the initial questioning the patient stated that he had stayed all the time of the voyage in Phuket where  minimal  and no cP. falciparum malaria that HIV-positive individuals have a temporarily increased viral load during acute malaria [P. knowlesi malaria. A baseline measurement before the acute malaria infection was not available as the HIV infection was diagnosed for the first time. Nevertheless, an increase in viral load was observed shortly after the P. knowlesi infection with a subsequent drop at follow-up examinations five and 12 weeks later without the initiation of antiretroviral therapy. These findings suggest a possible influence of acute P. knowlesi malaria on pre-existing HIV infection, resulting in a temporary increase in viral load as it has been described for acute P. falciparum malaria. However, as this is an observation in one patient it is necessary to assess more cases in order to confirm this finding.It has been shown for  malaria -36. Kubl malaria . A similP. falciparum co-infection and those of HIV-P. knowlesi co-infection. Most P. knowlesi-infected individuals develop clinically uncomplicated malaria, but severe cases with lethal outcomes have been described recently [P. falciparum malaria that HIV-positivity is a risk factor for the development of severe malaria and death [P. falciparum malaria to non-endemic countries showed that severe malaria was more frequent in patients with a CD4 cell count <350/μl, (odds ratio 2.5 and 3.2) [The described finding raises the question as to whether there are other similarities between clinical characteristics of HIV-recently . It has nd death -33. In and death . Two stuand 3.2) ,33.P. knowlesi malaria case described here was uncomplicated despite marked immunosuppression of the patient (115 CD4 positive cells/μl). Two research groups recently established the hight of parasitaemia as a good predictor for the risk of severe disease . The pafections ,40.P. knowlesi infection [P. knowlesi malaria improves on average 24 hours after successful parasite clearance, suggesting a possible relation between P. knowlesi parasitaemia and a decreased platelet count [P. knowlesi cases that are published without the development of thrombocytopaenia include a 11-year old boy with chronic myeloid leukaemia taking imatinib [P. knowlesi malaria. It is tempting to speculate that the HIV co-infection contributed to the absence of thrombocytopaenia in the presented case, however it is not possible to address this question on the basis of a single case report.Interestingly, thrombocytopaenia, which is nearly universal in patients with nfection -27, was imatinib  and two P. knowlesi malaria in an HIV-positive adult with advanced immunosuppression. The patient experienced a temporary elevation of the viral load that might have been due to the acute P. knowlesi malaria. Despite advanced immunosuppression the patient’s laboratory parameters were not suggestive of a severe P. knowlesi infection and he had an uncomplicated clinical course.The current case is the first description of Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.BLAST: Basic local alignment search tool; CD: Cluster of differentiation; HIV: Human immunodeficiency virus; PCR: Polymerase chain reaction.The authors declare that they have no competing interests.JE reviewed the literature, conducted the collection and interpretation of data and drafted the manuscript. AT helped with the data collection and data interpretation and revised the manuscript. PK provided conceptual advice. MF revised the manuscript, helped to interpret data, helped to draft the manuscript and has given final approval of the version to be published. All authors read and approved the final manuscript.


Plasmodium vivax contributes to almost half of the malaria cases in India, but the disease burden of HIV and P. vivax co-infection is unclear.Malaria and HIV co-infection adversely impact the outcome of both diseases and previous studies have mostly focused on falciparum malaria. Plasmodium falciparum and P. vivax.HIV-infected subjects (n=460) were randomly selected from the 4,611 individuals seen at a Voluntary Counseling and Testing Center in Chennai, India between Jan 2 to Dec 31 2008. Malaria testing was performed on stored plasma samples by nested PCR using both genus-specific and species-specific primers and immunochromatography-based rapid diagnostic test for detecting antibodies against Plasmodium vivax accounted for majority of the infections (60%) followed by P. falciparum (27%) and mixed infections (13%). Individuals with HIV and malaria co-infection were more likely to be men (p=0.01). Between those with and without malaria, there was no difference in age (p=0.14), CD4+ T-cell counts (p=0.19) or proportion CD4+ T-cell below 200/mL (p=0.51).Recent malaria co-infection, defined by the presence of antibodies, was detected in 9.8% (45/460) participants. P. vivax. However, the rate of P. falciparum infection was more than 6-fold higher among HIV-infected individuals than what would be expected in the general population in the region. Interestingly, individuals co-infected with malaria and HIV were not more likely to be immunosuppressed than individuals with HIV infection alone.Retrospective testing of stored plasma samples for malaria antibodies can facilitate identification of populations with high rates of co-infection, and in this southern India HIV-infected cohort there was a considerable burden of malaria co-infection, predominantly due to  Plasmodium falciparum in sub-Saharan Africa. Much less is known whether Plasmodium vivax has similar interactions with HIV. Therefore, this study was conducted to: i) determine the prevalence and risk factors of malaria co-infection in a cohort of HIV-infected individuals in southern India, a region with predominantly P. vivax malaria and ii) evaluate the strategy of using stored specimens for quick retrospective assessment of populations for co-infection burden.HIV, a sexually or parenterally transmitted viral infection, and malaria, a mosquito-borne parasitic disease, are two disparate and deadly diseases that are often brought together by overlapping geographic distributions. When HIV and malaria co-infection occur in the same individual, both diseases are adversely impacted. HIV-infected individuals are at increased risk of: acquiring malaria  developiThe subjects were randomly selected (10%) from the 4,611 HIV-1 positive individuals seen at the Voluntary Counseling and Testing center of Y. R. Gaitonde Center for AIDS Research and Education (YRGCARE) between Jan 2, 2008 and December 31, 2008. They were all newly diagnosed with HIV-1 infection and were not receiving antiretroviral therapy. The study was approved by the ethics boards of the University of California San Diego, YRGCARE, and the Indian Council of Medical Research. All volunteers provided written informed consent. Blood samples were processed immediately following collection and plasma stored in −70°C freezer for a period ranging from 18 to 24 months before being evaluated in the current study.Blood (6 mL) was collected in EDTA tubes  and plasma separated after centrifugation at 2,500 RPM for 12 min. Tests for diagnosing HIV were either Determine HIV 1/2 test (Abbott Laboratories), Signal HIV Rapid Test , or First Response HIV 1–2.0 . CD4+ T-cell counts were determined by flow cytometric panLeukogating method .Plasmodium spp. 18S small subunit ribosomal RNA genes [P. falciparum and P. vivax) and malaria negative individuals, was used as positive and negative control, respectively. To confirm the presence of amplifiable human DNA, 20% (90/460) samples were randomly selected and underwent PCR using primers for the human rRNA gene p53 [DNA was extracted from 200 μL of plasma sample using QIAamp DNA Blood Mini Kit . Nested PCR was done using both genus-specific and species-specific primers , was used for detecting antibodies  against merozoite surface protein (MSP) of both t-test was used for continuous variables and Chi-square test for categorical variables (IBM SPSS Statistics Version 19). Non-parametric test, Mann–Whitney U test, was used where appropriate. One-way ANOVA was done to compare characteristics of the 3 malaria positive subgroups.Student Plasmodium antibodies were detected in plasma from 45 of the 460 participants (9.8%). The majority of the infections were due to P. vivax (60%) followed by P. falciparum (27%) and mixed infections (13%). Three-fourths of the co-infected individuals were in the 30-50 year range, but there was no difference in average age between those with and without malaria , median CD4+ T-cell counts  or proportion CD4+ T-cell below 200/μL . The co-infected subjects, however, were more likely to be male . Further analysis of the malaria positive groups  did not show any significant difference in the above characteristics . Parasite DNA was not detected by PCR in any of the samples. Positive and negative controls were used with each batch of specimens processed and performed as expected. Amplifiable human DNA, tested using primers for the p53 gene, was found in 93% (84/90) of the randomly selected samples.Participant ages ranged from 21 to 68 years and 63% were male. Table P. vivax co-infection rate. The majority of the infections in this HIV-infected cohort were due to P. vivax (60%), which is similar to that in the general population in the state of Tamil Nadu [P. vivax (60% vs. 96%); higher proportion of P. falciparum (27% vs. 4%); and a considerable number of mixed infections (13%). The higher prevalence of falciparum malaria in HIV-infected individuals has implications for clinical