Large epidemic of a necrotic skin infection in the Democratic Republic of São Tomé and Principe: an epidemiological study

IntroductionCellulitis is a common infection of the skin associated with swelling, erythema, and pain of the affected area, ()often the lower limbs.(Björnsdóttir S. Gottfredsson M. Thórisdóttir A.S. Gunnarsson G.B. Ríkardsdóttir H. Kristjánsson M. Risk factors for acute cellulitis of the lower limb: a prospective case-control study.; Cannon J. Rajakaruna G. Dyer J. Carapetis J. Manning L. Severe lower limb cellulitis: defining the epidemiology and risk factors for primary episodes in a population-based case-control study.; Hirschmann J.V. Raugi G.J. Lower limb cellulitis and its mimics: part II. Conditions that simulate lower limb cellulitis.; McNamara D.R. Tleyjeh I.M. Berbari E.F. Lahr B.D. Martinez J.W. Mirzoyev S.A. Incidence of lower-extremity cellulitis: a population-based study in Olmsted county, Minnesota.) While mild cases can be prescribed oral treatment, more severe cases require intravenous treatment and hospitalisation.() Known risk factors for cellulitis are skin lesions, venous insufficiency and lymphedema. (Björnsdóttir S. Gottfredsson M. Thórisdóttir A.S. Gunnarsson G.B. Ríkardsdóttir H. Kristjánsson M. Risk factors for acute cellulitis of the lower limb: a prospective case-control study.; Dupuy A. Benchikhi H. Roujeau J.C. Bernard P. Vaillant L. Chosidow O. Risk factors for erysipelas of the leg (cellulitis): case-control study.; Association of athlete's foot with cellulitis of the lower extremities: diagnostic value of bacterial cultures of ipsilateral interdigital space samples.). The majority of cellulitis cases are caused by Gram-positive bacteria (eg beta-hemolitic streptococci and staphylococcus aureus). (Stevens D.L. Bisno A.L. Chambers H.F. Dellinger E.P. Goldstein E.J.C. Gorbach S.L. Practice guidelines for the diagnosis and management of skin and soft tissue infections: 2014 update by the Infectious Diseases Society of America.; ) Exposure to water can increase the risk of cellulitis caused by other bacteria such as Gram-negative Aeromonas hydrophila. (; Vally H. Whittle A. Cameron S. Dowse G.K. Watson T. Outbreak of Aeromonas hydrophila wound infections associated with mud football.) Cellulitis can progress to a necrotising skin infection, that can get to soft tissues. Necrotising fasciitis, a severe and rapidly progressing soft tissue polymicrobial infection that can be caused by the same bacteria that cause cellulitis, such as beta-hemolitic streptococci, as well as Gram-negative bacteria found in water such as Vibrio spp. It causes substantial morbidity, including substantial rates of skin graft, and a mortality up to 30%. (; Hakkarainen T.W. Kopari N.M. Pham T.N. Evans H.L. Necrotizing soft tissue infections: review and current concepts in treatment, systems of care, and outcomes.; Hayeri M.R. Ziai P. Shehata M.L. Teytelboym O.M. Huang B.K. Soft-Tissue Infections and Their Imaging Mimics: From Cellulitis to Necrotizing Fasciitis.; )

The Democratic Republic of São Tomé and Príncipe is a Portuguese-speaking archipelago of vulcanic origin located in th Gulf of Guinea, close to the Equator. It is constituted by two islands (i.e. São Tomé, and Príncipe) and numerous islets, has a total of around 200,000 inhabitants and a surface of 1,001 km2. In the last few years, cellulitis cases have been described there. However, from September 2016, hospital beds were quickly filled with patients with severe necrotic skin infection of unknown etiology requiring long hospital stays. Unlike necrotising fasciitis, this infection did not affect the fascia, and it was therefore referred as ‘necrotising cellulitis’, though this term does not exist in general clinical currency. A surveillance system and an epidemic investigation team was quickly set up. Here, we describe the epidemiology of this event, quantify its extent and investigate the etiology and risk factors for the disease, including climatic factors.

Methods Study design and methodology

After the notification of the hospital to the MoH of an increase in the number of cellulitis hospitalisations, disease surveillance was quickly set up in the 6 districts of São Tomé and in the autonomous region of Príncipe. Suspected cases were defined as any person who presented an inflammatory process circumscribed to a region of the body, painful or not, with or without injury, edema or lumps and / or serous or purulent secretion; probable cases were defined as any suspected case with extension of the inflammatory process or any person with epidermal detachment or cutaneous necrosis.

