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.

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.

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.

Antimicrobial resistance and clonality of Staphylococcus aureus causing bacteraemia in children admitted to the Manhiça District Hospital, Mozambique, over two decades.

Background: Staphylococcus aureus is one of the main causes of bacteraemia, associated with high mortality, mainly due to the occurrence of multidrug resistant (MDR) strains. Data on antibiotic susceptibility and genetic lineages of bacteraemic S. aureus are still scarce in Mozambique. The study aims to describe the antibiotic susceptibility and clonality of S. aureus isolated from blood cultures of children admitted to the Manhiça District Hospital over two decades (2001-2019). Methods: A total of 336 S. aureus isolates detected in blood cultures of children aged <5 years were analyzed for antibiotic susceptibility by disk diffusion or minimal inhibitory concentration, and for the presence of resistance determinants by PCR. The clonality was evaluated by SmaI-PFGE, spa typing, and MLST. The SCCmec element was characterized by SCCmec typing. Results: Most S. aureus (94%, 317/336) were resistant to at least one class of antibiotics, and one quarter (25%) showed a MDR phenotype. High rates of resistance were detected to penicillin (90%) and tetracycline (48%); followed by erythromycin/clindamycin (25%/23%), and co-trimoxazole (11%), while resistance to methicillin (MRSA strains) or gentamicin was less frequent (≤5%). The phenotypic resistance to distinct antibiotics correlated well with the corresponding resistance determinants (Cohen's κ test: 0.7-1.0). Molecular typing revealed highly diverse clones with predominance of CC5 (17%, 58/336) and CC8 (16%), followed by CC15 (11%) and CC1 (11%). The CC152, initially detected in 2001, re-emerged in 2010 and became predominant throughout the remaining surveillance period, while other CCs (CC1, CC5, CC8, CC15, CC25, CC80, and CC88) decreased over time. The 16 MRSA strains detected belonged to clones t064-ST612/CC8-SCCmecIVd (69%, 11/16), t008-ST8/CC8-SCCmecNT (25%, 4/16) and t5351-ST88/CC88-SCCmecIVa (6%, 1/16). Specific clonal lineages were associated with extended length of stay and high in-hospital mortality. Conclusion: We document the circulation of diverse MDR S. aureus causing paediatric bacteraemia in Manhiça district, Mozambique, requiring a prompt recognition of S. aureus bacteraemia by drug resistant clones to allow more targeted clinical management of patients.

Evaluation of Omnigene-Sputum for Preservation of Sputum Samples for Diagnosis of Mycobacterium tuberculosis.

In several low-income countries, the transport of sputa could take up to one week to reach the laboratories, resulting in increased contamination rates and a loss of growth. The aim of this study was to evaluate the effect of the OMNIgene-SPUTUM in preserving Mycobacterium tuberculosis on sputum samples simulating three hypothetical scenarios for conservation and/or decontamination: (1) sputum was mixed with OMN and conserved at room temperature for five days and then processed for culture (OMN); (2) sputum cultures followed the routine standing operating procedure at day 0 (STD); and (3) sputum samples were kept at room temperature for five days and mixed with the standard decontamination reagent (SDT5) and then processed for culture. The positivity rate based on smear microscopy was 36.4%, 29.1%, and 27.3% for STD, STD5, and OMN, respectively. The proportion of positive results by liquid culture (MGIT) was 39.1% (43/110) for STD, 26.4% (29/110) for STD5, and 20.0% for OMN (22/110). The overall concordance of liquid culture results was 51.8% (57/110): 37.3% (41/110) for negative results, 11.8% (13/110) for MTBC growth, and 2.7% (3/110) for contaminated results. The OMN arm showed better performance in solid culture than in liquid culture, with a notable reduction in contaminated results.

Genomic characterization of the rotavirus G3P[8] strain in vaccinated children, reveals possible reassortment events between human and animal strains in Manhiça District, Mozambique.