management. Incorrect identification of Plasmodium species can lead to inappropriate malaria treatment because chloroquine is still the first-line treatment for P. vivax[In India, the potential for intersection of malaria and HIV epidemics is great but data on co-infections are sparse. Two studies, one conducted in Chennai and the other in Mumbai, have identified co-infected individuals in hospital-based settings ,13. Howemil Nadu . In contmil Nadu , the co- P. vivax, but wou P. vivax.P. vivax and it is speculated that immunosuppression due to HIV may not increase the risk of vivax malaria co-infection, but more studies are needed to confirm this finding.There was a preponderance of men (80%) with a mean age of 40 ± 9 years in the co-infected group, which mirrors the trend seen in the general population . The higPlasmodium DNA in serum samples frozen up to 2½ years [P. falciparum and P. vivax, the two predominant Plasmodium species in the region and may have missed infections caused by other spp. like Plasmodium ovale and Plasmodium malariae although their contribution is unlikely given the antibody results.Malaria diagnosis was made by detection of malaria antibodies by the SD BIOLINE kit. Although, its sensitivity is much lower than microscopy or nested PCR and ranges from 47% to 69% ,21, this2½ years . The cur2½ years ,28, plas2½ years . Another2½ years  or simil2½ years  sensitivTaken together, although malaria antibody test cannot be used to make a clinical diagnosis, it is a preferred method for retrospective malaria prevalence studies in regions with unstable malaria transmission, as in this study region.The authors declare that they have no competing interests.ARB, DMS, SLL, and NK conceived the study, SS, VM, JS, and PB implemented the study. ARB and DMS undertook data analyses, interpretation of results and drafted the manuscript. SLL and NK were involved in critical revision of the manuscript. All authors read and approved the final manuscript.


Plasmodium berghei ANKA model of experimental cerebral malaria, the meeting did bring the two research communities closer together to identify further work to provide information needed to validate the model and revitalise the development of other animal models displaying features of human pathology. The driving force behind this was the desire to ensure better translation of experimental findings into effective treatments for severe malaria.In light of the recent controversies over the role of animal models for research into the development of new treatments for severe malaria, particularly cerebral disease, a group of scientists came together to discuss the relative merits of a range of animal models and their overlap with the complex clinical syndromes of human disease. While it was not possible to fully resolve differences over the utility of the Plasmodium berghei ANKA (PbA) mouse model of experimental CM (ECM) did not replicate the pathophysiology of cerebral disease in humans, and which was followed closely by a set of papers supporting the ECM approach In 2010 there were a number of publications discussing the relevance of rodent models for the study of human cerebral malaria (HCM). These were prompted by an initial discussion paper from Professor Nick White and colleagues  multi-organ failure, including kidney and liver dysfunction. For children, severe disease includes metabolic derangement, cerebral malaria, and severe malarial anaemia. Frank pulmonary oedema is less common in children. Severe malaria , although less frequent, can be caused by Plasmodium vivaxPlasmodium knowlesi cases also frequently develop severe malaria, with limited clinical data and autopsy findings from a single case report appearing to show similarities to P. falciparum pathology but without coma Most studies on severe malaria have focused on infection by the malaria parasite A principal feature of HCM from nearly all post-mortem studies is the packing of sequestered infected erythrocytes (infected red blood cells [iRBC]) in the brain microvasculature Mortality in paediatric HCM occurs very rapidly, with 50% of deaths being recorded within 24 hours of admission and nearly all fatalities by 48 hours. In adults a similar level of early mortality is observed, with about 50% of deaths occurring in the first 48 hours post admission, but subsequent deaths can take place later due to other complications such as renal failure or incidental pneumonia. Patients with HCM tend to have higher parasitaemias, and some studies have also suggested a larger parasite biomass . Given the multiple aetiologies of severe disease, this type of information will be critical in helping us to understand the relative contribution of different pathologies to the clinical signs observed in severe malaria. A consistent pathological finding in HCM at autopsy, unique to this disease, is the presence of sequestered iRBC that pack brain blood vessels. Indeed, patients who die soon after admission to a health care facility may have brain microvascular sequestration as the only significant pathological feature of CM. However, observation and correlation of neuropathological features with pre-mortem clinical diagnosis can be affected by multiple factors such as time to death, treatment, and other systemic complications of severe malarial infection, or may be compromised by misdiagnosis. In this regard there are some differences between adults and children, for example, in the degree of brain swelling and oedema observed using imaging techniques, and the accumulation of leukocytes and platelets in cerebral microvessels, with significant levels seen in some cases of paediatric HCM but very few observed in adults, despite clear evidence  of a vigorous pro-inflammatory host response. Astroglial activation and blood–brain barrier changes are also commonly observed in HCM, so inflammatory responses elicited within the brain parenchyma may be more subtle than merely accumulation of monocytes or other leukocytes Adhesion-related pathology in falciparum malaria is evident in placental infections in pregnant women. Here, placental sequestration of iRBC and the onset of fetal impairment and disease in women protected by immunity is linked to the appearance of a set of parasite variants expressing a class of PfEMP1 proteins known as VAR2CSA Plasmodium species have evolved multiple and redundant strategies to deflect and evade the host immune response, so it is unlikely that protection will be correlated with the ability to recognise and neutralise a single antigen P. falciparum, anti-disease immunity develops relatively quickly, perhaps after only a few infections, but this immunity is incomplete, still allowing lower density blood stage infection that is accompanied by either mild or no symptoms Two key questions, “what is the pathophysiology of severe disease?” and “how do people living in malaria endemic regions become protected from severe disease?”, cannot be answered fully by observations of infected humans. While studies on the clinical definition and histopathology of HCM and placental malaria have provided information regarding correlates of pathogenesis, the exact mechanisms whereby malaria parasites cause severe disease and how immunity protects against disease despite infection remain unknown. Parasites with complex life cycles such as Mechanistic studies in animal models may provide information on the processes of, and protection from, severe malarial disease, and there are good examples where they have provided insights into features and characteristics of human malaria infection. Although it is unlikely that any single model will reproduce the complexity and spectrum of disease and immunity observed in human malaria infections, there are parallels between some human and animal presentations of malaria infection (and disease). However, the choice of the host/parasite combination is important see .P. berghei ANKA–infected mice. Several groups provided data that supported the accumulation of iRBC in a number of organs, sometimes including the brain, and that higher parasite accumulation was seen in mice susceptible to ECM. However, the key piece of information missing was direct histological evidence of substantial parasitised erythrocyte sequestration packing brain microvessels in a similar pattern to that observed with HCM.In some ways, this is one of the most challenging questions for the malaria research community and accordingly has resulted in a degree of polarisation of viewpoints. The correct choice of a combination of experimental animal host and a malaria parasite able to appropriately mimic the disease pattern seen in humans is essential. However, it is also very clear that animal models allow for more detailed examination of multiple and specific patholphysiologic processes caused by malaria infection that is not possible for clinical studies of human malaria through the level and scope of experimental observation and intervention that can be applied to animals. In discussing the role of the PbA mouse model of HCM specifically, the issue that recurred throughout the meeting concerned the degree of sequestration of iRBC in the brains and other organs of P. berghei ECM, in contrast to HCM, is associated with marked accumulation of leukocytes, but not with prominent sequestration of cytoadherent mature trophozoite/schizont iRBC in brain vessels The consensus of most participants was that ECM is associated with increased accumulation of immune cells in the brain (particularly monocyte/macrophages and T cells). In particular, recruitment of CD8+ T cells to the brain late in disease pathogenesis, and