Mozambique is one of the four African countries which account for over half of all malaria deaths worldwide, yet little is known about the parasite genetic structure in that country. We performed P. falciparum amplicon and whole genome sequencing on 2251 malaria-infected blood samples collected in 2015 and 2018 in seven provinces of Mozambique to genotype antimalarial resistance markers and interrogate parasite population structure using genome-wide microhaplotyes. Here we show that the only resistance-associated markers observed at frequencies above 5% were pfmdr1-184F (59%), pfdhfr-51I/59 R/108 N (99%) and pfdhps-437G/540E (89%). The frequency of pfdhfr/pfdhps quintuple mutants associated with sulfadoxine-pyrimethamine resistance increased from 80% in 2015 to 89% in 2018 (p < 0.001), with a lower expected heterozygosity and higher relatedness of microhaplotypes surrounding pfdhps mutants than wild-type parasites suggestive of recent selection. pfdhfr/pfdhps quintuple mutants also increased from 72% in the north to 95% in the south (2018; p < 0.001). This resistance gradient was accompanied by a concentration of mutations at pfdhps-436 (17%) in the north, a south-to-north increase in the genetic complexity of P. falciparum infections (p = 0.001) and a microhaplotype signature of regional differentiation. The parasite population structure identified here offers insights to guide antimalarial interventions and epidemiological surveys.

Rift Valley fever virus (RVFV) is endemic in sub-Saharan Africa (SSA), with outbreaks reported in the Arabian Peninsula and throughout SSA. The natural reservoir for RVFV are ruminants, with livestock populations exceeding 50% exposure rates in some areas of SSA. Transmission to humans can occur through exposure to infected livestock products or multiple species of mosquito vectors. In 2013 and 2014, cross-sectional surveys occurred in two districts of Nacala-a-Velha and Mecubri in northern Mozambique, and participants provided blood samples for later serological assays. IgG against the N protein of RVFV was detected through multiplex bead assay (MBA). Of the 2,278 persons enrolled between the two surveys and study sites, 181 (7.9%, 95% confidence interval (CI): 6.9%-9.1%) were found to be IgG seropositive with increasing seroprevalence with older age and significantly higher seroprevalence in Nacala-a-Velha (10.5%, 8.8%-12.5%) versus Mecubri (5.7%, 4.5%-7.1%). Seroprevalence estimates were not significantly different between the 2013 and 2014 surveys. Significant spatial clustering of IgG positive persons were consistent among surveys and within the two districts, pointing toward the consistency of serology data for making population-level assumptions regarding RVFV seroprevalence. A subset of persons (n=539) provided samples for both the 2013 and 2014 surveys, and a low percentage (0.81%) of these were found to seroconvert between these two surveys. Including the RVFV N protein in an MBA antigen panel could assist elucidate RVFV exposure in SSA. IMPORTANCE Due to sporadic transmission, human contact with Rift Valley Fever Virus (RVFV) is difficult to ascertain at a population level. Detection of antibodies against RVFV antigens assist in estimating exposure as antibodies remain in the host long after the virus has been cleared. In this study, we show that antibodies against RVFV N protein can be detected from dried blood spot (DBS) samples being assayed by multiplex bead assay. DBS from two districts in northern Mozambique were tested for IgG against the N protein, and 7.9% of all enrolled persons were seropositive. Older persons, males, and persons residing closer to the coast had higher RVFV N protein seroprevalence. Spatial clustering of IgG positive persons was noted in both districts. These results show low exposure rates to RVFV in these two northern districts in Mozambique, and the ability to perform serology for the RVFV N protein from dried blood samples.

