Comprehensive infectious disease screening in a cohort of unaccompanied refugee minors in Germany from 2016 to 2017: A cross-sectional study.

BACKGROUND: Information regarding the prevalence of infectious diseases (IDs) in child and adolescent refugees in Europe is scarce. Here, we evaluate a standardized ID screening protocol in a cohort of unaccompanied refugee minors (URMs) in a municipal region of southwest Germany. METHODS AND
FINDINGS: From January 2016 to December 2017, we employed a structured questionnaire to screen a cohort of 890 URMs. Collecting sociodemographic information and medical history, we also performed a standardized diagnostics panel, including complete blood count, urine status, microbial stool testing, tuberculosis (TB) screening, and serologies for hepatitis B virus (HBV) and human immunodeficiency virus (HIV). The mean age was 16.2 years; 94.0% were male, and 93.6% originated from an African country. The most common health complaints were dental problems (66.0%). The single most frequent ID was scabies (14.2%). Of the 776 URMs originating from high-prevalence countries, 7.7% and 0.4% tested positive for HBV and HIV, respectively. Nineteen pathogens were detected in a total of 119 stool samples (16.0% positivity), with intestinal schistosomiasis being the most frequent pathogen (6.7%). Blood eosinophilia proved to be a nonspecific criterion for the detection of parasitic infections. Active pulmonary TB was identified in 1.7% of URMs screened. Of note, clinical warning symptoms (fever, cough >2 weeks, and weight loss) were insensitive parameters for the identification of patients with active TB. Study limitations include the possibility of an incomplete eosinophilia workup (as no parasite serologies or malaria diagnostics were performed), as well as the inherent selection bias in our cohort because refugee populations differ across Europe.
CONCLUSIONS: Our study found that standardized ID screening in a URM cohort was practicable and helped collection of relevant patient data in a thorough and time-effective manner. However, screening practices need to be ameliorated, especially in relation to testing for parasitic infections. Most importantly, we found that only a minority of infections were able to be detected clinically. This underscores the importance of active surveillance of IDs among refugees.

Introduction

In 2015, the European Union began experiencing a significant influx in refugees, especially from Africa and Asia. In the years since, ongoing migration has posed important economic, political, and healthcare challenges to the continent. Because the endemicities of many infectious diseases (IDs) vary globally, it seems reasonable to screen refugees for IDs that may be more prevalent in those coming from low- and middle-income countries. This includes infections with Mycobacterium tuberculosis, hepatitis B virus (HBV), human immunodeficiency virus (HIV), and parasitic diseases. Despite the potential for transmission, it is generally accepted that infected individuals do not represent a significant risk to populations in host countries [1,2]. Nevertheless, chronic infections negatively impact refugees’ general well-being while also increasing their morbidity and mortality. Furthermore, chronic parasitic infections may lead to additional problems such as anemia, nutrient deficiency, and stunting [3]. Often, the chronic and oligosymptomatic course of many IDs found in migrants leads to delays in diagnosis—delays compounded by language and cultural barriers, as well as by limited access to care. For these reasons, these diseases require active screening.

Most European countries have a low incidence of tuberculosis (TB; i.e., <20 cases/100,000 inhabitants/year). The majority of refugees, however, originate from low-income countries with high TB incidence (i.e., >100 cases/100,000 inhabitants/year). This is particularly an issue in the sub-Saharan region. In Gabon, for instance, TB incidence is approximately 60 times higher than it is in Germany [4]. The prevalence of chronic HBV infection in Africa overall (6.1%) is approximately four times higher than it is in Europe (1.5%) [5]. In relation to Germany (0.3%), the contrast with Africa is even more striking, with prevalence in Africa being 20-fold higher than it is in Germany [5,6]. Africa also has a high prevalence of HIV infection, (4.1% on average; >10% in some sub-Saharan countries), whereas European prevalence is approximately 0.4% (and, in Germany, 0.2%) [7].

During the peak period of 2015–2017, Germany had the highest immigration rate in Europe [8]. During this time, 1,444,225 asylum applications were filed with German authorities. Every fourth applicant was <18 years old, and among this group, 67,275 were entering the country as unaccompanied refugee minors (URMs), i.e., without their parents or other family members [8]. These child and adolescent refugees, exposed to hardships during travel via land or sea, represent a particularly vulnerable population. They are especially at risk for psychological trauma, malnutrition, and ID.

No systematic ID surveillance system for refugees exists in either Europe or Germany. Information on the ID burden among refugee minors, particularly URMs, is scarce. A few cross-sectional studies recently have been conducted in Germany [9-14]. However, none of them has adequately covered the abovementioned spectrum of ID in a URM cohort. Some of these reports have focused on urine and stool parasites [9-12], whereas others have focused on chronic hepatitis B prevalence [10-12] or on TB [13]. Three studies addressed a fuller range of relevant issues, but they included only a limited number of participants (102 and 154 URMs, respectively) [11,14], or else they focused on a select URM population (e.g., only Syrian refugees) [12].

In contrast to other world regions [15-17], until very recently, there were no EU-wide guidelines for ID screening of refugee minors. A review of international guidelines with experts’ recommendations from the European Academy of Paediatrics (EAP) only was released in August 2019 [18]. In 2015, a consensus paper on screening recommendations for refugee minors in Germany was published [19]. Due to lack of epidemiological data on ID in this group, screening recommendations were mainly based upon advice gathered from ID experts.

The goal of the study was to evaluate the performance and practicability of these screening recommendations [19]. We carried out a single-center, 2-year screening study of a cohort of URMs in southwest Germany.

Methods

Study design, reporting, and prespecified analysis plan

This 2-year (2016–2017) cross-sectional study followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guideline (S1 STROBE Checklist). The study’s prespecified analysis plan consisted of an algorithm for screening based upon the country of origin and clinical symptoms of each refugee minor (Fig 1).

Fig 1

Flowchart of applied screening algorithm in a cohort of unaccompanied refugee minors (n = 890).

*Participants refused screening or previously already screened; **comprehensive initial diagnostics in symptomatic patients (fever, cough >2 weeks, loss of weight); ***patients with gastrointestinal symptoms. Based on Pfeil and colleagues [ CXR, chest X-ray; HBV, hepatitis B virus; HIV, human immunodeficiency virus; IGRA, interferon gamma release assay; TB, tuberculosis.

Screening process

In order to standardize the screening protocol based upon history taking, physical examination, and laboratory investigations among URMs, we developed an electronic questionnaire to be used by the screening physician. The resulting data were directly importable into electronic database programs (S1 Text). This approach provided a structured, consistent format for patient screening and history taking while also facilitating data extraction and detailed data analysis. Screening was conducted at a single private pediatric practice in Freiburg, Germany. Here, in addition to pediatric care for the general population, a select team of pediatricians offered specific consultation hours for URMs. If a specific infection such as active TB was suspected during the initial visit, then the diagnostic workup was broadened to include non-prespecified analyses (e.g., chest CT scan). In accordance with German recommendations for the screening of refugee minors, all screening results subsequently were reviewed by pediatric ID specialists [19]. Referred through the regional reception center for URMs, patients were accompanied by a social worker and an interpreter. The study period lasted 24 months: from January 1, 2016, through December 31, 2017. Patients with pathological screening results were referred to the Pediatric Infectious Disease Department at the Freiburg University Medical Center for further evaluation. The structured patient history questionnaire included sociodemographic questions such as duration of transit, transit countries and route, and languages spoken, along with a set of medical history questions focused on underlying chronic conditions, drug use, present complaints, clinical signs of TB, and gastrointestinal infections. Mental health concerns were not systematically explored during the screening. However, if an underlying mental health condition was suspected during the general history taking or via indirect questions (e.g., sleep problems, mood instability, signs of anxiety, suicide intentions, or substance use), then patients were referred to the Department of Child and Adolescent Psychiatry at the Freiburg University Medical Center for additional psychological or psychiatric evaluation. The physical exam included a complete physical examination, as well as a basic vision test and audiometry. All patients underwent a full blood count in order to identify anemia, systemic inflammation, and eosinophilia (cutoff >500 eosinophils/μl) indicative of parasite infections [19].

