Plasmodium Mixed Infections Research Articles

Malaria in pregnancy in India: a 50-year bird's eye.

In 2021, India contributed for ~79% of malaria cases and ~ 83% of deaths in the South East Asia region. Here, we systematically and critically analyzed data published on malaria in pregnancy (MiP) in India. Epidemiological, clinical, parasitological, preventive and therapeutic aspects of MiP and its consequences on both mother and child were reviewed and critically analyzed. Knowledge gaps and solution ways are also presented and discussed. Several electronic databases including Google scholar, Google, PubMed, Scopus, Wiley Online library, the Malaria in Pregnancy Consortium library, the World Malaria Report, The WHO regional websites, and ClinicalTrials.gov were used to identify articles dealing with MiP in India. The archives of local scientific associations/journals and website of national programs were also consulted. Malaria in pregnancy is mainly due to Plasmodium falciparum (Pf) and P. vivax (Pv), and on rare occasions to P. ovale spp. and P. malariae too. The overall prevalence of MiP is ~0.1-57.7% for peripheral malaria and ~ 0-29.3% for placental malaria. Peripheral Pf infection at antenatal care (ANC) visits decreased from ~13% in 1991 to ~7% in 1995-1996 in Madhya Pradesh, while placental Pf infection at delivery unit slightly decreased from ~1.5% in 2006-2007 to ~1% in 2012-2015 in Jharkhand. In contrast, the prevalence of peripheral Pv infection at ANC increased from ~1% in 2006-2007 to ~5% in 2015 in Jharkhand, and from ~0.5% in 1984-1985 to ~1.5% in 2007-2008 in Chhattisgarh. Clinical presentation of MiP is diverse ranging from asymptomatic carriage of parasites to severe malaria, and associated with comorbidities and concurrent infections such as malnutrition, COVID-19, dengue, and cardiovascular disorders. Severe anemia, cerebral malaria, severe thrombocytopenia, and hypoglycemia are commonly seen in severe MiP, and are strongly associated with tragic consequences such as abortion and stillbirth. Congenital malaria is seen at prevalence of ~0-12.9%. Infected babies are generally small-for-gestational age, premature with low birthweight, and suffer mainly from anemia, thrombocytopenia, leucopenia and clinical jaundice. Main challenges and knowledge gaps to MiP control included diagnosis, relapsing malaria, mixed Plasmodium infection treatment, self-medication, low density infections and utility of artemisinin-based combination therapies. All taken together, the findings could be immensely helpful to control MiP in malaria endemic areas.

The Silent Threat: Unreported Mixed Plasmodium Infections and the Rising Vivax Malaria Challenge in India's Path to Elimination

The Silent Threat: Unreported Mixed Plasmodium Infections and the Rising Vivax Malaria Challenge in India's Path to Elimination

Effectiveness of pyronaridine-artesunate against Plasmodium malariae, Plasmodium ovale spp, and mixed-Plasmodium infections: a post-hoc analysis of the CANTAM-Pyramax trial

SummaryBackgroundHigh-quality evidence for the therapeutic efficacy and effectiveness of antimalarials for infections caused by Plasmodium malariae, Plasmodium ovale spp, and mixed-Plasmodium infections is scarce. In this study, we aimed to analyse the efficacy of pyronaridine–artesunate for the treatment of non-falciparum and mixed-species Plasmodium infections from a large phase 3b/4 clinical trial in central Africa.MethodsThis post-hoc analysis was done in a random subset of samples from two sites (in the Democratic Republic of the Congo and in Gabon) of the CANTAM-Pyramax trial assessing pyronaridine-artesunate therapy. We randomly selected paired dried blood spot samples from day 0 and day 28 (or unforeseen visit) and analysed them by quantitative PCR for mixed Plasmodium infections or non-falciparum mono-infections. Day 28 (or unforeseen visit) samples positive for non-falciparum malaria were re-assessed by microscopy to identify microscopic versus submicroscopic infections. Analyses were done on two sample sets: a per-protocol set and an intention-to-treat set.FindingsAmong 1502 randomly selected samples, 192 (12·8%) showed mixed-Plasmodium infections or non-falciparum mono-infections. We did not detect P vivax in the samples. For both the per-protocol and intention-to-treat sets, the overall day 28 cure rates for P malariae, P ovale curtisi, and P ovale wallikeri were 96·3% or higher (95% CIs from 81·0–99·9 to 95·7–100). Cure rates were consistently high in P malariae (99·2%, 95·7–100) and P ovale spp (97·9%, 88·7–99·9, for P ovale curtisi and 96·3%, 81·0–99·9, for P ovale wallikeri) infections.InterpretationThis post-hoc analysis provides important evidence supporting the high efficacy of pyronaridine-artesunate against mono-infections with P malariae, P ovale curtisi, or P ovale wallikeri and mixed-Plasmodium infections in a real-world setting.FundingMedicines for Malaria Venture.

