The American Journal of Tropical Medicine and Hygiene - Volume 68, Issue 5, 2003

The California Mosquito-Borne Virus Surveillance and Response Plan recently was developed to provide a semi-quantitative means for assessing risk for western equine encephalomyelitis (WEE) or St. Louis encephalitis (SLE) viruses and to provide intervention guidelines for mosquito control and public health agencies during periods of heightened risk for human infection. West Nile virus recently has arrived in California, and the response plan also will provide a baseline for assessing the risk for human and equine infection with this virus. In the response plan, overall risk is calculated by averaging risk due to 1) environmental conditions, 2) adult mosquito vector abundance, 3) vector infection rates, 4) sentinel chicken seroconversion rates, 5) equine cases (for WEE), 6) human cases, and 7) the proximity of virus activity to populated areas. Overall risk is categorized into three levels: normal season, emergency planning, or epidemic conditions. We evaluated this response plan using historical data from years with no, enzootic, and epidemic activity of WEE and SLE in several areas of California to determine whether calculated risk levels approximated actual conditions. Multiple methods of risk calculation were considered for both viruses. Assessed risk based on cumulative temperature, rainfall, and runoff levels over the entire season provided more or equally accurate assessments than biweekly assessments based solely on the previous half-month. For WEE, during years with enzootic activity or early-season periods of years with WEE epidemic activity, combining horse and human cases as a single risk factor improved the model’s ability to forecast pending WEE activity, but separating the two factors allowed a better indication of WEE activity during epidemics and periods with no activity. For SLE, assignment of higher risk to drier conditions as measured by rainfall and runoff yielded the most accurate representation of actual virus activity during all recent study periods.

A case-control study was carried out during 1990–1994 to identify risk factors associated with American cutaneous leishmaniasis (ACL) in Santiago del Estero, Argentina. The study subjects consisted of 171 cases and 308 controls matched by age, sex, and place of residence. The analysis was performed by conditional logistic regression. Risk factors found to be significantly associated with ACL were related to indoor transmission (few rooms in the house, dirt floor, and a permanent opening in lieu of a window); peridomestic transmission (presence of a pond or woodland within 150 m of the house and an agricultural area within 200 m of the house); and human behavior (sleeping in the backyard, collecting water, bathing, and performing agricultural activities). Most transmission appears to have occurred indoors and in the peridomicile. These environments should be included in further research and control policies.

Mucosal leishmaniasis (ML) is a disease characterized by intense activation of inflammatory cells and extensive tissue destruction. Among the cytokines involved in the immune response to ML, tumor necrosis factor-α (TNF-α) has attracted strong interest because of its roles in the modulation of the immune response. We studied 20 patients with ML who provided biopsy specimens before treatment and after lesion healing obtained by specific therapy. The biopsy specimens were subjected to immunohistochemical analysis for in situ quantification of cellular and extra-cellular TNF-α. The amount of TNF-α was significantly lower in the healed lesions compared with pretreatment biopsy specimens, although TNF-α persisted at the tissue level even after lesion healing. This relevant finding demonstrates for the first time an in situ tissue reduction of TNF-α after treatment and shows persistence of TNF-α in healed lesions may be related to the maintenance of an immunopathologic background for relapses observed in ML.

In April 2001, a second suspected outbreak of dengue hemorrhagic fever in the easternmost region of Indonesia was investigated in Merauke, a town located in the southeastern corner of Papua, by the Indonesian Ministry of Health and the U.S. Naval Medical Research Unit No. 2. Principal case criteria of hemorrhagic disease provided for a study enrollment of 15 clinically acute and 37 convalescing subjects. Additionally, 32 comparable age/sex controls were selected from neighboring households. Laboratory diagnosis involved three testing methodologies: virus isolation by cell culture, a reverse transcriptase–polymerase chain reaction (RT-PCR) assay, and serologic assays. Antibody (IgM) to dengue virus was detected in 27% of the acute clinical cases, 30% of the convalescing cases, and only 3% of the matched controls. Dengue 3 was the only viral serotype detected from acute serum samples by the RT-PCR. The mean ± SD age of the acute and convalescing cases was 7.8 ± 5.4 years. Overall hospital records accounted for 172 suspected outbreak cases, all urban residents of Merauke with no recent travel history outside the area. The estimated outbreak-associated case fatality rate among all suspected dengue cases was 1.2%. A seven-year retrospective review of hospital records in Merauke showed negligible disease reporting involving hemorrhagic disease prior to the outbreak.

