Pubmed Sorry! An Error Occurred While Rendering the Citation. Please Try Again Later.

Globally, diarrhea causes annually more 500,000 deaths in children under five years quondam. ^ane Fifty-fifty though diarrhea mortality has reduced >30% in 2000–2015, ⁱ the brunt of the disease is nonetheless enormous in low- and middle-income countries. ² Hither, the children under v years old experienced 1.ix billion and one.7 billion episodes of diarrhea in 1990 and 2010, respectively. ^two Knowing the causative agent is important for epidemiologic surveillance, for planning of preventive measures such as vaccines, and sometimes, for a specific antimicrobial treatment.

In the developing world, the etiology of diarrhea has been searched for mostly past using traditional assays. ^3–thirteen Some of these studies have included only children with diarrhea without a control group. ^{three , five , 7 , 9} In a recent review, rotavirus, calicivirus, enteropathogenic and enterotoxigenic Escherichia coli were estimated to cause more than than half of all diarrheal deaths in children under five years former. ¹⁴ Global Enteric Multicenter Written report (GEMS) found rotavirus, Cryptosporidium, enterotoxigenic E. coli and Shigella to be responsible for most cases of moderate-to-astringent diarrhea. ^eleven Even so, the importance of the pathogens varied according to the written report site. ^xi A multisite birth cohort study (MAL-ED) ended that in that location prevailed substantial heterogeneity in terms of the child's age, geography, flavour, rotavirus vaccine status and symptoms. ¹²

Obviously, methods with a wider capability to detect various agents must exist used. ^fifteen–17 On the other mitt, antibiotics, which are recommended but in encarmine diarrhea or severe cholera, are still frequently used and may render bacterial cultures negative. ¹⁸ Fortunately, recent years have brought diverse multiplexed molecular assays, which target enteric pathogens direct from fecal specimens. ^{16 , 19 , 20} Multiplex polymerase chain reaction (PCR) is another footstep forwards because information technology detects pathogens remaining unidentified by many conventional methods. ^{15 , 21} Five unlike laboratories used these tests for 15 mutual enteropathogens and found loftier sensitivity and good analytical and clinical functioning besides in childhood diarrhea. ¹⁷ Often, multiplex PCR reveals polymicrobial infections in diarrhea patients. ^{thirteen , 22 , 23} As enteropathogens are often establish also in children without diarrhea, quantitative analysis may aid distinguish clinically significant infections. ^{xiii , 17 , 24 , 25} A reanalysis of the GEMS written report using quantitative molecular methods improved characterization of the causes of diarrhea and indicated Shigella or enteroinvasive East. coli, rotavirus, adenovirus, enterotoxigenic E. coli, Cryptosporidium and Campylobacter as almost important enteropathogens. ²⁶

Republic of angola, a country in sub-Saharan Africa, has the highest reported under-v bloodshed in the globe, with 156.9 deaths per 1000 alive births. ²⁷ It is besides ane of the countries with highest burden of diarrhea in children under five years onetime, with estimated 3.five episodes per child-year and 18.000 almanac deaths. ²⁸ We aimed to investigate enteropathogens with multiplex PCR in children with and without diarrhea in Luanda, the capital of Republic of angola.

MATERIALS AND METHODS

Textile

The Pediatric Hospital of Luanda is a tertiary teaching and referral hospital. Well-nigh patients are self-referred and arrive from the greater metropolitan area. This study, approved by the institution's Ideals Committee, was carried out at the emergency department, which examines near 300 children each day; approximately 15% of these present with diarrhea. The report included children under 5 years of historic period whose diarrhea had lasted less than 2 weeks, who attended the Hospital in December 25, 2013, to Baronial 7, 2014, and of whom a stool sample could be nerveless.

Diarrhea was divers as three or more loose or liquid stools or at to the lowest degree one bloody stool a day. The command stool samples were nerveless during May xvi to October 5, 2014, from children with no diarrhea who visited the establishment for vaccinations, physiotherapy or a surgeon'south consultation. Rotavirus vaccination was implemented in Luanda in the end of 2022 only afterward our study. ESwab (Copan, Italia) liquid-based collection and transport organisation was used, and the samples were frozen in −70°C the day of collection. The samples were transported frozen to Republic of finland for multiplex PCR. Children were considered malnourished if their weight-for-height was less than −2 Z score.

