Data are available submitted as additional information S4.

Introduction {#sec005}
============

Diarrheal disease accounts for one in ten cases of death among children younger than five years \[[@pntd.0008448.ref001]\]. Cryptosporidiosis is caused by a coccidian parasite belonging to the genus *Cryptosporidium* and has been recognized as one of the major causes of diarrheal disease worldwide \[[@pntd.0008448.ref002]\]. Several species of *Cryptosporidium* are present in several host where they can cause disease. The First reports of human illness occured in 1976, when *Cryptosporidium* was identified in rectal biopsy specimens of a 3-year-old child \[[@pntd.0008448.ref003]--[@pntd.0008448.ref004]\]. *Cryptosporidium parvum and Cryptosporidium hominis* have been reported as the major causes of persistent diarrhea in developing countries, recognized as an opportunistic disease in HIV/AIDS patients, but also responsible for large outbreaks in immunocompetent individuals in developed countries \[[@pntd.0008448.ref005]--[@pntd.0008448.ref006]\]. *Cryptosporidium spp* therefore constitute a public health concern particularly due to reports of outbreaks in day care centers, immunocompromised patients and in waterborne transmissions \[[@pntd.0008448.ref007]--[@pntd.0008448.ref009]\]. The main symptoms of Cryptosporidiosis include; watery diarrhea which may be profused or prolonged, nausea, vomiting and low-grade fever \[[@pntd.0008448.ref010]--[@pntd.0008448.ref011]\]. In developing countries, other etiology related to the disease includes malnutrition and growth deficits among immunocompetent children and severe diarrhoea in immunocompromised persons \[[@pntd.0008448.ref012]\].

Numerous diagnostic techniques have been used to detect *Cryptosporidium* infection in humans and animals. They include the use of faecal smears stained by the modified Ziehl--Neelsen technique, Enzyme-linked immunosorbent assays (ELISA) and polymerase chain reaction (PCR) assays \[[@pntd.0008448.ref013]--[@pntd.0008448.ref014]\]. A comprehensive overview into the detection and molecular characterization of *Cryptosporidium* has been described elsewhere \[[@pntd.0008448.ref015]\]

The above mentioned methods have drawbacks such as being time-consuming and relatively expensive, as well as requiring well-equipped laboratories and well-trained or skilled personnel \[[@pntd.0008448.ref014]--[@pntd.0008448.ref016]\].

To overcome these limitations, antigens based-tests mostly relying on immuno-chromatographic assays have been marketed for rapid detection of *Cryptosporidium* antigens. RDTs have become increasingly popular tools and are highly suitable for point of care testing. They allow detection of antigens of one or more protozoan parasites in a single test format by lateral flow immunochromatographic assay. The advantage of such assay is that they are fast, easy to perform and interpret and thus can be used in low resource settings. Several of these RDTs have been in used over the years with varying sensitivities and specificities. In comparing the performance of 4 RDTs including; ImmunoCardSTAT! CGE, Crypto/Giardia Duo-Strip, RIDA QUICK *Cryptosporidium/Giardia/Entamoeba* Combi and *Giardia/Cryptosporidium* Quik Check the authors observed varying sensitivities ranging from 92 to 100% and 100% specificities for all RDT brands for the detection of *Cryptosporidium* when compared to ELISA, microscopy, and qPCR as gold standards \[[@pntd.0008448.ref017]\]. In addition, these RDTs demonstrate varying performances, and some of them are not well validated for the detection of less frequent non-parvum/hominis *Cryptosporidium* species \[[@pntd.0008448.ref002]--[@pntd.0008448.ref018]\].

The CerTest Crypto is a newly commercialized RDT with manufacturer reported sensitivity and specificity of \>99% respectively \[[@pntd.0008448.ref019]\]. No studies have so far evaluated the performance of this RDT in field settings including resource-limited settings such as ours. More so, most RDTs need to be refrigerated before use, which makes them unsuitable or difficult to use in settings with varying temperatures and humidity. The CerTest Crypto RDT however does not require refrigeration.

Consequently, there is a need for an extensive evaluation of this RDT in diverse field conditions in order to evaluate their diagnostic usefulness. The aim of this study was to assess the performance of CerTest Crypto against a composite reference standard of qPCR and RFLP-PCR.

Materials and methods {#sec006}
=====================

Study design, context and site {#sec007}
------------------------------

This prospective cross-sectional study, is part of a larger cross-sectional study entitled "Genetic determinants for transmission of *Cryptosporidium parvum*/*hominis* among humans and animals in Africa". The study was carried out from May 2017 to April 2018 in four sub-Saharan African countries (Lambaréné in Gabon, Agogo in Ghana, Antananarivo in Madagascar and Korogwe in Tanzania). All hospitals where participants were recruited are situated in semi-urban areas across all study sites. Samples used to evaluate the test performance of CerTest Crypto were collected from these health centers.

Study population {#sec008}
----------------

Stool samples were obtained from all children under 5 years of age presenting with diarrhoea or history of diarrhoea within the last 24h to the outpatient departments (OPDs) of the study hospitals of the four countries.

Field and Laboratory procedures {#sec009}
-------------------------------

Patients who provided a stool sample and an informed consent signed by the parents or legal guardian were enrolled in the study. Each child provided a fresh stool sample in a dry and leak-proof stool container. Prior to the start of the study, a general training was organized in Tanzania with participants from all the four study sites in attendance. Training was based on CerTest Crypto RDT testing and qPCR for Cryptosporidium. Laboratory technicians from all the four sites were trained on sample processing, time of testing, and protocols were developed and harmonized for use in all the four sites. The Standard Operation Pocedures (SOPs) for sample processing and testing with the CerTest Crypto RDT and qPCR were implemented in all the four sites. A small amount of stool sample (250 mg or 250 μl) was stored at -20°C for further analyses (with qPCR, RFLP-PCR).