The high risk of malarial recurrence in patients with Plasmodium-mixed infection after treatment with antimalarial drugs: a systematic review and meta-analysis

BackgroundMalaria mixed infections are often unrecognized by microscopists in the hospitals, and a delay or failure to treat Plasmodium-mixed infection may lead to aggravated morbidity and increased mortality. The present study aimed to quantify the pooled proportion and risk of malarial recurrences after the treatment of Plasmodium-mixed infection. The results of the study may provide benefits in the management of Plasmodium-mixed infection in co-endemic regions.MethodsThis systematic review and meta-analysis searched the international Prospective Register of Systematic Reviews (PROSPERO; ID = CRD42020199709), MEDLINE, Web of Science, and Scopus for potentially relevant studies in any language published between January 1, 1936, and July 20, 2020, assessing drug efficacy in patients with Plasmodium-mixed infection. The primary outcome was the pooled prevalence of Plasmodium parasitemia after initiating antimalarial treatment for Plasmodium-mixed infection. The secondary outcome was the pooled risk ratio (RR) of malarial recurrence in Plasmodium-mixed infection compared with those in Plasmodium falciparum and Plasmodium vivax mono-infection. The pooled analyses were calculated by random-effects meta-analysis. After the initial treatment in different days of recurrences (≤ 28 days or > 28 days), the risk of Plasmodium parasitemia was compared in subgroup analysis.ResultsOut of 5217 screened studies, 11 were included in the meta-analysis, including 4390 patients from six countries. The pooled prevalence of all recurrences of Plasmodium-mixed parasitemia was 30% (95% confidence interval (CI) 16–43; I2: 99.2%; 11 studies). The RR of malarial recurrence within 28 days after the initial treatment (clinical treatment failure) of Plasmodium-mixed parasitemia compared with the treatment of P. falciparum was 1.22 (p: 0.029; 95% CI 1.02–1.47; Cochran Q: 0.93; I2: 0%; six studies), while there was no significant difference in the risk of recurrence 28 days after initial treatment compared with the treatment of P. falciparum (p: 0.696, RR: 1.14; 95% CI 0.59–2.18; Cochran Q < 0.05; I2: 98.2%; four studies). The subgroup analysis of antimalarial drugs showed that significant malarial recurrence within 28 days was observed in patients treated with artemisinin-based combination therapies (ACTs) with no significant heterogeneity (p: 0.028, RR: 1.31; 95% CI 1.03–1.66; Cochran Q: 0.834; I2: 0%).ConclusionsThe present findings showed a high prevalence of malarial recurrence after the initial treatment of Plasmodium-mixed infection. Moreover, significant malaria recurrence of mixed infection occurred within 28 days after treatment with ACTs.Graphical

Detection of mixed infection of Plasmodium species in the Southern province of Rwanda. Case study: Ruhango, Bunyogombe and Kibilizi Health centres

Background: Malaria is a leading cause of mortality and morbidity in sub-Saharan Africa including Rwanda. Though the prevalence of malaria has been reduced due to the use of indoor residual sprayings and insecticide-treated bed nets, it is still a disease that kills many people annually. Many studies conducted revealed that in sub-Saharan Africa including Rwanda there is a high prevalence of Plasmodium falciparum. However, there is still a gap in the identification of the presence of mixed Plasmodium infection. This study was conducted to determine the overall prevalence of Plasmodium species as well as that of mixed plasmodium infection in Ruhango and Kibilizi Health centres.&#x0D; Methods: Descriptive cross-sectional study was conducted on 126 participants in Ruhango, Bunyogombe and Kibilizi health centres located in the southern province of Rwanda. The conventional sampling strategy was used for the selection of individuals who consented to participate in the study. Blood samples were used to detect Plasmodium species and the obtained data were analyzed using Microsoft excel and IBM SPSS version 21.&#x0D; Results: Among 126 participants presenting with signs and symptoms of malaria, the overall positive cases of Plasmodium species were 61(48.4%) and among the total positive cases 56 (44.5%) were infected with single Plasmodium species while 5 (4%) were infected with mixed Plasmodium species. Plasmodium falciparum was the most prevalent species infecting 49 (39%) participants while Plasmodium vivax was the least prevalent infection, detected in only 1(0.8%) participant.&#x0D; Conclusion: The study identified the significant prevalence of mixed-species of Plasmodium infection as well as the high prevalence of Plasmodium falciparum infection in the study population. These findings suggest that there is a need for continued monitoring of non-falciparum infection in this population and the introduction of species-specific RDTs that can be used for diagnostic purposes.

