Non-albicans Candida species are emerging in the nosocomial environment, with the multidrug-resistant (MDR) species Candida auris being the most notorious example. Consequently, rapid and accurate species identification has become essential. The objective of this study was to evaluate five commercially available chromogenic media for the presumptive identification of C. auris Two novel chromogenic formulations, CHROMagar Candida Plus (CHROMagar) and HiCrome C. auris MDR selective agar (HiMedia), and three reference media, CandiSelect (Bio-Rad), CHROMagar Candida (CHROMagar), and Chromatic Candida (Liofilchem), were inoculated with a collection of 9 genetically diverse C. auris strains and 35 strains from closely related comparator species. After 48 h of incubation, the media were evaluated for their ability to detect and identify C. auris All media had the same limitations in the differentiation of the more common species Candida dubliniensis and Candida glabrata Only on CHROMagar Candida Plus did C. auris colonies develop a species-specific coloration. Nevertheless, the closely related pathogenic species Candida pseudohaemulonii and Candida vulturna developed a similar appearance as C. auris on this medium. CHROMagar Candida Plus was shown to be superior in the detection and identification of C. auris, with 100% inclusivity for C. auris compared to 0% and 33% for the reference media and HiCrome C. auris MDR selective agar, respectively. Although C. vulturna and C. pseudohaemulonii can cause false positives, CHROMagar Candida Plus was shown to be a valuable addition to the plethora of mostly molecular methods for C. auris detection and identification.
HIV viral load (VL) testing is the recommended method for monitoring the response of people living with HIV and receiving antiretroviral therapy (ART). The availability of standard plasma VL testing in low- and middle-income countries (LMICs), and access to this testing, are limited by the need to use fresh plasma. Good specimen collection methods for HIV VL testing that are applicable to resource-constrained settings are needed. We assessed the diagnostic performance of the filtered dried plasma spot (FDPS), created using the newly developed, instrument-free VLPlasma device, in identifying treatment failure at a VL threshold of 1,000 copies/ml in fresh plasma. Performance was compared with that of the conventional dried blood spot (DBS). Venous blood samples from 201 people living with HIV and attending an infectious disease clinic in Malaysia were collected, and HIV VL was quantified using fresh plasma (the reference standard), FDPS, and DBS specimens. VL testing was done using the Roche Cobas AmpliPrep/Cobas TaqMan v2.0 assay. At a threshold of 1,000 copies/ml, the diagnostic performance of the FDPS was superior (sensitivity, 100% [95% confidence interval {CI}, 89.1 to 100%]; specificity, 100% [95% CI, 97.8 to 100%]) to that of the DBS (sensitivity, 100% [95% CI, 89.4 to 100%]; specificity, 36.8% [95% CI, 29.4 to 44.7%]) (P
This study demonstrates that the clinical sensitivity, specificity, and reproducibility of the novel cobas human papillomavirus (HPV) test on the cobas 6800 system for high-risk HPV types fulfills the criteria for use in population-based cervical screening. The criteria were formulated by an international consortium, using the cobas 4800 HPV test as a validated reference assay. The cobas HPV test detected over 98% of histologically confirmed cervical intraepithelial neoplasia grade 2+ (CIN2+) lesions in women age 30 years or older, with a specificity of 98.9% compared with the reference cobas 4800 test. Both the intra- and interlaboratory agreement for the cobas HPV test were 98%. The clinical performance of the cobas HPV test is comparable to those of longitudinally validated HPV assays and fulfills the criteria for its use in primary cervical screening.
Accumulating evidence shows a high prevalence of Clostridium difficile in Southeast Asia associated with a range of clinical presentations. However, severe infections are rarely reported. We investigated C. difficile infection (CDI) across four hospitals in Kuala Lumpur and Kota Bharu, Malaysia. Enzyme immunoassays for glutamate dehydrogenase (GDH) and toxin A or B were performed on diarrheal stool specimens collected from patients in 2015 and 2016. Specimens were also cultured and isolates of C. difficile characterized by PCR ribotyping and detection of toxin genes. In total, 437 specimens were collected and fecal toxin was detected in 3.0%. A further 16.2% of specimens were GDH positive and toxin negative. After culture, toxigenic strains were isolated from 10.3% and nontoxigenic strains from 12.4% of specimens. The most prevalent PCR ribotypes (RTs) were RT 017 (20.0%) and RT 043 (10.0%). The high prevalence of RT 017 and nontoxigenic strains in Malaysia and in neighboring Thailand and Indonesia suggests that they localize to the region of Southeast Asia, with an implication that they may mediate the burden of CDI in the region.
