Cryptosporidium spp. are associated with diarrheal disease which leads to nutritional deficiencies and significant morbidity and mortality in children and patients with immune defects. The continuous emergence and molecular diversity of Cryptosporidium spp. still remains a threat to human health in respect of diarrhea and malnutrition in children <5 years. The impact of the cryptosporidiosis is exacerbated by the spread of human immunodeficiency virus/AIDS, and water shortage in most part of Southern Africa and the burden of the disease is underestimated within the region. Both anthroponotic and zoonotic transmission are identified as a common transmission route. Molecular characterization of Cryptosporidium infection is still a challenge, and there is a paucity of information on the burden of the parasite in Southern Africa. Ongoing attempts to find appropriate drugs and vaccines to cure and prevent the spread of the disease have limitations; perhaps considering isolation of bioactive compounds from medicinal plants extracts for their effectiveness and potency may be an option in future to control and eliminate the disease. This review considered the prevalence and molecular diversity in respect of transmission routes and associated risk factors. Urgent assessment of public health significance of this parasite in Southern Africa and elsewhere is of utmost importance. It is apparent that there is a need for adequate surveillance in monitoring the pathogen and to control the spread of infection. Keywords: Animal, Cryptosporidium, epidemiology, human, prevalence, Southern Africa, waterborne
Burden of Human Immunodeficiency Virus, Diarrhea, and Malnutrition The estimated African population is expected to be around 1.2 billion people in few years. The Southern African countries make up about 35% of the total population of Africa with the prevalence rate of human immunodeficiency virus (HIV) between 1.0% and 26.5% in 2011. Adult HIV prevalence exceeded 20% in Swaziland, Botswana, and Lesotho. In terms of HIV case number, South Africa has the largest population of people living with HIV (6.3 million) in Southern Africa[1] [Figure 1].
Infectious diseases in developing countries cause enormous morbidity and mortality,[2] and diarrheal disease in Sub-Saharan Africa accounts for 16% of deaths in African children <5 years,[3] which is amplified by HIV epidemic.[4] Although Southern African region may have up to 71% of all people living with HIV, it is still spreading in the region and remains disproportionately affected by the epidemic. The same countries are home to 31% of people newly infected with HIV and 34% of people dying from AIDS-related causes.[5] Antiretroviral coverage increased from 24% to 54% in 2015, reaching a regional total of 10.3 million people.[6] For example, South Africa had nearly 3.4 million people on treatment more than any other country in the world. Botswana, Kenya, Malawi, Mozambique, South Africa, Swaziland, United Republic of Tanzania, Zambia, and Zimbabwe all increased treatment coverage by more than 25 percentage point between 2010 and 2015.[7] Part of sustainable development goals was ending all forms of malnutrition (stunting, wasting, and overweight) among children <5 years. Globally, in 2015, almost one in four children under 5 years of age (156 million children) were affected by stunting. and wasting was prevalent in the WHO South-East region.[7] Studies have demonstrated that poverty, malnutrition, and Cryptosporidium infection remain intricately connected,[8],[9] and there is a need to confirm such in Southern Africa. Studies have shown that stunted growth in the first 2 years has overbearing effects on growth and development.[9],[10],[11],[12],[13],[14] However, where Cryptosporidium is found in high prevalence among children, the best strategy is to treat infected people to reduce the spread of infection. The recommended drugs of choice for the treatment of cryptosporidiosis in immune-deficient human patients are nitazoxanide and paromomycin. Halofuginone is approved in Europe for prophylactic treatment of calves. Nitazoxanide has a broad spectrum of activity against many other gastrointestinal pathogens; however, it is not effective against Cryptosporidium in immunocompromised persons. This review attempts to address the public health significance of Cryptosporidium infection and its attendance implication in health care.