Fatal imported case of Plasmodium mixed infection: cerebral malaria, thrombocytopenia and multi organ dysfunctions (MODs)

Background: Plasmodium falciparum accounts for nearly all severe malaria cases among European travellers, even though, in areas of endemicity, Plasmodium vivax seems to cause severe malaria to a degree which is comparable with P. falciparum. Furthermore, unlike countries at high risk, the incidence of mixed infections among imported cases is very low. We report the case of a man from non-endemic country with a mixed infection diagnosed after a two-year stay in Guinea. Case Presentation: A 43-year-old male Italian patient developed hyperpyrexia and chills the day after his return to Italy. Within 7 days from fever onset, he was found unresponsive with vomiting and seizures. On admission to Emergency Room, the patient was hypotonic with flaccid tetraparesis, with a GCS score of 7. On the basis of personal and working history and clinical features, after exclusion of viral encephalitis, acute pyogenic meningitis, tubercular meningitis and pontine infarct, cerebral malaria infection was suspected. Rapid diagnostic test and peripheral blood smear were positive for plasmodium mixed infection (P. falciparum, P. vivax, Plasmodium ovale). Over the first 30 hours from admission, thrombocyte count dropped, and the patient developed disseminated intravascular coagulation. Renal failure required renal replacement therapy. Death occurred after 48 hours of ICU admission. Conclusion: The fatal course of the severe malaria with related cerebral impairment, lung and kidney failure, disseminated intravascular coagulation, severe acidosis, circulatory collapse with refractory septic shock and subsequent multi organ failure was likely due to delayed medical presentation by the patient and the mixed plasmodium infection. Diagnosis could only be established nine days after onset of symptoms.

Summary of discordant results between rapid diagnosis tests, microscopy, and polymerase chain reaction for detecting Plasmodium mixed infection: a systematic review and meta-analysis

Malaria rapid diagnostic tests (RDTs) are widely used to detect malaria parasites among patients who suspected malaria infections in malaria-endemic areas where microscopy is unavailable. Nevertheless, little is known about the performance of RDTs in detecting Plasmodium mixed infections. The present study aimed to evaluate the discordant results between RDTs and microscopy/polymerase chain reaction (PCR) in detecting Plasmodium mixed infections. The PubMed (MEDLINE), Web of Science, and Scopus databases were systematically reviewed to identify related studies that reported the performance of RDTs in detecting Plasmodium mixed infections. Studies were grouped according to the different RDT types including RDT type 2 (pf-HRP2/pan-aldolase), RDT type 3 (pf-HRP2/pan-pLDH), RDT type 4 (Pf-LDH/pan-pLDH), RDT type 5 (Pf/Pv-pLDH), and RDT type 6 (pf-HRP2/Pv-pLDH) for subgroup analysis. The estimates of the different proportions in each analysis group that were visually summarized in a forest plot showed the odds ratio (OR) and 95% confidence interval (CI). Plots were drawn using RevMan (version 5.3; Cochrane Community). Twenty-eight studies were included in the present study. Overall, the meta-analysis showed that RDTs could detect a significantly higher proportion of Plasmodium mixed infections than microscopy (p = 0.0007, OR = 3.33, 95% CI 1.66–6.68). Subgroup analysis demonstrated that only RDTs targeting Pf-specific histidine-rich protein 2 (HRP2)/pan-specific lactate dehydrogenase (LDH) could detect a significantly higher proportion of Plasmodium mixed infections than microscopy (p = 0.004, OR = 8.46, 95% CI 2.75–26.1). The subgroup analysis between RDTs and PCR methods demonstrated that RDTs targeting Pf-specific HRP2/Pv-specific LDH could detect a significantly lower proportion of Plasmodium mixed infections than PCR methods (p = 0.0005, OR = 0.42, 95% CI 0.26–0.68). This is the first study to summarize the discordant results between RDTs and microscopy/PCR in detecting Plasmodium mixed infections. Malaria RDTs targeting Pf-HRP2/pan-pLDH could detect a higher proportion of Plasmodium mixed infections than microscopy, while RDTs targeting Pf-HRP2/Pv-specific LDH could detect a lower proportion of Plasmodium mixed infections than PCR methods. The results of this study will support the selection and careful interpretations of RDTs for a better diagnosis of Plasmodium mixed-species infections and appropriate treatment of malaria patients in endemic and non-endemic settings.

