A conventional multiplex PCR for the detection of four common soil-transmitted nematodes in human feces: development and validation

@#Soil-transmitted helminth (STH) infections, mainly caused by Ascaris lumbricoides, Trichuris trichiura, and hookworms, are among the most common intestinal parasites that infect humans. The infections are widely distributed throughout tropical and subtropical countries, including Malaysia, particularly in underprivileged communities. Microscopic and culture techniques have been used as a gold standard for diagnostic techniques. However, these methods yield low sensitivity and specificity, laborious and time-consuming. Therefore, simple, rapid, and accurate alternative methods are needed for the simultaneous detection of STH infections. Although advanced technologies such as real-time multiplex PCR have been established, the use of this technique as a routine diagnostic is limited due to the high cost of the instrument. Therefore, a single-round multiplex conventional PCR assay for rapid detection of four STH species in the fecal sample was developed in this study. To perform the single-round multiplex PCR, each pair of species-specific primers was selected from target genes, including Ancylostoma duodenale (Internal Transcribed Spacer 2; accession No. AJ001594; 156 base pair), Necator americanus (ITS 2; accession No. AJ001599; 225 base pair), Ascaris lumbricoides (Internal Transcribed Spacer 1; accession No. AJ000895; 334 base pair) and Trichuris triciura (partial ITS 1, 5.8s rRNA and partial ITS 2; accession No. AM992981; 518 base pair). The results showed that the newly designed primers could detect the DNA of STH at low concentrations (0.001 ng/μl) with no cross-amplification with other species. This assay enables the differentiation of single infections as well as mixed infections. It could be used as an alternative and is a convenient method for the detection of STHs, especially for the differentiation of N. americanus and A. duodenale.

Clinical analysis of foreign-born patients with tuberculosis found in Malaysia.

In this retrospective study, we investigated 263 foreign patients who were diagnosed as having tuberculosis at the National Tuberculosis Center (NTBC) from January 2001 to December 2002. The age range was 14-72 years, with a mean of 33.3 +/- 9.95 years. The study subjects were predominantly males (60%) and females comprised 40%, where the greater impact of tuberculosis was observed in the young and active ones (up to 34 years of age), than middle-age (up to 54). A significantly higher percentage of these patients were from the Southeast Asian countries (87%) and particularly occurred in single male (47.5%) and married female (71.4%) patients (p<0.05). We also found that tuberculosis was significantly higher in female (50.5%) and male (64%) with smoking laborers (p<0.05). Fever (70%), cough (90.5%) and BCG vaccination status showed a significantly higher percentage in male patients (p<0.05), whereas lymphadenopathy (22%) was found in a significantly higher percentage in females (p<0.05). Overall, pulmonary disease (94.3%) occurred more commonly in males and the pleura (3.2%) was the most common site of disseminated tuberculosis. By contrast, the lymph node (11.4%) and miliary (4.8%) forms were the more common extrapulmonary tuberculosis in females. More males had higher percentage of treatment completed at > or = 6 (38%) and > or = 9 (13.3%) months in pulmonary tuberculosis, whilst, more females showed higher percentage of treatment completed (8.7%) in extrapulmonary tuberculosis. Surprisingly, more women showed non-compliance to the anti-tubercular therapy than their counterpart in this study.

Polypeptides associated with in vitro cyst formation of Blastocystis hominis.

The objective of this study was to characterize the polypeptides associated with cysts of Blastocystis hominis. This form is believed to be infective and plays a role in parasite resistance to anti-B. hominis drugs currently used for treatment of Blastocystis associated diarrhea. Cysts were induced through in vitro culture of the parasite in complete medium supplemented with bacterial extract with trypticase, metronidazole or doxycycline. SDS-PAGE analysis showed almost similar polypeptide patterns of parasite extracts obtained from in vitro cultured parasites before and after exposure with the three supplements. Polypeptide bands at 76, 58.5, 48, 45, 40, 38, 32, 25 and 22 kDa were constantly seen in all antigenic preparations and no specific cyst-associated polypeptide was present. However, on immunoblot analysis, 3 out of 16 blastocystosis human sera identified a cyst-associated polypeptide at 60 kDa in all parasite extracts prepared from cultures with the three supplements. In addition, there were associated morphological changes detected in these parasites stained with acridine orange and observed under fluorescence microscopy. Metronidazole induced cyst forms (reddish cells) as early as 12 hours post-exposure; more cyst production (with stronger immunoblot bands) occurred after 24 hours exposure. However, cysts rupture with release and destruction of B. hominis daughters cells occurred after 48 hours exposure. Doxycycline induced less cyst-like forms at 24 hours (weaker 60 kDa band) and less destruction of the cysts (60 kDa band still present at 72 hours post exposure). Bacterial extract and trypticase also induced cysts at 12 hours with increasing numbers up to 72 hours exposure (corresponding increase in intensity of 60 kDa band from samples harvested at 12 to 72 hours post exposure) without any sign of deleterious effect on the parasite.