Mozambique introduced the rotavirus vaccine (Rotarix®; GlaxoSmithKline Biologicals, Rixensart, Belgium) in 2015, and since then, the Centro de Investigação em Saúde de Manhiça has been monitoring its impact on rotavirus-associated diarrhea and the trend of circulating strains, where G3P[8] was reported as the predominant strain after the vaccine introduction. Genotype G3 is among the most commonly detected Rotavirus strains in humans and animals, and herein, we report on the whole genome constellation of G3P[8] detected in two children (aged 18 months old) hospitalized with moderate-to-severe diarrhea at the Manhiça District Hospital. The two strains had a typical Wa-like genome constellation (I1-R1-C1-M1-A1-N1-T1-E1-H1) and shared 100% nucleotide (nt) and amino acid (aa) identities in 10 gene segments, except for VP6. Phylogenetic analysis demonstrated that genome segments encoding VP7, VP6, VP1, NSP3, and NSP4 of the two strains clustered most closely with porcine, bovine, and equine strains with identities ranging from 86.9-99.9% nt and 97.2-100% aa. Moreover, they consistently formed distinct clusters with some G1P[8], G3P[8], G9P[8], G12P[6], and G12P[8] strains circulating from 2012 to 2019 in Africa (Mozambique, Kenya, Rwanda, and Malawi) and Asia (Japan, China, and India) in genome segments encoding six proteins (VP2, VP3, NSP1-NSP2, NSP5/6). The identification of segments exhibiting the closest relationships with animal strains shows significant diversity of rotavirus and suggests the possible occurrence of reassortment events between human and animal strains. This demonstrates the importance of applying next-generation sequencing to monitor and understand the evolutionary changes of strains and evaluate the impact of vaccines on strain diversity.

Prognostic accuracy of biomarkers of immune and endothelial activation in Mozambican children hospitalized with pneumonia.

Pneumonia is a leading cause of child mortality. However, currently we lack simple, objective, and accurate risk-stratification tools for pediatric pneumonia. Here we test the hypothesis that measuring biomarkers of immune and endothelial activation in children with pneumonia may facilitate the identification of those at risk of death. We recruited children <10 years old fulfilling WHO criteria for pneumonia and admitted to the Manhiça District Hospital (Mozambique) from 2010 to 2014. We measured plasma levels of IL-6, IL-8, Angpt-2, sTREM-1, sFlt-1, sTNFR1, PCT, and CRP at admission, and assessed their prognostic accuracy for in-hospital, 28-day, and 90-day mortality. Healthy community controls, within same age strata and location, were also assessed. All biomarkers were significantly elevated in 472 pneumonia cases versus 80 controls (p<0.001). IL-8, sFlt-1, and sTREM-1 were associated with in-hospital mortality (p<0.001) and showed the best discrimination with AUROCs of 0.877 (95% CI: 0.782 to 0.972), 0.832 (95% CI: 0.729 to 0.935) and 0.822 (95% CI: 0.735 to 0.908), respectively. Their performance was superior to CRP, PCT, oxygen saturation, and clinical severity scores. IL-8, sFlt-1, and sTREM-1 remained good predictors of 28-day and 90-day mortality. These findings suggest that measuring IL-8, sFlt-1, or sTREM-1 at hospital presentation can guide risk-stratification of children with pneumonia, which could enable prioritized care to improve survival and resource allocation.

Epidemiology and clinical presentation of community-acquired Staphylococcus aureus bacteraemia in children under 5 years of age admitted to the Manhiça District Hospital, Mozambique, 2001-2019.

Staphylococcus aureus bacteraemia (SAB) is one of the most common bloodstream infections globally. Data on the burden and epidemiology of community-acquired SAB in low-income countries are scarce but needed to define preventive and management strategies. Blood samples were collected from children < 5 years of age with fever or severe disease admitted to the Manhiça District Hospital for bacterial isolation, including S. aureus. Between 2001 and 2019, 7.6% (3,197/41,891) of children had bacteraemia, of which 12.3% corresponded to SAB. The overall incidence of SAB was 56.1 episodes/100,000 children-years at risk (CYAR), being highest among neonates (589.8 episodes/100,000 CYAR). SAB declined significantly between 2001 and 2019 (322.1 to 12.5 episodes/100,000 CYAR). In-hospital mortality by SAB was 9.3% (31/332), and significantly associated with infections by multidrug-resistant (MDR) strains (14.7%, 11/75 vs. 6.9%, 14/204 among non-MDR, p = 0.043) and methicillin-resistant S. aureus (33.3%, 5/15 vs. 7.6%, 20/264 among methicillin-susceptible S. aureus, p = 0.006). Despite the declining rates of SAB, this disease remains an important cause of death among children admitted to MDH, possibly in relation to the resistance to the first line of empirical treatment in use in our setting, suggesting an urgent need to review current policy recommendations.