Laboratory Methods

Samples and DNA Extraction

The stool samples in ESwab tube were extracted with NucliSENS EasyMAG (bioMérieux, France) co-ordinate to the manufacturer'due south protocol for Generic 2.0.ane program. For extraction of bacteria and viral nucleic acids, 70–100 μL of stool sample was added directly to the two mL of lysis buffer for off-board lysis step. Additional pretreatment protocol was executed for parasites. Shortly, 200 μL of stool samples and 1 mL of lysis buffer was added to the MagNA Lyser Greenish Beads (Roche, Switzerland) tube, and the samples were dewdrop beated in MagNA Lyser (Roche, Switzerland) musical instrument twice at 7000 oscillations per minute for 60 s having 90 south intermediate cooling steps later on each bead-beating pace. Samples were centrifuged at 10,000thousand for five min, and supernatant together with 1 mL of lysis buffer was transferred to the off-board lysis steps. Automatic extraction in NucliSENS EasyMAG was executed for all lysed samples using 100 μL as elution volume.

Real-Fourth dimension PCR and Assay

Samples were analyzed with 3 different real-time PCR assays. The Amplidiag Bacterial GE, Amplidiag Viral GE and Amplidiag Stool Parasites (Mobidiag, Finland) assays are developed for identification of diverse gastroenteritis-causing bacteria, viruses and parasites and are based on multiplexed existent-fourth dimension PCR distension in conjunction with fluorophore-labeled probes. The PCR reactions were conducted co-ordinate to the instructions of the each assays. Internal amplification control, positive PCR control and negative PCR control are included to the each examination serial. The PCR runs for bacteria and parasites were carried out in the Bio-Rad CFX qPCR Systems and CFX Manager Software (Bio-Rad, Hercules, CA). The PCR run for viruses was carried out in the ABI7500Fast real-fourth dimension PCR instrument and ABI7500Fast Software (Applied Biosystems, Waltham, MA). Run files were exported from the PCR software to the Amplidiag Analyzer (Mobidiag, Finland), which analyses and reports bacterial, viral and parasitic findings from each sample automatically. Semiquantitative estimates of pathogen load were analyzed based on threshold bike (C _t) values.

Statistical Analysis

All data were computed and analyzed with Statview five.1. The statistical tests comprised Fisher'south exact, χⁱⁱ, Student t test, Isle of man–Whitney U test, Kruskal–Wallis, Spearman correlation and logistic regression, as appropriate. Statistical significance was defined as P value beneath 0.05.

RESULTS

The analysis included 194 children less than 60 months of historic period, 98 children with and 96 without diarrhea. The median age was ten.five months [interquartile range (IQR), 17] overall, 8 months (IQR, 7) for children with and 18 months (IQR, 21) for those without diarrhea (P < 0.0001); 103 children were female and 91 male (P = 0.57).

Of the children with diarrhea, 30 were 0–v month old, 44 were vi–xi months quondam and 24 were 12–59 months old. Of them, 72% (70 of 97) showed also vomiting, 76% (71 of 94) fever and 59% (57 of 96) mucus in stools. Xix children complained abdominal pain and 5 had blood in stools. Likely diarrhea-caused dehydration was found in 57% (54 of 94) of children. Of children with diarrhea, 85% (79 of 93) had consulted earlier, 76% (71 of 94) had received some medication and 60% (52 of 87) also antibiotic treatment.

Running h2o at home was lacking more often in the diarrhea than in the not-diarrhea group (23 of fourscore, 29% vs. 39 of 87, 45%, P = 0.032). A tendency for less diarrhea was observed for children who used bottled or filtered drinking water and who had a toilet at home (Table 1). Malnourishment was more than common in the diarrhea than in the non-diarrhea group, 45% (44 of 97) versus 17% (sixteen of 94, P < 0.0001).

