Plasmodium knowlesi, a simian malaria parasite, has been reported in humans in many Southeast Asian countries. In Thailand, most of the limited numbers of cases reported so far were from areas near neighbouring countries, including Myanmar.P. knowlesi using nested PCR assays. Positive samples were also investigated by PCR for Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae and Plasmodium ovale, and were confirmed by sequencing the gene encoding the circumsporozoite protein (csp).Blood samples collected from 171 Thai and 248 Myanmese patients attending a malaria clinic in Ranong province, Thailand, located near the Myanmar border were investigated for P. knowlesi only. Nucleotide sequences of the csp gene derived from these two patients were identical and phylogenetically indistinguishable from other P. knowlesi sequences derived from monkeys and humans. Both patients worked in Koh Song, located in the Kawthoung district of Myanmar, which borders Thailand.Two samples, one obtained from a Thai and the other a Myanmese, were positive for P. knowlesi is occurring in the Ranong province of Thailand or the Kawthoung district of Myanmar. Further studies are required to assess the incidence of knowlesi malaria and whether macaques in these areas are the source of the infections.This study indicates that transmission of  Plasmodium that infect a variety of hosts, including reptiles, birds, rodents, primates and other mammals . Phylogenetic analysis of the non-repeat region indicated that these sequences were indistinguishable from those of P. knowlesi derived from monkey and human infections in Malaysia and Thailand  [b genes. Non-human primate malaria (P. inui and P. coatneyi) and Hepatocystis species were detected only in wild macaques in Ranong mangrove forests where anopheline mosquitoes were abundant [P. knowlesi was not detected in these macaques. Nevertheless, transmission of P. knowlesi from monkey to human cannot be ruled out.In Myanmar, a patient infected with  Myanmar . This pa or both . In Thair border . In addirovinces ,11. Intecularis) . Increascularis) . Malariaabundant . HoweverP. falciparum and P. vivax are most frequently detected in patients [P. vivax instead of P. malariae by microscopy. Thus, health care workers in this area who are responsible for identification of malaria species should be trained appropriately so that malaria species including P. knowlesi can be correctly identified.It is considered important to correctly identify the malaria species and clarify the mode of transmission for establishing appropriate preventive measures. In Thailand, patients . In thiscsp gene of P. knowlesi obtained from the two patients were identical. Comparison of these two P. knowlesi csp gene sequences and the other Plasmodium csp gene sequences deposited in GenBank confirmed that the P. knowlesi sequences obtained from the Thai and Myanmese patients were closely related to P. knowlesi derived from both monkeys and humans (Figure P. knowlesi and to understand the mode of transmission of this malaria parasite in the Thai-Myanmar border areas.The nucleotide sequences of the s Figure . Blood fP. knowlesi. Both of them work in the Kawthoung district of Myanmar that is close to Ranong province, Thailand and they visited Ranong to seek treatment. This indicates that this Thai-Myanmar border area is one of areas where P. knowlesi is being transmitted.Two blood samples, one obtained from a Thai and from a Myanmese, were each single infections of The authors declare that they have no competing interests.P. knowlesi positive control and helped in manuscript preparation. NS organized blood collection and helped in PCR analysis. MN provided some research funds and helped in study design. VV helped in writing the paper and discussion. All authors read and approved the submitted manuscript.NK carried out the PCR analysis and wrote the manuscript. BS provided


Plasmodium vivax is responsible for a significant proportion of malaria cases worldwide and is increasingly reported as a cause of severe disease. The objective of this study was to characterize severe vivax disease among children hospitalized in intensive care units (ICUs) in the Western Brazilian Amazon, and to identify risk factors associated with disease severity.P. falciparum monoinfection or P. falciparum/P. vivax mixed infection was diagnosed by microscopy in 10 cases, while P. vivax monoinfection was confirmed in the remaining 24 cases. Two of the 24 patients with P. vivax monoinfection died. Respiratory distress, shock and severe anemia were the most frequent complications associated with P. vivax infection. Ninety-one children hospitalized with P. vivax monoinfections but not requiring ICU were consecutively recruited in a tertiary care hospital for infectious diseases to serve as a reference population (comparators). Male sex (p = 0.039), age less than five years (p = 0.028), parasitemia greater than 500/mm3 (p = 0.018), and the presence of any acute (p = 0.023) or chronic (p = 0.017) co-morbidity were independently associated with ICU admission. At least one of the WHO severity criteria for malaria  was present in 23/24 (95.8%) of the patients admitted to the ICU and in 17/91 (18.7%) of controls, making these criteria a good predictor of ICU admission (p = 0.001). The only investigated criterion not associated with ICU admission was hyperbilirubinemia (p = 0.513)].In this retrospective study, clinical records of 34 children, 0–14 years of age hospitalized in the 11 public pediatric and neonatal ICUs of the Manaus area, were reviewed. P. vivax-associated severe disease in children, causing 72.5% of the malaria admissions to pediatric ICUs. WHO severity criteria demonstrated good sensitivity in predicting severe P. vivax infection in this small case series.Our study points to the importance of  Plasmodium species affecting humans, Plasmodium vivax was previously considered a relatively benign infection. Recent reports, however, have linked P. vivax to severe disease Malaria is one of the most important public health problems in the world, with almost half of the world's population at risk of disease and an estimated 800.000 deaths annually, mainly in children under five years of age P. vivax infection since the 1990's P. vivax), predominantly in the Amazon Region, representing 54.9% of all the malaria reported in the Americas Malaria in Brazil has been by and large attributed to P. vivax was considered a non-fatal infection. This perception, however, has changed in recent years, and vivax has become recognized as a cause of severe malarial disease. Severe clinical complications of P. vivax infection have been reported in Brazil for the past decade, especially in Manaus P. vivax disease have been published in Latin America. The current data on predictors of clinical outcomes originates from regions where P. falciparum and P. vivax are equally prevalent, which could make the clinical tools inappropriate for use in managing vivax monoinfections P. vivax is due to the fact that fatalities related to this species remain uncommon. According to the official statistics, between 1998 and 2008, only 234 deaths related to P. vivax infection were reported in the Brazilian Amazon Previously, P. falciparum infection, since, at that time, P. vivax was not thought to cause severe disease. Throughout the years, no effort has been made to validate or standardize specific severity criteria for vivax infections, and current definitions seem to be heterogeneous. This absence of consistency poses technical problems for literature review, meta-analyses and estimation of risk factors.The first attempt to assemble the available knowledge on severe malaria was conducted at an informal technical meeting convened by the World Health Organization (WHO) in Kuala Lumpur, Malaysia in 1985 Alarmingly, the incidence of severe infection appears to be increasing P. vivax hospitalized children not requiring intensive care.This study aimed to characterize all children under 14 years of age admitted with a malaria diagnosis to an ICU in the Manaus area from 2004 to 2009, to assess potential risk factors associated with ICU admission , and secondarily to address the applicability of WHO falciparum severity criteria to vivax infection, by comparing these cases to Fundação de Medicina Tropical Dr. Heitor Vieira Dourado , as well as by the ERB of the Health Secretariat of Manaus and the Health Secretariat of the Amazonas State. All data analyzed were anonymized. Since data were obtained exclusively from clinical charts, the ERB gave a waiver of informed consent.The review of clinical files was approved by the Ethics Review Board (ERB) of the Anopheles darlingi is the major malaria vector and the annual parasite index (API) was 11.5 cases/1,000 inhabitants, in 2009. The city has universal health assistance for citizens (Sistema Único de Saúde - SUS), and offers five public pediatric ICUs  and six neonatal ICUs  in 11 tertiary care pediatric hospitals and maternity wards. The infrastructure of all 11 hospitals is very similar and staff pediatricians are from the same cooperative society, which utilizes a single set of admission criteria and clinical management guidelines for ICU admission (severe compromise of respiratory and/or hemodynamic functions and/or coma). Since every febrile patient in Manaus is subject to a thick blood smear (TBS) and the symptoms of uncomplicated P. vivax and P. falciparum malaria are similar, treatment seeking bias seems unlikely. Since the access to tertiary care hospitals is relatively easy, we assume that all critically ill patients seeking health care were subsequently hospitalized in one of Manaus' ICUs. Patients in private ICUs in Manaus were excluded from this study because the patients are generally from non-malaria endemic areas. Patients infected with P. vivax that required an ICU admission were compared to children of the same age range infected with P. vivax not requiring an ICU admission but concurrently admitted to a tertiary care center for infectious diseases .The selection of cases was performed in the city of Manaus, capital of the Amazonas State, where This study is a retrospective review of clinical and laboratory records from hospital databases. Case files for children 0–14 years old admitted to any of the ICUs with a suspicion of malaria between January 2004 to December 2009 were reviewed for: (1) ICU logbooks , (2) hospital laboratory logbooks , and (3) SIVEP-Malaria . Cases were included if there was evidence of malaria diagnosis by microscopy in any of the three information systems searched. The study enrolled only patients after 2004 because of better reliability of the National Malaria Information System  and access to the clinical files, avoiding retrieval bias.Fundação de Medicina Tropical Dr. Heitor Vieira Dourado with a diagnosis of malaria were retrieved electronically from the computerized hospital records system (iDoctor®) and reviewed. With the exception of automated full blood count, routinely requested for all these hospitalized children, more specific laboratory tests  were requested only if clinical symptoms were suggestive of complications. As part of routine follow-up, all malaria admitted children were reassessed in the Outpatient Clinics seven days after discharge to guarantee adequate clinical and parasitological follow-up.As comparators, clinical and laboratory data from children of the same age group admitted between January 2009 and July 2010 to the 3); 1+ (301–500 parasites/mm3); 2+ ; 3+ ; and 4+ . The cut-off to define high parasitemia  was based on the distribution of semi-quantitative parasitemia results. All positive slides and 10% of negative slides are routinely reviewed in a reference unit with experienced microscopists. In case of divergence, the reviewed result is updated in the on-line system. PCR diagnosis for malaria was not routinely performed in these patients. Only children with a positive TBS for Plasmodium spp. are treated according to the Brazilian Anti-Malarial Treatment Guidelines, which recommends chloroquine (25 mg/kg over 3 days)+primaquine (0.5 mg/kg/day over 7 days) for P. vivax infection and artemether/lumefantrine for 3 days for non-severe P. falciparum infection. Severe P. falciparum infections are treated with parenteral