All patients diagnosed with somatic diseases other than IDs were referred for specialized care and appropriately treated, if indicated. This also was the case for URMs who were transferred to another region of Germany. In instances of HIV or HBV positivity, special attention was given to follow-up across regions. Nonmedical aspects of care for refugees such as education and translation were provided through the help of social workers closely looking after the refugees at the URM housing facilities.

Vaccination strategy

None of the URMs in our Freiburg cohort were able to bring immunization records with them. They therefore were considered to be vaccination-naive. Reimmunization according to the German recommendations for missed immunizations [19] was started on the day of screening. All URMs received a first dose of MMRV and DTPaIPV vaccines. During the winter months, URMs also received a dose of influenza vaccine at the time of the screening visit. Follow-up vaccinations (two doses of MMRV and three doses of DTPaIPV in total), as well as vaccinations against HBV (three doses), pneumococcus (one dose), and HPV (two to three doses, depending upon age), were deferred to the pediatricians/general practitioners who took over the URMs’ medical care in the communities in which the URMs later settled.

Laboratory examinations

In patients with eosinophilia or gastrointestinal symptoms, stool tests for parasites and helminths with a target sample of three independent stool samples per patient were performed. Fecal samples were diluted in saline and stained with methiolate–iodine–formalin (MIF; Parasite Concentration System, BioRepair). Ova, cysts, trophozoites, and adult worms were identified by their characteristic microscopic features. All patients received a urine dipstick test, primarily for the purpose of identifying microhematuria as an indicator for urogenital schistosomiasis. Serologies for HIV and HBV were performed in patients from high-prevalence countries (HIV: prevalence in country of origin of ≥1%; HBV: prevalence in country of origin of ≥8%) [5,7,19]. For HIV infection, HIV-1 and HIV-2 antibody and p24 antigen chemiluminescence microparticle immunoassays (CMIA, ACHITECT System, Abbott) were used as screening tests, and HIV-1 and HIV-2 antibody western blot as a confirmatory test. HBV screening included hepatitis B surface antigen and anti–hepatitis B surface antibodies (CMIA, ARCHITECT System, Abbott). Serology for hepatitis C infection was performed only in patients with HBV infection or else in cases of clinical suspicion. General TB screening was performed on an age-dependent basis. In children and adolescents ≤15 years of age, an immunodiagnostic screening with an interferon gamma release assay (IGRA) was conducted. In accordance with national German requirements [19,20], in all patients >15 years of age, a chest X-ray was performed, regardless of symptoms [19,20]. The planning, conduct, and reporting are in accordance with the Declaration of Helsinki, as revised in 2013.

Ethics

A legal guardian was assigned to each URM by the municipality that provided consent for the required diagnostic and therapeutic measurements. In a majority of cases, a registered interpreter informed the URMs about the planned healthcare checkup with the study. Because of the high rate of URM illiteracy, formal written consent was not obtained. In the rare instances when an interpreter was not immediately available, URMs were provided information by a person who spoke a language that the URM understood. Healthcare coverage and appropriate treatment of identified diseases were made possible through government-provided health insurance. This allowed URMs to obtain treatment comparable to that received by nonimmigrant children in Germany. Data analysis was able to be performed in an anonymized fashion because the data points from the electronic questionnaire were exported into a data spreadsheet without patient identifiers. The study protocol was submitted to the ethics committee of the University of Freiburg as a noninterventional clinical practice study, and approval was granted after detailed discussion (study identifier 340/18).

Statistical analyses and graphics

Statistical analyses were performed using R (v 3.4.4) and the dplyr (v 0.7.8) package. Graphics were generated using the open source R packages ggplot2 (v 3.1.0) and maps (v 3.3.0) for the map in Fig 2C, as well as Adobe Illustrator CS6, Microsoft Excel, and GraphPad Prism (Version 8). Comparison across patient subgroups was carried out using two-sample t test for continuous variables and chi-square tests for categorical variables. P values < 0.05 were considered significant.

Fig 2

Sociodemographic characteristics of the screened cohort of unaccompanied refugee minors (n = 890).

(A) Age distribution; (B) nutritional status; (C) route and (D) duration of migration, as well as country of origin; (E) clinical signs and symptoms. The gray scale indicates the frequency of refugees from a specific country (see legend on the right). The size of the arrow indicates the relative frequency of chosen route to Europe, with the central Mediterranean route being the most important transit route during the study period. BMI, body mass index.

Definition of variables

In relation to migration routes, URMs followed one of four main migration paths: the (1) western Mediterranean, (2) central Mediterranean, (3) eastern Mediterranean (Aegean Sea), or via land through the (4) Balkan route. Reference values for body mass index (BMI) were in accordance with those published by WHO [21]. Age-related local reference values for hemoglobin, median corpuscular volume (MCV), median hemoglobin concentration (MHC), and transaminase enzyme activity (ASAT, ALAT) were applied. The pathological threshold for eosinophils was set at 500 cells/μl.

Results

Sociodemographic data

In total, 890 URMs were screened following the algorithm described in the Methods section (Fig 1). Most were adolescents between 16 and 17 years old (80.8%, median age 16.2 years, IQR 15–17 years); 5.8% (n = 52) were <15 years old (Fig 2A). The stated date of birth must, however, be interpreted with caution because most refugees did not possess any personal identification documents, and statements they provided could not be independently verified. BMI as an indicator of nutritional status was known in 864 URMs (97.1%) and, in a majority of cases, was normal (82.9%; Fig 2B). Overweight status and obesity were more prevalent than underweight status (14.3% versus 2.8% [21]). The vast majority of URMs were male; only 6.0% (n = 54) of the cohort were female. The URMs originated from 35 different countries. As shown in Fig 2C (arrows) and S1 Table, 93.6% of the patients came from Africa (n = 830), whereas 6.0% (n = 53) came from Asia, and just a small number (n = 4, 0.5%) from southern Europe. The central Mediterranean route was the one most frequently used to reach Europe (n = 663, 92.0%), followed by the western Mediterranean route (n = 28, 3.9%) and the eastern Mediterranean route across the Aegean Sea (n = 25, 3.5%). Only five URMs entered Europe by land (Balkan route). Information on the duration of travel from the country of origin to Germany was available for 653 participants (Fig 2C). Over two-thirds of all URMs arrived after a minimum of 6 months of travel. The median time from the country of origin to Germany was 12 months (range 1–96 months; IQR 7–18 months; Fig 2D). The interval between arrival in Germany and performance of medical screening (information available from 257 URMs, i.e., 28.9%) was short (S1 Fig)—a median of 3 days (range 0–120 days; IQR 2–4 days).

