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Biomedical subjects

Chansuda Wongsrichanalai

Publications and source records attributed to Chansuda Wongsrichanalai.

At least 19 recordsLinked to original sources

Reader technique as a source of variability in determining malaria parasite density by microscopy.

BACKGROUND: Accurate identification and quantification of malaria parasites are critical for measuring clinical trial outcomes. Positive and negative diagnosis is usually sufficient for the assessment of therapeutic outcome, but vaccine or prophylactic drug trials require measuring density of infection as a primary endpoint. Microscopy is the most established and widely-used technique for quantifying parasite densities in the blood. METHODS: Results obtained by 24-27 expert malaria microscopists, who had independently read 895 slides from 35 donors, were analysed to understand how reader technique contributes to discrepancy in measurements of parasite density over a wide range of densities. RESULTS: Among these 35 donations, standard deviations ranged from 30% to 250% of the mean parasite density and the percent discrepancy was inversely correlated with the mean parasite density. The number of white blood cells indexed and whether parasites were counted in the thick film or thin film were shown to significantly contribute to discrepancy amongst microscopists. CONCLUSION: Errors in microscopy measurements are not widely appreciated or addressed but have serious consequences for efficacy trials, including possibly abandoning promising vaccine candidates.

Animals↗

Production and validation of durable, high quality standardized malaria microscopy slides for teaching, testing and quality assurance during an era of declining diagnostic proficiency.

BACKGROUND: Sets of Giemsa-stained, blood smear slides with systematically verified composite diagnoses would contribute substantially to development of externally validated quality assurance systems for the microscopic diagnosis of malaria. METHODS: whole blood from Plasmodium-positive donors in Cambodia and Indonesia and individuals with no history of risk for malaria was collected. Using standard operating procedures, technicians prepared Giemsa-stained thick and thin smears from each donor. One slide from each of the first 35 donations was distributed to each of 28 individuals acknowledged by reputation as having expertise in the microscopic diagnosis of malaria. These reference readers recorded presence or absence of Plasmodium species and parasite density. A composite diagnosis for each donation was determined based on microscopic findings and species-specific small subunit ribosomal RNA (ssrRNA) DNA polymerase chain reaction (PCR) amplification. RESULTS: More than 12,000 slides were generated from 124 donations. Reference readers correctly identified presence of parasites on 85% of slides with densities <100 parasites/microl, which improved to 100% for densities >350 parasites/microl. Percentages of agreement with composite diagnoses were highest for Plasmodium falciparum (99%), followed by Plasmodium vivax (86%). CONCLUSION: Herein, a standardized method for producing large numbers of consistently high quality, durable Giemsa-stained blood smears and validating composite diagnoses for the purpose of creating a malaria slide repository in support of initiatives to improve training and competency assessment amidst a background of variability in diagnosis is described.

Animals↗

Fever in patients with mixed-species malaria.

BACKGROUND: Clinical symptoms of mixed-species malaria infections have been variously reported as both less severe and more severe than those of single-species infections. METHODS: Oral temperatures were taken from and blood slides were prepared for 2308 adults who presented at outpatient malaria clinics in Tak Province (Thailand) during May-August 1998, May-July 1999, and May-June 2001 with malaria infections diagnosed by 2 expert research microscopists, each of whom was blinded to the other's reports. RESULTS: In each year, temperatures of patients with mixed Plasmodium vivax-Plasmodium falciparum infections were higher than temperatures of patients with P. vivax or P. falciparum infections. In every mixed-species case, P. falciparum parasitemia was higher than P. vivax parasitemia, but patient temperature was not correlated with the parasitemia of either species or with the total parasitemia. CONCLUSIONS: Among adults who self-report to malaria clinics in western Thailand, patients with mixed P. vivax-P. falciparum infections have higher fevers than patients with single-species infections, a distinction that cannot be attributed to differences in parasitemia. This observation warrants more detailed investigations, spanning wider ranges of ages and transmission environments.

Adult↗

Ensuring quality and access for malaria diagnosis: how can it be achieved?

The replacement of conventional antimalarial drugs with high-cost, artemisinin-based alternatives has created a gap in the successful management of malaria. This gap reflects an increased need for accurate disease diagnosis that cannot be met by traditional microscopy techniques. The recent introduction of rapid diagnostic tests (RDTs) has the potential to meet this need, but successful RDT implementation has been curtailed by poor product performance, inadequate methods to determine the quality of products and a lack of emphasis and capacity to deal with these issues. Economics and a desire for improved case management will result in the rapid growth of RDT use in the coming years. However, for their potential to be realized, it is crucial that high-quality RDT products that perform reliably and accurately under field conditions are made available. In achieving this goal, the shift from symptom-based diagnosis to parasite-based management of malaria can bring significant improvements to tropical fever management, rather than represent a further burden on poor, malaria-endemic populations and their overstretched health services.