Nipah virus, a paramyxovirus related to Hendra virus, first emerged in Malaysia in 1998. Clinical presentation ranges from asymptomatic infection to fatal encephalitis. Malaysia has had no more cases since 1999, but outbreaks continue to occur in Bangladesh and India. In the Malaysia-Singapore outbreak, transmission occurred primarily through contact with pigs, whereas in Bangladesh and India, it is associated with ingestion of contaminated date palm sap and human-to-human transmission. Bats are the main reservoir for this virus, which can cause disease in humans and animals. There are currently no effective therapeutics, and supportive care and prevention are the mainstays of management.
Acanthamoeba spp. and Balamuthia mandrillaris are causative agents of granulomatous amoebic encephalitis (GAE), while Naegleria fowleri causes primary amoebic meningoencephalitis (PAM). PAM is an acute infection that lasts a few days, while GAE is a chronic to subacute infection that can last up to several months. Here, we present a literature review of 86 case reports from 1968 to 2016, in order to explore the affinity of these amoebae for particular sites of the brain, diagnostic modalities, treatment options, and disease outcomes in a comparative manner.
Treponema pallidum infections can have severe complications if not diagnosed and treated at an early stage. Screening and diagnosis of syphilis require assays with high specificity and sensitivity. The Elecsys Syphilis assay is an automated treponemal immunoassay for the detection of antibodies against T. pallidum The performance of this assay was investigated previously in a multicenter study. The current study expands on that evaluation in a variety of diagnostic settings and patient populations, at seven independent laboratories. The samples included routine diagnostic samples, blood donation samples, samples from patients with confirmed HIV infections, samples from living organ or bone marrow donors, and banked samples, including samples previously confirmed as syphilis positive. This study also investigated the seroconversion sensitivity of the assay. With a total of 1,965 syphilis-negative routine diagnostic samples and 5,792 syphilis-negative samples collected from blood donations, the Elecsys Syphilis assay had specificity values of 99.85% and 99.86%, respectively. With 333 samples previously identified as syphilis positive, the sensitivity was 100% regardless of disease stage. The assay also showed 100% sensitivity and specificity with samples from 69 patients coinfected with HIV. The Elecsys Syphilis assay detected infection in the same bleed or earlier, compared with comparator assays, in a set of sequential samples from a patient with primary syphilis. In archived serial blood samples collected from 14 patients with direct diagnoses of primary syphilis, the Elecsys Syphilis assay detected T. pallidum antibodies for 3 patients for whom antibodies were not detected with the Architect Syphilis TP assay, indicating a trend for earlier detection of infection, which may have the potential to shorten the time between infection and reactive screening test results.
Mycobacterium abscessus subspecies classification has important clinical implications. We used phylogenomic network and amino acid analyses to provide evidence for the separation of Mycobacterium bolletii and Mycobacterium massiliense into two distinct subspecies which can potentially be differentiated rapidly by their protein signatures.
Hepatitis B virus (HBV) has been divided into 10 genotypes, A to J, based on an 8% nucleotide sequence divergence between genotypes. The conventional practice of using a single set of primers to amplify a near-complete HBV genome is hampered by its low analytical sensitivity. The current practice of using overlapping conserved primer sets to amplify a complete HBV genome in a clinical sample is limited by the lack of pan-primers to detect all HBV genotypes. In this study, we designed six highly conserved, overlapping primer sets to cover the complete HBV genome. We based our design on the sequences of 5,154 HBV genomes of genotypes A to I downloaded from the GenBank nucleotide database. These primer sets were tested on 126 plasma samples from Malaysia, containing genotypes A to D and with viral loads ranging from 20 to >79,780,000 IU/ml. The overall success rates for PCR amplification and sequencing were >96% and >94%, respectively. Similarly, there was 100% amplification and sequencing success when the primer sets were tested on an HBV reference panel of genotypes A to G. Thus, we have established primer sets that gave a high analytical sensitivity for PCR-based detection of HBV and a high rate of sequencing success for HBV genomes in most of the viral genotypes, if not all, without prior known sequence data for the particular genotype/genome.