CerTest Crypto RDT processing {#sec010}
-----------------------------

Fresh stool samples were tested using a commercially available RDT (CerTest BIOTEC S.L, Pol. Industial Rio Gallego II, Zaragoza Spain). The RDTs were stored at room temperature in all the sites according to manufacturer instructions. The stool samples were collected and transported in cool boxes (4°C) to the laboratory. All samples were analysed within 24 hours of sample collection. Stool sample aliquots were stored at -80°C for further analysis. Approximately 125 mg or 125 μl of stool sample was transferred into the diluent provided by the kit manufacturer by use of an applicator stick or pipette. The suspension was homogenized by shaking. Four drops of the diluted fecal material were dispensed into the circular window of the test card. The flow was allowed to run for 10 minutes followed by a visual interpretation. The test results were not recorded later than 10 minutes as instructed by manufacturer \[[@pntd.0008448.ref019]\].

The Certest Crypto Kit is supplied with quality control reagents. The quality control reagent consists of a known positive and negative sample. One negative and one positive control was run once a month in all the four sites and for each new batch of test kits. Additionally, the internal procedural control is included in the test, wherein the appearance of a green line in the control line result window is an indication of correct procedural technique and confirms enough specimen volume was used.

DNA isolation {#sec011}
-------------

A modified MO BIO-Qiagen stool DNA extraction protocol was performed incorporating six main steps including: sample treatment, cell lysis, inhibitors removal, DNA binding, a wash step, and DNA elution. Genomic DNA was extracted from 250 mg (or 250 μL) of stool with the use of DNeasy PowerSoil Kit (QIAGEN, Hilden Germany) formerly supplied by MO BIO (MO-BIO Carlsbad, CA, USA) as PowerSoil DNA Isolation Kit. All steps of the DNA extraction were performed following the manufacturer's instructions. The nucleic acids were eluted in 100 μL volume and 5 μL of the extract was used for qPCR.

Real-time PCR {#sec012}
-------------

DNA amplification was performed in a Rotor-Gene Q instrument (QIAGEN GmbH, Hilden-Germany) in 25 μL reactions using the HotStart Taq master mix kit (QIAGEN, Germany), 3.5 mM MgCl2, 500 nM forward primer (crypto-F) 5'- CGC TTC TCT AGC CTT TCA TGA -3', 500 nM reverse primer (crypto-R) 5'- CTT CAC GTG TGT TTG CCA AT-3', 175 nM Crypto probe 5'-ROX-CCA ATC ACA GAA TCA TCA GAA TCG ACT GGT ATC--BHQ2-3'. The primer and probe sequences and corresponding assession numbers have been published elsewhere \[[@pntd.0008448.ref020]\]. These primers and probe sequencies are specific for *Cryptosporidium parvum* and *C*. *hominis*, although their efficiency at detecting other *Cryptosporidium* species has not be evaluated.

All samples were tested in duplicate for *Cryptosporidium* species detection employing the following cycling protocol: one cycle at 95°C for 15 min (polymerase activation), followed by 45 cycles of 95°C for 15 seconds (denaturation), 67°C for 30 seconds (annealing) and 72°C for 30 seconds (extension), followed by a final cooling step at 40°C for 30 seconds. Phocin Herpes Virus (PhHV) Plasmid was incorporated into the master mix to control for PCR inhibitors and only samples with a cycle threshold \< = 38 were considered positive.

*Cryptosporidium* genotyping {#sec013}
----------------------------

All samples detected positive for *Cryptosporidium* species by qPCR were genotyped using a PCR--restriction fragment length polymorphism (RFLP) technique as described previously \[[@pntd.0008448.ref021]\]. To improve specificity, the PCR products from the restriction digest were sequenced and resulting sequences were blasted to identify the *Cryptosporidium* species.

Statistical analysis {#sec014}
--------------------

Statistical analyses were performed using Stata 14. Categorical variables were described as counts and percentages. Continuous variables were described by the median and interquartile range (IQR). To ensure a comparable set of observations for the analysis, observations with missing information on either the RDT or the PCR result were excluded from the analysis and the study period was restricted from May 2017 to April 2018. We calculated sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) to evaluate the performance of the CerTest Crypto RDT result versus the overall results observed by combining two PCR methods (PCR, RFLP-PCR) defined as a composite reference standard PCR. The performance characteristics of CerTest Crypto RDT were determined based on the ability of the CerTest to detect all species of *Cryptosporidium* (both human and animal species). Therefore, to assess the performance characteristics of this test to detect 2 of the most frequent species(*C*. *hominis* and *C*. *parvum*), eight observations that were PCR positive for "*C*. *meleagridis* "and "*C*. *xiaoi*/*bovis*" were excluded from the analysis. PCR was positive if either method yielded a positive result, otherwise negative. The test performance measures are presented as percentages along with the respective 95% confidence intervals (CI).

Ethical approval {#sec015}
----------------

Informed consent was obtained from the parents or legal guardian of the patient at the outpatient department (OPD) or inpatient department (IPD). The study protocol was approved by each Institutional Ethical Review Board of all four study sites. The National Health Research Ethics Committee (NatHREC) in Tanzania, Comité National d'Ethique du Gabon, the Committee On Human Research, Publications And Ethics, Kwame Nkrumah University Of Science And Technology of Kumasi, Ghana, the Medical Research Coordinating Committee of the National Institute for Medical Research, Tanzania (NIMR MRCC), the Ethical Committee of the Ministry of Health of the Republic of Madagascar and the Ärztekammer Hamburg, Hamburg, Germany.

Results {#sec016}
=======

A total of 596 stool samples from Ghana (N = 132), Gabon (N = 192), Madagascar (N = 83) and Tanzania (N = 189) was tested to assess the performance of CerTest Crypto RDT. [Table 1](#pntd.0008448.t001){ref-type="table"} provides the frequency distribution of all *Cryptosporidium* species from all study sites following analysis with PCR and RFLP. The diagnostic performance characteristics of *Cryptosporidium* CerTest Crypto RDT were analyzed using PCR as a reference standard in all 4 study sites. [Table 2](#pntd.0008448.t002){ref-type="table"} provides sensitivity, specificity, PPV, and NPV of the test to detect *Cryptosporidium* species in the four sites. The sensitivity of the test varied considerably in all study sites being highest in Madagascar (72.22%) in comparison to Gabon and Ghana which had 50 and 52% respectively and was lowest in Tanzania with 35.29%. Conversely, the specificity of the test was similar in all study sites ranging from 86 to 94%. However, subsequent re-analyses did not result in any changes in the performance characteristic of the cerTest across study sites ([Table 3](#pntd.0008448.t003){ref-type="table"}).

