Background: Soil-transmitted helminths (STH) are common in low and middle income countries where there is lack of access to clean water and sanitation. Effective diagnosis and treatment are essential for the control of STH infections. However, among STH parasites, Strongyloides stercoralis is the most neglected species, both in diagnostics and control strategies. Diagnostic methods cover different approaches, each with different sensitivities and specificities, such as serology, molecular techniques and microscopy based techniques. Of the later, the Baermann technique is the most commonly used procedure. In the literature, several ways have been described to perform the Baermann method, which illustrates the overall lack of a ‘(gold) reference standard’ method for the diagnosis of S. stercoralis infection. In this study we have evaluated the performance of three Baermann techniques in order to improve the reference standard for the microscopic diagnosis of S. stercoralis infection thereby facilitating individual case detection, mapping of the disease and proper evaluation of treatment responses. Methods/Principal findings: A community based cross sectional study was conducted at Zenzelima, Bahir Dar Zuria Ethiopia. A total of 437 stool samples were collected and analyzed by the following procedures: conventional Baermann (CB), modified Baermann (MB), and modified Baermann with charcoal pre-incubation (MBCI). The diagnostic sensitivity and Negative Predictive Value (NPV) of each technique was calculated using the combination of all the three techniques as a composite reference standard. Our result indicated that larvae of S. stercoralis were detected in 151 (34.6%) stool samples. The prevalence of S. stercoralis infection based on the three diagnostic methods was 9.6%, 8.0%, and 31.3% by CB, MB, and MBCI respectively. The sensitivity and NPV for CB, MB, and MBCI were 26.7% and 70.8%, 22.1% and 69.6%, and 87.0% and 93.2%, respectively. The MBCI showed significant difference (P- value = <0.001) in the sensitivity and NPV values when compared with CB and MB values. The agreement between CB, MB, and MBCI with the composite reference standard was 31.8%, 26.7%, 89.6%, respectively. Conclusion/Significance: Our results suggest the superior performance of MBCI. It is relatively easy to implement, simple to perform and comparatively cheaper. The CB is by far the commonly used method in routine diagnostic although this technique significantly underestimates the true burden of the disease and thereby contributing to the exclusion of S. stercoralis from the control strategies. Therefore, MBCI is recommended as a routine microscopy-based diagnostic test for S. stercoralis infection, particularly in settings where molecular procedures are not available.

Giardia duodenalis is an enteric parasite commonly detected in children. Exposure to this organism may lead to asymptomatic or symptomatic infection. Additionally, early-life infections by this protozoan have been associated with impaired growth and cognitive function in poor resource settings. The Global Enteric Multicenter Study (GEMS) in Mozambique demonstrated that G. duodenalis was more frequent among controls than in diarrhoeal cases (≥3 loosing stools in the previous 24 hours). However, no molecular investigation was conducted to ascertain the molecular variability of the parasite. Therefore, we describe here the frequency and genetic diversity of G. duodenalis infections in children younger than five years of age with and without diarrhoea from the Manhiça district in southern Mozambique enrolled in the context of GEMS. Genomic DNA from 757 G. duodenalis-positive stool samples by immunoassay collected between 2007–2012, were reanalysed by multiplex PCR targeting the E1-HP and C1-P21 genes for the differentiation of assemblages A and B. Overall, 47% (353) of the samples were successfully amplified in at least one locus. Assemblage B accounted for 90% (319/353) of all positives, followed by assemblage A (8%, 29/353) and mixed A+B infections (1%, 5/353). No association between the presence of a given assemblage and the occurrence of diarrhoea could be demonstrated. A total of 351 samples were further analysed by a multi-locus sequence genotyping (MLSG) approach at the glutamate dehydrogenase (gdh), ß-giardin (bg) and triose phosphate isomerase (tpi) genes. Overall, 63% (222/351) of samples were genotyped and/or sub-genotyped in at least one of the three markers. Sequence analysis revealed the presence of assemblages A (10%; 23/222) and B (90%; 199/222) with high molecular diversity at the nucleotide level within the latter; no mixed infections were identified under the MLSG scheme. Assemblage A sequences were assigned to sub-assemblages AI (0.5%, 1/222), AII (7%, 15/222) or ambiguous AII/AIII (3%, 7/222). Within assemblage B, sequences were assigned to sub-assemblages BIII (13%, 28/222), BIV (14%, 31/222) and ambiguous BIII/BIV (59%, 132/222). BIII/BIV sequences accumulated the majority of the single nucleotide polymorphisms detected, particularly in the form of double peaks at chromatogram inspection. This study demonstrated that the occurrence of gastrointestinal illness (diarrhoea) was not associated to a given genotype of G. duodenalis in Mozambican children younger than five years of age. The assemblage B of the parasite was responsible for nine out of ten infections detected in this paediatric population. The extremely high genetic diversity observed within assemblage B isolates was compatible with an hyperendemic epidemiological scenario where infections and reinfections were common. The obtained molecular data may be indicative of high coinfection rates by different G. duodenalis assemblages/sub-assemblages and/or genetic recombination events, although the exact contribution of both mechanisms to the genetic diversity of the parasite remains unknown.