A method for the rapid diagnosis of early dengue virus (DENV) infection is highly needed. Here, a prototype reverse transcription-recombinase polymerase amplification (RT-RPA) assay was developed. The assay detected DENV RNA in <20 min without the need for thermocycling amplification. The assay enabled the detection of as few as 10 copies of DENV RNA. The designed RT-RPA primers and exo probe detected the DENV genome of at least 12 genotypes of DENV circulating globally without cross-reacting with other arboviruses. We assessed the diagnostic performance of the RT-RPA assay for the detection of DENV RNA in 203 serum samples of patients with clinically suspected dengue. The sera were simultaneously tested for DENV using a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay, quantitative RT-PCR (qRT-PCR), and IgM- and IgG-capture enzyme-linked immunosorbent assays (ELISA). Acute DENV infection was confirmed in 130 samples and 61 of the samples (46.9%) were classified as viremic with qRT-PCR. The RT-RPA assay showed good concordance (κ of ≥0.723) with the RT-LAMP and qRT-PCR assays in detecting the dengue viremic samples. When used in combination with ELISA, both the RT-RPA and RT-LAMP assays increased the detection of acute DENV infection to ≥95.7% (≥45/47) in samples obtained within 5 days of illness. The results from the study suggest that the RT-RPA assay is the most rapid molecular diagnostic tool available for the detection of DENV. Hence, it is possible to use the RT-RPA assay in a laboratory to complement routine serology testing for dengue.
Human papillomavirus (HPV) is causally associated with anal cancer, as HPV DNA is detected in up to 90% of anal intraepithelial neoplasias and anal cancers. With the gradual increase of anal cancer rates, there is a growing need to establish reliable and clinically relevant methods to detect anal cancer precursors. In resource-limited settings, HPV DNA detection is a potentially relevant tool for anal cancer screening. Here, we evaluated the performance of the Hybribio GenoArray (GA) for genotyping HPV in anal samples, against the reference standard Roche Linear Array (LA). Anal swab samples were obtained from sexually active men who have sex with men. Following DNA extraction, each sample was genotyped using GA and LA. The overall interassay agreement, type-specific, and single and multiple genotype agreements were evaluated by kappa statistics and McNemar's χ(2) tests. Using GA and LA, 68% and 76% of samples were HPV DNA positive, respectively. There was substantial interassay agreements for the detection of all HPV genotypes (κ = 0.70, 86% agreement). Although LA was able to detect more genotypes per sample, the interassay agreement was acceptable (κ = 0.53, 63% agreement). GA had poorer specific detection of HPV genotypes 35, 42, and 51 (κ < 0.60). In conclusion, GA and LA showed good interassay agreement for the detection of most HPV genotypes in anal samples. However, the detection of HPV DNA in up to 76% of anal samples warrants further evaluation of its clinical significance.
We report a rare and unusual case of invasive Enterobius vermicularis infection in a fallopian tube. The patient was a 23-year-old Malaysian woman who presented with suprapubic pain and vaginal bleeding. A clinical diagnosis of ruptured right ovarian ectopic pregnancy was made. She underwent a laparotomy with a right salpingo-oophorectomy. Histopathological examination of the right fallopian tube showed eggs and adult remnants of E. vermicularis, and the results were confirmed using PCR and DNA sequencing.
Diarrheal diseases cause illness and death among children younger than 10 years in developing countries. Conventional testing for the detection of hemorrhagic bacteria takes 2 to 5 days to yield complete information on the organism and its antibiotic sensitivity pattern. Hence, in the present study, we developed a molecular-based diagnostic assay that identifies common hemorrhagic bacteria in stool samples. A set of specific primers were designed for the detection of Salmonella spp., Shigella spp., enterohemorrhagic Escherichia coli (EHEC), and Campylobacter spp., suitable for use in a one-tube PCR assay. The assay in the present study simultaneously detected five genes, namely, ompC for the Salmonella genus, virA for the Shigella genus, eaeA for EHEC, 16S rRNA for the Campylobacter genus, and hemA for an internal control. Specific primer pairs were successfully designed and simultaneously amplified the targeted genes. Validation with 20 Gram-negative and 17 Gram-positive strains yielded 100% specificity. The limit of detection of the multiplex PCR assay was 1 × 10(3) CFU at the bacterial cell level and 100 pg at the genomic DNA level. Further evaluation of the multiplex PCR with 223 bacterium-spiked stool specimens revealed 100% sensitivity and specificity. We conclude that the developed multiplex PCR assay was rapid, giving results within 4 h, which is essential for the identification of hemorrhagic bacteria, and it might be useful as an additional diagnostic tool whenever time is important in the diagnosis of hemorrhagic bacteria that cause diarrhea. In addition, the presence of an internal control in the multiplex PCR assay is important for excluding false-negative cases.
