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Background Minimally invasive tissue sampling (MITS) is an alternative to complete autopsy for determining causes of death. Multiplex molecular testing performed on MITS specimens poses challenges of interpretation, due to high sensitivity and indiscriminate detection of pathogenic, commensal, or contaminating microorganisms. Methods MITS was performed on 20 deceased children with respiratory illness, at 10 timepoints up to 88 hours postmortem. Samples were evaluated by multiplex molecular testing on fresh tissues by TaqMan® Array Card (TAC) and by histopathology, special stains, immunohistochemistry (IHC), and molecular testing (PCR) on formalin-fixed, paraffin-embedded (FFPE) tissues. Results were correlated to determine overall pathologic and etiologic diagnoses and to guide interpretation of TAC results. Results MITS specimens collected up to 3 days postmortem were adequate for histopathologic evaluation and testing. Seven different etiologic agents were detected by TAC in 10 cases. Three cases had etiologic agents detected by FFPE or other methods and not TAC; 2 were agents not present on TAC, and 2 were streptococci that may have been species other than those present on TAC. Result agreement was 43% for TAC and IHC or PCR, and 69% for IHC and PCR. Extraneous TAC results were common, especially when aspiration was present. Conclusions TAC can be performed on MITS up to 3 days after death with refrigeration and provides a sensitive method for detection of pathogens but requires careful interpretation in the context of clinicoepidemiologic and histopathologic findings. Interpretation of all diagnostic tests in aggregate to establish overall case diagnoses maximizes the utility of TAC in MITS.

High case counts after the Gamma (P. 1) variant of severe acute respiratory syndrome coronavirus 2 emerged in Brazil raised concerns that previously infected persons might become reinfected. Investigation of a cluster of coronavirus disease cases in Parintins, in the Brazilian Amazon, suggested household transmission but did not identify high rates of reinfection.

Background Diagnosis of invasive fungal infections from formalin-fixed paraffin-embedded (FFPE) tissues by PCR amplification is a developing technology. One of the difficulties of establishing a validated protocol for this testing is that the gold standard, culture, is much less sensitive than the test being validated. Objectives To validate FFPE PCR as a refence laboratory identification methodology in the absence of abundant gold standard specimens. Methods In this validation, PCR from FFPE tissue was compared to other diagnostic methods for genus/species identification. Four different groups of correlative data from FFPE tissues were used to validate this procedure. Thirteen specimens had culture or serology results and FFPE PCR results, 49 specimens had both immunohistochemistry (IHC) identification and FFPE PCR results, 118 specimens had histological evidence of fungal elements, 64 of which also had FFPE PCR results, and 36 fungal mock tissues or fungal negative tissues were used. Results The sensitivity determined from the tissues with positive fungal histopathology was 54%. The specificity of the cases for which there were both culture and FFPE PCR results was 100%. For the correlation with IHC, the specificity was 98%. For the mock tissues and fungal-negative tissues the calculated analytical sensitivity was 94%, specificity was 95% and accuracy was 94%. Conclusions By uniquely combining various data sources this study provides a comprehensive framework for how validation can be achieved in the absence of a gold standard and outlines the excellent performance of PCR from FFPE tissue, despite relatively the low sensitivity when compared to histopathology.