BACKGROUND: Malaria data reported through Mozambique's routine health information system are used to guide the implementation of prevention and control activities. Although previous studies have identified issues with the quality of aggregated data reported from public health facilities in the country, no studies have evaluated the quality of routine indicators recorded in health facility registries. This study addresses this issue by comparing indicators calculated from data from exit interviews and re-examinations of patients with data based on registry records from health facilities in order to measure the quality of registry data and data reporting in three provinces in Mozambique. METHODS: Data were collected from 1,840 outpatients from 117 health facilities in Maputo, Zambezia, and Cabo Delgado Provinces interviewed and examined as part of a malaria-specific health facility survey. Key indicators based on exit interview / re-examination data were compared to the same indicators based on records from health facility registries. Multivariable regression was performed to identify factors associated with indicators matching in re-examination / exit interview data and health facility registries. Aggregated indicators abstracted from facility registries were compared to those reported through the routine health management information system (HMIS) for the same time period. RESULTS: Sensitivity of exit interview / re-examination data compared with those recorded in facility registries was low for all indicators in all facilities. The lowest sensitivities were in Maputo, where the sensitivity for recording negative RDT results was 9.7%. The highest sensitivity was for recording positive RDT results in Cabo Delgado, at 75%. Multivariable analysis of factors associated with agreement between gold standard and registry data showed patients were less likely to be asked about having a fever in the triage ward in Maputo and Cabo Delgado (adjusted Odds Ratio 0.75 and 0.39 respectively), and in the outpatient ward in Cabo Delgado (aOR = 0.37), compared with the emergency department. Patients with positive RDT were also more likely to have RDT results recorded in all three provinces when patients had been managed according to national treatment guidelines during initial examination. Comparison of retrospective data abstracted from facility registries to HMIS data showed discrepancies in all three provinces. The proportion of outpatient cases with suspected and confirmed malaria were similar in registry and HMIS data across all provinces (a relatively low difference between registry and HMIS data of 3% in Maputo and Zambezia), though the total number of all-cause outpatient cases was consistently higher in the HMIS. The largest difference was in Maputo, where a total of 87,992 all-cause outpatient cases were reported in HMIS, compared with a total of 42,431 abstracted from facility registries. CONCLUSION: This study shows that care should be taken in interpreting trends based solely on routine data due to data quality issues, though the discrepancy in all-cause outpatient cases may be indicative that register availability and storage are important factors. As such, simple steps such as providing consistent access and storage of registers that include reporting of patient fever symptoms might improve the quality of routine data recorded at health facilities.

Routine incident malaria case data have become a pillar of malaria surveillance in sub-Saharan Africa. These data provide granular, timely information to track malaria burden. However, incidence data are sensitive to changes in care seeking rates, rates of testing of suspect cases, and reporting completeness. Based on a set of assumptions, we derived a simple algebraic formula to convert crude incidence rates to a corrected estimation of incidence, adjusting for biases in variable and suboptimal rates of care seeking, testing of suspect cases, and reporting completeness. We applied the correction to routine incidence data from Guinea and Mozambique, and aggregate data for sub-Saharan African countries from the World Malaria Report. We calculated continent-wide needs for malaria tests and treatments, assuming universal testing but current care seeking rates. Countries in southern and eastern Africa reporting recent increases in malaria incidence generally had lower overall corrected incidence than countries in Central and West Africa. Under current care seeking rates, the unmet need for malaria tests was estimated to be 160 million (M) (interquartile range [IQR]: 139-188) and for malaria treatments to be 37 M (IQR: 29-51). Maps of corrected incidence were more consistent with maps of community survey prevalence than was crude incidence in Guinea and Mozambique. Crude malaria incidence rates need to be interpreted in the context of suboptimal testing and care seeking rates, which vary over space and time. Adjusting for these factors can provide an insight into the spatiotemporal trends of malaria burden.

Like most malaria-endemic countries, Mozambique relies on tabulation of confirmed malaria test-positive febrile patients to track incidence of malaria. However, this approach is potentially biased by incidental malaria parasitemia in patients with fever of another etiology. We compared pan-Plasmodium aldolase and lactate dehydrogenase and Plasmodium falciparum HRP2 antigen concentrations measured using a laboratory bead-based assay of samples collected from 1,712 febrile and afebrile patients of all ages in Maputo, Zambezia, and Cabo Delgado provinces. We used a Bayesian latent class model to estimate the proportion of malaria-attributable fevers in malaria test-positive febrile patients. Depending on the antigen, estimated rates of malaria-attributable fever in malaria test-positive febrile patients were 100% in Maputo, 33-58% in Zambezia, and 63-74% in Cabo Delgado. Our findings indicate that most malaria test-positive febrile patients in the three provinces of Mozambique had a fever that was likely caused by the concurrent malaria infection. Counting malaria test-positive febrile patients for estimation of malaria incidence appears to be appropriate in this setting.

