A stool based qPCR for the diagnosis of TB in children and people living with HIV in Uganda, Eswatini and Mozambique (Stool4TB): a protocol for a multicenter diagnostic evaluation.

BACKGROUND: Tuberculosis (TB) is a major cause of mortality worldwide. Children and people living with HIV (PLHIV) have an increased risk of mortality, particularly in the absence of rapid diagnosis. The main challenges of diagnosing TB in these populations are due to the unspecific and paucibacillary disease presentation and the difficulty of obtaining respiratory samples. Thus, novel diagnostic strategies, based on non-respiratory specimens could improve clinical decision making and TB outcomes in high burden TB settings. We propose a multi-country, prospective diagnostic evaluation study with a nested longitudinal cohort evaluation to assess the performance of a new stool-based qPCR, developed by researchers at Baylor College of Medicine (Houston, Texas, USA) for TB bacteriological confirmation with promising results in pilot studies. METHODS: The study will take place in high TB/HIV burden countries (Mozambique, Eswatini and Uganda) where we will enroll, over a period of 30 months, 650 PLHIV (> 15) and 1295 children under 8 years of age (irrespective of HIV status) presenting pressumptive TB. At baseline, all participants will provide clinical history, complete a physical assessment, and undergo thoracic chest X-ray imaging. To obtain bacteriological confirmation, participants will provide respiratory samples (1 for adults, 2 in children) and 1 stool sample for Xpert Ultra MTB/RIF (Cepheid, Sunnyvale, CA, USA). Mycobacterium tuberculosis (M.tb) liquid culture will only be performed in respiratory samples and lateral flow lipoarabinomannan (LF-LAM) in urine following WHO recommendations. Participants will complete 2 months follow-up if they are not diagnosed with TB, and 6 months if they are. For analytical purposes, the participants in the pediatric cohort will be classified into "confirmed tuberculosis", "unconfirmed tuberculosis" and "unlikely tuberculosis". Participants of the adult cohort will be classified as "bacteriologically confirmed TB", "clinically diagnosed TB" or "not TB". We will assess accuracy of the novel qPCR test compared to bacteriological confirmation and Tb diagnosis irrespective of laboratory results. Longitudinal qPCR results will be analyzed to assess its use as treatment response monitoring. DISCUSSION: The proposed stool-based qPCR is an innovation because both the strategy of using a non-sputum based sample and a technique specially designed to detect M.tb DNA in stool. PROTOCOL REGISTRATION DETAILS: ClinicalTrials.gov Identifier: NCT05047315.

Detection of Enteric Viruses in Children under Five Years of Age before and after Rotavirus Vaccine Introduction in Manhiça District, Southern Mozambique, 2008-2019.

Enteric viruses are the leading cause of diarrhoea in children <5 years. Despite existing studies describing rotavirus diarrhoea in Mozambique, data on other enteric viruses remains scarce, especially after rotavirus vaccine introduction. We explored the prevalence of norovirus GI and GII, adenovirus 40/41, astrovirus, and sapovirus in children <5 years with moderate-to-severe (MSD), less severe (LSD) diarrhoea and community healthy controls, before (2008-2012) and after (2016-2019) rotavirus vaccine introduction in Manhiça District, Mozambique. The viruses were detected using ELISA and conventional reverse transcription PCR from stool samples. Overall, all of the viruses except norovirus GI were significantly more detected after rotavirus vaccine introduction compared to the period before vaccine introduction: norovirus GII in MSD (13/195, 6.7% vs. 24/886, 2.7%, respectively; p = 0.006) and LSD (25/268, 9.3% vs. 9/430, 2.1%, p < 0.001); adenovirus 40/41 in MSD (7.2% vs. 1.8%, p < 0.001); astrovirus in LSD (7.5% vs. 2.6%, p = 0.002); and sapovirus in MSD (7.1% vs. 1.4%, p = 0.047) and controls (21/475, 4.4% vs. 51/2380, 2.1%, p = 0.004). Norovirus GII, adenovirus 40/41, astrovirus, and sapovirus detection increased in MSD and LSD cases after rotavirus vaccine introduction, supporting the need for continued molecular surveillance for the implementation of appropriate control and prevention measures.