Health complaints

Based upon history taking and clinical examination, dental problems represented the most frequent complaints and were present in 65.8% of all URMs. The single most frequent ID in the cohort was scabies (14.2% of URMs). Mental health issues (e.g., sleep problems, mood instability, signs of anxiety, suicidal tendencies, or substance use) were noted in 24.6% of URMs (Fig 2E).

HBV and HIV infections

Study participants originating from countries with a high prevalence of chronic HBV infections (cutoff defined as ≥8%) were screened for hepatitis B antigen (HBsAg) [5,22]. Among the 776/890 participants tested, a total of 60 individuals (7.7% of all patients tested; Table 1) were identified with active HBV infection. Because most URMs were in our study area for a limited time period, we were only able to offer complete HBV diagnostics (including viral load) in 24/60 individuals (40.0%). In accordance with the European Association for the Study of the Liver (EASL) classification 2017 [22], the majority of tested URMs with active HBV infection were negative for hepatitis B e-antigen (HBeAg) (“inactive carrier”; i.e., 75.0%) and had low viral load in the blood (<104 IU/ml). Of note, the range of viremia differed substantially, ranging from <10 IU/ml to 1.2 × 109 IU/ml. In 5/26 of HBsAg-positive patients with known viral load, a viral load of >106 IU/ml was detected. Serum levels of aminotransferases did not correlate with viral load and often were normal in infected individuals (S2 Fig). Mode of transmission was unknown in all 60 patients. Most of the HBsAg-positive patients (15/21, 71.4%) had positive hepatitis A virus (HAV) serology indicative of previous HAV infection. No cases of combined HBV and HCV coinfection were identified. We did not initiate antiviral treatments in any of the patients with HBV infection, because the chronicity of infection could not be proven during the initial visit. Instead, the treatment mainstay was patient education regarding avoidance of hepatotoxic substances and sexual transmissibility of the disease. We also scheduled follow-up visits at our pediatric ID department and/or other appropriately specialized centers every 6 months.

Table 1

Screening for hepatitis B (n = 776), and HIV (n = 760).

Characteristic	Number of patients	Percent of tested patients
HBV status (known for 776/890; 87.2%)
HBsAg positivity	60	7.7%
Unknown mode of transmission	60	100.0%
Phase of HBV infection [22] (known for 24/60 patients)
HBeAg-positive HBV infection (“immune tolerant”)	5	20.8%
HBeAg-positive HBV infection (“immune active”)	1	4.2%
HBeAg-negative HBV infection (“inactive carrier”)	18	75.0%
HIV status (known for 760/890 patients)
Anti-HIV IgG positivity	3	0.4%

All URMs coming from high-prevalence countries of origin (HBsAg prevalence ≥8%) were screened for HBsAg. The positive participants were then tested for HBeAg. Serological screening for HIV was conducted in all URMs originating from countries with HIV prevalence >1%. HBeAg, hepatitis B e-antigen; HBsAg, hepatitis B antigen; HBV, hepatitis B virus; HIV, human immunodeficiency virus; IgG, immunoglobulin G; URM, unaccompanied refugee minor.

HIV infection was detected in 3/760 (0.4%) of URMs, all originating from sub-Saharan Africa. Of note, in all three patients, HIV infection was detected through screening and not by means of clinically indicated testing. In these three patients with HIV, antiretroviral treatment was initiated after extensive patient counseling about the disease.

Parasitic diseases

With a prevalence of 14.2% (n = 126) in the study cohort, scabies was the most frequent parasitic disease (Fig 2E), as well as the most frequent ID in the cohort overall.

Screening for stool parasites was performed in symptomatic patients (n = 7; 0.8%) and in patients with blood eosinophilia (n = 164; 18.8%). However, in a significant proportion of individuals with eosinophilia (32.3%, 53/164), stool samples could not be tested. In most cases, this was because the URMs became relocated to another part of Germany. Overall, the diagnostic yield of stool examinations was low. Only 19 pathogens could be detected in a total of 119 stool samples (16.0% positive tests), with intestinal schistosomiasis being the most frequent pathogen (n = 8). Six patients with urogenital schistosomiasis were identified via 4-hour midday urine microscopy. This was performed in response to the presence of microhematuria, for cases in which patients had a history of macrohematuria or dysuria without bacterial urinary tract infection, and in patients with blood eosinophilia along with negative stool investigation (Table 2).

Table 2

Screening of parasite infection.

Characteristic	Number of patients	Percent of tested patients
Eosinophil counts (known for 871/890 patients; 97.9%)
normal (<500/μl)	707	81.2%
500–1,000/μl	110	12.6%
>1,000/μl	54	6.2%
Stool parasites (n = 119 samples)
None	100	84.0%
Intestinal schistosomiasis	8	6.7%
Giardiasis	6	5.0%
Hook worm	2	1.7%
Taenia saginata	1	0.8%
Strongyloides stercoralis	1	0.8%
Amoebiasis	1	0.8%
Urinary parasites (n = 101 samples)
Urinary schistosomiasis	6	5.9%

Of 164 unaccompanied refugee minors with detected eosinophilia (>500/μl), stool tests were performed in 67.7% of cases. One to three stool samples per participant were tested. In the majority of cases (93.7%), two or three samples could be acquired. Besides eosinophilia, stool tests were performed when relevant gastrointestinal symptoms were present (n = 7). Coinfection of two stool parasites was rare (n = 1). In case of eosinophilia and negative stool parasitology (n = 94) and/or in patients with detected or reported hematuria (n = 4), microscopy of urine was performed.

As shown in Table 2, a significant number of URMs with eosinophilia tested negative for parasites (63/78, 80.8%), even when three stool samples were analyzed. Comparing eosinophil counts from patients with proven parasitic disease with those from patients with three negative stool samples and absence of hematuria revealed that eosinophilia was not specific for the presence of intestinal parasitic disease (p = 0.83, S3 Fig). This was not improved by raising the threshold to 1,000 eosinophils/μl, because only 7/19 (36.8%) patients with proven intestinal parasitosis had eosinophil counts of >1,000/μl.

Tuberculosis

Of the 890 patients eligible for screening, the majority (n = 751/874; 85.9%) were screened radiologically, whereas IGRA screening was conducted in 76 (8.7%, Table 3). IGRA screening was performed in patients under 15 years of age (n = 43), as well as in a select number of patients (n = 33) over 15 years old, (e.g., in case of pregnancy or when X-ray diagnostics already had been performed in another European transit country). In 47 patients, both X-ray diagnostics and IGRA were initially performed because of clinical or radiological suspicion of active TB.

Table 3

TB screening.