Cryptosporidium spp. are enteric protozoa with over 27 species described, but Cryptosporidium hominis and Cryptosporidium parvum most commonly infect humans.[15],[16],[17] There are over 40 genotypes with a high probability that many of these will eventually be given species status with increased biological and molecular characterization.[18] It is estimated that nearly 1.7 billion cases of diarrheal disease occur yearly, the leading cause of malnutrition in children under 5 years old and 2.6 billion lack improved sanitation and this may likely double by 2025.[19] However, diarrhea incidence and case-fatality ratios are much higher within low-income countries than in middle- and high-income countries. Fifteen countries in Africa account for 53% of the total episodes of diarrhea globally, 56% of severe episodes, and 74% of the total burden of diarrhea mortality in children <5 years.[20] Cryptosporidium is still an emerging pathogen and continuously identified as the cause of multiple diarrhea in developed and developing countries.[18],[21],[22],[23],[24],[25],[26] The Global Enteric Multicenter (GEM) assessed the cause, burden, clinical syndrome, and adverse outcomes of moderate-to-severe diarrhea in children at several sites in Sub-Saharan Africa and South Asia and identified Cryptosporidium as one of the four major contributions to moderate-to-severe diarrheal diseases during the first 2 years of life at all the sites.[27] Previous reports corroborate the GEM observation in Southern Africa and elsewhere.[28],[29],[30],[31],[32],[33],[34] Some longitudinal and cross-sectional studies have consistently shown that <5 years are associated with high-risk infection.[31],[33],[34] In addition, several authors have opined that unsafe water, sanitation, and hygiene conditions facilitate the transmission of enteric pathogens from infected individuals to a new susceptible host through direct contact and/or through the environment.[35],[36],[37],[38],[39],[40],[41],[42],[43],[44]
Despite so much knowledge of the distribution of Cryptosporidium spp. in the developed countries, there is still a gap in understanding the route of transmission, prevalence, and molecular epidemiology of the disease in most part of Sub-Saharan Africa, especially Southern African countries. One of the most common intestinal opportunistic parasites is Cryptosporidium and is listed as an AIDS-defining illness by the US Center for Disease Control and Prevention.[45],[46] Globally, the prevalence rate of Cryptosporidium infection may account for 10%–20% of the cases of diarrhea in HIV-infected patients living in developed countries and up to 50% in impecunious countries.[47],[48],[49],[50] Diarrhea has been recognized as a persistent, seasonally occurring disease in Chobe District, Botswana. Cryptosporidium infection frequently produces severe to chronic diarrhea with intestinal dysfunction leading to malnutrition even in healthy children.[29],[30] The parasite was implicated in an epidemiological review of enteropathogens with the prevalence rates of 2.2% in Gaborone, Botswana.[51] Another study[31] investigated mortality among nonbreastfed children during diarrhea outbreak and malnutrition; stool examination generated numerous pathogens of which 60% had Cryptosporidium (50% C. parvum and 41% C. hominis). According to the report, the effects of diarrhea were more pronounced in HIV-negative, not breastfed and malnourished children. These factors were considered important elements to their severe illness and death. Other studies reported various prevalence in children <5 years with more positive cases during diarrhea outbreak.[52],[53],[54] This indicates that Cryptosporidium infection is a potential cause of diarrheal disease in children within these communities and confirms that public health education must include an emphasis on improved sanitation practices in a household with diarrhea and surveillance should be directed at under and above 5 years age group in areas of high HIV prevalence. Shortage of quality water was identified as the major cause of diarrhea outbreak in Botswana. An extensive study from Madagascar[32] confirmed the predominance of C. hominis in children with diarrhea and the disease (cryptosporidiosis) accounted for 5.6% of the diarrhea cases in these group of children. The subtypes identified are Ia, Id, Ie, and IIc associated with anthroponotic transmission. However, there is a high possibility of identifying more species with large-scale study involving animals and environmental samples. Diarrhea, acute respiratory infection, malaria, and fever are among the leading causes of childhood mortality and morbidity in Malawi and elsewhere in Africa.[33],[44],[55] According to Morse et al.,[56] up to 10% of diarrhea in children aged <5 years was attributed to Cryptosporidium spp. infection with up to 21% reported in immunocompromised population. Evidence from a molecular diversity of Cryptosporidium infection was attributed to increased zoonotic exposure and malnutrition. This indicates the need to improve on water supply, sanitation, household hygiene, and animal control. Subsequently, molecular characterization identified C. hominis and C. parvum and sequence analysis established high intraspecific variations of C. hominis subtypes and anthroponotic transmission was associated with cryptosporidiosis.