Determination of the <i>P</i>. <i>falciparum</i> detection limit of the nested PCR system.

<p>The second PCR products were visualized on 2% agarose-TAE gel containing GelRed (Wako). The primer set used for the second PCR was P1 and F2. Lane L indicates a molecular marker (100-bp ladder). The letters shown below each lane indicate the template used for the second PCR reactions (lane P indicates the first PCR products amplified from the sequences of <i>P</i>. <i>falciparum</i>; lanes 10³, 10², 1, 10⁻¹, 10⁻², and 10⁻³ indicate the first PCR products amplified from the DNA extracted from the blood sample containing 10³, 10², 1, 10⁻¹, 10⁻², and 10⁻³ parasites/μL of blood, respectively; lane 0 indicates the first PCR products amplified from the DNA extracted from healthy blood; lane N indicates the diluted first PCR product from water; lane N’, water). The <i>P</i>. <i>falciparum-</i>specific PCR products are indicated with an arrow on the right side.</p

Sequences of the primers used in the present study.

<p>Sequences of the primers used in the present study.</p

Detailed conditions of the nested PCRs.

<p>Detailed conditions of the nested PCRs.</p

The PCR-targeted region of the SSU rRNA genes from 5 malaria parasite species.

<p>Partial sequences of the SSU rRNA genes from malaria parasites are indicated. Pf-S: The sequence from <i>P</i>. <i>falciparum</i> expressed at the sexual stage [GenBank: M19173]. Pf-A: The sequence from <i>P</i>. <i>falciparum</i> expressed at the asexual stage [GenBank: M19172]. Pv-S: The sequence from <i>P</i>. <i>vivax</i> expressed at the sexual stage [GenBank: U03080]. Pv-A: The sequence from <i>P</i>. <i>vivax</i> expressed at the asexual stage [GenBank: X13926]. Poc: The sequence from <i>P</i>. <i>ovale curtisi</i> [GenBank: L48986]. Pow: The sequence from <i>P</i>. <i>ovale wallikeri</i> [GenBank: AB182491] Pm: The sequence from <i>P</i>. <i>malariae</i> [GenBank: M54897]. Pk: The sequence from <i>P</i>. <i>knowlesi</i> [GenBank: AY327550]. The universal primers for the conserved region are highlighted in yellow (P1 and P2, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0191886#pone.0191886.t001" target="_blank">Table 1</a>). The inner-specific primers (F2, V3, M4, Oc4, Ow1, and K1 [see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0191886#pone.0191886.t001" target="_blank">Table 1</a>]) are highlighted in blue.</p

Results of the first PCR with the universal primers P1 and P2.

<p>The first PCR products were visualized on 2% agarose-TAE gel containing GelRed (Wako). Lane L indicates a molecular marker (100-bp ladder). The letters shown below each lane indicate the template DNA used for the first PCR reactions (lanes F, V, Oc, M, K, and Ow indicate the plasmid DNA containing each partial sequence of the SSU rRNA genes from <i>P</i>. <i>falciparum</i>, <i>P</i>. <i>vivax</i>, <i>P</i>. <i>ovale curtisi</i>, <i>P</i>. <i>malariae</i>, <i>P</i>. <i>knowlesi</i>, and <i>P</i>. <i>ovale wallikeri</i>, respectively; lane N indicates the negative control [water]). The PCR products are shown at 136–159 bp.</p

A novel PCR-based system for the detection of four species of human malaria parasites and <i>Plasmodium knowlesi</i>

<div><p>A microscopy-based diagnosis is the gold standard for the detection and identification of malaria parasites in a patient’s blood. However, the detection of cases involving a low number of parasites and the differentiation of species sometimes requires a skilled microscopist. Although PCR-based diagnostic methods are already known to be very powerful tools, the time required to apply such methods is still much longer in comparison to traditional microscopic observation. Thus, improvements to PCR systems are sought to facilitate the more rapid and accurate detection of human malaria parasites <i>Plasmodium falciparum</i>, <i>P</i>. <i>vivax</i>, <i>P</i>. <i>ovale</i>, and <i>P</i>. <i>malariae</i>, as well as <i>P</i>. <i>knowlesi</i>, which is a simian malaria parasite that is currently widely distributed in Southeast Asia. A nested PCR that targets the small subunit ribosomal RNA genes of malaria parasites was performed using a “fast PCR enzyme”. In the first PCR, universal primers for all parasite species were used. In the second PCR, inner-specific primers, which targeted sequences from <i>P</i>. <i>falciparum</i>, <i>P</i>. <i>vivax</i>, <i>P</i>. <i>ovale</i>, <i>P</i>. <i>malariae</i>, and <i>P</i>. <i>knowlesi</i>, were used. The PCR reaction time was reduced with the use of the “fast PCR enzyme”, with only 65 minutes required to perform the first and second PCRs. The specific primers only reacted with the sequences of their targeted parasite species and never cross-reacted with sequences from other species under the defined PCR conditions. The diagnoses of 36 clinical samples that were obtained using this new PCR system were highly consistent with the microscopic diagnoses.</p></div

The second PCR-targeted region of the SSU rRNA genes from variant parasite isotypes.