Impact of rotavirus vaccination on diarrheal hospitalizations in children younger than 5 years of age in a rural southern Mozambique.

BACKGROUND: Rotavirus vaccine(Rotarix®) was introduced in Mozambique through its Expanded Program of Immunization in September 2015. We assessed the impact of rotavirus vaccination on childhood gastroenteritis-associated hospitalizations post-vaccine introduction in a high HIV prevalence rural setting of southern Mozambique. METHODS: We reviewed and compared the trend of hospitalizations (prevalence) and incidence rates of acute gastroenteritis (AGE), and rotavirus associated-diarrhea (laboratory confirmed rotavirus) in pre- (January 2008-August 2015) and post-rotavirus vaccine introduction periods (September 2015-December 2020), among children <5 years of age admitted to Manhiça District Hospital. RESULTS: From January 2008 to December 2020, rotavirus vaccination was found to contribute to the decline of the prevalence of AGE from 19% (95% CI: 18.14-20.44) prior to the vaccine introduction to 10% (95% CI: 8.89-11.48) in the post-introduction period, preventing 40% (95 % IE: 38-42) and 84% (95 % IE: 80-87) of the expected AGE and laboratory confirmed rotavirus cases, respectively, among infants. Similarly, the overall incidence of rotavirus was 11.8-fold lower in the post-vaccine introduction period (0.4/1000 child-years-at-risk [CYAR]; 95% CI: 0.3-0.6) compared with the pre-vaccination period (4.7/1000 CYAR; 95% CI: 4.2-5.1) with the highest reduction being observed among infants (16.8-fold lower from the 15.1/1000 CYAR in the pre-vaccine to 0.9/1000 CYAR in the post-vaccine eras). CONCLUSIONS: We documented a significant reduction in all-cause diarrhea hospitalizations and rotavirus positivity after vaccine introduction demonstrating the beneficial impact of rotavirus vaccination in a highly vulnerable population.

The epidemiology of severe malaria at Manhiça District Hospital, Mozambique: a retrospective analysis of 20 years of malaria admissions surveillance data.

BACKGROUND: Most malaria burden estimates rely on modelling infection prevalence to case incidence data, with insufficient attention having been paid to the changing clinical presentation of severe disease and its relationship with changing transmission intensity. We present 20 years of longitudinal surveillance data to contribute to the understanding of the relationship between malaria transmission and the burden and clinical presentation of severe malaria and to inform policy. METHODS: This retrospective analysis of clinical surveillance hospital data included all children younger than 15 years admitted with malaria to Manhiça District Hospital (MDH), Mozambique, from July 1, 1997, to June 30, 2017. Case fatality ratios (CFRs) were calculated as the number of patients who died having a specific diagnosis or syndrome divided by the total number of patients with known outcome admitted with that diagnosis or syndrome. FINDINGS: Over the study period, 32 138 children were admitted to MDH with a malaria diagnosis. Malaria accounted for a large proportion of admissions, ranging from 4083 (76·9%) of 5307 admissions in 2000-01 to 706 (27·5%) of 2568 admissions in 2010-11. Since 2000-02, the absolute and relative number of malaria admissions and deaths presented a decreasing trend. The age pattern of patients with malaria shifted to older ages with a median age of 1·7 years (IQR 0·9-3·0) in 1997-2006 and 2·6 years (IQR 1·3-4·4) in 2006-17, although most malaria deaths (60-88% in 2009-17) still occurred in children younger than 5 years. The clinical presentation of severe malaria changed, with an increase in cerebral malaria and a decrease in severe anaemia and respiratory distress, leading to similar yearly cases for the three syndromes. CFRs for severe malaria fluctuated between 1·1% (2 of 186 in 2014-15) and 7·2% (11 of 152 in 2010-11), varying by severe malaria syndrome (3·3% [70 of 2105] for severe anaemia, 5·1% [191 of 3777] for respiratory distress, and 14·8% [72 of 487] for cerebral malaria). Overall malaria CFRs (1·8% [543 of 30 163]) did not vary by age group. INTERPRETATION: Despite the unprecedented scale up of malaria control tools, malaria still represented around 30-40% of paediatric hospital admissions in 2006-17. The age shift towards older children was not accompanied by an increase in severe malaria or deaths; however, control programmes should consider adapting their high-risk target groups to include older children. Malaria remains a leading cause of disease and health-care system use and the massive unfinished malaria control agenda warrants intensified efforts. FUNDING: Spanish Agency for International Cooperation and Development.

