The American Journal of Tropical Medicine and Hygiene - Current Issue

There is increasing interest in using dried blood spot (DBS) cards to extend the reach of global health and disease surveillance programs to hard-to-reach populations. Conceptually, DBS offers a cost-effective solution for multiple use cases by simplifying logistics for collecting, preserving, and transporting blood specimens in settings with minimal infrastructure. This review describes methods to determine both the reliability of DBS-based bioanalysis for a defined use case and the optimal conditions that minimize pre-analytical sources of data variability. Examples by the newborn screening, drug development, and global health communities are provided in this review of published literature. Sources of variability are linked in most cases, emphasizing the importance of field-to-laboratory standard operating procedures that are evidence based and consider both stability and efficiency of recovery for a specified analyte in defining the type of DBS card, accessories, handling procedures, and storage conditions. Also included in this review are reports where DBS was determined to not be feasible because of technology limitations or physiological properties of a targeted analyte.

Leptospirosis is a neglected zoonotic disease with worldwide endemicity and continues to be a significant public health burden on resource-limited populations. Previously, we produced three highly purified recombinant antigens (rLipL32, rLipL41, and rLigA-Rep) and evaluated their performance of detecting Leptospira-specific antibodies in enzyme-linked immunosorbent assay (ELISA) as compared with the microscopic agglutination test (MAT). The overall sensitivity of this assay approached 90%. Recently, another recombinant antigen (rLigB-Rep) was prepared. We tested each individual antigen and a 1:1:1:1 mixture of these four antigens for the detection of Leptospira-specific antibodies in ELISA. The performance of these recombinant antigens was evaluated with a much larger febrile patient panel (337 MAT-confirmed positive sera and 92 MAT-negative sera from febrile patients). Combining the detection results of immunoglobulin M and immunoglobulin G from these four individual antigens, the overall sensitivity was close to 90% but the specificity was only 66%, based on the MAT reference method. The overall sensitivity and specificity of the four-antigen mixture were 82% and 86%, respectively. The mixture of four antigens also exhibited a broader reactivity with MAT-positive samples of 18 serovars from six major pathogenic Leptospira species. Given the limitations of MAT, the data were further analyzed by Bayesian latent class model, showing that ELISA using a 1:1:1:1 mixture still maintained high sensitivity (79%) and specificity (88%) as compared with the sensitivity (90%) and specificity (83%) of MAT. Therefore, ELISA using a mixture of these four antigens could be a very useful test for seroprevalence studies.

Leptospira licerasiae serovar Varillal, a group II intermediate pathogen species/serovar discovered in the Peruvian Amazon city of Iquitos, is commonly recognized in this region by sera from humans (at least 40% seroprevalence) without a known clinical history of leptospirosis. This high frequency of human seroreactivity remains unexplained. To test the hypothesis that the oral route of infection might explain the high rate of human seroreactivity against L. licerasiae, an experimental infection model using Rattus norvegicus was developed, given that rats were one of the original reservoir hosts identified as being colonized by this leptospire. Sprague–Dawley rats were experimentally exposed via mucosa, direct gastric gavage, or parenteral inoculation with nine different isolates of L. licerasiae originally isolated from Peruvian humans, peridomiciliary rodents, and wildlife. As shown by quantitative polymerase chain reaction of kidney tissue, Leptospira infection via these routes of infection was equally successful. Importantly, the data show that L. licerasiae infects R. norvegicus via the oral route, leading to renal colonization. Not only do these findings confirm the infectiousness of group II Leptospira, but also they underscore the potential importance of oral as well as mucosal and transcutaneous routes of Leptospira infection.

We investigated the in vitro activity of a novel fifth-generation cephalosporin–tazobactam combination, ceftolozane–tazobactam against Burkholderia pseudomallei, the etiological agent of melioidosis. Using both disc diffusion and minimum inhibitory concentration (MIC) strip techniques against 56 clinical isolates and an national collection of type cultures (NCTC) strain, the MIC to ceftolozane–tazobactam was found to be between 0.75 and 4 mcg/mL. The MIC50 was found to be 1.5 mcg/mL and MIC90 was 2.0 mcg/mL. This study provides initial evidence of ceftolozane–tazobactam as a novel agent in the management of melioidosis.

