Prevalence of malaria resistance-associated mutations in Plasmodium falciparum circulating in 2017-2018, Bo, Sierra Leone.

Introduction: In spite of promising medical, sociological, and engineering strategies and interventions to reduce the burden of disease, malaria remains a source of significant morbidity and mortality, especially among children in sub-Saharan Africa. In particular, progress in the development and administration of chemotherapeutic agents is threatened by evolved resistance to most of the antimalarials currently in use, including artemisinins. Methods: This study analyzed the prevalence of mutations associated with antimalarial resistance in Plasmodium falciparum from 95 clinical samples collected from individuals with clinically confirmed malaria at a hospital in Bo, Sierra Leone between May 2017 and December 2018. The combination of polymerase chain reaction amplification and subsequent high throughput DNA sequencing was used to determine the presence of resistance-associated mutations in five P. falciparum genes - pfcrt, pfmdr1, pfdhfr, pfdhps and pfkelch13. The geographic origin of parasites was assigned using mitochondrial sequences. Results: Relevant mutations were detected in the pfcrt (22%), pfmdr1 (>58%), pfdhfr (100%) and pfdhps (>80%) genes while no resistance-associated mutations were found in the pfkelch13 gene. The mitochondrial barcodes were consistent with a West African parasite origin with one exception indicating an isolate imported from East Africa. Discussion: Detection of the pfmdr1 NFSND haplotype in 50% of the samples indicated the increasing prevalence of strains with elevated tolerance to artemeter + lumefantrine (AL) threatening the combination currently used to treat uncomplicated malaria in Sierra Leone. The frequency of mutations linked to resistance to antifolates suggests widespread resistance to the drug combination used for intermittent preventive treatment during pregnancy.

Use of real-time multiplex PCR, malaria rapid diagnostic test and microscopy to investigate the prevalence of Plasmodium species among febrile hospital patients in Sierra Leone.

BACKGROUND: Malaria continues to affect over 200 million individuals every year, especially children in Africa. Rapid and sensitive detection and identification of Plasmodium parasites is crucial for treating patients and monitoring of control efforts. Compared to traditional diagnostic methods such as microscopy and rapid diagnostic tests (RDTs), DNA based methods, such as polymerase chain reaction (PCR) offer significantly higher sensitivity, definitive discrimination of Plasmodium species, and detection of mixed infections. While PCR is not currently optimized for routine diagnostics, its role in epidemiological studies is increasing as the world moves closer toward regional and eventually global malaria elimination. This study demonstrates the field use of a novel, ambient temperature-stabilized, multiplexed PCR assay in a small hospital setting in Sierra Leone. METHODS: Blood samples from 534 febrile individuals reporting to a hospital in Bo, Sierra Leone, were tested using three methods: a commercial RDT, microscopy, and a Multiplex Malaria Sample Ready (MMSR) PCR designed to detect a universal malaria marker and species-specific markers for Plasmodium falciparum and Plasmodium vivax. A separate PCR assay was used to identify species of Plasmodium in samples in which MMSR detected malaria, but was unable to identify the species. RESULTS: MMSR detected the presence of any malaria marker in 50.2% of all tested samples with P. falciparum identified in 48.7% of the samples. Plasmodium vivax was not detected. Testing of MMSR P. falciparum-negative/universal malaria-positive specimens with a panel of species-specific PCRs revealed the presence of Plasmodium malariae (n = 2) and Plasmodium ovale (n = 2). The commercial RDT detected P. falciparum in 24.6% of all samples while microscopy was able to detect malaria in 12.8% of tested specimens. CONCLUSIONS: Wider application of PCR for detection of malaria parasites may help to fill gaps existing as a result of use of microscopy and RDTs. Due to its high sensitivity and specificity, species coverage, room temperature stability and relative low complexity, the MMSR assay may be useful for detection of malaria and epidemiological studies especially in low-resource settings.

Seroprevalence of hepatitis B surface antigen (HBsAg) in Bo, Sierra Leone, 2012-2013.

OBJECTIVE: The aim of this study was to determine the prevalence of hepatitis B surface antigen (HBsAg) among febrile individuals tested at Mercy Hospital Research Laboratory (MHRL) in Bo, Sierra Leone. RESULTS: A total of 860 febrile individuals ages 5 years and older were tested by MHRL between July 2012 and June 2013 with a Standard Diagnostics Bioline HBsAg rapid diagnostic test. The overall HBsAg prevalence rate was 13.7%, including a rate of 15.5% among males and 12.6% among females. The HBsAg rate did not differ by child or adult age group (p > 0.5). The prevalence rate in Bo was similar to the 11-15% HBsAg prevalence rates reported in the past decade from other studies across West Africa. Scaling up the infant hepatitis B vaccination program in Sierra Leone will be important for reducing the future burden of disease and premature death attributable to chronic viral hepatitis B disease.