Local mortality impacts due to future air pollution under climate change scenarios.

The health impacts of global climate change mitigation will affect local populations differently. However, most co-benefits analyses have been done at a global level, with relatively few studies providing local level results. We aimed to quantify the local health impacts due to fine particles (PM2.5) under the governance arrangements embedded in the Shared Socioeconomic Pathways (SSPs1-5) under two greenhouse gas concentration scenarios (Representative Concentration Pathways (RCPs) 2.6 and 8.5) in local populations of Mozambique, India, and Spain. We simulated the SSP-RCP scenarios using the Global Change Analysis Model, which was linked to the TM5-FASST model to estimate PM2.5 levels. PM2.5 levels were calibrated with local measurements. We used comparative risk assessment methods to estimate attributable premature deaths due to PM2.5 linking local population and mortality data with PM2.5-mortality relationships from the literature, and incorporating population projections under the SSPs. PM2.5 attributable burdens in 2050 differed across SSP-RCP scenarios, and sensitivity of results across scenarios varied across populations. Future attributable mortality burden of PM2.5 was highly sensitive to assumptions about how populations will change according to SSP. SSPs reflecting high challenges for adaptation (SSPs 3 and 4) consistently resulted in the highest PM2.5 attributable burdens mid-century. Our analysis of local PM2.5 attributable premature deaths under SSP-RCP scenarios in three local populations highlights the importance of both socioeconomic development and climate policy in reducing the health burden from air pollution. Sensitivity of future PM2.5 mortality burden to SSPs was particularly evident in low- and middle- income country settings due either to high air pollution levels or dynamic populations.

Molecular Epidemiology of Rotavirus Strains in Symptomatic and Asymptomatic Children in Manhiça District, Southern Mozambique 2008-2019.

Group A rotaviruses remain the leading cause of diarrhoea in children aged <5 years. Mozambique introduced rotavirus vaccine (Rotarix®) in September 2015. We report rotavirus genotypes circulating among symptomatic and asymptomatic children in Manhiça District, Mozambique, pre- and post-vaccine introduction. Stool was collected from enrolled children and screened for rotavirus by enzyme-immuno-sorbent assay. Positive specimens were genotyped for VP7 (G genotypes) and VP4 (P genotypes) by the conventional reverse transcriptase polymerase chain reaction. The combination G12P[8] was more frequently observed in pre-vaccine than in post-vaccine introduction, in moderate to severe diarrhoea (34%, 61/177 vs. 0, p < 0.0001) and controls (23%, 26/113 vs. 0, p = 0.0013) and mixed genotypes (36%, 24/67 vs. 7% 4/58, p = 0.0003) in less severe diarrhoea. We observed changes in post-vaccine compared to pre-vaccine introduction, where G3P[4] and G3P[8] were prevalent in moderate to severe diarrhoea (10%, 5/49 vs. 0, p = 0.0002; and 14%, 7/49 vs. 1%, 1/177, p < 0.0001; respectively), and in less severe diarrhoea (21%, 12/58 vs. 0, p = 0.003; and 24%, 14/58 vs. 0, p < 0.0001; respectively). Our surveillance demonstrated the circulation of similar genotypes contemporaneously among cases and controls, as well as switching from pre- to post-vaccine introduction. Continuous surveillance is needed to evaluate the dynamics of the changes in genotypes following vaccine introduction.

Initiation and adherence to isoniazid preventive therapy in children under 5 years of age in Manhiça, Southern Mozambique.

The WHO recommends preventive treatment for all pediatric contacts of a confirmed TB case, but coverage remains low in many high TB burden countries. We aimed to assess the coverage and adherence of the isoniazid preventive therapy (IPT) program among children under 5 years of age with household exposure to an adult pulmonary TB case in a rural district of Southern Mozambique. The estimated IPT coverage was 11.7%. A longer distance to the health center and lower age of the children hindered IPT initiation. Among patients who started IPT, 12/18 (69.9%) were adherent to the 6-month treatment.

Performance of Xpert MTB/RIF Ultra for tuberculosis diagnosis in the context of passive and active case finding.