artemether or artesunate for 7 days.TBSs are routinely performed for the diagnosis of malaria in Brazil, prepared as recommended by the Walker technique 2∶FiO2<200 mmHg. The data retrieved were reviewed by another member of the study team and discrepancies resolved by a third member if necessary.Data for each admitted individual was systematically retrieved from the medical charts by the same member of the study team, and included the following variables: sex, age, malarial species, semi-quantitative parasitemia, duration of disease (in days) prior to the admission to the ICU or ward, outcome, presence of a given acute co-morbidity other than malaria or a chronic disease confirmed through a reliable diagnostic method, and presence of any WHO-defined severe malaria criteria 2, FiO2, base excess in arterial blood, need for mechanical ventilation at any time during the first hour in ICU, elective admission, and presence of concomitant diagnoses In all ICUs, collection of venous blood samples for aerobic culture and arterial blood for gas analysis was routinely carried out on admission prior to administration of antibiotics. From all the patients hospitalized in the ICU, the Pediatric Index of Mortality (PIM) score was calculated with data from the first hour of admission, based on the systolic blood pressure, pupillary reactions to bright light, PaOOdds Ratio (OR) with its respective 95% Confidence Interval (95% CI) was determined considering the admission to ICU as the dependent variable. Logistic regression was used for the multivariate analyses and the adjusted Odds Ratios with 95% CI were also calculated. A multivariate logistic regression was performed with admission to the ICU as the outcome, using an automated backward and forward stepwise estimation. All variables that were associated with admission to the ICU at a significance level of p<0.10 in the univariate analysis were included in the multivariate analysis. Statistical significance was considered if p<0.05 in the Hosmer-Lemeshow goodness-of-fit test.Data were analyzed using SPSS® version 16.0 for Windows . Proportions of fatality were compared by Fisher exact test (corrected by Yates' test if necessary); differences were considered statistically significant for p<0.05. The crude P. vivax infections), as shown in P. vivax  and P. falciparum , but a much higher relative risk for mixed infections .According to the official reporting systems, during the period of 2004–2009, 64,032 children 0–14 years of age were diagnosed with malaria in Manaus . The review of logbooks disclosed that 40 of these children were admitted with malaria to one of the 11 pediatric or neonatal ICUs in Manaus. Thirty-four clinical files were actually retrieved from these 40 cases . Altogether, respiratory distress (7/10) and shock (7/10) were the most common clinical complications in these children and the case fatality rate was 30.0% (3/10) . No other concurrent cause for ICU admission was found in 40.0% (4/10) of the children while in the other six children, acute and/or chronic conditions were probably acting synergistically to produce the complications. Only one concomitant bacterial infection was diagnosed (Pseudomonas aeruginosa) in a child with P. falciparum monoinfection. In all 10 patients, at least one of the WHO criteria for severe malaria was present.P. vivax monoinfection admitted to the ICU are presented in 3), and therefore 500 parasites/mm3 (the lower bound of this range) was chosen as the cut-off to define high parasitemia in our analysis, enabling similar number of patients in both categories (low and high parasitemia). Respiratory distress (16/24) and shock (13/24) were also the most common complications associated with this species. Three out of 16 patients with respiratory distress had ARDS and 8/16 presented with simultaneous metabolic acidosis. No other apparent concurrent cause for ICU admission was found in 10/24 (41.6%) children, while in the remaining 14 children there was evidence of the participation of acute and/or chronic conditions in the occurrence of complications. All children were treated with chloroquine (through nasogastric catheter when the child was intubated), primaquine after discharge, and one or more empiric antibiotic regimens. Although, only two patients  presented positive blood cultures during the admission ; (p = 0.018)], the presence of acute  and/or chronic co-morbidities  were all independently associated with the risk of developing life-threatening P. vivax monoinfection requiring admission to an ICU. None of the children not admitted to the ICU developed severe disease after their discharge, as reviewed in their electronic files from follow-up visits in the outpatient clinics.P. vivax monoinfections with hospitalized P. vivax patients not requiring ICU admission. Of all variables assessed for both groups, the odds of being admitted to the ICU increased in patients fulfilling at least one WHO severity criterion, particularly in those with severe anemia or coma. The mean of the PIM, a validated score that parallels bad prognosis in pediatric ICUs, was higher in children with shock and coma. P. falciparum or P. falciparum/P. vivax infected children (30.0%) died more frequently than those infected with P. vivax monoinfection (8.3%), but this difference was not significant . P. falciparum monoinfection showed higher fatality  when compared to P. vivax monoinfection , but this difference likewise was not significant (p = 0.086).P. vivax during the period of study, confirming the public health impact of this infection 3, and the presence of any acute or chronic co-morbidity were independently associated with ICU admission. At least one of the WHO severity criteria for malaria  was present in most of the patients admitted to the ICU. WHO severity criteria may also serve as predictors of severity for P. vivax patients. Further studies, however, are needed to confirm this finding considering the small number of cases presented in our series.This study showed that in Manaus most of the malaria-associated hospitalizations in ICUs were related to A significant male sex predominance among children admitted to the ICU in our study contrasts with data from Indonesia and India, where the female sex was associated with severity in vivax infection P. vivax infection, and had a particularly poor prognosis. In P. falciparum malaria, respiratory distress arises secondary to either primary pulmonary pathology mediated by sequestration of parasitized erythrocytes in the lung microvasculature or from the drive to compensate metabolic acidosis. For P. vivax malaria, however, the pathophysiology behind respiratory distress is not well understood. Studies performed in uncomplicated vivax patients suggest that progressive alveolar-capillary dysfunction occurs after treatment, and this inflammatory response is proportional to initial parasite burden in P. vivaxex vivo adhesion of P. vivax-parasitized red blood cells to human lung endothelial cells was demonstrated for the first time Similar to what was observed by Tjitra and colleagues in Papua, Indonesia P. vivax as a cause of severe morbidity in the early infancy. Severe anemia can also be seen, not triggered by the parasite itself, in patients with G6PD deficiency, as a complication of the use of primaquine for the radical cure of hypnozoites, as observed in three patients described here (two with pure P. vivax and one with mixed infection). If followed by acute renal failure (ARF), it constitutes the syndromic presentation of blackwater fever P. malariae infection, a species known to cause acute or delayed renal complications seems highly improbable, as this species is not detected in Manaus − variant among males has been estimated at 3% The occurrence of severe anemia also paralleled data from other endemic areas P. vivax infection was both frequent and severe, requiring the use of vasopressors, with or without a confirmed positive blood culture for aerobes. Even for P. falciparum malaria, the etiology of this complication is uncertain and the potential role of septic shock as a concomitant entity has been proposed Circulatory collapse  associated with P. vivax per se or resulted from a concomitant liver infection.The presence of hyperbilirubinemia is becoming recognized recently as an inappropriate criterion of severe disease if found in isolation Coma was also reported in our series, but in 3 out of 5 children with coma, additional explanations other than cerebral malaria could be found, such as viral encephalitis in one, and decompensation of previous neurological sequelae in two. In one patient hypoglycemia could also be a superimposed factor. Cerebral vivax malaria actually seems to be more common in children P. vivax. This was also observed in an almost identical proportion (6/10) of P. falciparum patients, which leads to the conclusion that the presence of co-morbidities may contribute to or facilitate the appearance of complications in all malaria. Importantly, this aspect seems to have been specially underreported in prospective studies on vivax severity P.vivax-triggered anemia could be related to parvovirus B19 infection P. vivax (4 cases) and P. falciparum (2 cases) children. Despite the lack of local data on rotavirus infection, the worldwide-recognized increased severity in those less than 5 years of age could in part explain the younger age of the ICU hospitalized children. Furthermore, in our children, septic shock with confirmed bacteremia was observed in three cases. In Mozambique, the systematic performance of blood cultures on hospital admissions showed that a positive culture for aerobes in children with malaria was associated with an increased risk of death Plasmodium species. The finding of four malnourished children among vivax cases admitted to the ICU does not seem negligible, and should be taken into consideration as a potential confounding factor when severity frequencies are compared across the world P. vivax can surely independently cause severe or even life threatening episodes in adults and children, the relative importance of co-morbidities or other chronic existing conditions in accelerating the progression to a severe disease cannot be disregarded.It is noteworthy that acute or chronic co-morbidities were detected in 58% (14/24) of the patients with P. vivax malaria as patients with severe P. falciparum/P. vivax malaria, due to the possibility of submicroscopic parasitemia of the first species, the Brazilian Guidelines for Antimalarial Treatment have only recently changed. During the study period of the present study, chloroquine was prescribed to all patients. Resistance to chloroquine was described for the first time in Latin America in Manaus in vivo resistance in this locality is estimated at 10% P. vivax is still chloroquine. Ecological studies suggest that severity in vivax infection could be related to chloroquine-resistance Despite the WHO recommendation to treat patients with severe P. vivax patients hospitalized in the ICU (8.3%) was not insignificant, even in the Brazilian scenario where diagnosis and treatment of malaria are free and universally available. These numbers could be far larger, however, if a reasonably established health system with tertiary care facilities is not in place. Our small sample was unable to show that P. falciparum children died more frequently than those infected with P. vivax. Actually data from other settings point to a similar fatality rate between the two species The fatality rate among P. vivax monoinfections may in fact be mixed cases. Although possible, this seems unlikely, as a preliminary analysis from our laboratory shows that only 5% of the positive TBSs for P. vivax identify mixed infections, based on real-time PCR (data not shown). Another limitation is the lack of information on total number of patients hospitalized due to malaria in Manaus in the period.An important limitation of this study is its retrospective design, and all analyzed information is based on the available registration in clinical charts. Besides that, the absence of PCR confirmation of malarial infections is an additional limitation. Species-specific PCR increases the diagnostic accuracy and can detect submicroscopic infections that may have been disregarded only after standard microscopy. It is possible therefore, that some of our assumed P. vivax infection was responsible for most of the ICU admissions of children 0–14 years old due to severe malaria , and that the clinical complications were very similar to those seen in P. falciparum in this same endemic area. Despite the fact that peripheral parasitemia greater than 500/mm3 was associated with ICU hospitalization, the positive predictive value of this cut-off for predicting need for ICU admission is extremely low, given the tiny fraction of malaria cases requiring ICU in the general population. Severe vivax patients should also trigger the routine screening of additional infectious diseases in the daily practice in the tropics. From the public health perspective, decision-makers from national malaria programs should re-orientate clinical management algorithms in order to improve treatment, diminish the burden of hospitalization, decrease costs and reduce malaria-associated mortality.In the present study we showed that, in absolute numbers, P. vivax infection, for which mechanisms are poorly understood even in the case of severe P. falciparum. More studies are needed to evaluate the applicability of these findings in other age groups. Due to the fact that studies on the clinical aspects and biology of P. vivax have been neglected during most of the 20th century, the large knowledge gaps regarding this parasite From the basic science perspective, pathogenesis studies in severe vivax should carefully rule out other diseases, which may alter the immune response