<p><b>(A)</b> Partial sequences of the variant SSU rRNA genes from malaria parasites. Pf, Pv, Po, and Pm indicate <i>P</i>. <i>falciparum</i>, <i>P</i>. <i>vivax</i>, <i>P</i>. <i>ovale</i> and <i>P</i>. <i>malariae</i> respectively. Pf-standard: The sequence from <i>P</i>. <i>falciparum</i> [GenBank: M19172]. Pf isotype-1 and isotype-2: The identified variant sequences from <i>P</i>. <i>falciparum</i> [Genbank: KJ170099.1 and JQ627151.1]. Pv-standard: The sequence from <i>P</i>. <i>vivax</i> [GenBank: X13926]. Pv isotype-1, isotype-2 and isotype-3: The identified variant sequences from <i>P</i>. <i>vivax</i> [Genbank: U83877.1, KC750244.1 and AF145335.1]. Poc standard: The sequence from <i>P</i>. <i>ovale curtisi</i> [GenBank: L48986]. Poc isotype-1, isotype-2, isotype-3 and isotype-4: The identified variant sequences from <i>P</i>. <i>ovale curtisi</i> [Genbank: KF696376.1, KC633228.1, KJ871671.1 and KF696371.1]. Pow standard: The sequence from <i>P</i>. <i>ovale wallikeri</i> [GenBank: AB182491] Pm standard: The sequence from <i>P</i>. <i>malariae</i> [GenBank: M54897]. Pm isotype-1 and isotype-2: The identified variant sequences from <i>P</i>. <i>malariae</i> [Genbank: KJ619947.1 and KJ170106.1]. All variant sequences from each species are indicated as multiple alignment comparisons. The universal P1 primer region is highlighted in yellow (P1). The inner-specific primers (F2, V3, Oc4 and M4,) are highlighted in blue. The nucleotide changes identified in each variant are highlighted in red. <b>(B)</b> Results of the PCR with the universal primer P1 and the inner-specific primers using the variant DNAs as templates. The PCR reactions were performed with the universal P1 primer and each species-specific primer. The PCR conditions were same as those of the second PCR (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0191886#sec002" target="_blank">Materials and Methods</a>). In each reaction mix, 0.1 ng of the synthesized DNA of each variant sequence was included as template. The products were visualized on 2% agarose-TAE gel containing GelRed (Wako). Lane L indicates a molecular marker (100-bp ladder). The letters shown below each lane indicate the specific primer used for the second PCR reactions (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0191886#pone.0191886.t001" target="_blank">Table 1</a>). Arrows indicate the PCR products (100–106 bp). The template DNAs are indicated below the gels.</p

Clinical characteristics and epidemiology of intestinal tapeworm infections over the last decade in Tokyo, Japan: A retrospective review

<div><p>Background</p><p>Tapeworm (cestode) infections occur worldwide even in developed countries and globalization has further complicated the epidemiology of such infections. Nonetheless, recent epidemiological data on cestode infections are limited. Our objectives were to elucidate the clinical characteristics and epidemiology of diphyllobothriosis and taeniosis in Tokyo, Japan.</p><p>Methodology/Principal findings</p><p>We retrospectively reviewed 24 cases of human intestinal cestode infection from January 2006 to December 2015 at a tertiary referral hospital in Tokyo, Japan. The patients included were diagnosed with cestode infection based on morphological and/or molecular identification of expelled proglottids and/or eggs and treated in our hospital. Fifteen and 9 patients were diagnosed with diphyllobothriosis and taeniosis, respectively. The median patient age was 31 years (interquartile range [IQR]: 26–42 years), and 13 (54%) were male. Most of the patients (91.7%) were Japanese. All patients were successfully treated with praziquantel without recurrence. Diphyllobothriosis was caused by <i>Diphyllobothrium nihonkaiense</i> in all patients. Taeniosis was due to infection of <i>Taenia saginata</i> in 8 [88.9%] patients and <i>T</i>. <i>asiatica</i> in 1 [11.1%] patient. All patients with taeniosis were infected outside Japan, as opposed to those with diphyllobothriosis, which were domestic. The source locations of taeniosis were mostly in developing regions. The median duration of the stay of the patients with taeniosis at the respective source location was 1 month (IQR: 1–8).</p><p>Conclusions/Significance</p><p>The cestode infection, especially with <i>D</i>. <i>nihonkaiense</i>, has frequently occurred, even in Japanese cities, thereby implicating the probable increase in the prevalence of diphyllobothriosis among travelers, as the number of travelers is expected to increase owing to the Tokyo Olympics/Paralympics in 2020. In addition, medical practitioners should be aware of the importance of providing advice to travelers to endemic countries of taeniosis, including the potential risks of infection and preventive methods for these infections.</p></div

Demographic and clinical characteristics of patients with diphyllobothriosis and taeniosis.

<p>Demographic and clinical characteristics of patients with diphyllobothriosis and taeniosis.</p

Logistic regression analysis of variables in the prediction of enteric fever diagnosis.

<p>Logistic regression analysis of variables in the prediction of enteric fever diagnosis.</p