Serotype Distribution of Clinical Streptococcus pneumoniae Isolates before the Introduction of the 13-Valent Pneumococcal Conjugate Vaccine in Cambodia.

Childhood vaccination with the 13-valent pneumococcal conjugate vaccine (PCV13) was introduced in Cambodia in January 2015. Baseline data regarding circulating serotypes are scarce. All microbiology laboratories in Cambodia were contacted for identification of stored isolates of Streptococcus pneumoniae from clinical specimens taken before the introduction of PCV13. Available isolates were serotyped using a multiplex polymerase chain reaction method. Among 166 identified isolates available for serotyping from patients with pneumococcal disease, 4% were isolated from upper respiratory samples and 80% were from lower respiratory samples, and 16% were invasive isolates. PCV13 serotypes accounted for 60% (95% confidence interval [CI] 52-67) of all isolates; 56% (95% CI 48-64) of noninvasive and 77% (95% CI 57-89) of invasive isolates. Antibiotic resistance was more common among PCV13 serotypes. This study of clinical S. pneumoniae isolates supports the potential for high reduction in pneumococcal disease burden and may serve as baseline data for future monitoring of S. pneumoniae serotypes circulation after implementation of PCV13 childhood vaccination in Cambodia.

Prevalence of markers of HIV infection among febrile adults and children in Bo, Sierra Leone, 2012-2013.

OBJECTIVE: The goal of this study was to examine the prevalence of HIV among febrile patients seeking care in Mercy Hospital, Bo, Sierra Leone, in 2012-2013. RESULTS: A total of 1207 febrile persons were tested for HIV with Determine™ and SD Bioline rapid diagnostic tests kits that detect the presence of HIV antibodies and HIV p24 antigens. The overall prevalence of HIV among the tested patients was 8.9%, which is considerably higher than the < 2% prevalence of HIV reported previously in the general population. While these results are not sufficient to prove a causal relationship, the obtained data imply that HIV positive individuals may be more likely to suffer from febrile infectious diseases than individuals without HIV infection. Increasing the availability and use of HIV testing services will allow antiretroviral therapy to be accessed in a timely manner and improve health status among people living with HIV.

Surveillance of Vector-Borne Infections (Chikungunya, Dengue, and Malaria) in Bo, Sierra Leone, 2012-2013.

Malaria remains a significant cause of morbidity and mortality in West Africa, but the contribution of other vector-borne infections (VBIs) to the burden of disease has been understudied. We used rapid diagnostic tests (RDTs) for three VBIs to test blood samples from 1,795 febrile residents of Bo City, Sierra Leone, over a 1-year period in 2012-2013. In total, 24% of the tests were positive for malaria, fewer than 5% were positive for markers of dengue virus infection, and 39% were positive for IgM directed against chikungunya virus (CHIKV) or a related alphavirus. In total, more than half (55%) of these febrile individuals tested positive for at least one of the three VBIs, which highlights the very high burden of vector-borne diseases in this population. The prevalence of positives on the Chikungunya IgM and dengue tests did not vary significantly with age (P > 0.36), but higher rates of malaria were observed in children < 15 years of age (P < 0.001). Positive results on the Chikungunya IgM RDTs were moderately correlated with rainfall (r2 = 0.599). Based on the high prevalence of positive results on the Chikungunya IgM RDTs from individuals Bo and its environs, there is a need to examine whether an ecological shift toward a greater burden from CHIKV or related alphaviruses is occurring in other parts of Sierra Leone or the West African region.

Complete Genome Sequences of Five Zika Virus Isolates.

Zika virus is an emerging human pathogen of great concern due to putative links to microcephaly and Guillain-Barre syndrome. Here, we report the complete genomes, including the 5' and 3' untranslated regions, of five Zika virus isolates, one from the Asian lineage and four from the African lineage.

Multicountry prospective clinical evaluation of two enzyme-linked immunosorbent assays and two rapid diagnostic tests for diagnosing dengue fever.