Shigella is a major cause of severe diarrhea in children less than the age of 5 years in sub-Saharan Africa. The aim of this study was to describe the (sub-)serotype distribution and antimicrobial susceptibility of Shigella serogroups from Centrafrican patients with diarrhea between 2002 and 2013. We collected 443 Shigella isolates in total. The most common serogroups were Shigella flexneri (N = 243, 54.9%), followed by Shigella sonnei (N = 90, 20.3%) and Shigella dysenteriae (N = 72, 16.3%). The high diversity of (sub-)serotypes of S. flexneri and S. dysenteriae may impede the development of an efficient vaccine. Rates of resistance were high for ampicillin, chloramphenicol, tetracycline, and cotrimoxazole but low for many other antimicrobials, confirming recommendations for the use of third-generation cephalosporins (only one organism resistant) and fluoroquinolones (no resistance). However, the detection of one extended-spectrum beta-lactamase–producing Shigella organism highlights the need for continued monitoring of antimicrobial drug susceptibility.

Severe fever with thrombocytopenia syndrome (SFTS) is a tick-borne viral disease with a high mortality rate. Infection can also occur through close contact with an infected patient. Scrub typhus is an acute febrile illness caused by Orientia tsutsugamushi, a bacterium transmitted to humans through chigger mite bites. South Korea is an endemic region of SFTS and scrub typhus. In this study, we confirmed that a patient was coinfected with SFTS virus and two (Boryong and Taguchi) genotypes of O. tsutsugamushi.

Presently, few studies have investigated the role of domestic cats (Felis catus) in the recrudescence of flea-borne rickettsioses in California and the southern United States. In this study, we aimed to investigate the presence of Rickettsia typhi or Rickettisa felis in domestic cats (F. catus) and the fleas (primarily Ctenocephalides felis, the cat flea) associated with these cats in Riverside County, California. Thirty cats and 64 pools of fleas collected from these cats were investigated for rickettsial infections. Three cats and 17 flea pools (from 10 cats) tested positive for rickettsial infections. polymerase chain reaction and DNA sequencing indicated that one of the cats was positive for R. felis infections, whereas two were positive for Candidatus Rickettsia senegalensis infection. In addition, 12 of the flea pools were positive for R. felis, whereas five were positive for Ca. R. senegalensis. By contrast, no cats or their associated fleas tested positive for R. typhi. Finally, eight sera from these cats contained spotted fever group Rickettsia (SFGR) antibodies. The detection of R. felis and SFGR antibodies and the lack of R. typhi and TGR antibodies support R. felis as the main rickettsial species infecting cat fleas. The detection of Ca. R. senegalensis in both fleas and cats also provides additional evidence that cats and their associated fleas are infected with other R. felis–like organisms highlighting the potential risk for human infections with R. felis or R. felis–like organisms.

Ehrlichia spp. are emerging infectious pathogens, especially in the Americas. Although Ehrlichia canis is primarily a parasite of dogs, polymerase chain reaction-confirmed human infections have been reported from Mexico, Venezuela, and Costa Rica. This study reports the presence of E. canis DNA in 13.7% of 205 dogs from urban areas in Peru and of those, five were analyzed for phylogenetic variation using the Tandem Repeat Protein 36 (TRP36) gene. The use of the TRP36 gene for such analysis was validated against 16S rRNA and heat shock protein genes using Shannon’s entropy bioinformatic approach. When compared with other E. canis strains previously reported, three unique and novel E. canis strains were detected. In addition, the TRP36 amino acid tandem repeat sequences of the Peruvian strains share close similarity to an E. canis strain detected from four human blood bank samples in Costa Rica. This study reports for the first time domestic dogs infected with E. canis strains closely related to a zoonotic strain, which may be of public health concern as dogs can be chronically infected with this pathogen.

We report five cases of Q fever among cadets during a training program for Military Firefighters Academy in the state of Rio de Janeiro, Brazil. This cluster confirms the significance of Coxiella burnetii as an infectious agent in Brazil, where the occurrence of this zoonosis is poorly documented and highlights the potential risk for Q fever transmission in rural areas or farms with infected animals.