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. Enterocytozoon bieneusi is an obligate intracellular parasite that infects a wide range of vertebrate hosts. It is the most important etiological agent of human microsporidiasis. Most clinical and epidemiological studies conducted to date have focused on immunodeficient or immunosuppressed individuals including HIV+ patients and solid organ transplant recipients, as in those E. bieneusi infection causes life-threatening chronic diarrhoea. In contrast, latent microsporidia infections in immunocompetent individuals have received far less attention. Molecular epidemiological studies in humans and animals have revealed that E. bieneusi encompasses a very large diversity of genetic variants (genotypes) with marked differences in host specificity and even geographical distribution. In Mozambique, as in many other African countries, the epidemiology of E. bieneusi is completely unknown. Therefore, to identify the occurrence and genetic diversity of this pathogen in Mozambique stool samples were obtained from children, including apparently healthy and symptomatic, in Zambézia and Maputo provinces and tested for E. bieneusi by molecular methods. Results demonstrated the presence of E. bieneusi genotypes for the first time in Mozambique. Four genotypes were identified, three genotypes that have previously been reported in humans (Peru11, Type IV, and S2) and a novel genotype (HhMzEb1). Two of the genotypes Peru11, Type IV have also been frequently identified in animals indicating that potentially zoonotic E. bieneusi genotypes are inadvertently circulating in the surveyed populations. Additional population genetic studies are needed to elucidate the actual extent of the epidemiology and transmission dynamics of E. bieneusi in Mozambique.

Background: Soil-transmitted helminths (STH) are targeted for control through mass drug-administration campaigns to prevent morbidity affecting at-risk groups in endemic regions. Although broadly successful, the use of albendazole and mebendazole achieved variable progress, with deficiencies against Trichuris trichiura and a predictable low efficacy against Strongyloides stercoralis. Novel drug combinations offer a potential solution, providing they can be delivered safely and maintain efficacy against all STH species. Here we present the protocol of a clinical trial to evaluate a fixed-dose combination (FDC) tablet containing albendazole and ivermectin that will be compared against albendazole against STH. Methods: An adaptive phase II/III randomized controlled trial will be undertaken in STH endemic sites in Ethiopia, Kenya and Mozambique to evaluate an oral FDC of 400 mg albendazole and either 9- or 18 mg ivermectin. FDC will be administered as a single dose or single doses over three-consecutive days and assessed against a single dose of 400 mg albendazole. In the phase II trial, 126 T. trichiura-infected children weighting 15 to 45 kg will be treated in a dose-escalation manner to determine safety objectives. In the phase III trial, 1097 participants aged 5 to 18 years old infected with T. trichiura, hookworm and S. stercoralis will be recruited to determine safety and efficacy. The trial will be open-label with blinded outcome assessors. Cure rate measured 21-days after-treatment in duplicate Kato-Katz is the primary efficacy outcome. Secondary objectives include efficacy evaluation by quantitative polymerase chain reaction (PCR) as an outcome measurement, description of pharmacokinetic parameters, palatability and acceptability evaluations, and monitoring of anthelmintic resistance. Conclusions: This trial with registrational goals seeks to evaluate an innovative fixed-dose combination of albendazole and ivermectin co-formulated tablets, with the goal of providing an anthelmintic regimen with improved efficacy and spectrum of coverage against STH. ClinicalTrials.gov registration: NCT05124691 (18/11/2021).