10.1371/journal.pntd.0008448.t001

###### Frequency distribution of *Cryptosporidium* rapid diagnostic test results by study sits.

![](pntd.0008448.t001){#pntd.0008448.t001g}

  ----------------------------------------------------------------------------------------
  Country                   C. hominis\   C. meleagridis\   C. parvum\   C. xiaoi/bovis\
                            n (%)         n (%)             n (%)        n (%)
  ------------------------- ------------- ----------------- ------------ -----------------
  **Overall (N = 482)**     91 (18.9)     7 (1.5)           15 (3.1)     1 (0.2)

  **Ghana (N = 107)**       15 (14)       2 (1.9)           7 (6.5.)     1 (0.9)

  **Gabon (N = 154)**       31 (20.1)     0                 7 (4.5)      0

  **Madagascar (N = 65)**   14 (21.5)     4 (6.2)           0            0

  **Tanzania (N = 156)**    31 (19.9)     1 (0.6)           1 (0.6)      0
  ----------------------------------------------------------------------------------------

10.1371/journal.pntd.0008448.t002

###### Overall and country specific test evaluation results of the RDT when compared to qPCR and RFLP as reference standard.

![](pntd.0008448.t002){#pntd.0008448.t002g}

  Country                   Sensitivity \[95% CI\]   Specificity \[95% CI\]   Positive predictive value \[95% CI\]   Negative predictive value \[95% CI\]
  ------------------------- ------------------------ ------------------------ -------------------------------------- --------------------------------------
  **Overall (N = 596)**     49.57 \[40.11, 59.04\]   92.52 \[89.79, 94.70\]   61.29 \[50.62, 71.22\]                 88.47 \[85.35, 91.13\]
  **Ghana (N = 132)**       52.00 \[31.31, 72.20\]   94.39 \[88.19, 97.91\]   68.42 \[43.45, 87.42\]                 89.38 \[82.18, 94.39\]
  **Gabon (N = 192)**       50.00 \[33.38, 66.62\]   94.81 \[90.02, 97.73\]   70.37 \[49.82, 86.25\]                 88.48 \[82.60, 92.92\]
  **Madagascar (N = 83)**   72.22 \[46.52, 90.31\]   86.15 \[75.34, 93.47\]   59.09 \[36.35, 79.29\]                 91.80 \[81.90, 97.28\]
  **Tanzania (N = 189)**    35.29 \[19.75, 53.51\]   91.61 \[86.08, 95.46\]   48.00 \[27.80, 68.69\]                 86.59 \[80.40, 91.40\]

10.1371/journal.pntd.0008448.t003

###### Overall and country specific test evaluation results of the RDT when compared to qPCR and RFLP as reference standard, excluding test results of *Cryptosporidium* meleagridis", "*Cryptosporidium* xiaoi/bovis".

![](pntd.0008448.t003){#pntd.0008448.t003g}

  Country                   Sensitivity \[95% CI\]   Specificity \[95% CI\]   Positive predictive value \[95% CI\]   Negative predictive value \[95% CI\]
  ------------------------- ------------------------ ------------------------ -------------------------------------- --------------------------------------
  **Overall (N = 588)**     49.53 \[39.72, 59.37\]   92.52 \[89.79, 94.70\]   59.55 \[48.62, 69.83\]                 89.18 \[86.12, 91.77\]
  **Ghana (N = 129)**       54.55 \[32.21, 75.61\]   94.39 \[88.19, 97.91\]   66.67 \[40.99, 86.66\]                 90.99 \[84.06, 95.59\]
  **Gabon (N = 192)**       50.00 \[33.38, 66.62\]   94.81 \[90.02, 97.73\]   70.37 \[49.82, 86.25\]                 88.48 \[82.60, 92.92\]
  **Madagascar (N = 79)**   71.43 \[41.90, 91.61\]   86.15 \[75.34, 93.47\]   52.63 \[28.86, 75.55\]                 93.33 \[83.80, 98.15\]
  **Tanzania (N = 188)**    36.36 \[20.40, 54.88\]   91.61 \[86.08, 95.46\]   48.00 \[27.80, 68.69\]                 87.12 \[80.98, 91.84\]

Further analyses of all cerTest negative samples from all study sites was performed using PCR-RFLP.

To further evaluate the stability throughout the year and batches variabilities of the CerTest Crypto against PCR reference standard, we looked at the performance of the test over the course of the year in all study sites (Figs [1](#pntd.0008448.g001){ref-type="fig"}--[4](#pntd.0008448.g004){ref-type="fig"}). Overall the false netative (FN) cases were detected almost every month in Gabon from the start to the end of the study period. Meanwhile, in Ghana, FN cases were detected more frequently during the first 5 months (May to September 2017) and then during the last 2 months (March to April 2018). While in Madagascar and Tanzania, the FN cases were mostly found in the beginning and towards the end of the study period. These numbers of FN observed at the four sites are not consistent and do not represent a clear pattern to suggest any batch effect on the test performance from the different study sites.

![Frequencies of diagnostic test evaluation (RDT vs. PCR as reference standard) from all patients for Ghana.](pntd.0008448.g001){#pntd.0008448.g001}

![Frequencies of diagnostic test evaluation (RDT vs. PCR as reference standard) from all patients for Gabon.](pntd.0008448.g002){#pntd.0008448.g002}

![Frequencies of diagnostic test evaluation (RDT vs. PCR as reference standard) from all patients for Madagascar.](pntd.0008448.g003){#pntd.0008448.g003}

![Frequencies of diagnostic test evaluation (RDT vs. PCR as reference standard) from all patients for Tanzania.](pntd.0008448.g004){#pntd.0008448.g004}

Finally, there is no evidence of the effect of gender, rainfall, sampling period as well as age group on *Cryptosporidium* infection across the study sites. However, there is a high proportion of *Cryptosporidium* infection occurring during the first two years of age accros countries ([S1 Table](#pntd.0008448.s004){ref-type="supplementary-material"} and [S1 Fig](#pntd.0008448.s006){ref-type="supplementary-material"}).

