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Abstract. Chikungunya virus (CHIKV) often co-circulates with dengue virus (DENV). A cross-sectional surveillance study was conducted at a tertiary hospital in Manila, Philippines, to describe the prevalence and characteristics of DENV and CHIKV infections among patients seeking care for dengue-like illness. Acute blood samples from patients ≥ 6 months of age clinically diagnosed with dengue from November 2012 to December 2013 underwent reverse transcription polymerase chain reaction (RT-PCR) to detect DENV and CHIKV RNA. A total of 118 patients with clinically diagnosed dengue (age range = 1-89 years, mean = 22 years; male-to-female ratio = 1.51) were tested by DENV RT-PCR; 40 (34%) were DENV PCR-positive (age range = 1-45 years, mean = 17 years). All DENV serotypes were detected: 11 (28%) DENV-1, 6 (15%) DENV-2, 6 (15%) DENV-3, and 17 (42%) DENV-4. Of 112 patients clinically diagnosed with dengue and tested by CHIKV RT-PCR, 11 (10%) were CHIKV PCR-positive (age range = 2-47 years, mean = 20.3 years). No coinfections were detected. Presenting signs/symptoms did not differ between DENV-and CHIKV-positive cases. Sequencing of envelope 1 gene from two CHIKV PCR-positive samples showed Asian genotype. This study highlights the potential for misdiagnosis of medically attended CHIKV infections as DENV infection and the difficulty in clinically differentiating dengue and chikungunya based on presenting signs/symptoms alone. This underscores the necessity for diagnostic laboratory tests to distinguish CHIKV infections in the background of actively co-circulating DENV.

Buruli ulcer, a disease caused by Mycobacterium ulcerans, causes ulcerative skin disease likely generated by a toxin that mediates apoptosis. We analyzed paraffin-embedded sections of surgically excised Buruli ulcer lesions (two ulcers and one edematous plaque) and adjacent non-lesional skin samples (n = 9) for apoptosis by an indirect immunofluorescent terminal deoxynucleotide transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) assay. All samples were stained for acid-fast bacilli (AFB) and cultured for mycobacteria, and most were analyzed with an M. ulcerans-specific diagnostic polymerase chain reaction (PCR). TUNEL (+) bodies were numerous in both ulcers and the plaque, and sparse or absent in adjacent non-lesional skin. The AFB tissue stains and cultures for M. ulcerans were positive only in the three lesions. The result of the PCR for M. ulcerans was positive in all three lesions and in four of six non-lesional tissue samples; three contained sparse TUNEL (+) bodies. An abundance of TUNEL (+) bodies in the three AFB stain (+), culture (+), and PCR (+) Buruli ulcer lesional samples, but not in nearby AFB stain (-), culture (-), and PCR (+) non-lesional skin samples, strengthen the evidence that apoptosis is an important tissue destruction mechanism in human lesions closely associated with viable M. ulcerans.

a b s t r a c tBackground: AFRIMS longitudinal dengue surveillance in Thailand depends on the nested RT-PCR and the dengue IgM/IgG ELISA. Objective: To examine and improve the sensitivity of the nested RT-PCR using a panel of archived samples collected during dengue surveillance. Study design: A retrospective analysis of 16,454 dengue IgM/IgG ELISA positive cases collected between 2000 and 2013 was done to investigate the sensitivity of the nested RT-PCR. From these cases, 318 acute serum specimens or extracted RNA, previously found to be negative by the nested RT-PCR, were tested using TaqMan real-time RT-PCR (TaqMan rRT-PCR). To improve the sensitivity of nested RT-PCR, we designed a new primer based on nucleotide sequences from contemporary strains found to be positive by the TaqMan rRT-PCR. Sensitivity of the new nested PCR was calculated using a panel of 87 samples collected during 2011-2013. Results and conclusion: The percentage of dengue IgM/IgG ELISA positive cases that were negative by the nested RT-PCR varied from 17% to 42% for all serotypes depending on the year. Using TaqMan rRT-PCR, dengue RNA was detected in 194 (61%) of the 318 acute sera or extracted RNA previously found to be negative by the nested RT-PCR. The newly designed DENV-1 specific primer increased the sensitivity of DENV-1 detection by the nested RT-PCR from 48% to 88%, and of all 4 serotypes from 73% to 87%. These findings demonstrate the impact of genetic diversity and signal erosion on the sensitivity of PCR-based methods.Published by Elsevier B.V.

BackgroundNew recruits within military barracks present conditions favorable for the spread of respiratory pathogens. However, respiratory pathogen transmission in such confined settings in the tropics has not been well studied.MethodsRecruits in four successive Royal Thai Army basic training classes living in military barracks were monitored for the symptoms of influenza-like illness (ILI) or upper respiratory illness (URI). Classes 1 and 2 were also monitored after basic training. Nasal/throat swabs from acute illnesses were collected and tested by influenza RT-PCR (all four classes). In addition, class 1 had multiplex PCR performed along with the analysis of bed locations within the barracks.ResultsInfluenza-like illness/upper respiratory illness rates ranged from 4·7 to 6·9 per 100 recruit-weeks in the four classes and generally decreased during the course of basic training (P < 0·05 in three of four classes). Rates during basic training were 1·7 (95% CI: 1·29, 2·29) and 2·5 (95% CI: 1·5, 4·1) times higher than after basic training (classes 1 and 2, respectively). In class 1, coronavirus, parainfluenza virus, and rhinovirus were the most commonly identified respiratory pathogens; only one influenza PCR-positive infection was detected in all four classes. Bed locations of URI/ILI cases in class 1 tended to be in closer proximity to each other.ConclusionBasic training recruits in military barracks in the tropics had high rates of acute respiratory illnesses with illness patterns consistent with external seeding followed by substantial internal transmission. Our findings may contribute to control measures in similar confined settings both within and outside the military.