Rapid Diagnostic Tests for Malaria Diagnosis in the Peruvian Amazon: Impact of pfhrp2 Gene Deletions and Cross-Reactions

Background

In the Peruvian Amazon, Plasmodium falciparum and Plasmodium vivax malaria are endemic in rural areas, where microscopy is not available. Malaria rapid diagnostic tests (RDTs) provide quick and accurate diagnosis. However, pfhrp2 gene deletions may limit the use of histidine-rich protein-2 (PfHRP2) detecting RDTs. Further, cross-reactions of P. falciparum with P. vivax-specific test lines and vice versa may impair diagnostic specificity.

Methods

Thirteen RDT products were evaluated on 179 prospectively collected malaria positive samples. Species diagnosis was performed by microscopy and confirmed by PCR. Pfhrp2 gene deletions were assessed by PCR.

Results

Sensitivity for P. falciparum diagnosis was lower for PfHRP2 compared to P. falciparum-specific Plasmodium lactate dehydrogenase (Pf-pLDH)- detecting RDTs (71.6% vs. 98.7%, p<0.001). Most (19/21) false negative PfHRP2 results were associated with pfhrp2 gene deletions (25.7% of 74 P. falciparum samples). Diagnostic sensitivity for P. vivax (101 samples) was excellent, except for two products. In 10/12 P. vivax-detecting RDT products, cross-reactions with the PfHRP2 or Pf-pLDH line occurred at a median frequency of 2.5% (range 0%–10.9%) of P. vivax samples assessed. In two RDT products, two and one P. falciparum samples respectively cross-reacted with the Pv-pLDH line. Two Pf-pLDH/pan-pLDH-detecting RDTs showed excellent sensitivity with few (1.0%) cross-reactions but showed faint Pf-pLDH lines in 24.7% and 38.9% of P. falciparum samples.

Conclusion

PfHRP2-detecting RDTs are not suitable in the Peruvian Amazon due to pfhrp2 gene deletions. Two Pf-pLDH-detecting RDTs performed excellently and are promising RDTs for this region although faint test lines are of concern.     

Detection of histidine-rich protein 2- and/or 3-deleted Plasmodium falciparum using the automated hematology analyzer XN-31: A proof-of-concept study

Rapid diagnostic tests (RDTs) based on immunochromatographic detection of Plasmodium falciparum histidine-rich protein 2 (HRP2) have been frequently used for malaria diagnosis. The HRP2-based RDTs are highly sensitive and easy to use; however, their sensitivity may be low in detecting P. falciparum strains carrying deletion of the pfhrp2 and pfhrp3 genes encoding HRP2 and HRP3, respectively. The automated hematology analyzer XN-31, developed by Sysmex (Kobe, Japan) to aid in malaria diagnosis, has higher sensitivity than RDTs owing to a unique automated nucleic acid staining technology that has shown great potential in clinical settings. In this study, we compared the performance of the XN-31 analyzer and two RDTs to detect pfhrp2- and/or pfhrp3-deleted parasites cultured in vitro. The analyses showed that the analyzer was not only as sensitive to pfhrp2- and/or pfhrp3-deleted strains as it was to the wild-type strain but also had higher sensitivity than the RDTs. These results suggested that the XN-31 analyzer is useful for rapid and reliable detection of pfhrp2- and/or pfhrp3-deleted parasites in clinical settings.     

Temporal association of acute hepatitis A and Plasmodium falciparum malaria in children

Background

In sub-Saharan Africa, Plasmodium falciparum and hepatitis A (HAV) infections are common, especially in children. Co-infections with these two pathogens may therefore occur, but it is unknown if temporal clustering exists.

Materials and Methods

We studied the pattern of co-infection of P. falciparum malaria and acute HAV in Kenyan children under the age of 5 years in a cohort of children presenting with uncomplicated P. falciparum malaria. HAV status was determined during a 3-month follow-up period.

Discussion

Among 222 cases of uncomplicated malaria, 10 patients were anti-HAV IgM positive. The incidence of HAV infections during P. falciparum malaria was 1.7 (95% CI 0.81–3.1) infections/person-year while the cumulative incidence of HAV over the 3-month follow-up period was 0.27 (95% CI 0.14–0.50) infections/person-year. Children with or without HAV co-infections had similar mean P. falciparum asexual parasite densities at presentation (31,000/µL vs. 34,000/µL, respectively), largely exceeding the pyrogenic threshold of 2,500 parasites/µL in this population and minimizing risk of over-diagnosis of malaria as an explanation.