Discussion {#sec017}
==========

There is a need for efficient diagnostic methods for *Cryptosporidium* infection in settings where the disease is prevalent. Consequently, RDTs appear as a unique opportunity for point-of-care diagnosis in the absence of routine stool microscopy and advanced diagnostic tools. CerTest Crypto test is a commercially available RDT that is easy to perform, does not require refrigeration and saves time for the detection of *Cryptosporidium* spp. According to previous studies, the performance levels' variability of RDTs can depend on differences in commercial products, dissimilar methodologies employed and genetic diversity of *Cryptosporidium* across geographical regions \[[@pntd.0008448.ref018]\]. The cerTest Crypto RDT, whose high-performance parameters (Sensitivity and specificity \> 99%) has been claimed by the manufacturer, was evaluated in this study. Our observations on the performance characteristics of cerTest Crypto showed high specificity (Tables [1](#pntd.0008448.t001){ref-type="table"} and [2](#pntd.0008448.t002){ref-type="table"}). Our findings suggest that this RDT is reliable for detecting samples with no *Cryptosporidium*. As mentioned in previous studies, this result could also mean no cross-reaction with other pathogens causing diarrhoea \[[@pntd.0008448.ref018]--[@pntd.0008448.ref022]\].

In contrast, there are different sensitivities in each study site, ranging from 35.29% to 72.22% for the detection of *Cryptosporidium* species and from 36.36% to 71.43% for the detection of *C*. *parvum* and *C*. *hominis*. This indicates that this test performs poorly in the detection of all *Cryptosporidium* species. The overall number of true positives (TP) was generally low for all four sites. Meanwhile, the number of false negative did not vary considerably by month. The trend of the distribution of *Cryptosporidium spp*. that we observed amongst the younger children is similar to that reported by Current and Garcia in 1991 \[[@pntd.0008448.ref023]\], suggesting that the demographics could not influence the performance characteristics of cerTest Crypto ([S1 Table](#pntd.0008448.s004){ref-type="supplementary-material"}).

Therefore, the observed low sensitivity could be due to several other factors such as low parasite densities as reported elsewhere \[[@pntd.0008448.ref024]--[@pntd.0008448.ref025]\]. Moreover, our findings show a lower apparent sensitivity of the test kit that may be as a result of comparing the test to PCR, which can detect parasite DNA remaining in a patient's after an infection has been cleared as previously mentioned by Boyce and O'Meara in 2017 \[[@pntd.0008448.ref024]\]. Contrary to the study conducted by Bouyou-Akotet \[[@pntd.0008448.ref025]\] in which the authors suggest that low sensitivity was correlated to the lower parasite load in a test that targets a specific species \[[@pntd.0008448.ref026]\], our finding revealed a lower overall sensitivity with CerTest RDT which was not specific to one particular species.

Furthermore, our finding highlighted the overall decrease in PPV due to the very low number of TP observed from the four sites. Although the NPV of 88.47% can be considered high enough, this is not comparable to manufacturer reported \> 99% NPV. A considerable number of FN was observed from all study sites. Without any microscopy data to correlate the observed oocyst concentrations (infection intensities) and percentage of FN, it is difficult to determine the effect of parasite load on the detection of all species of *Cryptosporidium* (Tables [1](#pntd.0008448.t001){ref-type="table"} and [2](#pntd.0008448.t002){ref-type="table"}). More so, some *Cryptosporodium* genotypes may lack part or all of the protein targeted by the CerTest RDT which could explain the observed numbers of FN. Additionally, the PPV of 61.29% is due to very low numbers of TP and a considerable number of FP observed from all sites.

The evaluation of the sensitivity of RDTs used in the detection of malaria parasites has been reported to be influenced by changing seasons \[[@pntd.0008448.ref027]\]. In light of this, the observed differences in our case could be correlated with the fluctuation of temperatures in all study areas. Thus, most cases of FN and FP found in different months were likely due to the incidence of Cryptosporidiosis that varies with changing seasons and across geographical areas. Moreover, other reports suggest that temperatures above 30°C can affect the overall performance of the RDTs \[[@pntd.0008448.ref028]\] in the case of malaria parasites detection. Taken together, the accuracy of any RDT result may depend on several factors such as the quality of the RDT, storage, transport and end-user performance.

Our study had two main limitations. Firstly, due to the absence of microscopy data on oocyst concentrations, we could not correlate *Cryptosporidium* oocyst load and the test performance. However, according to the manufacturer, the current test does not detect low concentration antigens in the stool. Hence a sample that tests negative with the RDT is unlikely to be positive microscopically. Taken together, we could not report the cause of all FN reported from all study sites. Secondly, with no additional investigation on protozoan pathogens causing diarrhea, we cannot exclude the possibility of cross-reacting species as the cause of all FP cases in this study. Our demographic data did not not provide any substantial evidence on the effect of temperature and humidity on the test kit. Thus the variation in performance of the test kit that we observed in this study is not related to the handling and storage of the kits.

In conclusion, CerTest Crypto RDT has been designed to detect *Cryptosporidium species*. Although this coproantigen detection assay is rapid, its sensitivity is low for the detection of *Cryptosporidium* spp as well as *C*. *parvum* and *C*. *hominis* in particular. Therefore, additional research is needed to evaluate the performance of the CerTest Crypto RDT with particular emphasis on light intensity infections whereby the concentrations of antigens present in the sample are below the detection limit of the test that may result in an increase number of false negatives.

Supporting information {#sec018}
======================

###### STARD-checklist for CerTest Crypto RDT study.

(DOCX)

###### 

Click here for additional data file.

###### STARD diagram for participants flow in CerTest Crypto RDT study.

(PNG)

###### 

Click here for additional data file.