Background: Rapid diagnostic tests (RDTs) that detect the Plasmodium falciparum-specific histidine-rich protein 2 (PfHRP2) antigen are the primary method for malaria diagnosis in Mozambique. However, these tests do not detect infections with non-falciparum malaria or Pfhrp2/3-deleted P. falciparum parasites.Methods: To assess the appropriateness of conventional PfHRP2-only RDTs for malaria diagnosis in Mozambique, samples collected during a health facility survey conducted in three provinces of Mozambique were screened using antigen detection methods and further characterized by molecular techniques. Samples from 1861 outpatients of all ages and symptoms attending 117 randomly-selected public health facilities in 2018 were analyzed with an ultra-sensitive bead-based immunoassay for the presence of PfHRP2, pan-Plasmodium Aldolase (pAldo), and pan-Plasmodium lactate dehydrogenase (pLDH). Presence of PfHRP2 in patient blood detected using the bead-based assay was compared to the results of PfHRP2-based RDTs performed during the routine health facility consult and during the survey re-examination at exit interview. Samples with discordant antigen profiles (negative for PfHRP2 but positive for pAldo and/or pLDH) were further characterized by photo-induced electron transfer (PET)-PCR.Results: Using the bead-based laboratory assay as the gold standard, the sensitivity of the conventional RDTs administered during the routine health facility consult and the exit interview was 90% and 83%, respectively, and specificity was 91% and 97%. Of 710 samples positive for at least one antigen, 704 (99.2%) were positive for PfHRP2. Six (0.8% of total) discordant samples lacked PfHRP2 but were positive for pAldo and/or pLDH; 3 of these (0.4% of total) were P. ovale mono-infections or co-infections where P. ovale was the dominant species. The remaining 3 discordant samples were negative by PET-PCR.Conclusions: The sensitivity and specificity of the conventional RDTs performed in the routine health facility consults and survey exit interviews were acceptable, and there was no evidence of Pfhrp2/3-deleted parasites. Mono-infections with non-falciparum malaria species comprised <1% of total malaria infections. Nearly all malaria antigen-positive patients had detectable PfHRP2, confirming this antigen remains an appropriate malaria diagnostic target in the surveyed provinces.

BACKGROUND: Fever associated with malaria is the leading cause of health care-seeking in Mozambique, yet there is limited evidence on the quality of malaria case management. This study evaluated the quality of malaria service provision offered in public health facilities in Mozambique. METHODS: A cross-sectional assessment was conducted in April-May 2018 in three provinces of Mozambique: Maputo Province (low malaria burden), Cabo Delgado (high), and Zambezia (high). The study included all secondary and tertiary facilities and a random sample of primary facilities in each province. Data collection included exit interviews and re-examinations of 20 randomly selected outpatient service patients, interviews with up to five health care providers and the health facility director, a stockroom inventory and routine data abstraction. RESULTS: A total of 319 health care providers and 1840 patients from 117 health facilities were included. Of these, 1325 patients (72%) had suspected malaria (fever/history of fever) and 550 (30%) had febrile, confirmed malaria with the highest burden in Cabo Delgado (43%), followed by Zambezia (34%) and Maputo Province (2%). Appropriate management of malaria cases, defined as testing malaria suspects and treating confirmed cases with the correct dose of anti-malarial, was highest in Zambezia and Cabo Delgado where 52% (95% CI 42-62) and 49% (42-57) of febrile malaria cases were appropriately managed, respectively. Only 14% (5-34) of febrile cases in Maputo Province were appropriately managed. The biggest gap in the malaria case management pathway was failure to test febrile patients, with only 46% of patients with this indication tested for malaria in Maputo Province. Additionally, anti-malarial treatment of patients with a negative malaria test result was common, ranging from 8% (2-23) in Maputo Province to 22% (14-32) of patients with a negative test in Zambezia. Only 58-62% of patients prescribed an anti-malarial correctly recited dosing instructions. Provider training and malaria knowledge was low outside of Zambezia and supervision rates were low in all provinces. Factors associated with correct case management varied by province and included patient age, facility type, treatment and testing availability, supervision, and training. CONCLUSION: These findings underscore the need to strengthen provider testing of all patients with fever, provider adherence to negative test results, and effective counselling of patients across epidemiological settings in Mozambique.