Improving tuberculosis case detection through contact risk stratification by Xpert MTB/RIF Ultra and spatial parameters: Evaluation of an innovative active case finding strategy in Mozambique (Xpatial-TB).

Prompt diagnosis is critical for tuberculosis (TB) control, as it enables early treatment which in turn, reduces transmission and improves treatment outcomes. We investigated the impact on TB diagnosis of introducing Xpert Ultra as the frontline diagnostic test, combined with an innovative active-case finding (ACF) strategy (based on Xpert Ultra semi-quantitative results and spatial parameters), in a semi-rural district of Southern Mozambique. From January-December 2018 we recruited incident TB-cases (index cases, ICs) and their household contacts (HCs). Recruitment of close community contacts (CCs) depended on IC´s Xpert Ultra results, and the population density of their area. TB-contacts, either symptomatic or people living with HIV, were asked to provide a spot sputum for lab-testing. Trends on TB case notification were compared to the previous years and to those of two districts in the south of the Maputo province (control area), using an interrupted time series analysis with and without control (CITS/ITS). A total of 1010 TB ICs (37.1% laboratory-confirmed) were recruited; 3165 HCs and 4730 CCs were screened for TB. Eighty-nine additional TB cases were identified through the ACF intervention (52.8% laboratory-confirmed). The intervention increased by 8.2% all forms of TB cases detected in 2018. Xpert Ultra trace positive results accounted for a high proportion of laboratory confirmations in the ACF cohort (51.1% vs 13.7% of those passively diagnosed). The Number Needed to Screen to find a TB case differed widely among HCs (55) and CCs (153). During the intervention period, a reversal of the previous negative trend in lab-confirmed case notifications was observed in the district. However, the CITS model did not show any statistically significant difference compared to the control area. Paediatric population benefited the most from the ACF strategy and HCs screening seemed an effective intervention to find microbiological confirmed cases in early stages of the disease.

Performance of a stool-based quantitative PCR assay for the diagnosis of tuberculosis in adolescents and adults: a multinational, prospective diagnostic accuracy study.

BACKGROUND: Despite increasing availability of rapid molecular tests for the diagnosis of tuberculosis in high-burden settings, many people with tuberculosis are undiagnosed. Reliance on sputum as the primary specimen for tuberculosis diagnostics contributes to this diagnostic gap. We evaluated the diagnostic accuracy and additive yield of a novel stool quantitative PCR (qPCR) assay for the diagnosis of tuberculosis in three countries in Africa with high tuberculosis burdens. METHODS: We undertook a prospective diagnostic accuracy study in Eswatini, Mozambique, and Tanzania from Sept 21, 2020, to Feb 2, 2023, to compare the diagnostic accuracy for tuberculosis of a novel stool qPCR test with the current diagnostic standard for Mycobacterium tuberculosis DNA detection from sputum and stool, Xpert-MTB/RIF Ultra (Xpert Ultra). Sputum, stool, and urine samples were provided by a cohort of participants, aged 10 years or older, diagnosed with tuberculosis. Participants with tuberculosis (cases) were enrolled within 72 h of treatment initiation for tuberculosis diagnosed clinically or following laboratory confirmation. Participants without tuberculosis (controls) consisted of household contacts of the cases who did not develop tuberculosis during a 6-month follow-up. The performance was compared with a robust composite microbiological reference standard (CMRS). FINDINGS: The cohort of adolescents and adults (n=408) included 268 participants with confirmed or clinical tuberculosis (cases), 147 (55%) of whom were living with HIV, and 140 participants (controls) without tuberculosis. The sensitivity of the novel stool qPCR was 93·7% (95% CI 87·4-97·4) compared with participants with detectable growth on M tuberculosis culture, and 88·1% (81·3-93·0) compared with sputum Xpert Ultra. The stool qPCR had an equivalent sensitivity as sputum Xpert Ultra (94·8%, 89·1-98·1) compared with culture. Compared with the CMRS, the sensitivity of the stool qPCR was higher than the current standard for tuberculosis diagnostics on stool, Xpert Ultra (80·4%, 73·4-86·2 vs 73·5%, 66·0-80·1; p=0·025 on paired comparison). The qPCR also identified 17-21% additional tuberculosis cases compared to sputum Xpert Ultra or sputum culture. In controls without tuberculosis, the specificity of the stool qPCR was 96·9% (92·2-99·1). INTERPRETATION: In this study, a novel qPCR for the diagnosis of tuberculosis from stool specimens had a higher accuracy in adolescents and adults than the current diagnostic PCR gold standard on stool, Xpert-MTB/RIF Ultra, and equivalent sensitivity to Xpert-MTB/RIF Ultra on sputum. FUNDING: National Institutes of Health (NIH) Allergy and Infectious Diseases, and NIH Fogarty International Center.