[57],[58] Few studies have identified Cryptosporidium in some provinces of South Africa[59],[60],[61],[62],[63],[64],[65] as the causative agent of diarrheal disease in immunocompetent, HIV-positive children and adults. From such studies, Cryptosporidium was found more in children <5 years and sometimes coexist with Giardia infection.[61],[66] Furthermore, Leav et al.[67] identified species and genotypes of Cryptosporidium isolates from HIV-infected children in Durban and 15 isolates of genotype I allele (Ia, Ib, Ic, and Id) and 5 displayed a novel genotype Ie of C. parvum. Some of the isolates manifested unusual chimeric genotype Ib, Ic, or Ic/II allele, raising concern about the possibility of sexual recombination within and between parasite genotypes. In Limpopo Province, Cryptosporidium was found among children, and hospital patients and C. hominis were predominantly common than C. parvum,[68] but genotypic identification was unaccomplished to confirm zoonotic or anthroponotic transmission. The importance of Cryptosporidium and Giardia infection was confirmed in another survey.[23] Such finding confirms that both organisms are highly associated with diarrhea in some parts of South Africa. Molecular analyses from four provinces identified C. hominis (76.0%) with subtypes families (Ia, Ib, Id, Ie, and If), C. parvum (20%) with three subtypes family (IIc, IIe, and IIb), and subtype IIc being the most common.[69] In contrast, Cryptosporidium meleagridis of subtype family IIId was identified in other studies, a parasite originally described in turkeys and humans.[70],[71],[72],[73] Thus Confirming C. hominis and anthroponotic C. parvum subtypes are common in South Africa. Collectively from these studies, anthroponotic and zoonotic transmissions are common in some of the provinces studied. Considering the level of HIV infection in Swaziland, information on the burden of cryptosporidiosis is still inadequate. The first report of Cryptosporidium infection in Swaziland gave an infection rate of 4.2% from the diarrheic stool of pediatric outpatients.[74] In our view, there is lack of information about cryptosporidiosis infection in this country considering the level of HIV/AIDS. In contrast, Moyo et al.[75] reported the prevalence of 18.9% Cryptosporidium infection in children hospitalized for diarrhea in Tanzania which was higher than that of Cegielski et al.[76] The high rate of infection was attributed to the presence of C. parvum and other enteropathogenic organisms. Another study examined 108 women and infants in rural/semi-rural Tanzania[77] and noted the high level of maternal Cryptosporidium infection (63%) among the participants. It was noted that Cryptosporidium in infants increased dramatically between 3 and 6 months of age, a period that corresponds to changes in breastfeeding practices. Reiterating the fact that infants residing in rural and semi-rural areas are more susceptible to Cryptosporidium infection in early infancy with approximately 1/3 of infants showing evidence of infection by 6 months of age.[75],[78] Invariably, concerted efforts must be geared toward understanding the public health significance of the pathogenic parasite to humans and animals in these countries. Annual rainfall reductions in the Southern Africa region and increased human consumption have contributed immensely to water shortage. Seasonal peaks in cryptosporidiosis have been reported in several Sub-Saharan countries. In a report from Durban, South Africa,[79]Cryptosporidium infection was associated with seasonal variation, especially during the dry and wet season. High prevalence of infection was associated low-income settings and rainy season. The nonavailability of good drinking water during dry season may compound the spread of infection, thereby necessitating people within a community to consume water from protected and unprotected water sources that may have been contaminated with this parasite and other enteric pathogens. Cryptosporidium infection is greatly compounded by HIV infection and malnutrition in Sub-Saharan Africa. Among HIV-positive children in Zambia[34] with persistent diarrhea and malnutrition, C. parvum was the dominant species when compared with other intestinal infectious agents. Based on previous reports, the prevalence rate of infection ranged from 18% to 32%.[80],[81],[82],[83],[84],[85] The astonishingly high rate in children with persistent diarrhea, malnutrition syndrome, particularly the impact of cryptosporidiosis, and salmonellosis contributes to pathophysiology among the studied population. Within the same region, C. parvum was found in 18.0% of children who presented with diarrhea; most of the children had acute illnesses.[86],[87] Another study determined the prevalence, incidence, and seasonal variation of Cryptosporidium and Giardia intestinalis from preschool children in Kafue, Zambia,[82]Cryptosporidium (30.75%) and Giardia (29.0%) infections were documented, and diarrhea was significantly associated with cryptosporidiosis than giardiasis. The prevalence of more than 80% of Zambian children being parasitized with Cryptosporidium is remarkable and supported a seasonal increase in cryptosporidiosis during the rainy season.[79],[80]