We evaluated four dengue diagnostic devices from Alere, including the SD Bioline Dengue Duo (nonstructural [NS] 1 Ag and IgG/IgM), the Panbio Dengue Duo Cassette (IgM/IgG) rapid diagnostic tests (RDTs), and the Panbio dengue IgM and IgG capture enzyme-linked immunosorbent assays (ELISAs) in a prospective, controlled, multicenter study in Peru, Venezuela, Cambodia, and the United States, using samples from 1,021 febrile individuals. Archived, well-characterized samples from an additional 135 febrile individuals from Thailand were also used. Reference testing was performed on all samples using an algorithm involving virus isolation, in-house IgM and IgG capture ELISAs, and plaque reduction neutralization tests (PRNT) to determine the infection status of the individual. The primary endpoints were the clinical sensitivities and specificities of these devices. The SD Bioline Dengue Duo had an overall sensitivity of 87.3% (95% confidence interval [CI], 84.1 to 90.2%) and specificity of 86.8% (95% CI, 83.9 to 89.3%) during the first 14 days post-symptom onset (p.s.o.). The Panbio Dengue Duo Cassette demonstrated a sensitivity of 92.1% (87.8 to 95.2%) and specificity of 62.2% (54.5 to 69.5%) during days 4 to 14 p.s.o. The Panbio IgM capture ELISA had a sensitivity of 87.6% (82.7 to 91.4%) and specificity of 88.1% (82.2 to 92.6%) during days 4 to 14 p.s.o. Finally, the Panbio IgG capture ELISA had a sensitivity of 69.6% (62.1 to 76.4%) and a specificity of 88.4% (82.6 to 92.8%) during days 4 to 14 p.s.o. for identification of secondary dengue infections. This multicountry prospective study resulted in reliable real-world performance data that will facilitate data-driven laboratory test choices for managing patient care during dengue outbreaks.

Little evidence of subclinical avian influenza virus infections among rural villagers in Cambodia.

In 2008, 800 adults living within rural Kampong Cham Province, Cambodia were enrolled in a prospective cohort study of zoonotic influenza transmission. After enrollment, participants were contacted weekly for 24 months to identify acute influenza-like illnesses (ILI). Follow-up sera were collected at 12 and 24 months. A transmission substudy was also conducted among the family contacts of cohort members reporting ILI who were influenza A positive. Samples were assessed using serological or molecular techniques looking for evidence of infection with human and avian influenza viruses. Over 24 months, 438 ILI investigations among 284 cohort members were conducted. One cohort member was hospitalized with a H5N1 highly pathogenic avian influenza (HPAI) virus infection and withdrew from the study. Ninety-seven ILI cases (22.1%) were identified as influenza A virus infections by real-time RT-PCR; none yielded evidence for AIV. During the 2 years of follow-up, 21 participants (3.0%) had detectable antibody titers (≥ 1:10) against the studied AIVs: 1 against an avian-like A/Migratory duck/Hong Kong/MPS180/2003(H4N6), 3 against an avian-like A/Teal/Hong Kong/w312/97(H6N1), 9 (3 of which had detectible antibody titers at both 12- and 24-month follow-up) against an avian-like A/Hong Kong/1073/1999(H9N2), 6 (1 detected at both 12- and 24-month follow-up) against an avian-like A/Duck/Memphis/546/74(H11N9), and 2 against an avian-like A/Duck/Alberta/60/76(H12N5). With the exception of the one hospitalized cohort member with H5N1 infection, no other symptomatic avian influenza infections were detected among the cohort. Serological evidence for subclinical infections was sparse with only one subject showing a 4-fold rise in microneutralization titer over time against AvH12N5. In summary, despite conducting this closely monitored cohort study in a region enzootic for H5N1 HPAI, we were unable to detect subclinical avian influenza infections, suggesting either that these infections are rare or that our assays are insensitive at detecting them.

Contributions of the Global Emerging Infections Surveillance and Response System Network to global health security in 2011.

In its 15th year, the Global Emerging Infections Surveillance and Response System (GEIS) continued to make significant contributions to global public health and emerging infectious disease surveillance worldwide. As a division of the US Department of Defense's Armed Forces Health Surveillance Center since 2008, GEIS coordinated a network of surveillance and response activities through collaborations with 33 partners in 76 countries. The GEIS was involved in 73 outbreak responses in fiscal year 2011. Significant laboratory capacity-building initiatives were undertaken with 53 foreign health, agriculture and/or defense ministries, as well as with other US government entities and international institutions, including support for numerous national influenza centers. Equally important, a variety of epidemiologic training endeavors reached over 4,500 individuals in 96 countries. Collectively, these activities enhanced the ability of partner countries and the US military to make decisions about biological threats and design programs to protect global public health as well as global health security.

Evidence for avian H9N2 influenza virus infections among rural villagers in Cambodia.