Rotavirus disease burden pre-vaccine introduction in young children in Rural Southern Mozambique, an area of high HIV prevalence

Rotavirus vaccines have been adopted in African countries since 2009, including Mozambique (2015). Disease burden data are needed to evaluate the impact of rotavirus vaccine. We report the burden of rotavirus-associated diarrhea in Mozambique from the Global Enteric Multicenter Study (GEMS) before vaccine introduction. Methods: A case-control study (GEMS), was conducted in Manhiça district, recruiting children aged 0-59 months with moderate-to-severe diarrhea (MSD) and less-severe-diarrhea (LSD) between December 2007 and November 2012; including 1-3 matched (age, sex and neighborhood) healthy community controls. Clinical and epidemiological data and stool samples (for laboratory investigation) were collected. Association of rotavirus with MSD or LSD was determined by conditional logistic regression and adjusted attributable fractions (AF) calculated, and risk factors for rotavirus diarrhea assessed. Results: Overall 915 cases and 1,977 controls for MSD, and 431 cases and 430 controls for LSD were enrolled. Rotavirus positivity was 44% (217/495) for cases and 15% (160/1046) of controls, with AF = 34.9% (95% CI: 32.85-37.06) and adjusted Odds Ratio (aOR) of 6.4 p< 0.0001 in infants with MSD compared to 30% (46/155) in cases and 14% (22/154) in controls yielding AF = 18.7%, (95% CI: 12.02-25.39) and aOR = 2.8, p = 0.0011 in infants with LSD. The proportion of children with rotavirus was 32% (21/66) among HIV-positive children and 23% (128/566) among HIV-negative ones for MSD. Presence of animals in the compound (OR = 1.9; p = 0.0151) and giving stored water to the child (OR = 2.0, p = 0.0483) were risk factors for MSD; while animals in the compound (OR = 2.37, p = 0.007); not having routine access to water on a daily basis (OR = 1.53, p = 0.015) and washing hands before cooking (OR = 1.76, p = 0.0197) were risk factors for LSD. Conclusion: The implementation of vaccination against rotavirus may likely result in a significant reduction of rotavirus-associated diarrhea, suggesting the need for monitoring of vaccine impact.

Risk factors for death among children 0-59 months of age with moderate-to-severe diarrhea in Manhiça district, southern Mozambique

In spite of very significant decreasing trends in the last three decades, childhood mortality remains unacceptably high globally, with 5.6 million children dying every year before reaching their 5th birthday, the majority of which in low and middle-income countries (LMIC) [1]. Diarrheal diseases still represent a major cause of morbidity and mortality in childhood, and are believed to account for 499,000­526,000 annual child deaths, nearly 9% of all under five global mortality [2, 3], in spite of the good intake of life-saving interventions such as oral rehydration solution (ORS) [3] and the rotavirus vaccine [4]. Deaths in sub-Saharan Africa and Southeast Asia account for ~ 78% of deaths due to diarrhea worldwide [5, 6], underscoring the inequities related to this particular syndrome. The Global Enteric Multi-center Study (GEMS) reported that most episodes of moderate-to-severe diarrhea (MSD) among children under 5 years old were primarily due to four pathogens: Rotavirus, Cryptosporidium, Enterotoxigenic Escherichia coli (ST-ETEC), and Shigella [7]. The risk of dying from diarrheal disease was reported to be higher among children younger than 2 years of age, albeit with relatively different rates from one region to another [7]. The advent of the HIV/AIDS pandemic has also changed the incidence [8], clinical presentation and outcome of diarrheal diseases, as the immunosuppression derived from the infection favors a higher incidence of gastrointestinal infections, not only from "classical" diarrhea pathogens, but also from more aggressive opportunistic infections, typical of the immunocompromised host. This has resulted in significant changes in the last decades in the spectrum, clinical presentation, duration and prognosis of diarrheal episodes in those countries where HIV is highly prevalent [9, 10].