###### Data base_comb_reduced.

(DTA)

###### 

Click here for additional data file.

###### Proportion of *Cryptosporidium*-PCR positives and demographic data across the four study sites.

(DOCX)

###### 

Click here for additional data file.

###### Proportion of *Cryptosporidium*-PCR positives and demographic data across the four study sites.

(DOCX)

###### 

Click here for additional data file.

###### Distribution of infected stools across sites and age in months.

(DOCX)

###### 

Click here for additional data file.

We thank all participants, laboratory staff and physicians for their professionalism and dedication.

10.1371/journal.pntd.0008448.r001

Decision Letter 0

Ferreira

Marcelo U.

Deputy Editor

Bartelt

Luther A

Guest Editor

© 2020 Ferreira, Bartelt

2020

Ferreira, Bartelt

This is an open access article distributed under the terms of the

Creative Commons Attribution License

, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

13 Nov 2019

Dear Prof. Dr. Adegnika:

Thank you very much for submitting your manuscript \"Performance of a rapid diagnostic test for the detection of Cryptosporidium spp. in African children admitted to hospital with diarrhea\" (\#PNTD-D-19-01374) for review by PLOS Neglected Tropical Diseases. Your manuscript was fully evaluated at the editorial level and by independent peer reviewers. The reviewers appreciated the attention to an important problem, but raised some substantial concerns about the manuscript as it currently stands. These issues must be addressed before we would be willing to consider a revised version of your study. We cannot, of course, promise publication at that time.

We therefore ask you to modify the manuscript according to the review recommendations before we can consider your manuscript for acceptance. Your revisions should address the specific points made by each reviewer.

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\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*

In response to the important methodological and results questions raised by the reviewers, please also address whether cycle-time of Cryptosporidium detection by PCR had any relationship to the rapid antigen diagnostic result. (ie. were there more false-negative RDT at higher cycle time qPCR detections).

Reviewer\'s Responses to Questions

**Key Review Criteria Required for Acceptance?**

As you describe the new analyses required for acceptance, please consider the following:

**Methods**

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer \#1: This is an interesting study. The authors state that evaluating the accuracy of CerTest Crypto, a commercialized rapid diagnostic test (RDT) for the detection of Cryptosporidium antigens in the stool of children presenting with diarrhoea. A cross-sectional study was conducted in four study sites in Sub-Saharan Africa. All 596 specimens were processed and analyzed using CerTest Crypto RDT against a composite diagnostic panel involving two polymerase chain reaction (PCR) tests (qPCR and RFLP-PCR,) as the gold standard. GP60 sequencing was used to study potential Cryptosporidium subtype-dependent variation of the performance of the RDT.\'

They didn't mention anything regarding storage of their stool samples for CerTest Crypto. What was the timing of the test after collecting the stool samples? Did the test run on the same day of collecting stool samples. More details need to be given about this CerTest. From where they procured this test.

The primer-probe set used in this study was specific for C.Parvum and not for Cryptosporidium species,

Reviewer \#2: Authors need to review some aspects in materials and methods as indicated below:

Ethical approval: Some important details are needed about the lab tech training both on CerTest Crypto RDT using and PCR test (one training including the lab tech of all sites? Or one training per site? The training was followed by trainer's assessment or not?).

No mention of quality control and quality assurance in the materials and methods part. Why? Kindly clarify.

Lines 114-116: "All hospitals where participants were recruited are situated in semi-urban areas across all study sites. All samples collected within this study were used to evaluate the test performance of CerTest Crypto compared to the composite reference standard of qPCR and RFLP-PCR". Please, give provide clarifications (or rephrase) since this is not observed in the results (cf Tables 1 and 2).

Line 122: As indicated in Ethical approval it is better to write: The parents or legal guardian of the patient who provided the signed informed consent .....

Line 180: Please add: of Ghana (the Committee On Human Research, Publications And Ethics, Kwame Nkrumah University Of Science And Technology of Ghana, ...)

Reviewer \#3: See \"Summary and General Comments Section\" for detailed comments

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated? YES

-Is the study design appropriate to address the stated objectives? YES

-Is the population clearly described and appropriate for the hypothesis being tested? NO (description inadequate)

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested? YES

-Were correct statistical analysis used to support conclusions? YES

-Are there concerns about ethical or regulatory requirements being met? YES

\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\--

**Results**

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer \#1: The result is poorly written, the quality of experimental data need to be improved . Frequency distribution of RDT test results in Table 1. Presentation of Table 2 and Table 3 are not very clear. The authors are requested to compare . Sensitivity, Specificity, Positive Predictive Value (PPV) and, Negative Predictive Value (NPV) of RDT when compared with qPCR assay should be mentioned.

Reviewer \#2: The analysis presented match the analysis plan. However many complementary informations will need to clarify some points.

Lines 185-188: Please, remove this sentence here and completed materials and methods with: To ensure a comparable ..... to April 2018.

Line 192: Please, replace all 4 sites by the four sites.

Lines 196-200: Please remove "These performance characteristics ...were excluded from the analysis". Better in materials and methods part.

Discussion

Your study shows intra sites sensitivity compared to Rapid test specificity (The sensitivity of the test varied considerably in all study sites being highest in Madagascar (72.22%) in comparison to Gabon and Ghana which had 50 and 52 % respectively and was lowest in Tanzania with 35.29 %). Kindly, give some eventual raisons (training? cerTest Crypto RDT conservation? cerTest Crypto RDT manufacturing? samples conservation????).

Limitations

The absence of microscopic data is a real limitations of these results

The absence of QC and QA will need to be discussed if it's the case.

References

Please, updated the references. For example, the references below should be helpful

Bouzid et al, Clin Microbiol Rev. 2013: 115--134. doi: 10.1128/CMR.00076-12

Garcia et al, Clin Microbiol Rev. 2018: e00025-17. doi: 10.1128/CMR.00025-17

Cunha et al, Rev Inst Med Trop Sao Paulo. 2019; 61: e28. doi: 10.1590/S1678 9946201961028

Sylvia Afriyie Squire, Una Ryan, Parasit Vectors. 2017; 10: 195. doi: 10.1186/s13071-017-2111-y

Moore et al, PLoS Negl Trop Dis. 2016 Jul; 10(7): e0004822. Published online 2016 Jul 7. doi: 10.1371/journal.pntd.0004822

..........................