For both case control studies, cases were probable “necrotising cellulitis” cases recruited from the regional hospital in Príncipe for the case control study in Príncipe, or the Ayres de Menezes hospital or other health unit with hospitalisation capacity in São Tomé island for the case control study in São Tomé. In the latter, controls were matched by age (intervals of 2 years for <18 y/o, intervals of 5 years for ≥18 y/o), sex and week of hospital admission. Controls were patients hospitalised for any other disease than cellulitis, but no microbiological investigation was available neither for cases nor for controls. For the Príncipe case control study, because of the limited number of cases available, no formal sample size calculation was done and a case control ratio of 1:4 was chosen to improve power. For the São Tomé case control study, using an expected odds ratio of 3 for the estimated least frequent independent variable in the population, a power of 80%, a confidence level of 5% and a case control ratio 1:2, the sample size calculation predicted the need of 44 cases and 88 controls. Exclusion criteria for cases and controls were: being less than 5 y/o, having chronic conditions (eg diabetes, alcoholism) or having other skin lesions diagnosed as the diabetic foot, harseniasis, ulcer or other superinfected chronic lesions.

 Laboratory procedures

Microbiological investigation was done on a limited number of samples in December 2016 and March 2017. In March 2017, a field laboratory was set up with the qPCR tests for the following organisms based on the potential causes of cellulitis and necrotising fasciitis: Staphylococcus aureus, Streptococcus pyogenes, Mycobacterium ulcerans and marinum, Pseudomonas aeruginosa, Aspergillus spp., Vibrio spp. The following additional qPCR tests were available to serve as positive and negative controls: Brucella sp., Bacteroides sp., Rickettsia sp, Legionella sp. In addition to the above tests, the lab was able to undertake the following untargeted testing approaches on the MinIon sequencing platform: i) full length (V1-6) and partial (V1-3)16S - to identify bacterial species without culture, ii) Fungal ITS for zygomycetes and pan-fungal primers; iii) Full bacterial genome sequencing of cultured isolates (supplementary Figure 1).

In addition to the mobile capabilities, testing capabilities back in the UK included a next generation sequencing platform (Illumina HiSeq 2500) and metagenomics, selectively performed on key samples.

 Statistical analysis

Logistic regression was used to investigate risk factors associated to the disease within the case control studies and to hospitalisation using the database of the notified cases. The following variables were studied in the case control studies: age, sex, drinking alcohol every day, having recurring health problems, having had a severe skin lesion in the past. In addition, the following were noted if they happened in the 2 weeks prior hospital admission: having had skin lesions/injuries, having visited a water source, having been at the beach or in the sea, having lived close to a river, having had contact with stagnating water, preparing fish for selling or eating, having fished, having had contact with a person with a severe skin lesion, farming pigs or having been scratched/injured/bitten by a pig or other animal, having taken drugs, having travelled or having taken drugs.

We retrieved information by month on temperature, relative humidity, mm of rainfalls and monthly number of rainy days from September 2013 until October 2018 registered by the only weather station in São Tomé and Príncipe, located close to São Tomé international airport.

Seasonal and temporal trends in occurrence of cases were evaluated using an approach similar to that used by Jensen et al.,(Jensen E.S. Lundbye-Christensen S. Pedersen L. Sørensen H.T. Schønheyder H.C. Seasonal variation in meningococcal disease in Denmark: relation to age and meningococcal phenotype.) with construction of a negative binomial regression model incorporating month and year, and accounting for seasonality trends.

For all models, variables, unless forced, were retained in the final model if they had a p<0.1. Data were entered in Epi Info software (version 7) and the statistical analyses were done using R (version 3.5.4) or STATA version 14.0.