Postdischarge Mortality Prediction in Sub-Saharan Africa

Background: Although the burden of postdischarge mortality (PDM) in low-income settings appears to be significant, no clear recommendations have been proposed in relation to follow-up care after hospitalization. We aimed to determine the burden of pediatric PDM and develop predictive models to identify children who are at risk for dying after discharge. Methods: Deaths after hospital discharge among children aged <15 years in the last 17 years were reviewed in an area under demographic and morbidity surveillance in Southern Mozambique. We determined PDM over time (up to 90 days) and derived predictive models of PDM using easily collected variables on admission. Results: Overall PDM was high (3.6%), with half of the deaths occurring in the first 30 days. One primary predictive model for all ages included young age, moderate or severe malnutrition, a history of diarrhea, clinical pneumonia symptoms, prostration, bacteremia, having a positive HIV status, the rainy season, and transfer or absconding, with an area under the curve of 0.79 (0.75-0.82) at day 90 after discharge. Alternative models for all ages including simplified clinical predictors had a similar performance. A model specific to infants <3 months old was used to identify as predictors being a neonate, having a low weight-for-age z score, having breathing difficulties, having hypothermia or fever, having oral candidiasis, and having a history of absconding or transfer to another hospital, with an area under the curve of 0.76 (0.72-0.91) at day 90 of follow-up. Conclusions: Death after discharge is an important although poorly recognized contributor to child mortality. A simple predictive algorithm based on easily recognizable variables could readily be used to identify most infants and children who are at a high risk of dying after discharge.

Rotavirus a strains obtained from children with acute gastroenteritis in mozambique, 2012-2013: g and p genotypes and phylogenetic analysis of vp7 and partial vp4 genes

In Mozambique rotavirus (RV) was shown to be the greatest cause of acute diarrhoea in infants from 0 to 11 months, and in 2015, national rotavirus vaccination was introduced. As with other developing countries, there is very limited active strain characterisation. Rotavirus positive clinical specimens, collected between 2012 and 2013, have now provided information on the genotypes circulating in southern Mozambique prior to vaccine introduction. Genotypes G2 (32.4%), G12 (28.0%), P[4] (41.4%) and P[6] (22.9%) (n = 157) strains were commonly detected with G2P[4] (42.3%) RVs being predominant, specifically during 2013. Phylogenetic evaluation of the VP7 and VP8* encoding genes showed, for the majority of the Mozambican strains, that they clustered with other African strains based on genotype. RVA/Human-wt/MOZ/0153/2013/G2P[4], RVA/Human-wt/MOZ/0308/2012/G2P[4] and RVA/Human-wt/MOZ/0288/2012/G12P[8] formed separate clusters from the other Mozambican strains with similar genotypes, suggesting possible reassortment. Amino acid substitutions in selected epitope regions also supported phylogenetic clustering. As expected, the VP7 and VP8* genes from the Mozambican strains differed from both the RotaTeq® (SC2-9) G2P[5] and Rotarix® (A41CB052A) G1P[8] genes. This study provides information on the genetic diversity of rotavirus strains prior to vaccine introduction and generates baseline data for future monitoring of any changes in rotavirus strains in response to vaccine pressure.