AIMS: We present a field evaluation of the diagnostic accuracy of Xpert MTB/RIF ("Xpert") and Xpert MTB/RIF Ultra ("Ultra") using two cohorts in a high tuberculosis/HIV burden setting in Southern Mozambique. METHODS: Single respiratory specimens from symptomatic adults accessing healthcare services (passive case finding (PCF) cohort) and from household and community close contacts (active case finding (ACF) cohort) were tested by smear microscopy, culture, Xpert and Ultra. Liquid and solid culture served as a composite reference standard. We explored the impact of trace results on specificity via their recategorisation to negative (in all and just among those previously treated individuals). RESULTS: 1419 and 252 participants were enrolled in the PCF and ACF cohorts, respectively. For the PCF cohort, Ultra showed higher sensitivity than Xpert overall (0.95 (95% CI 0.90-0.98) versus 0.88 (96% CI 0.82-0.93); p<0.001) and among smear-negative patients (0.84 (96% CI 0.71-0.93) versus 0.63 (96% CI 0.48-0.76)). Ultra's specificity was lower than Xpert's (0.96 (96% CI 0.95-0.97) versus 0.98 (96% CI 0.97-0.99); p=0.008). For ACF, sensitivities were the same (0.67 (95% CI 0.22-0.96) for both tests), although Ultra detected a higher number of microbiologically confirmed samples than Xpert (4.7% (12 out of 252) versus 2.7% (seven out of 252)). Conditional recategorisation of trace results among previously treated participants maintained differences in specificity in the PCF cohort. CONCLUSION: These results add evidence on the improved sensitivity of Ultra and support its use in different case finding scenarios.

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

BACKGROUND: 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.

Biomarkers to Distinguish Bacterial From Viral Pediatric Clinical Pneumonia in a Malaria-Endemic Setting.

BACKGROUND: Differential etiologies of pediatric acute febrile respiratory illness pose challenges for all populations globally, but especially in malaria-endemic settings because the pathogens responsible overlap in clinical presentation and frequently occur together. Rapid identification of bacterial pneumonia with high-quality diagnostic tools would enable appropriate, point-of-care antibiotic treatment. Current diagnostics are insufficient, and the discovery and development of new tools is needed. We report a unique biomarker signature identified in blood samples to accomplish this. METHODS: Blood samples from 195 pediatric Mozambican patients with clinical pneumonia were analyzed with an aptamer-based, high-dynamic-range, quantitative assay (~1200 proteins). We identified new biomarkers using a training set of samples from patients with established bacterial, viral, or malarial pneumonia. Proteins with significantly variable abundance across etiologies (false discovery rate <0.01) formed the basis for predictive diagnostic models derived from machine learning techniques (Random Forest, Elastic Net). Validation on a dedicated test set of samples was performed. RESULTS: Significantly different abundances between bacterial and viral infections (219 proteins) and bacterial infections and mixed (viral and malaria) infections (151 proteins) were found. Predictive models achieved >90% sensitivity and >80% specificity, regardless of number of pathogen classes. Bacterial pneumonia was strongly associated with neutrophil markers-in particular, degranulation including HP, LCN2, LTF, MPO, MMP8, PGLYRP1, RETN, SERPINA1, S100A9, and SLPI. CONCLUSIONS: Blood protein signatures highly associated with neutrophil biology reliably differentiated bacterial pneumonia from other causes. With appropriate technology, these markers could provide the basis for a rapid diagnostic for field-based triage for antibiotic treatment of pediatric pneumonia.

Low frequency of enterohemorrhagic, enteroinvasive and diffusely adherent Escherichia coli in children under 5 years in rural Mozambique: a case-control study.