Conclusion

The observed temporal association between acute HAV and P. falciparum malaria suggests that co-infections of these two hepatotrophic human pathogens may result from changes in host susceptibility. Testing this hypothesis will require larger prospective studies.     

Performance of local light microscopy and the ParaScreen Pan/Pf rapid diagnostic test to detect malaria in health centers in Northwest Ethiopia

Background

Diagnostic tests are recommended for suspected malaria cases before treatment, but comparative performance of microscopy and rapid diagnostic tests (RDTs) at rural health centers has rarely been studied compared to independent expert microscopy.

Methods

Participants (N = 1997) with presumptive malaria were recruited from ten health centers with a range of transmission intensities in Amhara Regional State, Northwest Ethiopia during October to December 2007. Microscopy and ParaScreen Pan/Pf® RDT were done immediately by health center technicians. Blood slides were re-examined later at a central laboratory by independent expert microscopists.

Results

Of 1,997 febrile patients, 475 (23.8%) were positive by expert microscopists, with 57.7% P.falciparum, 24.6% P.vivax and 17.7% mixed infections. Sensitivity of health center microscopists for any malaria species was >90% in five health centers (four of which had the highest prevalence), >70% in nine centers and 44% in one site with lowest prevalence. Specificity for health center microscopy was very good (>95%) in all centers. For ParaScreen RDT, sensitivity was ≥90% in three centers, ≥70% in six and <60% in four centers. Specificity was ≥90% in all centers except one where it was 85%.

Conclusions

Health center microscopists performed well in nine of the ten health centers; while for ParaScreen RDT they performed well in only six centers. Overall the accuracy of local microscopy exceeded that of RDT for all outcomes. This study supports the introduction of RDTs only if accompanied by appropriate training, frequent supervision and quality control at all levels. Deficiencies in RDT use at some health centers must be rectified before universal replacement of good routine microscopy with RDTs. Maintenance and strengthening of good quality microscopy remains a priority at health center level.     

Comparative Evaluation of Bivalent Malaria Rapid Diagnostic Tests versus Traditional Methods in Field with Special Reference to Heat Stability Testing in Central India

Background

Malaria presents a diagnostic challenge in areas where both Plasmodium falciparum and P.vivax are co-endemic. Bivalent Rapid Diagnostic tests (RDTs) showed promise as diagnostic tools for P.falciparum and P.vivax. To assist national malaria control programme in the selection of RDTs, commercially available seven malaria RDTs were evaluated in terms of their performance with special reference to heat stability.

Methodology/Principal Findings

This study was undertaken in four forested districts of central India (July, 2011– March, 2012). All RDTs were tested simultaneously in field along with microscopy as gold standard. These RDTs were stored in their original packing at 25°C before transport to the field or they were stored at 35°C and 45°C upto 100 days for testing the performance of RDTs at high temperature. In all 2841 patients with fever were screened for malaria of which 26% were positive for P.falciparum, and 17% for P.vivax. The highest sensitivity of any RDT for P.falciparum was 98% (95% CI; 95.9–98.8) and lowest sensitivity was 76% (95% CI; 71.7–79.6). For P.vivax highest and lowest sensitivity for any RDT was 80% (95% CI; 94.9 - 83.9) and 20% (95% CI; 15.6–24.5) respectively. Heat stability experiments showed that most RDTs for P.falciparum showed high sensitivity at 45°C upto 90 days. While for P.vivax only two RDTs maintained good sensitivity upto day 90 when compared with RDTs kept at room temperature. Agreement between observers was excellent for positive and negative readings for both P.falciparum and P.vivax (Kappa >0.6–0.9).

Conclusion

This is first field evaluation of RDTs regarding their temperature stability. Although RDTs are useful as diagnostic tool for P.falciparum and P.vivax even at high temperature, the quality of RDTs should be regulated and monitored more closely.     