Plasmodium vivax accounts for 86% of the cases circulating in the country. The extra-Amazonian region, where transmission does not occur, recorded about 566 cases imported from the Amazonian area in Brazil and South America, from Central America, Asia and African countries. Prolonged incubation periods have been described for P. vivax malaria in temperate climates. The diversity in essential biological characteristics is traditionally considered as one possible explanation to the emergence of relapse in malaria and to the differences in the duration of the incubation period, which can also be explained by the use of chemoprophylaxis. Studying the reported cases of P. vivax malaria in Rio de Janeiro, where there is no vector transmission, has made it possible to evaluate the extension of the incubation period and to notice that it may be extended in some cases.In 2010, Brazil recorded 3343,599 cases of malaria, with 99.6% of them concentrated in the Amazon region. Descriptive study of every malaria patients who visited the clinic in the last five years. The mean, standard deviation, median, minimum and maximum of all incubation periods were analysed.P. vivax. Between those, seven had an estimated incubation period varying from three to 12 months and were returned travellers from Brazilian Amazonian states (6) and Indonesia (1). None of them had taken malarial chemoprophylaxis.From the total of 80 patients seen in the clinic during the study time, with confirmed diagnosis of malaria, 49 (63%) were infected with The authors emphasize that considering malaria as a possible cause of febrile syndrome should be a post-travel routine, independent of the time elapsed after exposure in the transmission area, even in the absence of malaria chemoprophylaxis. They speculate that, since there is no current and detailed information about the biological cycle of human malaria plasmodia's in Brazil, it is possible that new strains are circulating in endemic regions or a change in cycle of preexisting strains is occurring. Considering that a prolonged incubation period may confer advantages on the survival of the parasite, difficulties in malaria control might arise. Anopheles mosquito) and the manifestation of the first clinical sign or symptom. Usually, these periods vary depending on the species of Plasmodium causing malaria. The average incubation period is 9-14 days for Plasmodium falciparum, 12-17 days for infections by Plasmodium vivax and 18-40 days for infections caused by Plasmodium malariae  proe (1946)  demonstre (1946) .P. vivax strains could be explained by the assumption that, in varying proportions, all strains of P. vivax produce two types of sporozoites: one eliciting short prepatent periods (Type I) and the other lying dormant or developing slowly to give rise to long prepatent periods (Type II). In this model, the latter type would greatly predominate in "temperate strains", but not in tropical ones. It was thought that relapses of P. vivax could in reality correspond to a delayed parasitaemia arising from Type II sporozoites. In the same year, Garnham stated that the length of the incubation period was considered the major biological difference between Dutch, Madagascar, and USSR strains, and although there was no evidence of specifically dormant forms, it was believed that if certain sporozoites failed to develop in the normal time, they could be reactivated by an unknown factor one year or more after inoculation [According to Shute , the difculation .P. vivax in humans, based on the concept that temperatures persistently above a minimum of 23.9°C were required for sporozoite maturation [et al in Korea [In 1980, Warwick  proposedin Korea ,11.P. vivax malaria in Rio de Janeiro, where there is no vector transmission, has made it possible to detect and to evaluate certain peculiar aspects of the natural evolution of the disease in human beings. One main aspect was the extension of time required for the parasites to progress through liver schizogony and produce symptoms by their propagation in the bloodstream.The opportunity to study some cases of Plasmodium vivax infections with prolonged periods of incubation and no association with malaria prophylaxis in patients from the Amazon region in Brazil and in one patient from Indonesia are presented.P. vivax interactions.In addition to demonstrating an interesting clinical situation and the need for clinicians to consider the diagnosis of malaria in a patient presenting symptoms a long time after exposure, even in the absence of chemoprophylaxis, our cases raise questions regarding the understanding of the biology of the host/This is a descriptive study conducted at the Acute Febrile Disease Outpatient Clinics of IPEC, Fiocruz, a specialized post-travel care clinic located in Rio de Janeiro, from January, 2005, to February, 2010.P. falciparum and 12 days for P. vivax) [All malaria patients presenting clinical signs or symptoms of malaria and positive thick blood smears were enrolled in the study. The following variables of interest were recorded: estimated incubation period, place and year of infection, date of diagnosis, previous malaria history and year of the first malarial infection. No patients had malaria prophylaxis, had not received blood transfusions nor had close contact with a person with malaria after departure from the endemic area. No patients had haemoglobinopathies. Because it was not possible to determine the date of exposure to the infective mosquito bites, the minimum incubation period was estimated based on literature , Fiocruz (number 0020.0.009.000-07), maintaining strict secrecy and confidentiality of the information obtained.Plasmodium asexual and sexual stages, according to standard procedures [Thin and thick blood smears were stained with Giemsa and analysed by light microscopy using an immersion oil lens (X100 objective magnification) to identify the parasite species and determine the density of ocedures . Each smAll information was recorded on a standardised form for study and subsequently entered into a database using Statistical Package for Social Sciences (SPSS). SPSS-WIN 16.0 was also used for data analysis.P. vivax malaria, 20 (25%) with P. falciparum malaria, eight (10%) with mixed  infection and two (2.5%) with P. malariae. All patients were travellers, most of them (51) from the Amazon region, in Brazil; 17 travelled from Africa, 11 were from South and Central America, and one was from Indonesia.During the study period, 80 malarious patients were diagnosed and treated. Of them, 50 (62.5%) presented with P.vivax than for Plasmodium falciparum and is illustrated in Table Time between the day of return from the malaria transmission area and the first day of onset of symptoms recorded for all patients diagnosed in the Acute Febrile Diseases Clinic Rio de Janeiro (2005 until January 2010) was four times longer for P. vivax malaria  of the patients with 4% of theet al [Plasmodium development; blood transfusions; close contact with a person with malaria after departure from the endemic area or haemoglobinopathies) were eliminated. The observation of a longer incubation period (≥90 days) in 14% of the P. vivax malaria patients seen at IPEC, in Rio de Janeiro, may indicate the importance of monitoring these characteristics worldwide, as it may represent an evolutionary change in P. vivax behaviour. The average incubation period of P. vivax malaria presented here was approximately twelve times longer than the classical period described in the literature. In this study, the extended incubation time occurred in both prime-infected (130 days) and non-prime-infected (131 days) patients, so the possibility of relapse among non-prime-infected patients cannot be ruled out. However, in two patients previously infected with malaria, the period between the last infection and the current clinical manifestation was five and six years, by far exceeding the maximum period of relapse reported for P. vivax (three years) [This is the second report of prolonged incubation period of malaria in patients without chemoprophylaxis coming from an endemic area in Brazil. A recent paper by one of the authors of this report (Tauil PL) described three cases of vivax malaria originating from the Amazon region and diagnosed in Brasilia, Federal District, six months after departure from the endemic region in 2008 . Two of et al . All posP. vivax malaria diagnosed outside the endemic area were observed between these cases with prolonged incubation periods. Regardless, the postulate that extended incubation periods may represent an adaptation of the species to overcome cold temperatures, thereby conferring advantages for the survival of the parasite, does not seem to fit the reality of tropical areas, where the temperature is rarely below 10°C. Although the role of strain-specific variation in prolonged incubation periods has been questioned by some authors [Plasmodium are circulating in tropical areas, especially in the Amazon, which is a region frequently visited by foreigners and which has seen the movements of troops.During the five years of surveillance (2005-2010) no seasonal differences in the prevalence of clinical  authors , it is pFever is one of the most common clinical signs in returning travellers -20. The Anopheles spp. by mechanisms yet to be defined. Therefore, new molecular tools need to be used for investigation of biological characteristics and origin of the Plasmodium strains that presents a prolonged incubation time in Brazilian patients that have never visited the temperate zone.It is classically considered that the co-existence of short and long-term incubation periods may imply that prolongation of this phase is either a genetically regulated feature of parasites or is controlled within Plasmodium vivax, responsible for 86% of malaria cases in Brazil [n Brazil , has lonn Brazil . The chaThe authors declare that they have no competing interests.PB - responsible for conception and design of the work, interpretation of data and drafting the manuscript.APC - analyzed data, made the literature review and helped drafting the manuscript.RSP - helped analyzing the data and reviewed the text.CSB - responsible for the production of data and helped reviewing the text.SS - carried out the parasitological examinations and helped in the literature review.PLT - helped in interpretation of data, literature review and reviewing the manuscript.CTDR - helped in the design of the work and reviewed the text up to the final version to be published.All authors read and approved the final manuscript.This work was supported by CGLAB from the Secretaria de Vigilância em Saúde to the Centro de Pesquisa Diagnóstico e Treinamento em Malária , Fiocruz, Ministério da Saúde, Brazil.