Reviewer \#3: See \"Summary and General Comments Section\" for detailed comments

-Does the analysis presented match the analysis plan? YES

-Are the results clearly and completely presented? YES

-Are the figures (Tables, Images) of sufficient quality for clarity? YES

\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\--

**Conclusions**

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer \#1: Conclusion is not very clear by the data presented.

Reviewer \#2: The conclusions of this study supported the data presented but the limitations of analysis didn\'t clearly described.

The authors discuss how these data can be helpful to advance our understanding of the topic under study and the public health relevance was addressed

Reviewer \#3: See \"Summary and General Comments Section\" for detailed comments

-Are the conclusions supported by the data presented? YES

-Are the limitations of analysis clearly described? PARTIALLY

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study? YES

-Is public health relevance addressed? YES

\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\--

**Editorial and Data Presentation Modifications?**

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend "Minor Revision" or "Accept".

Reviewer \#1: Many changes will be required

Reviewer \#2: No comment

Reviewer \#3: See \"Summary and General Comments Section\" for detailed comments

\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\--

**Summary and General Comments**

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer \#1: This is paper of interest but, poorly written and need to be revised

Reviewer \#2: Most human infections causing diarrhea are attributed to two species: Crytosporidium parvum (zoonotic transmission) and C. hominis (anthroponotic transmission). Overall this is an important paper for improving epidemiology and diagnostic of Cryptosporidium parasites using CerTest Crypto rapid diagnostic test; therefore could lead to better and faster management of diarrhea cases in our hospitals.

I have a few general comments. Please add in the introduction the background of the different cryptosporidium species in the four countries and clearly update diagnostic tests validated by the WHO to date. Although well readable, the text would benefit from a review by a professional editors include as co-authorships. The English is not fluent in several passages and it looks like the text has been translated from French.

Reviewer \#3: Summary statement: The authors presents an analysis of the efficacy of a novel Cryptosporidium rapid diagnostic test. The manuscript is clear and addresses an important concern -- specifically, that RDTs may be efficacious in the lab but their reliability and utility in the field is more important. The authors qualitatively evaluate the potential 'batch effect' variability in the assay; however, relevant details about the study and study design should be included. There may be a demographic bias rather than batch bias of the test. For example, the assay may be more efficacious when testing in young infant (i.e. using age as a proxy for parasitemia), vs. children and adults. Alternatively, it might be more effective in children who are at low risk for multiple infections rather than high risk for multiple infections (i.e. addressing the author's concern about off target organisms). By presenting more of the study design and cohort demographic data, these considerations could be evaluated.

Major concerns:

1\) Is this the first report of this cross-sectional study (or the cohort represented in the study)? If so, greater methodological detail is necessary. If not, please include a citation.

2\) Please include an analysis of patient demographics, per my point in the summary statement.

3\) Please clarify in the 'Real-time PCR' section which species could be detected with these pan-Crypto primers.

4\) Table 1 -- what do the dashes mean? If 0, please replace with 0.

5\) Is the protein targeted in the CerTest assay known? Can it be BLAST-ed to identify which organisms might be responsible for the hypothesized cross-reaction from other protozoa?

6\) On lines 96-98, the authors mention that RDTs tend to have reduced sensitivity with uncommon species. What species are defined as 'uncommon'? Naming the species would make it clear if this problem motivated Table 2.

7\) Line 308 -- without knowing more about the RDT, the following statement is not clear: 'observed color intensity on the test window in case of light infections'. It sounds as if the authors are suggesting that the intensity of the test color band could be difficult to see in light infections. The manufacturer's instructions available online do not include any text related to interpreting presence/absence of the band in cases where intensity is questionable. The authors should update the methods section to include details of the training provided to field workers related to interpretation of the CerTest results.

Minor concerns:

1\) Please add references for claims about the assay (Around line 100)

2\) Line 133 -- Please provide a reference for the manufacturer's instructions.

3\) Line 135 -- 'modified MO BIO-Qiagen stool DNA extraction protocol' Were both the MO-BIO and Qiagen versions of the kit used, and if so could there be an extraction bias with the different versions?

Grammar/copy edit suggestions:

Lines 71-73: 'Cryptosporidiosis which is caused by a coccidian parasite belonging to the genus Cryptosporidium has been recognized as one of the major causes of diarrheal disease worldwide (2).' -- this is grammatically incorrect. I'd suggest replacing it with 'Cryptosporidiosis is caused by a coccidian parasite belonging to the genus Cryptosporidium and has been recognized as one of the major causes of diarrheal disease worldwide (2).'

Lines 82-84: 'All of these methods have some drawbacks that include; being time-consuming and relatively expensive, require well-equipped laboratories and well-trained or skilled personnel' -- Inappropriate use of semi colon. I'd suggest replacing this text with 'All of these methods have drawbacks including being time-consuming and relatively expensive, as well as requiring well-equipped laboratories and well-trained or skilled personnel'

line 98 -- Please add new paragraph character.

Line 125: define SOP.

Line 136 -- 'Inhibitors' should be 'inhibitors'.

A number of abbreviations are presented that are unnecessary (i.e. not ever used again).

Cryptosporidium is periodically not italicized.

Line 304 -- 'species' should be 'species'.

\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\--

PLOS authors have the option to publish the peer review history of their article ([what does this mean?](https://journals.plos.org/plosntds/s/editorial-and-peer-review-process#loc-peer-review-history)). If published, this will include your full peer review and any attached files.

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**Do you want your identity to be public for this peer review?** For information about this choice, including consent withdrawal, please see our [Privacy Policy](https://www.plos.org/privacy-policy).