Results Clinical presentation of suspected and probable necrotising cellulitis and treatment of choiceReports from international infectious disease experts that visited a limited number of cases (n=35) meeting the definition of suspected and probable necrotising cellulitis between October 2016 and February 2017 underlined the main common clinical features. A skin lesion, deep or superficial, was noticed in most cases. The start of the disease was most often brutal, with high fever (>38°C) and inflammatory oedema of variable extension and depth and at times presence of lumps, either evolving to desquamation (suspected cases, Figure 1, Panel A) or evolving to superficial and localised necrotic plaques or even large and diffused necrotic plaques in 4-10 days but without involvement of the fascia, and without heavily affecting the general state of the patient (probable cases, Figure 1, Panel B). Most new cases did not have any epidemiological link to previous cases and there was no evidence for person-to-person transmission. Localisation was the lower limb in almost all cases (Figure 1). The delay between the initial wound and the symptoms varied from 48 hours to 2 months. Interval between onset of symptoms and admission varied from 8 days to 3 months.Figure 1

Figure 1Typical clinical presentation of suspected (A) and probable cases of necrotic skin infection (B).

Recommended first-line treatment during the acute phase for mild cases (not allergic to penicillins) was clavamox 875 mg or Amoxicilin 1 g or Ampicillin 500 mg. For more severe cases, Clavamox 1g or Ampicillin plus Clindamicin 600 mg. Second-line treatment during the acute phase was Floxapen 1g + Cefriaxone 1g. In the late phase (ulcer phase), Clavamox 1g was raccomanded.

 Descriptive epidemiologyFrom October 2016, the MoH started notifying suspected and clinically probable necrotizing cellulitis cases. Until February 2017, the median number of cases per week was 61 (interquartile range, IQR 40-79), with a notification peak at week 52 (108 cases). The second wave of the epidemic, from March 2017 to October 2018, registered a median number of cases per week of 18 (IQR 15-25). From November 2019 (until February 2019), the median number of cases went down to 6 (IQR 4-8); this roughly corresponded to the background hospitalization rate for cellulitis, that a retrospective analysis estimated at around 20 a month for the main hospital in São Tomé (Figure 2). Therefore, we analysed cases, divided in suspected and probable, from the first and the second wave of the epidemic only, excluding cases notified after week 44-2018.Figure 2

Figure 2Epidemic curve of the suspected and probable cases of necrotic skin infection in Sao Tome, 2016-2018. After October 2018 (asterisk), the monthly incidence of hospitalisations returned to background levels.

Of the overall notifications, we had demographic data and information on hospitalisation for 2441/2841 (85.9%). Of the cases, 52.9% were males (1501/2839). Twenty-one per cent were children (<15 y/o, 605/2836), 42.5% were young adults (15-44 y/o,1206/2836), 23.9% were 45-64 y/o (679/2836) and 12.2% were ≥65 y/o (346/2836). Of the 2441 cases with information on hospitalization, 50% were hospitalized for at least 24 hours (n=1176).

Cases came from all seven districts (6 in São Tomé and one in Príncipe). One third of the cases was notified during the first wave of the epidemic (35.3%) while the rest was notified during the second wave (64.7%).

Age distribution did not differ between suspected and probable cases, while males were more common among suspected cases (53.8%) than among probable cases (46.9%,). Hospitalisation rate was higher among probable cases than suspected cases (63.0% versus 45.9% Table 1).

Table 1Characteristics of suspected and probable necrotizing cellulitis cases (N=2841). Missing data for age=5; sex=2; district=1; consultation period=0; hospitalization=400 (14%).

By the end of the epidemic, the country cumulative incidence was 15.5 per 1,000 inhabitants. It was highest in Caué (28.5 per 1,000 inhabitants) and Cantagalo (24.2 per 1,000) and lowest in Agua Grande and Príncipe (10.2 and 12.0 per 1,000, respectively, Figure 3).Figure 3

Figure 3Cumulative incidence of suspected and probable cases per 1,000 inhabitants of necrotic skin infection by health district.

Of the total, only 28 patients have benefited of surgical infections, of which 19 were skin grafts. No single death directly caused by necrotising cellulitis was reported during the epidemic.