Plasmodium knowlesi causes severe and fatal malaria in Malaysia. Microscopic misdiagnosis is common and may delay appropriate treatment. P. knowlesi can cross-react with "species-specific" parasite lactate dehydrogenase (pLDH) monoclonal antibodies used in rapid diagnostic tests (RDTs) to detect P. falciparum and P. vivax. At one tertiary-care hospital and two district hospitals in Sabah, we prospectively evaluated two combination RDTs for malaria diagnosis by using both a pan-Plasmodium-pLDH (pan-pLDH)/P. falciparum-specific-pLDH (Pf-pLDH) RDT (OptiMAL-IT) and a non-P. falciparum VOM-pLDH/Pf-HRP2 RDT (CareStart). Differential cross-reactivity among these combinations was hypothesized to differentiate P. knowlesi from other Plasmodium monoinfections. Among 323 patients with PCR-confirmed P. knowlesi (n = 193), P. falciparum (n = 93), and P. vivax (n = 37) monoinfections, the VOM-pLDH individual component had the highest sensitivity for nonsevere (35%; 95% confidence interval [CI], 27 to 43%) and severe (92%; CI, 81 to 100%) P. knowlesi malaria. CareStart demonstrated a P. knowlesi sensitivity of 42% (CI, 34 to 49%) and specificity of 74% (CI, 65 to 82%), a P. vivax sensitivity of 83% (CI, 66 to 93%) and specificity of 71% (CI, 65 to 76%), and a P. falciparum sensitivity of 97% (CI, 90 to 99%) and specificity of 99% (CI, 97 to 100%). OptiMAL-IT demonstrated a P. knowlesi sensitivity of 32% (CI, 25 to 39%) and specificity of 21% (CI, 15 to 29%), a P. vivax sensitivity of 60% (CI, 42 to 75%) and specificity of 97% (CI, 94 to 99%), and a P. falciparum sensitivity of 82% (CI, 72 to 89%) and specificity of 39% (CI, 33 to 46%). The combination of CareStart plus OptiMAL-IT for P. knowlesi using predefined criteria gave a sensitivity of 25% (CI, 19 to 32%) and specificity of 97% (CI, 92 to 99%). Combining two RDT combinations was highly specific for P. knowlesi malaria diagnosis; however, sensitivity was poor. The specificity of pLDH RDTs was decreased for P. vivax and P. falciparum because of P. knowlesi cross-reactivity and cautions against their use alone in areas where P. knowlesi malaria is endemic. Sensitive P. knowlesi-specific RDTs and/or alternative molecular diagnostic tools are needed in areas where P. knowlesi malaria is endemic.
Misidentifications of Burkholderia pseudomallei as Burkholderia cepacia by Vitek 2 have occurred. Multidimensional scaling ordination of biochemical profiles of 217 Malaysian and Australian B. pseudomallei isolates found clustering of misidentified B. pseudomallei isolates from Malaysian Borneo. Specificity of B. pseudomallei identification in Vitek 2 and potentially other automated identification systems is regionally dependent.
Plasmodium knowlesi can cause severe and fatal human malaria in Southeast Asia. Rapid diagnosis of all Plasmodium species is essential for initiation of effective treatment. Rapid diagnostic tests (RDTs) are sensitive for detection of uncomplicated and severe falciparum malaria but have not been systematically evaluated in knowlesi malaria. At a tertiary referral hospital in Sabah, Malaysia, we prospectively evaluated the sensitivity of two combination RDTs for the diagnosis of uncomplicated and severe malaria from all three potentially fatal Plasmodium species, using a pan-Plasmodium lactate dehydrogenase (pLDH)-P. falciparum histidine-rich protein 2 (PfHRP2) RDT (First Response) and a pan-Plasmodium aldolase-PfHRP2 RDT (ParaHIT). Among 293 hospitalized adults with PCR-confirmed Plasmodium monoinfection, the sensitivity of the pLDH component of the pLDH-PfHRP2 RDT was 74% (95/129; 95% confidence interval [CI], 65 to 80%), 91% (110/121; 95% CI, 84 to 95%), and 95% (41/43; 95% CI, 85 to 99%) for PCR-confirmed P. knowlesi, P. falciparum, and P. vivax infections, respectively, and 88% (30/34; 95% CI, 73 to 95%), 90% (38/42; 95% CI, 78 to 96%), and 100% (12/12; 95% CI, 76 to 100%) among patients tested before antimalarial treatment was begun. Sensitivity in severe malaria was 95% (36/38; 95% CI, 83 to 99), 100% (13/13; 95% CI, 77 to 100), and 100% (7/7; 95% CI, 65 to 100%), respectively. The aldolase component of the aldolase-PfHRP2 RDT performed poorly in all Plasmodium species. The pLDH-based RDT was highly sensitive for the diagnosis of severe malaria from all species; however, neither the pLDH- nor aldolase-based RDT demonstrated sufficiently high overall sensitivity for P. knowlesi. More sensitive RDTs are needed in regions of P. knowlesi endemicity.