High Frequency of Cryptosporidium hominis Infecting Infants Points to A Potential Anthroponotic Transmission in Maputo, Mozambique

Cryptosporidium is one of the most important causes of diarrhea in children less than 2 years of age. In this study, we report the frequency, risk factors and species of Cryptosporidium detected by molecular diagnostic methods in children admitted to two public hospitals in Maputo City, Mozambique. We studied 319 patients under the age of five years who were admitted due to diarrhea between April 2015 and February 2016. Single stool samples were examined for the presence of Cryptosporidium spp. oocysts, microscopically by using a Modified Ziehl–Neelsen (mZN) staining method and by using Polymerase Chain Reaction and Restriction Fragment Length Polymorphism (PCR-RFLP) technique using 18S ribosomal RNA gene as a target. Overall, 57.7% (184/319) were males, the median age (Interquartile range, IQR) was 11.0 (7–15) months. Cryptosporidium spp. oocysts were detected in 11.0% (35/319) by microscopy and in 35.4% (68/192) using PCR-RFLP. The most affected age group were children older than two years, [adjusted odds ratio (aOR): 5.861; 95% confidence interval (CI): 1.532–22.417; p-value < 0.05]. Children with illiterate caregivers had higher risk of infection (aOR: 1.688; 95% CI: 1.001–2.845; p-value < 0.05). An anthroponotic species C. hominis was found in 93.0% (27/29) of samples. Our findings demonstrated that cryptosporidiosis in children with diarrhea might be caused by anthroponomic transmission.


Introduction
Diarrhea is the one main causes of mortality among children less than 5 years old in low and middle-income countries (LMIC) [1,2]. In Mozambique, it was estimated that 11% Pathogens 2021, 10, 293 2 of 12 of pediatric diseases were due to diarrhea [3]. Rotavirus remains the main etiological agent of diarrhea in children, followed by Cryptosporidium spp. [1].
Cryptosporidium spp. is an apicomplexan enteric pathogenic parasite protozoan related to water and foodborne outbreaks worldwide [4][5][6][7]. It easily spreads in the environment, through soil, drinking and recreation water (swimming pool, surface waters) or even directly by person-to-person contact and contact with objects surfaces with oocysts [4]. Cryptosporidium is also considered an opportunistic parasite that can infect immunocompetent and immunocompromised people [8]. It can also infect wild and domestic animals [4,9,10], which facilitates spread in the environment.
Currently, more than 40 species of Cryptosporidium are recognized, some of which were described recently [9,10]. Even with increased efforts and improved laboratory detection, unknown Cryptosporidium species remain to be identified [9]. Among humans, the species most frequently associated with cryptosporidiosis are C. hominis (anthroponotic species) and C. parvum (anthroponotic and zoonotic species) [9,11,12]. Moreover, C. parvum is known as having the broadest range of hosts and is otherwise an important zoonotic species [9][10][11][12].
The Global Enteric Multicenter Study (GEMS) showed that Cryptosporidium spp. is the second most attributable pathogen moderate-to-severe diarrhea (MSD) requiring medical attention among young infants [1,13,14]. In Mozambique, several studies demonstrated the presence of this parasite in different populations and/or regions of the country. Additionally, a study in rural Mozambique indicated that Cryptosporidium spp. is one of the two pathogens associated with an increased risk of death in children with MSD [15]. Studies of children under 14 years with diarrhea reported different frequencies of Cryptosporidium spp. The highest frequency was observed in children aged 0-11 months at 20.0%, followed by 19.0% in children aged 12-23 months, 9.0% in children aged 24-59 months [16], 12.0% in children aged 0-168 months [17] detected by a commercial immunoassay method, and 3.4% to 34.0% in all individuals with diarrhea using microscopy [18,19]. A community study detected Cryptosporidium spp. in less than 5.0% of the study population, aged 0 to 48 months old living in poor environment sanitation [20].
Currently, due to its zoonotic and anthroponotic features, it is becoming more important to determine the molecular epidemiology of Cryptosporidium spp. Polymerase Chain Reaction (PCR) is a molecular-based method commonly used to study this parasite [10][11][12]. Additionally, this method provides information about the occurrence and distribution of Cryptosporidium species, contributing to a better understanding of the parasite.
Moreover, risk factors also play an important role in infection dynamics. Infection with human immunodeficiency virus (HIV) and consequently development of diarrhea is one of the risk factors that contribute to the chronic diarrhea profile [4,6]. There are few studies in Mozambique that report the risk factors in children with diarrhea and/or that used molecular tools to characterize Cryptosporidium [1,15,21]. Altogether, the current analyses were performed with the aim of determining the frequency, risk factors, and molecular diagnostic of Cryptosporidium by using a PCR Restriction Fragment Length Polymorphism (PCR-RFLP) in children hospitalized in Maputo City within the context of National Surveillance of Acute Diarrhea (ViNaDiA).