BACKGROUND: Southeast Asia remains a critical region for the emergence of novel and/or zoonotic influenza, underscoring the importance of extensive sampling in rural areas where early transmission is most likely to occur. METHODS: In 2008, 800 adult participants from eight sites were enrolled in a prospective population-based study of avian influenza (AI) virus transmission where highly pathogenic avian influenza (HPAI) H5N1 virus had been reported in humans and poultry from 2006 to 2008. From their enrollment sera and questionnaires, we report risk factor findings for serologic evidence of previous infection with 18 AI virus strains. RESULTS: Serologic assays revealed no evidence of previous infection with 13 different low-pathogenic AI viruses or with HPAI avian-like A/Cambodia/R0404050/2007(H5N1). However, 21 participants had elevated antibodies against avian-like A/Hong Kong/1073/1999(H9N2), validated with a monoclonal antibody blocking ELISA assay specific for avian H9. CONCLUSIONS: Although cross-reaction from antibodies against human influenza viruses cannot be completely excluded, the study data suggest that a number of participants were previously infected with the avian-like A/Hong Kong/1073/1999(H9N2) virus, likely due to as yet unidentified environmental exposures. Prospective data from this cohort will help us better understand the serology of zoonotic influenza infection in a rural cohort in SE Asia.

Dynamic of H5N1 virus in Cambodia and emergence of a novel endemic sub-clade.

In Cambodia, the first detection of HPAI H5N1 virus in birds occurred in January 2004 and since then there have been 33 outbreaks in poultry while 21 human cases were reported. The origin and dynamics of these epizootics in Cambodia remain unclear. In this work we used a range of bioinformatics methods to analyze the Cambodian virus sequences together with those from neighboring countries. Six HA lineages belonging to clades 1 and 1.1 were identified since 2004. Lineage 1 shares an ancestor with viruses from Thailand and disappeared after 2005, to be replaced by lineage 2 originating from Vietnam and then by lineage 3. The highly adapted lineage 4 was seen only in Cambodia. Lineage 5 is circulating both in Vietnam and Cambodia since 2008 and was probably introduced in Cambodia through unregistered transboundary poultry trade. Lineage 6 is endemic to Cambodia since 2010 and could be classified as a new clade according to WHO/OIE/FAO criteria for H5N1 virus nomenclature. We propose to name it clade 1.1A. There is a direct filiation of lineages 2 to 6 with a temporal evolution and geographic differentiation for lineages 4 and 6. By the end of 2011, two lineages, i.e. lineages 5 and 6, with different transmission paths cocirculate in Cambodia. The presence of lineage 6 only in Cambodia suggests the existence of a transmission specific to this country whereas the presence of lineage 5 in both Cambodia and Vietnam indicates a distinct way of circulation of infected poultry.

Epidemiological characteristics, clinical presentation and diagnosis at point-of-care during the first wave of the H1N1 influenza pandemic in Cambodia.

We conducted clinic-based surveillance for influenza virus among cases with acute febrile illness at 9 medical clinics in south-central Cambodia during 2006-2009. Patients greater than or equal to 24 months old presenting with acute fever (> 38 degrees C) were enrolled. In late July 2009, the study identified its first case of pandemic H1N1 (pH1N1) influenza virus infection. The prevalence of pH1N1 infections increased rapidly during August and September and by October, pH1N1 infections had peaked replacing H3N2 as the dominant subtype. The incidence of pH1N1 subsequently decreased, with only one case identified in late December. From late July through December 2009, 42.4% of all influenza cases were caused by pH1N1. Except for headache, less frequently reported among pH1N1-infected patients, patients infected with the pH1N1 reported symptoms (eg, cough, diarrhea, vomiting and nausea) similar to seasonal H3N2 and B virus infections. Among children 6 to 12 years old, there was a higher number of hospitalizations campared to other age groups. Identification of influenza virus types A and B using the QuickVue rapid diagnostic test was found to be equally sensitive for pH1N1 (50.4%), H3N2 (51.7%) and influenza B (53.9%) viruses, although the sensitivity was low among all subtypes. The pH1N1 virus rapidly became the dominant virus subtype in 2009 in Cambodia, but no symptoms consistently distinguished the pandemic strain from other influenza virus subtypes. The QuickVue test was as sensitive for detecting pH1N1 viral as well as other circulating seasonal influenza viruses.

Genetic characterization of Zika virus strains: geographic expansion of the Asian lineage.

