Juan Castillo Mewa

In 2016, Panama's Surveillance Program for Influenza and Other Respiratory Viruses received 4041 samples with suspected viral respiratory infection, of which 68.78% tested positive on the respiratory viral panel, while the remaining 31.22% resulted in no etiological diagnosis. Given the need to determine which other viruses could cause these respiratory infections, a retrospective search for endemic coronaviruses and Enterovirus D68 was conducted, with the primary objective of detecting them in negative samples from that year. One hundred and twenty-seven nasopharyngeal swab samples were retrospectively selected, all of which had been previously tested using a routine respiratory viral panel for pandemic Influenza viruses, Respiratory Syncytial Virus (RSV), Human Metapneumovirus (hMPV), Parainfluenza viruses (PIV-1, PIV-2, PIV-3), Adenovirus (AdV), and Rhinovirus (RV). Of these, 70% were negative and 30% positive for at least one respiratory virus, and were subsequently classified by age group, clinical outcome and geographic location. Nucleic acids were extracted using automated equipment and amplified by Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR). The human RNase P gene was used as an internal control. Samples and controls were considered positive with a Ct value ≤ 35 cycles. The statistical analysis was performed using Pearson's chi-square, considering a p value < 0.05 as statistically significant. The human coronavirus 229E (HCoV-229E) (n = 3), human coranvirus OC-43 (HCoV-OC43) (n = 3) and EV-D68 (n = 17) viruses were detected, presenting viral co-detections between HCoV-229E/pandemic Influenza A/H1, HCoV-OC43/Parainfluenza type 1, Enterovirus D68/Rhinovirus, Enterovirus D68/Respiratory Syncytial Virus and Enterovirus D68/Parainfluenza type 3. Overall, 78.26% (n = 18/23) of the cases were hospitalized patients and 21.74% (n = 5/23) were outpatients. Regarding age, 52.17% (n = 12/23) of the cases corresponded to children under 5 years old, 13.04% (n = 3/23) to patients between 5 and 18 years old, and 34.78% (n = 8/23) to adults over 18 years old. The circulation of HCoV-229E, HCoV-OC43, and EV-D68 was confirmed, causing both single respiratory infections and co-detections with other viruses from the routine diagnostic panel. This is why it is crucial to expand our knowledge of the epidemiology of respiratory viruses that are not currently part of routine surveillance systems, but which could be contributing significantly to the burden of disease in the population.

Low-frequency mutations associated with drug resistance have been related to virologic failure in subjects with no history of pre-treatment and recent HIV diagnosis. In total, 78 antiretroviral treatment (ART)-naïve subjects with a recent HIV diagnosis were selected and followed by CD4+ T lymphocytes and viral load tests to detect virologic failure. We sequenced the basal samples retrospectively using next-generation sequencing (NGS), looking for low-frequency mutations that had not been detected before using the Sanger sequencing method (SSM) and describing the response to ART. Twenty-two subjects developed virologic failure (VF), and thirteen of them had at least one drug-resistance mutation associated with Reverse Transcriptase Inhibitors (RTI) and Protease Inhibitors (PIs) at frequency levels ≤ 1%, not detected previously in their basal genotyping test. No resistance mutations were observed to Integrase Strand Transfer Inhibitors (INSTIs). We identified a possible cause of VF in ART-naïve subjects with low-frequency mutations detected. To our knowledge, this is the first evaluation of pre-existing drug resistance for HIV-1 minority variants carried out on ART-naïve people living with HIV/AIDS (PLWHA) by analyzing the HIV-1 pol gene using NGS in the country.

A total of 123 DNA samples from Panamanian patients with cutaneous leishmaniasis (CL) lesions were evaluated. These samples were previously confirmed with CL by a specific KDNA-Viannia PCR but had a negative parasitological diagnosis (Group A). Epidemiological variables, such as age, sex, geographic origin, evolution time, and the number and location of the lesions, were analyzed. No significant differences (p < 0.05) were found when these variables were evaluated against a control panel of 123 CL lesion samples from CL patients with positive parasitological diagnoses (Group B). Of the 123 samples (Group A), 67% (82/123) gave positive results when re-analyzed by PCR-hsp70. An analysis of 69 of these samples via PCR-hsp70-RFLP showed that 59.4% (41/69) of the found restriction patterns corresponded to Leishmania (Viannia) panamensis and 40.6% (28/69) to Leishmania (Viannia) guyanensis. Finally, the sequence and phylogenetic analysis of 32 of the samples confirmed the species in 21 (65.6%, 21/32) samples, originally characterized as L. (V.) panamensis. However, 11 samples (34.4%, 11/32), initially identified via RFLP-Hsp70 as L. (V.) guyanensis, matched the sequence of a genetic variant known as Leishmania sp.1. These results point out the species/genetic variants of Leishmania in the case of CL lesions with an apparently low parasite load.

Isolates from 475 cutaneous leishmaniasis (CL) patients from three endemic regions were studied by three typing techniques. The molecular analysis from lesion scrapings based on hsp70 PCR-restriction fragment length polymorphism (RFLP) showed that 78.1% (371/475) restriction patterns corresponded to Leishmania (Viannia) panamensis, 19% (90/475) to Leishmania (Viannia) guyanensis, and 3.0% (14/475) to Leishmania (Viannia) braziliensis. Promastigotes isolated by culture from lesions of 228 patients (48.0%, 228/475) were identified by multi-locus enzyme electrophoresis. Of them, 95.2% (217/228) were typified as L. (V.) panamensis, 1.3% (3/228) as L. (V.) guyanensis, 2.2% (5/228) as L. (V.) braziliensis, and 1.3% (3/228) as hybrids (L. [V.] braziliensis/L. [V.] panamensis). However, a partial sequencing analysis of the hsp70 gene from 77 selected samples showed 16.9% (13/77) typified as L. (V.) panamensis, 68.8% (53/77) as Leishmania (V.) sp., 1, 3.9% (3/77) as L. (V.) guyanensis, 1.3% (1/77) as L. (V.) braziliensis outlier, 2.6% (2/77) as Leishmania (Viannia) naiffi, 2.6% as (2/77) Leishmania (V.) sp., and 2 and 3.9% (3/77) hybrid isolates of L. (V.) braziliensis/L. (V.) guyanensis. These results confirm L. (V.) panamensis as the predominant species and cause of CL lesions in Panama and that L. (V.) guyanensis, L. (V.) braziliensis, and L. (V.) naiffi are circulating to a lower degree. Furthermore, the determination of parasite isolates belonging to atypical clusters and hybrid isolates suggests the circulation of genetic variants with important implications for the epidemiology and clinical follow-up of CL in Panama. No evidence of the existence of parasites of the Leishmania (Leishmania) mexicana complex in Panamanian territory was found in this study.

Human leishmaniasis is a neglected disease caused by parasites of the genus Leishmania. Clinical aspects of this disease can vary significantly, reflecting the wide range of parasites in the genus Leishmania. Knowing accurately the Leishmania species infecting humans is important for clinical case management and evaluation of epidemiological risk. Calmodulin is an essential gene in trypanosomatids that modulates the calcium metabolism in various cellular activities. Despite its strong conservation in trypanosomatids, it has been recently observed that its untranslated regions (UTR) diverge among species.