Characteristic	n of patients	Percent of tested patients
Screening modality (n = 874/890 screened patients; 98.2%)
Chest X-ray only	751	85.9%
IGRA only	76	8.7%
Chest X-ray + IGRA1	47	5.4%
Abnormal TB screening2	75/874	8.6%
Abnormal chest X-ray	34/798	4.3%
Positive IGRA	58/123	47.2%
Final TB workup
Negative screening	799/874	91.4%
Lost to follow-up	11/874	1.3%
Latent TB infection	38/123	30.9%
Pulmonary TB	15/874	1.7%

URMs originating from countries with high prevalence of TB (TB prevalence ≥20/100,000) were screened for TB. URMs <15 years of age were screened by IGRA, and URMs ≥15 years of age were screened by chest X-ray. In case of positivity, a second diagnostic mean (IGRA/chest X-ray) was added, and further diagnostics were performed. When active TB was suspected clinically1, both tests were performed in parallel.2 Abnormal TB screening was defined as IGRA positivity, X-ray abnormalities, or both.

Abbreviations: IGRA, interferon gamma release assay; TB, tuberculosis; URM, unaccompanied refugee minor

The rate of latent TB infection (LTBI) patients among the URMs screened by IGRA was high (38/123, 30.9%). Overall, we diagnosed 15 patients, all originating from sub-Saharan Africa, with pulmonary TB (1.7% of all screened URMs). Acid-fast bacilli only could be detected microscopically in the sputum of 3/15 TB patients (20.0%). Classical clinical symptoms (fever, cough >2 weeks, and weight loss) were neither sensitive nor specific for the identification of patients with active TB (S2 Table). Although more detailed analyses were hampered by the relatively small cohort of patients with pulmonary TB (n = 15) or LTBI (n = 38), we found that a positive history of cough for 2 weeks or longer was more common among TB patients than among URMs who had had a negative TB screening (26.4% versus 5.5%). However, coughing was also commonly reported among patients with a final diagnosis of LTBI (18.4%; S2 Table). The ratio of URMs who missed TB screening was low (n = 16; 1.8%), with half of them stating that they already had been screened with a chest X-ray elsewhere in Europe prior to coming to Germany. All patients with pulmonary TB were started on standard treatment regimens (isoniazid/rifampicin/pyrazinamide ± ethambutol) after cultural isolation of mycobacteria by sputum analysis, bronchoscopy, or endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) whenever possible. For LTBI patients, we recommended chemoprophylaxis (isoniazid ± rifampicin) in adolescents younger than 17 years or in older patients if treatment adherence (and safety regarding liver toxicity) was warranted.

Discussion

Here, we present the results of a cross-sectional study on ID screening in a cohort of 890 URMs in Germany during 2016–2017. Scabies (14%) and chronic hepatitis B (8% of URMs from high-prevalence countries) were the most frequently identified IDs, whereas active TB (1.7%) and HIV (0.4%) were diagnosed in a small minority of refugees. Importantly, the vast majority of IDs (apart from scabies) were not recognized clinically, either because the URMs were asymptomatic or else because the symptoms displayed were nonspecific. This underscores the need for symptom-independent screening approaches.

Sociodemographics

We found that a majority of the URMs originated from Africa and entered Europe via the central Mediterranean route. The sociodemographic composition of our cohort differs from other studies of the region [1,14]. In a retrospective study, Pohl and colleagues reported on 93 young patients (median age 5.7 years, originating mainly from Eritrea, Syria, and Afghanistan) who were hospitalized in a tertiary medical center in Switzerland [1]. In a study from southeast Germany, URMs described in an outpatient care setting were older (median age 16 years) and originated mainly from Somalia, Eritrea, and Afghanistan [14]. Our finding that approximately one-third of URMs underwent a journey lasting over 1 year underscores the potential for enormous physical and psychological impacts.

HBV and HIV

The overall prevalence of HBsAg positivity within our cohort (7.7%) was high in comparison to other studies [10,12,13]. Because only URMs originating from high-prevalence countries (8%) were selected for screening [5, 19], a selection bias must be taken into account. This approach differs significantly from the American, Canadian, and Australian guidelines on ID screening in new immigrants, as these countries additionally recommend performing HBsAg screening in individuals arriving from countries with intermediate prevalence (≥2%) [15,16] or else in all URMs [17]. In our cohort, the high number of HBV-infected URMs justified continuous screening. The high viral load present in some patients suggested an urgent need for patient counseling, especially with regard to sexual practices, as well as for close follow-up to confirm diagnosis and to avoid losing track of the patient. Vaccination of nonimmune people working and living together with URMs should be provided to prevent transmission. The best possible prevention strategy would be rigorous implementation of national vaccination programs in the URMs’ countries of origin, as this is likely to demonstrably lower HBV prevalence [23]. The low number of URMs infected with HIV (0.4%) is in line with previous studies [14,24]. Despite this low HIV prevalence, HIV screening still should be offered to all refugee minors in order to minimize transmission risks [19]. Internationally, approaches to HIV screening of URMs vary. Some authorities recommend HIV screening for all refugee minors [16], whereas others screen only unaccompanied adolescents [17] or adolescents originating from countries with an HIV prevalence of ≥1% [15]. Because the number of URMs tested for HCV was low in our cohort, our data interpretation here was limited.

We found scabies to be the most prevalent ID (14.2%) in our cohort. However, because diagnosis was based upon clinical evaluation and additional diagnostic tests were not performed, disease prevalence may have been overestimated.

Overall, it was only in a low proportion of patients with eosinophilia that parasitic infection was able to be detected by stool and urine analyses (21/119, 21.0%). Although we cannot definitively rule out parasitic infections in participants with negative stool and urine diagnostics, our data support the possibility that eosinophilia testing may not be suitable for the purpose of identifying patients who require workup for parasitic disease [25-27]. However, because routine serological testing for schistosomiasis and strongyloidiasis was lacking, this may have led to an underestimation of the helminth load and, therefore, may have skewed our data interpretation. Moreover, we may have missed some patients with asymptomatic malaria, although Malaria tropica could be ruled out due to the travel time after leaving endemic regions.

Our screening approach may explain this low diagnostic yield because only symptomatic individuals and those with eosinophilia >500/μl received additional testing (stool and urine examination), and no serologies for schistosomiasis or strongyloidiasis were performed. Some non-European authorities have suggested presumptive treatment of parasitic infection and a focus on specific serologies [15-17,28].

The overall prevalence of active TB (1.7%) in our cohort was low. This was well in line with findings from other screening studies [29-31]. Most URMs with active TB originated from the Horn of Africa, and all were from sub-Saharan African countries. Most URMs with TB were asymptomatic. As other studies also have shown [31], clinical data relating to weight loss, fever, and chronic cough did not help identify those with active TB disease versus LTBI or TB-naive patients (S2 Table). This finding confirms the need for active screening in order to achieve timely TB diagnoses [32].

In our cohort, there were only three TB sputum–positive patients (0.3%)—a finding that reconfirms the minimal risk URMs represent for the domestic population with regard to onward TB transmission. As compared with other published data [32], the proportion of URMs lost to follow-up during the workup of pathological TB screening was low (1.3%).

The currently recommended screening approach [19] focuses on the search for active TB and enforces screening for LTBI only in those <15 years of age. This recommendation is based upon data showing that young children with LTBI are at higher risk for developing active TB disease [33]. This elevated TB risk is less likely in adolescents and adults. In Europe, compliance and chemoprophylaxis completion rates among migrants with LTBI identified via screening have been reported to be low [34]. When treatment adherence is low, necessary follow-up visits get missed, regular blood tests are not regularly performed, and treatment safety is reduced. In such circumstances, LTBI screening may seem unjustified. However, in the subgroup of URMs <15 years old, the LTBI rate reached 23.3% in our study. For this age group, LTBI screening appears to be medically justified.