BACKGROUND: Zika virus (ZIKV) is a mosquito-borne flavivirus distributed throughout much of Africa and Asia. Infection with the virus may cause acute febrile illness that clinically resembles dengue fever. A recent study indicated the existence of three geographically distinct viral lineages; however this analysis utilized only a single viral gene. Although ZIKV has been known to circulate in both Africa and Asia since at least the 1950s, little is known about the genetic relationships between geographically distinct virus strains. Moreover, the geographic origin of the strains responsible for the epidemic that occurred on Yap Island, Federated States of Micronesia in 2007, and a 2010 pediatric case in Cambodia, has not been determined. METHODOLOGY/PRINCIPAL FINDINGS: To elucidate the genetic relationships of geographically distinct ZIKV strains and the origin of the strains responsible for the 2007 outbreak on Yap Island and a 2010 Cambodian pediatric case of ZIKV infection, the nucleotide sequences of the open reading frame of five isolates from Cambodia, Malaysia, Nigeria, Uganda, and Senegal collected between 1947 and 2010 were determined. Phylogenetic analyses of these and previously published ZIKV sequences revealed the existence of two main virus lineages (African and Asian) and that the strain responsible for the Yap epidemic and the Cambodian case most likely originated in Southeast Asia. Examination of the nucleotide and amino acid sequence alignments revealed the loss of a potential glycosylation site in some of the virus strains, which may correlate with the passage history of the virus. CONCLUSIONS/SIGNIFICANCE: The basal position of the ZIKV strain isolated in Malaysia in 1966 suggests that the recent outbreak in Micronesia was initiated by a strain from Southeast Asia. Because ZIKV infection in humans produces an illness clinically similar to dengue fever and many other tropical infectious diseases, it is likely greatly misdiagnosed and underreported.

Infectious etiologies of acute febrile illness among patients seeking health care in south-central Cambodia.

The agents of human febrile illness can vary by region and country suggesting that diagnosis, treatment, and control programs need to be based on a methodical evaluation of area-specific etiologies. From December 2006 to December 2009, 9,997 individuals presenting with acute febrile illness at nine health care clinics in south-central Cambodia were enrolled in a study to elucidate the etiologies. Upon enrollment, respiratory specimens, whole blood, and serum were collected. Testing was performed for viral, bacterial, and parasitic pathogens. Etiologies were identified in 38.0% of patients. Influenza was the most frequent pathogen, followed by dengue, malaria, and bacterial pathogens isolated from blood culture. In addition, 3.5% of enrolled patients were infected with more than one pathogen. Our data provide the first systematic assessment of the etiologies of acute febrile illness in south-central Cambodia. Data from syndromic-based surveillance studies can help guide public health responses in developing nations.

Short report: Rapid-test based identification of influenza as an etiology of acute febrile illness in Cambodia.

Influenza can be manifested as an acute febrile illness, with symptoms similar to many pathogens endemic to Cambodia. The objective of this study was to evaluate the Quickvue influenza A+B rapid test to identify the etiology of acute febrile illness in Cambodia. During December 2006-May 2008, patients enrolled in a study to identify the etiology of acute febrile illnesses were tested for influenza by real-time reverse transcriptase PCR (RT-PCR) and Quickvue influenza A+B rapid test. The prevalence of influenza was 19.7% by RT-PCR. Compared with RT-PCR, the sensitivity and specificity of the rapid test were 52.1% and 92.5%, respectively. The influenza rapid test identified the etiology in 10.2% of enrollees and ≥ 35% during peak times of influenza activity. This study suggests that rapid influenza tests may be useful during peak times of influenza activity in an area where several different etiologies can present as an acute febrile illness.

Dual infection of novel influenza viruses A/H1N1 and A/H3N2 in a cluster of Cambodian patients.

During the early months of 2009, a novel influenza A/H1N1 virus (pH1N1) emerged in Mexico and quickly spread across the globe. In October 2009, a 23-year-old male residing in central Cambodia was diagnosed with pH1N1. Subsequently, a cluster of four influenza-like illness cases developed involving three children who resided in his home and the children's school teacher. Base composition analysis of internal genes using reverse transcriptase polymerase chain reaction and electrospray ionization mass spectrometry revealed that specimens from two of the secondary victims were coinfected with influenza A/H3N2 and pH1N1. Phylogenetic analysis of the hemagglutinin genes from these isolated viruses showed that they were closely related to existing pH1N1 and A/H3N2 viruses circulating in the region. Genetic recombination was not evident within plaque-purified viral isolates on full genome sequencing. This incident confirms dual influenza virus infections and highlights the risk of zoonotic and seasonal influenza viruses to coinfect and possibly, reassort where they cocirculate.