production of the cytolytic molecules granzyme B and perforin, is critical for ECM development ECM is clearly an inflammatory syndrome with local vascular endothelial activation with inflammatory cytokines playing an essential role, which has obvious differences and some similarities to the clinical and pathological features of HCM, for example, the signs of vascular inflammation/damage seen in some cases of paediatric HCM One of the benefits of the discussion about ECM studies at the meeting was a clarification of how inhibitor or intervention studies of adjuvant treatments in mice should be interpreted. In most studies, the inhibitors/drugs are given before the development of neurological symptoms, thereby providing information on disease processes in ECM but not necessarily on the identification of viable therapies. The validity of carrying out human clinical trials, often in underpowered studies, purely on the basis of this type of rodent malaria data is questionable. On the other hand, human trials carried out on the basis of pathophysiology studies in humans  is influenced by  differences between isolates/strains and cloned laboratory lines  may create an in vivo screening system for testing inhibitors that block P. falciparum cytoadherence. While this “double transgenic” model is not available yet, human graft systems have been investigated using SCID mice, as transient models allowing perfusion of P. falciparum iRBC in human dermal tissue in a format where detailed microscopy is facilitated P. falciparum infections in NOD/SCID mice have also been described With questions raised about the relevance of the PbA mouse model for HCM, a consideration of alternative rodent models was undertaken. Improvements based on genetic manipulations to the mouse model were discussed, such as a humanised rodent system. Schizonts of Given the greater genetic relatedness of primate hosts and primate parasites, the importance of NHP and malaria parasite combinations as appropriate systems for researchers to study ECM was also discussed at the workshop. Further, it was noted that a degree of reluctance to use NHP models in malaria research has led to a reduction in the resources needed to support such research. If NHP systems are capable of providing models of HCM and other types of severe disease in human malaria, then significant investment will be required to grow and sustain this research community, train young researchers, and provide the resources for wider participation of clinical and laboratory oriented investigators.Plasmodium coatneyi is a simian parasite that in Japanese (Macaca fuscata) and rhesus (Macaca mulatta) macaques exhibits phenotypic characters that mimic P. falciparum iRBC sequestration, rosetting, and clinically severe disease Plasmodium fragile iRBC profoundly sequester by cytoadhesion in the vasculature of various tissues and organs and produce severe disease in rhesus monkeys P. knowlesi, but not P. berghei and other rodent malarias, have var-like gene families that exhibit antigenic variation and adhesion properties P. coatneyi malaria Several host/parasite combinations were discussed : PlasmodP. knowlesi infections in M. mulatta produce “severe disease” that is almost always lethal to this host. Mature P. knowlesi iRBC in rhesus monkeys partially sequester in a variety of tissue sites via the variant-antigen products of the large var-like SICAvar multi-gene family P. knowlesi rhesus macaque model can mimic some clinical syndromes of P. falciparum severe disease in humans, it is questionable whether it is a useful model for HCM, as neither adhesion of iRBC to cerebral vessels or clinical signs of HCM have been noted. Although it is unclear if P. knowlesi in rhesus monkeys is a satisfactory model for HCM, from initial reports it is certain to be a model for human severe disease caused by this parasite P. knowlesi in the olive baboon (Paplio anubis) P. falciparum will also infect and cause severe disease  and death in neotropical monkeys such as various species of Aotus (owl monkeys).P. falciparum.There are, however, limitations to this primate model since symptoms of CM and cerebral vascular sequestration have not been noted in New World monkeys infected with var multi-gene family in rodent and some primate malaria species  means that the roles of the encoded variant antigens in placental sequestration cannot be investigated. However, NHP models such as P. coatneyi in rhesus monkeys P. falciparum in Aotus monkeys are also available; and P. coatneyi expresses the comparable variant antigen multi-gene family . Furthermore, analysis of the effects of inflammatory responses and parasite accumulation/sequestration in the placenta and foetus may provide insights into the role of pro-inflammatory cytokines in malarial placental pathology Plasmodium chabaudi, and the ability of some host/parasite systems to develop almost complete immunity after a single infection. Investigation of the effects of malaria on multiple pregnancies is therefore not possible in most rodent models except in drug-induced chronic P. berghei infection in mice, which is one of the best described models for rodent placental malaria. A “humanised” PbA mouse model may also be developed for analysing sequestration properties during pregnancy. Transgenic P. berghei parasites may be generated that express Pf VAR2CSA or CSA binding domains on the surface of iRBC. Such parasites in combination with a “humanised placental malaria mouse model” may offer a screening system for in vivo testing inhibitors  that block P. falciparum sequestration.At the meeting, brief mention was made of models for placental malaria, acute respiratory distress (ARDS), anaemia, and immunity. The lack of the P. berghei in mice can be models to investigate malaria-associated lung pathology Several recent studies indicate that P. chabaudiP. yoelii 17xNL in mice and P. berghei in Brown Norway rats  were briefly mentioned as possible models for malarial anaemia. However, the lack of a real chronic infection and the rapid acquisition of sterilising immunity preclude the use of many rodent models to study anaemia in chronic infection, which would be more reflective of severe malaria in children. However, mechanisms of disruption of bone marrow haematopoeisis Models for anaemia were also considered to be important to support human clinical studies. P. coatneyi in acute infections produce profound anaemia that is life-threatening due to clearance of uninfected erythrocytes and a dyserythropoesis that does not allow a compensatory reticulocytosis , as well as severe anaemia later on during chronic severely recrudescing infections that can last for months or over a year . P. cynomolgi, like P. vivax, also produces a profound anaemia during acute infections . Severe lethal anaemia also develops in semi-immune neotropical primates with P. falciparum infections going into chronic infection and upon reinfection or challenge of animals induced into a semi-immune state . P. vivax infections in neotropical monkeys also develop severe anaemia during acute and chronic infections Based on mainly unpublished reports, several NHP models offer opportunities for studying severe anaemia in both acute and chronic infection in regards to clarifying the mechanisms and interventions for clearance dynamics of uninfected RBC, bone marrow differentiation, and dyserythropoesis in severe malarial anaemia. Simian malarias such as P. chabaudi and P. yoelii in mice and P. berghei in Brown Norway rats show self-resolving infections with low peak parasitaemias and anaemia. It is generally assumed that immunity in humans and NHPs develops slowly over many months and incompletely, in contrast to rodents where immunity develops rapidly and is frequently sterilising. Only P. chabaudi in mice P. berghei in rats exhibit low density chronic infections for prolonged periods (2–3 months), whereas in humans and NHPs single infections may last years and decades. One feature in common to human, NHP, and rodent infections is that protective immune responses are generally species-, genotype-, and variant-specific, although in the case of variant-specific immunity some of the antigens that are products of multi-gene families  exhibit evolutionarily distinct sequences and characteristics in the species and strains where they exist. Humans are generally exposed to different strains, genotypes, and variants of the parasite, whereas in most experimental model studies the same cloned parasite line or a single strain/isolate is used for repeated infections. It was emphasised that when humans are similarly re-exposed to the same strain/genotype, acquisition of immunity is generally similar to that of mice. A recommendation therefore was that studies on immunity to re-infection and the effectiveness of immunological memory should be carried out in mice or monkeys using different strains of the same parasite species. More immunological studies in mouse/rat peripheral blood, for comparison with humans, would also be valuable. Another important point of experimental model studies was that direct injection of infected erythrocytes to initiate infections was normal practice in the study of immune mechanisms and immunopathology. However, this does not reproduce the sequence of the natural infection where infection starts with a mosquito bite and inoculation of sporozoites. It was discussed here that blood stage infections of some Plasmodium species may look very different and be considerably less virulent when initiated via the natural route compared with injection of iRBC. This should be taken into account when translating