Rapid diagnostic tests (RDTs) have become an essential tool in the contemporary malaria control and management programmes in the world. This study aims to evaluate the performance of two commonly used RDTs for malaria diagnosis in the China-Myanmar border area.A total 606 febrile patients in the China-Myanmar border were recruited to this study and were diagnosed for malaria infections by microscopy, two RDTs tests  and nested PCR.Plasmodium falciparum, Plasmodium vivax and P. falciparum/P. vivax mixed infections, respectively. Compared to microscopy, the sensitivity of the Pf/Pan device was 88.6% for P. falciparum and 69.9% for P. vivax with the specificity of 90.4%. For a subset of 350 patients, the sensitivity of the Pf/Pan device and Pv/Pf device for detection of P. falciparum was 87.5% and 91.7%, respectively; and for detection of P. vivax was 72.0% and 73.8%, respectively. The specificity of the Pf/Pan device and Pv/Pf device was 94.3% and 96.5%, respectively. Nested PCR detected malaria parasites in 174 of 606 samples, of which 67, 79, two and 26 were P. falciparum, P. vivax, P. ovale and P. falciparum/P. vivax mixed infections, respectively. Compared to nested PCR, all other methods had sensitivity below 80%, suggesting that a significant number of cases were missed.Malaria parasites were found in 143 patients by microscopy, of which 51, 73, and 19 were P. falciparum diagnosis, but performed worse for P. vivax diagnosis. Other RDT products should be selected with higher sensitivity (and good specificity) for both P. falciparum and P. vivax diagnosis.Compared to PCR, both microscopy and RDTs had lower sensitivities. RDTs had similar performance to microscopy for  Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale and Plasmodium knowlesi), which have different geographical distributions. Plasmodium falciparum causes the most severe form of the disease and tends to predominate in tropical areas. Plasmodium vivax is the predominant species outside Africa. In recent years, it has been increasingly recognized that P. vivax is also associated with severe symptoms[P. vivax infections each year and the significant lag in research and prevention justifies considering P. vivax malaria as a neglected tropical disease[P. vivax occurs sympatrically with P. falciparum. Since these two parasites require different drug treatments, accurate diagnosis is required to differentiate these two species in areas of co-existence.Malaria is a highly prevalent disease in tropical and subtropical regions, affecting half of the world’s population in 108 countries, resulting in almost one million deaths annually[symptoms, which c disease,4. In maP. vivax is more challenging, because parasite density during P. vivax infections is often low. Recently, the use of molecular methods such as PCR, for the diagnosis of malaria has proved to be highly sensitive and specific[Microscopic examination of Giemsa-stained blood smears under a light microscope remains the gold standard method for malaria diagnosis. However, this technique requires a relatively long observation time and well-trained microscopists. Misdiagnosis often happens in samples with low parasitaemia, especially when drugs are taken inappropriately,6. Furthspecific,8, but dspecific-11. Malaspecific, especiaspecific. HoweverP. falciparum-specific histidine-rich protein-2 (PfHRP2) and parasite lactate dehydrogenase (pLDH). However, the performance of RDTs is easily affected by humidity and extreme temperatures. In addition, persistence of antigens that may remain in the circulation of a patient after treatment may give false positive results[P. falciparum and P. vivax being the predominant species. In this region, cases of human infected by the monkey malaria parasite P. knowlesi were also reported[P. falciparum and P. vivax in this area: a Pf/Pan test device and a Pv/Pf test device. However, their performance for malaria diagnosis under this specific endemic setting has not been evaluated. In this study, the performance of the two RDTs was compared with that of microscopy and PCR for the diagnosis of P. falciparum and P. vivax malaria.RDTs are designed using antibodies against parasite species-specific or genus-specific antigens, such as  results. In malareported,16. Severeported,18. In rP. falciparum and P. vivax infections, is perennial with distinct seasonality, which occurs mostly in the rainy season from April to November. A total of 606 patients attending local malaria clinics and hospital were recruited into the study based on the following criteria: suspected of uncomplicated malaria, having fever with axillary temperature above 37.5°C at the time of examination and willing to participate in the study. The study population has a sex ratio of ~1. Their ages ranged from six months to 88 years with a median of 20.3 years. The relative proportions of patients under 15 years, between 16 and 50 years, and >50 years were 52.9, 39.3, and 7.8%, respectively. The study protocol was reviewed and approved by the Institutional Review Board of Kunming Medical University. All participants or legal guardians gave written informed consent before entering the study. Finger-prick blood was obtained for blood films, RDTs and PCR.The study was conducted in 2011 in Laiza township area along the China-Myanmar border. Malaria in this region, caused mainly by Thick and thin blood films were prepared from peripheral blood. The slides were stained with Giemsa and screened for the presence of parasites and identification of parasite species. Stained blood films were examined with (a 100×) oil immersion lens. Parasite density was determined by counting the parasites and leucocytes, assuming 8,000 leucocytes/μL of blood. Blood fP. falciparum and all human Plasmodium species, respectively. The Pv/Pf test device is based on PfHRP2 antigen and Pv-pLDH antigen, which are specific for P. falciparum and P. vivax, respectively. Five microlitres of fresh whole blood was added to the card pad, and three drops of specific lying agent were added. The RDT result was read in 15–20 min according to the manufacturer’s instructions and immediately recorded. The test was considered valid when the control line on the immune-chromatographic test strip was shown. For the Pf/Pan test device, it was counted as P. falciparum-positive if the line detecting P. falciparum-specific PfHRP2 was positive or if the lines detecting both PfHRP2 and pan-pLDH were positive; as non-falciparum if only the pan-pLDH line was positive. For the Pv/Pf test device, it was counted as P. falciparum- or P. vivax-positive if one of both specific test lines were positive. The readers of RDTs were blinded to the results of microscopy and PCR. All 606 cases in this study were diagnosed by the Malaria Pf/Pan test device. Later, the Malaria Pv/Pf test device was added as a comparison and 350 cases were diagnosed by both RDTs.The RDTs used in this study are One Step Malaria Pf/Pan test  and Malaria Pv/Pf test device . The major target antigens of the Pf/Pan test device are PfHRP2 and pan-pLDH, which are specific for Plasmodium species was slightly modified based on previously published work using the small subunit (SSU ) rRNA gene[Plasmodium genus-specific primers were shown in Table P. knowlesi in separate reaction tubes. After another 30 cycles of amplification , the PCR products were separated in 1% and 2% agarose gels for primary and nested PCR, respectively. DNA bands were stained with ethidium bromide and visualized under a UV light. The primers and expected sizes of the PCR fragments of the SSU rRNA genes are shown in Table Fresh blood samples were spotted on Whatman 3 paper, air-dried at room temperature, and stored individually in a zipper plastic bag at −20°C until use to prevent DNA degradation. Genomic DNA was extracted from dried blood spots using QIAamp DNA Micro Kit (Qiagen) according to the manufacturer’s instruction. Nested PCR for RNA gene. PlasmodThe RDT results were compared with those from microscopy and nested PCR. Sensitivity and specificity were calculated with 95% confidence intervals (C.I.) in two separate analyses: (1) diagnostic performance of RDTs in comparison with the microscopic method, and 2) comparative diagnostic performances of RDTs in comparison with the microscopy and PCR as the references. Based on the microscopic results, the RDT results were considered true positive (TP), true negative (TN), false positive (FP), and false negative (FN) using the interpretation criteria presented in Table  comparatP. falciparum, P. vivax and P. falciparum/P. vivax mixed infections, respectively , 56 non-falciparum cases (only the pan-pLDH line visible) and 70 P. falciparum infections/potentially mixed infections (both lines visible)  than those with a density below 500 parasites/μL (<75.0%)  Table . HoweverP. falciparum, P. vivax, P. ovale and P. falciparum/P. vivax mixed infections, respectively. No P. knowlesi infections were detected by PCR . Re-examination of the P. ovale slides by microscopy did detect P. ovale-parasitized erythrocytes . Only one P. falciparum case with higher parasite density  was not detected by the Pf/Pan test device, but was positive by the Pv/Pf test device. Five out of eight P. vivax cases were lower parasite density (<560 parasites/μL). The remaining three P. vivax cases with higher parasite density  were not detected by the Pf/Pan test device. In the subset of 350 samples examined by the Pv/Pf test device  were false negative. Nine P. vivax cases were the false negative, of which three had lower parasite density (<320 parasites/μL), and three cases  were positive by the Pf/Pan test device. The other three cases with higher parasite density  were not detected by both RDTs.According to the microscopy results corrected by PCR, four and eight cases were false negative in 606 samples examined by the Pf/Pan test device for y Table . When miP. vivax case, one P. ovale case, and eight negative cases showed the PfHRP2 line. Ten P. vivax cases showed not only the pan-pLDH line, but the PfHRP2 line. Seven negative cases showed both PfHRP2 and pan-pLDH lines  showed not only the PfHRP2 line, but also the Pv-pLDH line  with the Pv-pLDH line visible showed that cross reactions between different species occurred although parasite densities of some P. falciparum infections were not high. From these comparisons, the specificities of these devices need further improvement for areas with both P. falciparum and P. vivax malaria.False positive and wrong species identification were observed with both RDTs. In all 606 samples  examined by the Pf/Pan test device, one s Table . In the e Table . It has LDH line-25. For P. falciparum and P. vivax infections are treated with different drugs, it would be important to compare the different methods for detecting mixed species infections. PCR, microscopy and Pv/Pf test device detected 21, 11, and eight mixed-species infections in the 350 subset samples . Both RDTs had similar levels of detection limits, and their performance was poor for infections with parasite densities below 500 parasites/μL for P. falciparum infections. Besides, since both devices rely on the PfHRP2 antigen for the detection of P. falciparum, pfhrp2 gene deletions will lead to false-negative results[P. falciparum parasites with pfhrp2 deletions in South America[RDTs are playing an essential role in the current malaria control/elimination campaign worldwide. In this study, the performance of two RDT devices commonly used at the China-Myanmar border area was evaluated. Compared to diagnosis by microscopy, both the Pf/Pan and Pv/Pf devices showed higher sensitivity (>87%) for detecting  results. A recen America indicateP. vivax infections (<64%), although the specificity of the all detection methods remained above 92%. In addition, the sensitivity of conventional microscopy was also low, ranging from 71.0% to 77.4% for detecting both parasite species. This result is worrisome; since nearly 30% of the malaria cases was not correctly