Our study contains certain limitations: For parasite screening, no serology-based testing was performed; thus, some infections may have been overlooked. Moreover, the TB screening approach was a mixture of screening for active TB in older URMs as well as LTBI in younger URMs. Therefore, true LTBI prevalence remains unknown for our study cohort. With the exception of HBV and HIV, screening for sexually transmitted diseases was not included in our study. Because of the small number of female URMs, no conclusions regarding health state differences between the sexes can be stated.

Most importantly, our results cannot be generalized to the overall child and adolescent refugee population and/or to other areas of Europe, because the characteristics of accompanied refugee minors are likely to differ given the diverse countries of origin and ethnicity of refugees across Europe [8].

As we have shown, our screening approach was practicable and had relatively low drop-out rates. However, some changes should be considered, especially in screening for parasitic diseases. Blood eosinophilia detection, together with stool and urine investigation, pose logistical challenges. They are also expensive and provide relatively unreliable results [35]. Our data indicate that a general screening for blood eosinophilia as a test for parasitic diseases is not immediately helpful. For this reason, studies are needed that address presumptive parasitic treatment or a combination of serology for schistosomiasis and strongyloidiasis [27,28,35,36] with symptom-based treatment in pediatric refugees. Given the unreliable data on disease prevalence in conflict zones, screening of all children and adolescents for active TB, hepatitis B, and HIV should be considered, especially if the numbers of migrants arriving in Europe continue to go down. Of note, the evaluated screening approach [19] was intended for a situation in which an unusually high number of children were migrating to Germany, as was the case during 2015 and 2016. Hence, our screening approach was restricted as compared with other established screening procedures [15,16,17]. Screening modalities for refugees and migrants inside the European Union need to become more clearly defined. For this, European evidence-based guidelines are urgently needed [15,18]. We plan to use the data from this study in order to revise screening recommendations—especially with respect to screening for parasitic infections.

In addition, in most cases, we had little (if any) reliable information from our patients regarding previous screening investigations before they arrived in Germany. Many of our URMs had healthcare contacts at their first ports of entry into Europe. This suggests that at least some of the investigations are likely to have been repeated and therefore could have been avoided. Some patients with active TB reported probable tuberculostatic treatment in other European countries before arriving in Germany. Unfortunately, however, their treatment was discontinued before completion, usually because the patient migrated or became transferred to another country. This suggests that the need for inter-European healthcare communication regarding these patients is urgent. Electronic health records would be ideal. In order to improve upon the follow-up of refugees with chronic infections, as well as to reduce frequency of repeat tests and thus rationalize screening and healthcare procedures, a more efficient transfer of healthcare records and information is needed.

Checklist of items that should be included in reports of cross-sectional studies.

STROBE, Strengthening the Reporting of Observational Studies in Epidemiology.

(DOC)

Click here for additional data file.

Electronic questionnaire for data acquisition (translated English version).

The questionnaire was completed by the physician performing the screening visit.

(PDF)

Click here for additional data file.

Delay of screening after arrival in Germany in unaccompanied refugee minors within the cohort; indicated is the screening time point in days after arrival to Germany.

(EPS)

Click here for additional data file.

Distribution of aminotransferase levels in HBsAg-positive patients.

Normal values are depicted with dotted lines. HBsAg, hepatitis B antigen.

(EPS)

Click here for additional data file.

Comparison of eosinophil distributions in patients with or without detected stool parasites.

Only patients with >500 eosinophils/μl and three analyzed stool samples were included. Analyzed by unpaired t test (Welch correction).

(EPS)

Click here for additional data file.

Countries of origin of unaccompanied refugee minors within the cohort (n = 890).

(DOCX)

Click here for additional data file.

Clinical warning signs for active tuberculosis infection in screened unaccompanied refugee minors (n = 874).

(DOCX)

Click here for additional data file.

19 Nov 2019

Dear Dr. Elling,

Thank you very much for submitting your manuscript "Comprehensive infectious disease screening among a cohort of 890 unaccompanied refugee minors in Germany from 2016 to 2017" (PMEDICINE-D-19-03543) for consideration at PLOS Medicine for our upcoming special issue on refugee and migrant health.

Your paper was discussed among the editorial team and sent to independent reviewers, including a statistical reviewer. The reviews are appended at the bottom of this email and any accompanying reviewer attachments can be seen via the link below:

[LINK]

In light of these reviews, we will not be able to accept the manuscript for publication in the journal in its current form, but we would like to invite you to submit a revised version that fully addresses the reviewers' and editors' comments. You will appreciate that we cannot make a decision about publication until we have seen the revised manuscript and your response, and we expect to seek re-review by one or more of the reviewers.

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Please let me know if you have any questions. Otherwise, we look forward to receiving your revised manuscript soon.

Sincerely,

Richard Turner, PhD

Senior Editor, PLOS Medicine

rturner@plos.org

-----------------------------------------------------------

Requests from the editors:

Please provide a fuller account of the ethical situation, in which you discuss the responsible party for unaccompanied minors; the issue of informed consent not having been obtained; and the accountability for providing appropriate treatment for the diseases identified.

Please remove "890" from your title, and add a study descriptor to the title following a colon, e.g. "... : a cross-sectional study".

We ask you to combine the "aims" subsection of your abstract with the preceding "background" subsection.

Please combine the "methods" and "findings" subsections of your abstract, and add a new final sentence to the new combined subsection summarizing the study's main limitations.

Please make that "sociodemographic" at line 35, and "January" at line 38.

At line 48, please start the sentence with "In this study, we found that ..." or similar.

After your abstract, we will need to ask you to add a new and accessible "author summary" section in non-identical prose. You may fine it helpful to consult one or two recently published research papers in PLOS medicine to get a sense of the preferred style.

Please remove the text at line 104-107, or move it to the discussion section.

Early in the methods section of your main text, please state whether or not the study had a protocol or prespecified analysis plan, and if so attach the relevant document(s) as a supplementary file. Please highlight analyses that were not prespecified.

The first paragraph of your discussion section should summarize the study's findings, and we ask you to expand the text at lines 293-297 to achieve this.

Please remove trade marks throughout your ms.

Please add a completed checklist for the most appropriate reporting guideline, e.g., STROBE, as a supplementary document, and refer to this early in your methods section. In the checklist, individual items should be referred to by section (e.g., "Methods") and paragraph number rather than by line or page numbers, as the latter generally change in the event of publication.

Comments from the reviewers:

*** Reviewer #1:

I confine my remarks to statistical aspects of this paper. These were quite simple, but appropriately so. However, they were not adequately described in the methods section. The "statistical analysis" section is now 3 lines long. It needs to say what was done and how it was done, even if it's simple. It also needs to describe how variables were operationalized. E.g. what were the "3 routes" to Europe? How were people assigned to one of them? What is "nutritional status" (from the results, it seems like it is BMI ... which isn't really a measure of nutrition, it's a rather poor measure of obesity but a person can be obese and malnourished)/ This sort of thing should be done for all the variables.

More specific comments:

Lines 92-93 These adjectives "limited number" and "select population" apply to all studies including this one. Please give details and say why your paper is better.