findings from animal models to humans.It was generally agreed that rodent models can provide relevant information on mechanisms of host defence and immunity to all stages of the infection. A recent example of this is the demonstration of suppression of liver stage malaria parasite development in the presence of a blood stage infection At the meeting the use of animal models for development and testing of vaccines and drugs was only briefly mentioned. Discussion of this area was outside the scope of the meeting, but it was acknowledged that animal models have played a major role in the development of anti-malarial therapies, including those targeting different stages in the parasite life cycle There was universal support for stronger interactions between groups working on human and animal studies, both to improve current models and to promote more realistic translation of laboratory findings into clinical studies.P. falciparum. In the absence in the PbA mouse ECM model of this hallmark of human CM, and based on observations of pathologic changes to endothelial cells on interaction with iRBC, it was the view of some of the participants that the mouse/PbA system was not a useful model of HCM. However, it was pointed out that we do not know that P. falciparum cytoadherence causes HCM and its role in pathology may have been exaggerated. At the same time, comparisons of ECM and HCM have revealed many similarities , and therefore the PbA mouse model can provide a platform for detailed mechanistic experiments in this field. Thus, the overriding conclusion of this meeting was that there are presently genuine differences of opinion on this topic, but this was coupled with the genuine willingness indicated by all parties at the meeting to try overcoming these differences by collaborating. Specifically, these centered around two major areas: 1) A more careful use of the term “drug” when applied to interventions used in ECM, particularly when these were applied prior to the physiological signs of cerebral disease in mice, although the validity of the use of inhibitors to understand pathological processes was appreciated. 2) A bi-directional exchange of resources and knowledge to provide better information about the complex pattern of human disease to scientists using animal models and better access for this latter community to human tissues in order to validate findings obtained from model systems. The major issues raised at the meeting are summarised in The importance of animal models in malaria research was recognised by all participants. However, it was not possible to reach a consensus on the role of the PbA mouse model for studies on HCM. The basis of the disagreement centred on the relative importance of iRBC adhesion to brain endothelial cells in causing HCM pathology. There is evidence to support the presence of PbA iRBC in cerebral vessels in ECM, but this accumulation represents a minor (if any) concentration of iRBC compared to the peripheral parasitaemia, unlike the levels of adherent iRBC seen with The mouse and human malaria research communities need to work together more closely to support better translation of laboratory studies into clinical leads.P. berghei ANKA mouse model for human cerebral malaria, particularly in the area of cytoadherence-associated pathology. It was agreed that researchers using the P. berghei model should be cautious in their interpretation of inhibitor studies, particularly where the intervention is given prior to symptoms.It was not possible to resolve the disagreements over the use of the The spectrum of malarial disease in humans is broad and we do not fully understand the pathological mechanisms.Development and standardisation of animal models, including renewed investment in non-human primate systems, is a priority.The main recommendations from the meeting were:(i) Coordinated pathological studies across speciesAs with many disciplines, one of the major confounders of research is the lack of standardisation. This is particularly clear with the mouse/PbA model, where differences between parasite lines and environmental factors, such as host diet, can influence features of pathology. Thus, there was a general consensus that tissue specimens from animal models of CM produced in different laboratories need to be analysed using uniform methods of histology and quantification. While it was questioned whether the mouse/PbA model is appropriate to derive cytoadhesion-based therapies for CM, it does not preclude this model as a vehicle to investigate possible inflammatory processes in severe disease, including CM. Further research is needed to link animal and human studies to allow a comparison of the pathology of disease across a spectrum of clinical syndromes and thereby to develop the much needed animal models that can drive research into the development of interventions for severe malaria. The mouse model may be useful in studying tissues where inflammation is known to play a significant role in human pathology, such as the lung. But in some clinically important syndromes of human malaria such as malaria-associated acute renal failure in adults, very little data are available on the clinical or pathological correlates in animal models. However, NHP infections in rhesus monkeys involving several simian malaria species can cause acute renal failure.(ii) More use of primates and better access to primatesP. falciparum and P. vivax (as well as P. knowlesi). Infections with P. fragile, P. coatneyi, and P. knowlesi in certain macaque species are potential models of CM, anaemia, placental malaria, and ARDS and other severe malaria syndromes in humans P. coatneyi in Japanese or rhesus macaques, exhibit cerebral sequestration but it is not clear how similar they are to HCM.Strengths of studies on NHPs are that their physiology and acquisition of immunity to infection are more similar to humans than mice, and their size may also permit a wider range of studies. Also, within limits, neotropical (New World) primates can be infected with human NHP models of vaccine efficacy  have been reasonably predictive of human clinical trials . Unlike rodent models, they do provide access to some of the life cycle stages that are not available in vitro, including hypnozoites.NHP studies for malaria research require specialised scientific centres with the regulatory framework, access to NHP animals, and expertise to conduct such research. The group recognised that investment in existing NHP research centres would be a valuable and cost-effective way forward, coupled with a scheme to provide “placements” for scientists from collaborating laboratories. This would ensure an enhanced and vibrant scientific environment for malaria research within the NHP centres and disseminate the potential for NHP research to the community.(iii) Clear discrimination between intervention and mechanistic studiesOne of the more fundamental problems of animal models highlighted by recent publications has been the confusion between studies aimed at collecting mechanistic information on disease processes and studies aimed at screening for potential therapies against severe disease, perhaps most graphically demonstrated by the figures of 38 of 42 interventions for ECM working but none of 16 being successful in HCM. The limitations associated with the application of therapies before clinical signs, often used in PbA mouse model studies, need to be recognised. Relevant animal models are highly desirable, as studies of humans with severe malaria are by definition limited to situations where clinical interventions are the priority. Thus, interpretations of the result of therapies preventing CM must be done in the context of anti-malarial drug treatment and other supportive measures.(iv) Facilities to support research on severe malariaOne of the major outcomes of the workshop was the requirement for further development of facilities for research. We need to understand more about human disease and immunity and communicate more effectively with researchers working on animal studies so that we can identify appropriate models for detailed mechanistic studies. Apart from a general need for more work in this area, two specific areas were identified for further development:(v) Tissue biobankingOur lack of knowledge of the detailed pathology of human disease is part of the problem in identifying relevant animal models. To address this, the creation of a tissue biobank was proposed. This resource would make material from humans with malaria available to the broader research community so that validation of initial observations from well-characterised clinical specimens could be examined from a variety of perspectives using novel technologies. There are clearly ethical and logistic issues associated with this, but the benefit accrued in terms of validating animal studies by having good access to human tissues was deemed to outweigh the effort that would be required to set this up properly. The use of tissue microarrays, where multiple cases can be examined on a single slide, may facilitate this approach.(vi) Standardisation of animal modelsIt was proposed that a repository of standard parasite lines and protocols relating to the maintenance/passage of parasite lines and their hosts should be created. There should also be an opportunity for members of the research community to share information (particularly unpublished data that is not widely available), including data such as a comparison of parasite sequences.Table S1Animal host/malaria parasite combinations for the study of human malaria pathogenesis.(DOC)Click here for additional data file.