Frequency of Cryptosporidium spp. Infection
During the period of the study, 319 stool samples were collected, examined, and tested for presence of Cryptosporidium spp. It was possible to test all 319 samples using the modified Ziehl-Neelsen (mZN) staining method and 192 samples using a PCR method. Only samples with a sufficient stool amount were included for PCR analysis, regardless of results from the staining.
As shown in Figure 1, it was not possible to perform molecular analyses on all the 319 samples, because 127 samples had not sufficient quantity for further testing (the remaining 192 samples tested using PCR). In our PCR-based protocol, it was not possible to amplify all genetic material, because 4 positives for mZN failed to amplify.
In the restriction with SspI digestion products for C. parvum, C. hominis and the mixed infection showed an identical restriction pattern with three visible bands of 111 bp, 254 bp, and 449 bp. A different pattern was seen with AseI digestion products, where C. parvum had two visible bands of 104 bp and 268 bp. C. hominis also had two visible bands of

Risk Factors for Cryptosporidium spp. Infection
Older children were more likely to be infected with Cryptosporidium spp. The most susceptible group was children older than two years, compared to younger than seven months (p-value < 0.05; adjusted odds ratio (aOR): 5.861, 95% CI: 1.532-22.417) ( Table 2).

Risk Factors for Cryptosporidium spp. Infection
Older children were more likely to be infected with Cryptosporidium spp. The most susceptible group was children older than two years, compared to younger than seven months (p-value < 0.05; adjusted odds ratio (aOR): 5.861, 95% CI: 1.532-22.417) ( Table 2).
Being male (crude odds ratio (