Tabular array i.:

Characteristics of Children With and Without Diarrhea

Of the stool samples, enteropathogens overall and bacteria, viruses and parasites were detected in 176 (91%), 152 (78%), 97 (50%) and 48 (25%), respectively (Table ii). When comparing the diarrhea versus the non-diarrhea groups, positive samples were institute significantly more than often in the onetime grouping when testing for all pathogens combined (95 vs. 81, P = 0.003), for leaner solitary (86 vs. 66, P = 0.001) and for viruses alone (64 vs. 33, P < 0.0001; Table 2). In contrast, no such departure was found for parasites (28 vs. 20, P = 0.21). Mixed infections with more than 1 pathogen group (bacteria, viruses or parasites) were found in 101 of 176 (57%) of all positive samples, in 67 of 95 (71%) of children with and in 34 of 81 (42%) of those without diarrhea (P = 0.0001). Furthermore, more than one pathogen was found in 131 of 176 (74%) of all positive samples and in 83 of 95 (87%) of children with and in 48 of 81 (59%) of those without diarrhea (P < 0.0001). In the diarrhea versus the non-diarrhea groups, the median number (IQR) of pathogens detected was 3 (2) versus i.5 (two; P < 0.0001); for bacteria, the numbers were ii (2) versus ane (ii; P < 0.0001); for viruses, one (i) versus 0 (1; P < 0.0001); and for parasites, 0 (1) versus 0 (0; P = 0.24).

TABLE 2.:

Results of Multiplex PCR of Stool Samples of Children With and Without Diarrhea

Regarding the children showing a positive PCR (of any kind), in that location was an association with age ≥ six months (P = 0.002), not being exclusively breastfed (P = 0.036) and eating solid foods (P = 0.036). A positive PCR for bacteria alone related likewise with age ≥ 6 months (P = 0.002), not being exclusively breastfed (P = 0.004) and eating solid foods (P = 0.002), whereas a positive PCR for viruses solitary associated only with age < 12 months (P = 0.003). Interestingly, no clear associations were found for parasites. The number of bacteria had a weak negative correlation with the child's weight for age Z score (Rho corrected for ties −0.155; P = 0.034). In the diarrhea group, the age correlated with the number of detected pathogen groups (Rho corrected for ties, 0.285; P = 0.005), with the number of detected pathogens (Rho corrected for ties, 0.375; P = 0.0002), with the number of bacteria (Rho, 0.293; P = 0.004) and with the number of parasites (Rho, 0.213; P = 0.036), merely non with the number of viruses. In all children, this kind of correlation with age, albeit negative, was plant just for viruses (Rho, −0.152; P = 0.035).

Enteropathogenic Eastward. coli was detected in 100 (of 194, 52%) samples, enterotoxigenic E. coli in 54 (28%), enteroaggregative E. coli in 89 (46%), enteroinvasive East. coli/Shigella in 29 (15%), Campylobacter in 29 (15%) and Salmonella in two (1%) cases (Tabular array ii). Of viruses, PCR for rotavirus was positive in xl (21%), for adenovirus in seven (4%), for norovirus in 43 (22%), for astrovirus in 7 (iv%) and for sapovirus in 22 (eleven%) samples. Of parasites, Giardia was found in 27 (14%), Cryptosporidium in 22 (11%) and Dientamoeba in 7 (4%) samples.

Diarrhea was found to be associated with the detection of enterotoxigenic (P < 0.0001) and enteroaggregative (P = 0.0005) E. coli, Campylobacter (P = 0.0008), rotavirus (P = 0.0005), sapovirus (P = 0.0004) and Cryptosporidium (P = 0.0004; Table 2). When age equally a confounding variable was taken into account, odds ratio for diarrhea with enterotoxigenic Due east. coli was 3.95 [95% confidence interval (CI): i.84–8.48; P = 0.0004]; for enteroaggregative Eastward. coli, 2.21 (95% CI: 1.16–4.22; P = 0.016); for Shigella or enteroinvasive E. coli, 3.seventy (95% CI: 1.38–9.92; P = 0.009) and for Campylobacter, iv.72 (95% CI: 1.68–thirteen.27; P = 0.003; Fig. i). Of viruses, rotavirus had an odds ratio for diarrhea of 2.33 (95% CI: ane.02–5.33; P = 0.046) and sapovirus of 8.46 (95% CI: 3.00–32.58; P = 0.002). Of parasites, only Cryptosporiudium correlated with diarrhea (odds ratio 5.96; 95% CI: i.66–21.40; P = 0.006).

Effigy 1.:

Odds ratios and 95% confidence intervals for diarrhea of pathogens with pregnant results.