Plasmodium spp. mixed infection leading to severe malaria: a systematic review and meta-analysis

Mixed Plasmodium malaria infections can lead to severe malaria. This systematic review and meta-analysis aimed to explore the prevalence of severe mixed Plasmodium malaria infection and to compare it with the prevalence of severe P. falciparum malaria mono-infection across the included studies. Original English-language research articles from PubMed, Scopus, and ISI Web of Science were identified and screened. Articles reporting the number of mixed infections and the number of severe mixed infections were used to determine the main outcome of this study, while the number of P. falciparum infections and the number of severe P. falciparum infections were used to determine the secondary outcome of this study. For the main outcome, the pooled prevalence and 95% confidence interval (CI) of severe mixed infections was analysed using STATA software version 15.0 (Stata Corp, College Station, TX, USA). For the secondary outcome, the rate of severe mixed infections compared to severe P. falciparum infections was analysed using the meta-analysis approach, and summary odds ratios (ORs) and 95% CIs were calculated. Random-effects models were used to produce the summary ORs. The Mantel–Haenszel method and calculated I2 were also reported to test whether there was heterogeneity among the included studies. Publication bias was also assessed using funnel plots. The meta-analysis of secondary outcomes was conducted using Review Manager 5.3 software (Cochrane Community). A total of 894,561 malaria patients were reported in all 16 included studies. Overall, a pooled analysis showed that 9% (2,006/35,768, 95% CI 7.0–12.0%) of patients with mixed Plasmodium infection had severe mixed infection. A meta-analysis of 14 studies demonstrated that patients with mixed Plasmodium infection (1,999/35,755) and patients with P. falciparum malaria (9,249/294,397) had an equal risk of developing severe malaria (OR 0.93, 95% CI 0.59–1.44). Both mixed infection and P. falciparum mono-infection showed a similar trend of complications in which severe anaemia, pulmonary failure, and renal impairment were the three most common complications found. However, patients with mixed infection had a higher proportion of severe anaemia and pulmonary complications than those with P. falciparum infection. Moreover, patients with mixed infection had a higher proportion of multiple organ failure than those with P. falciparum mono-infection. Mixed Plasmodium spp. infections were common but often unrecognized or underestimated, leading to severe complications among these malaria patients. Therefore, in routine clinical laboratories, using an accurate combination of diagnostic procedures to identify suspected patients with mixed infections is crucial for therapeutic decisions, prompt treatment, and effective patient management.

Recurrence of Plasmodium malariae and P. falciparum Following Treatment of Uncomplicated Malaria in North Sumatera With Dihydroartemisinin-Piperaquine or Artemether-Lumefantrine.

BackgroundWe assessed the efficacy of artemisinin-based combination therapies for treatment of uncomplicated falciparum malaria, with or without co-infecting Plasmodium spp., in Sumatera, Indonesia.MethodsFebrile patients aged >6 months with uncomplicated P. falciparum were randomized to receive dihydroartemisinin-piperaquine or artemether-lumefantrine, plus single-dose primaquine, and were followed for 42 days. Mixed Plasmodium infections were included; P. vivax infections received 14 days of primaquine. We retrospectively restricted the analysis to cases with polymerase chain reaction (PCR)–confirmed parasitemia. Recurrent parasitemia in follow-up was identified by species-specific nested PCR.ResultsOf the 3731 participants screened, 302 were enrolled and randomized. In the dihydroartemisinin-piperaquine arm, P. falciparum infections were confirmed by PCR in 59 participants, with mixed infections in 23 (39.0%). In the artemether-lumefantrine arm, P. falciparum infections were confirmed by PCR in 55 participants, with mixed infections in 16 (29.0%). Both regimens were well tolerated, and symptoms improved rapidly in all treated participants. In the dihydroartemisinin-piperaquine arm, 1 P. falciparum recurrence (on day 7) and 6 P. malariae recurrences (1 had a mixed infection with P. falciparum) were identified during days 3–42 of follow-up. In the artemether-lumefantrine arm, 1 P. falciparum/P. malariae/P. vivax recurrence occurred on day 35. Submicroscopic persistence occurred during follow-up in 21 (37%) of 57 receiving dihydroartemisinin-piperaquine and 20 (39%) of 51 receiving artemether-lumefantrine.ConclusionsIn Sumatera, both regimens effectively cleared initial parasitemia, but P. falciparum and P. malariae persisted in some individuals. Molecular species detection should be deployed in antimalarial efficacy trials in Indonesia.Trial registrationNCT02325180.

