Diagnosing Polyparasitism in a High-Prevalence Setting in Beira, Mozambique: Detection of Intestinal Parasites in Fecal Samples by Microscopy and Real-Time PCR

Background: Many different intestinal parasite species can co-occur in the same population. However, classic diagnostic tools can only frame a particular group of intestinal parasite species. Hence, one or two tests do not suffice to provide a complete picture of infecting parasite species in a given population. The present study investigated intestinal parasitic infections in Beira, Mozambique, i.e. in the informal settlement of Inhamudima. Diagnostic accuracy of five classical microscopy techniques and real-time PCR for the detection of a broad spectrum of parasites was compared. Methodology/Principal Findings: A cross-sectional population-based survey was performed. One stool sample per participant (n = 303) was examined by direct smear, formal-ether concentration (FEC), Kato smear, Baermann method, coproculture and real-time PCR. We found that virtually all people (96%) harbored at least one helminth, and that almost half (49%) harbored three helminths or more. Remarkably, Strongyloides stercoralis infections were widespread with a prevalence of 48%, and Ancylostoma spp. prevalence was higher than that of Necator americanus (25% versus 15%), the hookworm species that is often assumed to prevail in East-Africa. Among the microscopic techniques, FEC was able to detect the broadest spectrum of parasite species. However, FEC also missed a considerable number of infections, notably S. stercoralis, Schistosoma mansoni and G. intestinalis. PCR outperformed microscopy in terms of sensitivity and range of parasite species detected. Conclusions/Significance: We showed intestinal parasites—especially helminths—to be omnipresent in Inhamudima, Beira. However, it is a challenge to achieve high diagnostic sensitivity for all species. Classical techniques such as FEC are useful for the detection of some intestinal helminth species, but they lack sensitivity for other parasite species. PCR can detect intestinal parasites more accurately but is generally not feasible in resource-poor settings, at least not in peripheral labs. Hence, there is a need for a more field-friendly, sensitive approach for on-the-spot diagnosis of parasitic infections

Diagnosing Polyparasitism in a High-Prevalence Setting in Beira, Mozambique: Detection of Intestinal Parasites in Fecal Samples by Microscopy and Real-Time PCR

<div><p>Background</p><p>Many different intestinal parasite species can co-occur in the same population. However, classic diagnostic tools can only frame a particular group of intestinal parasite species. Hence, one or two tests do not suffice to provide a complete picture of infecting parasite species in a given population. The present study investigated intestinal parasitic infections in Beira, Mozambique, i.e. in the informal settlement of Inhamudima. Diagnostic accuracy of five classical microscopy techniques and real-time PCR for the detection of a broad spectrum of parasites was compared.</p><p>Methodology/Principal Findings</p><p>A cross-sectional population-based survey was performed. One stool sample per participant (n = 303) was examined by direct smear, formal-ether concentration (FEC), Kato smear, Baermann method, coproculture and real-time PCR. We found that virtually all people (96%) harbored at least one helminth, and that almost half (49%) harbored three helminths or more. Remarkably, <i>Strongyloides stercoralis</i> infections were widespread with a prevalence of 48%, and <i>Ancylostoma</i> spp. prevalence was higher than that of <i>Necator americanus</i> (25% <i>versus</i> 15%), the hookworm species that is often assumed to prevail in East-Africa. Among the microscopic techniques, FEC was able to detect the broadest spectrum of parasite species. However, FEC also missed a considerable number of infections, notably <i>S</i>. <i>stercoralis</i>, <i>Schistosoma mansoni</i> and <i>G</i>. <i>intestinalis</i>. PCR outperformed microscopy in terms of sensitivity and range of parasite species detected.</p><p>Conclusions/Significance</p><p>We showed intestinal parasites—especially helminths—to be omnipresent in Inhamudima, Beira. However, it is a challenge to achieve high diagnostic sensitivity for all species. Classical techniques such as FEC are useful for the detection of some intestinal helminth species, but they lack sensitivity for other parasite species. PCR can detect intestinal parasites more accurately but is generally not feasible in resource-poor settings, at least not in peripheral labs. Hence, there is a need for a more field-friendly, sensitive approach for on-the-spot diagnosis of parasitic infections.</p></div

Number of helminth species found per person.

<p>Prevalence of infection is based on the composite reference standard for <i>S</i>. <i>stercoralis</i>, <i>A</i>. <i>lumbricoides</i>, <i>T</i>. <i>trichiura</i>, and <i>S</i>. <i>mansoni</i>, and on PCR for hookworm—<i>Ancylostoma</i> spp. and <i>N</i>. <i>americanus</i> (n = 303).</p

Ct values in PCR-positives: microscopy-negative <i>versus</i> -positive samples.

<p>Total number of PCR-positives per species is indicated between brackets. ‘-’ indicates microscopy-negative and ‘+’ microscopy-positive samples. Horizontal lines indicate median Ct values. Differences in Ct values between microscopy-positive and–negative samples were all significant p≤0.007, ^a microscopy cannot differentiate the two hookworm species.</p

Prevalence of intestinal parasitic infections in the study population according to different diagnostic methods.

<p>Whiskers indicate 95% confidence intervals of the observed prevalence. Percentages are based on observations in 303 individuals. (A) Prevalence of helminth infections. <i>Strongyloides stercoralis</i> infection was not determined (ND) in Kato smears, hookworm was not determined by the Baermann method, while <i>A</i>. <i>lumbricoides</i>, <i>T</i>. <i>trichiura</i>, and <i>S</i>. <i>mansoni</i> were not determined by the Baermann method or coproculture. <i>Trichuris trichiura</i> was not determined by PCR either, and the composite reference standard (CRS) for this infection was consequently based on microscopic results only. (B) Prevalence of intestinal protozoan infections. Feces were examined by both microscopy and PCR for <i>G</i>. <i>intestinalis</i>, and <i>E</i>. <i>histolytica</i> complex spp. (one observation was missing for PCR, and consequently for the CRS). Only PCR data was used for <i>C</i>. <i>parvum/C</i>. <i>hominis</i> (one observation missing) and for, <i>E</i>. <i>bieneusi</i> and <i>Encephalitozoon</i> spp. (two observations missing).* While microscopy cannot differentiate between the pathogenic species <i>Entamoeba histolytica</i> and the nonpathogenic species of the <i>E</i>. <i>histolytica</i> complex, PCR is specific for the pathogenic species (<i>E</i>. <i>histolytica</i>).</p

Discordance between microscopy and PCR in microscopy-positives.

<p>Discordance between microscopy and PCR in microscopy-positives.</p

Sensitivities of the different diagnostic methods for the detection of intestinal parasitic infections.

<p>Whiskers indicate 95% confidence intervals of observed sensitivities (n = 303). <i>Strongyloides stercoralis</i> infection was not determined (ND) in Kato smears, hookworm was not determined by the Baermann method, while <i>A</i>. <i>lumbricoides</i>, <i>T</i>. <i>trichiura</i>, and <i>S</i>. <i>mansoni</i> were not determined by the Baermann method or coproculture. <i>Trichuris trichiura</i> was not determined by PCR, and for this species the sensitivity was therefore based on microscopic results only. <i>Giardia intestinalis</i> was not determined by Kato smear, the Baermann method or coproculture (one observation missing).</p

Diagnostic methods performed and definitions of composite reference standards.

<p>Diagnostic methods performed and definitions of composite reference standards.</p

Flow diagram of the selection of the study population and diagnostic procedures.

<p>Flow diagram of the selection of the study population and diagnostic procedures.</p