Hematologic and clinical indices of malaria in a semi-immune population of western Thailand

This study examines hematologic profiles of persons with acute Plasmodium falciparum or P. vivax infection in Maesod on Thailand's western border with Myanmar compared with febrile, non-parasitemic persons also reporting to malaria clinics. Nine hundred seventy-nine subjects were malaria-negative, 414 were infected with P. falciparum, and 646 were infected with P. vivax. Persons with patent parasitemia tended to have significantly lower white blood cell, red blood cell, platelet, and hemoglobin levels than those who were malaria-negative. For the first time, a parallel trend in thrombocytopenia with parasitemia was found to be associated with both P. falciparum, and P. vivax infection. Using logistic regression, persons with platelet counts < 150,000/microL were 12-15 times more likely to have malaria than persons with platelet counts > or = 150,000/microL. This study supplements previous literature on the hematologic effects of malaria and helps define those alterations for a semi-immune population. Thrombocytopenia is identified as a key indicator of malaria in these febrile patients.     

In vitro antimalarial activity of azithromycin, artesunate, and quinine in combination and correlation with clinical outcome

Azithromycin when used in combination with faster-acting antimalarials has proven efficacious in treating Plasmodium falciparum malaria in phase 2 clinical trials. The aim of this study was to establish optimal combination ratios for azithromycin in combination with either dihydroartemisinin or quinine, to determine the clinical correlates of in vitro drug sensitivity for these compounds, and to assess the cross-sensitivity patterns. Seventy-three fresh P. falciparum isolates originating from patients from the western border regions of Thailand were successfully tested for their drug susceptibility in a histidine-rich protein 2 (HRP2) assay. With overall mean fractional inhibitory concentrations of 0.84 (95% confidence interval [CI]=0.77 to 1.08) and 0.78 (95% CI=0.72 to 0.98), the interactions between azithromycin and dihydroartemisinin, as well as quinine, were classified as additive, with a tendency toward synergism. The strongest tendency toward synergy was seen with a combination ratio of 1:547 for the combination with dihydroartemisinin and 1:44 with quinine. The geometric mean 50% inhibitory concentration (IC50) of azithromycin was 2,570.3 (95% CI=2,175.58 to 3,036.58) ng/ml. The IC50s for mefloquine, quinine, and chloroquine were 11.42, 64.4, and 54.4 ng/ml, respectively, suggesting a relatively high level of background resistance in this patient population. Distinct correlations (R=0.53; P=0.001) between quinine in vitro results and parasite clearance may indicate a compromised sensitivity to this drug. The correlation with dihydroartemisinin data was weaker (R=0.34; P=0.038), and no such correlation was observed for azithromycin. Our in vitro data confirm that azithromycin in combination with artemisinin derivatives or quinine exerts additive to synergistic interactions, shows no cross-sensitivity with traditional antimalarials, and has substantial antimalarial activity on its own.     

Ex Vivo Drug Susceptibility Testing and Molecular Profiling of Clinical Plasmodium falciparum Isolates from Cambodia from 2008 to 2013 Suggest Emerging Piperaquine Resistance