Animals↗

Ensuring quality and access for malaria diagnosis: how can it be achieved?

The replacement of conventional antimalarial drugs with high-cost, artemisinin-based alternatives has created a gap in the successful management of malaria. This gap reflects an increased need for accurate disease diagnosis that cannot be met by traditional microscopy techniques. The recent introduction of rapid diagnostic tests (RDTs) has the potential to meet this need, but successful RDT implementation has been curtailed by poor product performance, inadequate methods to determine the quality of products and a lack of emphasis and capacity to deal with these issues. Economics and a desire for improved case management will result in the rapid growth of RDT use in the coming years. However, for their potential to be realized, it is crucial that high-quality RDT products that perform reliably and accurately under field conditions are made available. In achieving this goal, the shift from symptom-based diagnosis to parasite-based management of malaria can bring significant improvements to tropical fever management, rather than represent a further burden on poor, malaria-endemic populations and their overstretched health services.

Antigens, Protozoan↗

In vivo sensitivity monitoring of mefloquine monotherapy and artesunate-mefloquine combinations for the treatment of uncomplicated falciparum malaria in Thailand in 2003.

OBJECTIVE: To monitor the efficacy of anti-malarial treatments in Thailand. METHOD: A 28-day in vivo study in nine provinces along international borders in 2003. The first group comprised 164 patients from four provinces: Mae Hong Son, Chiang Mai, Ratchaburi and Ubon Ratchathani. These patients received 15 mg/kg mefloquine as a single dose. The second group, 58 patients from Kanchanaburi, were treated with 15 mg/kg mefloquine plus artesunate (12 mg/kg). The third group, 196 patients from provinces with high-level mefloquine resistance (Tak, Ranong, Chanthaburi and Trat), received 25 mg/kg of mefloquine plus 12 mg/kg artesunate. In all arms, follow-up blood smears were scheduled for days 1, 2, 3, 7, 14, 21 and 28. All patients tolerated the regimens well. RESULTS: The percentage of adequate clinical and parasitological response to mefloquine monotherapy was 62.0% in Mae Hong Son, 75.0% in Chiang Mai, 94.0% in Ratchaburi and 89.7% in Ubon Ratchathani. In Kanchanaburi, the percentage of adequate clinical and parasitological response to the artesunate-mefloquine combination was 94.2%. In the third group, this response exceeded 90%, except in Trat, where it was only 78.6% (44 patients). CONCLUSION: Mefloquine monotherapy must urgently be replaced in Mae Hong Son and Chiang Mai. The markedly reduced efficacy of the artesunate-mefloquine combination used in Trat raises questions about the future of this therapy on the southeastern border of Thailand with Cambodia. It is very worrying because no practical and affordable alternative is yet available.

Administration, Oral↗

Effective treatment of uncomplicated Plasmodium falciparum malaria with azithromycin-quinine combinations: a randomized, dose-ranging study.

Azithromycin, the most potent antimalarial macrolide antibiotic, is synergistic with quinine against Plasmodium falciparum in vitro. We assessed combinations of azithromycin and quinine against uncomplicated P. falciparum malaria at the Armed Forces Research Institute of Medical Sciences-Kwai River Clinical Center along the Thailand-Myanmar border, an area with a high prevalence of multidrug-resistant P. falciparum. Four regimens were assessed in an open-label dose-ranging design involving 61 volunteers. All received oral quinine (Q; 30 mg/kg/day divided every 8 hours for 3 days) with oral azithromycin (Az; 500 mg twice a day for 3 days, 500 mg twice a day for 5 days, or 500 mg three times a day for 3 days). A comparator group received quinine and doxycycline (Dx; 100 mg twice a day for 7 days). Study observation was 28 days per protocol. Sixty volunteers completed the study. Seven days of QDx cured 100% of the volunteers. One failure occurred in the lowest QAz regimen (on day 28) and none occurred in either of the two higher Az regimens. Cinchonism occurred in nearly all subjects. Overall, the azithromycin regimens were well tolerated, and no volunteers discontinued therapy. Three- and five-day azithromycin-quinine combination therapy appears safe, well tolerated, and effective in curing drug-resistant P. falciparum malaria. Further evaluation, especially in pediatric and obstetric populations, is warranted.

Administration, Oral↗

White blood cell counts and malaria.