BACKGROUND: Multiplex bead assays (MBA) that measure IgG antibodies to the carboxy-terminal 19-kDa sub-unit of the merozoite surface protein 1 (MSP119) are currently used to determine malaria seroprevalence in human populations living in areas with both stable and unstable transmission. However, the species specificities of the IgG antibody responses to the malaria MSP119 antigens have not been extensively characterized using MBA. METHODS: Recombinant Plasmodium falciparum (3D7), Plasmodium malariae (China I), Plasmodium ovale (Nigeria I), and Plasmodium vivax (Belem) MSP119 proteins were covalently coupled to beads for MBA. Threshold cut-off values for the assays were estimated using sera from US citizens with no history of foreign travel and by receiver operator characteristic curve analysis using diagnostic samples. Banked sera from experimentally infected chimpanzees, sera from humans from low transmission regions of Haiti and Cambodia (N = 12), and elutions from blood spots from humans selected from a high transmission region of Mozambique (N = 20) were used to develop an antigen competition MBA for antibody cross-reactivity studies. A sub-set of samples was further characterized using antibody capture/elution MBA, IgG subclass determination, and antibody avidity measurement. RESULTS: Total IgG antibody responses in experimentally infected chimpanzees were species specific and could be completely suppressed by homologous competitor protein at a concentration of 10 mug/ml. Eleven of 12 samples from the low transmission regions and 12 of 20 samples from the high transmission area had antibody responses that were completely species specific. For 7 additional samples, the P. falciparum MSP119 responses were species specific, but various levels of incomplete heterologous competition were observed for the non-P. falciparum assays. A pan-malaria MSP119 cross-reactive antibody response was observed in elutions of blood spots from two 20-30 years old Mozambique donors. The antibody response from one of these two donors had low avidity and skewed almost entirely to the IgG3 subclass. CONCLUSIONS: Even when P. falciparum, P. malariae, P. ovale, and P. vivax are co-endemic in a high transmission setting, most antibody responses to MSP119 antigens are species-specific and are likely indicative of previous infection history. True pan-malaria cross-reactive responses were found to occur rarely.

BACKGROUND: Universal coverage with long-lasting insecticidal nets (LLINs) is a primary control strategy against Plasmodium falciparum malaria. However, its impact on the three other main species of human malaria and lymphatic filariasis (LF), which share the same vectors in many co-endemic areas, is not as well characterized. The recent development of multiplex antibody detection provides the opportunity for simultaneous evaluation of the impact of control measures on the burden of multiple diseases. METHODOLOGY/PRINCIPAL FINDINGS: Two cross-sectional household surveys at baseline and one year after a LLIN distribution campaign were implemented in Mecuburi and Nacala-a-Velha Districts in Nampula Province, Mozambique. Both districts were known to be endemic for LF; both received mass drug administration (MDA) with antifilarial drugs during the evaluation period. Access to and use of LLINs was recorded, and household members were tested with P. falciparum rapid diagnostic tests (RDTs). Dried blood spots were collected and analyzed for presence of antibodies to three P. falciparum antigens, P. vivax MSP-119, P. ovale MSP-119, P. malariae MSP-119, and three LF antigens. Seroconversion rates were calculated and the association between LLIN use and post-campaign seropositivity was estimated using multivariate regression. The campaign covered 68% (95% CI: 58-77) of the population in Nacala-a-Velha and 46% (37-56) in Mecuburi. There was no statistically significant change in P. falciparum RDT positivity between the two surveys. Population seropositivity at baseline ranged from 31-81% for the P. falciparum antigens, 3-4% for P. vivax MSP-119, 41-43% for P. ovale MSP-119, 46-56% for P. malariae MSP-119, and 37-76% for the LF antigens. The seroconversion rate to the LF Bm33 antigen decreased significantly in both districts. The seroconversion rate to P. malariae MSP-119 and the LF Wb123 and Bm14 antigens each decreased significantly in one of the two districts. Community LLIN use was associated with a decreased risk of P. falciparum RDT positivity, P. falciparum CSP and LSA-1 seropositivity, and P. malariae MSP-119 seropositivity, but not LF antigen seropositivity. CONCLUSIONS/SIGNIFICANCE: The study area noted significant declines in LF seropositivity, but these were not associated with LLIN use. The MDA could have masked any impact of the LLINs on population LF seropositivity. The LLIN campaign did not reach adequately high coverage to decrease P. falciparum RDT positivity, the most common measure of P. falciparum burden. However, the significant decreases in the seroconversion rate to the P. malariae antigen, coupled with an association between community LLIN use and individual-level decreases in seropositivity to P. falciparum and P. malariae antigens show evidence of impact of the LLIN campaign and highlight the utility of using multiantigenic serological approaches for measuring intervention impact.