Lines 163-164 I'm a little confused here. What is "documented date of birth"? How was it determined? What about undocumented DOB - just ask the person their birthday?

Line 166-167 Certainly 6.0% girls seems low, but how do you know it is "underrepresented"? It's at least theoretically possible that nearly all URMs are boys. Do you have evidence one way oir the other?

Line 175 and 178 Please give interquartile range, as well

Figure 2 D - This isn't really a good graph. Why was this unusual format chosen? Maybe just text about this (it's only 3 pieces of information).

Peter Flom

*** Reviewer #2:

Overall this is an important descriptive paper for a large population of URM who live in Germany and throughout the EU.

Introduction:

- 2nd & 3rd lines of 1st paragraphs would be strengthened by: removing the 2nd line or adding what kinds of challenges: economic, educational, health etc. For the 3rd line, I would recommend reframing to the fact that infectious diseases have varying endemicities globally rather than framing it as it is reasonable to assume that the endemicity is higher in place where refugees are migrating from.

-Please more explicitly state the research question/objective

Methods:

-Please describe the types of patients cared for at a private practice in southwest Germany.

-Figure E. Please revise "mental problems" to say "mental health concerns"

Results:

Very well described, thorough discussion. Well used tables and figures.

Discussion:

It would be helpful to further describe a brief line or two on how the study is similar or different to Switzerland and SE Germany as described, in demographics, screening or ID prevalence?

Incorporation of the findings of this paper on Hepatitis B by Yun et al includes a large cohort of youth from E. Africa and would be a fitting for comparison, in addition to their discussion on immunization.

Increasing Hepatitis B Vaccine Prevalence Among Refugee Children Arriving in the United States, 2006-2012.

Yun K, Urban K, Mamo B, Matheson J, Payton C, Scott KC, Song L, Stauffer WM, Stone BL, Young J, Lin H.

Am J Public Health. 2016 Aug;106(8):1460-2. doi: 10.2105/AJPH.2016.303203. Epub 2016 Jun 16.

The findings of eosinophilia not being predictive of parasitic disease which supports the rationale has been described in a few studies in North America that would further strengthen the discussion on this topic, and may suggest the need for an alternative approach to parasitic disease screening vs. presumptive treatment among URM in Germany. Potential Papers to consider:

Eosinophilia: A poor predictor of Strongyloides infection in refugees.

Naidu P, Yanow SK, Kowalewska-Grochowska KT.

Can J Infect Dis Med Microbiol. 2013 Summer;24(2):93-6.

Eosinophilia and the seroprevalence of schistosomiasis and strongyloidiasis in newly arrived pediatric refugees: an examination of Centers for Disease Control and Prevention screening guidelines.

Dawson-Hahn EE, Greenberg SLM, Domachowske JB, Olson BG.

J Pediatr. 2010 Jun;156(6):1016-1018.e1. doi: 10.1016/j.jpeds.2010.02.043. Epub 2010 Apr 18.

High prevalence and presumptive treatment of schistosomiasis and strongyloidiasis among African refugees.

Posey DL, Blackburn BG, Weinberg M, Flagg EW, Ortega L, Wilson M, Secor WE, Sanders-Lewis K, Won K, Maguire JH.

Clin Infect Dis. 2007 Nov 15;45(10):1310-5. Epub 2007 Oct 11.

The discussion of the strong need to weave together surveillance systems and results was well described in a recent paper about migrants in the UE by Kunst et al and team and would further strengthen the basis for your argument in this area:

Int J Tuberc Lung Dis. 2017 Aug 1;21(8):840-851. doi: 10.5588/ijtld.17.0036.

Tuberculosis and latent tuberculous infection screening of migrants in Europe: comparative analysis of policies, surveillance systems and results.

Kunst H1, Burman M1, Arnesen TM2, Fiebig L3, Hergens MP4, Kalkouni O5, Klinkenberg E6, Orcau À7, Soini H8, Sotgiu G9, Zenner D10, de Vries G11.

*** Reviewer #3:

PLOS Medicine Review PMEDICINE-D-19-03543

Comprehensive infectious disease screening among a cohort of 890 unaccompanied refugee minors in Germany from 2016-2017

Thank you for the opportunity to review the above manuscript. The authors outline infectious diseases screening on a large cohort of predominantly male adolescent unaccompanied refugee minors (URM) presenting in transit to a paediatric practice in South West Germany. Given the lack of symptoms in many URMs, their finding appear to support universal infection screening regardless of clinical symptoms. It should be noted however that the clinicians did not complete full screening when compared to other published international refugee health management guidelines but an abridged version (local German guidelines).

The comments below are intended to improve the clarity of the manuscript and thus the message for the wider readership.

MAJOR COMMENTS

(1) It would be useful to comment upon the structure of the paediatric practice (which undertook screening) and how the infectious diseases screening of the URM was incorporated into the holistic health assessment of the cohort. Specifically, how were the patients managed with respect to treatment of identified diseases/infections and subsequently followed up?

(2) The authors state that the German recommendations were not based on local data; the Australian (2009 and revised 2016; Chaves et al) and Canadian (2011 Pottie et al) guidelines are evidence based guidelines with specific details relating to epidemiology of similar cohorts screened (e.g Mutch et al 2012 Journal of Paediatrics and Child Health, Paxton et al 2019 JPCH). Given this fact, I am querying why malaria was not included in the screening protocols, particularly as many URMs appeared to originate from (or transit through) malaria endemic regions. This would also be important to consider in your findings of peripheral eosinophilia and is a limitation of this paper.

(3) Was broader STI screening considered in this cohort (e.g. HCV, syphilis, chlamydia, gonorrhoea)? This is a high risk/vulnerable population (especially for the small percentage of URM females): other intentional screening recommendations include routine testing of this cohort due to heightened risks related to gender based violence, which are often unreported.

(4) The authors stated that 'no antiviral treatment was instituted" (Results section HBV and HIV infection line 210) - does this specifically apply only to the cohort that were HBV infected or inclusive of the HIV positive patients? If no treatment was commenced, how was this clinically justified for the HIV cohort? What follow-up, counselling and referrals were put in place?

(5) Did the practice undertake review of immunisation status (e.g. rubella, varicella, HBV, HPV)? What catch up immunisation processes were put in place for this cohort? Specifically for the HBV non-immune cohort, how did you commence catch up regimens (in the context of high prevalence of HBV infected URMs in this cohort)?

(6) It is unclear how nutritional status was ascertained. Was this based on normal haemoglobin and MCV levels on the FBP and normal BMI status? Were iron and vitamin D levels assessed in this cohort? There are data pertaining to children with LTBI who have low vitamin D levels, having an increased risk of progression to active TB. Given that 30% of the cohort with an IGRA were found to have LTBI, this is an important consideration. Were (1) vitamin D levels in this cohort and (2) how did you institute prophylactic or active therapy in this cohort?

(7) I note that almost a quarter of the cohort have "mental health" symptoms at first assessment. How were the wider components of refugee health assessment (e.g. education, dental health, immunisation, trauma, malnutrition, haemoglobinopathy screening, non-communicable diseases etc) dealt with in this practice or were they all referred on to other providers?