Variation in Plasmodium falciparum Histidine-Rich Protein 2 (Pfhrp2) and Plasmodium falciparum Histidine-Rich Protein 3 (Pfhrp3) Gene Deletions in Guyana and Suriname

Guyana and Suriname have made important progress in reducing the burden of malaria. While both countries use microscopy as the primary tool for clinical diagnosis, malaria rapid diagnostic tests (RDTs) are useful in remote areas of the interior where laboratory support may be limited or unavailable. Recent reports indicate that histidine-rich protein 2 (PfHRP2)-based diagnostic tests specific for detection of P. falciparum may provide false negative results in some parts of South America due to the emergence of P. falciparum parasites that lack the pfhrp2 gene, and thus produce no PfHRP2 antigen. Pfhrp2 and pfhrp3 genes were amplified in parasite isolates collected from Guyana and Suriname to determine if there were circulating isolates with deletions in these genes. Pfhrp3 deletions were monitored because some monoclonal antibodies utilized in PfHRP2-based RDTs cross-react with the PfHRP3 protein. We found that all 97 isolates from Guyana that met the inclusion criteria were both pfhrp2- and pfhrp3-positive. In Suriname (N = 78), 14% of the samples tested were pfhrp2-negative while 4% were pfhrp3-negative. Furthermore, analysis of the genomic region proximal to pfhrp2 and pfhrp3 revealed that genomic deletions extended to the flanking genes. We also investigated the population substructure of the isolates collected to determine if the parasites that had deletions of pfhrp2 and pfhrp3 belonged to any genetic subtypes. Cluster analysis revealed that there was no predominant P. falciparum population substructure among the isolates from either country, an indication of genetic admixture among the parasite populations. Furthermore, the pfhrp2-deleted parasites from Suriname did not appear to share a single, unique genetic background.     

Hepatitis C virus infection may lead to slower emergence of P. falciparum in blood

Background

Areas endemic for Plasmodium falciparum, hepatitis B virus (HBV) and hepatitis C virus (HCV) overlap in many parts of sub-Saharan Africa. HBV and HCV infections develop in the liver, where takes place the first development stage of P. falciparum before its further spread in blood. The complex mechanisms involved in the development of hepatitis may potentially influence the development of the liver stage of malaria parasites. Understanding the molecular mechanisms of these interactions could provide new pathophysiological insights for treatment strategies in Malaria.

Methodology

We studied a cohort of 319 individuals living in a village where the three infections are prevalent. The patients were initially given a curative antimalarial treatment and were then monitored for the emergence of asexual P. falciparum forms in blood, fortnightly for one year, by microscopy and polymerase chain reaction.

Principal Findings

At inclusion, 65 (20.4%) subjects had detectable malaria parasites in blood, 36 (11.3%) were HBV chronic carriers, and 61 (18.9%) were HCV chronic carriers. During follow-up, asexual P. falciparum forms were detected in the blood of 203 patients. The median time to P. falciparum emergence in blood was respectively 140 and 120 days in HBV- and HBV+ individuals, and 135 and 224 days in HCV- and HCV+ individuals. HCV carriage was associated with delayed emergence of asexual P. falciparum forms in blood relative to patients without HCV infection.

Conclusions

This pilot study represents first tentative evidence of a potential epidemiological interaction between HBV, HCV and P. falciparum infections. Age is an important confounding factor in this setting however multivariate analysis points to an interaction between P. falciparum and HCV at the hepatic level with a slower emergence of P. falciparum in HCV chronic carriers. More in depth analysis are necessary to unravel the basis of hepatic interactions between these two pathogens, which could help in identifying new therapeutic approaches against malaria.     

Identifying residual foci of Plasmodium falciparum infections for malaria elimination: the urban context of Khartoum, Sudan

Background

Identifying the location and size of residual foci of infections is critical where malaria elimination is the primary goal. Here the spatial heterogeneity of Plasmodium falciparum infections within the urban extent of Khartoum state in Sudan is investigated using data from cross-sectional surveys undertaken from 1999 to 2008 to inform the Khartoum Malaria Free Initiative (KMFI).

Methods

From 1999–2008 the KMFI undertook cross-sectional surveys of 256 clusters across 203 random samples of residential blocks in the urban Khartoum state in September of each year. Within sampled blocks, at least five persons, including at least one child under the age of five years, were selected from each household. Blood smears were collected from the sampled individuals to examine the presence of P. falciparum parasites. Residential blocks were mapped. Data were analysed for spatial clustering using the Bernoulli model and the significance of clusters were tested using the Kulldorff scan statistic.