BACKGROUND: Rapid diagnostic test (RDT) positivity is supplanting microscopy as the standard measure of malaria burden at the population level. However, there is currently no standard for externally validating RDT results from field surveys. METHODS: Individuals' blood concentration of the Plasmodium falciparum histidine rich protein 2 (HRP2) protein were compared to results of HRP2-detecting RDTs in participants from field surveys in Angola, Mozambique, Haiti, and Senegal. A logistic regression model was used to estimate the HRP2 concentrations corresponding to the 50 and 90% level of detection (LOD) specific for each survey. RESULTS: There was a sigmoidal dose-response relationship between HRP2 concentration and RDT positivity for all surveys. Variation was noted in estimates for field RDT sensitivity, with the 50% LOD ranging between 0.076 and 6.1 ng/mL and the 90% LOD ranging between 1.1 and 53 ng/mL. Surveys conducted in two different provinces of Angola using the same brand of RDT and same study methodology showed a threefold difference in LOD. CONCLUSIONS: Measures of malaria prevalence estimated using population RDT positivity should be interpreted in the context of potentially large variation in RDT LODs between, and even within, surveys. Surveys based on RDT positivity would benefit from external validation of field RDT results by comparing RDT positivity and antigen concentration.

Malaria-endemic countries have to decide how much of their limited resources for vector control to allocate toward implementing long-lasting insecticidal nets (LLINs) versus indoor residual spraying (IRS). To help the Mozambique Ministry of Health use an evidence-based approach to determine funding allocation toward various malaria control strategies, the Global Fund convened the Mozambique Modeling Working Group which then used JANUS, a software platform that includes integrated computational economic, operational, and clinical outcome models that can link with different transmission models (in this case, OpenMalaria) to determine the economic value of vector control strategies. Any increase in LLINs (from 80% baseline coverage) or IRS (from 80% baseline coverage) would be cost-effective (incremental cost-effectiveness ratios <= $114/disability-adjusted life year averted). However, LLIN coverage increases tend to be more cost-effective than similar IRS coverage increases, except where both pyrethroid resistance is high and LLIN usage is low. In high-transmission northern regions, increasing LLIN coverage would be more cost-effective than increasing IRS coverage. In medium-transmission central regions, changing from LLINs to IRS would be more costly and less effective. In low-transmission southern regions, LLINs were more costly and less effective than IRS, due to low LLIN usage. In regions where LLINs are more cost-effective than IRS, it is worth considering prioritizing LLIN coverage and use. However, IRS may have an important role in insecticide resistance management and epidemic control. Malaria intervention campaigns are not a one-size-fits-all solution, and tailored approaches are necessary to account for the heterogeneity of malaria epidemiology.

Detection of histidine-rich protein 2 (HRP2) from the malaria parasite Plasmodium falciparum provides evidence for active or recent infection, and is utilized for both diagnostic and surveillance purposes, but current laboratory immunoassays for HRP2 are hindered by low sensitivities and high costs. Here we present a new HRP2 immunoassay based on antigen capture through a bead-based system capable of detecting HRP2 at sub-picogram levels. The assay is highly specific and cost-effective, allowing fast processing and screening of large numbers of samples. We utilized the assay to assess results of HRP2-based rapid diagnostic tests (RDTs) in different P. falciparum transmission settings, generating estimates for true performance in the field. Through this method of external validation, HRP2 RDTs were found to perform well in the high-endemic areas of Mozambique and Angola with 86.4% and 73.9% of persons with HRP2 in their blood testing positive by RDTs, respectively, and false-positive rates of 4.3% and 0.5%. However, in the low-endemic setting of Haiti, only 14.5% of persons found to be HRP2 positive by the bead assay were RDT positive. Additionally, 62.5% of Haitians showing a positive RDT test had no detectable HRP2 by the bead assay, likely indicating that these were false positive tests. In addition to RDT validation, HRP2 biomass was assessed for the populations in these different settings, and may provide an additional metric by which to estimate P. falciparum transmission intensity and measure the impact of interventions.