(8) How do the German guidelines align with the international guidelines (Aust/Canada) which recommend routine screening for all refugee children and adolescents across all of the above domains (not just infectious diseases focused)?

(9) What changes to clinical practice guidelines will be (or have been implemented) as a result of these findings? This is an important aspect of this study to include in your discussion, including whether broader screening should be implemented e.g helminth serology, other clinical domains (in keeping with other international processes).

MINOR COMMENTS

(1) Limitations; the lack of malaria testing and serology for schistosomiasis and strongyloidiasis is am important consideration and likely underestimates the helminth load demonstrated in your results. Many refugees are asymptomatic with respect to infection; this would also impact on your assessment of eosinophilia (used as an adjunct to serology).

(2) What proportion of the URMs had a protracted refugee transit (> 5 years)? Did this have any bearing on the prevalence of infections (e.g. scabies, malnutrition, dental caries, HBV)?

(3) Some typographical errors are noted in the paper; particularly in the abstract. It would also be better to use the term "practical" rather than "practicable" for ease of reading.

(5) How did you address language and literacy barriers within the screening process?

(6) In your introduction, the authors state that chronic parasitic infections may lead to problems such as anaemia, nutrient deficiency or stunting. What was the correlation in this cohort?

(7) Where there any differences in the infection profiles between female and male URMs?

(8) Data analyses (plural) is the correct term; use of analysis incorrectly through the manuscript is noted.

(9) Age distribution is skewed (ie non-normally distributed). Median and interquartile range would be more appropriate to use rather than mean age.

(10) The authors are incorrect in stating that no patients were connected with HCV as it was only tested for in a very small subset (21 patients). This should be corrected and noted in limitations.

(11) Looking at BMI status of patients with respect to TB status, it appears non-significant; can the authors confirm this interpretation?

***

Any attachments provided with reviews can be seen via the following link:

[LINK]

17 Dec 2019

Submitted filename: PBP_final.docx

Click here for additional data file.

3 Feb 2020

Dear Dr. Elling,

Thank you very much for re-submitting your manuscript "Comprehensive infectious disease screening among a large cohort of unaccompanied refugee minors in Germany: a cross-sectional study" (PMEDICINE-D-19-03543R1) for consideration at PLOS Medicine.

I have discussed the paper with editorial colleagues and the guest editors for the special issue, and it was also seen again by two reviewers. I am pleased to tell you that, once the remaining editorial and production issues are dealt with, we hope to be able to accept the paper for publication in the journal.

The remaining issues that need to be addressed are listed at the end of this email. Any accompanying reviewer attachments can be seen via the link below. Please take these into account before resubmitting your manuscript:

[LINK]

Our publications team (plosmedicine@plos.org) will be in touch shortly about the production requirements for your paper, and the link and deadline for resubmission. DO NOT RESUBMIT BEFORE YOU'VE RECEIVED THE PRODUCTION REQUIREMENTS.

***Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.***

In revising the manuscript for further consideration here, please ensure you address the specific points made by each reviewer and the editors. In your rebuttal letter you should indicate your response to the reviewers' and editors' comments and the changes you have made in the manuscript. Please submit a clean version of the paper as the main article file. A version with changes marked must also be uploaded as a marked up manuscript file.

Please also check the guidelines for revised papers at http://journals.plos.org/plosmedicine/s/revising-your-manuscript for any that apply to your paper. If you haven't already, we ask that you provide a short, non-technical Author Summary of your research to make findings accessible to a wide audience that includes both scientists and non-scientists. The Author Summary should immediately follow the Abstract in your revised manuscript. This text is subject to editorial change and should be distinct from the scientific abstract.

We hope to receive your revised manuscript within 1 week. Please email us (plosmedicine@plos.org) if you have any questions or concerns.

We ask every co-author listed on the manuscript to fill in a contributing author statement. If any of the co-authors have not filled in the statement, we will remind them to do so when the paper is revised. If all statements are not completed in a timely fashion this could hold up the re-review process. Should there be a problem getting one of your co-authors to fill in a statement we will be in contact. YOU MUST NOT ADD OR REMOVE AUTHORS UNLESS YOU HAVE ALERTED THE EDITOR HANDLING THE MANUSCRIPT TO THE CHANGE AND THEY SPECIFICALLY HAVE AGREED TO IT.

Please ensure that the paper adheres to the PLOS Data Availability Policy (see http://journals.plos.org/plosmedicine/s/data-availability), which requires that all data underlying the study's findings be provided in a repository or as Supporting Information. For data residing with a third party, authors are required to provide instructions with contact information for obtaining the data. PLOS journals do not allow statements supported by "data not shown" or "unpublished results." For such statements, authors must provide supporting data or cite public sources that include it.

Please let me know if you have any questions in the meantime. Otherwise, we look forward to receiving your revised manuscript shortly.

Kind regards,

Richard Turner, PhD

Senior Editor, PLOS Medicine

rturner@plos.org

------------------------------------------------------------

Requests from Editors:

Please remove the word "large" (cohort) from the title; and add a date range for when the study was done.

Please also remove "large" from line 35.

At line 47, please make that "criterion".

At line 56, please make that "were detected clinically" (assuming this is correct).

We suggest noting the prevalence of mental health disorders in your abstract.

At line 64, please substitute the word "fleeing" with "migrating" or similar.

At lines 79 and 388, would adapting the wording to "... were not detected clinically." be appropriate?

At line 83, please remove the word "completely".

Please adapt the sentence at line 115 to make it clear that the prevalence is lower in Germany (e.g., "In Germany ... a twenty-fold lower prevalence.").

At line 162, please make that "non-prespecified".

At line 169, please substitute "transit" (or similar) for "flight".

At lines 170 and 173, please substitute "drug [substance] use" (or similar) for "drug [substance] abuse".

Please reword the text at line 209 to make it clear whether the legal guardian or municipality provided consent. Please make it clear whether consent was informed, and how it was established (written/verbal). The wording in the text seems to contradict the note in your response to editorial points that it was "not possible to prospectively obtain informed consent" and we ask you to make the actual situation clear in the revised ms.

At line 213, please make that "Data analysis was performed in an anonymized fashion." (assuming this is correct).

At line 234, we suggest making the information less specific, e.g. "... the youngest ... was 9 years of age".

At line 268, can you be more specific about "mental health issues", i.e., were disorders noted?

At line 282, should that be "anti-HBV immunity"?

At line 387, please amend the text to "... were diagnosed in a small minority of refugees" or similar.

At line 388, please make that "were not recognized clinically".

At line 389, please adapt the text to "because they showed" or similar.

At line 399, please substitute "endure an escape" with "underwent a journey" or similar, and remove the word "flight".

At line 413, please avoid the word "subjects" (in favour of "people" or similar). Would "provided" be a good substitute for "vigorously controlled"?

At line 420, please substitute "countries" or similar for COI.

At line 435, please expand on "M.tropica", as this does not appear to occur earlier in your ms.

At line 452, we suggest revisiting the text "reconfirms the minimal risk". It may be that noting the small proportion of sputum positive patients could suggest a low risk of onward transmission. You might wish to briefly mention local policies on BCG vaccination, which could be relevant to this point.

At line 462, would that be "... reached 23.3% in this study"?

At line 483, please substitute "migrating" for "immigrating".

