Molecular Analysis of Rabies Virus Using RNA Extracted from Used Lateral Flow Devices.

Molecular analysis of rabies virus can provide accurate diagnosis and information on its genetic diversity. The transportation of rabies brain samples from remote areas to a central laboratory is challenging owing to biohazard risks and decomposability. We investigated the utility of used lateral flow devices (LFDs) for subsequent molecular analysis and assessed the necessary storage temperatures. Using RNA extracted from used LFD strips, we performed conventional reverse transcription-PCR (RT-PCR) using an LN34 primer set to amplify short fragments (165 bp) for rabies virus detection and the P1-304 primer set to amplify long fragments of the entire N gene amplicon (1,506 bp) for phylogenetic analysis. Among 71 used LFDs stored in a refrigerator and 64 used LFDs stored at room temperature, the LN34 assay showed high sensitivities (96.2% and 100%, respectively) for the diagnosis of rabies, regardless of the storage temperature. A significant reduction in the sensitivity of rabies diagnosis was observed when using the P1-304 primer set for used LFDs stored at room temperature compared to those stored at refrigeration temperature (20.9% versus 100%; P < 0.05). Subsequent sequencing and phylogenetic analysis were successfully performed using the amplicons generated by the P1-304 RT-PCR assays. Used LFDs are thus promising resources for rabies virus RNA detection and sequence analysis. Virus detection via RT-PCR, amplifying a short fragment, was possible regardless of the storage temperature of the used LFDs. However, refrigerated storage is recommended for RT-PCR amplification of long fragments for phylogenetic analysis.

Evaluation of lateral flow devices for postmortem rabies diagnosis in animals in the Philippines: a multicenter study.

Expansion of the use of lateral flow devices (LFD) for animal rabies diagnosis can help mitigate the widespread underreporting of rabies. However, this has been hindered by the limited number and small sample size of previous studies. To overcome this limitation, we conducted a multicenter study with a larger sample size to assess the diagnostic accuracy of the ADTEC LFD for postmortem rabies diagnosis in animals. Thirteen governmental animal diagnostic laboratories in the Philippines were involved in this study, and 791 animals suspected of having rabies were tested using both the direct fluorescence antibody test (DFAT) and ADTEC LFD between August 2021 and October 2022. The LFD demonstrated a sensitivity of 96.3% [95% confidence interval (CI): 94.1%-97.9%] and a specificity of 99.7% (95% CI: 98.4%-100%). Notably, false-negative results were more likely to occur in laboratories with lower annual processing volumes of rabies samples in the previous years (adjusted odds ratio 4.97, 95% CI: 1.49-16.53). In this multicenter study, the high sensitivity and specificity of the LFD for the diagnosis of animal rabies, compared to that of the DFAT, was demonstrated, yet concerns regarding false-negative results remain. In areas with limited experience in processing rabies samples, it is essential to provide comprehensive training and careful attention during implementation.

Puppies as the primary causal animal for human rabies cases: three-year prospective study of human rabies in the Philippines.

Introduction: While rabies remains a global concern, detailed studies on human rabies, particularly regarding causal animals and the reasons for not receiving postexposure prophylaxis (PEP), are lacking. Methods: We conducted a 3-year prospective study (October 2019-September 2022) at the Philippines' largest rabies referral center. We interviewed patients with suspected rabies and their families. We used LN34 qRT-PCR and rapid fluorescent focus inhibition test on saliva samples. We also compared our findings with two retrospective studies at the same hospital. Results: We enrolled 151 patients, including 131 with potential rabies exposure. Similar to retrospective studies, the participants were predominantly males (75.5%), adults (76.8%), low-income individuals (91.4%), and rural dwellers (62.3%). The causal animals were mainly dogs (97.0%), with similar incubation periods, clinical symptoms, and a high proportion not receiving vaccines or immunoglobulins (93.2%). Most causal animals were owned by either the patients' households or their neighbors (60.2%), with a significant proportion being puppies (58.8%). Most patients had knowledge of rabies; however, reasons for not seeking PEP included misconceptions about minor bites not causing rabies (51.3%), beliefs in traditional healers (33.9%), and economic constraints (22.6%). Despite completing the WHO regimen, two PEP failures were observed. LN34 qRT-PCR detected 98 positive cases (sensitivity, 64.9%; 95% CI 56.7-72.5). These strains belong to the Southeast Asia 4 subclade. Discussion: In conclusion, this study highlights the role of puppies as primary causal animals and the presence of misconceptions that preclude patients from acquiring PEP.