An ongoing pandemic of coronavirus disease (COVID-19) is caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Characterization of the histopathology and cellular localization of SARS-CoV-2 in the tissues of patients with fatal COVID-19 is critical to further understand its pathogenesis and transmission and for public health prevention measures. We report clinicopathologic, immunohistochemical, and electron microscopic findings in tissues from 8 fatal laboratory-confirmed cases of SARS-CoV-2 infection in the United States. All cases except 1 were in residents of long-term care facilities. In these patients, SARS-CoV-2 infected epithelium of the upper and lower airways with diffuse alveolar damage as the predominant pulmonary pathology. SARS-CoV-2 was detectable by immunohistochemistry and electron microscopy in conducting airways, pneumocytes, alveolar macrophages, and a hilar lymph node but was not identified in other extrapulmonary tissues. Respiratory viral co-infections were identified in 3 cases; 3 cases had evidence of bacterial co-infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes respiratory disease in mink similar to human COVID-19. We characterized the pathological findings in 72 mink from US farms with SARS-CoV-2 outbreaks, localized SARS-CoV-2 and its host cellular receptor angiotensin-converting enzyme 2 (ACE2) in mink respiratory tissues, and evaluated the utility of various test methods and specimens for SARS-CoV-2 detection in necropsy tissues. Of SARS-CoV-2-positive animals found dead, 74% had bronchiolitis and diffuse alveolar damage (DAD). Of euthanized SARS-CoV-2-positive animals, 72% had only mild interstitial pneumonia or minimal nonspecific lung changes (congestion, edema, macrophages); similar findings were seen in SARS-CoV-2-negative animals. Suppurative rhinitis, lymphocytic perivascular inflammation in the lungs, and lymphocytic infiltrates in other tissues were common in both SARS-CoV-2-positive and SARS-CoV-2-negative animals. In formalin-fixed paraffin-embedded (FFPE) upper respiratory tract (URT) specimens, conventional reverse transcription-polymerase chain reaction (cRT-PCR) was more sensitive than in situ hybridization (ISH) or immunohistochemistry (IHC) for detection of SARS-CoV-2. FFPE lung specimens yielded less detection of virus than FFPE URT specimens by all test methods. By IHC and ISH, virus localized extensively to epithelial cells in the nasal turbinates, and prominently within intact epithelium; olfactory mucosa was mostly spared. The SARS-CoV-2 receptor ACE2 was extensively detected by IHC within turbinate epithelium, with decreased detection in lower respiratory tract epithelium and alveolar macrophages. This study expands on the knowledge of the pathology and pathogenesis of natural SARS-CoV-2 infection in mink and supports their further investigation as a potential animal model of SARS-CoV-2 infection in humans.

Through active surveillance and contact tracing from outpatients, we aimed to identify and characterize SARS-CoV-2 variants circulating in Porto Velho-Rondônia, a city in the Brazilian Amazon. As part of a prospective cohort, we gathered information from 2,506 individuals among COVID-19 patients and household contacts. Epidemiological data, nasopharyngeal swabs, and blood samples were collected from all participants. Nasopharyngeal swabs were tested for antigen rapid diagnostic test and reverse transcription-polymerase chain reaction (RT-PCR) followed by genomic sequencing. Blood samples underwent ELISA testing for IgA, IgG, and IgM antibody levels. From 757 specimens sequenced, three were identified as Mu variant, none of the individuals carrying this variant had a travel history in the previous 15 days before diagnosis. One case was asymptomatic and two presented mild symptoms. Two infected individuals from different households caring viruses with additional amino acid substitutions ORF7a P45L and ORF1a T1055A compared to the Mu virus reference sequence. One patient presented IgG levels. Our results highlight that genomic surveillance for SARS-CoV-2 variants can assist in detecting the emergency of SARS-CoV-2 variants in the community, before its identification in other parts of the country.

Ebola virus disease (EVD) is associated with elevated cytokine levels, and hypercytokinemia is more pronounced in fatal cases. This type of hyperinflammatory state is reminiscent of 2 rheumatologic disorders known as macrophage activation syndrome and hemophagocytic lymphohistiocytosis, which are characterized by macrophage and T-cell activation. An evaluation of 2 cohorts of patients with EVD revealed that a marker of macrophage activation (sCD163) but not T-cell activation (sCD25) was associated with severe and fatal EVD. Furthermore, substantial immunoreactivity of host tissues to a CD163-specific antibody, predominantly in areas of extensive immunostaining for Ebola virus antigens, was observed in fatal cases. These data suggest that host macrophage activation contributes to EVD pathogenesis and that directed antiinflammatory therapies could be beneficial in the treatment of EVD.

Summary Background Zika virus is an arthropod-borne virus that is a member of the family Flaviviridae transmitted mainly by mosquitoes of the genus Aedes . Although usually asymptomatic, infection can result in a mild and self-limiting illness characterised by fever, rash, arthralgia, and conjunctivitis. An increase in the number of children born with microcephaly was noted in 2015 in regions of Brazil with high transmission of Zika virus. More recently, evidence has been accumulating supporting a link between Zika virus and microcephaly. Here, we describe findings from three fatal cases and two spontaneous abortions associated with Zika virus infection. Methods In this case series, formalin-fixed paraffin-embedded tissue samples from five cases, including two newborn babies with microcephaly and severe arthrogryposis who died shortly after birth, one 2-month-old baby, and two placentas from spontaneous abortions, from Brazil were submitted to the Infectious Diseases Pathology Branch at the US Centers for Disease Control and Prevention (Atlanta, GA, USA) between December, 2015, and March, 2016. Specimens were assessed by histopathological examination, immunohistochemical assays using a mouse anti-Zika virus antibody, and RT-PCR assays targeting the NS5 and envelope genes. Amplicons of RT-PCR positive cases were sequenced for characterisation of strains. Findings Viral antigens were localised to glial cells and neurons and associated with microcalcifications in all three fatal cases with microcephaly. Antigens were also seen in chorionic villi of one of the first trimester placentas. Tissues from all five cases were positive for Zika virus RNA by RT-PCR, and sequence analyses showed highest identities with Zika virus strains isolated from Brazil during 2015. Interpretation These findings provide strong evidence of a link between Zika virus infection and different congenital central nervous system malformations, including microcephaly as well as arthrogryposis and spontaneous abortions. Funding None.