Dengue fever and scrub typhus are common infections in Asia that often present as acute febrile illness of unclear etiology. We prospectively evaluated febrile adults presenting to the outpatient department of a hospital in northern Thailand to attempt to identify distinguishing characteristics between the two infections. Fifty-four patients were infected with scrub typhus and 35 were infected with dengue virus. Dengue virus infection was associated with hemorrhagic manifestations, particularly bleeding from the gums, which was reported by 27% of the dengue patients, but by none of the scrub typhus patients (P < 0.001, by Fisher’s exact test). A low platelet count (< 140,000/mm3) and low white blood cell count (< 5,000/mm3) were strongly associated with dengue infections: odds ratio = 26.3 (95% confidence interval [CI] = 7.4–93.2) for platelet count and 8.2 (95% CI = 2.6–25.5) for leukocyte count. Prospective evaluations of the usefulness of these simple criteria to differentiate scrub typhus from dengue infection are needed in other areas, particularly where rapid confirmatory diagnostic tests are not available.

Dengue serotype 2 (DEN-2) viruses with the potential to cause dengue hemorrhagic fever have been shown to belong to the Southeast (SE) Asian genotype. These viruses appear to be rapidly displacing the American genotype of DEN-2 in the Western Hemisphere. To determine whether distinct genotypes of DEN-2 virus are better adapted to mosquito transmission, we classified 15 viral strains of DEN-2 phylogenetically and compared their ability to infect and disseminate in different populations of Aedes aegypti mosquitoes. Envelope gene nucleotide sequence analysis confirmed that six strains belonged to the American genotype and nine strains were of the SE Asian genotype. The overall rate of disseminated infection in mosquitoes from Texas was 27% for the SE Asian genotype versus 9% for the American genotype. This pattern of infection was similar in another population of mosquitoes sampled from southern Mexico (30% versus 13%). Together, these findings suggest that Ae. aegypti tends to be more susceptible to infection by DEN-2 viruses of the SE Asian genotype than to those of the American genotype, and this may have epidemiologic implications.

Measles is a major cause of mortality in complex emergencies. Both high vaccination coverage and vaccine efficacy are required to prevent major epidemics of measles in such situations. Evaluation of field vaccine efficacy is a critical but underutilized component of program monitoring in emergencies, and is particularly important in rural areas where the integrity of the cold chain is difficult to guarantee. In July 2000, we evaluated the field vaccine efficacy for measles vaccination by comparing the incidence of cases in vaccinated and unvaccinated groups during a two-stage cluster survey of 563 children in Ethiopia. Approximately 30% of the measles cases occurred in vaccinated children. Estimated field vaccine efficacy for measles was 66.9% in children 9–36 months old. The finding of a field vaccine efficacy for measles less than 80% warrants formal assessment of measles vaccine efficacy, particularly in famine emergencies where measles is associated with a high case fatality rate.

A total of 1,345 Ixodes persulcatus ticks collected from northeastern China were investigated for the presence of Anaplasma phagocytophila and Borrelia burgdorferi by a nested polymerase chain reaction (PCR). Sixty-two (4.6%) ticks were positive for A. phagocytophila and 454 (33.8%) were positive for B. burgdorferi. Seven (0.5%) were coinfected with both agents. Sequence analysis of 919-basepair PCR amplicons revealed three types of A. phagocytophila. Type 1 was identical to the published sequences of A. phagocytophilas responsible for human granulocytic ehrlichiosis (HGE). The other two variants differed from the HGE agent sequence at one and four positions, respectively. These findings imply that infection with A. phagocytophila poses a potential health threat to both humans and animals in northeastern China, and that ehrlichiosis should be considered in the differential diagnosis of febrile patients with a history of tick bite, particularly when clinical manifestations are atypical for Lyme disease.

We have identified for the first time Rickettsia africae, and the ticks that harbored them, in Kenya. A total of 5,325 ticks were collected from vegetation, livestock, and wild animals during two field trips to southwestern Kenya. Most were immature forms (85.2%) belonging to the genera Amblyomma or Rhipicephalus. The adults also included representatives from the genus Boophilus. Ticks were assessed for rickettsial DNA by a polymerase chain reaction (PCR) using primers for the spotted fever group (SFG)–specific rickettsial outer membrane protein A (rompA) gene, and positive amplicons were sequenced. While none of the immature ticks tested positive by PCR, 15.8% of the adult Amblyomma variegatum and less than 1% of the Rhipicephalus spp. were SFG positive. Sequences of amplified products were identified as R. africae. These findings extend the known range of R. africae.