A prospective observational study of community-acquired bacterial bloodstream infections in Metro Manila, the Philippines.

Community-acquired bacterial bloodstream infections are caused by diverse pathogens with changing antimicrobial-resistance patterns. In low-middle income countries in Southeast Asia, where dengue fever is endemic and a leading cause of fever, limited information is available about bacterial bloodstream infections due to challenges of implementing a blood culture service. This study describes bacterial bloodstream pathogens and antimicrobial-resistance patterns in Metro Manila, the Philippines. We aimed to identify the proportion of patients with a positive blood culture, the bacteria isolated and their antimicrobial resistance patterns, and the clinical characteristics of these patients, in this dengue endemic area. We conducted a prospective observational study in a single hospital enrolling febrile patients clinically suspected of having a community-acquired bacterial bloodstream infection between 1st July 2015 and 30th June 2019. Each patient had a blood culture and additional diagnostic tests according to their clinical presentation. We enrolled 1315 patients and a significant positive blood culture was found in 77 (5.9%) including Staphylococcus aureus (n = 20), Salmonella enterica Typhi (n = 18), Escherichia coli (n = 16), Streptococcus pneumoniae (n = 3) and Burkholderia pseudomallei (n = 2). Thirty-four patients had meningococcal disease diagnosed by culture (n = 8) or blood PCR (n = 26). Additional confirmed diagnoses included leptospirosis (n = 177), dengue virus infection (n = 159) and respiratory diphtheria (n = 50). There were 79 (6.0%, 95%CI 4.8%-7.4%) patients who died within 28 days of enrollment. Patients with a positive blood culture were significantly more likely to die than patients with negative culture (15.2% vs 4.4%, P<0.01). Among S. aureus isolates, 11/20 (55%) were methicillin-resistant (MRSA) and ST30: USA1100 was dominant sequence type (88.9%). Antimicrobial-susceptibility was well preserved in S. enterica Typhi. Among hospitalized patients with clinically suspected community-acquired bacterial bloodstream infection in Metro Manila, the Philippines, 5.9% had a blood culture confirmed infection of whom 15.6% died. S. aureus, including a significant number of MRSA (USA1100 clones), S. enterica Typhi, E.coli and Neisseria meningitidis were frequently identified pathogens.

Performance of MALDI-TOF Mass Spectrometry in the Philippines.

Identification of the causative pathogen in infectious diseases is important for surveillance and to guide treatment. In low- and middle-income countries (LMIC), conventional culture and identification methods, including biochemical methods, are reference-standard. Biochemical methods can lack sensitivity and specificity and have slow turnaround times, causing delays in definitive therapy. Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) is a rapid and accurate diagnostic method. Most studies comparing MALDI-TOF MS and biochemical methods are from high-income countries, with few reports from LMIC with tropical climates. The aim of this study was to assess the performance of MALDI-TOF MS compared to conventional methods in the Philippines. Clinical bacterial or fungal isolates were identified by both MALDI-TOF MS and automated (VITEK2) or manual biochemical methods in the San Lazaro Hospital, Metro Manila, the Philippines. The concordance between MALDI-TOF MS and automated (VITEK2) or manual biochemical methods was analyzed at the species and genus levels. In total, 3530 bacterial or fungal isolates were analyzed. The concordance rate between MALDI-TOF MS and biochemical methods was 96.2% at the species level and 99.9% at the genus level. Twenty-three isolates could not be identified by MALDI-TOF MS. In this setting, MALDI-TOF MS was accurate compared with biochemical methods, at both the genus and the species level. Additionally, MALDI-TOF MS improved the turnaround time for results. These advantages could lead to improved infection management and infection control in low- and middle-income countries, even though the initial cost is high.