The role of HIV infection in the etiology and epidemiology of diarrheal disease among children aged 0­59 months in Manhiça District, Rural Mozambique

The Global Enteric Multicenter Study enrolled children with moderate-to-severe diarrhea (MSD) and less-severe diarrhea (LSD) between December 2007 and November 2012. One to three controls for MSD cases and one per LSD case were enrolled and matched by age, sex, and neighborhood. All children were tested for HIV. Clinical data, anthropometric data, and stool samples were collected. Follow-up was performed at 60 days. Results Two hundred and fourteen MSD cases and 418 controls, together with 349 LSD cases and 214 controls were tested. HIV prevalence was 25% among MSD cases (4% for matched controls) and 6% among LSD cases (6% among matched controls). HIV-infected children were more likely to have MSD (odds ratio 5.6, p < 0.0001). Mortality rates were higher among HIV-infected children than among the uninfected (34 vs. 5 per 1000 child-weeks at risk; p = 0.0039). Cryptosporidium, Giardia, and enteroaggregative Escherichia coli (aatA only) were more prevalent among HIV-infected MSD cases than among uninfected ones.

Nasopharyngeal carriage of Streptococcus pneumoniae among HIV-infected and -uninfected children <5 years of age before introduction of pneumococcal conjugate vaccine in Mozambique

Nasopharyngeal carriage is a precursor for pneumococcal disease and can be useful for evaluating pneumococcal conjugate vaccine (PCV) impact. We studied pre-PCV pneumococcal carriage among HIV-infected and -uninfected children in Mozambique. Between October 2012 and March 2013, we enrolled HIV-infected children age <5 years presenting for routine care at seven HIV clinics in 3 sites, including Maputo (urban-south), Nampula (urban-north), and Manhiça (rural-south). We also enrolled a random sample of HIV-uninfected children <5 years old from a demographic surveillance site in Manhiça. A single nasopharyngeal swab was obtained and cultured following enrichment in Todd Hewitt broth with yeast extract and rabbit serum. Pneumococcal isolates were serotyped by Quellung reaction and multiplex polymerase chain reaction. Factors associated with pneumococcal carriage were examined using logistic regression. Overall pneumococcal carriage prevalence was 80.5% (585/727), with similar prevalences among HIV-infected (81.5%, 339/416) and HIV-uninfected (79.1%, 246/311) children, and across age strata. Among HIV-infected, after adjusting for recent antibiotic use and hospitalization, there was no significant association between study site and colonization: Maputo (74.8%, 92/123), Nampula (83.7%, 82/98), Manhiça (84.6%, 165/195). Among HIV-uninfected, report of having been born to an HIV-infected mother was not associated with colonization. Among 601 pneumococcal isolates from 585 children, serotypes 19F (13.5%), 23F (13.1%), 6A (9.2%), 6B (6.2%) and 19A (5.2%) were most common. The proportion of serotypes included in the 10- and 13-valent vaccines was 44.9% and 61.7%, respectively, with no significant differences by HIV status or age group. Overall 36.9% (n = 268) of children were colonized with a PCV10 serotype and 49.7% (n = 361) with a PCV13 serotype. Pneumococcal carriage was common, with little variation by geographic region, age, or HIV status. PCV10 was introduced in April 2013; ongoing carriage studies will examine the benefits of PCV10 among HIV-infected and-uninfected children.

Molecular Characterization of Community Acquired Staphylococcus aureus Bacteremia in Young Children in Southern Mozambique, 2001-2009

The emergence of community-acquired Staphylococcus aureus infections is increasingly recognized as life threating problem worldwide. In Manhiça district, southern Mozambique, S. aureus is the leading cause of community-acquired bacteremia in neonates. Methods: Eighty-four S. aureus isolates from children less than 5 years admitted to Manhiça District Hospital from 2001 to 2009 were randomly selected and genetically characterized by DNA microarray and spa typing. Antimicrobial susceptibility was determined by VITEK 2. Results: Thirty-eight different spa types and 14 clonal complexes (CC) were identified. Spa-type t084 (n = 10; 12%) was the most predominant while CC8 (n = 18; 21%) and CC15 (n = 14; 16%) were the most frequent CCs. Mortality tended to be higher among children infected with CC45 (33.3%, 1/3) and CC8 (27.8%, 5/18). The majority of isolates possessed the accessory gene regulator I (45%) and belonged to either capsule type 8 (52%) or 5 (47%). Panton valentine leukocidin (PVL) encoding genes were detected in 30%. Antibiotic resistance was high for penicillin (89%), tetracycline (59%) and Trimethoprim Sulfamethoxazole (36%) while MRSA was uncommon (8%). Conclusions: Although MRSA were uncommon, we found high genetic diversity of methicillin susceptible S. aureus causing bacteremia in Mozambican children, associated with high resistance to the most available antibiotics in this community. Some CCs are likely to be more lethal indicating the need for prompt recognition and appropriate treatment.