P. falciparum genetic diversity and multiplicity of infection during the malaria transmission season of October 2011 to February 2012 in these areas were described.There have been few investigations evaluating the burden of malaria disease at district level in the Republic of Congo since the introduction of artemisinin-based combination therapies (ACTs). The main objective of this study was to document laboratory-confirmed cases of malaria using microscopy and/or rapid diagnostic tests (RDTs) in children and pregnant women attending selected health facilities in Brazzaville and Pointe Noire, the two main cities of the country. Secondly, Three and one health facilities were selected in Brazzaville and Pointe-Noire as sentinel sites for malaria surveillance. Children under 15 years of age and pregnant women were enrolled if study criteria were met and lab technicians used RDT and/or microscopy to diagnose malaria. In order to determine the multiplicity of infection, parasite DNA was extracted from RDT cassette and msp2 P.falciparum genotyped.P. falciparum genetic diversity determination using msp2 gene marker ranged from 9 to 20 alleles and remains stable while multiplicity of infection  and parasite densities in clinical isolates were lower than previously reported.Malaria prevalence among more than 3,000 children and 700 pregnant women ranged from 8 to 29%, and 8 to 24% respectively depending on health center locality. While health workers did not optimize use of RDTs, microscopy remained a reference diagnostic tool. Quality control of malaria diagnosis at the reference laboratory showed acceptable health centre performances. These findings are consistent with a reduction of malaria transmission in the two areas. This study raises the issue of targeted training for health workers and sustained availability of RDTs in order to improve quality of care through optimal use of RDTs. Deployment of various control tools has boosted malaria control in sub-Saharan Africa. This includes wide deployment of artemisinin-combination therapies (ACTs), insecticide-treated nets and in some places targeted indoor residual spraying (IRS) of insecticides. Consequently, the burden of malaria has declined in some countries , while oThe World Health Organization (WHO) recommends parasitological confirmation of malaria using microscopy or RDTs in all malaria suspected patients before starting them on anti-malarial treatment . TreatmePublished data on the burden of malaria from the Republic of Congo since drug policy change in 2002 is limited, making it difficult to evaluate the impact of implemented malaria interventions. Malaria remains the main cause of hospital consultations, accounting for 49 to 51.5% of hospital admissions and 35.4% of children deaths at health facilities in Brazzaville and Pointe-Noire districts  × 100. The specificity of the test was determined as Number of true negatives/ Number of true negatives + number of false positives] ×100.Positive predictive values (PPVs) and negative predictive values (NPVs) were calculated as described by Iqbal et al. .2 test was used to investigate any significant differences between microscopy and RDT or PCR and RDT. Fisher's Exact test was used to compare malaria prevalence between health centers and also between children and pregnant women. Differences were considered significant if p value<0.05.McNemar-χThe kappa value (κ), representing the proportion of agreement beyond chance, was used to quantify the level of agreement or concordance between both methods; a kappa value of 0.21-0.60 was considered moderate agreement, 0.61-0.80 good agreement and κ ≥ 0.8 was of high concordance .P. falciparum species was detected by microscopy. Four-hundred and four (404) children had malaria infection, representing 12% of consulting cases and reflecting the number of children who received antimalarials. In Brazzaville, CSI Fleuve Congo which is close to Congo river presented the highest malaria prevalence in children: 29% (76/263) compared to 8% (159/2037) and 19% (108/578) at the hospital Talangai and CSI Intendance which are located on a hill. In Pointe-Noire: Out of a total of 412 children, 61 were found microscopy or TDR positive for Plasmodium falciparum. The median age of children on consultation at different health care facilities was similar (5 years).A total of 3,290 children were recruited into the study from four health facilities in Brazzaville and Pointe-Noire Table ; out of P. falciparum infection in pregnant women ranged from 8 to 24% for all health facilities  and showed malaria prevalence in pregnant women to be similar to that observed in children under 15 years of age in all sites. In details, a total of 750 pregnant women distributed as 737 and 13 were recruited at Brazzaville and Pointe-Noire respectively from October 2011 to February 2012. The mean age of recruited women was 24 years in all selected sites. The prevalence of 31 cases CSI and CSI Ndaka Sossou preferentially utilized microscopy and RDT for the diagnosis of malaria respectively. Because of this, both district centers were assessed for the microscopy, RDT or PCR at the reference molecular biology laboratory of the Faculty of Health Sciences/University M. Ngouabi in Brazzaville as shown in Table Plasmodium falciparum clones per infected individual, genetic diversity and parasite densities were determined for children consulting in Brazzaville and Pointe-Noire health facilities. MOI was similar (about 1.7) in both health centers. The distribution of 3D7 and FC27 allelic families of msp2 Plasmodium falciparum show the presence of 11 and 22 msp2 alleles in clinical isolates from patients from Brazzaville (CSI Intendance) and Pointe Noire (CSI ndaka Sossou) respectively The genetic diversity was much higher in isolates from Pointe-Noire and parasite densities higher in patients from Brazzaville (Table The multiplicity of infection (MOI) referred as the mean number of To bridge knowledge gaps due to limited data from the Republic of Congo on the burden of malaria infection and on utility of available diagnostic tools, the present study documents the burden of uncomplicated malaria and utility of laboratory diagnostics for malaria case management at selected health facilities in Brazzaville and Pointe-Noire, where RDTs and microscopes are already provided. To the best of our knowledge, this is the first study since change of National malaria treatment policy in 2002.et al.[The malaria prevalence of 9% reported in children from two health districts in Northern part of the capital is similar to that reported by Koukouikila et al. from SouIt is interesting noting that at two health centers where quality control for microscopy and interpretation of RDT were done, utility of RDTs as a diagnostic tool was poor particularly in Brazzaville even though laboratory technicians knew. It is unlikely that poor adherence was due to lack of experience. Informally, many technicians were reluctant to use RDTs because their availability was not sustainable and microscope slides were still abundant. Studies conducted in Tanzania -17 correPlasmodium falciparum field isolates in Northern Brazzaville and in Pointe-Noire reveals limited and high genetic diversity but not surprising considering that CSI Intendance is located on a hill. In comparison the genetic diversity in Southern part of Brazzaville where malaria transmission is intense, the MOI and genetic diversity [The genetic diversity among iversity  was highiversity . Unfortuiversity  and reduiversity -21. ThisWe recommend that laboratory technicians should be trained on the importance and advantages of using RDTs compared to microscopy such as simplicity, patient time saved and its modest reliability. Indeed, malaria is over-diagnosed and over-treated in many African countries through clinical diagnosis only, while other clinicians do not honor laboratory results -24. WhilP.falciparum genetic diversity remains stable.The study has shown the need to provide a targeted training to health workers to enhance acceptability of RDTs and improve quality of care. It also revealed that MOI and parasite densities in clinical Congolese isolates from the two main cities are low and the RDT: Rapid diagnostic test; IRS: Indoor residual spray; PCR: Polymerase chain reaction; SP: Sulfadoxine pyrimethamine; IPTp: Intermittent preventive treatment during pregnancy; MOI: Multiplicity of infection.The authors declare no competing interest.FN, JCV and AS contributed to the study design, data analysis and drafting of the manuscript. ML and RI performed the lab work. JCV performed the statistical analysis. FN ensured the supervision of the all work. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/13/135/prepub