Reviewer \#1: No

Reviewer \#2: No

Reviewer \#3: No

###### 

Submitted filename: Reviewer Comment.docx

###### 

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10.1371/journal.pntd.0008448.r002

Author response to Decision Letter 0

20 Dec 2019

###### 

Submitted filename: Rebutal letter_PLOS neglected Tropical Diseases.docx

###### 

Click here for additional data file.

10.1371/journal.pntd.0008448.r003

Decision Letter 1

Ferreira

Marcelo U.

Deputy Editor

Bartelt

Luther A

Guest Editor

© 2020 Ferreira, Bartelt

2020

Ferreira, Bartelt

This is an open access article distributed under the terms of the

Creative Commons Attribution License

, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

20 Feb 2020

Dear Prof. Dr. Adegnika,

Thank you very much for submitting your manuscript \"Performance of a rapid diagnostic test for the detection of Cryptosporidium spp. in African children admitted to hospital with diarrhea\" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.  

When you are ready to resubmit, please upload the following:

\[1\] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript. 

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

\[2\] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don\'t hesitate to contact us if you have any questions or comments.

Sincerely,

Luther A Bartelt

Guest Editor

PLOS Neglected Tropical Diseases

Marcelo Ferreira

Deputy Editor

PLOS Neglected Tropical Diseases

\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*

Reviewer\'s Responses to Questions

**Key Review Criteria Required for Acceptance?**

As you describe the new analyses required for acceptance, please consider the following:

**Methods**

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer \#2: Authors described clearly the study methods with objectives clearly articulated. Study population well described with adequate sample size power. Endeed, the conclusions were supported by correct statistical analysis.

Reviewer \#3: Yes

\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\--

**Results**

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer \#2: In this study all analysis match the analysis plan and the results are presented accordingly with good tables and figures.

Reviewer \#3: Yes

\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\--

**Conclusions**

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer \#2: In this reviewed version, the conclusions are supported by data presented and authors discuss how data can be helpful to advance the study topic understanding. However some limitations will need to be clarify (ex. microscopic data)

Reviewer \#3: Yes

\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\--

**Editorial and Data Presentation Modifications?**

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend "Minor Revision" or "Accept".

Reviewer \#2: This manuscript can be accepted for publication in PloS NTD.

Reviewer \#3: (No Response)

\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\--

**Summary and General Comments**

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer \#2: No comment

Reviewer \#3: We find the scientific content of the manuscript acceptable in its current state, but clarify that our previous concern about demographics bias was raised because there is substantial variation in the incidence of crypto infection even within the first five years of life. Cryptosporidium detection, and cryptosporidium-positive stools, are heavily biased towards the first year of life and incidence is well known to be higher earlier in life (see Cryptosporidiosis, Current & Garcia, Clinical Microbiology Reviews 1991). There is no scientific value of discussion of batch effects if it does not also discuss potential demographic biases (with known associations with crypto incidence) across the batches. We recommend including demographic summaries across sites for factors known to be associated with incidence of crypto (gender balance, average age at sampling, average monthly rainfall at each site).

\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\--

PLOS authors have the option to publish the peer review history of their article ([what does this mean?](https://journals.plos.org/plosntds/s/editorial-and-peer-review-process#loc-peer-review-history)). If published, this will include your full peer review and any attached files.

If you choose "no", your identity will remain anonymous but your review may still be made public.

**Do you want your identity to be public for this peer review?** For information about this choice, including consent withdrawal, please see our [Privacy Policy](https://www.plos.org/privacy-policy).

Reviewer \#2: No

Reviewer \#3: No

Figure Files:

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, [https://pacev2.apexcovantage.com. PACE](https://pacev2.apexcovantage.com. PACE) helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at <figures@plos.org>.

Data Requirements:

Please note that, as a condition of publication, PLOS\' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here: <http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5>.

Reproducibility:

To enhance the reproducibility of your results, PLOS recommends that you deposit laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see <http://journals.plos.org/plosntds/s/submission-guidelines#loc-materials-and-methods>

###### 

Submitted filename: Reviewercomment.docx

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10.1371/journal.pntd.0008448.r004

Author response to Decision Letter 1

12 Mar 2020

###### 

Submitted filename: Rebuttal letter_PLOS neglected Tropical Diseases_MM.docx

###### 

Click here for additional data file.

10.1371/journal.pntd.0008448.r005

Decision Letter 2

Ferreira

Marcelo U.

Deputy Editor

Bartelt

Luther A

Guest Editor

© 2020 Ferreira, Bartelt

2020

Ferreira, Bartelt

This is an open access article distributed under the terms of the

Creative Commons Attribution License

, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

23 Mar 2020

Dear Prof. Dr. Adegnika,

Thank you very much for submitting your manuscript \"Performance of a rapid diagnostic test for the detection of Cryptosporidium spp. in African children admitted to hospital with diarrhea\" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.  

When you are ready to resubmit, please upload the following:

\[1\] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript. 

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

\[2\] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don\'t hesitate to contact us if you have any questions or comments.

Sincerely,

Luther A Bartelt

Guest Editor

PLOS Neglected Tropical Diseases

Marcelo Ferreira

Deputy Editor

PLOS Neglected Tropical Diseases

\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*

Reviewer\'s Responses to Questions

**Key Review Criteria Required for Acceptance?**

As you describe the new analyses required for acceptance, please consider the following:

**Methods**

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer \#3: Demographic information as reported in the new Supplementary Table S4 are inconsistent. Based on sex only being indicated for \~10% of each population and age for less than \~20% for each population, it appears the authors have only indicated demographic information for the crypto-positive children. Demographic information from all tested children is necessary to determine whether differences in age or sex across cohorts influenced test performance. The authors should indicate the total number of male and female subjects within each site, note just the number of males and females that were PCR-positive. A similar breakdown should be included for age. Additionally, a simple histogram with \~2 month resolution would easily demonstrate whether there are substantial differences in the distribution of subject age in each cohort.

\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\--

**Results**

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer \#3: Yes

\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\--

**Conclusions**

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer \#3: Yes, except for the statements related to Age and Sex differences across cohorts.

\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\--

**Editorial and Data Presentation Modifications?**

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend "Minor Revision" or "Accept".