Epidemiological analysis of malaria prevalence in Jiangsu Province in 2015

Objective To understand the malaria epidemic situation and characteristics in Jiangsu Province in 2015, so as to provide the evidence for malaria elimination. Methods The data of malaria cases in Jiangsu Province in 2015 were collected from China's routine diseases surveillance information system. Results Totally 405 imported cases were reported in Jiangsu Province in 2015, and the cases increased by 14.08% compared with those in 2014. All the malaria cases were imported, and 5 cases (1.23%) were from Southeast Asia and 400 (98.77%) were from 25 African countries or regions. The imported malaria cases were reported in 13 cities across Jiangsu Province, where Taizhou, Lianyungang, Nantong, Huaian and Yangzhou cities accounted for 68.64% of all the cases in the province. Jiangsu Institute of Parasitic Diseases (JIPD) reference lab checked all the cases and classified 299 falciparum malaria cases, 13 vivax malaria cases, 18 quartan malaria cases, 71 ovale malaria cases, and 4 mixed Plasmodium infection cases. Conclusions In Jiangsu Province, there are no local malaria cases for 4 consecutive years, but the imported cases are on the rise. Compared to 2014, the imported vivax cases increased significantly in 2015. It should be the key points to strengthen the surveillance of imported malaria cases, improve malaria diagnosis and treatment abilities for all levels of medical institutions, and promote the abilities of Plasmodium check, and focus survey and disposal of all Center for Disease Control (CDC) staffs across Jiangsu Province.

Epidemic situation of imported malaria in Chengdu City from 2012 to 2016

Objective To understand the malaria epidemic situation in Chengdu City, so as to provide the evidence for effectively carrying out the malaria elimination work. Methods The data of imported malaria in Chengdu City from 2012 to 2016 were collected from the Disease Surveillance Information Reporting System of Chinese Center for Disease Control and Prevention and analyzed respectively. Results A total of 240 imported malaria cases were reported in Chengdu City from 2012 to 2016. In detail, 68.75% of the cases were falciparum malaria cases, 20.41% were vivax malaria cases, 2.50% were quartan malaria cases, 6.25% were ovale malaria cases, and 2.08% were mixed Plasmodium infections. Among them three patients died, with a mortality of 1.25%. All of the cases were infected overseas, among which 224 patients returned from Africa and 16 patients from Southeast Asia. The annual average morbidity was 0.28/100 000, and there was a significantly difference among annual average morbidities (χ2 = 23.87, P < 0.001). There were malaria cases reported in every month, and no significantly difference among seasons (χ2 = 0.833, P > 0.05). Conclusion The number of overseas imported malaria in Chengdu City increases year by year. The effective measures to control the overseas imported malaria should be strengthened to consolidate the achievements of malaria elimination.

Major Burden of Severe Anemia from Non-Falciparum Malaria Species in Southern Papua: A Hospital-Based Surveillance Study

The burden of anemia attributable to non-falciparum malarias in regions with Plasmodium co-endemicity is poorly documented. We compared the hematological profile of patients with and without malaria in southern Papua, Indonesia. Clinical and laboratory data were linked for all patients presenting to a referral hospital between April 2004 and December 2012. Data were available on patient demographics, malaria diagnosis, hemoglobin concentration, and clinical outcome, but other potential causes of anemia could not be identified reliably. Of 922,120 patient episodes (837,989 as outpatients and 84,131 as inpatients), a total of 219,845 (23.8%) were associated with a hemoglobin measurement, of whom 67,696 (30.8%) had malaria. Patients with P. malariae infection had the lowest hemoglobin concentration (n = 1,608, mean = 8.93 [95% CI 8.81-9.06]), followed by those with mixed species infections (n = 8,645, mean = 9.22 [95% CI 9.16-9.28]), P. falciparum (n = 37,554, mean = 9.47 [95% CI 9.44-9.50]), and P. vivax (n = 19,858, mean = 9.53 [95% CI 9.49-9.57]); p-value for all comparisons <0.001. Severe anemia (hemoglobin <5 g/dl) was present in 8,151 (3.7%) patients. Compared to patients without malaria, those with mixed Plasmodium infection were at greatest risk of severe anemia (adjusted odds ratio [AOR] 3.25 [95% CI 2.99-3.54]); AORs for severe anaemia associated with P. falciparum, P. vivax, and P. malariae were 2.11 (95% CI 2.00-2.23), 1.87 (95% CI 1.74-2.01), and 2.18 (95% CI 1.76-2.67), respectively, p<0.001. Overall, 12.2% (95% CI 11.2%-13.3%) of severe anemia was attributable to non-falciparum infections compared with 15.1% (95% CI 13.9%-16.3%) for P. falciparum monoinfections. Patients with severe anemia had an increased risk of death (AOR = 5.80 [95% CI 5.17-6.50]; p<0.001). Not all patients had a hemoglobin measurement, thus limitations of the study include the potential for selection bias, and possible residual confounding in multivariable analyses. In Papua P. vivax is the dominant cause of severe anemia in early infancy, mixed P. vivax/P. falciparum infections are associated with a greater hematological impairment than either species alone, and in adulthood P. malariae, although rare, is associated with the lowest hemoglobin concentration. These findings highlight the public health importance of integrated genus-wide malaria control strategies in areas of Plasmodium co-endemicity.