diagnosed by microscopy or RDTs and subsequently treated, these patients may constitute an important reservoir for transmission. Whereas low parasite density might be the most important limiting factor for microscopic diagnosis[P. falciparum only and showed highly satisfactory result[P. falciparum and P. vivax infections[When PCR was used as the gold standard, the detection sensitivity of both RDTs was reduced especially for the detection of ections <4%, althoiagnosis, the variagnosis. In the fections. TherefoP. falciparum infections, but the sensitivity for detecting P. vivax was much lower. This could be due to lower parasite density for P. vivax since this parasite selectively invades reticulocytes. However, comparison of the two RDTs showed that the sensitivities for P. vivax detection were not different between the high and low parasite densities (Table P. vivax. In addition, the sensitivity to P. vivax may be affected by other factors. Therefore, better RDTs with significantly improved sensitivity for P. vivax are needed.Overall, both RDTs have good sensitivity for detecting s Table . This obP. falciparum. The advantage of the Pf/Pan test is that it is designed to detect four human parasite species, which co-exist in the GMS. However, this device failed to detect the two P. ovale infections, suggesting that further improvement in sensitivity for detecting other malaria species is needed. It is also worth to note that the PCR method failed to identify any P. knowlesi infections, which is in stark contrast to the eaerlier report of some 30% of malaria cases in this region as mixed infections with P. knowlesi[P. ovale and P. malariae suggest that such an advantage is hardly of any importance in this region. In comparison, the Pv/Pf test is specific for P. falciparum and P. vivax, which make up the majority of malaria infections in the GMS, although it would certainly miss detecting P. malariae and P. ovale infections. Because P. falciparum and P. vivax require different antimalarial treatments, correct diagnosis of mixed infections should be an important criterion for the selection of an RDT. Even though the Pv/Pf device is able to detect P. falciparum and P. vivax mixed infection, the detection rate was low and the eight mix infections detected by this device were all misdiagnosed when compared to the PCR result. This result may be due to reduced density of one parasite species in the presence of another due to competition in the same host. Consequently, with the current anti-malarial drug policy of this region, misdiagnosis of mixed species infections as single species infections would lead to improper drug treatments.One important criterion for the selection of RDTs in the GMS is the ability to detect other human malaria species in addition to  knowlesi. TherefoP. falciparum infections but reduced sensitivity for detecting P. vivax infections. Neither of them had satisfactory results in detecting mixed species infections. Therefore, methods with improved sensitivity for diagnosing P. vivax malaria are needed. False negative results for the diagnosis of P. falciparum malaria call for further investigations on potential deletion of pfhrp2 gene in this malaria-endemic region.The aim of this study was to evaluate the performance of two RDTs for malaria diagnosis at the China-Myanmar border area. PCR method was much more sensitive than microscopy and RDTs. Compared to microscopy, both RDTs demonstrated similar sensitivities for detecting The authors declare that they have no competing interests.YJ, LN, WX, LP and ZZ carried out the experimental work and data analysis.WL, LS and ZG participated in data analysis. YJ performed manuscript writing. YC, QF and LC conceived the study and participated in the design of the study. All authors read and approved the final manuscript.


P < 0.0001). Severity of retinopathy increased with severity of disease (P for trend < 0.0001), and renal failure, acidosis, and moderate/severe retinopathy were independent predictors of mortality by both ophthalmoscopic techniques. Direct ophthalmoscopy by non-ophthalmologists is an important clinical tool to aid diagnosis and prognosis in adults with severe malaria, and indirect ophthalmoscopy by non-ophthalmologists, although more sensitive, provides minimal additional prognostic information.To establish if assessment of malarial retinopathy in adult malaria using ophthalmoscopy by non-ophthalmologists has clinical and prognostic significance, 210 Bangladeshi adults were assessed by both direct and indirect ophthalmoscopy; 20 of 20 healthy subjects and 20 of 20 patients with vivax malaria showed no retinal changes, whereas in patients with falciparum malaria, indirect ophthalmoscopy revealed malarial retinopathy  in 18 of 21 (86%) fatal, 31 of 75 (41%) cerebral, 16 of 64 (25%) non-cerebral but severe, and 1 of 31 (3%) uncomplicated cases. Direct ophthalmoscopy missed retinopathy in one of these cases and found fewer retinal hemorrhages (mean difference = 3.09; 95% confidence interval = 1.50–4.68;  Every year, more than 1 million people die of severe and cerebral malaria.This observational study was conducted at Chittagong Medical College Hospital, a large, 1,000-bed teaching hospital in Chittagong, Bangladesh from May 2007 to December 2007.Plasmodium falciparum malaria were recruited if written informed consent was obtained from them or an attending relative. These patients were divided into the following groups: cerebral malaria , severe non-cerebral malaria , severe corneal scarring or cataracts in both eyes precluding ophthalmoscopy, diabetes mellitus, hypertension, intracranial space occupying lesion(s), epilepsy, alcoholism, head injury, and chronic renal failure.Consecutive adult patients (> 16 years) with peripheral blood slide- or rapid diagnostic test -confirmed On admission, a full history and examination were carried out. Blood samples were obtained for hemoglobin, hematocrit, parasitemia, platelet count, leukocyte count, serum glucose, and full biochemistry. Eye examination included pupillary reaction to light and direct and indirect ophthalmoscopy. Because indirect ophthalmoscopy is more sensitive with a wider field of view and is much better for examining the peripheral retina , it was used as the gold standard and was always performed after direct ophthalmoscopy to minimize bias. Observations were made by two independent observers who underwent 2 weeks of intensive training to supplement their limited prior experience of direct and indirect ophthalmoscopy. In case of non-matching findings, the patient was reexamined, and findings were scored by consensus between the observers. Ophthalmoscopy was performed on admission, and in those who had changes on day 1, it was repeated 3 days after admission and at discharge. Two drops of tropicamide 0.5% or 1% eye drops, with the addition of 2.5% phenylephrine, were used for mydriasis. Retinal changes were graded as mild or moderate to severe by simplified criteria based on the classification of Beare and othersAntimalarial medications and supportive treatments were in accordance with the 2006 World Health Organization (WHO) guidelinesP value for trend. Numbers of retinal hemorrhages were compared using independent samples t test and Kruskal–Wallis test as appropriate. All the tests were two-sided, and P < 0.05 was considered to be statistically significant. Independent predictors of mortality were identified using a stepwise logistic regression procedure. Only variables with a P value of less than 0.05 were retained in the final model.Statistical analysis was carried out using the SPSS version 16.0 and Stata version 10. Correlation of malaria severity with retinopathy severity was by A total of 210 subjects were studied, of whom 75 had cerebral malaria, 64 had severe malaria but no coma, 31 had uncomplicated falciparum malaria, 20 had vivax malaria and 20 subjects served as healthy controls .The distribution of presenting severity features is shown in P. vivax malaria, and 0 of 20 healthy volunteers  .Features compatible with malarial retinopathy were found in 47 of 139 patients (34%) with severe malaria, 18 of 21 patients (86%) with a fatal course, 31 of 75 patients (41%) with cerebral malaria, 16 of 64 patients (25%) with non-cerebral severe malaria, 1 of 31 patients (3%) with uncomplicated malaria, 0 of 20 patients (0%) with  0.0001) . The preP < 0.0001). In all but 10 patients, the overall severity of retinopathy was the same by direct and indirect ophthalmoscopy. In seven patients (3%), retinopathy was scored as severe by indirect ophthalmoscopy and mild by direct ophthalmoscopy, and in one patient, retinopathy was scored as mild by indirect ophthalmoscopy and absent by direct ophthalmoscopy; this was because of differences in the numbers of retinal hemorrhages seen. In three patients (1%), papilloedema was seen by direct ophthalmoscopy but not by indirect ophthalmoscopy.A comparison of findings by direct and indirect ophthalmoscopy is shown in P < 0.0001 for indirect ophthalmoscopy; mean = 4.2, 95% CI = 3.0–5.4 versus mean = 1.3, 95% CI = 0.9–1.8; P < 0.0001 for direct ophthalmoscopy). Of the patients with papilloedema, 8 of 9 (89%) also had other retinal findings (7 of 9 [78%] had hemorrhages and 2 of 9 [22%] had whitening), 7 of 9 (78%) of which were moderate to severe.Indirect ophthalmoscopy findings in patients with fatal and non-fatal severe malaria are shown in P = 0.252). Retinal whitening was found in 6 of 16 patients, and no vessel changes were seen. In only two of these patients was the retinal whitening found by indirect ophthalmoscopy, and in one of these patients, it was found by direct ophthalmoscopy. Both of these patients had marked changes in and around the macula. Papilloedema was not observed on the photographs.Sixteen unselected patients with severe malaria underwent retinal photography when sufficiently well, and two examples are shown in The mean duration of admission for patients with malaria was 5.5 days (95% CI = 5.2–5.9 days); 42 patients with malaria and retinal changes on admission  were reexamined on day 3, and 30 of these were also examined at discharge. By day 3, the grade of retinal changes had improved in 1 of 42 patients  and worsened in 0 patients. By the time of discharge, malarial retinopathy had fully cleared in 11 of 30 patients (37%), had improved in 2 of 30 patients (7%), and was the same as on admission in 17 of 30 patients (57%) who were assessed.This study showed that over one-third of adult patients with severe malaria have malarial retinopathy on admission when assessed by non-expert direct and indirect ophthalmoscopy. Retinal hemorrhages are the most easily visible manifestation of malaria in the retina and as such, have the greatest potential as a bedside diagnostic and prognostic sign. This has been proposed for the diagnosis of pediatric cerebral malaria in high transmission settings in sub-Saharan Africa.The current study compared bedside direct and indirect ophthalmoscopy for the assessment of malaria retinopathy. There was little advantage gained from indirect ophthalmoscopy by a non-expert compared with direct ophthalmoscopy regarding overall sensitivity to detect malarial retinopathy. However, it is likely that, with increased training, indirect ophthalmoscopy would be more sensitive. In the current study, both techniques had similar prognostic value for a fatal course of the disease, suggesting that simpler direct ophthalmoscopy is an adequate tool to assess disease severity, although this was based predominantly on numbers of retinal hemorrhages alone. Both methods found significantly higher numbers of hemorrhages in fatal versus non-fatal infections. This was despite the number of retinal hemorrhages seen by indirect ophthalmoscopy being around double those seen by direct ophthalmoscopy.The present study used a simplified version of the classification of malarial retinopathy used previously in children, which allowed direct comparison of findings across