Viewing Plasmodium in Romanovsky-stained blood has long been considered the gold standard for diagnosis and a cornerstone in management of the disease. This method however, requires a subjective evaluation by trained, experienced diagnosticians and establishing proficiency of diagnosis is fraught with many challenges. Reported here is an evaluation of a diagnostic system  that evaluates scanned images of standard Giemsa-stained slides and reports species and parasitaemia.The device was challenged with two independent tests: a 55 slide, expert slide reading test the composition of which has been published by the World Health Organization (“WHO55” test), and a second test in which slides were made from a sample of consenting subjects participating in a malaria incidence survey conducted in Equatorial Guinea (EGMIS test). These subjects’ blood was tested by malaria RDT as well as having the blood smear diagnosis unequivocally determined by a worldwide panel of a minimum of six reference microscopists. Only slides with unequivocal microscopic diagnoses were used for the device challenge, n = 119.On the WHO55 test, the device scored a “Level 4” using the WHO published grading scheme. Broken down by more traditional analysis parameters this result was translated to 89% and 70% sensitivity and specificity, respectively. Species were correctly identified in 61% of the slides and the quantification of parasites fell within acceptable range of the validated parasitaemia in 10% of the cases. On the EGMIS test it scored 100% and 94% sensitivity/specificity, with 64% of the species correct and 45% of the parasitaemia within an acceptable range. A pooled analysis of the 174 slides used for both tests resulted in an overall 92% sensitivity and 90% specificity with 61% species and 19% quantifications correct.In its current manifestation, the device performs at a level comparable to that of many human slide readers. Because its use requires minimal additional equipment and it uses standard stained slides as starting material, its widespread adoption may eliminate the current uncertainty about the quality of microscopic diagnoses worldwide. Plasmodia gametocytes or the rate of haemozoin-containing macrophages, can be assessed [Despite tremendous recent gains, the World Health Organization (WHO) still reports over 225 million cases and nearly 800,000 deaths in its most recent report . In addiassessed , and d) assessed .“Expert microscopy” as an entity, however, is not always consistent with respect to a number of aspects that can have a direct effect on the results obtained. An important example of this inconsistency is in the length of time a slide is reviewed. A review of 88 studies demonstrated as much as 20-fold variations in the measures of the time spent reviewing a slide such as high power fields or leucocytes amassed . The seqMansonella perstans filariasis in several of the patients involved in this study. They were found and identified by the microscopists, but were undetectable by malaria RDTs. To ensure quality malaria diagnosis by microscope, significant challenges are involved not only in pre-service training, but also in maintaining the proficiency of human microscopists, both in endemic settings and as well as in the parts of the world with imported malaria.Malaria microscopy is a skill which requires considerable training, experience and practice to achieve and maintain proficiency. Even to make the measurement of that proficiency consistent and fair is a daunting task. While examining a blood film, the well-trained microscopist looks at and integrates other aspects of the haematology being examined into the diagnosis. A relevant example is the identification of Given these challenges and inherent variability, there has long been a quest for alternative, if not less subjective malaria diagnostic methods, and a wealth of tools have been developed, some even experimentally evaluated. Recently the prospect of using scanning technology and a computer algorithm to analyze images captured from the microscopic examination of stained blood smears to find, identify and quantify malaria parasites has emerged. In a review in 2008 John Frean first pointed out the suitability of image analysis for enumerating malaria parasites . A year Purwar et al.  reportedThis paper, reports on the evaluation of a similar algorithm, however one which uses both thick and/or thin blood films and is coupled with an automated scanner. The coupling of the scanner with a PC comprises a completely automated system which is referred to here as a “device.” This device was evaluated using slides in accordance with WHO published testing standards and under field conditions within the Bioko Island Malaria Control Project (BIMCP) in Equatorial Guinea.The device was challenged with two independent tests. The first was a 55 slide expert microscopy test using validated archived slides (WHO55 test). The second challenge came from 140 slides garnered from a malaria prevalence survey, the Equatorial Guinea Malaria Indicator Survey (EGMIS). The two tests are reported both separately and as pooled data. In Figures The WHT software technology uses digital images made from standard Giemsa or Field’s stained microscope slides. Either digital microscopes or imaging scanners may be used to acquire the images that are stored and subsequently serve as the input for the algorithm to locate, identify and count the parasites. Localization, recognition and enumeration of the salient constituents of the scans (parasites and leucocytes) are based on pattern, color and shape recognition of parasites in red blood cells (RBCs) of a thin film and/or parasites that remain in a thick film after lysis of RBCs. The algorithms were developed with the help of pathologists experienced in malaria, and are able to distinguish malaria parasites from other blood constituents and artifacts. As the database of such images increases, the software will be enhanced to be more proficient at distinguishing interferences and anomalies from parasites, thereby improving the accuracy of the diagnosis.To use the WHT system, a slide is placed in the scanner (with an automated scanner many slides can be placed at one time), and the scanner captures images at the selected magnification. Two scanners were used for this study, IScan Coreo Gold®  and Doctor’s Choice®, a custom portable device made to WHT specifications Figure , a and bThe software performs three major operations: pre-scan, removal of artifacts, and a full software analysis.(i) Pre-scan: the software compares all the images against a set of “tight criteria” which are parameters designed to find the malaria parasites, if present. In order to be deemed positive, a pre-set threshold level of items matching these tight criteria must be found. If the threshold criterion is met, the software executes the next step.(ii) Removal of artifacts in the scanned digital image: blood components that may be confused with malaria parasites, including platelets, stain crystals and other interfering images are digitally “removed.” An iterated threshold is used that defines the parameters used to form a binary image. Pixel intensity is used as a part of this iterative threshold, which then separates foreground from background and therefore, cellular blood constituents from artifact. Despite ignoring these potential confounds in the subsequent processing, the software does not alter the originally acquired scan. At the end of this operation the “cleaned” scan is now ready for the final step.(iii) Full software scan: the resulting ‘images-of-interest’ are compared to a wide range of parameters that describe the parasite by employing a databased library. It is the augmentation of this library that will allow the performance that has been recorded in this study to be improved over time as relevant shapes are added. This is the step that identifies the cellular constituents of interest, whether in thick or thin film and therefore allows the counting of all the parasites and white blood cells (WBCs), and calculates the infection density. Density is determined by establishing a ratio of parasites to the observed number of white blood cells (WBC). Because WBCs and parasites are not homogeneously distributed across the slide , these tin situ may be “seen” and evaluated by the algorithm. Therefore visual cues  can be “seen” by the software. The software also patches together all 800 tiled digital images to provide a zoomable image, much like a virtual microscope, thereby making human review of the entire slide possible.In a very comparable sequence of events to that carried out by human readers, the scanner collects images from the thin film in order to identify the parasite species after processing the images collected from the thick film. Unlike the thick film, the thin film is methanol fixed during the preparation of the slide. In the thin film, the intact RBCs are visible and the morphology of parasites This challenge was selected because it reflects a testing standard published by the World Health Organization (WHO) , pp 31–4Slides from the Malaria Research and Reagent Reference Resource Center (MR4) archive comprised 49 of the 55 slide test set. The remaining six slides were selected from the National Archive of Malaria Slides (NAMS) of Equatorial Guinea. Both archives were prepared under the guidance of Hydas World Health  and adhered to the slide archiving principles that they pioneered for the MR4 archive . Fifty-fPlasmodium falciparum infections ranging from 75 to 165,500 parasites/μL. The remaining 10 positive slides were non-P. falciparum and mixed species infections in the following proportions: Four were Plasmodium vivax with densities between 355 and 14,809 parasites/μL, two Plasmodium malariae with 164 and 3,275 parasites/μL and four mixed  with densities between 31 K and 129 K parasites/μL (combined parasite count). In mixed species infections each species was present in excess of 40 parasites/μL. All slides were protected by cover slips, one shielding the entire thick and thin film (25x50mm) and the other covering the label (25x25mm).The test consisted of 40 slides which were to be qualitatively analyzed as positive or negative along with the species, if positive. Twenty of these 40 slides were true negatives and of the remaining 20, ten were In addition to a qualitative assessment of positivity or negativity, it is important on a slide reading test to assess a diagnostician’s ability to quantify an infection. For this aspect, the WHO 55 test calls for an additional 15 slides that were marked as “counting slides.” Normally, examinees are told that these 15 slides are pf positive and that their only task is to quantify and report the severity of the infection as parasites/μL. The WHT software however, quantifies all positive slides as a matter of course. To make the evaluation truly equivalent to that of a human reader, only the reported parasitaemia for the 15 “counting” slides were used to evaluate its quantifying ability using the “level 1,2,3,4” system promulgated by the WHO. Analysis of the WHO55 test slides was carried out in the WHT facility in Columbus OH using the equipment described above. Figure The Equatorial Guinea household based Malaria Indicator Survey is part of the Bioko Island Malaria Control Project (BIMCP) that has been implemented by Medical Care Development International (MCDI) in collaboration with the country’s National Malaria Control Programme since 2003. The BIMCP is funded by a consortium led by Marathon Oil Corporation  and the Government of Equatorial Guinea. Slides collected from donors in the EGMIS were used for two reasons. First, they provided a source of blood smears from asymptomatic patients. The hypothesis that asymptomatic donors may well provide infections with a lower parasitaemia was borne out by the median of 673 parasites/μL among the 13 positive slides collected. Second, they represented slides that were prepared by the Equatoguinean technicians in the normal course of their clinical duties. The IScan Coreo Gold® scanner was transported to Equatorial Guinea, set up in a clinical laboratory and acquired images that were analysed by a laptop running the WHT software . In addition to this RDT, a random sub-sample of these individuals also had a blood smear made that were used in this study. Further details of the Equatoguinean malaria indicator survey have been previously described . In 2010P. falciparum and non- P. falciparum species. Since cost and logistics prevented the microscope validation of all of the nearly 6.5 thousand EGMIS samples, only a sub-sample (approximately 150) participants had microscopy slides made from their finger prick blood , and a blood sample was collected on filter paper for PCR analysis if needed. The microscope slides were kept in a closed container and Giemsa-stained at the Malabo Central Hospital malaria laboratory by hospital laboratory staff within 24 hours of collection, in accordance with their Giemsa staining standard operating procedures. After staining, all slides were protected with coverslips using Poly-Mount®  as the mounting cement.For the household survey, participants’ blood was collected by Equatoguinean technicians in the field by finger-prick and immediately analyzed by the ICT Rapid Diagnostic Test . The ICT RDT is a HRP-2/aldolase based immunochromatographic strip that provides results for Approximately two thirds of the Equatoguinean slides were scanned by the WHT device in the US and one third were scanned at the Malabo Central Hospital using the equipment described above. Ten of the original 150 slides prepared during the survey were lost or broken during preparation, WHT scanning or shipment to HWH for validation. Therefore, a total of 140 subjects had their blood subjected to the three methods of diagnosis: RDT, a computer image analysis using the system developed by WHT and classical microscopy by a panel of proven expert microscopists .For the reference microscopy, the 140 slides were sent to a minimum of six expert microscopists, each of which provided his/her diagnosis in turn. It was this “composite microscopy diagnosis” (CMD) that served as the “gold standard” against which the WHT system was compared. HWH used a pool of 9 individuals as reference readers to validate the results for this EGMIS slide set. Six of the 9 served as reference readers for the MR4 archive project  and scora priori, that in the case of divergent reads by the microscopists, a minimum of 80% of the reads must be qualitatively concordant. Thus, more than one divergent reference read was interpreted as a slide on which the experts disagreed and a CMD was not possible. These slides were not included in the analysis. Discrepant microscope diagnoses occurred in 21 of the 140 cases, leaving 119 slides with evaluable results , and by the Ethics Review Committee of the Equatorial Guinea Ministry of Health and Social Welfare. Preparation of slides for the MR4 archive was approved by the Indonesian Ministry of Health National Institute of Health Research and Development ethics review board (reference KS.02.01.2.1-4090) and the Naval Medical Research Unit #2 Institutional Review Board (reference DoD#30873). Therefore, all slides used for this test were collected from subjects covered by an ethics board approved minimal risk human use protocol and all subjects gave informed written consent in compliance with the Helsinki Declaration.The WHO Malaria Quality Assurance Manual uses the 55-slide core level test to score three areas: detection of parasitaemia, species identification, and parasite quantification . The resn to 55 and the resulting sensitivity increases to 89% (95% CI 73 to 97) while the specificity remains unchanged at 70%. (95% CI 46 to 88). The values used in the analysis of 40 non-counting slides only are in Table In addition to the WHO promulgated “Level” score however, further information can be gleaned from this WHO55 test. Using the traditional measures of sensitivity and specificity the results of the WHT automated scanning analysis on this slide set yielded a sensitivity of 80% (95% CI 56 to 94) and a specificity of 70% (95% CI 46 to 88) using only the non-counting slides (n = 40). However, since the program cannot be cognizant of the “counting slides” being pf positive, a case can be made for including those analyses in the sensitivity and specificity calculation. This raises the  95% CI 7 to 97 whLikewise, the percentage of correctly identified species was 9, using as denominator only the 20 positive, non-counting slides to arrive at the score of 45%. By considering the 15, counting slides and not penalizing a false negative as an incorrect species, this percentage jumps to 20 correct out of a total of 32 possible or 63%; (95% CI 44 to 79).The quantification of parasitaemia reported by the software was compared to that derived from a median value from the reference readers used to validate the slide. For each slide used in the test, a minimum of 12 reference reader valuations comprised the median value, used as the “truth” with respect to parasitaemia. As noted above and seen in Table P. falciparum count seen by two or more of the readers and the remainder of the validators who reported the slide as negative . In the case of slide # 74, three readers reported P. vivax (mean density 136 parasites/μL) while four others identified the parasites as P. ovale (mean density 77 parasites/μL). The reference readers were blinded to the reads of others and were not aware that the geographic origin of the slides was West Africa. The CMD of slides #74 and #78 was, therefore, deemed to be “Positive” in both cases, and this result was used only in the sensitivity/specificity calculations. Therefore the results reported here are predicated on 119 evaluable results, 106 were negative and 13 positives for sensitivity/specificity calculations while only 11 EGMIS positives were used in species accuracy calculations  and 94% (95% CI 87 to 98) respectively Table . The RDT5 to 100 All of the unambiguously confirmed positives were identified by the reference microscopists as pf. The WHT device correctly identified seven of the 11 as pf, while the remaining four were called mixed infections of pfpm. Therefore, a species score of 64% (95% CI 31 to 89) was achieved on the EGMIS.With respect to quantitation of the infections, the WHT device’s parasite densities for the 11 evaluable EGMIS positive slides are shown in Table n and therefore presumably a more robust statistic.The four evaluated parameters are summarized for each test in the