Cross-reactive anti-PfCLAG9 antibodies in the sera of asymptomatic parasite carriers of Plasmodium vivax

The PfCLAG9 has been extensively studied because their immunogenicity. Thereby, the gene product is important for therapeutics interventions and a potential vaccine candidate. Antibodies against synthetic peptides corresponding to selected sequences of the Plasmodium falciparum antigen PfCLAG9 were found in sera of falciparum malaria patients from Rondônia, in the Brazilian Amazon. Much higher antibody titres were found in semi-immune and immune asymptomatic parasite carriers than in subjects suffering clinical infections, corroborating original findings in Papua Guinea. However, sera of Plasmodium vivax patients from the same Amazon area, in particular from asymptomatic vivax parasite carriers, reacted strongly with the same peptides. Bioinformatic analyses revealed regions of similarity between P. falciparum Pfclag9 and the P. vivax ortholog Pvclag7. Indirect fluorescent microscopy analysis showed that antibodies against PfCLAG9 peptides elicited in BALB/c mice react with human red blood cells (RBCs) infected with both P. falciparum and P. vivax parasites. The patterns of reactivity on the surface of the parasitised RBCs are very similar. The present observations support previous findings that PfCLAG9 may be a target of protective immune responses and raises the possibility that the cross reactive antibodies to PvCLAG7 in mixed infections play a role in regulate the fate of Plasmodium mixed infections.

Concurrent infection of Japanese encephalitis and mixed plasmodium infection

Japanese encephalitis (JE) and malaria would coexist in the areas where both illnesses are endemic with overlapping clinical pictures, especially in a case of febrile encephalopathy with hepatosplenomegaly. However, there are no published data till date showing concurrent infection of these two agents despite both diseases being coendemic in many areas. We report a case of concurrent infection of JE and mixed plasmodium infection, where the case, initially diagnosed as cerebral malaria did not improve on antimalarials and alternative diagnosis of JEV encephalitis was thought which was confirmed by a serological test. To the best of our knowledge, this is the first case report of concurrent Japanese encephalitis with mixed plasmodium infection. We report a case of 3-year-old male child, who presented with febrile encephalopathy with hepatosplenomegaly. Based on a rapid diagnostic test and peripheral smear examination, a diagnosis of mixed P.Vivax and P.falciparum infection was made and the patient was treated with quinine and doxycycline. However, besides giving antimalarials the patient did not improve and an alternative diagnosis of JE was considered as the patient was from the endemic zone of Japanese encephalitis. Cerebrospinal fluid (CSF) of the patient was sent for a virological study which came out to be positive for JE IgM in CSF, which is confirmatory of JE infection. In a patient with febrile encephalopathy with hepatosplenomegaly especially in areas coendemic for JE and malaria, the possibility of mixed infection should be kept in mind.

Concurrent infection of Japanese encephalitis and mixed plasmodium infection

Concurrent infection of Japanese encephalitis and mixed plasmodium infection

Validation of a four-primer real-time PCR as a diagnostic tool for single and mixed Plasmodium infections

Although microscopy remains the reference standard for malaria diagnosis, molecular tools are attracting increasing interest. To improve the detection of mixed infections, we developed a four-primer real-time PCR with four Plasmodium species-specific forward primers, based on the pan-primer design with universal Plasmodium primers as described previously. After validation for analytical sensitivity, specificity and reproducibility, the four-primer PCR was evaluated on 351 blood samples from patients presenting at the outpatient clinic of the Institute of Tropical Medicine (Belgium). With the four-primer PCR, we identified 188 Plasmodium falciparum (Pf), 54 Plasmodium vivax (Pv), 52 Plasmodium ovale (Po) and 13 Plasmodium malariae (Pm) single infections, 27 mixed infections (14 Pf + Pm; 12 Pf + Po; one Pv + Pm) and 17 negative specimens. We found lower cycle threshold values than with the pan-primer PCR, with a mean difference of 2.23, a higher analytical sensitivity (in asexual parasites/µL: Pf/Pv, 0.02; Po, 0.004; Pm, 0.006) and 15 extra mixed infections. As compared with microscopy, 17 extra mixed infections were detected and Plasmodium species were identified in four microscopy-positive samples in which species identification was not possible. Additionally, the PCR corrected 13 species mismatches between Po and Pv, and in 11 cases detected Pf as a second species that was not identified by microscopy and in five of them was not detected by rapid diagnostic tests (RDTs). PCR confirmed the presence of Pf in 30/46 histidine-rich protein-2-positive samples that were microscopy-negative. We conclude that the presently developed four-primer real-time PCR is complementary to standard malaria diagnostic tests in clinical laboratories, with an added value for simultaneous identification of the four Plasmodium species and the detection of mixed infections.

