Genetic Polymorphisms and Drug Susceptibility in Four Isolates of Leishmania tropica Obtained from Canadian Soldiers Returning from Afghanistan

Cutaneous leishmaniasis (CL) is a vector-borne parasitic disease transmitted by the bite of sandflies, resulting in sores on the skin. No vaccines are available, and treatment relies on chemotherapy. CL has been frequently diagnosed in military personnel deployed to Afghanistan and returning from duty. The parasites isolated from Canadian soldiers were characterized by pulsed field gels and by sequencing conserved genes and were identified as Leishmania tropica. In contrast to other Leishmania species, high allelic polymorphisms were observed at several genetic loci for the L. tropica isolates that were characterized. In vitro susceptibility testing in macrophages showed that all isolates, despite their genetic heterogeneity, were sensitive to most antileishmanial drugs (antimonials, miltefosine, amphotericin B, paromomycin) but were insensitive to fluconazole. This study suggests a number of therapeutic regimens for treating cutaneous leishmaniasis caused by L. tropica among patients and soldiers returning from Afghanistan. Canadian soldiers from this study were successfully treated with miltefosine

Comparison of the Directigen Flu A+B Test, the QuickVue Influenza Test, and Clinical Case Definition to Viral Culture and Reverse Transcription-PCR for Rapid Diagnosis of Influenza Virus Infection

The diagnostic performances of the clinical case definition of influenza virus infection based on the combination of fever and cough and of two rapid influenza diagnostic tests, the Directigen Flu A+B test (Directigen; BD Diagnostic Systems, Sparks, Md.) and the QuickVue influenza test (QuickVue; Quidel, San Diego, Calif.), were compared to those of viral culture and an in-house reverse transcription (RT)-PCR during the 2000-2001 flu season. Two hundred consecutive nasopharyngeal aspirates were analyzed from 192 patients, including 122 adults and 70 children. Viral culture identified influenza virus A in 16 samples and influenza virus B in 55 samples, whereas RT-PCR identified influenza virus A in 21 samples and influenza virus B in 64 samples. When RT-PCR was used as the reference standard, the likelihood ratios for a positive test were 40.0 for Directigen, 8.6 for QuickVue, and 1.4 for the combination of fever and cough, whereas the likelihood ratios for a negative test were 0.22, 0.16, and 0.48, respectively. Our study suggests that (i) the poor specificity (35 to 58%) and the poor positive predictive value (41 to 60%) of the clinical case definition of influenza preclude its use for prediction of influenza virus infections during epidemics, especially when infection control decision making in the hospital setting is considered; (ii) Directigen has a higher diagnostic yield than QuickVue but is associated with a larger number of invalid results; (iii) the sensitivities of the rapid diagnostic tests are significantly lower with samples from adults than with samples from children, with the rates of false-negative results reaching up to 29%; and (iv) RT-PCR detects more cases of influenza than viral culture, and this greater accuracy makes it a more useful reference standard

Single nucleotide polymorphisms within <i>Leishmania</i> species for 10 genes^a.

<p>ND – not done.</p>a<p>For each gene, the number of heterozygous sites is indicated.</p>*<p>The <i>CYTB</i> gene is located on kinetoplast DNA.</p><p>Abreviations: <i>PTR1</i> – pteridine reductase 1; <i>NH1</i> – nucleoside hydrolase 1; <i>DHFR-TS</i> – dihydrofolate reductase-thymidylate synthase; <i>SAD</i> – stearic acid desaturase; <i>GPI</i> – glucose-6-phosphate isomerise; <i>PGD</i> – 6-phosphogluconate dehydrogenase; <i>ASAT</i> – aspartate aminotransferase; <i>G6PDH</i> – glucose-6-phosphate dehydrogenase; <i>MPI</i> – mannose phosphate isomerise; <i>CYTB</i> – Cytochrome B.</p

Karyotypes of Afghan and Iranian <i>Leishmania tropica</i> isolates as characterized by PFGE.

<p>Cells were embedded and lyzed in agarose and their chromosomes were electrophoresed and stained with ethidium bromide. <b>A.</b> 600–1300 kb electrophoresis <b>B.</b> 100–500 kb electrophoresis. The field isolates 017102, 431462, 072218 and 18693 from Afghanistan have closely related karyotype to the <i>L. tropica</i> reference strains 175 and MHOM/SU/74/K27. M, yeast chromosomes molecular weight marker (BioRad).</p

Paromomycin susceptibility of <i>Leishmania tropica</i> field isolates recovered from soldiers suffering from CL.

<p>The four <i>L. tropica</i> isolates from Afghanistan are sensitive to paromomycin as intracellular amastigotes. Intracellular parasites were incubated for 4 days with the indicated concentrations of paromomycin. The mean of two independent experiments done in duplicate is shown. Values are represented as numbers of relative light units (RLU).</p

Polymorphisms in <i>L. tropica</i> isolates^a^,^b.

a<p>Nucleotide polymorphisms and corresponding amino acid changes are shown.</p>b<p>The polymorphisms in bold cause an amino acid substitution.</p>c<p>The polymorphisms indicated were common to every <i>L. tropica</i> isolates analyzed, except for <i>PGD</i>, <i>G6PDH</i>, and <i>SAD</i>, which were not polymorphic in <i>L. tropica</i> MHOM/SU/74/K27.</p>d<p>For nucleotide numbering, +1 corresponds to the A of the ATG translation initiation codon.</p><p><i>PTR1</i> – pteridine reductase 1; <i>NH1</i> – nucleoside hydrolase 1; <i>DHFRTS</i> – dihydrofolate reductase-thymidylate synthase; <i>SAD</i> – stearic acid desaturase; <i>GPI</i> – glucose-6-phosphate isomerase; <i>PGD</i> – 6-phosphogluconate dehydrogenase; <i>ASAT</i> – aspartate aminotransferase; <i>G6PDH</i> – glucose-6-phosphate dehydrogenase.</p

Susceptibility of <i>L. tropica</i> clinical isolates to antileishmanial drugs.

a<p>Determined as amastigotes in THP-1 cells.</p

Phylogenetic analysis of the PTR1 sequences.

<p>Amino acid sequences were aligned using the ClustalW algorithm. The resulting multiple alignment was subjected to phylogenetic analysis by using the neighbor-joining method with Poisson correction as implemented in the MEGA3.1 software. The field isolates 017102, 431462, 072218, and 18693 from Afghanistan are clustering with the <i>L. tropica</i> reference strains 175 and MHOM/SU/74/K27. The reliabilities of each branch point were assessed by the analysis of 1000 bootstrap replicates.</p