Anaemia in hospitalised preschool children from a rural area in Mozambique: a case control study in search for aetiological agents

Young children bear the world's highest prevalence of anaemia, the majority of which is of multifactorial aetiology, which in turn hampers its successful prevention. Even moderate degrees of anaemia are associated with increased mortality and morbidity. Despite this evidence, there is a lack of effective preventive programs and absence of consensus in the safety of iron supplementation in malaria areas, which reflects the poor understanding of the contribution of different aetiologies to anaemia. In order to reduce the anaemia burden in the most vulnerable population, a study to determine the aetiology of anaemia among pre-school Mozambican children was performed. Methods: We undertook a case-control study of 443 preschool hospitalized children with anaemia (haemoglobin concentration <11 g/dl) and 289 community controls without anaemia. Inclusion criteria were: age 1-59 months, no blood transfusion in the previous month, residence in the study area and signed informed consent. Both univariable and multivariable logistic regression analyses were performed to identify factors associated with anaemia and adjusted attributable fractions (AAF) were estimated when appropriate. Results: Malaria (adjusted odds ratio (AOR) = 8.39, p < 0.0001; AAF = 37%), underweight (AOR = 8.10, p < 0.0001; AAF = 43%), prealbumin deficiency (AOR = 7.11, p < 0.0001; AAF = 77%), albumin deficiency (AOR = 4.29, p = 0.0012; AAF = 30%), HIV (AOR = 5.73, p = 0.0060; AAF = 18%), and iron deficiency (AOR = 4.05, p < 0.0001; AAF = 53%) were associated with anaemia. Vitamin A deficiency and α-thalassaemia were frequent (69% and 64%, respectively in cases) but not independently related to anaemia. Bacteraemia (odds ratio (OR) = 8.49, p = 0.004), Parvovirus-B19 (OR = 6.05, p = 0.017) and Epstein-Barr virus (OR = 2.10, p = 0.0015) infections were related to anaemia only in the unadjusted analysis. Neither vitamin B12 deficiency nor intestinal parasites were associated with anaemia. Folate deficiency was not observed. Conclusions: Undernutrition, iron deficiency, malaria, and HIV are main factors related to anaemia in hospitalised Mozambican preschool children. Effective programs and strategies for the prevention and management of these conditions need to be reinforced. Specifically, prevention of iron deficiency that accounted in this study for more than half of anaemia cases would have a high impact in reducing the burden of anaemia in children living under similar conditions. However this deficiency, a common preventable and treatable condition, remains neglected by the international public health community.

Hypoglycemia and risk factors for death in 13 years of pediatric admissions in mozambique

Abstract. Hypoglycemia is a life-threatening complication of several diseases in childhood. We describe the prevalence and incidence of hypoglycemia among admitted Mozambican children, establishing its associated risk factors. We retro spectively reviewed clinical data of 13 years collected through an ongoing systematic morbidity surveillance in Manhiça District Hospital in rural Mozambique. Logistic regression was used to identify risk factors for hypoglycemia and death. Minimum community-based incidence rates (MCBIRs) for hypoglycemia were calculated using data from the demo graphic surveillance system. Of 49,089 children < 15 years hospitalized in Manhiça District Hospital, 45,573 (92.8%) had a glycemia assessment on admission. A total of 1,478 children (3.2%) presented hypoglycemia (< 3 mmol/L), of which about two-thirds (972) were with levels < 2.5 mmol/L. Independent risk factors for hypoglycemia on admission and death among hypoglycemic children included prostration, unconsciousness, edema, malnutrition, and bacteremia. Hypoglycemic children were significantly more likely to die (odds ratio [OR] = 7.11; P < 0.001), with an associated case fatality rate (CFR) of 19.3% (245/1,267). Overall MCBIR of hypoglycemia was 1.57 episodes/1,000 child years at risk (CYAR), sig nificantly decreasing throughout the study period. Newborns showed the highest incidences (9.47 episodes/1,000 CYAR, P < 0.001). Hypoglycemia remains a hazardous condition for African children. Symptoms and signs associated to hypogly cemia should trigger the verification of glycemia and the implementation of life-saving corrective measures.