age groups and settings. The prevalences of retinopathy in the present study are around one-half of those detected by objective expert assessment using retinal photography in a smaller study in the same patient populationThe highly specific and common (around 50% of adults)The prognostic significance of retinal hemorrhages was independent of other well-established prognostic indicators, such as metabolic acidosis and renal failure, and is in accordance with previous studies Thailand. This in2The frequency of papilloedema in this study was similar to the findings in the African studies, reporting a prevalence of 15% in pediatric cerebral malaria compared with 4% in children with severe malaria anemia.In children, macular and peripheral whitening are frequent findings, being present in 44% and 46%, respectively, of those with cerebral malaria in one Malawian study.No patients in this study had visible vessel discoloration. This includes those who underwent retinal photography. This finding agrees with the previous study in Bangladesh that used retinal photography.30Just under two-thirds of adult patients with malarial retinopathy in this study still had retinopathy at the time of discharge, and most retinal abnormalities did not improve during the admission. In Malawian children, it was shown that malaria retinopathy takes from 1 to 4 weeks to resolve. Additional studies in adults will be needed to carefully assess the dynamics of retinal pathology over time. This is particularly important for patients readmitted with a febrile illness after an episode with severe malaria to determine whether findings could persist from this previous episode.Limitations of this study include its dependence on the skill of the examining doctor, because ophthalmoscopy is operator-dependent and thus, subjective. Study subjects were examined by non-ophthalmologists with relatively limited training in ophthalmoscopy. However, this should provide a realistic picture of what might be achieved by similarly skilled non-specialists at the bedside. In the 16 patients who underwent retinal photography, the findings closely matched those recorded by indirect ophthalmoscopy, with the exception that much of the retinal whitening seen on the photographs was missed by indirect ophthalmoscopy and papilloedema was overdiagnosed by direct ophthalmoscopy. In addition, direct and indirect ophthalmoscopy was performed by the same individuals on each patient, thus potentially biasing the second examination to match the first. For this reason, less sensitive direct ophthalmoscopy was always performed first.In conclusion, non-expert direct ophthalmoscopy is an important prognostic tool in adults with severe malaria in the Bangladeshi setting. There is little additional information to be gained from non-expert indirect ophthalmoscopy in this setting.


The children aged under 5 years from vast African areas badly suffer from falciparum malaria and many of them die of this disease. Therapeutic efficacy of anti-malaria drugs, especially pyrimethamine-sulfadoxine (PS) and chloroquine (CQ) to falciparum malaria is frequently evaluated and reported in recent 10 years. Unfortunately, to date, these widespread materials and researches have not been systematically collected and analyzed. In our study, two investigators were employed to widely and independently gather researches on efficacy of PS vs. CQ mono-therapy of falciparum malaria in children aged below 5 years in unpublished and published databases. Meta-analyses were conducted in categories of PS group and CQ group respectively. Pooled OR of PS vs. CQ was 0.11 . PS showed higher therapeutic efficacy to falciparum malaria in less-than-5-year children than CQ. Random model was chosen to analyze for the heterogeneity existence between different studies. Subgroup analyses were performed, but heterogeneity was still presented. Heterogeneity might be caused by different resistance of falciparum malaria to PS and CQ in different settings. Malaria type associated with parasite species, basic information of PS and CQ, and PS & CQ resistant malaria control measures were demonstrated and discussed respectively in detail in this article. Plasmodium parasites which is transmitted by the bite of any one of the 50 species of Anopheles mosquitoes. Malaria is one of the most important diseases in the world. It exacts an enormous toll social life, medical costs and productivity in tropical regions of Africa, Asia, and Central and South Americas , mefloquine (MQ) of aryl amino alcohols, amodiaquine (AQ), chloroquine (CQ) of 4-aminoquinolines, pyrimethamine-sulfadoxine (PS) of folate synthesis inhibitors, primaquine (PQ) of 8-aminoquinolines, clindamycin (CL), dox-ycyclinean (D), tetracycline (T) of antimicrobials, and artemether-lumefantrine (AL), artesunate (AS), dihydroartemisinin (DHA) of peroxides. Two or more same or different kinds of them are recommended to use in combination, except CQ therapy of  regions –11. Amon regions –15. Simi regions . The resn Medani . In the n Medani , 19. By n Medani . Since te chosen . CurrentCQ or PS . AQ is wCQ or PS –26. AN iCQ or PS  and has Although efficacy failure of PS and CQ to malaria is generally reported, PS & CQ are still extensively used to combat malaria for its low cost, especially in developing countries, such as countries from Africa. In this continent, CQ and PS remain the first-line and second-line therapies of uncomplicated malaria in some areas. In this study, we reviewed effect of PS vs. CQ on uncomplicated falciparum malaria in children aged below 5 years old in recent 10 years through meta-analysis. Before that, we looked back to some basic information about the invention, use history, and resistance of PS and CQ.The antimalarial efficacy of sulfa drugs was discovered in 1940s. They inhibit the malarial parasites multiplication and development by blocking nucleic acid synthesis . In 1950Use history and study of PS as an antimaliarial drug were described per decade. During 1970-1979, PS for antimalarial was mainly reported in South East Asia, especially Thailand , 31, andIn 1891, dye methylene blue was discovered to be able to kill malaria parasites. During World War I, German scientist used it to synthesize the prototype of antimalarials. In 1932, other dye atabrine was employed to synthesize another antimalarials. German scientists found sontoquine in Tunis, and then sontoquine was modified to be chloroquine. Sontoquine and chloroquine were patented for prophylaxis and control of malaria in the United States in 1941. CQ could quickly control the symptom of susceptible malaria with minimal toxicity. In 1949, CQ was approved as antimalarial by the FDA .vivax malaria  experiences oxidative stress, and this infection changes the anti-oxidative defense system of the host. When CQ introduced, the anti-oxidative defense system returns near normal levels . Literatures that we required were available in grey literature databases(unpublished literature) and published literature databases such as PubMed, British Medicine Association (BMA), Cambridge Science Abstract (CSA), Global health, Conference Proceedings Citation Index (CPCI) and Wanfang data (a Chinese database with published paper and unpublished data that is mainly from conference proceedings).P<0.01). The other statistic indicator of 95% CI of OR whose value range excluded the cut-off value 1 also could be utilized to support this conclusion.Studies we screened occurred in Africa except a study performed in Myanmar of the Southeast Asia. These areas endured intense and widespread malaria incidence, especially among children aged less than 5 years. The total amount of 1911 patients, 919 for CQ group, 992 for PS group, with sample sizes ranging from 33 to 683, were included in 10 studies for analysis. 3 studies had less than 100 patients in sample size –125. Mor2=71.72, P<0.1; I2=87%)  . The ran I2=87%) . FollowiCombined data from different studies was useless due to the heterogeneity. We transferred to tell therapeutic efficacy of PS vs. CQ to treat malaria disease one by one. ORs of PS vs. CQ efficacy against malaria in ten studies ranged from 0 to 0.43 –132. TheMany studies reported different effect of PS vs. CQ for fighting against malaria. Some studies showed PS and CQ all had good effect on malaria, some denoted PS moderate efficacy to malaria, and other studies supported mild or little effect of PS and CQ on malaria. Judged from single paper or a study, we could not conclude the real efficacy of PS vs. CQ to combat malaria because of published bias. Until now, only a study for meta-analysis had revealed comparison of PS vs. CQ efficacy for the treatment of malaria in pregnant women and children. This study more concentrated on PS vs. CQ efficacy to women. Children's age did not refer in this study . Our metThe screened studies were controlled clinical trials, not randomized controlled trials. Drugs were randomly assigned to the enrolled patients from clinicians or experimenter. Nonetheless, we could not find that these study designs included allocation concealment and random sample. There was only one study following blind method using random number. Sample size should be calculated scientifically in a good study design. Most of our screened studies were performed well in sample size calculation regarding of precision, type I error risk, estimated value (failure rate) and loss to follow-up. Blood smear test for parasite density and PCR test for reinfection or recrudescence in late clinical failure/response were aided to assess end/surrogate point of PS vs. CQ effect on children patients. Good laboratory test really reflected the valid clinical response to drugs in patients. Some of our recruited studies involved laboratory quality management. In a word, our enrolled studies designs were not seriously conducted except sample size calculation.Patients who were characterized by loss to follow-up, reinfection, PCR results inconclusive, or other factors which affected to reflect real efficacy of PS vs. CQ to malaria, were absolutely withdrawn. Total withdrawal didn't exceed 10% of enrolled patients. Patient individuals were recruited to well test efficacy of PS vs. CQ against malaria.Clinical response of PS vs. CQ to malaria was evaluated by percent of total failure in all patients. Recommended by WHO, anti-malaria drugs response to malaria are subgrouped by total failure which is equal to sum of clinical failure, early treatment failure and late treatment failure , and adequate clinical and parasitological response . SynthetMalaria is widely resistant to the first line anti-malaria CQ in African countries, South East Asia and Latin America because of the long-time application for combating malaria –141. PS As resistance to anti-malarial mono-therapy becomes a serious problem, chemotherapeutic strategy for control of malaria caused by a parasite should be reconsidered. Combination therapy with drugs having different mechanisms of act and biological target in this parasite may be way out . It is iAnopheles mosquitoes are main vector of malaria pathogens. At least fifty Anopheles species contribute to transmit these pathogens. Population can avoid malaria infection by eradication of mosquitoes. Mosquito prevention and control methods prevail in many articles. At present, many mosquito controls are divided into chemical measurement, biological treatment, habitat management and application of appliance for prevention and control in mosquito bite and annoyance, such as bed-net, mosquito-killer magnet. These control measures are usually used in combination . If persons achieved no mosquito bite by these methods, malaria will end among them.Other measures in malaria control are to prevent mosquito bite in addition to intake of anti-malaria drugs. To date, PS and CQ can also be used to fight against malaria in some areas. However, PS showed higher therapeutic efficacy to falciparum malaria in less-than-5-year children than CQ in our systematic review.