BACKGROUND: During 2004-2013 in Mozambique, 455,600 HIV-positive adults (≥15 years old) initiated antiretroviral therapy (ART). We evaluated trends in patient characteristics and outcomes during 2004-2013, outcomes of universal treatment for pregnant women (Option B+) implemented since 2013, and effect on outcomes of distributing ART to stable patients through Community ART Support Groups (CASG) since 2010. METHODS: Data for 306,335 adults starting ART during 2004-2013 at 170 ART facilities were analyzed. Mortality and loss to follow-up (LTFU) were estimated using competing risks models. Outcome determinants were estimated using proportional hazards models, including CASG participation as a time-varying covariate. RESULTS: Compared with ART enrollees in 2004, enrollees in 2013 were more commonly female (55% vs. 73%), more commonly pregnant if female (<1% vs. 30%), and had a higher median baseline CD4 count (139 vs. 235/microL). During 2004-2013, observed 6-month mortality declined from 7% to 2% but LTFU increased from 24% to 30%. Pregnant women starting ART with CD4 count >350/microL and WHO stage I/II under Option B+ guidelines in 2013 had low 6-month mortality (0.1%) but high 6-month LTFU (38%). During 2010-2013, 6,766 patients joined CASGs. In multivariable analysis, compared with non-participation in CASG, CASG participation was associated with 35% lower LTFU but similar mortality. CONCLUSIONS: Initiation of ART at earlier disease stages in later calendar years might explain observed declines in mortality. Retention interventions are needed to address trends of increasing LTFU overall and the high LTFU among Option B+ pregnant women specifically. Further expansion of CASG could help reduce LTFU.

To examine human gene expression during uncomplicated P. falciparum malaria, we obtained three samples (acute illness, treatment, and recovery) from 10 subjects and utilized each subject's recovery sample as their baseline. At the time of acute illness (day 1), subjects had upregulation of innate immune response, cytokine, and inflammation-related genes (IL-1beta, IL-6, TNF, and IFN-gamma), which was more frequent with parasitemias >100,000 per muL and body temperatures ≥39 degrees C. Apoptosis-related genes (Fas, BAX, and TP53) were upregulated acutely and for several days thereafter (days 1-3). In contrast, the expression of immune-modulatory (transcription factor 7, HLV-DOA, and CD6) and apoptosis inhibitory (c-myc, caspase 8, and Fas Ligand G) genes was downregulated initially and returned to normal with clinical recovery (days 7-10). These results indicate that the innate immune response, cytokine, and apoptosis pathways are upregulated acutely in uncomplicated malaria with concomitant downregulation of immune-modulatory and apoptosis inhibitory genes.

OBJECTIVE: Universal coverage with insecticide-treated bed nets is a cornerstone of modern malaria control. Mozambique has developed a novel bed net allocation strategy, where the number of bed nets allocated per household is calculated on the basis of household composition and assumptions about who sleeps with whom. We set out to evaluate the performance of the novel allocation strategy. METHODS: 1,994 households were visited during household surveys following two universal coverage bed net distribution campaigns in Sofala and Nampula Provinces in 2010-2013. Each sleeping space was observed for the presence of a bed net, and the sleeping patterns for each household were recorded. The observed coverage and efficiency were compared to a simulated coverage and efficiency had conventional allocation strategies been used. A composite indicator, the product of coverage and efficiency, was calculated. Observed sleeping patterns were compared with the sleeping pattern assumptions. RESULTS: In households reached by the campaign, 93% (95% CI: 93-94%) of sleeping spaces in Sofala and 84% (82-86%) in Nampula were covered by campaign bed nets. The achieved efficiency was high, with 92% (91-93%) of distributed bed nets in Sofala and 93% (91-95%) in Nampula covering a sleeping space. Using the composite indicator, the novel allocation strategy outperformed all conventional strategies in Sofala and was tied for best in Nampula. The sleeping pattern assumptions were completely satisfied in 66% of households in Sofala and 56% of households in Nampula. The most common violation of the sleeping pattern assumptions was that male children 3-10 years of age tended not to share sleeping spaces with female children 3-10 or 10-16 years of age. CONCLUSIONS: The sleeping pattern assumptions underlying the novel bed net allocation strategy are generally valid, and net allocation using these assumptions can achieve high coverage and compare favorably with conventional allocation strategies.

