Molecular characterization of hookworm spp . isolated from food handlers , Khartoum , Sudan : A cross-sectional study

Hookworms infect the intestines, cause an itchy rash, Background: respiratory and gastrointestinal problems, and eventually iron deficiency (anaemia) due to the ongoing loss of blood. The objectives of this study were to assess the prevalence and molecular characterization of hookworms isolated from food handlers attending the Public Health Laboratories in Khartoum state, Sudan, for annual check-ups, and to assess the efficiency of PCR as molecular probe for hookworm infection. A total of 350 foods handlers’ participant's stool samples who Methods: were not suspected to be infected with hookworms were studied. Conventional methods were applied to make an early diagnosis. Stool samples were collected from public health laboratories (the public health lab in the Medical Commission) of Khartoum State; Omdurman locality, Khartoum North locality and Khartoum locality between October 2016 and April 2017. Specific identification was made by PCR on specimens identified as positive by Baermann’s technique, which were then sequence and genotyped The prevalence of hookworms in the stool samples of Results: food-handlers was 1.43%. One larval specimen recovered by Baermann’s technique was confirmed to be by PCR. PCR also Necator americanus confirmed that was the common species isolated from Necator americanus four further specimens. The results of DNA sequencing for Necator were deposited in NCBI GenBank under the following americanus accession numbers: sample 91, ; sample 92, ; MH035824 MH035825 sample 294, ; and sample 319 . MH035826 MH035827 PCR was found to be effective for confirmation of the Conclusion: diagnosis of hookworm infection and can aid the clinician in initiating prompt and appropriate antiparasite therapy. 1 2 3


Introduction
Ancylostomatidae and strongyl nematodes were known to pose a burden among a variety of mammalian hosts, including humans 1 .Ancylostoma duodenale and Necator americanus are the species recorded as being most responsible for human infection.They are found in mostly in warm and tropical areas 2 , and infect around 1.3 billion people worldwide 3 .A. duodenale are located in Central, Eastern and Northern Africa, India, Australia and Europe 4 .Further, N. americanus is present in Sub-Saharan Africa, Eastern Asia and Southeast Asia 4 .A zoonotic species in Asia (A. ceylanicum), also causes human infection; however, this is of limited significance and its exact geological appropriation has not been depicted.The most widespread of all hookworms are A. caninum, a parasite that infects dogs, and has lately been affirmed to survive in the human gut (but without developing sexually) 5 .Laboratory diagnosis of hookworm infection, routinely based on the presence of eggs in faeces by direct microscopy and/or concentration techniques 6 .Sometimes after gathering, through faeces culture tests (after 24 hours), hookworm ova may have hatched and rhabditiform larvae might be visible; due to morphological similarity between the two, these larvae must be differentiated from Strongyloides larvae.The severity of disease can depend on the number of ova that are counted in faeces.Furthermore, in some cases, adult hookworms may be found 6 .Use of molecular techniques (such as PCR) for parasitic detection and identification is more accurate and effective than conventional methods, and requires DNA of the hookworms for detection.For example, identifying both types of internal transcript spacer (ITS1 and ITS2, individually) of ribosomal DNA (rDNA) are proven genetic markers of parasitic nematodes, including A. duodenale and N. americanus.
The aim of this study was to estimate the prevalence and molecular characterization of hookworms isolated from the stool of food handlers attending Public Health Laboratories in Khartoum state, Sudan, for annual check-ups.

Samples
A total of 350 stool samples were collected.A previously described formula was used to determine sample size 7 .
Food handlers working in food facilities in Khartoum State, annually medically checked in public health laboratories and willing to participate were included in this study irrespective of their age, gender and nationality.The public health laboratories were located in Khartoum State, Omdurman locality, Khartoum North locality and Khartoum locality, with sample collection conducted between October 2016 and April 2017.The age of participant ranged from 16 to 68 years, with an average age of 32 years; 46% of the participants were less than 29 years old compared with 54% of being 29 or above.The majority of participants were males (83.19%), with 16.81% being female.Distribution of samples according to the residence data showed that 101 participants were from Khartoum north (28.9%), 160 participants were from Omdurman (45.7%) and 89 participants were from Khartoum (25.4%).
In the first stage, the collected specimens were examined using a microscope (Olympus CX22, Japan), the formol-ether concentration technique and Baermann's technique, as described previously 8 .Positive detected samples (those that included hookworm ova/larvae) were examined using PCR and DNA sequencing techniques for genotyping.Fresh stool specimens were collected from public health labs in Khartoum State.Samples that were found to be positive for hookworms eggs by direct examination, or for larvae by Baermann's technique, were selected for PCR testing (five samples).The stool samples that were negative for parasites by direct smear or the formol-ether concentration technique on three consecutive stool samples were used as negative controls.For molecular examinations, all stool samples were preserved in 70% ethanol at −20°C.The third-stage larvae that were recovered by Baermann's technique (Filariform) were collected and preserved.The extracted DNA from filariform larvae were used as control DNA during the molecular assay using Biotechnology G-Spin тм Total DNA Extraction Kit (iNtRON Biotechnology, Inc.), according to the manufacturer's protocol.