Murine typhus (MT) is an important cause of febrile illness in endemic areas, and there is an epidemiologic resurgence of this infection currently transpiring in Texas and California. Fatal cases and severe neurological complications are rare. A fatal case of MT in a middle-aged man is reported with a course culminating in multi-organ failure and refractory status epilepticus. An autopsy revealed hemorrhagic pneumonia, acute tubular necrosis, and ischemic necrosis in the liver, adrenals, and brain. We have also reviewed the neurologic complications of MT.

A quantifiable, stool-based, Mycobacterium tuberculosis (Mtb) test has potential complementary value to respiratory specimens. Limit of detection (LOD) was determined by spiking control stool. Clinical test performance was evaluated in a cohort with pulmonary tuberculosis (TB) (N = 166) and asymptomatic household TB child contacts (N = 105). Stool-quantitative polymerase chain reaction (qPCR) results were compared with sputum acid-fast bacilli (AFB) microscopy, GeneXpert MTB/RIF (Xpert MTB/RIF), and cultures. In Mtb stool-spiking studies, the LOD was 96 colony-forming units/50 mg of stool (95% confidence interval [CI]: 84.8–105.6). Among specimens collected within 72 hours of antituberculosis treatment (ATT) initiation, stool qPCR detected 22 of 23 (95%) of culture-positive cases. Among clinically diagnosed cases that were Xpert MTB/RIF and culture negative, stool qPCR detected an additional 8% (3/37). Among asymptomatic, recently TB-exposed participants, stool PCR detected Mtb in two of 105 (1.9%) patients. Two months after ATT, the Mtb quantitative burden in femtogram per microliters decreased (Wilcoxon signed-rank P < 0.001) and persistent positive stool PCR was associated with treatment failure or drug resistance (relative risk 2.8, CI: 1.2–6.5; P = 0.012). Stool-based qPCR is a promising complementary technique to sputum-based diagnosis. It detects and quantifies low levels of stool Mtb DNA, thereby supporting adjunct diagnosis and treatment monitoring in pulmonary TB.

Human immunodeficiency virus (HIV) infection is a major risk factor for the development of active tuberculosis (TB), one of the deadliest infectious diseases globally. The high mortality associated with the disease can be reduced by early diagnosis and prompt antituberculous treatment initiation. Facilities in TB-endemic regions are increasing the use of nucleic acid amplification (e.g., GeneXpert), which provides rapid results but may have suboptimal sensitivity in HIV-associated TB. Our objective was to evaluate the current practices for TB diagnosis at Edendale Hospital, a large regional hospital in KwaZulu-Natal, South Africa—a TB-endemic region with high HIV prevalence. In this cross-sectional study, all adult inpatients newly started on TB treatment at Edendale were identified over a 6-week period. Demographics, clinical information, diagnostic test results, and outcomes were documented. Pulmonary TB (PTB), extrapulmonary TB (EXTB), and PTB + EXTB were defined as disease evidence in the lungs, other organs, or both, respectively. Ninety-four cases were identified, of which 83% were HIV-associated. Only 30% of all TB patients were microbiologically confirmed, consisting of 7/16 (44%) HIV-uninfected and 21/78 (27%) HIV-infected TB patients. Smear microscopy and mycobacterial culture were seldom ordered. Ultrasound was performed in about one-third of suspected EXTB cases and was valuable in identifying abdominal TB. In this clinical setting with a high incidence of HIV-associated TB, TB diagnosis was more commonly based on clinical assessment and imaging results than on mycobacterial gold standard test confirmation.

Tetanus remains a significant burden in many low- and middle-income countries. The tetanus toxin acts within the central nervous system and intrathecal antitoxin administration may be beneficial, but there are safety concerns, especially in resource-limited settings. We performed a pilot study to assess the safety and feasibility of intrathecal human tetanus immunoglobulin in five adults with tetanus before the conduct of a large randomized controlled trial. Intrathecal injection via lumbar puncture was given to all patients within a median 140 (range 100–165) minutes of intensive care unit (ICU) admission. There were no serious adverse effects associated with the procedure although three patients had probably related minor adverse events which resolved spontaneously. Median ICU length of stay was 14 (range 5–17) days. Two patients required mechanical ventilation and one developed a deep vein thrombosis. Within 240 days of hospital discharge, no patients died and all patients returned to work.