In quantitative analysis of reactive samples, we found a pregnant difference only for Giardia: children with diarrhea had lower median C _t values (IQR) than those without diarrhea [32 (5) vs. 38 (6); P = 0.028]. In case of enteropathogenic Eastward. coli, just children with diarrhea had C _t below the cutoff value of nineteen.5 (seven/53 vs. 0/48; P = 0.013).

DISCUSSION

In our study, multiplex PCR detected enteropathogens in stool samples of nearly all children, whether with diarrhea (97%) or non (84%). In Rwandan children, the detection rate of PCR was equal, 94% in diarrhea patients and 79% in controls. ²⁴ In Mainland china, PCR for 10 bacterial and viral pathogens was positive in 82% of children with diarrhea and in 47% of control children. ^thirteen The GEMS, which used mostly traditional methods, identified at to the lowest degree one putative pathogen in 83% of children with moderate to severe diarrhea and in 72% of controls. ¹¹ In a cohort study in eight sites in the developing world (MAL-ED), where mostly traditional testing was used, at to the lowest degree 1 pathogen was detected in 77% of diarrheal stools and 65% of not-diarrheal stools of children 0–24 months of historic period. ¹²

In our study, PCR identified more pathogens, leaner or viruses in diarrhea than in solid stools. If a specimen was positive, more than ane pathogen was detected in diarrhea in 87% and in 59% in non-diarrhea. In Rwanda, the corresponding rates for the PCR-positive cases were 67% and 67%. ²⁴ 2 or more agents were identified in 53% of the diarrhea cases versus in 43% of controls in the GEMS ¹¹ and in 53% and 45% in the MAL-ED study, ¹² respectively. In Bengo province, Republic of angola, of traditionally tested diarrhea samples, merely 37% of the positive samples had 2 or more than pathogens identified. ³ There is no doubt that the mod methodology vastly improves detection of possible enteropathogens.

In our study, diarrhea correlated with the detection of enterotoxigenic and enteroaggregative Eastward. coli, Shigella or enteroinvasive E. coli, Campylobacter, rotavirus, sapovirus and Cryptosporiudium. In Rwanda, enterotoxigenic E. coli, Shigella, rotavirus and, contrary to Luanda, also norovirus were associated with symptomatic infection. ²⁴ Unlike our report, in the GEMS report, enteroaggregative Eastward. coli did non correlate with diarrhea just enteropathogenic E. coli did. ¹¹ In our study, diarrhea correlated with high quantity of enteropathogenic E. coli. In the MAL-ED study, Campylobacter, rotavirus and norovirus associated with diarrhea in children 0–24 months of historic period, Cryptosporidium just in the first yr of life and Shigella and astrovirus in the 2d year of life. ¹² In our study, the number of detected pathogens increased with age in children with diarrhea; in the MAL-ED study, this correlation was seen in all children, peculiarly during the first yr of life. ¹²

In the multicenter study in which 15 enteropathogens were searched for and the analytical cutoff was set at 35 quantification cycles, Campylobacter, enteropathogenic E.coli, enterotoxigenic E.coli, Shigella/enteroinvasive E.coli, Vibrio cholerae, rotavirus and Cryptosporidium associated with diarrhea, and the association was clearer at high pathogen loads. ¹⁷ Of interest, Giardia was more common in control children, ¹⁷ whereas in our study, diarrhea correlated with loftier Giardia load. In Tanzanian children with quantitative detection with TaqMan array cards, no pathogens were significantly associated with diarrhea. Even so, when pathogen quantity was taken into business relationship, an clan was observed for Shigella/EIEC, rotavirus and astrovirus. ²⁵ Here, sapovirus was associated with diarrhea, as was the case in India for the 24- to 59-month-old children. ^eleven Again, rather dissimilar findings were observed in Rwanda and Liu's multicenter study, where sapovirus was more mutual in control children. ^{17 , 24}

Our study has limitations. The number of the samples examined was restricted because of express resources. The children without diarrhea were older than children with diarrhea. Therefore, nosotros tested the relevance of dissimilar pathogens causing diarrhea, taking into business relationship age as confounding variable. The stool samples of children without diarrhea were nerveless later in the year than from those with diarrhea. Nosotros practise not, nonetheless, think this skewed the results because the findings remained rather abiding throughout all months of the year. All clinical data were not recorded, just we believe this happened randomly and did, thus, not distort the results. Differences in case definitions tin brand between-report comparisons hard. For example, the nowadays written report, and the MAL-ED study, ¹² included all children with diarrhea (3 or more loose stools or at to the lowest degree 1 encarmine stool a day), while in the GEMS study, the episode had to exist moderate to severe. ¹¹