DNA extraction
DNA was extracted from stool as per manufacturer's instruction used iNtRON Biotechnology G-Spin тм Total DNA Extraction Kit (iNtRON Biotechnology, Inc.) according to the manufacturer's instructions.
The partial ITS1, full-length 5.8S gene, and partial ITS2 ribosomal DNA regions were amplified from larvae and ova using PCR.Amplicon sizes were approximately 485 bp (if it typical to N. americanus) or 380 bp (if it typical to Ancylostoma spp.).The procedure for single-round PCR amplification was performed according to Maxime PCR premix kit (iNtRON Biotechnology, Inc.) REF technique.Briefly 5 µl of DNA extract was added to PCR premix (Maxime PCR premix kit i-Taq), containing i-TagTM DNA polymerase, dNTP mixture and reaction buffer.Next, 2 µl primer (forward and reverse) was added alongside 13 µl of nuclease-free water.The reaction mixture was initially denatured at 94°C for 5 min, followed by 30 cycles of denaturation at 94°C for 30 secs, annealing at 65°C for 30 secs and extension at 72°C for 30 sec.This was followed by a final extension step for 10 min at 72°C in a thermal cycler (SensoQuest GmbH, Germany).
Approximately 5µl of PCR product was elecrophoresed on a 1.5% agarose gel (containing 1.5 µg/100 ml ethidium bromide) in Tris-borate-EDTA buffer, along with the tracking dye bromophenol blue, initially at 120 V and 35 A for 60 min.Thereafter, bands were visualized under UV light and an amplicon of 485 bp was considered positive for hookworm DNA.

DNA sequencing
DNA sequencing was carried out to confirm identification of the pathogen.Owing to the limited amount of DNA generated from one sample, only four samples of PCR products (485 bp) were sequenced using Sanger sequencing (Macrogen, Inc., Korea).The DNA fragment was 485 bp (if from N. americanus) or 380 bp (if it typical of Ancylostoma spp) from an internal sequence of the amplicon of single-round PCR were obtained using the specific primers.Two sequence fragments were generated for five samples, which were edited manually to correct possible base errors using BIOEDIT 7.09.They were then subsequently joined to reconstruct a fragment of 485 bp or 380 bp spanning genes for hookworm spp.

Bioinformatics analysis
DNA sequences were compared with the NCBI database to check DNA sequencing quality and specificity using the nucleotide BLAST server.Sequences were submitted in sequence alignment form to Clustal W (online tool) for multiple sequence alignment.

Sequences similarity and alignment
Firstly, before uploading the sequences to NCBI, we proofread the nucleotide chromatogram using Finch TV software version 1.4.0 to ensure that all ambiguous sites were correctly called and to determine the overall quality.Next, nucleotides sequences were searched for sequence similarity using nucleotide BLAST 10 Highly similar sequences were retrieved from NCBI and subjected to multiple sequence alignment using BIOEDIT software 11 .

Ethical considerations
Samples were collected from participants after provision of informed consent.Ethical approval was obtained from the National Committee for Research, Ministry of Health, Khartoum State.

Results
In this study, the stool samples of 350 participants were investigated for hookworms; five samples were found to be positive (1.43%) using the formol-ether concentration technique.One sample was found to be positive using Baermann's technique, which was used as the positive control.Five samples were found to positive by PCR, as shown in Figure 1.

Discussion
The present study indicates that the prevalence of hookworms in food-handlers who attended for annual check-ups in Khartoum State, Sudan was 1.43%.To the best of our knowledge, this is the first study to identify hookworm infection in Sudan using molecular techniques; it can therefore serve as a baseline for studies of hookworms in Sudan.Molecular techniques are more advantageous for hookworm identification as they are rapid and more sensitive.DNA was extracted from larva and ova using the iNtRON Biotechnology G-Spin тм Total DNA    Extraction Kit.Partial ITS1, full-length 5.8S and partial ITS2 rDNA regions were amplified and then sequenced.Molecular analysis was used to confirm human infections with one species of human hookworms, namely, N. americanus.The nucleotide sequences were analyzed by nucleotide BLAST searching and DNA was aligned using Clustal W 13 .Amplicon sizes were approximately 485 bp (typical of N. americanus).Sequences showed extremely high similarities (97-100%) with hookworm sequences in the GenBank database.The 4 samples studied were N. americanus.They were similar to N. americanus LC036565.1 (Japan), KM891738.1 (China) and LC036563.1 (Southern Vietnam).All sequences obtained in this study were deposited in the GenBank database.The results also indicate that PCR may be considered as a sensitive method for confirming a diagnosis of hookworm infection, and can aid the clinician in initiating prompt and appropriate antiphrastic therapy.
The differences in hookworm species populations causing human disease in different areas may possibly be related to parasite manner, ethnicity, atmosphere, temperature, and ecological factors 2,14 .The finding of N. americanus in Sudan goes well with other finding reported from Peninsular Malaysia 15 and Thailand 9 , where N. americanus was more common than A. ceylanicum.Their findings also supported this study that A. duodenale infection was not found.