Enteric opportunistic parasites are associated with HIV/AIDS, chronic diarrhea, education, occupation and residence in a slum, exposure to pets and animals, antiretroviral therapy, use of public toilets, water and practicing unsafe homosexual activity.[83],[84],[85],[86],[87],[88],[89] Few studies in Congo[89],[90],[91] determined the prevalence and species spectrum of intestinal parasites (IP) in hospitalized AIDs patients. Diarrhea was a common syndrome in 49.7% patients hospitalized, 26.9% harbor an IP, and 15.4% were infected with at least one opportunistic IP and 9.7% with Cryptosporidium infection. Unsurprisingly, coinfection of Cryptosporidium spp. with helminths was observed in some HIV-patients in Kinshasa, DRC,[91] suggesting the need for improved epidemiological surveillance and hygiene to reduce transmission and level of infection. The complex picture of Cryptosporidium transmission in recent years continues to emerge because of the increase use of molecular tools for diagnosis. In a study that cuts across four countries (Kenya, Malawi, Brazil, and Vietnam),[92] four genotypes of Cryptosporidium were identified; C. parvum human genotypes, C. parvum bovine genotype, C. meleagridis genotype, and Cryptosporidium muris genotype from HIV patients were documented. This finding is significant because the broad range of these species confirms that they are environmentally ubiquitous. The natural reservoirs of these species are believed to be poultry, rodents, and cattle. However, the results are difficult to interpret because of the small number of cases identified. In addition, when HIV-infected individuals are perceived to be more likely to have zoonotic genotypes than non-HIV infected individuals, there is close human proximity with animals. This is a common scenario in most parts of rural settings in Africa. Subsequently, Cranendonk et al.[93] assessed the importance of C. parvum and I. belli (actually designated Cystoisopora belli) infections as a cause of diarrhea among HIV/AIDS patients with 11% positive for Cryptosporidium. Infection was significantly higher in the cohort with diarrhea than in those without diarrhea, confirming the dominance of C. parvum infection which is significantly associated with diarrhea among population studied. Similar study in Madagascar[94] also associated C. parvum and I.belli with diarrhea in AIDS patients but one of the limitation of the study was the non-characterization of the various species to ascertain the molecular epidemiology of the parasite. Future studies must consider a holistic approach to understand the molecular epidemiology of cryptosporidiosis in most of these areas. In Limpopo Province, South Africa,[95] seroprevalence of Cryptosporidium in HIV-infected adults and local University students confirmed the high prevalence of Cryptosporidium (75.3%) in HIV-infected individuals compared with student volunteers (32.8%). Suggesting that there is possibility of widespread exposure to the parasite in both groups and the underlying risk factor maybe the source of drinking water. Numerous opportunistic infections occur in HIV-infected patients as result of the downregulation of the immune system. To determine if there was any correlation between Cryptosporidium infection and other IP associated with diarrhea, Samie et al.[96] reported corroborated the coexistence of Cryptosporidium and IP among HIV patients using loop-mediated isothermal amplification technique. Cryptosporidium spp. was found in 26.4%, which supports data from previous reports that Cryptosporidium and other IP are associated with diarrhea.[83],[97],[98] Principal risk factors seem to coincide with residing in rural areas, consumption of nonpiped water, close contact with cow dung and anthroponotic transmission. Moreover, another study implicated farm animals as a source of human infection and Cryptosporidium from calves to humans was reported in 18 cases among 82 farm workers and 207 households. The study identified C. parvum (75%) in farm workers, household members (60%) and in calves (62%). Of the positive calves, 62% were infected with C. parvum indicating zoonotic transmission among the workers.[99] Similarly, Mtapuri-Zinyowera et al.[100] reported enteric IP coexisting with Cryptosporidium. C. parvum (7.6%) and C. cayetanensis (22.1%) showed a remarkable high rate of infection among HIV patients. Interestingly, 11 (36.6%) water bodies had protozoan parasites associated with diarrhea including C. parvum (3.3%). This water sources used by rural populace are not treated and therefore pose a risk for acquiring such organisms. This emphasizes the need for early detection and treatment of such infection in HIV-infected patients to reduce morbidity. Some of the prevalence and molecular epidemiology studies are summarized in [Table 1].