Results

A total of 128,510 malaria slide examinations were undertaken during the study period. In 1999, overall prevalence was 2.5%, rising to 3.2% in 2000 and consistently staying below 1% in subsequent years. From 2006, over 90% of all surveyed clusters reported no infections. Spatial clustering of infections was present in each year but not statistically significant in the years 2001, 2002, 2004 and 2008. Spatial clusters of high infection were often located at the junction of the Blue and White Niles.

Conclusion

Persisting foci of malaria infection in Khartoum are likely to distort wide area assessments and disproportionately affect future transmission within the city limits. Improved investments in surveillance that combines both passive and active case detection linked to a geographic information system and a more detailed analysis of the location and stability of foci should be undertaken to facilitate and track malaria elimination in the state of Khartoum.     

Rapid Antigen Detection Tests for Malaria Diagnosis in Severely Ill Papua New Guinean Children: A Comparative Study Using Bayesian Latent Class Models

Background

Although rapid diagnostic tests (RDTs) have practical advantages over light microscopy (LM) and good sensitivity in severe falciparum malaria in Africa, their utility where severe non-falciparum malaria occurs is unknown. LM, RDTs and polymerase chain reaction (PCR)-based methods have limitations, and thus conventional comparative malaria diagnostic studies employ imperfect gold standards. We assessed whether, using Bayesian latent class models (LCMs) which do not require a reference method, RDTs could safely direct initial anti-infective therapy in severe ill children from an area of hyperendemic transmission of both Plasmodium falciparum and P. vivax.

Methods and Findings

We studied 797 Papua New Guinean children hospitalized with well-characterized severe illness for whom LM, RDT and nested PCR (nPCR) results were available. For any severe malaria, the estimated prevalence was 47.5% with RDTs exhibiting similar sensitivity and negative predictive value (NPV) to nPCR (≥96.0%). LM was the least sensitive test (87.4%) and had the lowest NPV (89.7%), but had the highest specificity (99.1%) and positive predictive value (98.9%). For severe falciparum malaria (prevalence 42.9%), the findings were similar. For non-falciparum severe malaria (prevalence 6.9%), no test had the WHO-recommended sensitivity and specificity of >95% and >90%, respectively. RDTs were the least sensitive (69.6%) and had the lowest NPV (96.7%).

Conclusions

RDTs appear a valuable point-of-care test that is at least equivalent to LM in diagnosing severe falciparum malaria in this epidemiologic situation. None of the tests had the required sensitivity/specificity for severe non-falciparum malaria but the number of false-negative RDTs in this group was small.     

Toward a surrogate marker of malaria exposure: modeling longitudinal antibody measurements under outbreak conditions

Background

Biomarkers of exposure to Plasmodium falciparum would be a useful tool for the assessment of malaria burden and analysis of intervention and epidemiological studies. Antibodies to pre-erythrocytic antigens represent potential surrogates of exposure.

Methods and Findings

In an outbreak cohort of U.S. Marines deployed to Liberia, we modeled pre- and post-deployment IgG against P. falciparum sporozoites by immunofluorescence antibody test, and both IgG and IgM against the P. falciparum circumsporozoite protein by enzyme-linked immunosorbant assay. Modeling seroconversion thresholds by a fixed ratio, linear regression or nonlinear regression produced sensitivity for identification of exposed U.S. Marines between 58–70% and specificities between 87–97%, compared with malaria-naïve U.S. volunteers. Exposure was predicted in 30–45% of the cohort.

Conclusion

Each of the three models tested has merits in different studies, but further development and validation in endemic populations is required. Overall, these models provide support for an antibody-based surrogate marker of exposure to malaria.     

Antibody-mediated growth inhibition of Plasmodium falciparum: relationship to age and protection from parasitemia in Kenyan children and adults

Background

Antibodies that impair Plasmodium falciparum merozoite invasion and intraerythrocytic development are one of several mechanisms that mediate naturally acquired immunity to malaria. Attempts to correlate anti-malaria antibodies with risk of infection and morbidity have yielded inconsistent results. Growth inhibition assays (GIA) offer a convenient method to quantify functional antibody activity against blood stage malaria.