 Laboratory investigationMicrobial investigation was largely unavailable throughout the epidemic. International laboratory investigation teams travelled to São Tomé and Príncipe and took a limited number of samples. Infections with Mycobacterium ulcerans was soon excluded, as 68 samples from October 2016 were negative by Ziehl/Nielsen stain and/or PCR . Those from a mission in April 2017 (46 samples from 21 patients) identified polymicrobial infections, with S. pyogenes in 12/21 cases, S.aureus in 15/21 cases and co-infections with both in half of the cases (11/21), as well as other opportunistic infections in most cases. C. dyphteriae was not isolated (Gram-negative were not attempted for these samples) but it was identified by metagenomics in 9/12 tested samples (Figure 4). The strains identified by shotgun metagenomics are reported in Supplementary table 1.Figure 4

Figure 4Sample results summary matrix The results below summarise the qPCR, 16S, full genome and metagenomics analysis. Results are binary i.e. they simply display presence of absence of an organism. Metagenomics included robust negative controls to identify possible environmental contamination namely three negative control extractions, prepared alongside the patient samples.

 Risk factors for necrotising cellulitis and hospitalisation

The first case control study was conducted from October 1st, 2016 until February 28th, 2018 in Sao Tomé, and from January 1st, 2017 until April 15th, 2018 in Príncipe. Forty-five cases and 90 controls were recruited in São Tomé island and 27 cases and 108 controls were recruited from in Príncipe island. The lesion was in the lower leg in 100% of cases with known location (16/16 in Príncipe).

Presence of an injury in the 2 weeks before disease onset (adjusted odds ratio, aOR 7.7, 95% CI 3.2-18.6 in São Tomé, aOR 8.3, 95% CI 2.8-23.2 in Príncipe), and living close to a river (aOR 2.59, 95% CI 1.01-6.87 in São Tomé only) were identified as risk factors, while having a recurrent health problem was identified as a protective factor (aOR 0.38, 95% CI 0,16-0,90 in São Tomé, aOR 0.31, 95% CI 0,10-0,88 in Príncipe). In addition, increased age group was identified as a risk factor (in Príncipe only, p=0.01). Having had contact with a person with a severe skin lesion was not found to be a risk factor.

Multivariable analysis using the database of all suspected and probable necrotic skin infectionsfrom October 2016 until October 2018 to investigate risk factors for hospitalisation found a lower risk of hospitalisation for suspected cases, as compared to probable cases (aOR 0.50, 95% CI 0.38-0.67), no difference in risk between males and females (p=0.53), and an increased risk of hospitalisation in increasing age groups (p<0.001, supplementary Table 2).

 Environmental risk factors for increase in the monthly number of cases

Because the increase of the number of cases appeared to be correlated with the amount of rain in São Tomé, we investigated if climatic factors had a potential role in this epidemic.

We collected minimum, maximum and average temperatures, precipitations (mm), number of rainy days and relative humidity by month for the last 5 rainy seasons, including the two associated with the necrotising cellulitis epidemic, from the only São Tomé weather station (Supplementary Table 3). São Tomé is crossed by the equator and the mean temperature for the period under study ranged from 23.8 and 27 degrees (minimum 18,5, maximum 32,7 degrees).

From October to February, the period corresponding to the first big epidemic wave, the average amount of monthly rain was 273.6 mm (range 142.6-435.0) for the pre-epidemic seasons (2013/2014 until 2015/2016). The same period of rainy season 2016/2017 registered a substantial increase in the amount of rainfalls (640.5 mm, 134% increase as compared to the mean mm rain for the seasons 2013/2014 to 2015/2016, or 160% increase as compared to the amount for the previous rainy season). The period from October 2017 until February 2018 registered 438.3 mm rain, i.e. a 60% increase as compared to the mean mm rain for the seasons 2013/2014 to 2015/2016, or 50% decrease as compared to the previous rainy season, 2016/2017).

We studied time trends, seasonality and weather effects using aggregate monthly cases. The overall goodness of fit of the time trend model was good (Chi2 22.0; pTable 2). We found that a one percent increase in the relative humidity results to a 5% increase in the monthly number of cases. Furthermore, the mean maximum temperature below the median (i.e. 30.9 degrees) in the previous month resulted in double the number of notification for the following month (Table 2). Similarly, compared to lowest amount of monthly rain (0-49 mm), middle amount (50-149 mm) and highest amount of monthly rain (≥150 mm) were associated with a one-month lagged higher number of cases (IRR 1.41, 95% CI 1.13-1.77 and IRR 1.50, 95% CI 1.12-1.99, respectively) (Table 2).

Table 2Incidence rate ratio estimates from the time-series model taking environmental factors into account.

Discussion

We described a large epidemic of a necrotic skin infection with a big first peak of infections overlapping the first peak of rainy season 2016/2017, characterized by unusually abundant rains.

