Usefulness of Plasmodium falciparum-specific rapid diagnostic tests for assessment of parasite clearance and detection of recurrent infections after artemisinin-based combination therapy.

BACKGROUND: Rapid diagnostic test (RDT) is an important tool for parasite-based malaria diagnosis. High specificity of RDTs to distinguish an active Plasmodium falciparum infection from residual antigens from a previous infection is crucial in endemic areas where residents are repeatedly exposed to malaria. The efficiency of two RDTs based on histidine-rich protein 2 (HRP2) and lactate dehydrogenase (LDH) antigens were studied and compared with two microscopy techniques (Giemsa and acridine orange-stained blood smears) and real-time polymerase chain reaction (PCR) for assessment of initial clearance and detection of recurrent P. falciparum infections after artemisinin-based combination therapy (ACT) in a moderately high endemic area of rural Tanzania. METHODS: In this exploratory study 53 children < five years with uncomplicated P. falciparum malaria infection were followed up on nine occasions, i.e., day 1, 2, 3, 7, 14, 21, 28, 35 and 42, after initiation of artemether-lumefantrine treatment. At each visit capillary blood samples was collected for the HRP2 and LDH-based RDTs, Giemsa and acridine orange-stained blood smears for microscopy and real-time PCR. Assessment of clearance times and detection of recurrent P. falciparum infections were done for all diagnostic methods. RESULTS: The median clearance times were 28 (range seven to >42) and seven (two to 14) days for HRP2 and LDH-based RDTs, two (one to seven) and two (one to 14) days for Giemsa and acridine orange-stained blood smear and two (one to 28) days for real-time PCR. RDT specificity against Giemsa-stained blood smear microscopy was 21% for HRP2 on day 14, reaching 87% on day 42, and ≥96% from day 14 to 42 for LDH. There was no significant correlation between parasite density at enrolment and duration of HRP2 positivity (r = 0.13, p = 0.34). Recurrent malaria infections occurred in ten (19%) children. The HRP2 and LDH-based RDTs did not detect eight and two of the recurrent infections, respectively. CONCLUSION: The LDH-based RDT was superior to HRP2-based for monitoring of treatment outcome and detection of recurrent infections after ACT in this moderately high transmission setting. The results may have implications for the choice of RDT devices in similar transmission settings for improved malaria case management. TRIAL REGISTRATION: Clinicaltrials.gov, NCT01843764.

Rapid diagnostic tests for molecular surveillance of Plasmodium falciparum malaria -assessment of DNA extraction methods and field applicability.

BACKGROUND: The need for new malaria surveillance tools and strategies is critical, given improved global malaria control and regional elimination efforts. High quality Plasmodium falciparum DNA can reliably be extracted from malaria rapid diagnostic tests (RDTs). Together with highly sensitive molecular assays, wide scale collection of used RDTs may serve as a modern tool for improved malaria case detection and drug resistance surveillance. However, comparative studies of DNA extraction efficiency from RDTs and the field applicability are lacking. The aim of this study was to compare and evaluate different methods of DNA extraction from RDTs and to test the field applicability for the purpose of molecular epidemiological investigations. METHODS: DNA was extracted from two RDT devices (Paracheck-Pf® and SD Bioline Malaria Pf/Pan®), seeded in vitro with 10-fold dilutions of cultured 3D7 P. falciparum parasites diluted in malaria negative whole blood. The level of P. falciparum detection was determined for each extraction method and RDT device with multiple nested-PCR and real-time PCR assays. The field applicability was tested on 855 paired RDT (Paracheck-Pf) and filter paper (Whatman® 3MM) blood samples (734 RDT negative and 121 RDT positive samples) collected from febrile patients in Zanzibar 2010. RDT positive samples were genotyped at four key single nucleotide polymorphisms (SNPs) in pfmdr1 and pfcrt as well as for pfmdr1 copy number, all associated with anti-malarial drug resistance. RESULTS: The P. falciparum DNA detection limit varied with RDT device and extraction method. Chelex-100 extraction performed best for all extraction matrixes. There was no statistically significant difference in PCR detection rates in DNA extracted from RDTs and filter paper field samples. Similarly there were no significant differences in the PCR success rates and genotyping outcomes for the respective SNPs in the 121 RDT positive samples. CONCLUSIONS: The results support RDTs as a valuable source of parasite DNA and provide evidence for RDT-DNA extraction for improved malaria case detection, molecular drug resistance surveillance, and RDT quality control.