Cambodia''s first-line artemisinin combination therapy, dihydroartemisinin-piperaquine (DHA-PPQ), is no longer sufficiently curative against multidrug-resistant Plasmodium falciparum malaria at some Thai-Cambodian border regions. We report recent (2008 to 2013) drug resistance trends in 753 isolates from northern, western, and southern Cambodia by surveying for ex vivo drug susceptibility and molecular drug resistance markers to guide the selection of an effective alternative to DHA-PPQ. Over the last 3 study years, PPQ susceptibility declined dramatically (geomean 50% inhibitory concentration [IC₅₀] increased from 12.8 to 29.6 nM), while mefloquine (MQ) sensitivity doubled (67.1 to 26 nM) in northern Cambodia. These changes in drug susceptibility were significantly associated with a decreased prevalence of P. falciparum multidrug resistance 1 gene (Pfmdr1) multiple copy isolates and coincided with the timing of replacing artesunate-mefloquine (AS-MQ) with DHA-PPQ as the first-line therapy. Widespread chloroquine resistance was suggested by all isolates being of the P. falciparum chloroquine resistance transporter gene CVIET haplotype. Nearly all isolates collected from the most recent years had P. falciparum kelch13 mutations, indicative of artemisinin resistance. Ex vivo bioassay measurements of antimalarial activity in plasma indicated 20% of patients recently took antimalarials, and their plasma had activity (median of 49.8 nM DHA equivalents) suggestive of substantial in vivo drug pressure. Overall, our findings suggest DHA-PPQ failures are associated with emerging PPQ resistance in a background of artemisinin resistance. The observed connection between drug policy changes and significant reduction in PPQ susceptibility with mitigation of MQ resistance supports reintroduction of AS-MQ, in conjunction with monitoring of the P. falciparum mdr1 copy number, as a stop-gap measure in areas of DHA-PPQ failure.     

Simple histidine-rich protein 2 double-site sandwich enzyme-linked immunosorbent assay for use in malaria drug sensitivity testing

A simple double-site sandwich enzyme-linked immunosorbent assay (ELISA) for Plasmodium falciparum in vitro drug sensitivity tests based on measuring histidine-rich protein 2 (HRP2) is presented. The ELISA uses two commercial monoclonal antibodies and provides a drastically cheaper alternative to the test kits previously used in the HRP2 drug sensitivity test. The assay is simple to establish and perform. The sensitivity is comparable and the drug sensitivity results very closely match those obtained with the commercial ELISA kits (R(2) = 0.979; P < 0.001; mean log difference at the 50% inhibitory concentration = 0.07).     

Rapid diagnostic devices for malaria: field evaluation of a new prototype immunochromatographic assay for the detection of Plasmodium falciparum and non-falciparum Plasmodium

The NOW ICT Malaria P.f./P.v. for Whole Blood (Binax, Inc., Portland, ME) is a new malaria rapid diagnostic device that represents a technical advance over previous assays, such as ICT Malaria P.f./P.v. and ICT Malaria P.f.. We evaluated this device in March 2001 in symptomatic patients at malaria clinics in Maesod, Thailand. Microscopic examination of Giemsa-stained blood smears was the reference standard. In 246 patients, microscopy showed 32 (13.0%) infected with Plasmodium falciparum, 63 (25.6%) with P. vivax, 6 (2.4%) with mixed infections of P. falciparum and P. vivax, 5 (2.0%) with P. malariae, and 140 (56.9%) negative. Sensitivity for P. falciparum was 100% and specificity was 96.2% (200 of 208; 95% confidence interval [CI] = 92-98). For P. vivax, sensitivity was 87.3% (55 of 63; 95% CI = 77-93) and specificity was 97.7% (173 of 177; 95% CI = 95-99), but all the four false-positive results were microscopically positive for P. malariae; thus, specificity for non-falciparum Plasmodium was 100%. These results suggest improved performance over NOW ICT predecessors.     

Ex Vivo Activity of Endoperoxide Antimalarials,Including Artemisone and Arterolane,against Multidrug-Resistant Plasmodium falciparum Isolates from Cambodia