Hygienic conditions in São Tomé and Príncipe are limited and skin infections are common in this kind of setting. Poverty is widespread, with over 50% of the population leaving under the threshold of poverty and 15% in extreme poverty (as of 2001), and population is growing very fast in the last few years(

General Direction of the Environment, Ministry of Public Infrastructures and Natural Resources, UNDP. Second National Communication, Democratic Republic of Sao Tome and Principe 2011.

) The pathogens identified were a range of virulent, multi-drug resistant bacterial strains. Characterized strains and antimicrobial resistance pattern differed by whole genome sequencing analysis, pointing at enhanced transmission of virulent strains rather than the epidemic being caused by one single strain. These included among other tetracyclin-resistant S. pyogenes, Methicillin-sensitive, Panton Valentin (PVL) toxin positive S.aureus, Methicillin-resistant S.aureus (MRSA) and Corynebacterium diphteriae, identified only by metagenomics in three quarters of the samples that were tested. It is also possible thatthe epidemic captured here wasinstead two overlapping outbreaks of skin and soft tissue infection over a ∼24-month period, with a "first wave" potentially being driven by cutaneous diphtheria (the usual manifestations of which fit the clinical case description of the reported condition quite neatly) and the "second wave" representing a potentially climate-associated increased incidence of ubiquitous bacterial strains superinfecting skin wounds. Hovewer, vaccination against diphtheria started in 1977 in Sao Tomé and vaccine coverage for the third dose of the pentavalent vaccine (DTP-HepB-Hib) in 2016 was 93% (95% CI 88-96%) in 12-23 months old children, with a dropout rate of ),Despite this microbiological evidence, because clinical and epidemiological data of these patients was not well characterised, we cannot exclude that one unidentified strain was the cause of the epidemic, and that human skin commensals have increased the pathogenesis of it, as described for Staphyloccucus aureus, (Boldock E. Surewaard B.G.J. Shamarina D. Na M. Fei Y. Ali A. Human skin commensals augment Staphylococcus aureus pathogenesis.) or that regulatory networks had a role in host-pathogen interactions, as described for S. pyogenes.(Kreikemeyer B. McIver K.S. Podbielski A. Virulence factor regulation and regulatory networks in Streptococcus pyogenes and their impact on pathogen-host interactions.)Results from the case control studies identified the presence of a wound and/or intertrigo in the two weeks prior to the disease, used by ubiquitous bacteria as an entry site, a known risk factor for the diagnosis of cellulitis with common superinfection with S. pyogenes. (Dupuy A. Benchikhi H. Roujeau J.C. Bernard P. Vaillant L. Chosidow O. Risk factors for erysipelas of the leg (cellulitis): case-control study.; Ferretti J. Stevens D. Fischietti V. Streptococcus pyogenes : Basic Biology to Clinical Manifestations.) The protective effect of having a recurrent health problem was most likely because controls were more likely to have a recurrent health problem and/or health seeking behaviour than the general population. Rather than patients admitted to the hospital for other medical conditions, it would have been more appropriate to select geographically, age and sex matched community-based individuals as controlsEnvironmental investigation confirmed the hypothesis that favourable climatic conditions for bacterial survival and growth played a role in the increased transmission of bacterial strains, including virulent ones, in the community. An increase in relative humidity was associated to a larger number of cases, which suggests that it may have helped the infection to occur, often in the presence of a skin lesion, most often located in the legs. Rainfall in São Tomé and Príncipe can be sudden and heavy, and these factors can facilitate the spread of bacteria in the environment. People from São Tomé and Príncipe often walk barefoot or only with flip flops, and rivers are used for several activities including washing clothes. Seasonality of cellulitis, associated to warmer months, has been described. (Haydock S.F. Bornshin S. Wall E.C. Connick R.M. Admissions to a U.K. teaching hospital with nonnecrotizing lower limb cellulitis show a marked seasonal variation.; Macario-Barrel A. Zeghnoun A. Young P. Froment L. Levesque H. Caron F. Influence of environmental temperature on the occurrence of non-necrotizing cellulitis of the leg.; Manning L. Cannon J. Dyer J. Carapetis J. Seasonal and regional patterns of lower leg cellulitis in Western Australia.; Peterson R.A. Polgreen L.A. Sewell D.K. Polgreen P.M. Warmer Weather as a Risk Factor for Cellulitis: A Population-based Investigation.) Total rainfall was also recently associated to cellulitis incidence in Taiwan; weather conditions such as temperature and humidity may interfere with the skin barrier and promote bacterial growth. (Hsu R.-J. Chou C.-C. Liu J.-M. Pang S.-T. Lin C.-Y. Chuang H.-C. The association of cellulitis incidence and meteorological factors in Taiwan.)In São Tomé and Príncipe, rainy season is lightly warmer than the dry season, but the temperature differences between seasons are limited, as the country lies on the equator. Islands can be more susceptible to climate change when they host fragile ecosystems. Between 1951 until 2010, precipitation in São Tomé and Príncipe have decreased of an average of 1.7 mm per year from 1950 until 2000 and temperature has increased of 1.2°C from 1978 until 2000. (