Discussion
This study aimed to determine the frequency and risk factors for Cryptosporidium spp. infection in children hospitalized in Maputo City through the National Surveillance of Acute Diarrhea (ViNaDiA).
The frequency of Cryptosporidium spp. found in our study was higher than those in studies conducted in different areas (urban, peri-urban and/or rural) and regions from Mozambique (north, south and/or central). Those studies indicate lower frequencies of Cryptosporidium spp. if we consider our PCR results (35.4%): 12% in children hospitalized with diarrhea by using ELISA [17], 3.4% in children admitted in pediatric ward in one north central hospital by using mZN and rapid test [18] and 34% in children from ViNaDiA tested before the current study by using mZN, between 2013 and 2015 [19]. The differences in frequencies among these studies can be attributed to different study designs, population characteristics [20,22], diagnostic tools (for instance only microscopy, serology or PCRbased methods) and region of the country (north, south and/or central).
Children older than six months showed higher risk of infection. This trend has been reported in other African countries [23][24][25][26] and also in one of the studies conducted in Mozambique [22]. Higher frequencies in older children (>six months) may be explained by feeding practices, mobility of the child and/or age at which the child has high contact with other children when playing. In Mozambique, exclusive breastfeeding is recommended for the first six months, and then, complementary food is added to the child's diet. It was previously observed that breastfeeding has a protective effect against any protozoan infection, including Cryptosporidium in children from 0 to 48 months old in Maputo, Mozambique [20,27]. The introduction of potentially contaminated complementary foods and the increased mobility of the child expose them to other possible sources of infection, such as soil and animal contact, increasing the risk of infection among older children before mature immunity achieved [4].
Children from illiterate caregivers were more susceptible to infection by Cryptosporidium spp. Empirically, literate caregivers suggest that the household is in a higher wealth quintile, compared to those that did not go to school (illiterate). It is reasoned that caregivers who learned better hygiene practices at school are more aware of health risks and practice improved sanitary and hygiene behaviors.
In our analysis, we found no association between Cryptosporidium infection and HIVstatus, although more than one third of the children had unknown HIV status. Conversely, in Tanzania [28] and Kenya [25], strong associations between Cryptosporidium infection and HIV-status have been observed, with higher frequencies of Cryptosporidium infection in HIV-positive children.
Animal contact was not a predictor for Cryptosporidium spp. infection in children. Cryptosporidium infection can be acquired through animal contact but can also be transmitted through an anthroponotic route. In our sample, we observed that the majority of the PCR-tested samples contained C. hominis. This is commonly reported in Africa, and its acquisition is related to person-to-person transmission [14,21,23,24,28,29], suggesting that animal contact plays a smaller role in infection.
The increased occurrence of C. hominis is corroborated with one hospital-based study of adults with diarrhea in Maputo City which used the 60-kDa glycoprotein gene (gp60) as a target [21]. Occurrence of anthroponomical transmission in children, as described in our study, suggests empiric circulation of the parasite if we consider that adults assist children. An infected adult can easily transmit the parasite to the child, and/or the child can pass it onto another adult or other children.
On the other hand, the PCR-RFLP targeting SSU rRNA (18S rRNA gene) used in this study was the first attempt to molecularly characterize Cryptosporidium in children with diarrhea in Mozambique. The target used is the most used among investigators, because this region is less polymorphic, presenting five copies per genome [7,10]. Although gene sequencing could enrich the findings, we did not have the technical conditions at the time of the study. As there was no prior knowledge of the molecular epidemiology of Cryptosporidium sp. in our country, we opted for PCR-RFLP to conduct a survey of the circulating species and genotypes. Furthermore, we analyzed a single stool samples instead of the optimal multiple (at least 3) consecutive approach, which could result in underestimation. We also applied the PCR technique, which is a highly sensitive diagnostic approach [30,31]. Additionally, this was a hospital-based analysis, meaning that our findings can only be extrapolated to the population from the sites included. However, in four samples mZN-positive, the presence of DNA was not identified. This may have been a consequence of DNA degradation due to suboptimal temperature during transportation and storage, due to the presence of inhibitors [32] or due to a different species with a mutation in the primer's region [10].
There are few studies in Mozambique [1,19], reporting the risk factors for Cryptosporidium infection in the children with diarrhea and/or using molecular tools. The findings of this study should receive attention, since the high frequency of C. hominis in children observed may be a result of anthroponotic transmission. There is a need to expand the analysis to other provinces of the country and complement it with sequencing tools to better characterize the species in circulation. It is also worth noting that other hosts may be participating in the transmission routes, which is corroborated by the identification of C. parvum isolates.

Ethics Statements
The data used in the present analysis were provided by the ViNaDiA in children. The related protocol was approved by the Mozambique National Bioethics Committee for Health (IRB00002657, reference Nr. 348/CNBS/13). Written informed consent was obtained from children's parents or legal guardians before questionnaire administration and sample collection.

Study Design, Site and Population
Cross-sectional, hospital-based surveillance was conducted between April 2015 and February 2016 in Hospital Geral de Mavalane (HGM) and Hospital Geral José Macamo (HGJM). These hospitals were selected as sentinel sites because they receive patients from Maputo City and surrounding areas. Both have pediatric out-and inpatient wards. The HGM and HGJM are referral hospitals for both Mavalane and José Macamo health areas and cover fourteen (14) and nine (9) health centers, respectively.
In each sentinel site, focal points (laboratory technicians, physicians and nurses) were identified and trained to screen diarrhea cases, administer the questionnaire, collect and send stool samples to Instituto Nacional de Saúde (INS) where the samples were processed. Children up to 60 months old who presented in the sentinel sites with acute diarrhea, defined as three or more loose or liquid stools within 24 h and less than 14 days, were included [33].

Sample Size Calculation
Minimum sample size expected was calculated using OpenEpi [34], with 95% confidence interval (CI), desired precision of 3.0% and an estimated frequency of 4.8% for Cryptosporidium spp. from a previous study in children aged up to five years with diarrhea [16]. We obtained a minimum sample size of at least 196.