Assessing the cost-benefit effect of a Plasmodium falciparum drug resistance mutation on parasite growth in vitro.

Plasmodium falciparum mutations associated with antimalarial resistance may be beneficial for parasites under drug pressure, although they may also cause a fitness cost. We herein present an in vitro model showing how this combined effect on parasite growth varies with the drug concentration and suggest a calculated drug-specific cost-benefit index, indicating the possible advantage for mutated parasites. We specifically studied the D-to-Y change at position 1246 encoded by the pfmdr1 gene (pfmdr1 D1246Y) in relation to amodiaquine resistance. Susceptibilities to amodiaquine, desethylamodiaquine, and chloroquine, as well as relative fitness, were determined for two modified isogenic P. falciparum clones differing only in the pfmdr1 1246 position. Data were used to create a new comparative graph of relative growth in relation to the drug concentration and to calculate the ratio between the benefit of resistance and the fitness cost. Results were related to an in vivo allele selection analysis after amodiaquine or artesunate-amodiaquine treatment. pfmdr1 1246Y was associated with decreased susceptibility to amodiaquine and desethylamodiaquine but at a growth fitness cost of 11%. Mutated parasites grew less in low drug concentrations due to a predominating fitness cost, but beyond a breakpoint concentration they grew more due to a predominating benefit of increased resistance. The cost-benefit indexes indicated that pfmdr1 1246Y was most advantageous for amodiaquine-exposed parasites. In vivo, a first drug selection of mutant parasites followed by a fitness selection of wild-type parasites supported the in vitro data. This cost-benefit model may predict the risk for selection of drug resistance mutations in different malaria transmission settings.

Assessment of perfusion in normal carpal bones with dynamic gadolinium-enhanced MRI at 3 Tesla.

PURPOSE: To investigate the normal enhancement patterns of the scaphoid, lunate, and capitate bones with dynamic contrast-enhanced magnetic resonance imaging (MRI). MATERIALS AND METHODS: The study was approved by the hospital's Ethics Committee. Nineteen volunteers (13 female, 6 male; mean age 38 years) were examined and all gave written consent. Perfusion was assessed at 3 Tesla using dynamic contrast-enhanced MRI. After two-dimensional (2D) motion correction of the data set, regions of interest were placed in the capitate, lunate, and distal and proximal pole of scaphoid bone and from the mean signal intensities (SI), the enhancement was computed. The four locations were compared for time to peak, delay time, maximum enhancement, and maximum slope using Friedman's two-way analysis of variance. RESULTS: Typical SI versus time curves revealed two components: a faster component with strong contrast enhancement and a slow component with prolonged enhancement. The mean value (standard deviation, SD) for maximum enhancement was 51 (33)% in the capitate, 54 (25)% in the lunate, 51 (34)% in the proximal pole and 51 (28)% in the distal pole of the scaphoid. The result of the Friedman test showed no significant difference (P < 0.05) in the perfusion variables between the capitate, lunate, and distal and proximal scaphoid bones. CONCLUSION: Assessment of perfusion in normal carpal bone using contrast-enhanced MRI is possible. Optimization of the method and understanding of the normal perfusion may allow evaluation of pathological conditions such as osteonecrosis.

System effectiveness of a targeted free mass distribution of long lasting insecticidal nets in Zanzibar, Tanzania.