The purpose of this study was to determine the prevalence of IgM and IgG antibodies against Bartonella henselae and B. quintana in a healthy Greek population using a commercially available immunofluorescent test (Focus test). Five hundred healthy individuals were divided by sex into four age groups and three groups according to contact with cats. IgM antibodies were not detected in any of the subjects examined, while 99 (19.8%) and 75 (15%) were IgG seropositive to B. henselae and to B. quintana, respectively. No statistical difference in the seropositivity was observed among these groups. The IgG antibody titers ranged from 1/64 to 1/256 for B. henselae and from 1/64 to 1/512 for B. quintana. A high percentage (12.4%) of cross-reactivity between the two species was observed. Our data show that the prevalence of both Bartonella species in Greece is high. However, low IgG antibody levels are not sufficient evidence of active infection.

While a number of microbial infections induce a transient burst in viral load in individuals infected with human immunodeficiency virus-1 (HIV-1), a recent study has suggested that scrub typhus may suppress HIV-1 infection. We investigated the effects of Orientia tsutsugamushi on HIV-1 infection. In vitro HIV-1 infection experiments were conducted using peripheral blood mononuclear cells (PBMC) acutely infected with R5 and X4 HIV-1 or PBMC derived from patients receiving highly active antiretroviral therapy (HAART) whose plasma viral load was undetectable. Stimulation of PBMC with O. tsutsugamushi induced production of proinflammatory cytokines and β-chemokines, and markedly down-regulated expression of CCR5. Although pretreatment with O. tsutsugamushi rendered PBMC resistant to R5 HIV-1, it otherwise enhanced HIV-1 replication. Stimulation by O. tsutsugamushi induced HIV-1 replication in PBMC from patients receiving HAART. These findings suggest that scrub typhus does not necessarily suppress HIV-1 infection and does have potential to enhance HIV-1 replication.

Dermatologic manifestations are quite common in patients with adult T cell leukemia/lymphoma and myelopathy/tropical spastic paraparesis associated with infection with human T cell lymphotropic virus type-1 (HTLV-1). In this study, we evaluated the dermatologic lesions of eligible blood donors in the state of Minas Gerais in Brazil who were seropositive but asymptomatic for infection with HTLV-1. The study population was composed of 128 HTLV-1-seropositive individuals and 108 seronegative controls. All individuals underwent a dermatologic evaluation. Biopsy specimens were obtained from abnormal and normal skin samples of seropositive individuals in an attempt to detect HTLV-1 in tissue samples by a polymerase chain reaction. Dermatologic alterations were observed more frequently in the seropositive group (adjusted odds ratio [OR] = 8.77, 95% confidence interval [CI] = 4.11–18.71). The most common skin diseases were dermatophytoses (adjusted OR = 3.32, 95% CI = 1.50–7.35), seborrheic dermatitis (OR = 3.53, 95% CI = 0.67–24.66), and acquired ichthyosis (P = 0.001). Virus was detected more frequently in abnormal skin samples. Dermatologic lesions probably related to HTLV-1 infection were diagnosed in eligible blood donors who were infected with this virus, who were previously considered to be asymptomatic carriers of HTLV-1.

The twenty-seventh documented case of human Mesocestoides infection, which corresponds to the seventh documented case in the United States, is reported. The case had its origin in Alexandria, Louisiana in the summer of 1998. The patient was a 19-month-old boy. The strobila consisted of 35 proglottids that included mature as well as gravid segments containing a ventral genital pore and a parauterine organ. After a detailed microscopic examination, the tapeworm was identified as belonging to the genus Mesocestoides. Mesocestoides variabilis is the probable species responsible for the infection, since the six cases previously reported in the United States were identified as this species. After the treatment with a single dose of praziquantel (10 mg/kg), the tapeworm segments were no longer detectable in the child’s feces. A food-borne origin of this infection derived from culinary customs of the Acadian and Creole communities in Louisiana is proposed.