BACKGROUND: Holes in netting provide potential routes for mosquitoes to enter ITNs. Despite this, there is little information on how mosquitoes respond to holes in bed nets and how their responses are affected by hole size, shape and orientation or by ambient conditions around the net. METHODS: Female Anopheles gambiae (G3) were recorded in a simulated bed net consisting of two sizes of untreated netting-covered behavioural arenas placed above and beside (to simulate the bed net roof and sides respectively) the experimenter who was a source of host cues from 'inside' the net. A round hole of 9 mm or 13 mm diameter was cut into the centre of the netting of each arena. Videos of unfed female mosquitoes in arenas were analysed for time spent flying, walking and standing still and for exit through the hole. The effects of the experimenter on temperature and relative humidity around the simulated net were also measured. RESULTS: Mosquitoes were significantly more active in overhead arenas than in arenas to the side. Hole passage was significantly more likely in smaller arenas than larger ones and for larger holes than smaller ones. In arenas to the side, hole passage rate through small holes was about 50 % less likely than what could be explained by area alone. Passage rate through holes in overhead arenas was consistent with hole area. Temperature in arenas did not strongly reflect the experimenter's presence in the simulated net. Relative humidity and absolute humidity in overhead arenas, but not in arenas to the side, were immediately affected by experimenter presence. CONCLUSIONS: Higher levels of activity in overhead arenas than in arenas to the side were likely due to the rising heat and humidity plume from the experimenter. Lower than expected passage rates through smaller vertically oriented holes may have been be due to an edge effect that does not apply to horizontally oriented holes. Results suggest that current methods of assessing the importance of physical damage to ITNs may not accurately reflect mosquito entry risk in all cases.

West Nile virus has caused several outbreaks among humans in the Phoenix metropolitan area (Arizona, southwest USA) within the last decade. Recent ecologic studies have implicated Culex quinquefasciatus and Culex tarsalis as the mosquito vectors and identified three abundant passerine birds-great-tailed grackle (Quiscalus mexicanus), house sparrow (Passer domesticus), and house finch (Haemorhous mexicanus)-as key amplifiers among vertebrates. Nocturnal congregations of certain species have been suggested as critical for late summer West Nile virus amplification. We evaluated the hypothesis that house sparrow (P. domesticus) and/or great-tailed grackle (Q. mexicanus) communal roost sites (n = 22 and n = 5, respectively) in a primarily suburban environment were spatially associated with West Nile virus transmission indices during the 2010 outbreak of human neurological disease in metropolitan Phoenix. Spatial associations between human case residences and communal roosts were non-significant for house sparrows, and were negative for great-tailed grackle. Several theories that explain these observations are discussed, including the possibility that grackle communal roosts are protective.

BACKGROUND: Malaria is the leading cause of death in Mozambique in children under five years old. In 2009, Mozambique developed a novel bed net distribution model to increase coverage, based on assumptions about sleeping patterns. The coverage and impact of a bed net distribution campaign using this model in four districts in Sofala Province, Mozambique was evaluated. METHODS: Paired household, cross-sectional surveys were conducted one month after the 2010 distribution of 140,000 bed nets and again 14 months after the campaign in 2011. During household visits, malaria blood smears were performed and haemoglobin levels were assessed on children under five and data on bed net ownership, access and use were collected; these indicators were analysed at individual, household and community levels. Logistic regression was used to evaluate predictors of malaria infection and anaemia. RESULTS: The campaign reached 98% (95% CI: 97-99%) of households registered during the precampaign listing, with 81% (95% CI: 77-85%) of sleeping spaces covered by campaign bed nets and 85% (95% CI: 81-88%) of the population sleeping in a sleeping space with a campaign bed net designated for the sleeping space. One year after the campaign, 65% (95% CI: 57-72%) of sleeping spaces were observed to have hanging bed nets. The proportion of sleeping spaces for which bed nets were reported used four or more times per week was 65% (95% CI: 56-74%) in the wet season and 60% (95% CI: 52-68%) in the dry season. Malaria parasitaemia prevalence in children under five years old was 47% (95% CI: 40-54%) in 2010 and 36% (95% CI: 27-45%) in 2011. Individual-level malaria infection and anaemia were significantly associated with community-level use of bed nets. CONCLUSIONS: The campaign using the novel distribution model achieved high coverage, although usage was not uniformly high. A significant decrease in malaria parasitaemia prevalence a year after the campaign was not observed, but community-level use of bed nets was significantly associated with a reduced risk for malaria infection and anaemia in children under five.

High-quality laboratory space to support basic science, clinical research projects, or health services is often severely lacking in the developing world. Moreover, the construction of suitable facilities using traditional methods is time-consuming, expensive, and challenging to implement. Three real world examples showing how shipping containers can be converted into modern laboratories are highlighted. These include use as an insectary, a molecular laboratory, and a BSL-3 containment laboratory. These modular conversions have a number of advantages over brick and mortar construction and provide a cost-effective and timely solution to offer high-quality, user-friendly laboratory space applicable within the developing world.