Methods

A treatment-time-to-infection study was conducted over 12-weeks in a malaria holoendemic area of Kenya. Plasma collected from healthy individuals (98 children and 99 adults) before artemether-lumefantrine treatment was tested by GIA in three separate laboratories.

Results

Median GIA levels varied with P. falciparum line (D10, 8.8%; 3D7, 34.9%; FVO, 51.4% inhibition). The magnitude of growth inhibition decreased with age in all P. falciparum lines tested with the highest median levels among children <4 years compared to adults (e.g. 3D7, 45.4% vs. 30.0% respectively, p = 0.0003). Time-to-infection measured by weekly blood smears was significantly associated with level of GIA controlling for age. Upper quartile inhibition activity was associated with less risk of infection compared to individuals with lower levels (e.g. 3D7, hazard ratio = 1.535, 95% CI = 1.012–2.329; p = 0.0438). Various GIA methodologies had little effect on measured parasite growth inhibition.

Conclusion

Plasma antibody-mediated growth inhibition of blood stage P. falciparum decreases with age in residents of a malaria holoendemic area. Growth inhibition assay may be a useful surrogate of protection against infection when outcome is controlled for age.     

Helminth Infection and Eosinophilia and the Risk of Plasmodium falciparum Malaria in 1- to 6-Year-Old Children in a Malaria Endemic Area

Background

Helminth infection is common in malaria endemic areas, and an interaction between the two would be of considerable public health importance. Animal models suggest that helminth infections may increase susceptibility to malaria, but epidemiological data has been limited and contradictory.

Methodology/Principal Findings

In a vaccine trial, we studied 387 one- to six-year-old children for the effect of helminth infections on febrile Plasmodium falciparum malaria episodes. Gastrointestinal helminth infection and eosinophilia were prevalent (25% and 50% respectively), but did not influence susceptibility to malaria. Hazard ratios were 1 for gastrointestinal helminth infection (95% CI 0.6–1.6) and 0.85 and 0.85 for mild and marked eosinophilia, respectively (95% CI 0.56–1.76 and 0.69–1.96). Incident rate ratios for multiple episodes were 0.83 for gastro-intestinal helminth infection (95% CI 0.5–1.33) and 0.86 and 0.98 for mild and marked eosinophilia (95% CI 0.5–1.4 and 0.6–1.5).

Conclusions/Significance

There was no evidence that infection with gastrointestinal helminths or urinary schistosomiasis increased susceptibility to Plasmodium falciparum malaria in this study. Larger studies including populations with a greater prevalence of helminth infection should be undertaken.     

Associations between Antibodies to a Panel of Plasmodium falciparum Specific Antigens and Response to Sub-Optimal Antimalarial Therapy in Kampala,Uganda

Background

Antibodies are important in the control of blood stage Plasmodium falciparum infection. It is unclear which antibody responses are responsible for, or even associated with protection, partly due to confounding by heterogeneous exposure. Assessment of response to partially effective antimalarial therapy, which requires the host to assist in clearing parasites, offers an opportunity to measure protection independent of exposure.

Methods

A cohort of children aged 1–10 years in Kampala, Uganda were treated with amodiaquine+sulfadoxine-pyrimethamine for uncomplicated malaria. Serum samples from the time of malaria diagnosis and 14 days later were analyzed for total IgG to 8 P. falciparum antigens using a quantitative indirect ELISA. Associations between antibody levels and risk of treatment failure were estimated using Cox proportional hazard regression.

Results

Higher levels of antibodies to apical membrane antigen 1 (AMA-1), but to none of the other 7 antigens were significantly associated with protection against treatment failure (HR 0.57 per 10-fold increase in antibody level, CI 0.41–0.79, p = 0.001). Protection increased consistently across the entire range of antibody levels.

Conclusions

Measurement of antibody levels to AMA-1 at the time of malaria may offer a quantitative biomarker of blood stage immunity to P. falciparum, a tool which is currently lacking.     