The TREAT Asia Quality Assessment Scheme (TAQAS) was developed as a quality assessment programme through expert education and training, for laboratories in the Asia-Pacific and Africa that perform HIV drug-resistance (HIVDR) genotyping. We evaluated the programme performance and factors associated with high-quality HIVDR genotyping.agreement with panel-specific consensus sequence, and detection of drug-resistance mutations (DRMs) and mixtures of wild-type and resistant virus (mixtures). High-quality performance was defined as detection of ≥95% DRMs.Laboratories used their standard protocols to test panels of human immunodeficiency virus (HIV)-positive plasma samples or electropherograms. Protocols were documented and performance was evaluated according to a newly developed scoring system, p<0.001) and number of DRMs as mixtures (p<0.001); and laboratory performance factors – detection of mixtures (p<0.001) and agreement with consensus sequence (p<0.001), were associated with high performance; sample format (plasma or electropherogram), subtype and genotyping protocol were not.Over 4.5 years, 23 participating laboratories in 13 countries tested 45 samples  in nine panels. Median detection of DRMs was 88–98% in plasma panels and 90–97% in electropherogram panels. Laboratories were supported to amend and improve their test outcomes as appropriate. Three laboratories that detected <80% DRMs in early panels demonstrated subsequent improvement. Sample complexity factors – number of DRMs (High-quality HIVDR genotyping was achieved in the TAQAS collaborative laboratory network. Sample complexity and detection of mixtures were associated with performance quality. Laboratories conducting HIVDR genotyping are encouraged to participate in quality assessment programmes. Over the past decade, combined international efforts have achieved a more than 20-fold increase in access to antiretroviral (ARV) treatment for individuals infected with the human immunodeficiency virus (HIV) in resource-limited settings (RLS) . IncreasWhether using commercial or in-house HIVDR testing, laboratory participation in external quality assessment (EQA) is recommended by expert committees –7. AvailTREAT Asia , a progIn 2006, TREAT Asia was funded by the Dutch Ministry of Foreign Affairs to build surveillance and monitoring capacity for HIVDR in Asia. The resulting TREAT Asia Studies to Evaluate Resistance (TASER)  are partWe previously described findings from the initial implementation of TAQAS within 10 laboratories over a 19-month period in 2005–2007, and demonstrated improvement or maintenance of high standards of genotyping outcomes [n=19 in 11 Asian countries) and PASER (n=3 in 2 African countries) sites  sites . A VirolOver the 4.5-year study period (12/5–6/10), nine 5-sample TAQAS panels (45 samples) were distributed to and tested by participating laboratories, using their standard protocols. Panels were prepared by, shipped from and results returned to the NRL , an independent quality assurance provider.pol electropherograms, derived from ARV-treated individuals, and used to account for inter-laboratory sequence production variation in HIVDR genotyping outcome. Panels varied in the number of DRMs; number of mixtures, defined as >1 nucleotide base at one position; and number of DRMs present as mixtures (n=35) had a viral load >1000 copies/mL; most (31/35) >10,000 copies/mL.Samples in seven panels were either: (i) plasma sourced from HIV-1-patients (27/35), or (ii) culture-amplified virus in HIV-negative plasma (8/35) . Most samixtures . All plapol sequences; (ii) lists of DRMs; and (iii) predicted susceptibility to a standardized list of ARVs. Detailed genotyping information on testing methods was collected via an electronic “Protocol Questionnaire” (Supplementary File 1).Samples were shipped biannually frozen on dry ice (plasma panels) or sent electronically (electropherogram panels) to TAQAS participants after obtaining necessary country-specific permits. Within five weeks of panel receipt, participants were required to return three result outputs: (i) FASTA nucleotide Sequences returned underwent quality control and phylogenetic analyses to examine within-sample clustering using Sequence Quality Analysis Tool (SQUAT) . OutlierHIVDR genotyping performance was evaluated against a panel-specific consensus sequence, termed the “target genotype” (TG), deduced by aligning sample-specific FASTA nucleotide sequences returned by all participants and applying an algorithm to identify the most likely consensus quasi-species detectable by DNA sequencing . The algLaboratory performance was scored using a system composed of eight criteria . PerformAt the conclusion of each panel nucleotide sequence alignments, analyses and scores of all participants’ were made available on a password-protected website. Participants were encouraged to review and compare results, and, if necessary, amend and improve their methods. Sequence alignments were provided for participants whose performance was not scored and they were alerted to the reason. Resubmission of results was not permitted.To provide support and follow-up, annual workshops were conducted for technical and scientific staff directly involved with HIVDR genotyping from participating laboratories, with detailed discussions and expert presentations of laboratory methods. This also facilitated development of a regional network of operators with a range of expertise. When suboptimal performance was identified, laboratories were asked to describe initiatives to address identified problems. In two cases, expert laboratory personnel from within the TAQAS network assisted in setting up HIVDR genotyping protocols or addressed recurring problems.panel complexity and laboratory performance were examined. Panel complexity was defined by: (i) number of DRMs in the TG; and (ii) number of DRMs present as mixtures in the TG; (iii) sample subtype (B or non-B); and (iv) sample format (plasma or electropherogram). Laboratory performance was defined by: (i) length of nucleotide sequence; (ii) nucleic acid agreement with TG; (iii) detection of DRMs in the TG; (iv) detection of mixtures in the TG; and (v) elements of HIVDR genotyping protocol . Mixed-effect logistic regression models were used to derive the endpoint using panel complexity and laboratory performance as predictors. All results, including those from incomplete result sets, were included in the models. As laboratories participated in varying panels, and incomplete result sets were included, random-effect models were used to take into account variation both within and between laboratories. The final model included predictors that were significant at 0.10 level (two-sided). Data management and statistical analyses were performed using Stata .To evaluate participants’ HIVDR genotyping outcome, a binary endpoint was defined as detection of ≥95%  vs. <95%  of consensus DRMs in the TG. Factors potentially associated with Nine TAQAS panels were delivered to 19 laboratories in the Asia-Pacific, three in Africa and one in the United States . Eight lThe Questionnaire, which was completed between the fifth and sixth panels by all but one laboratory (Lab 11 ceased HIVDR testing after Panel VI), demonstrated a wide variability in HIVDR genotyping experience. Laboratories were conducting HIVDR testing for a median of six years ; the median testing throughput was 348 tests per annum ; and the median per sample turnaround time was 14 days . Fourteen laboratories required staff qualifications of a bachelor degree or higher and three required training in molecular biological technique (five did not respond). Time pressure to complete HIVDR testing was perceived by 9 out of 22 laboratories.® one laboratory; ViroSeq® three laboratories). The impact on test outcome of participants’ sequencing practices was assessed. Only a few laboratories (6/22) outsourced sequencing. Most laboratories (20/22) used an automatic base calling software and all reported manual checking and editing of automated base calls. In most laboratories (16/22), more than one person was involved in sequence editing. The peak height to call mixed bases was set at 20–30% by 19 laboratories. It was policy in most laboratories (15/22) to review sequence data at sites associated with ARV resistance. Twelve laboratories reported controlling for contamination using software such as Clustal  or Mega (www.megasoftware.net).The majority of laboratories (18/22) used locally assembled protocols, with wide variations in primers, sequencing probes and input sample volume (data not shown). These aspects of the protocols were not compared. Four laboratories used commercial kits  or two (n=1) panels. Late submission of results was recorded and these data sets were included in analyses. One hundred and thirty six data sets (107 plasma and 29 electropherograms) were suitable for assessment. Eight data sets were removed from analyses because they were derived from less than four out of five samples per panel due to sequence amplification failures (two datasets); production of sequence deemed of poor quality (three data sets); or did not satisfy quality control analyses (three data sets)  . On follThe median detection of DRMs in the TG in the seven plasma panels ranged between 88% and 98% . In mostThe median detection of DRMs in the electropherogram panels was 97% and 90%. Again, detection of mixtures varied . Labs 2 and 5 detected low levels of mixtures in the electropherogram panels . In addip<0.05).Data sets from all participants were quantitatively evaluated using the scoring system . High-qulaboratory performance that had an impact on HIVDR genotyping outcome included mixture detection  and level of agreement with the TG  , and number of DRMs present as mixtures . Subtype (B or non-B) and sample format (plasma or electropherogram) were not associated with genotyping performance.Factors associated with p<0.001) . NeitherWe report extended results from TAQAS, an EQA programme for HIVDR genotyping by a group of laboratories in Asia-Pacific and Africa . Twenty-Similar to other EQA programmes, TAQAS participants varied in testing experience and access to technical resources , 29. In EQA programmes should aim to assess the HIVDR laboratory testing process using clinically relevant sample types. The predominant use of plasma samples in TAQAS enabled assessment of detection of viral mixtures, important in DRM detection , 20, 32,Subtle differences in EQA programmes that reported higher levels of DRM detection than reported by TAQAS participants are noted , 29–31. Panel complexity, detection of mixtures and level of sequence agreement were found to predict HIVDR genotyping quality, confirming previous reports , 34, 35.The electropherogram samples used in two TAQAS panels showed that variation in detection of mixtures can occur because of inter-laboratory differences post-sequence production, rather than genotyping extraction, amplification and sequencing protocols. Sequence editing has been suggested to contribute to disparity in HIVDR genotyping outcomes particularly with respect to the mixtures detection . HoweverObjective assessment of EQA performance outcomes enables intra- and inter-laboratory and between-panel comparison, and potentially comparison between EQA programmes , 31, 37.Some previously reported limitations of TAQAS  have beeTAQAS was established to build capacity for and assure quality of HIVDR genotyping to support clinical care and research in Asia and Africa. Participation by 22 laboratories over an extended timeframe confirmed their HIVDR testing proficiency. Several reasons are proffered as to the high-quality test outcomes of this complicated test: diligent and conscientious attitude of participants, provision of complex clinical samples, comprehensive and comparative analysis of results, follow-up initiatives after suboptimal performance, and support and information dissemination by TREAT Asia and NRL. TAQAS facilitated successful application of a quantitative measure of laboratory performance of HIVDR genotyping and identification of predictors of test quality. Both tools can improve utility of future EQA programmes. The importance of continuous EQA participation to maintain and improve HIVDR genotyping outcome has been validated , 25, 30.