In October 2000, 71,187 persons were treated for lymphatic filariasis using albendazole and diethylcar-bamazine (DEC) or DEC alone in Leogane, Haiti. We documented the frequency of adverse reactions, severity and cost of treatment. Adverse reactions were classified as minor, moderate, or severe. Overall, 24% (17,421) of the treated persons reported one or more adverse reactions. There were 15,916 (91%) minor and 1502 (9%) moderate adverse reaction reports. Men outnumbered women 2:1 in reporting moderate problems. Three patients, representing roughly one in 25,000 persons treated, were hospitalized with severe adverse reactions judged to be treatment-associated by physician review. The cost per person treated for adverse reactions was more than twice the cost per person treated for lymphatic filariasis ($1.60 versus $0.71). Severe adverse reactions to lymphatic filariasis treatment using DEC with or without albendazole are uncommon. Minor and moderate reactions are more commonly reported and their management represents a challenge to lymphatic filariasis elimination programs.

The polymerase chain reaction (PCR) is a potentially interesting diagnostic tool for detecting congenital Trypanosoma cruzi infection at birth. We have compared the sensitivity and capacity of a group of T. cruzi PCR primers in detecting the complete spectrum of known T. cruzi lineages, and to improve and simplify the detection of infection in neonatal blood. We found that the two primers, Tcz1/Tcz2 and Diaz1/Diaz2, which target the 195-basepair satellite repeat, detected all parasitic lineages with the same sensitivity. However, the intensity of the amplicon was somewhat higher with Tcz1/Tcz2. For other tested primers (nuclear DNA primers BP1/BP2, O1/O2, Pon1/Pon2, and Tca1/Tca2 and kinetoplast DNA primers S35′/S36′ and 121/122), either the intensity of amplicons varied according to T. cruzi lineages or the PCR assay was less sensitive. The use of the Tcz1/Tcz2 primers, which target a tandem repetitive sequence, requires a careful determination of the appropriate amount of Taq polymerase to avoid the formation of smears and multiple amplicon bands. The Tcz1/Tcz2 primers resulted in an intense 200-basepair amplicon with DNA extracted from blood equivalent to 0.02 parasites per assay when used with a simple DNA extraction method and of a low amount of Taq polymerase from a standard PCR kit. To better assess such PCR protocol, we assayed 311 samples of neonatal blood previously tested by parasitologic methods. The reliability of our PCR test was demonstrated, since all the 18 blood samples from newborns with congenital T. cruzi infection were positive, whereas the remaining samples (30 from control newborns of uninfected mothers and 262 of 263 from babies born to infected mothers) were negative. Since our PCR method is simple, reliable, robust, and inexpensive, it appears suitable for the detection of T. cruzi infection in neonatal blood, even in laboratories that are not equipped for performing the PCR.

Malaria and leptospirosis are both common in the tropics. Simultaneous infections are possible, although not previously reported. We report two cases of malaria from an area of Thailand on the Thailand-Myanmar border with compelling serologic evidence of simultaneous acute leptospirosis. One was a case of infection with Plasmodium falciparum with acute and convalescent microscopic agglutination test (MAT) titers for Leptospira serovar icterohaemorrhagiae of 1:200 and 1:1,600, respectively. The other was a case of infection with P. vivax that seroconverted to a titer of 1:3,200 for Leptospira serovar bataviae. Additionally, there were five probable cases of leptospirosis with patent malaria parasitemia (three P. falciparum and two P. vivax) detected. Management of dual infections is complicated by their similar clinical presentations, and because the confirmatory diagnosis of malaria is readily available as opposed to that of leptospirosis. Treatment focusing on malaria mono-infections instead of dual infections could result in a delay of specific therapy for leptospirosis and possible consequences of serious complications.

A total of 70 Plasmodium falciparum isolates were tested in vitro against pyrimethamine (PYR), tri-methoprim (TRM), sulfadoxine (SDX), and sulfamethoxazole (SMX), and their dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genotypes were determined. dhfr genotypes correlated with PYR and TRM drug responses (r = 0.93 and 0.85). Isolates with wild-type alleles showed mean half inhibitory concentrations (IC50 ± SD) of 0.10 ± 0.10 and 0.15 ± 0.06 μg/100 μl for PYR and TRM. Parasites with mutations at codons 108 and 51 alone or combined with codon 59 have IC50 of 11.46 ± 0.86 (PYR) and 2.90 ± 0.59 μg/100 μl (TRM). For both drugs, the differences in the mean IC50 between wild and mutant parasites were statistically significant (P < 0.001). Isolates with mixed wild and mutant alleles showed an intermediate level of susceptibility. Our data show partial cross-resistance between PYR/TRM and SDX/SMX (r = 0.85 and 0.65). Correlation was not observed between different dhps genotypes and the in vitro outcome to SDX and SMX (r = 0.30 and 0.34). The lack of correlation could be due to folates and para-aminobenzoic acid in the RPMI medium and the serum used to supplement the cultures.