The Caribbean island of Hispaniola is targeted for malaria elimination. Currently, this is the only island with ongoing transmission of malaria in the Caribbean. In 2015, six patients from Puerto Rico and one from Massachusetts, who traveled to Punta Cana, Dominican Republic, were confirmed to be infected with Plasmodium falciparum. Additional molecular analysis was performed at the Centers for Disease Control and Prevention to characterize the drug-resistant alleles and Plasmodium population genetic markers. All specimens carried wildtype genotypes for chloroquine, sulfadoxine-pyrimethamine, and artemisinin resistance genetic markers. A mutation in codon 184 (Y/F) of Pfmdr-1 gene was observed in all samples and they shared an identical genetic lineage as determined by microsatellite analysis. This genetic profile was similar to one previously reported from Hispaniola suggesting that a clonal P. falciparum residual parasite population present in Punta Cana is the source population for these imported malaria cases.

This study was supported by the Amazon Malaria Initiative, which is funded by the United States Agency for International Development (USAID) Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Ananindeua, PA, Brasil. Centers for Disease Control and Prevention. Centers for Global Health. Division of Parasitic Diseases and Malaria. Malaria Branch. Atlanta, GA, United States of America. Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Ananindeua, PA, Brasil. Centers for Disease Control and Prevention. Centers for Global Health. Division of Parasitic Diseases and Malaria. Malaria Branch. Atlanta, GA, United States of America. Secretaria Estadual de Sa?de do Acre. Hemon?cleo Cruzeiro do Sul. Cruzeiro do Sul, AC, Brasil. Secretaria Estadual de Sa?de do Acre. Hemon?cleo Cruzeiro do Sul. Cruzeiro do Sul, AC, Brasil. Secretaria Estadual de Sa?de do Acre. Hemon?cleo Cruzeiro do Sul. Cruzeiro do Sul, AC, Brasil. Coordena??o Geral do Programa Nacional de Controle da Mal?ria e Doen?as Transmitidas pelo Aedes. Bras?lia, DF, Brasil. Coordena??o Geral do Programa Nacional de Controle da Mal?ria e Doen?as Transmitidas pelo Aedes. Bras?lia, DF, Brasil. Centers for Disease Control and Prevention. Centers for Global Health. Division of Parasitic Diseases and Malaria. Malaria Branch. Atlanta, GA, United States of America. Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Ananindeua, PA, Brasil. Centers for Disease Control and Prevention. Centers for Global Health. Division of Parasitic Diseases and Malaria. Malaria Branch. Atlanta, GA, United States of America. Conventional molecular methods, such as nested polymerase chain reaction (PCR), are very sensitive for detection of malaria parasites, but require advanced laboratory equipment and trained personnel. Real-time loop-mediated isothermal amplification (RealAmp), a loopmediated isothermal amplification-based molecular tool (LAMP), facilitates rapid target amplification at a single temperature setting, reducing the need for sophisticated equipment. We evaluated the performance of a field-adapted RealAmp assay for malaria diagnosis in Cruzeiro do Sul, Acre State, Brazil, a remote area in Brazil with limited laboratory capabilities. We enrolled 1,000 patients with fever (axillary temperature 37.5 C) or history of fever in last 24 h presenting for malaria diagnosis from February through June 2015. DNA was extracted from dried blood spots using a boil and spin method (heat treatment) at the sample processing site, and also using commercial kits at a Brazilian national reference laboratory. RealAmp was performed for Plasmodium genus, P. falciparum, and P. vivax identification. In addition, Giemsa-stained blood smears were prepared and examined by two independent well-trained study microscopists. A combination of Real-time PCR and nested PCR was used as reference test. The sensitivity and specificity of RealAmp in the field site laboratory were 94.1% (95% confidence interval [CI]: 90.1?96.8) and 83.9% (95% CI: 81.1?86.4), respectively. The sensitivity and specificity of local microscopy were 87.7% (95% CI: 82.6? 91.7) and 98.9% (95% CI: 97.8?99.4), respectively, while study microscopy showed sensitivity of 96.4% (95% CI: 93.0?98.4) and specificity of 98.2% (95% CI: 97.0?99.0). None of the three tests detected 20 P. falciparum and P. vivax mixed infections identified by the reference test. Our findings highlight that it is possible to implement simple molecular tests in facilities with limited resources such as Cruzeiro do Sul in Brazil. RealAmp sensitivity was similar to that of microscopy performed by skilled professionals; both RealAmp and study microscopy performed poorly in detection of mixed infection. Attempts to develop and evaluate simpler molecular tools should continue, especially for the detection of malaria infection in remote areas.