BACKGROUND: Insecticide-treated nets (ITN) and long-lasting insecticidal treated nets (LLIN) are important means of malaria prevention. Although there is consensus regarding their importance, there is uncertainty as to which delivery strategies are optimal for dispensing these life saving interventions. A targeted mass distribution of free LLINs to children under five and pregnant women was implemented in Zanzibar between August 2005 and January 2006. The outcomes of this distribution among children under five were evaluated, four to nine months after implementation. METHODS: Two cross-sectional surveys were conducted in May 2006 in two districts of Zanzibar: Micheweni (MI) on Pemba Island and North A (NA) on Unguja Island. Household interviews were conducted with 509 caretakers of under-five children, who were surveyed for socio-economic status, the net distribution process, perceptions and use of bed nets. Each step in the distribution process was assessed in all children one to five years of age for unconditional and conditional proportion of success. System effectiveness (the accumulated proportion of success) and equity effectiveness were calculated, and predictors for LLIN use were identified. RESULTS: The overall proportion of children under five sleeping under any type of treated net was 83.7% (318/380) in MI and 91.8% (357/389) in NA. The LLIN usage was 56.8% (216/380) in MI and 86.9% (338/389) in NA. Overall system effectiveness was 49% in MI and 87% in NA, and equity was found in the distribution scale-up in NA. In both districts, the predicting factor of a child sleeping under an LLIN was caretakers thinking that LLINs are better than conventional nets (OR = 2.8, p = 0.005 in MI and 2.5, p = 0.041 in NA), in addition to receiving an LLIN (OR = 4.9, p < 0.001 in MI and in OR = 30.1, p = 0.001 in NA). CONCLUSIONS: Targeted free mass distribution of LLINs can result in high and equitable bed net coverage among children under five. However, in order to sustain high effective coverage, there is need for complimentary distribution strategies between mass distribution campaigns. Considering the community's preferences prior to a mass distribution and addressing the communities concerns through information, education and communication, may improve the LLIN usage.

HIV-infected African parents living in Stockholm, Sweden: disclosure and planning for their children's future.

In Sweden, most HIV-infected parents are of African origin. The present study explored the frequency of HIV-infected African parents' disclosure of their status to their children and custody planning for their children's future to identify support needs among these families. Semistructured interviews were conducted with 47 parents (41 families). The study population included first-generation immigrants, with a total of 87 children less than 18 years of age. Only women had disclosed their HIV status, and only to eight of 59 children older than six. Half of the parents had talked to someone about future custody arrangements. These parents had more contact with a social worker at the social welfare office and with a medical social worker at the HIV clinic. Most parents (30) wanted their children to be cared for by a relative in Sweden or by their HIV-negative partner. Neither disclosure nor custody planning was associated with clinical status or antiretroviral treatment. This study highlights the low HIV-disclosure rate to children of HIV-infected African immigrant parents and the importance of support from social workers.

Willingness and ability to pay for artemisinin-based combination therapy in rural Tanzania.

BACKGROUND: The aim of this study was to analyse willingness to pay (WTP) and ability to pay (ATP) for ACT for children below five years of age in a rural setting in Tanzania before the introduction of artemisinin-based combination therapy (ACT) as first-line treatment for uncomplicated malaria. Socio-economic factors associated with WTP and expectations on anti-malaria drugs, including ACT, were also explored. METHODS: Structured interviews and focus group discussions were held with mothers, household heads, health-care workers and village leaders in Ishozi, Gera and Ishunju wards in north-west Tanzania in 2004. Contingent valuation method (CVM) was used with "take-it-or-leave-it" as the eliciting method, expressed as WTP for a full course of ACT for a child and households' opportunity cost of ACT was used to assess ATP. The study included descriptive analyses with multivariate adjustment for potential confounding factors. RESULTS: Among 265 mothers and household heads, 244 (92%, CI = 88%-95%) were willing to pay Tanzanian Shillings (TSh) 500 (US$ 0.46) for a child's dose of ACT, but only 55% (49%-61%) were willing to pay more than TSh 500. Mothers were more often willing to pay than male household heads (adjusted odds ratio = 2.1, CI = 1.2-3.6). Socio-economic status had no significant effect on WTP. The median annual non-subsidized ACT cost for clinical malaria episodes in an average household was calculated as US$ 6.0, which would represent 0.9% of the average total consumption expenditures as estimated from official data in 2001. The cost of non-subsidized ACT represented 7.0% of reported total annual expenditure on food and 33.0% of total annual expenditure on health care."Rapid effect," "no adverse effect" and "inexpensive" were the most desired features of an anti-malarial drug. CONCLUSION: WTP for ACT in this study was less than its real cost and a subsidy is, therefore, needed to enable its equitable affordability. The decision taken in Tanzania to subsidize Coartem fully at governmental health care facilities and at a consumer price of TSh 300-500 (US$ 0.28-0.46) at special designated shops through the programme of Accredited Drug Dispensing Outlets (ADDOs) appears to be well founded.