Previous studies in animal models have revealed an association between interferon-γ (IFN- γ), produced by CD8+ T cells and irradiated sporozoite-induced sterile immunity. To determine whether IFN-γ can serve as a marker of pre-erythrocytic protective immunity in individuals naturally exposed to malaria, we characterized IFN-γ and lymphocyte proliferative responses to previously defined CD8+ cytotoxic T lymphocyte (CTL) epitopes from six pre-erythrocytic stage antigens in 107 children six months to two years old from a community-based birth cohort in western Kenya. We found that IFN- γ positive responders had higher hemoglobin (Hb) levels and significantly reduced prevalence of severe malarial anemia one month after the test compared with IFN- γ non-responders, suggesting that IFN- γ immune responses to these pre-erythrocytic antigens were associated with protection against malarial anemia. Children who responded by lymphocyte proliferation had a significantly longer time to first documented malaria parasitemia after birth; however, there was no correlation between the presence of lymphocyte proliferative response and higher Hb levels. We propose that IFN- γ production could be used as a potential marker of protective immunity against malaria associated anemia in young children living in malaria holoendemic areas.

From 1992–1999, we have assessed the therapeutic efficacy of three malaria treatment regimens (chloroquine 25 mg/kg over three days, pyrimethamine/sulfadoxine 1.25/25 mg/kg in one dose, and quinine 25–30 mg/kg daily in three oral doses over a four-, five-, or seven-day period) in 1,189 children under age 10 at Malabo Regional Hospital in Equatorial Guinea. Of those children, 958 were followed up clinically and parasitologically for 14 days. With chloroquine, the failure rate varied from 55% in 1996 to 40% in 1999; the early treatment failure rate increased progressively over the years, from 6% in 1992 to 30% in 1999. With pyrimethamine/sulfadoxine, the failure rate varied from 0% in 1996 to 16% in 1995. The short quinine treatment regimens used in 1992 and 1993 (4 and 5 days, respectively) resulted in significantly higher failure rates (19% and 22%, respectively) than the 7d regimen (3–5.5%). We conclude that: a) failure rates for chloroquine are in the change period (>25%), and urgent action is needed; b) pyrimethamine/ sulfadoxine failure rates are in the alert period (6–15%), and surveillance must be continued; and c) quinine failure rates are in the grace period (<6%), so quinine can be recommended.

Parasitologic and entomologic cross-sectional surveys were carried out during an outbreak of malaria between December 1998 and August 2000 in forest villages near the Mohkhed Primary Health Center in the Chhindwara District of Madhya Pradesh in central India. In December 1998, surveys showed that more than 70% of the fever cases had malaria, with 87% of the malaria caused by Plasmodium falciparum. The rate of enlarged spleens in children was 74.5%. In November 1999, 58% of the inhabitants were infected with malaria, with 80% of these cases caused by P. falciparum. Chloroquine resistance was seen in 23% of the cases. Anopheles culicifacies was the dominant mosquito species in all surveys (70–85%) and was resistant to DDT. The results indicate that the incidence of malaria in Chhindwara has increased gradually from 0.31 per 1,000 in 1990 to 6.75 per 1,000 in 2000. Improved access to treatment facilities, combination therapy, and vector control using an effective insecticide appear to be the most promising methods for controlling malaria in this region.

In the Amazon Basin of Peru, more than 50% of patients with uncomplicated Plasmodium falciparum malaria fail to respond to treatment with chloroquine or sulfadoxine-pyrimethamine. To assist the National Malaria Control Program in identifying an alternative first-line therapy for this region, we conducted a trial of the safety and efficacy of mefloquine (MQ) compared with mefloquine-artesunate (MQ-AS) combination therapy. Patients with uncomplicated P. falciparum infections between the ages of 5 and 50 years were randomly assigned to be treated with either MQ (15 mg/kg in a single oral dose) or MQ (15 mg/kg) plus AS (4 mg/kg/day for three days). A total of 98 patients were enrolled and followed for 28 days. None of the 47 patients who received MQ alone or the 51 patients who received MQ-AS combination therapy had recurrences of parasitemia during the 28-day follow-up period. Asexual parasite densities decreased significantly more rapidly and the proportion of patients with gametocytes was significantly lower on days 3–21 in the MQ-AS group than in patients treated with MQ alone. All patients tolerated the medication well. Based on the results of this study and with the objective of slowing the development of resistance, the Peruvian Ministry of Health has decided to revise its malaria treatment policy and recommend combination therapy with MQ-AS as the new first-line treatment of uncomplicated P. falciparum malaria in the Amazon region.