Malaria has always been an important public health problem in Brazil. The early history of Brazilian malaria and its control was powered by colonisation by Europeans and the forced relocation of Africans as slaves. Internal migration brought malaria to many regions in Brazil where, given suitable Anopheles mosquito vectors, it thrived. Almost from the start, officials recognised the problem malaria presented to economic development, but early control efforts were hampered by still developing public health control and ignorance of the underlying biology and ecology of malaria. Multiple regional and national malaria control efforts have been attempted with varying success. At present, the Amazon Basin accounts for 99% of Brazil's reported malaria cases with regional increases in incidence often associated with large scale public works or migration. Here, we provide an exhaustive summary of primary literature in English, Spanish and Portuguese regarding Brazilian malaria control. Our goal was not to interpret the history of Brazilian malaria control from a particular political or theoretical perspective, but rather to provide a straightforward, chronological narrative of the events that have transpired in Brazil over the past 200 years and identify common themes.

The rapid emergence of drug-resistant malaria parasites during the course of an infection remains a major challenge for providing accurate treatment guidelines. This is particularly important in cases of malaria treatment failure. Using a previously well-characterized case of malaria treatment failure, we show the utility of using next-generation sequencing for early detection of the rise and selection of a previously reported atovaquone-proguanil (malarone) drug resistance-associated mutation.

Background Ebola virus disease (EVD) is associated with elevated cytokine levels that are more pronounced in fatal cases. This type of hyperinflammatory state is reminiscent of other inflammatory disorders, such as macrophage activation syndrome (MAS) or hemophagocytic lymphohistiocytosis (HLH). These are both part of a spectrum of rheumatologic phenomena characterized by both macrophage and T-cell activation. These disorders can be secondary to infection, malignancy, underlying rheumatologic disorder, or, paradoxically, immune deficiency. Methods Two cohorts of EVD patients were evaluated with respect to common plasma markers of HLH/MAS. Immunohistochemistry was used to evaluate tissue macrophages and viral antigens in various tissues from fatal cases of EVD. Results Neither fibrinogen nor soluble IL-2 receptor were significantly different between fatal and nonfatal cases. However, elevated levels of triglycerides, ferritin and sCD163, a marker of macrophage activation were noted in patients with EVD and they correlated with disease severity and a fatal outcome. Furthermore, significant immunoreactivity for CD163 + cells in host tissues was observed in fatal cases, predominantly in areas of extensive immunostaining for EBOV antigens. Conclusion These data suggest that host macrophage activation contributes to EVD pathogenesis and that directed anti-inflammatory therapies could be beneficial in the treatment of EVD. Disclosures All authors: No reported disclosures.

Ebola virus disease (EVD) is associated with elevated cytokine levels, that are more pronounced in fatal cases. This type of hyperinflammatory state is reminiscent of other inflammatory disorders, such as macrophage activation syndrome (MAS) or hemophagocytic lymphohistiocytosis (HLH). These are both part of a spectrum of rheumatologic phenomena characterized by both macrophage and T cell activation. These disorders can be secondary to infection, malignancy, underlying rheumatologic disorder, or, paradoxically, immune deficiency. An evaluation of two cohorts of patients with EVD revealed that several of the common laboratory features of MAS and HLH are shared by EVD. Neither fibrinogen nor soluble IL-2 receptor were significantly different between fatal and non-fatal cases. However, elevated levels of triglycerides, ferritin and sCD163, a marker of macrophage activation were noted in patients with EVD and they correlated with disease severity and a fatal outcome. Furthermore, significant immunoreactivity for CD163 + cells in host tissues was observed in fatal cases, predominantly in areas of extensive immunostaining for EBOV antigens. These data suggest that host macrophage activation contributes to EVD pathogenesis and that directed anti-inflammatory therapies could be beneficial in the treatment of EVD.