Malaria, Oromia Regional State, Ethiopia, 2001-2006

To the Editor: In Ethiopia, malaria is unstable and commonly occurs as intraannual and interannual epidemics. Transmission is associated with altitude, temperature, and rainfall, generally peaking twice a year, after the 2 rainy seasons (March–May and July–September) (1). Cases are caused by Plasmodium falciparum and P. vivax. Anopheles arabiensis mosquitoes are the main vector for both species. Although malaria is the most common communicable disease in Ethiopia (2), few longitudinal case data has been published (3). We report a retrospective analysis of outpatient data for July 2001–June 2006 obtained from all secondary and tertiary government-run health facilities (152 health centers and 25 hospitals) in Oromia Regional State. Oromia has 17 administrative zones and 297 districts. Data were reported monthly on paper forms by health facility staff at district level to the Oromia Regional Health Bureau Zonal Health Offices, which aggregated zonal data before forwarding them to the Oromia Regional Health Bureau Malaria Control Department. Data obtained included number of outpatient cases (i.e., patients attending the health facility grouped by age 5 years); number of clinical malaria cases (i.e., patients with fever grouped by age and sex); number of clinical cases confirmed by microscopy; and number of cases caused by P. falciparum and P. vivax. If no outpatient data were reported, the case number was changed from zero to missing. The data were entered into Microsoft Excel (Microsoft, Redmond, WA, USA) and analyzed by using Stata version 9.0 (StataCorp LP, College Station, TX, USA). During 2001–2006, a total of 8,786,088 outpatient consultations were reported. A total of 905,467 and 562,996 clinical and confirmed malaria cases, respectively, were reported. Patients were predominantly seen at health centers rather than at hospitals, with 80.2% clinical and 72.2% confirmed malaria cases seen at health centers. Clinical malaria accounted for 10.3% of outpatient consultations in all facilities. However, this percentage varied between years (6.1%–16.0%) and zones (1.3%–21.9%) (Technical Appendix Figure 1). Of clinical malaria cases, 16.5% were in children <5 years of age (range between years [RBY] 14.0%–18.3%, range between zones [RBZ] 10.9%–61.0%) and 54.3% were in male patients (RBY 52.2%–55.6%, RBZ 50.1%–66.8%). Of clinical malaria cases, 49.2% were confirmed by microscopy (RBY 37.1%–58.0%, RBZ 15.3%–98.4%), and 58.5% (RBY 46.4%–63.4%, RBZ 12.1%–82.4%), and 41.2% (RBY 36.3%–53.4%, RBZ 17.6%–87.9%) of confirmed cases were caused by P. falciparum and P. vivax, respectively. Of confirmed cases, 0.4% were caused by mixed Plasmodium infections (RBY 0.2%–0.5%, RBZ 0.0%–1.1%). The average incidence of clinical malaria per 100,000 population per month ranged from 14 in February 2002 to 122 in November 2003, and there was considerable variation between months, years, and administrative zones (Technical Appendix Figure 2). We found that up to 29.0% of outpatient visits to health facilities in certain administrative zones during high transmission years were for malaria. The incidence of malaria is likely to be underestimated because only ≈30% of the population accessed health facilities at that time (4). There appeared to be only 1 annual peak of transmission in September–January (Technical Appendix Figure 1). Clinical and confirmed disease varied between zones; 5 of the 15 zones in Oromia (East Hararge, East Shoa, East Wellega, Jimma, West Hararge) reported >75% of the clinical cases seen at health facilities during 2001–2006. Malaria incidence varied between years: clinical and confirmed cases increased in 2003, the last epidemic year recorded in Oromia (5), before decreasing to 2001 levels in 2004 (Technical Appendix Figure 1). The P. falciparum to P. vixax ratio changed geographically and temporally (Technical Appendix Figure 1), and increases in the proportion of P. falciparum cases coincided with the peak malaria transmission season. In the epidemic year of 2003, the proportion of P. falciparum cases was larger than in other years, and children <5 years of age were disproportionately affected (Technical Appendix Figure 1). Contrary to previous reports (6), our data did not indicate a change in the P. falciparum to P. vivax ratio after artemether/lumefantrine was introduced in 2005. Health facility data can have many caveats (7), including concerns about data representativeness (e.g., if only a small number of facilities are assessed); data validity, particularly if, as was the case during that time, only limited diagnostic quality assurance was available (8); and analytical approaches used. Our analysis comprised all Oromia secondary and tertiary facilities; only 3.4% of health centers and 13.0% of hospitals surveyed had no data, suggesting that given the extensive data reported, these missing data would have only marginally affected the temporal and spatial trends observed. Our data complement those of recent cross-sectional surveys (9) and provide a useful baseline to assess scale-up of malaria prevention and control efforts. Unlike cross-sectional and small-scale facility surveys (6), our comprehensive longitudinal monthly data monitored disease trends spatially and temporally, showing that malaria still represented a major health services problem until 2006.