Staphylococcal infections are among the most common foodborne diseases. We performed the antibiotic susceptibility and molecular characterization of S. aureus from milk samples of dairy cows in Manhiça District. We observed a high frequency of S. aureus (41%, 58/143), in which 71% (41/58) were from commercial farms and 29% (17/58) from smallholder farms. Half of the isolates (50%, 29/58) were resistant to at least one antibiotic, with higher rates of resistance to penicillin (43%, 25/58), followed by tetracycline (16%, 9/58). Multidrug-resistant and methicillin-resistant S. aureus isolates were rare (5%, 3/58 and 3%, 2/58, respectively). The genetic diversity was low, with predominance of human-adapted strains being: ST1/CC1-t5388 (78%) and ST152-t1299 (10%), followed by ST8/CC8-t1476 (5%) and ST5/CC5-t002 (3%) and lastly, ST508/CC45-t331 and ST152-t355, with 2% each. The Panton–Valentine leukocidin (PVL) gene was detected among 14% (8/58) of the isolates, while genes encoding staphylococcal enterotoxins were scarce (3%, 2/58). Our findings revealed a high frequency of S. aureus, with high rates of resistance to the antibiotics commonly used in veterinary and human medicine. Further investigations focusing on the molecular epidemiology of S. aureus from cattle and farmers will provide detailed insights on the genetic relatedness between the strains.

Background: Following the World Health Organization (WHO) recommendation, 38/47 countries have introduced rotavirus vaccines into the program of immunization in the WHO Regional Office for Africa (WHO/AFRO). Initially, two vaccines (Rotarix and Rotateq) were recommended and recently two additional vaccines (Rotavac and Rotasiil) have become available. However, the global supply challenges have increasingly forced some countries in Africa to switch vaccine products. Therefore, the recent WHO pre-qualified vaccines (Rotavac, Rotasiil) manufactured in India, offer alternatives and reduce global supply challenges related to rotavirus vaccines; Methods: Using a questionnaire, we administered to the Program Managers, Expanded Program for Immunization, we collected data on vaccine introduction and vaccine switch and the key drivers of the decisions for switching vaccines products, in the WHO/AFRO. Data was also collected fromliterature review and the global new vaccine introduction status data base maintained by WHO and other agencies. Results: Of the 38 countries that introduced the vaccine, 35 (92%) initially adopted Rotateq or Rotarix; and 23% (8/35) switched between products after rotavirus vaccine introduction to either Rotavac (n = 3), Rotasiil (n = 2) or Rotarix (n = 3). Three countries (Benin, Democratic Republic of Congo and Nigeria) introduced the rotavirus vaccines manufactured in India. The decision to either introduce or switch to the Indian vaccines was predominately driven by global supply challenges or supply shortage. The withdrawal of Rotateq from the African market, or cost-saving for countries that graduated or in transition from Gavi support was another reason to switch the vaccine; Conclusions: The recently WHO pre-qualified vaccines have offered the countries, opportunities to adopt these cost-effective products, particularly for countries that have graduated or transitioning from full Gavi support, to sustain the demand of vaccines products.