We detected potential enteropathogens more than has been the example in well-nigh other studies. Also, here, more than i pathogen was detected with high frequency. Fifty-fifty if Angola is considered an upper middle-income country, wealth is distributed unevenly, and most children served by the Pediatric Hospital represent poorer population. In this kind of low-resource settings with overcrowding and poor hygiene, the children are repeatedly exposed to multiple pathogens.

In a setting in which several potential pathogens are detected in the same sample, and the aforementioned putative pathogens are found also in children without diarrhea, the role of each single pathogen in terms of causing diarrhea is hard to determine. PCR has high sensitivity and tin can detect pathogens in low quantities with unclear significance, whereas quantitative PCR may help to distinguish clinically of import infection from asymptomatic wagon. ^{25 , 26} It is likely that the load of each pathogen is important. It is as well thought that subclinical infections may cause physiologic and structural alterations of the gut, which lead to repeated episodes of diarrhea, impaired gut part and growth harm. ^{12 , 29}

REFERENCES

i. Liu L, Oza S, Hogan D, et al. Global, regional, and national causes of under-5 mortality in 2000-fifteen: an updated systematic analysis with implications for the Sustainable Development Goals. Lancet. 2016;388:30273035.

2. Fischer Walker CL, Perin J, Aryee MJ, Boschi-Pinto C, Black RE. Diarrhea incidence in low- and middle-income countries in 1990 and 2010: a systematic review. BMC Public Health. 2012;12:220.

3. Gasparinho C, Mirante MC, Centeno-Lima S, et al. Etiology of diarrhea in children younger than five years attending the Bengo Full general Hospital in Angola. Pediatr Infect Dis J. 2016;35:e28e34.

iv. Albert MJ, Faruque As, Faruque SM, et al. Case-control study of enteropathogens associated with childhood diarrhea in Dhaka, Bangladesh. J Clin Microbiol. 1999;37:34583464.

v. Mandomando IM, Macete EV, Ruiz J, et al. Etiology of diarrhea in children younger than 5 years of historic period admitted in a rural hospital of southern Mozambique. Am J Trop Med Hyg. 2007;76:522527.

6. Reither K, Ignatius R, Weitzel T, et al. Astute childhood diarrhoea in northern Ghana: epidemiological, clinical and microbiological characteristics. BMC Infect Dis. 2007;7:104.

• Cited Here |
• PubMed

7. Jafari F, Garcia-Gil LJ, Salmanzadeh-Ahrabi South, et al. Diagnosis and prevalence of enteropathogenic leaner in children less than 5 years of age with acute diarrhea in Tehran children'south hospitals. J Infect. 2009;58:2127.

8. Samie A, Guerrant RL, Barrett L, et al. Prevalence of intestinal parasitic and bacterial pathogens in diarrhoeal and non-diarroeal homo stools from Vhembe commune, South Africa. J Health Popul Nutr. 2009;27:739745.

• Cited Here |
• PubMed

nine. van Eijk AM, Brooks JT, Adcock PM, et al. Diarrhea in children less than 2 years of age with known HIV condition in Kisumu, Republic of kenya. Int J Infect Dis. 2010;14:e220e225.

10. Bonkoungou IJ, Haukka Yard, Österblad Grand, et al. Bacterial and viral etiology of childhood diarrhea in Ouagadougou, Burkina Faso. BMC Pediatr. 2013;13:36.

• Cited Here |
• PubMed

xi. Kotloff KL, Nataro JP, Blackwelder WC, et al. Brunt and aetiology of diarrhoeal illness in infants and immature children in developing countries (the Global Enteric Multicenter Study, GEMS): a prospective, case-control study. Lancet. 2013;382:209222.

12. Platts-Mills JA, Babji S, Bodhidatta Fifty, et al. Pathogen-specific burdens of community diarrhoea in developing countries: a multisite birth cohort study (MAL-ED). Lancet Glob Health. 2015;3:e564e575.