Humans acquire Cryptosporidium infection through several transmission routes, such as anthroponotic (persontoperson transmission), zoonotic (animal transmission), or contaminated fomites, or foodborne or water contaminated with infected feces. The different role of animals in the transmission of human cryptosporidiosis in most developing countries requires adequate attention. In several studies in Southern Africa, most of the Cryptosporidium strains found to have infected humans are nonzoonotic; however, C. hominis, C. parvum, C. meleagridis, Ctenocephalides felis, Ctenocephalides canis, Cryptosporidium cuniculus. Chlamydia suis, C. muris, Cryptosporidium Andersoni, and Cryptosporidium ubiquitum have been associated with human diseases.[101],[102] These infectious agents pose one of the greatest threats to endangered species and a greater risk of transmission from humans to wildlife. They should be considered a public health concern in tourism. An increased anthropogenic impact on primate populations may result in high level of interaction between parasite, humans, and primates. To assess such impact in the Central African Republic,[103] a study evaluated the influence of close contact with humans and potential transmission zoonotic protists in western lowland Gorillas (Gorilla gorilla gorilla) on microsporidia, Cryptosporidium and Giardia infections at different stages of the habituation, humans, and other wildlife. Corynebacterium bovis was detected in gorilla and humans, and only G. intestinalis subgroup AII in gorilla. In other wild and domestic animals, E. cuniculi, G. intestinalis assemblage E and C. muris TS03 were identified, suggesting high potential of zoonotic transmission The observed possibility of human–gorilla transmission of parasites emphasized need to improve on hygiene where there is an increased human–gorilla contact. Moreover, Cryptosporidium is a persistent challenge to livestock production. Recently, molecular epidemiology in Madagascar[104] confirmed C. suis as the dominant species infecting humans, cattle, pigs, and rodents. The broad species range of C. suis and lack of common cattle species (C. parvum and C. andersoni) are astonishing. Therefore, transmission of C. suis maybe widespread in humans and cattle than previously envisaged in the studied environment. Other studies have also documented the presence of Cryptosporidium in various animals in Madagascar and Malawi.[105],[106] The prevalence rate was between 5.6%-37% and occurrence was throughout all seasons. Species identified were C. hominis, C. parvum, C. bovis, and/or C. ryanae from various animals, indicating the presence of zoonotic and probably anthroponotic transmission in Malawi. Moreover, studies have considered cattle a common source of zoonotic cryptosporidiosis since they are considered as major host of C. parvum worldwide. However, C. andersoni, C. bovis, and C. ryannae also infect cattle, and their occurrence is related to the age of the host.[107] Recently, Abu Samra et al.[108] identified C. parvum from elephant (Loxodonta africana), buffalo (Syncerus caffer), and impala (Aepyceros melampus) from Kruger National Park with different prevalence rates. This demonstrated transmission between wildlife, domestic animals and humans should be of concern. Within the same Park, Abu Samra et al.[109] identified C. andersoni in calves, C. ubiquitum in impala and C. bovis in one buffalo. C. ubiquitum has been reported to infect a wide array of hosts; wild and domesticated ruminants, rodents, carnivores, primates, and humans. Supporting previous studies on zoonotic transmission of Cryptosporidium in South Africa, the parasite was detected in cats and dogs. High level of infection was found more in stray dogs and cats which could have public health implication for humans and other animals.[110] There is abundant evidence that zoonotic transmission of this parasite in animals and game reserves have serious implication in public health where tourism is considered.[111],[112],[113],[114],[115],[116],[117],[118],[119],[120],[121],[122],[123],[124],[125] Nevertheless, understanding how environmental conditions and social and behavioral factors influence transmission is important. The paper on ecology and epidemiology of Cryptosporidium in humans, nonhuman primates, and livestock confirms a complex molecular epidemiology between humans, baboons and a subset of chimpanzees infected with C. hominis subtype IfA12G2; another subset of chimpanzees was infected with C. xiaoi. Despite the high degree of habitat overlap established, the dominance of C. hominis subtype IfA12G2 among humans and nonhuman primates suggest cross-species and anthropozoonotic transmission in the system studied.[123] Cryptosporidiosis causes enormous economic losses in animals’ husbandry and livestock production. To obtain information on the occurrence of cryptosporidiosis in lamb and other livestock, and the potential zoonotic role of Cryptosporidium isolates, C. suis and C. parvum was identified in lambs and goat kids which could be a potential reservoir for human infection in Zambia.[126] Recently, Cryptosporidium and Giardia were found in pigs from Zambia and 6.9% had a mixed infection. Presence of the pathogens may have potential zoonotic effects on humans and public health hazard in Zambia[127] and elsewhere.