OBJECTIVE: Acute hepatitis B virus (HBV) infections have been reported in long-term care facilities (LTCFs), primarily associated with infection control breaks during assisted blood glucose monitoring. We investigated HBV outbreaks that occurred in separate skilled nursing facilities (SNFs) to determine factors associated with transmission. DESIGN: Outbreak investigation with case-control studies. SETTING: Two SNFs (facilities A and B) in Durham, North Carolina, during 2009-2010. PATIENTS: Residents with acute HBV infection and controls randomly selected from HBV-susceptible residents during the outbreak period. METHODS: After initial cases were identified, screening was offered to all residents, with repeat testing 3 months later for HBV-susceptible residents. Molecular testing was performed to assess viral relatedness. Infection control practices were observed. Case-control studies were conducted to evaluate associations between exposures and acute HBV infection in each facility. RESULTS: Six acute HBV cases were identified in each SNF. Viral phylogenetic analysis revealed a high degree of HBV relatedness within, but not between, facilities. No evaluated exposures were significantly associated with acute HBV infection in facility A; those associated with infection in facility B (all odds ratios >20) included injections, hospital or emergency room visits, and daily blood glucose monitoring. Observations revealed absence of trained infection control staff at facility A and suboptimal hand hygiene practices during blood glucose monitoring and insulin injections at facility B. CONCLUSIONS: These outbreaks underscore the vulnerability of LTCF residents to acute HBV infection, the importance of surveillance and prompt investigation of incident cases, and the need for improved infection control education to prevent transmission.

In 2010, Arizona experienced an unusually early and severe outbreak of West Nile virus (WNV) centered in the southeast section of Maricopa County. Entomological data were collected before and during the outbreak, from May 25 through July 31, 2010, using the CO-baited light trap monitoring system maintained by Maricopa County Vector Control. In the outbreak area, the most abundant species in the Town of Gilbert and in the area covered by the Roosevelt Water Conservation District was Culex quinquefasciatus, constituting 75.1% and 71.8% of the total number of mosquitoes collected, respectively. Vector index (VI) profiles showed that the abundance of infected Cx. quinquefasciatus peaked prior to human cases, suggesting that this species was involved in the initiation of the outbreak. In contrast, the VI profiles for Cx. tarsalis were consistently low, suggesting limited involvement in initiating and sustaining transmission. Taken together, the higher abundance and the VI profiles strongly suggest that Cx. quinquefasciatus was the primary vector for this outbreak. The VI profiles consistently showed that the abundance of infected mosquitoes peaked 1 to 2 wk before the peaks of human cases, suggesting that VI could have successfully been utilized to predict the WNV outbreak in Maricopa County, AZ, in 2010.

West Nile virus (WNV) is the leading cause of mosquito-borne disease in the United States; however, risk factors for infection are poorly defined. We performed a case-control study to identify modifiable risk factors for WNV infection. Case-patients (N = 49) had laboratory evidence of recent WNV infection, whereas control-subjects (N = 74) had negative WNV serology. We interviewed participants, surveyed households, and assessed environmental data. WNV infection was associated with living in or near Water District X within Gilbert Township (adjusted odds ratio [aOR] 5.2; 95% confidence interval [95% CI] = 1.5-18.1), having water-holding containers in their yard (aOR 5.0; 95% CI = 1.5-17.3), and not working or attending school outside the home (aOR 2.4; 95% CI = 1.1-5.5). During this outbreak, WNV infection was likely primarily acquired peri-domestically with increased risk associated with potential mosquito larval habitats around the home and neighborhood.

We used a whole-genome scanning technique to identify the NADH dehydrogenase gamma subunit (nuoG) primer set that is sensitive and specific enough to detect a diverse number of Bartonella species in a wide range of environmental samples, yet maintained minimal cross-reactivity to mammalian host and arthropod vector organisms.

Ectoparasites, including chigger mites (genera Leptotrombidium, Schoengastia, and Blankarrtia) and one tick (genus Haemaphysalis) collected from wild-caught rodents in Thailand, were assessed for the presence of Bartonella DNA by using a polymerase chain reaction assay targeting the 16S-23S intergenic spacer region and citrate synthase gene (gltA). Of the 41 pooled samples tested, 34 were positive for Bartonella DNA. Sequence analysis demonstrated that DNA detected in 33 chigger mite pools and one tick pool was similar to Bartonella tamiae sequences previously isolated from three patients in Thailand. This is the first report of the detection of B. tamiae DNA in chigger mites; additional field and experimental investigations are required to determine the role of chigger mites as potential vectors of B. tamiae.