• Cited Here

13. Li LL, Liu N, Humphries EM, et al. Aetiology of diarrhoeal disease and evaluation of viral-bacterial coinfection in children nether v years quondam in China: a matched case-control study. Clin Microbiol Infect. 2016;22:381.e9381.e16.

14. Lanata CF, Fischer-Walker CL, Olascoaga AC, et al.; Child Wellness Epidemiology Reference Group of the World Wellness Arrangement and UNICEF. Global causes of diarrheal disease mortality in children <5 years of age: a systematic review. PLoS One. 2013;viii:e72788.

15. McAuliffe GN, Anderson TP, Stevens G, et al. Systematic application of multiplex PCR enhances the detection of leaner, parasites, and viruses in stool samples. J Infect. 2013;67:122129.

16. Humphries RM, Linscott AJ. Laboratory diagnosis of bacterial gastroenteritis. Clin Microbiol Rev. 2015;28:331.

17. Liu J, Kabir F, Manneh J, et al. Evolution and assessment of molecular diagnostic tests for 15 enteropathogens causing childhood diarrhoea: a multicentre study. Lancet Infect Dis. 2014;14:716724.

eighteen. Carter East, Bryce J, Perin J, et al. Harmful practices in the management of babyhood diarrhea in low- and middle-income countries: a systematic review. BMC Public Health. 2015;xv:788.

xix. Antikainen J, Tarkka E, Haukka K, et al. New 16-plex PCR method for rapid detection of diarrheagenic Escherichia coli directly from stool samples. Eur J Clin Microbiol Infect Dis. 2009;28:899908.

20. Antikainen J, Kantele A, Pakkanen SH, et al. A quantitative polymerase chain reaction assay for rapid detection of nine pathogens directly from stools of travelers with diarrhea. Clin Gastroenterol Hepatol. 2013;eleven:13001307.e3.

21. Ajjampur SS, Rajendran P, Ramani S, et al. Closing the diarrhoea diagnostic gap in Indian children by the awarding of molecular techniques. J Med Microbiol. 2008;57(pt 11):13641368.

22. Sinha A, SenGupta Due south, Guin Due south, et al. Culture-independent real-fourth dimension PCR reveals extensive polymicrobial infections in hospitalized diarrhoea cases in Kolkata, Republic of india. Clin Microbiol Infect. 2013;nineteen:173180.

23. Lääveri T, Pakkanen SH, Antikainen J, et al. Loftier number of diarrhoeal co-infections in travellers to Benin, Due west Africa. BMC Infect Dis. 2014;14:81.

• Cited Hither |
• PubMed

24. Kabayiza JC, Andersson ME, Nilsson Due south, et al. Real-fourth dimension PCR identification of agents causing diarrhea in Rwandan children less than v years of age. Pediatr Infect Dis J. 2014;33:10371042.

25. Platts-Mills JA, Gratz J, Mduma E, et al. Association between stool enteropathogen quantity and disease in Tanzanian children using TaqMan array cards: a nested case-control study. Am J Trop Med Hyg. 2014;90:133138.

• Cited Here |
• PubMed

26. Liu J, Platts-Mills JA, Juma J, et al. Use of quantitative molecular diagnostic methods to place causes of diarrhoea in children: a reanalysis of the GEMS case-command study. Lancet. 2016;388:12911301.

27. You D, Hug Fifty, Ejdemyr South, et al.; United Nations Inter-agency Group for Kid Mortality Estimation (UN IGME). Global, regional, and national levels and trends in under-5 bloodshed between 1990 and 2015, with scenario-based projections to 2030: a systematic analysis past the UN Inter-bureau Group for Child Mortality Estimation. Lancet. 2015;386:22752286.

28. Das JK, Salam RA, Bhutta ZA. Global burden of childhood diarrhea and interventions. Curr Opin Infect Dis. 2014;27:451458.

29. Das JK, Bhutta ZA. Global challenges in acute diarrhea. Curr Opin Gastroenterol. 2016;32:1823.

Keywords:

diarrhea; children; developing countries; etiology; multiplex real-time PCR

Copyright © 2022 The Author(s). Published by Wolters Kluwer Health, Inc.

kleinevines.blogspot.com

Source: https://journals.lww.com/pidj/Fulltext/2018/05000/Potential_Diarrheal_Pathogens_Common_Also_in.10.aspx

Share this post