White blood cells (WBCs) were counted in 4697 individuals who presented to outpatient malaria clinics in Maesod, Tak Province, Thailand, and Iquitos, Peru, between 28 May and 28 August 1998 and between 17 May and 9 July 1999. At each site and in each year, WBC counts in the Plasmodium falciparum-infected patients were lower than those in the Plasmodium vivax-infected patients, which, in turn, were lower than those in the uninfected patients. In Thailand, one-sixth of the P. falciparum-infected patients had WBC counts of <4000 cells/microL. Leukopenia may confound population studies that estimate parasite densities on the basis of an assumed WBC count of 8000 cells/microL. For instance, in the present study, use of this conventional approach would have overestimated average asexual parasite densities in the P. falciparum-infected patients in Thailand by nearly one-third.

Adolescent↗

pfmdr1 genotyping and in vivo mefloquine resistance on the Thai-Myanmar border.

Molecular markers have been proposed as a method of monitoring malaria drug resistance and could potentially be used to prolong the life span of antimalarial drugs. Single nucleotide polymorphisms (SNPs) in the Plasmodium falciparum gene pfmdr1 and increased gene copy number have been associated with in vitro drug resistance but have not been well studied in vivo. In a prospective cohort study of malaria patients receiving mefloquine treatment on the Thai-Myanmar border, there was no significant association between either pfmdr1 SNPs or in vitro drug sensitivity and mefloquine resistance in vivo. Increased pfmdr1 gene copy number was significantly associated with recrudescence (relative risk 2.30, 95% CI 1.27-4.15). pfmdr1 gene copy number may be a useful surveillance tool for mefloquine-resistant falciparum malaria in Thailand.

ATP-Binding Cassette Transporters↗

Measuring allelic heterogeneity in Plasmodium falciparum by a heteroduplex tracking assay.

We developed a novel Plasmodium falciparum genotyping strategy based on the heteroduplex tracking assay (HTA) method commonly used to genotype viruses. Because it can detect both sequence and size polymorphisms, we hypothesized that HTA is more sensitive than current methods. To test this hypothesis, we compared the ability of HTA and a nested polymerase chain reaction (PCR) to detect genetic diversity in 17 Thai samples. The HTA detected more MSP1 sequence variants in eight isolates (47%), less sequence variants in three isolates (18%), and an equal number of sequence variants in six isolates (35%), suggesting that HTA is equal to or more sensitive than the nested PCR. This study is a proof of concept that HTA is a sensitive allelic discrimination method able to determine genetic diversity in P. falciparum and warrants its use in studies of antimalarial drug efficacy.

Alleles↗

Sources of variability in determining malaria parasite density by microscopy.

Enumeration of parasites by microscopic examination of blood smears is the only method available for quantifying parasitemia in infected blood. However, the sources and scale of error inherent in this technique have not been systematically investigated. Here we use data collected in outpatient clinics in Peru and Thailand to elucidate important sources of variation in parasite density measurements. We show that discrepancies between readings from two independent microscopists and multiple readings from a single microscopist are inversely related to the density of the infection. We present an example of how differences in reader technique, specifically the number of white blood cells counted, can contribute to the differences between readings. We discuss the implications of this analysis for field studies and clinical trials.

Animals↗

Randomized, controlled, double-blind trial of daily oral azithromycin in adults for the prophylaxis of Plasmodium vivax malaria in Western Thailand.

We assessed the prophylactic efficacy of azithromycin (250 mg/day) against malaria in 276 adults in western Thailand in a randomized, double-blind, placebo-controlled trial. After antimalarial suppressive treatment, volunteers were randomized in a 2:1 ratio to either the azithromycin or placebo, respectively. Study medication was given for an average of 74 days. The azithromycin group (n = 179) had five endpoint parasitemias (1 Plasmodium vivax and 4 P. falciparum), and the placebo group (n = 97) had 28 endpoint parasitemias (21 P. vivax, 5 P. falciparum, and 2 mixed infections). Adverse events and compliance and withdrawal rates were similar in both groups. The protective efficacy (PE) of azithromycin was 98% for P. vivax (95% confidence interval [CI] = 88-100%). There were too few cases to reliably estimate the efficacy of azithromycin for P. falciparum (PE =71%, 95% C =-14-94%). We conclude that daily azithromycin was safe, well-tolerated, and had a high efficacy for the prevention of P. vivax malaria.

Adult↗

Bartonella species in rodents and shrews in the greater Jakarta area.