Reviewer \#3: (No Response)

\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\--

**Summary and General Comments**

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer \#3: (No Response)

\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\--

PLOS authors have the option to publish the peer review history of their article ([what does this mean?](https://journals.plos.org/plosntds/s/editorial-and-peer-review-process#loc-peer-review-history)). If published, this will include your full peer review and any attached files.

If you choose "no", your identity will remain anonymous but your review may still be made public.

**Do you want your identity to be public for this peer review?** For information about this choice, including consent withdrawal, please see our [Privacy Policy](https://www.plos.org/privacy-policy).

Reviewer \#3: No

Figure Files:

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, [https://pacev2.apexcovantage.com. PACE](https://pacev2.apexcovantage.com. PACE) helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at <figures@plos.org>.

Data Requirements:

Please note that, as a condition of publication, PLOS\' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here: <http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5>.

Reproducibility:

To enhance the reproducibility of your results, PLOS recommends that you deposit laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see <http://journals.plos.org/plosntds/s/submission-guidelines#loc-materials-and-methods>

10.1371/journal.pntd.0008448.r006

Author response to Decision Letter 2

30 Mar 2020

###### 

Submitted filename: Rebuttal letter_PLOS neglected Tropical Diseases_MM.docx

###### 

Click here for additional data file.

10.1371/journal.pntd.0008448.r007

Decision Letter 3

Ferreira

Marcelo U.

Deputy Editor

Bartelt

Luther A

Guest Editor

© 2020 Ferreira, Bartelt

2020

Ferreira, Bartelt

This is an open access article distributed under the terms of the

Creative Commons Attribution License

, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

7 May 2020

Dear Prof. Dr. Adegnika,

Thank you very much for submitting your manuscript \"Performance of a rapid diagnostic test for the detection of Cryptosporidium spp. in African children admitted to hospital with diarrhea\" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

Thank you for your most recent revision. Please update the rebuttal letter and address questions in the supplementary material as requested by the reviewer.

I apologize for the time delays in our response as Covid-19 operations in our hospital have been very time-consuming.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.  

When you are ready to resubmit, please upload the following:

\[1\] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript. 

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

\[2\] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don\'t hesitate to contact us if you have any questions or comments.

Sincerely,

Luther A Bartelt

Guest Editor

PLOS Neglected Tropical Diseases

Marcelo Ferreira

Deputy Editor

PLOS Neglected Tropical Diseases

\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*

Thank you for your most recent revision. Please update the rebuttal letter and address questions in the supplementary material as requested by the reviewer.

I apologize for the time delays in our response as Covid-19 operations in our hospital have been very time-consuming.

Figure Files:

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, [https://pacev2.apexcovantage.com. PACE](https://pacev2.apexcovantage.com. PACE) helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at <figures@plos.org>.

Data Requirements:

Please note that, as a condition of publication, PLOS\' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here: <http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5>.

Reproducibility:

To enhance the reproducibility of your results, PLOS recommends that you deposit laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see <http://journals.plos.org/plosntds/s/submission-guidelines#loc-materials-and-methods>

10.1371/journal.pntd.0008448.r008

Author response to Decision Letter 3

8 May 2020

###### 

Submitted filename: Rebutal letter_PLOS neglected Tropical Diseases.docx

###### 

Click here for additional data file.

10.1371/journal.pntd.0008448.r009

Decision Letter 4

Ferreira

Marcelo U.

Deputy Editor

Bartelt

Luther A

Guest Editor

© 2020 Ferreira, Bartelt

2020

Ferreira, Bartelt

This is an open access article distributed under the terms of the

Creative Commons Attribution License

, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

2 Jun 2020

Dear Prof. Dr. Adegnika,

We are pleased to inform you that your manuscript \'Performance of a rapid diagnostic test for the detection of Cryptosporidium spp. in African children admitted to hospital with diarrhea\' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

Should you, your institution\'s press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Luther A Bartelt

Guest Editor

PLOS Neglected Tropical Diseases

Marcelo Ferreira

Deputy Editor

PLOS Neglected Tropical Diseases

\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*

Reviewer\'s Responses to Questions

**Key Review Criteria Required for Acceptance?**

As you describe the new analyses required for acceptance, please consider the following:

**Methods**

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer \#3: (No Response)

\*\*\*\*\*\*\*\*\*\*

**Results**

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer \#3: (No Response)

\*\*\*\*\*\*\*\*\*\*

**Conclusions**

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer \#3: (No Response)

\*\*\*\*\*\*\*\*\*\*

**Editorial and Data Presentation Modifications?**

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend "Minor Revision" or "Accept".

Reviewer \#3: (No Response)

\*\*\*\*\*\*\*\*\*\*

**Summary and General Comments**

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer \#3: The authors have addressed all of our concerns

\*\*\*\*\*\*\*\*\*\*

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Reviewer \#3: No

10.1371/journal.pntd.0008448.r010

Acceptance letter

Ferreira

Marcelo U.

Deputy Editor

Bartelt

Luther A

Guest Editor

© 2020 Ferreira, Bartelt

2020

Ferreira, Bartelt

This is an open access article distributed under the terms of the

Creative Commons Attribution License

, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

30 Jun 2020

Dear Prof. Dr. Adegnika,

We are delighted to inform you that your manuscript, \"Performance of a rapid diagnostic test for the detection of Cryptosporidium spp. in African children admitted to hospital with diarrhea,\" has been formally accepted for publication in PLOS Neglected Tropical Diseases.

We have now passed your article onto the PLOS Production Department who will complete the rest of the publication process. All authors will receive a confirmation email upon publication.

The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any scientific or type-setting errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript. Note: Proofs for Front Matter articles (Editorial, Viewpoint, Symposium, Review, etc\...) are generated on a different schedule and may not be made available as quickly.

Soon after your final files are uploaded, the early version of your manuscript will be published online unless you opted out of this process. The date of the early version will be your article\'s publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers.

Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Shaden Kamhawi

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

Paul Brindley

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

[^1]: The authors have declared that no competing interests exist.