Congenital malaria is defined as malaria parasitaemia in the first week of life. The reported prevalence of congenital malaria in sub-Saharan Africa is variable (0 - 46%). Even though the clinical significance of congenital malaria parasitaemia is uncertain, anti-malarial drugs are empirically prescribed for sick newborns by frontline health care workers. Data on prevalence of congenital malaria in high-risk newborns will inform appropriate drug use and timely referral of sick newborns.Blood samples of untreated newborns less than 1 week of age at the time of referral to Korle Bu Teaching hospital in Accra, Ghana during the peak malaria seasons (April to July) of 2008 and 2010 were examined for malaria parasites by, i) Giemsa-stained thick and thin blood smears for parasite count and species identification, ii) histidine-rich protein- and lactic dehydrogenase-based rapid diagnosis tests, or iii) polymerase chain reaction amplification of the merozoite surface protein 2 gene, for identification of sub-microscopic parasitaemia. Other investigations were also done as clinically indicated.Plasmodium falciparum parasitaemia were diagnosed by microscopy in 405 (2.2%) newborns. All the nine newborns had low parasite densities (≤50 per microlitre). In 2010, there was no case of parasitaemia by either microscopy or rapid diagnosis tests in 522 newborns; however, 56/467 (12%) cases of P. falciparum were detected by polymerase chain reaction.In 2008, nine cases of Congenital malaria is an uncommon cause of clinical illness in high-risk untreated newborns referred to a tertiary hospital in the first week of life. Empirical anti-malarial drug treatment for sick newborns without laboratory confirmation of parasitaemia is imprudent. Early referral of sick newborns to hospitals with resources and skills for appropriate care is recommended. Malaria in pregnancy is a major underlying cause of neonatal morbidity in endemic regions -3. The bCongenital malaria, defined as malaria parasitaemia in the first week of life can be acquired transplacentally or during the time of birth. The prevalence in sub-Saharan Africa has been reported to vary from 0 – 46% ; howeverThe prevalence of congenital malaria in a community based prospective study in infants in southern Ghana in 1998 was 13.6% by polymerase chain reaction (PCR) and 2.5% by microscopy ,15.Clinical malaria in the newborn usually manifests after the first week of life (but has been reported on day 1) with symptoms such as fever, poor feeding, lethargy, anaemia, hepatosplenomegaly, jaundice, irritability, and drowsiness. These clinical features are similar to the manifestations of neonatal sepsis and other neonatal conditions that more commonly cause morbidity and mortality in the newborn period ,16,17. MMalaria is endemic in Ghana and World Health Organization (WHO) guidelines for empirical treatment of malaria in suspected cases is widely adhered to . This prThis cross-sectional study was done at the newborn unit and malaria research laboratory of the Department of Child Health, Korle Bu Teaching Hospital, in Accra during the peak malaria seasons (April – July) of 2008 and 2010. The 55-bed newborn unit serves as a tertiary referral centre for sick and low birth weight or preterm newborns from southern regions of Ghana. About 2000 newborns are admitted annually. Ethical approval was given by the Ethical and Protocol Review Committee of the University of Ghana Medical School.All newborns hospitalized at the newborn unit in the first week of life during the study period were eligible for inclusion. Newborns with clinical indications that required blood sampling e.g. signs of illness or risk factors for infection were enrolled. Stable newborns e.g. infants of diabetic mothers or well preterm infants of hypertensive mothers who were referred for observation and did not require laboratory investigations, were excluded. All clinical procedures and laboratory investigations for hospitalized newborns were done according to standard and departmental guidelines.A structured questionnaire was used to collect clinical data on newborn and maternal health during pregnancy, including use of insecticide treated nets and intermittent preventive treatment. Other clinical data was extracted from the ward register and hospital files. Sample size calculation was based on an estimated prevalence of a 20% risk of congenital malaria in high-risk newborns in the first week of life .Newborn venous blood (1 ml) was collected into EDTA tubes for haematological investigations. Total white blood cell count (WBC) with differential, platelet count and red cell distribution width was measured by an automated haematology analyzer (Sysmex KX-21) according to manufacturer’s instructions. Thick and thin blood smears prepared from the EDTA sample were stained with Giemsa. Thin smears were used for malaria parasite species identification, and the number of asexual parasites per 200 white blood cells (WBC) on the thick smear was multiplied by the measured WBC count to obtain the parasite count per microlitre. An experienced technologist conducted periodic controls of blood slides for quality control. This traditional method of microscopy was used in 2008 and 2010. In 2010, a rapid diagnosis test (RDT) for identification of malaria parasite histidine rich protein 2 and lactate dehydrogenase , and a nested PCR methods were used to identify sub-microscopic parasitaemia.Plasmodium falciparum and other plasmodial species. It contains a membrane strip pre-coated with two monoclonal antibodies as two separate lines across a test strip. One monoclonal antibody is pan-specific to lactate dehydrogenase of the Plasmodium species and the other monoclonal antibody is specific to histidine-rich protein 2 of P. falciparum and [10.5 × 109 (range 4.2 – 19.4)] respectively. There was no statistical difference in the haematological indices of congenital malaria cases as compared with non-affected newborns (see Table Nine (2.2%) of the 405 newborns tested in 2008 had P. falciparum. The prevalence of LBW in the PCR parasitaemia-positive newborns was 55% as compared to 41.9% in those who were PCR parasitaemia-negative . The mean haemoglobin (14.6 g/dl vs. 15.3 g/dl) and WBC (14.6 × 109 vs. 16.4 × 109) in those with parasitaemia vs. those without parasitaemia respectively was not statistically different.None of the 522 newborns recruited in 2010 had parasitaemia by microscopy or RDT. All 522 samples were tested by PCR, results were obtained in 467/522 and 56 (12%) newborns were positive for Data collected from mothers about their health and malaria preventive practices (use of insecticide treated nets and intermittent preventive treatment) during pregnancy were not analyzed because the information could not be verified.This study shows that patent malaria parasitaemia was uncommon in untreated high-risk newborns referred to Korle Bu Teaching hospital in southern Ghana during the peak seasons for malaria in 2008, and 2010. The 2.2% congenital malaria prevalence by microscopy in the 2008 cohort was lower than rates from longitudinal studies from neighbouring countries, 5.1% in Nigeria and 4.7% in Ivory Coast . The datSeveral hospital-based studies from the West African sub-region have reported high prevalence of malaria in newborns ,24. The P. falciparum malaria parasites in the placenta as detected by PCR are more likely to result in congenital malaria in the infant during the first three months of life [The PCR method diagnoses congenital malaria more frequently because of its ability to detect parasite macromolecules and not necessarily live parasites . The cli of life .P. falciparum hrp2 gene. The sensitivity and specificity of this method has been shown to range from 90-95% and 85-92% respectively. However, relative to thick smear microscopy, recent studies have shown that false negative RDT results occur in persons with asymptomatic P. falciparum infections as in our patients. The false negatives are thought to result from spontaneous deletion of the histidine-rich repeat region of the hrp2 gene [Most widely used RDTs are based on the detection of the histidine-rich protein 2 (HRP2) product of the rp2 gene .The data from this study and previous community-based study on congenital malaria in Ghana  shows thThis study has some limitations. Though one of the objectives was to identify the impact of maternal malarial preventive practices in pregnancy on the prevalence of congenital malaria, we could not collect accurate data on maternal practices. It was difficult to verify that those who said they had used insecticide treated nets during pregnancy actually did so. Also, the information mothers gave on intermittent anti-malarial prophylaxis was not always recorded in the available maternal health records.The data from this study does not support empirical treatment for malaria in sick newborns in Ghana. Laboratory confirmation of malaria in newborns should be done and other causes of clinical illness excluded before anti-malarial drugs are prescribed for newborns especially in the first week of life. The prevalence of malaria as detected by PCR in this study could be used to make a case for improving malaria preventive practices during pregnancy thus reducing placental malaria - a known cause of LBW and a pre-requisite for congenital malaria. Early referral of sick newborns to health facilities with adequate skills for early diagnosis and appropriate management is recommended. Concerted effort to reduce empirical prescription of anti-malarial drugs for newborns is needed. Data such as presented in this study could be used in continuing educational programs to improve knowledge and influence the practice of frontline health care providers.LBW: Low birth weight; PCR: Polymerase chain reaction; RDT: Rapid diagnostic test; WBC: White blood cells; WHO: World Health Organization.The authors have no competing interest to declare. None of the authors received any remuneration for this work. The laboratory investigations of this study were partly supported by DANIDA Denmark (DFC grant number 09-080RH).CEL conceived the study and worked with GOA and BM on the design and data acquisition. GOA, BM, ND and NBQ did the laboratory work. CEL drafted the manuscript and all 5 authors participated in the several revisions of the manuscript and gave approval for the final version for publication.