Novel synthetic endoperoxides are being evaluated as new components of artemisinin combination therapies (ACTs) to treat artemisinin-resistant Plasmodium falciparum malaria. We conducted blinded ex vivo activity testing of fully synthetic (OZ78 and OZ277) and semisynthetic (artemisone, artemiside, artesunate, and dihydroartemisinin) endoperoxides in the histidine-rich protein 2 enzyme-linked immunosorbent assay against 200 P. falciparum isolates from areas of artemisinin-resistant malaria in western and northern Cambodia in 2009 and 2010. The order of potency and geometric mean (GM) 50% inhibitory concentrations (IC₅₀s) were as follows: artemisone (2.40 nM) > artesunate (8.49 nM) > dihydroartemisinin (11.26 nM) > artemiside (15.28 nM) > OZ277 (31.25 nM) > OZ78 (755.27 nM). Ex vivo activities of test endoperoxides positively correlated with dihydroartemisinin and artesunate. The isolates were over 2-fold less susceptible to dihydroartemisinin than the artemisinin-sensitive P. falciparum W2 clone and showed sensitivity comparable to those with test endoperoxides and artesunate, with isolate/W2 IC₅₀ susceptibility ratios of <2.0. All isolates had P. falciparum chloroquine resistance transporter mutations, with negative correlations in sensitivity to endoperoxides and chloroquine. The activities of endoperoxides (artesunate, dihydroartemisinin, OZ277, and artemisone) significantly correlated with that of the ACT partner drug, mefloquine. Isolates had mutations associated with clinical resistance to mefloquine, with 35% prevalence of P. falciparum multidrug resistance gene 1 (pfmdr1) amplification and 84.5% occurrence of the pfmdr1 Y184F mutation. GM IC₅₀s for mefloquine, lumefantrine, and endoperoxides (artesunate, dihydroartemisinin, OZ277, OZ78, and artemisone) correlated with pfmdr1 copy number. Given that current ACTs are failing potentially from reduced sensitivity to artemisinins and partner drugs, newly identified mutations associated with artemisinin resistance reported in the literature and pfmdr1 mutations should be examined for their combined contributions to emerging ACT resistance.     

Sensitivity and specificity of an antigen detection ELISA for malaria diagnosis

Enzyme-linked immunosorbent assays (ELISAs) allow for the testing of large numbers of samples within a short time frame. We tested the sensitivity and specificity of a histidine-rich protein 2 (HRP2)-based, commercially available ELISA antigen detection assay for Plasmodium falciparum (Malaria Antigen CELISA; Cellabs, Sydney, Australia). A total of 700 whole blood samples obtained from symptomatic outpatients of malaria clinics along the Thai-Myanmar border were tested relative to blinded duplicate expert microscopy adjusted with species-specific polymerase chain reaction (PCR). PCR-adjusted microscopy showed that 79 (11.3%) were infected with P. falciparum, 118 (16.9%) with P. vivax, 1 (0.1%) with P. malariae, 7 (1.0%) with mixed infections (P. falciparum and P. vivax), and 495 (70.7%) were negative. The geometric mean parasite density for P. falciparum was 7547/muL (range: 12-363,810/muL). The overall sensitivity of the HRP2 ELISA for P. falciparum malaria was 98.8% (95% CI, 93.6-100%) and the specificity was 100% (95% CI, 99.5-100%). The positive and negative predictive values for the ELISA were 100% (95% CI, 96.5-100%) and 99.8% (95% CI, 99.1-100%), respectively. The results for P. falciparum were clearly superior to expert microscopy alone, particularly in mixed infections. Microscopy combined with ELISA reaches a sensitivity and specificity similar to PCR-adjusted microscopy for the diagnosis of P. falciparum while being considerably less expensive and faster. We conclude that ELISA serves as an excellent tool to augment microscopy as the gold standard for P. falciparum diagnosis in research settings and should be further evaluated for screening in blood banks.     

Causes of fever in adults on the Thai-Myanmar border

A hospital-based study was conducted along the Thai-Myanmar border to provide greater knowledge of the causes of febrile illness and to determine what zoonotic and vector-borne emerging infectious diseases might be present. A total of 613 adults were enrolled from June 1999 to March 2002. Cases were classified based on clinical findings and laboratory results. An etiologic diagnosis was made for 48% of subjects. Malaria was the most common diagnosis, accounting for 25% of subjects, with two-thirds Plasmodium falciparum. Serologic evidence for leptospirosis was found in 17% of subjects. Other etiologic diagnoses included rickettsial infections, dengue fever, and typhoid. The most frequent clinical diagnoses were nonspecific febrile illness, respiratory infections, and gastroenteritis. Clinical associations were generally not predictive of etiologic diagnosis. Apparent dual diagnoses were common, particularly for malaria and leptospirosis. Findings have been used to modify treatment of unspecified febrile illness in the area.