Evaluation of three PCR-based diagnostic assays for detecting mixed Plasmodium infection

BackgroundOne of the most commonly used molecular test for malaria diagnosis is the polymerase chain reaction (PCR)-based amplification of the 18S ribosomal DNA (rDNA) gene. Published diagnostic assays based on the 18S gene include the "gold standard" nested assay, semi-nested multiplex assay, and one tube multiplex assay. To our knowledge, no one has reported whether the two multiplex methods are better at detecting mixed Plasmodium infections compared to the nested assay using known quantities of DNA in experimentally mixed cocktails.FindingsHere we evaluated three PCR assays (nested, semi-nested multiplex, and one-tube multiplex) for the simultaneous detection of human malaria parasites using experimentally mixed cocktails of known quantities of laboratory derived DNA. All three assays detected individual species with high sensitivity and specificity when DNA was from any one single species; however, experimentally mixed DNA cocktails with all four species present were correctly identified most consistently with the nested method. The other two methods failed to consistently identify all four species correctly, especially at lower concentrations of DNA -subclinical levels of malaria (DNA equivalent to or less than 10 parasites per microliter).ConclusionsThe nested PCR method remains the method of choice for the detection of mixed malaria infections and especially of sub-clinical infections. Further optimization and/or new molecular gene targets may improve the success rate of detecting multiple parasite species simultaneously using traditional PCR assays.

A nucleotide-constrained single base extension method for improved detection of minority alleles in Plasmodium

In genetically mixed Plasmodium infections, minority alleles may have a significant role in drug resistance and other responses to selective pressures. Many available methods to detect single nucleotide polymorphisms representing minority alleles either are not sensitive enough to detect these rare alleles or are limited in the number of loci to be screened in a single reaction. In order to achieve highly sensitive, multiplex SNP genotyping, we have developed a nucleotide-constrained method based on the traditional single base extension approach. Here, we report results when using standard and nucleotide-constrained reactions to determine alleles at nine SNP loci in the trap gene of Plasmodium relictum. This innovative method offers great improvements in detection limits while maintaining the accuracy and multiplex capabilities of single base extension SNP genotyping.

Plasmodium vivax and Mixed Infections Are Associated with Severe Malaria in Children: A Prospective Cohort Study from Papua New Guinea

BackgroundSevere malaria (SM) is classically associated with Plasmodium falciparum infection. Little information is available on the contribution of P. vivax to severe disease. There are some epidemiological indications that P. vivax or mixed infections protect against complications and deaths. A large morbidity surveillance conducted in an area where the four species coexist allowed us to estimate rates of SM among patients infected with one or several species.Methods and FindingsThis was a prospective cohort study conducted within the framework of the Malaria Vaccine Epidemiology and Evaluation Project. All presumptive malaria cases presenting at two rural health facilities over an 8-y period were investigated with history taking, clinical examination, and laboratory assessment. Case definition of SM was based on the World Health Organization (WHO) criteria adapted for the setting (i.e., clinical diagnosis of malaria associated with asexual blood stage parasitaemia and recent history of fits, or coma, or respiratory distress, or anaemia [haemoglobin < 5 g/dl]). Out of 17,201 presumptive malaria cases, 9,537 (55%) had a confirmed Plasmodium parasitaemia. Among those, 6.2% (95% confidence interval [CI] 5.7%–6.8%) fulfilled the case definition of SM, most of them in children <5 y. In this age group, the proportion of SM was 11.7% (10.4%–13.2%) for P. falciparum, 8.8% (7.1%–10.7%) for P. vivax, and 17.3% (11.7%–24.2%) for mixed P. falciparum and P. vivax infections. P. vivax SM presented more often with respiratory distress than did P. falciparum (60% versus 41%, p = 0.002), but less often with anaemia (19% versus 41%, p = 0.0001).ConclusionP. vivax monoinfections as well as mixed Plasmodium infections are associated with SM. There is no indication that mixed infections protected against SM. Interventions targeted toward P. falciparum only might be insufficient to eliminate the overall malaria burden, and especially severe disease, in areas where P. falciparum and P. vivax coexist.