General Direction of the Environment, Ministry of Public Infrastructures and Natural Resources, UNDP. Second National Communication, Democratic Republic of Sao Tome and Principe 2011.

)

Though hospital may have played a role in the spread of virulent strains and possibly in the severity of the disease, the infection causing first symptoms mostly happened in the community. Unfortunately, Data on the follow-up of the course of the infection was suboptimal for the great majority of cases, and they were not systematically requested by the surveillance system. Moreover, the case definition for suspected patients was sensitive and included any skin inflammation whatever the cause (trauma, burn, wound), as patients with lymphangitis of any infectious cause, ulcer linked to varicosity, ulcer related to underlying disease such as diabetes were rarely properly diagnosed and therefore excluded from the counts. Therefore, an overestimation of the number of cases designated as “necrotising cellulitis” is likely to have occurred. According to the visit to the São Tomé hospital of international medical experts who reviewed admitted patients (n=20) during the peak of the epidemic, only 60% were actual necrotising infections. Interestingly, cellulitis was almost never getting to the soft tissue, and no cases of necrotising fasciitis were reported. This is in line with zero mortality that would be very unusual for necrotising fasciitis.

The lack of microbiological capacity was amongst the biggest challenges of the response.

Human expertise was present, but reagents were most of the times unavailable and basic microbiology was seldom done. The laboratory investigation of the subset of samples that were analysed abroad were limited by the lack of medical and epidemiological information attached to the samples.

Similarly, despite presence of strict guidelines for treatment of cases, antibiotic treatment of choice was irregularly prescribed also because it was often unavailable. Drug susceptibility testing was unavailable for most cases, and response to antibiotic treatment was not formally documented apart from very few exceptions. It is also surprising that despite the severity and the extent of the epidemic, no deaths were reported. Although deaths may have been underestimated, clinicians in Sao Tomé and Principe agreed that the great majority of patients recovered, even if morbidity and DALYs count would be high, if quantified, as skin surgical treatment and skin graft were practically not available, and many patients are likely to have had sequelae.

Control measures that were taken included improved hygiene in the hospitals and messages from the MoH on different media such as newspapers and television to improve personal hygiene in the community.

Changes in environmental conditions are likely to have played a role in this large-scale event, with relative humidity having favoured infection and abundant rainfalls likely to have had a negative effect on general hygienic conditions on the Islands. Though it is possible that most severe cases were caused by the same highly virulent strain, we could not identify it due to a lack of microbiological capacity. This experience calls on investing in diagnostics capacity, especially now that climate change has clearly started to increase the burden of infectious diseases in fragile environments such as Subsaharan Africa and island countries.

Acknowledgments

We acknowledge the participants to the study for their collaboration. We also thank Timothée Dub for technical support with the time-series analysis.

Author contributions

LS, AS, IB, EA, BCS, ABD, CADC, FS, MTP, MC and VSG performed field data collection. VL, JQ and LM performed laboratory investigations. LS and JL collected the climatic data and performed the time-series analysis. LS, AP, MHD and ISF performed data analysis and interpretation. LS wrote the manuscript and all authors revised the manuscript and approved its final version.

Ethics approval

The study followed international guidelines for Ethics in research such as the universal declaration of bioethics and human rights and the Helsinki declaration, and in agreement with São Tomé and Príncipe legislations. An informed consent was signed by every participant of the case control studies. These were coordinated by the Ministry of Health (MoH), with the technical support of WHO.

Funding source

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

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