BACKGROUND: Diarrheagenic Escherichia coli (DEC) are among the leading pathogens associated with endemic diarrhea in low income countries. Yet, few epidemiological studies have focused the contribution of enterohemorrhagic E. coli (EHEC), enteroinvasive E. coli (EIEC) and diffusely adherent E. coli (DAEC). METHODS: We assessed the contribution of EHEC, EIEC and DAEC isolated from stool samples from a case-control study conducted in children aged < 5 years in Southern Mozambique between December 2007 and November 2012. The isolates were screened by conventional PCR targeting stx1 and stx2 (EHEC), ial and ipaH (EIEC), and daaE (DAEC) genes. RESULTS: We analyzed 297 samples from cases with less-severe diarrhea (LSD) matched to 297 controls, and 89 samples from cases with moderate-to-severe diarrhea (MSD) matched to 222 controls, collected between November 3, 2011 and November 2, 2012. DEC were more common among LSD cases (2.7%, [8/297] of cases vs. 1.3% [4/297] of controls; p = 0.243]) than in MSD cases (0%, [0/89] of cases vs. 0.4%, [1/222] of controls; p = 1.000). Detailed analysis revealed low frequency of EHEC, DAEC or EIEC and no association with diarrhea in all age strata. Although the low frequency, EIEC was predominant in LSD cases aged 24-59 months (4.1% for cases vs. 0% for controls), followed by DAEC in similar frequency for cases and controls in infants (1.9%) and lastly EHEC from one control. Analysis of a subset of samples from previous period (December 10, 2007 and October 31, 2011) showed high frequency of DEC in controls compared to MSD cases (16.2%, [25/154] vs. 11.9%, [14/118], p = 0.383, respectively). Among these, DAEC predominated, being detected in 7.7% of cases vs. 17.6% of controls aged 24-59 months, followed by EIEC in 7.7% of cases vs. 5.9% of controls for the same age category, although no association was observed. EHEC was detected in one sample from cases and two from controls. CONCLUSIONS: Our data suggests that although EHEC, DAEC and EIEC are less frequent in endemic diarrhea in rural Mozambique, attention should be given to their transmission dynamics (e.g. the role on sporadic or epidemic diarrhea) considering that the role of asymptomatic individuals as source of dissemination remains unknown.

First identification of genotypes of Enterocytozoon bieneusi (Microsporidia) among symptomatic and asymptomatic children in Mozambique.

Enterocytozoon bieneusi is a human pathogen with a broad range of animal hosts. Initially, E. bieneusi was considered an emerging opportunistic pathogen in immunocompromised, mainly HIV-infected patients, but it has been increasingly reported in apparently healthy individuals globally. As in other African countries, the molecular epidemiology of E. bieneusi in Mozambique remains completely unknown. Therefore, we undertook a study to investigate the occurrence and genetic diversity of E. bieneusi infections in children with gastrointestinal symptoms as well as in asymptomatic children in Mozambique. Individual stool specimens were collected from 1,247 children aged between 0 and 14 years-old living in urban and rural settings in Zambézia (n = 1,097) and Maputo (n = 150) provinces between 2016 and 2019. Samples were analysed for E. bieneusi by nested-PCR targeting the internal transcribed spacer (ITS) region of the rRNA gene. All positive amplicons were confirmed and genotyped. Penalised logistic regression (Firth) was used to evaluate risk associations. The overall prevalence of E. bieneusi in this children population was 0.7% (9/1,247). A 10-fold higher prevalence was found in Maputo (4.0%; 6/150) than in Zambézia (0.3%; 3/1,097). All E. bieneusi-positive samples were from children older than 1-year of age, and most (8/9) from asymptomatic children. Nucleotide sequence analysis of the ITS region revealed the presence of four genotypes, three previously reported (Peru11, n = 1; Type IV, n = 2, and S2, n = 2) and a novel genotype (named HhMzEb1, n = 4). Novel genotype HhMzEb1 was identified in both asymptomatic (75%, 3/4) and symptomatic (25%, 1/4) children from a rural area in Maputo province in southern Mozambique. Genotypes HhMzEb1, Peru11, S2, and Type IV belonged to the Group 1 that includes genotypes with low host specificity and the potential for zoonotic and cross-species transmission. Being infected by enteric protozoan parasites and no handwashing were identified as risk associations for E. bieneusi infection. This study reports the first investigation of E. bieneusi genotypes in Mozambique with the identification of three previously reported genotypes in humans as well as a novel genotype (HhMzEb1). Findings highlight the need to conduct additional research to elucidate the epidemiology of E. bieneusi in the country, especially in rural areas where poor hygiene conditions still prevail. Special attention should be paid to the identification of suitable animal and environmental reservoirs of this parasite and to the characterization of transmission pathways.

Post-malarial anemia in Mozambican children treated with quinine or artesunate: A retrospective observational study.