Plasmodium falciparum malaria challenge by the bite of aseptic Anopheles stephensi mosquitoes: results of a randomized infectivity trial

Background

Experimental infection of malaria-naïve volunteers by the bite of Plasmodium falciparum-infected mosquitoes is a preferred means to test the protective effect of malaria vaccines and drugs. The standard model relies on the bite of five infected mosquitoes to induce malaria. We examined the efficacy of malaria transmission using mosquitoes raised aseptically in compliance with current Good Manufacturing Practices (cGMPs).

Methods and Findings

Eighteen adults aged 18–40 years were randomized to receive 1, 3 or 5 bites of Anopheles stephensi mosquitoes infected with the chloroquine-sensitive NF54 strain of P. falciparum.Seventeen participants developed malaria; fourteen occurring on Day 11. The mean prepatent period was 10.9 days (9–12 days). The geometric mean parasitemia was 15.7 parasites/µL (range: 4–70) by microscopy. Polymerase chain reaction (PCR) detected parasites 3.1 (range: 0–4) days prior to microscopy. The geometric mean sporozoite load was 16,753 sporozoites per infected mosquito (range: 1,000–57,500). A 1-bite participant withdrew from the study on Day 13 post-challenge and was PCR and smear negative.

Conclusions

The use of aseptic, cGMP-compliant P. falciparum-infected mosquitoes is safe, is associated with a precise prepatent period compared to the standard model and appears more efficient than the standard approach, as it led to infection in 100% (6/6) of volunteers exposed to three mosquito bites and 83% (5/6) of volunteers exposed to one mosquito bite.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00744133","term_id":"NCT00744133"}}NCT00744133     

Surveillance for malaria elimination in Swaziland: a national cross-sectional study using pooled PCR and serology

Background

To guide malaria elimination efforts in Swaziland and other countries, accurate assessments of transmission are critical. Pooled-PCR has potential to efficiently improve sensitivity to detect infections; serology may clarify temporal and spatial trends in exposure.

Methodology/Principal Findings

Using a stratified two-stage cluster, cross-sectional design, subjects were recruited from the malaria endemic region of Swaziland. Blood was collected for rapid diagnostic testing (RDT), pooled PCR, and ELISA detecting antibodies to Plasmodium falciparum surface antigens. Of 4330 participants tested, three were RDT-positive yet false positives by PCR. Pooled PCR led to the identification of one P. falciparum and one P. malariae infection among RDT-negative participants. The P. falciparum-infected participant reported recent travel to Mozambique. Compared to performing individual testing on thousands of samples, PCR pooling reduced labor and consumable costs by 95.5%. Seropositivity was associated with age ≥20 years (11·7% vs 1·9%, P<0.001), recent travel to Mozambique (OR 4.4 [95% CI 1.0–19.0]) and residence in southeast Swaziland (RR 3.78, P<0.001).

Conclusions

The prevalence of malaria infection and recent exposure in Swaziland are extremely low, suggesting elimination is feasible. Future efforts should address imported malaria and target remaining foci of transmission. Pooled PCR and ELISA are valuable surveillance tools for guiding elimination efforts.     

Malaria Prevalence in Endemic Districts of Bangladesh

Background

Following the 1971 ban of DDT in Bangladesh, malaria cases have increased steadily. Malaria persists as a major health problem in the thirteen south-eastern and north-eastern districts of Bangladesh. At present the national malaria control program, largely supported by the Global Fund for AIDS, Tuberculosis and Malaria (GFATM), provides interventions including advocacy at community level, Insecticide Treated Net (ITN) distribution, introduction of Rapid Diagnostic Tests (RDT) and combination therapy with Coartem. It is imperative, therefore, that baseline data on malaria prevalence and other malaria indicators are collected to assess the effectiveness of the interventions and rationalize the prevention and control efforts. The objective of this study was to obtain this baseline on the prevalence of malaria and bed net use in the thirteen malaria endemic districts of Bangladesh.

Methods and Principal Findings

In 2007, BRAC and ICDDR,B carried out a malaria prevalence survey in thirteen malaria endemic districts of Bangladesh. A multi-stage cluster sampling technique was used and 9750 blood samples were collected. Rapid Diagnostic Tests (RDT) were used for the diagnosis of malaria. The weighted average malaria prevalence in the thirteen endemic districts was 3.97%. In five south-eastern districts weighted average malaria prevalence rate was 6.00% and in the eight north-eastern districts weighted average malaria prevalence rate was (0.40%). The highest malaria prevalence was observed in Khagrachari district. The majority of the cases (90.18%) were P. falciparum infections. Malaria morbidity rates in five south-eastern districts was 2.94%. In eight north-eastern districts, morbidity was 0.07%.