Conclusion
PCR is a valuable tool for the laboratory diagnosis of parasites.It was found to be effective, sensitive and confirmatory for the diagnosis of hookworm infection and can aid the clinician in initiating prompt and appropriate antiparasite therapy.We confirmed that the major hookworm species infecting humans in Sudan is N. americanus.
I recommend someone that is an expert in the molecular diagnostics of hookworm infections to carefully evaluate the methods-to the best of my knowledge the methods are sound.

Major comments:
Please include values for the proportion infected and the 95% confidence intervals for all proportions infected/detected reported in your data.I think that the discussion section could be expanded a bit more to include other studies of molecular diagnostics of hookworm and compare the prevalence and detection rates of those studies.Since in Dataset 1 you have complete positive/negative results of each technique, you may also be able to do statistical tests of agreement.
In your conclusion, you state that 'We confirmed that the major hookworm species infecting humans in Sudan is .'I think that a sample size of 350 stool samples is insufficient to make that N. americanus conclusion.You can say that in your sample population, you confirmed that the major hookworm species infecting humans in Sudan is N. Americans.

Competing Interests:
Reviewer Expertise: Disease ecology, wildlife pathology, theoretical disease ecology, vector borne and zoonotic diseases I have read this submission.I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
It is not necessary to mention the collection period of the samples in the abstract.In the results of the abstract, I suggest that the authors mention the absolute number of infected persons after the percentage: "The prevalence of hookworms in the stool samples of food-handlers was 1.43% (5/350)." It is not necessary to specify the accession numbers of the GenBank sequences in the abstract.
At the conclusion of the abstract, the authors do not say anything about the percentage of parasitized individuals.What is the impact of this prevalence on food handlers?What are the risks to consumers?

Introduction
The citation of reference 3 is outdated.The most current prevalence of hookworm infection can be seen in Pullan RL, Smith JL, Jasrasaria R, Brooker SJ 2014.Global numbers of infection and disease burden of soil transmitted helminth infections in 2010.Parasit Vectors 7:37.

Methodology
It is not clear to me whether all 350 samples were analyzed by formaldehyde-ether concentration technique and Baermann's technique.If all samples were analyzed by these two methods, this should also be clear in the abstract.
The concentrations of the PCR components and DNA must be provided.The authors only provide the volumes used, but not the concentration.For reproduction of the technique, other researchers need the same conditions.I did not understand why the authors mentioned that the "amplicon of 485 bp was considered positive for hookworm DNA".What if the amplicon had 380 bp (corresponding to the species Ancylostoma ssp.)?

Discussion
The authors should be more cautious about discussing molecular results.Although it is true that PCR is a more sensitive technique than conventional coproparasitological methods, the results of the work do not allow to conclude this.The authors do not screen all samples comparing the different methods.In addition, the authors could discuss the possibility of cross-reaction.How specific are these primers so that they can amplify only the genetic material of hookworms and not that of other nematodes?How feasible is the use of PCR for diagnosis in Sudan?Although PCR is a more sensitive technique, the ideal for the reality of many localities is still the combination of different coproparasitological methods, which, although requiring expertise, are inexpensive techniques.Some molecular techniques such as RFLP-PCR and qPCR have already been standardized for the determination of hookworm species.The authors should discuss something about these other techniques.
Although the prevalence of hookworm infection has been low, the authors discuss nothing about the impact of parasitosis on food handlers.There are many papers that discuss the risks of this infection.Some speculation can be made.

Conclusion
To conclude that Necator americanus is the most prevalent hookworm in Sudan based only on these results is very strong.The analysis of only 350 samples, with the molecular confirmation of only four of these samples does not allow to conclude something so strong.

Is the study design appropriate and is the work technically sound? Yes
Are sufficient details of methods and analysis provided to allow replication by others?Partly If applicable, is the statistical analysis and its interpretation appropriate?Not applicable Are all the source data underlying the results available to ensure full reproducibility?Yes

Are the conclusions drawn adequately supported by the results? Partly
No competing interests were disclosed.

Competing Interests:
We have read this submission.We believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however we have significant reservations, as outlined above.
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Minor comments: First sentence Introduction-change 'were' to 'are' Change' age of participant' to 'age of participants' Fig 1-is the low molecular weight band a primer dimer?Is the work clearly and accurately presented and does it cite the current literature?Partly Partly Is the study design appropriate and is the work technically sound?Yes Are sufficient details of methods and analysis provided to allow replication by others?Yes If applicable, is the statistical analysis and its interpretation appropriate?Partly Are all the source data underlying the results available to ensure full reproducibility?Yes Are the conclusions drawn adequately supported by the results?Partly No competing interests were disclosed.