At line 485, should reference citations be substituted for "(Pottie; CDC; Chaves)"?

In reference 20, please abbreviate "World Health Organization" as "WHO", or not at all.

Please revisit reference 31, as the title appears to be partially duplicated.

In figure 2, please remove the word "escape" (route to Europe).

Comments from Reviewers:

*** Reviewer #1:

The authors have addressed my concerns. However they wrote

<<<

We agree with reviewer 2 that the representation is unconventional and changed Figure 2D to a more classical graph (pie chart).

>>>

I'm not a fan of pie charts. See my blog post https://statisticalanalysisconsulting.com/graphics-for-univariate-data-pie-is-delicious-but-not-nutritious/

If the editors want to publish as is, that's OK, but I'd rather delete the pie chart and just describe the data (it's only 3 points of information)

Peter Flom

*** Reviewer #2:

appreciate the authors additional consideration of the editors and 3 reviewers comments. I think altogether they have strengthened this paper. This paper is important as very little is understood about URM populations globally, however, I think the paper still requires some additional revisions for clarity and placing the papers in the context of the existing literature. Some additional suggestions follow:

-Reviewer 1 asked a question about the documented date of birth, and it was answered in a way as to clarify this question. The authors response should be added to the paper for clarity for the reader.

-Reviewer 2 recommended adding additional citations to the text. It's noted that these citations were added, however, additional sentences were not added placing this new study in the context of these previous studies. That component is very important. Additional comparison and potential emphasis or disagreement with these prior papers is needed to place this new study in context.

-Reviewer 3 queried about STI testing. Given this study is focusing on ID it is important that this discussion and additional commentary from the authors is included in the discussion of this topic. Many unaccompanied refugee minors have experienced trafficking and are at high risk for it. Like many adolescents, they may not reveal their sexual history particularly for non-consensual sex, or that differs from their definition of sex. Arguably they are at high risk and more discussion of STI testing should be included in the discussion as well as acknowledged in limitations.

-Reviewer 3 raised a very important concern about language and literacy barriers. In the body of the text the more detailed description of the interpretation should be added as well as the use of interpreter rather than translator.

-Reviewer 3 raises an excellent point about chronic parasitic infection contributing anemia, nutrient deficiency or stunting being included in the introduction this sentence would benefit from a citation. Further, the description provided by the authors should be included in the paper. Please change infest to infect, throughout this comment and the paper

-Reviewer 3 raises an important point about the data as it relates to the few female refugees in the study. Please add your response to the comment to the limitations section.

Introduction:

-For line 99, "...it seems reasonable to screen refugees for ID that bay me more prevalent in this population." Please provide more context for this assertion. I imagine that the authors mean that ID is higher prevalence in low and middle income countries and more refugees originate from these countries, however, I think this additional context is needed.

-In the conclusion to paragraph 1, I think it is important to also include that limited/no access to care may also contribute to delays in diagnosis

-Paragraph 2: It would be helpful to provide some context as to why Europe and Africa as continents are being compared specifically. I imagine this is due to the high number of URM from Africa. Further, please add language to reflect that these difference in prevalence relate to being low and high resource places rather than something inherent to the continents.

Methods:

-It is not clear what language the survey was written or administered in and if it was interviewer/interpreter assisted in completing this. were the questions developed with language and culturally concordat team members? Please provide additional context here.

Results:

-Please remove line 234-235 about the youngest study team member as this may be identifying.

-Figure 2: There are errors in the key as I believe C and E are flipped. Additionally it may be fitting to call it systems of clinical signs and symptoms, or systems and diagnoses given it relates to body systems and then scabies

-For Line 276-277, the majority should be placed in the context of the fact that it was only patients among the 24 that had additional testing. This would not reflect the majority overall, and is therefore misleading.

-Table 1, please move the section related to the 24 of 60 patients to another table or to another column as it is important that the % are interpreted to be reflective of the 24 patients not the overall 776 that had testing for HBV

-Line 341 please remove the frame "surprisingly" high, since this is the results section it should not include such descriptive words and opinions of the authors. Similarly please remove interestingly.

-Line 343 Please remove the line that TB patients were not contagious, or elaborate on the context. Clinicians commonly say that anyone with active disease is considered contagious.

-Line 346-347: Please remove the idea that there was a tendency toward lower BMI in TB patient as p=0.6 is far from significant.

-Paragraph 375-381 may fit best in the methods section as it does not seem like it follows from the results as it is not about something that was screened for

Discussion:

-Line 399-400 please add the citation that the finding is in reference to.

-Line 403-404 please provide additional context as to why it si worth noting that the URM are predominantly of African origin. Is it similar or different from prior studies?

-Line 410-413: Please revise these lines or remove them as it is inaccurate as currently framed. The viral load being high in 20% of patients is 5 patients given that there were only 24 patients who had their viral loads evaluated. This is 0.6% of the population of those tested for HBV. Additionally, it would strengthen the discussion to add that HBV may be acquired through vertical transmission, blood or sex. It would be helpful to provide some additional context around what other forms of prevention might be helpful to consider as compared to large scale prevention in the country of origin

-Line 455-456 please include a citation for the recommendation around LTBI vs. active TB disase screening

-Line 475-476 Please add context as to how these findings are similar or different than the studies cited

-I recommend removing Table 4, the sample size is <5 in the majority of the cells (making it difficult to interpret), and individuals with latent TB do not have symptoms suggesting that those that have cough, weight loss or fever have another illness.

***

Any attachments provided with reviews can be seen via the following link:

[LINK]

17 Feb 2020

Submitted filename: Reply to editors Feb17.docx

Click here for additional data file.

25 Feb 2020

Dear Dr. Elling,

On behalf of my colleagues and the academic editor, Dr. Paul Spiegel, I am delighted to inform you that your manuscript entitled "Comprehensive infectious disease screening in a cohort of unaccompanied refugee minors in Germany from 2016 to 2017: a cross-sectional study" (PMEDICINE-D-19-03543R2) has been accepted for publication in PLOS Medicine.

PRODUCTION PROCESS

Before publication you will see the copyedited word document (in around 1-2 weeks from now) and a PDF galley proof shortly after that. The copyeditor will be in touch shortly before sending you the copyedited Word document. We will make some revisions at the copyediting stage to conform to our general style, and for clarification. When you receive this version you should check and revise it very carefully, including figures, tables, references, and supporting information, because corrections at the next stage (proofs) will be strictly limited to (1) errors in author names or affiliations, (2) errors of scientific fact that would cause misunderstandings to readers, and (3) printer's (introduced) errors.

If you are likely to be away when either this document or the proof is sent, please ensure we have contact information of a second person, as we will need you to respond quickly at each point.

PRESS

A selection of our articles each week are press released by the journal. You will be contacted nearer the time if we are press releasing your article in order to approve the content and check the contact information for journalists is correct. If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact.

PROFILE INFORMATION

Now that your manuscript has been accepted, please log into EM and update your profile. Go to https://www.editorialmanager.com/pmedicine, log in, and click on the "Update My Information" link at the top of the page. Please update your user information to ensure an efficient production and billing process.

Thank you again for submitting the manuscript to PLOS Medicine. We look forward to publishing it.

Best wishes,

Richard Turner, PhD

Senior Editor

PLOS Medicine

plosmedicine.org