In February 2004, we captured 221 rodents and shrews in the Greater Jakarta area as part of a study to determine the prevalence of rodent-associated vector-borne infections. Microscopic examination of blood smears revealed 6% (13/218) to be positive for Bartonella spp. The corresponding DNA samples, either from blood blots or frozen spleen pieces and from fleas collected on these animals, were tested for evidence of Bartonella infection by PCR, targeting the portions: 378bp and 930bp of the citrate synthase gene (g/tA). The sequences from our sample clusters with a Peruvian entity, B. phoceensis, B. rattimassiliensis and B. elizabethae, the latter species has been associated with endocarditis and neuroretinitis in humans. As previous analyses have shown, there appears to be little geographic or host consistency with phylogenetic placement. The public health significance of these findings remains to be determined.

Animals↗

A new method for detection of pfmdr1 mutations in Plasmodium falciparum DNA using real-time PCR.

BACKGROUND: Surveillance for drug-resistant Plasmodium falciparum should be a component of malaria control programmes. Real-time PCR methods for the detection of parasite single-nucleotide polymorphisms (SNPs) and gene amplification could be useful survellance tools. METHODS: A real-time PCR assay has been developed that identifies single nucleotide polymorphisms (SNPs) at amino acids 86, 184, 1034 and 1042 in the P. falciparum multi-drug resistant (pfmdr 1) gene that may be associated with anti-malarial drug resistance. RESULTS: This assay has a sensitivity and specificity of 94% and 100% when compared to traditional PCR methods for genotyping. Only 54 of 68 (79%) paired pre- and post-culture DNA samples were concordant at all four loci. CONCLUSION: Real-time PCR is a sensitive and specific method to detect SNP's in pfmdr 1. Genotypes of parasites after in vitro culture may not reflect that seen in vivo.

ATP-Binding Cassette Transporters↗

Efficacy of the merozoite surface protein 1 of Plasmodium vivax as an antigen for ELISA to diagnose malaria.

Malaria is still a major health problem in Thailand and its incidence is currently rising in Korea. To identify a useful antigen for the diagnosis of malaria patients, a cDNA expression library from malaria parasites was constructed and screened out immunologically. One clone was selected in view of its predominant reactivity with the patient sera. The recombinant malaria parasite antigen (Pv30) with 27 kDa as a C-terminal His-tag fusion protein that was produced in Escherichia coli was identified through immunoblot analysis. The deduced amino acid sequence had the sequence homology with the merozoite surface protein 1 (MSP1) genes of Plasmodium falciparum and P. yoelii, each by 41% and 42%, respectively. Measurement of serum IgG and IgM antibody to Pv30 by enzyme-linked immunosorbent assay (ELISA) was evaluated as a serodiagnostic test for malaria patients in Thailand (endemic area) and Korea (recently reemerging area). The sensitivity of P. vivax, P. falciparum, and P. malariae was 96.3% (26 /27), 90.6% (29/32), and 100% (6/6), respectively, and the specificity was 63.5% (40/63) in Thailand samples. The sensitivity of P. vivax was 98.8% (88/89), and the specificity was 96.6% (86/89) in Korean samples. Pv30 appears to be a good and reliable recombinant antigen for serodiagonosis of malaria in a nonendemic area.

Amino Acid Sequence↗

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.

Adult↗

A study of'febrile illnesses on the Thai-Myanmar border: predictive factors of rickettsioses.

We have performed a case-control analysis to determine the significance of clinical, laboratory and epidemiological features as predictive factors of rickettsioses among patients in Sangkhla Buri, Thailand (Thai-Myanmar border). Fifteen serologically-confirmed rickettsiosis patients including Spotted Fever Group (SFG) rickettsioses, scrub typhus, and murine typhus were classified as 'cases'; one hundred and sixty-three acutely febrile patients presenting to the same hospital during the same time period, who had no serological evidence of acute rickettsiosis, were classified as 'controls'. Patients' report of rash/arthropod bite [Odds ratio (OR) 22.90, 95% CI (confidence interval) 6.23, 84.13] and history of jungle trips (OR 5.30, 95% CI 1.69-16.62) were significant risk factors. Elevated ALT (OR 3.04, 95% CI 1.04, 8.88) and depressed platelet count (OR 3.38, 95% CI 1.13, 10.10) were also useful differentiating markers of rickettsioses in this population. Definitive diagnosis of rickettsioses is difficult without specialized diagnostic capabilities that are rarely available in remote areas such as Sangkhla Buri, where other acute febrile illnesses with similar presentation are commonly found. The relative importance of predictive factors presented here may provide clinicians with some useful guidance in distinguishing rickettsioses from other acute febrile illnesses. Timely administration of empiric treatment in highly suspicious cases can deter potential morbidity from these arthropod-borne infections.

Adult↗