diagram of Figure Inevitably, the performance of a device in a test such as this begs the question, “how do the scores compare to existing diagnosticians.” Although difficult to answer, the increased attention to all aspects of malaria control over the past decade has provided some data with which to illuminate this question. A cross sectional survey of 17 medical treatment facilities in Kenya revealed microscope diagnosis under operational conditions to have a sensitivity and specificity of 69% and 62%, respectively . The comThe performance of the WHT device to diagnose standard malaria blood smears, both thick and thin films was at least comparable to these data. For the pooled data in our evaluation, sensitivity and specificity both above 90% is noteworthy considering the fact that the device functions with minimal need for operator intervention. Despite the fact that the operators need not make the ultimate diagnosis they can, if they so desire, review any and all of the scans and view the items that the algorithm detects as malaria because the machine circles these on the stored image Figure .P. ovale diagnosis on a P. vivax positive slide. These two species share many phenotypic similarities and their misidentification is an error frequently made by microscopists.With only a total of 10 non-pf positives in this evaluation, the device cannot be considered to have been exhaustively tested with respect to species identification. However, having correctly identified half of them, to include 2 mixed infections, is a promising result. A sixth result was a 2 value, at 0.82 indicates that the correlation to the regression line could be much better, the slope of 1.027 shows that there was a relationship between the machine and human counts in which higher parasitaemias resulted in higher machine counts. The y-intercept of 23 in the regression line is a mathematical indication that the machine tends to overestimate the parasitaemias. This finding is also evidenced by a plot of the difference between the two counts plotted on the ordinate against their mean on the abscissa also known as a Tukey mean difference, or Bland/Altman test [The weakest parameter examined for the device was quantitation, which happens to be the most difficult aspect for most human slide readers as well, a fact evidenced by the criteria of the WHO grading scheme Tables  and 8. Eman test . This grOne explanation for these data is, of course, that artifacts, such as stain crystals or even platelets, are being mistakenly identified by the algorithm as parasites. The y-intercept of the regression cannot be interpreted as the projected reading for a slide with 0 parasites however, since the number of false positives was comparatively low. These data, taken together, point to the importance of the pre-scan function and its inherent threshold value.Blood was collected on filter paper for PCR if needed, and this can still be done. However, it is felt that the benefit may not outweigh the cost and effort in this case, especially since certain conclusions can be drawn from the data as they stand. A closer examination of the 21 slides that were removed from the analysis in the EGMIS test reveals them to be slides with a very low parasitaemia . Only three of these were called positive by the WHT device  is noteworthy. It is also of note that this level of performance was reached without the need for highly specialized equipment. The device used standard Giemsa-stained slides and required only a personal computer and a means of digitizing the microscope derived high power fields (scanner). Although it generated 12 false positives (7% of cases), considering the multitude of artifacts that could be mistaken for parasites; we consider this also to be a promising performance. The four false negative results (3% of cases) occurred at an average parasitaemia of 140 parasites/μL which is an indication of the limit of reliable detection.Save the operator identification of the scanning area, the system has been designed to operate without operator intervention. However, the fact that the digital scans are saved and can be reviewed is an important aspect which will allow for rapid improvement of the algorithm. An additional advantage of this system is the fact that it functions in a very analogous way to human slide readers. Unlike other devices described to date ,10, the A few of the improvements already being worked on by the developers of this system include:· X/Y coordinate location of parasites,· composite images of specific species for human confirmation,· self confirming diagnoses between parasites in thick and thin film of the same slide,· a “confidence” value to flag those specimens that require microscopist review· exploring the trade-off between the increased scanning time and file size versus the (presumably) improved performance (sensitivity and specificity) by using a 100x, oil immersion objective to scan the slide as opposed to the 40x ‘high, dry’ lens used in this evaluation.The quality of malaria diagnosis and reporting is not an issue restricted to tropical countries in which the disease is endemic – among 278 US laboratory personnel surveyed, 90% reported that their labs include malaria diagnosis among their capabilities, and among those, 85% report that such diagnoses are performed in-house  . The labA study which did address proficiency test performance was a review of the American Proficiency Institute's (API) Parasitology programme. The fact that the programme sends participating laboratories three slides per year is a testament for the logistical difficulties of evaluating the microscopic diagnosis of malaria. However, irrespective of the rigor of the program, the fact that the proficiency test results over a 10-year period from 1999–2008, revealed that half of the participating laboratories failed to report a “full workup” is in itself telling. In other words, half of the laboratories failed to report either species determination, parasite quantification, or both .By comparison, the WHT device has an objectively measured level of accuracy that is continually being improved to make it comparable to that of human slide readers. Its ability to scan slides rapidly would greatly assist laboratories in meeting the final three of the CSLI criteria, which include the number of thin and thick films examined and the speed at which reports can be provided to clinicians. For a first screening of clinical samples, even a relatively low specificity score, when combined with high sensitivity, results in the elimination from further consideration of the many correctly identified negative specimens, allowing technicians to concentrate on those the device marks as positive. For laboratories in non-endemic countries, such as North America, Europe or Australia, where the preponderance of slides will in fact be negative, the work saved by such screening would be significant. Further, the use of an automated diagnostic device would eliminate the uncertainties currently surrounding the question of diagnostic proficiency, which is extremely difficult to assess due to logistics and the limited availability of standardized slide sets.BIMCP, Bioko Island Malaria Control Project; CI, Confidence interval; CSLI, Clinical and Laboratory Standards Institute; CMD, Composite microscopy diagnosis”; EGMIS, Equatorial Guinea Malaria Indicator Survey; HWH, Hydas World Health; LSHT, London School of Hygiene and Tropical Medicine; MR4, Malaria Research and Reagent Reference Resource Center; MCDI, Medical Care Development International; μL, microliter; NAMS, National Archive of Malaria Slides; PCR, Polymerase Chain Reaction; QBC, qualitative buffy coat microscopy; RBC, Red blood cells; RR, Reference Reader; STARD, Standards for Reporting of Diagnostic Accuracy; WBCs, White blood cells; WHO, World Health Organization; WHT, World Health Technology.MP and JB are the inventors and developers of the device that has been tested in this publication and therefore may not be considered to be unbiased. They supplied not only the equipment and their programs but also contributed to the methods section of this paper to describe the workings of the system. The remaining authors have no commercial interest in the findings of this study.WP and RJ conceived of the study, analyzed the data and prepared the manuscript. VC contributed to data analysis. MG, IK and CS reviewed the manuscript and provided valuable suggestions toward data analysis. MM and MT coordinated the collection and preparation of all slides for the EGMIS portion of the study as well as that of the Equatoguinean archive slides that contributed to the WHO55 sets. All authors read and approved the final manuscript.WP and RJ work for Hydas World Health (HWH), a non-profit which initiated the concept of National Archives of Malaria Slides (NAMS) and, with the US Navy’s NAMRU-2 overseas laboratory, created the proof-of-concept MR4 archive, the use of which is administered by NIH. To date, HWH has been involved in the creation of NAMS in Equatorial Guinea and Ghana, and is beginning their creation in Ethiopia and Zambia.