OBJECTIVES: This retrospective analysis performed in Manhiça, Southern Mozambique, aimed to describe the frequency of post-malarial anemia (measured as a decrease of hematocrit ≥10%) and the need for blood transfusions in children with severe malaria treated with intravenous quinine or parenteral artesunate. METHODS: All children <15 years admitted with a parasitologically-confirmed diagnosis of malaria from 1st January 2003 to 31st December 2017, alive at hospital discharge, and with at least one measurement of hematocrit within 28 days after hospital discharge, detected by passive case detection, were included. RESULTS: The overall prevalence of post-malarial anemia observed in the study was 23.13%, with an estimated incidence rate of 288.84 episodes/1,000 children-month at risk in the follow-up period (28 days after discharge). There were no differences between treatment groups, although the study showed a higher association between blood transfusions and artesunate treatment. CONCLUSIONS: In this setting, children with severe malaria frequently present a meaningful decrease of hematocrit (>=10%) in the first weeks after their episode, sometimes requiring blood transfusions. Because of the high underlying prevalence of anemia in malaria-endemic settings, all children with severe malaria need to be actively followed up, irrespective of the treatment received.

Diarrhoeal disease and subsequent risk of death in infants and children residing in low-income and middle-income countries: analysis of the GEMS case-control study and 12-month GEMS-1A follow-on study.

BACKGROUND: The Global Enteric Multicenter Study (GEMS) was a 3-year case-control study that measured the burden, aetiology, and consequences of moderate-to-severe diarrhoea (MSD) in children aged 0-59 months. GEMS-1A, a 12-month follow-on study, comprised two parallel case-control studies, one assessing MSD and the other less-severe diarrhoea (LSD). In this report, we analyse the risk of death with each diarrhoea type and the specific pathogens associated with fatal outcomes. METHODS: GEMS was a prospective, age-stratified, matched case-control study done at seven sites in Africa and Asia. Children aged 0-59 months with MSD seeking care at sentinel health centres were recruited along with one to three randomly selected matched community control children without diarrhoea. In the 12-month GEMS-1A follow-on study, children with LSD and matched controls, in addition to children with MSD and matched controls, were recruited at six of the seven sites; only cases of MSD and controls were enrolled at the seventh site. We compared risk of death during the period between enrolment and one follow-up household visit done about 60 days later (range 50-90 days) in children with MSD and LSD and in their respective controls. Approximately 50 pathogens were detected using, as appropriate, classic bacteriology, immunoassays, gel-based PCR and reverse transcriptase PCR, and quantitative real-time PCR (qPCR). Specimens from a subset of GEMS cases and controls were also tested by a TaqMan Array Card that compartmentalised probe-based qPCR for 32 enteropathogens. FINDINGS: 223 (2·0%) of 11 108 children with MSD and 43 (0·3%) of 16 369 matched controls died between study enrolment and the follow-up visit at about 60 days (hazard ratio [HR] 8·16, 95% CI 5·69-11·68, p<0·0001). 12 (0·4%) of 2962 children with LSD and seven (0·2%) of 4074 matched controls died during the follow-up period (HR 2·78, 95% CI 0·95-8·11, p=0·061). Risk of death was lower in children with dysenteric MSD than in children with non-dysenteric MSD (HR 0·20, 95% CI 0·05-0·87, p=0·032), and lower in children with LSD than in those with non-dysenteric MSD (HR 0·29, 0·14-0·59, p=0·0006). In children younger than 24 months with MSD, infection with typical enteropathogenic Escherichia coli, enterotoxigenic E coli encoding heat-stable toxin, enteroaggregative E coli, Shigella spp (non-dysentery cases), Aeromonas spp, Cryptosporidium spp, and Entamoeba histolytica increased risk of death. Of 61 deaths in children aged 12-59 months with non-dysenteric MSD, 31 occurred among 942 children qPCR-positive for Shigella spp and 30 deaths occurred in 1384 qPCR-negative children (HR 2·2, 95% CI 1·2-3·9, p=0·0090), showing that Shigella was strongly associated with increased risk of death. INTERPRETATION: Risk of death is increased following MSD and, to a lesser extent, LSD. Considering there are approximately three times more cases of LSD than MSD in the population, more deaths are expected among children with LSD than in those with MSD. Because the major attributable LSD-associated and MSD-associated pathogens are the same, implementing vaccines and rapid diagnosis and treatment interventions against these major pathogens are rational investments. FUNDING: Bill & Melinda Gates Foundation.