Data Collection
Demographic, clinical and epidemiological data were assessed by interviewing children's caregivers with a semi-standardized questionnaire. Information regarding sex, age, animal contact (defined as having physical contact with an animal or their excrements) [16] and caregiver education status were collected. HIV status was self-reported by the children's caregivers and confirmed in the children's vaccination cards. If unknown, permission was asked to collect blood samples and tested according to the national testing algorithm. The children newly diagnosed as HIV-positive were followed by the physician at hospital and referred to their neighborhood health facilities for routine assistance after discharge.

Sample Collection and Management
A single stool sample from each child was collected after inclusion. In cases of liquid diarrhea, non-absorbent diapers were used instead of ordinary diapers. Samples were transferred to sterile polystyrene tubes, kept refrigerated in cooler boxes (approximately 2 • C to 8 • C), without preservative and sent to the Laboratory of Parasitology in INS, Maputo.
A smear was made from fresh stool, and an aliquot was kept in the original tube under 2 to 8 • C for concentration and subsequent microscopic examination for Cryptosporidium spp. oocysts. A second aliquot was placed in a vial without preservative, stored under −40 • C and was specifically intended for extracting and purifying genomic deoxyribonucleic acid (DNA) for molecular analysis. A set of previously frozen sub-samples (approximately 0.5 mL) was shipped to Laboratório Interdisciplinar de Pesquisas Médicas, Instituto Oswaldo Cruz/Fundação Oswaldo Cruz in Rio de Janeiro, Brazil, under dry ice for DNA extraction, detection and genetic characterization of Cryptosporidium species.

Direct Microscopy
The presence of Cryptosporidium oocysts in stools was first established using the modified Ziehl-Neelsen (mZN) staining method as described by the World Health Organization (WHO) [35]. Briefly, thin smears of fresh samples and concentrated pellet from formolether concentration technique were prepared on the same glass side and air-dried before mZN staining. Samples were read using a microscope; the results were recorded as a positive for those where oocysts of the parasite were visualized under 100X magnification. All stool samples were collected, labeled, processed and stored following the laboratory standard procedures, including Good Laboratory Practice (GLP) recommendations. For the parasitological assay, each internal quality control was made, and all sample readings were double-checked for concordance by two technicians. In case of discordant results, a third observation was required.

DNA Extraction
DNA was extracted from frozen stool sample using a commercial QIAmp stool Mini Kit (Qiagen, Hilden, Germany) following the manufacturer's protocol with the following modification: the lysis temperature was raised to 95 • C and the DNA was eluted in 100 µL. The pre-treating of the sample and usage of the QIAmp stool Mini Kit was done in a standard procedure as in similar studies, and with this there is no need to freeze or apply thawing cycles [32]. DNA extracts were stored at −20 • C until use.

Molecular Detection by Conventional Polymerase Chain Reaction (PCR)
The 18S rRNA gene was amplified for all samples (positive and negative by mZN staining) by conventional polymerase chain reaction. The forward primer 5'-AACCTGGTTGATC CTGCCAGTAGTC-3' and reverse primer 5'-TGATCCTTCTGCAGGTTCACCTACG-3' as described by Xiao et al. [36] were used. Briefly, the PCR contained 10X PCR buffer (MgCl 2 ) at a final concentration of 1X, 5mM MgCl 2 , 200 mM (each) deoxynucleotides triphosphate (dNTP), 1.0 U of Taq polymerase (Invitrogen Life Technologies, São Paulo, SP-Brasil), 100 nM (each) primer (Extend, SP-Brasil) and 2.5 µL of DNA template in a total 25 µL reaction mixture. The following parameter was adjusted in our study: MgCl 2 concentration, Taq polymerase quantity when compared with the study that was used as reference. Each PCR had small adjustments; annealing was set to 61 • C for 45 s and extension to 72 • C for 7 min. The PCR efficacy and the identification of the Cryptosporidium genetic material from samples were verified through electrophoresis in agarose gel (1.2%).
Stool samples were considered positive if oocysts with typical characteristics (approximately 4 µm and 6 µm in diameter; stained bright pink within a clear halo under green field) or Cryptosporidium DNA with expected base pair (bp) were detected by conventional PCR.