Using two polymorphic genetic markers, the merozoite surface protein-3α (MSP-3α) and the circumsporozoite protein (CSP), we investigated the population diversity of Plasmodium vivax in Mae Sod, Thailand from April 2000 through June 2001. Genotyping the parasites isolated from 90 malaria patients attending two local clinics for the dimorphic CSP gene revealed that the majority of the parasites (77%) were the VK210 type. Genotyping the MSP3-α gene indicated that P. vivax populations exhibited an equally high level of polymorphism as those from Papua New Guinea, a hyperendemic region. Based on the length of polymerase chain reaction products, three major types of the MSP-3α locus were distinguished, with frequencies of 74.8%, 18.7%, and 6.5%, respectively. The 13 alleles distinguished by restriction fragment length polymorphism analysis did not show a significant seasonal variation in frequency. Genotyping the MSP-3α and CSP genes showed that 19.3% and 25.6% of the patients had multiple infections, respectively, and the combined rate was 35.6%. Comparisons of MSP-3α sequences from nine clones further confirmed the high level of genetic diversity of the parasite and also suggested that geographic isolation may exist. These results strongly indicate that P. vivax populations are highly diverse and multiple clonal infections are common in this malaria-hypoendemic region of Thailand.

An open randomized controlled study of mefloquine-artesunate and mefloquine-primaquine for the treatment of uncomplicated Plasmodium falciparum malaria was carried out in Kanchanaburi in the Saiyok District in western Thailand. Weekly parasite counts from thick and thin blood films were done for six weeks. The gametocyte carriage rate was calculated and compared between the two treatment groups. Gametocytes on presentation, recrudescent infection, and reinfection were the significant factors associated with subsequent development of gametocytemia. It is the increased propensity of recrudescent infections to produce gametocytes that drives drug resistance. The results of this study confirmed that the complete eradication of asexual forms of P. falciparum by effective antimalarial treatment, but not by combination treatment with primaquine, is the most effective means to prevent subsequent gametocytemia.

The OptiMAL test detects both Plasmodium falciparum and P. vivax malaria infections. In this study, we evaluated the performance of the OptiMAL test at the Basic Health Units (BHUs) and the District Health Quarter (DHQ) Center in rural villages of Punjab, Pakistan that provide minimal health services. Two sets of blood specimens obtained from 930 suspected malaria patients attending these BHUs were tested at BHUs and the DHQ Center by microscopy and the OptiMAL test. At the BHUs, 231 (25%) of the patients were positive by microscopy and 278 (30%) patients tested positive by the OptiMAL test. At the DHQ Center, microscopic analysis of a second set of specimens from the same patients confirmed the malaria infection in 386 (42%) patients and the OptiMAL test result was positive in 300 (32%) patients. To determine the performance of OptiMAL test at the BHUs and the DHQ Center, all data were compared with microscopy results obtained at the DHQ Center. The OptiMAL test results for P. falciparum at the BHUs were comparable to those of the OptiMAL test at the DHQ Center. However, the sensitivity, positive predictive value (PPV), and negative predictive value (NPV) of the OptiMAL test were considerably lower for P. vivax infections than for P. falciparum infections, irrespective of whether the test was performed at the BHUs or at the DHQ Center (P. falciparum: sensitivity = 78–85%, PPV = 89–97%, NPV = 96–98%; P. vivax: sensitivity = 61–76%, PPV = 88–95%, NPV = 90–93%). The OptiMAL test also detected a number of false-positive and false-negative results at both the BHUs and the DHQ Center. The false-positive results ranged from 1% to 2%; however, the number of false-negative results was much higher (BHUs: P. falciparum = 22%, P. vivax = 39%; DHQ Center: P. falciparum = 15%, P. vivax = 24%). In conclusion, these results, when combined with other advantages of the OptiMAL test, suggest that this test can be used by relatively inexperienced persons to diagnose malaria infection in rural areas where facilities for microscopy are not available.