Conclusion and Significance

Bangladesh has hypoendemic malaria with P. falciparum the dominant parasite species. The malaria situation in the five north-eastern districts of Bangladesh in particular warrants urgent attention. Detailed maps of the baseline malaria prevalence and summaries of the data collected are provided along with the survey results in full, in a supplemental information     

PfHRP2-PfHRP3 diversity among Kenyan isolates and comparative evaluation of PfHRP2/pLDH malaria RDT with microscopy and nested PCR methodologies

Rapid diagnostic tests (RDT) are valuable tools that support prudent and timely use of antimalarial drugs, particularly if reliable microscopy is not available. However, the performance and reliability of these tests vary between and within geographical regions. The present study evaluated the performance of routine malaria RDT in Kenyan febrile patients in Busia County, Kenya. A cross sectional study design was employed to recruit febrile patients attending health facilities between August and November 2016. A total of 192 febrile patients who were slide positive and negative were evaluated for their infection status by nested PCR and RDTs (PfHRP2/pLDH). In addition, P. falciparum diversity of the histidine-rich proteins 2 and 3, that influences the RDT test results were determined. All individuals were P. falciparum positive. Among the investigated 192 febrile patients, 76 (40%) were positive by microscopy, 101 (53%) by RDTs and 80 (42%) were PCR positive. The performance of the CareStart? HRP2/pLDH (pf) RDTs was better than microscopy (Sensitivity 94%; Specificity 75%) and Nucleic acid testing (sensitivity 95%, specificity 77%) with high negative predictive values, indicating the suitability of the RDT in routine practice. Specific pfhrp2/pfhrp3 deletions shown to associate with RDT false negativity was not observed. However, high genetic diversity among pfhrp2 gene was observed. Eleven new PfHRP2 and nine PfHRP3 repeats were observed. False positivity by microscopy and under reporting of infections may thus be a barrier in malaria control and elimination programs. The HRP2/pLDH(Pf) based RDT yet demonstrate to be an effective tool for malaria surveillance program.     

Declining responsiveness of Plasmodium falciparum infections to artemisinin-based combination treatments on the Kenyan coast

Background

The emergence of artemisinin-resistant P. falciparum malaria in South-East Asia highlights the need for continued global surveillance of the efficacy of artemisinin-based combination therapies.

Methods

On the Kenyan coast we studied the treatment responses in 474 children 6–59 months old with uncomplicated P. falciparum malaria in a randomized controlled trial of dihydroartemisinin-piperaquine vs. artemether-lumefantrine from 2005 to 2008. (ISRCTN88705995)

Results

The proportion of patients with residual parasitemia on day 1 rose from 55% in 2005–2006 to 87% in 2007–2008 (odds ratio, 5.4, 95%CI, 2.7–11.1; P<0.001) and from 81% to 95% (OR, 4.1, 95%CI, 1.7–9.9; P = 0.002) in the DHA-PPQ and AM-LM groups, respectively. In parallel, Kaplan-Meier estimated risks of apparent recrudescent infection by day 84 increased from 7% to 14% (P = 0.1) and from 6% to 15% (P = 0.05) with DHA-PPQ and AM-LM, respectively. Coinciding with decreasing transmission in the study area, clinical tolerance to parasitemia (defined as absence of fever) declined between 2005–2006 and 2007–2008 (OR body temperature >37.5°C, 2.8, 1.9–4.1; P<0.001). Neither in vitro sensitivity of parasites to DHA nor levels of antibodies against parasite extract accounted for parasite clearance rates or changes thereof.

Conclusions

The significant, albeit small, decline through time of parasitological response rates to treatment with ACTs may be due to the emergence of parasites with reduced drug sensitivity, to the coincident reduction in population-level clinical immunity, or both. Maintaining the efficacy of artemisinin-based therapy in Africa would benefit from a better understanding of the mechanisms underlying reduced parasite clearance rates.

Trial Registration

Controlled-Trials.com ISRCTN88705995