Characterization of Cryptosporidium spp. Isolates by Nested PCR and Restriction Fragment Polymorphism (RFLP) Analysis
All samples that were previously positive for 18S rRNA gene amplification were genotyped by a PCR-RFLP technique using genomic DNA as template. Firstly, a PCR product of approximately 1325 bp of the SSU rRNA gene was amplified by a nested PCR using the following primers 5'-TTCTAGAGCTAATACATGCG-3' and 5'-CCCTAATCCTTCGAAACAG GA-3' [11,12]. The first PCR contained 1X PCR buffer (MgCl 2 ), 6 mM of MgCl 2 , 2.5 U of Taq polymerase, 200 µM of each primer concentration (500 nM each) and 2 µL of DNA template in a total volume of the reaction mixture (50 µL). The initial denaturation was 94 • C for 3 min, followed by the amplification performed in 35 total cycles: 94 • C for 45 s for denaturation, 58 • C for 45 s for annealing and 72 • C for 60 s for extension. The final extension was 72 • C for 7 min. For secondary PCR, for a product of 826 to 864 bp, it was done by using the following primers 5'-GGAAGGGTTGTATTTATTAGATAAAG-3' and 5'-AAGGAGTAAGGAACAACCTC CA-3' [11]. A total volume of the reaction mixture (50 µL), Taq DNA amount (1 U) and the primary PCR product was optimized (0.5 µL diluted at 1:20 or 1 µL non-diluted product). Amplification condition for secondary PCR was as follows: 94 • C for 45 s, 59 • C for 30 s and 72 • C for 45 s in 25 cycles with an initial hot start at 94 • C for 3 min and a final extension at 72 • C for 7 min. The PCR efficacy and the identification of the Cryptosporidium genetic material from samples were verified through electrophoresis in agarose gel (1.2%).
Genotype identification was made through the analysis of pattern of the secondary product after restriction digestion with the enzymes SspI and AseI (New England BioLabs Inc., Beverly, MA, USA) as described by Xiao et al. [11]. The AseI enzyme has the same digestion function and pattern as the enzyme VspI. Each set of experiments included a negative PCR control (laboratory-grade distilled water). For the restriction, 20 µL of the second product of the nested PCR, 10 U of SspI or AseI, 5 µL pf specific enzyme buffer was digested in a 50 µL of the reaction by 37 • C for one hour under conditions recommended by the supplier manufacturer. Aliquots of amplified and digested fragments were separated and visualized under Ultra-Violet light translucent (Bio-Rad, Milan, Italy) after separation in 1.5% to 2% agarose gel (Invitrogen, Aukland, New Zealand) by electrophoresis stained with 3X GelRed (Biotium, San Francisco, CA, USA). The expected band for each enzyme varied according to the species detected were the most observed highlighted in bold (Table 3). . Categorical variables were summarized as frequencies and continuous variables were summarized as medians and Inter-quartile Range (IQR). Contingency tables were built between dependent and independent variables. Crude and adjusted odds ratio were estimated through simple and multiple logistic regression models. Independent variables with p-values ≤ 0.2 in the simple logistic regression were included in the multiple logistic regression model in order to obtain adjusted odds ratio. Goodness-of-fit was assesses using the Hosmer and Lemeshow test for the multiple logistic regression model. A p-value less than 0.05 was considered evidence of statistical significance and all analyses were performed considering a 95% confidence interval (95% CI).

Conclusions
This study showed a high frequency of Cryptosporidium spp. infection detected by PCR-RFLP among children admitted to two public hospitals in Maputo City due to acute diarrhea. Child age (25 to 60 months) and caregiver literacy status were predictors for infection. Our findings demonstrated that the infection is mostly due to C. hominis. This suggests mainly anthroponomic transmission, with great public health implications. The present study demonstrated the need for improved public health recommendations by including Cryptosporidium spp. in routine testing among children with diarrhea in Mozambique. Moreover, the study presented the need for an establishment of more accurate molecular characterization platform of the parasite in a national context, in order to understand which species occur in the country, routes of infection (if anthroponotic and/or zoonotic) and/or regional patterns, informing public health programs.  Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author. The data are not publicly available due to the ethical reasons.