Water is the major transmission route of Cryptosporidium and Giardia, the oocysts and cysts can resist and remain infective for long periods and resistant to conventional water treatment.[128],[129],[130],[131],[132],[133],[134],[135],[136],[137],[138] The infective dose required by Cryptosporidium and Giardia varies according to authors.[133],[134],[135] Nevertheless, most chemicals used for disinfection are not practical outside the laboratory and high concentrations that significantly reduce oocyst infectivity are either very expensive or quite toxic. In several countries in Southern Africa, unavailability of good drinking water is still a major challenge and common sources of drinking water in rural settings include rivers, reservoirs, canals, and low land reservoirs. Farm animals, humans, and wild animals are considered major contributors of Cryptosporidium and Giardia (oo) cysts contamination of surface water. Few reports have indicated that dual infection with Giardia cysts and Cryptosporidium oocyst(s) in all types of water tested including surface water, sewage, or treated effluents in South Africa.[136],[137],[138] The two pathogens were found in a significant percentage. The drawback of some of these small-scale studies was that molecular characterization was not performed to determine exposure route. River water has been identified as a significant source of Cryptosporidium and other pathogens in most developing countries.[139],[140],[141] Emphasizing that detection of both organisms in water sources is vital because of its public health implications. Dungeni and Momba[142] assessed the effectiveness of four wastewater treatments plants of the Gauteng Province and identified C. parvum and G. intestinalis as major pathogens. Thus, confirming that both pathogens are ubiquitous in the influents and raises serious public health concern in rural and urban settings.[143] The World Health Organization estimated that 80% of all diarrheal cases and illnesses are attributed to unsafe water supply and sanitation.[144] In rural and some urban settings such as those found in Zimbabwe and other African countries, drinking water is obtained from protected and unprotected water bodies representing important risk factors for waterborne gastroenteritis.[96],[145] Proper identification of pathogens is extremely important, not only because of clinical implications but for epidemiological purposes. It is important that more studies be undertaken in these countries because there is glaring evidence of the inadequate source of good drinking water and the need to implement systemic monitoring programs for both protozoa in Southern Africa region. Cryptosporidiosis is a major infectious agent associated with diarrhea in most low-income countries; however, there are insufficient data on Cryptosporidium species contamination of water sources and the need to assess species/genotypes identification in respect of human infection.
The major obstacles to human health in most developing countries are associated unsafe water, poor sanitation, and personal hygiene. All these factors facilitate the transmission of Cryptosporidium and other protozoans associated with diarrhea in Southern Africa. Couple with the high prevalence of HIV/AIDS in this region, and the emergence of intestinal opportunistic parasites in humans should be of concern to public health officials and scientists. Despite the importance of cryptosporidiosis in childhood health, there is still lack of adequate attention to transmission of this parasite in Southern Africa. Considering the poor sanitation, hygiene, close proximity with animals, contamination of water bodies, effective control measures are needed to reduce the prevalence of this pathogen and other protozoans associated with diarrhea. Evidence has shown from the review that both anthroponotic and zoonotic transmission is common. There is still a wide gap in understanding the transmission route and molecular epidemiology in Southern Africa in relation to a population infected with HIV/AIDS and inadequate source of drinking water. Molecular characterization of most identified species is still lacking because facilities for such are inadequate or unavailable in most countries. The absence of effective drug(s) or vaccine to cure or prevent cryptosporidiosis is still a challenge. It is appropriate to search for alternative drugs of choice by considering isolation of bioactive compounds from medicinal plants for novel lead drugs. Although molecular studies have enhanced our understanding of Cryptosporidium transmission in developed countries, however, there are insufficient data in Southern Africa. Acknowledgment The authors are grateful to the Directorate of Research Development, University of the Free State for Postdoctoral Fellowship of Dr. Ojuromi. We also acknowledge the support from the management of Lagos State University, Ojo, Lagos, Nigeria. The authors also wish to acknowledge the efforts of Prof. Olga Matos of Unidade de Parasitologia Medica, Institute de Hygiene e Medicina Tropical, Universidade Nova de Lisboa, Portugal, for her thoughtful comments during the review of this manuscript. Financial support and sponsorship Nil. Conflicts of interest There are no conflicts of interest.
Source of Support: None, Conflict of Interest: None
[Figure 1]
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