On July 16 2015, the Puerto Rico Department of Health (PRDH) was notified of a case of malaria, diagnosed by a hospital parasitology laboratory in a student who had traveled to Punta Cana, Dominican Republic, during late June for a school-organized graduation trip. Malaria is a mosquito-borne parasitic infection, characterized by fever, shaking chills, headaches, muscle pains, nausea, general malaise, and vomiting (1). Malaria can be clinically difficult to distinguish from other acute febrile illnesses, and a definitive diagnosis requires demonstration of malaria parasites using microscopy or molecular diagnostic tests. The student's initial diagnosis on July 10 was suspected dengue virus infection. Puerto Rico eliminated local malaria transmission during the mid-1950s (2); however, reintroduction remains a risk because of the presence of a competent vector (Anopheles albimanus) and ease of travel to areas where the disease is endemic, including Hispaniola, the island shared by the Dominican Republic and Haiti, and the only island in the Caribbean with endemic malaria (3). During 2014, the Dominican Republic reported 496 confirmed malaria cases and four associated deaths; Haiti reported 17,662 confirmed cases and nine deaths (4). During 2000-2014, Puerto Rico reported a total of 35 imported malaria cases (range = 0-7 per year); three cases were imported from Hispaniola. During June-August 2015, eight confirmed malaria cases among travelers to the Dominican Republic were reported to CDC's National Malaria Surveillance System (CDC, unpublished data, 2015).

Zika virus is a mosquito-borne flavivirus that is related to dengue virus and transmitted primarily by Aedes aegypti mosquitoes, with humans acting as the principal amplifying host during outbreaks. Zika virus was first reported in Brazil in May 2015 (1). By February 9, 2016, local transmission of infection had been reported in 26 countries or territories in the Americas.* Infection is usually asymptomatic, and, when symptoms are present, typically results in mild and self-limited illness with symptoms including fever, rash, arthralgia, and conjunctivitis. However, a surge in the number of children born with microcephaly was noted in regions of Brazil with a high prevalence of suspected Zika virus disease cases. More than 4,700 suspected cases of microcephaly were reported from mid-2015 through January 2016, although additional investigations might eventually result in a revised lower number (2). In response, the Brazil Ministry of Health established a task force to further investigate possible connections between the virus and brain anomalies in infants (3).

Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Ananindeua, PA, Brasil. Centers for Disease Control and Prevention. Center for Global Health. Malaria Branch, Division of Parasitic Diseases and Malaria. Atlanta, GA, USA / Atlanta Research and Education Foundation. Decatur, GA, USA. Centers for Disease Control and Prevention. Center for Global Health. Malaria Branch, Division of Parasitic Diseases and Malaria. Atlanta, GA, USA / Atlanta Research and Education Foundation. Decatur, GA, USA. Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Ananindeua, PA, Brasil. Centers for Disease Control and Prevention. Center for Global Health. Malaria Branch, Division of Parasitic Diseases and Malaria. Atlanta, GA, USA. Centers for Disease Control and Prevention. Center for Global Health. Malaria Branch, Division of Parasitic Diseases and Malaria. Atlanta, GA, USA. Centers for Disease Control and Prevention. Center for Global Health. Malaria Branch, Division of Parasitic Diseases and Malaria. Atlanta, GA, USA. Centers for Disease Control and Prevention. Center for Global Health. Malaria Branch, Division of Parasitic Diseases and Malaria. Atlanta, GA, USA. Pan American Health Organization. La Paz, BO. Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Ananindeua, PA, Brasil. Universidade da S?o Paulo. Instituto de Ci?ncias Biom?dicas 5. S?o Paulo, SP, Brazil / Faculdade S?o Lucas. Porto Velho, RO, Brasil. Secretaria Estadual de Sa?de do Acre. Hemon?cleo Cruzeiro do Sul. Cruzeiro do Sul, AC, Brasil. Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Ananindeua, PA, Brasil. Centers for Disease Control and Prevention. Center for Global Health. Malaria Branch, Division of Parasitic Diseases and Malaria. Atlanta, GA, USA. Centers for Disease Control and Prevention. Center for Global Health. Malaria Branch, Division of Parasitic Diseases and Malaria. Atlanta, GA, USA. More than 80% of available malaria rapid diagnostic tests (RDTs) are based on the detection of histidine-rich protein-2 (PfHRP2) for diagnosis of Plasmodium falciparum malaria. Recent studies have shown the genes that code for this protein and its paralog, histidine-rich protein-3 (PfHRP3), are absent in parasites from the Peruvian Amazon Basin. Lack of PfHRP2 protein through deletion of the pfhrp2 gene leads to false-negative RDT results for P. falciparum. We have evaluated the extent of pfhrp2 and pfhrp3 gene deletions in a convenience sample of 198 isolates from six sites in three states across the Brazilian Amazon Basin (Acre, Rondonia and Para) and 25 isolates from two sites in Bolivia collected at different times between 2010 and 2012. Pfhrp2 and pfhrp3 gene and their flanking genes on chromosomes 7 and 13, respectively, were amplified from 198 blood specimens collected in Brazil. In Brazil, the isolates collected in Acre state, located in the western part of the Brazilian Amazon, had the highest percentage of deletions for pfhrp2 25 (31.2%) of 79, while among those collected in Rondonia, the prevalence of pfhrp2 gene deletion was only 3.3% (2 out of 60 patients). In isolates from Para state, all parasites were pfhrp2-positive. In contrast, we detected high proportions of isolates from all 3 states that were pfhrp3-negative ranging from 18.3% (11 out of 60 samples) to 50.9% (30 out of 59 samples). In Bolivia, only one of 25 samples (4%) tested had deleted pfhrp2 gene, while 68% (17 out of 25 samples) were pfhrp3-negative. Among the isolates tested, P. falciparum pfhrp2 gene deletions were present mainly in those from Acre State in the Brazilian Amazon. These results indicate it is important to reconsider the use of PfHRP2-based RDTs in the western region of the Brazilian Amazon and to implement appropriate surveillance systems to monitor pfhrp2 gene deletions in this and other parts of the Amazon region.

The drug combination atovaquone-proguanil (AP), trade name Malarone (GlaxoSmithKline, Brentford, UK), was the most commonly prescribed drug for malaria prophylaxis in US travelers in 2011 [1], and it was the most frequently used drug for the treatment of uncomplicated malaria in the United States in 2011 [2]. Atovaquone-proguanil is also the drug most frequently used for acute uncomplicated Plasmodium falciparum malaria in France and other countries in Europe [3, 4]. Despite high efficacy, there have been sporadic reports of AP treatment failures since AP was first introduced for malaria prophylaxis and treatment in 2000 [5–15]. Resistance to cycloguanil, the active metabolite of proguanil, is conferred by the accumulation of multiple mutations in the P falciparum dihydrofolate reductase (pfdhfr) gene. These mutations also confer resistance to pyrimethamine and have become fixed in many malaria-endemic areas worldwide. In contrast, resistance to atovaquone can be conferred by just 1 single-nucleotide polymorphism (SNP) in the P falciparum cytochrome B gene (Pfcytb), encoding the molecular target of atovaquone. Unlike pfdhfr mutations, Pfcytb mutations that are associated with AP treatment failures are rarely found in pretreatment isolates [16–21], but they can arise after treatment with AP. Monotherapy with atovaquone has one of the highest rates of inducing resistant parasites during therapy [22], with up to 30%–40% of P falciparum infections exhibiting resistant recrudescent parasites after treatment [23, 24]. Surprisingly, AP has remained highly effective, even in areas with a high prevalence of proguanil-resistant genotypes. This result is probably due to proguanil acting as a possible synergistic potentiator of atovaquone activity [25]. Reported AP treatment failures therefore tend to follow a specific pattern, in which some parasites that are originally proguanil-resistant but atovaquone-sensitive develop de novo mutations that confer resistance during the acute infection, leading to late recrudescence after treatment. To date, all reported mutations directly associated with resistance to the atovaquone in AP treatment failures have occurred in codon 268 of the pfcytb gene [5–12]. Most commonly, the mutations replace a tyrosine with a serine (Y268S) or a cysteine (Y268C), but they can also substitute an asparagine (Y268N). In this study, we present 2 cases of AP treatment failures in US travelers who stayed in the same compound for foreign workers near Port Harcourt in Nigeria, 7 months apart. Although the parasites from 1 recrudescent infection exhibited the previously described Y268S mutation in Pfcytb, the parasites from the other infection exhibited a novel mutation in cytb (I258M).