Infecções invasivas por salmonella entre crianças da zona rural de moçambique, 2001­2014

Invasive nontyphoidal Salmonella (iNTS) has emerged as a cause of bacteremia in African children and HIV-infected adults, which is associated with high mortality. Epidemiological data and burden of iNTS infections in resource-constrained settings are needed to better define preventive and curative strategies. Methods. Blood and, if appropriate, cerebrospinal fluid, were collected from children <15 years of age with fever or severe disease admitted to the Manhiça District Hospital and cultured for NTS; isolates were then characterized. Results. From January 2001 to December 2014, 41 668 of the 51 878 admitted children had a blood culture performed. Invasive NTS was isolated from 670 (1.6%) specimens collected from 41 668 patients; 69 (10.3% died). Salmonella enterica subspecies enterica serovar Typhi or Salmonella enterica subspecies enterica serovar Paratyphi A or C were only isolated in 14 (0.03%) patients. A total of 460 of 620 (74.2%) NTS isolates serotyped were Salmonella enterica subspecies enterica serovar Typhimurium (45% [116/258] of which were multilocus sequence type 313). The incidence of iNTS was 61.8 (95% confidence interval, 55.4­68.9) cases per 100 000 child-years, being highest among infants (217.7 cases/100 000 child-years). The incidence of iNTS declined significantly (P < .0001) over time, but the case fatality ratio remained constant at approximately 10%. Antimicrobial resistance of iNTS against most available antimicrobials has steadily increased, with a predominance of multidrug-resistant strains. Conclusions. The decreasing but still high incidence of iNTS, its high associated case fatality ratio, and the common detection of multidrug-resistant strains call for a need to improve treatment and prevention strategies for iNTS.

Changing Trends in P. falciparum Burden, Immunity, and Disease in Pregnancy

Background: Prevention of reinfection and resurgence is an integral component of the goal to eradicate malaria. However, the adverse effects of malaria resurgences are not known. Methods: We assessed the prevalence of Plasmodium falciparum infection among 1819 Mozambican women who delivered infants between 2003 and 2012. We used microscopic and histologic examination and a quantitative polymerase-chain-reaction (qPCR) assay, as well as flow-cytometric analysis of IgG antibody responses against two parasite lines. Results: Positive qPCR tests for P. falciparum decreased from 33% in 2003 to 2% in 2010 and increased to 6% in 2012, with antimalarial IgG antibody responses mirroring these trends. Parasite densities in peripheral blood on qPCR assay were higher in 2010-2012 (geometric mean [±SD], 409±1569 genomes per microliter) than in 2003-2005 (44±169 genomes per microliter, P=0.02), as were parasite densities in placental blood on histologic assessment (50±39% of infected erythrocytes vs. 4±6%, P<0.001). The malaria-associated reduction in maternal hemoglobin levels was larger in 2010-2012 (10.1±1.8 g per deciliter in infected women vs. 10.9±1.7 g per deciliter in uninfected women; mean difference, -0.82 g per deciliter; 95% confidence interval [CI], -1.39 to -0.25) than in 2003-2005 (10.5±1.1 g per deciliter vs. 10.6±1.5 g per deciliter; difference, -0.12 g per deciliter; 95% CI, -0.67 to 0.43), as was the reduction in birth weight (2863±440 g in women with past or chronic infections vs. 3070±482 g in uninfected women in 2010-2012; mean difference, -164.5 g; 95% CI, -289.7 to -39.4; and 2994±487 g vs. 3117±455 g in 2003-2005; difference, -44.8 g; 95% CI, -139.1 to 49.5). Conclusions: Antimalarial antibodies were reduced and the adverse consequences of P. falciparum infections were increased in pregnant women after 5 years of a decline in the prevalence of malaria. (Funded by Malaria Eradication Scientific Alliance and others).