**3. Application of the newly developed platform for diagnosing tropical diseases**

The developed platform has been applied to the following 3 tropical diseases to evaluate its performance as a practical diagnostic system.

#### **3.1 Malaria and human African trypanosomiasis**

Malaria is 1 of the 3 major infectious disease endemics in most tropical countries. More than 500 million people have been infected, and more than 1 million people die from malaria each year, mostly infants and pregnant women. Of the 4 malaria causing species, *Plasmodium falciparum* often causes severe, acute, and fatal malaria. In most developing countries, malaria is confirmed mainly by a blood smear test, although the sensitivity of the test is not sufficient to detect the parasites in patients with early-stage malaria.

Dried LAMP reagents using *P. falciparum* (Pf)-specific primers and pan genus (Pg) primers were developed in this study. The Pg LAMP primers were designed on the basis of the homogeneous sequence shared by all the 4 malaria species, thus providing the same primer specificity for all the 4 species. The Pf-specific LAMP primers were designed based on mutations between the Pf sequence and the other 3 sequences, making the primer specific only to *P. falciparum*. If both Pf and Pg LAMP assays give positive results, it can be interpreted that the patient is infected by *P. falciparum*. On the other hand, if only the Pg LAMP assay gives positive results, the patient can be diagnosed with malaria caused by 1 or more of the other 3 malarial parasites.

The sensitivity of PURE and malaria-LAMP have been evaluated by using of cultured *P. falciparum* parasites obtained from the American Type Culture Collection (ATCC). As shown in figure 5, both Pf and Pg malaria-LAMP assays can detect down to 1 parasite in 1 μl blood, processed by the PURE method. This sensitivity of 1 parasite/μl blood is much higher than that of smear microscopy test (~50 parasites/μl blood for routine tests in an endemic area (Moody, 2002)). It has been reported that the initial malarial symptoms appear after the accumulation of approximately 1,000 parasites/ml of blood (Andrews et al., 2005). Therefore, PURE-malaria-LAMP is sensitive enough to detect parasites in patients who present with the initial symptoms of malaria.

Human African trypanosomiasis (HAT) is one of the most neglected disease endemics in central African countries (Hotez, 2007). HAT is caused by an infection of protozoa

Novel Molecular Diagnostic Platform for Tropical Infectious Diseases 453

To enhance sensitivity, the LAMP primers for HAT are designed on a multi-copy gene named RIME, which contains 80–250 copies in a parasite genome (Njiru et al., 2008). The efficacy of the PURE-HAT-LAMP assay was also evaluated by using uninfected control blood spike with cultured parasites. Positive results have been successfully obtained from samples with a parasite density of 100/ml, indicating that the overall analytical sensitivity of PURE-HAT-LAMP can be estimated to be approximately 100 parasites/ml blood. The sensitivities of the currently available smear microscopy tests have been reported to be between 100 and 10,000 parasites/ml blood (Chappuis., 2005). The sensitivity of PURE-HAT-LAMP has been found to be over 10 times higher than that of the simple Giemsastained smear microscopy test, which is one of the most common diagnostic tests in HAT endemic areas, and is almost comparable with that of the mini-anion-exchange

Tuberculosis (TB) is one of the most threatening airborne diseases worldwide. One-third of the world's population is thought to be infected with *Mycobacterium tuberculosis* and new infections occur at a rate of about 1/second. Since the TB patients are concentrated in developing counties, including many tropical countries, TB is considered as a major

The sputum direct smear microscopy test is the only diagnostic method available for the detection of TB at peripheral laboratories in developing countries (Keeler et al., 2006). Because of the low sensitivity of the smear test, few patients with a low number of infected TB cells are often misdiagnosed as negative, thus preventing eradication of TB. In order to overcome this situation, we attempted to apply the novel platform PURE-LAMP system for

As summarized in Table 1, PURE-TB-LAMP provided positive results for both smear- and culture-positive sputum samples collected from patients suspected with TB. Furthermore,

Negative Negative 9/91 9.9

Sixty microliters of sputa obtained from patients suspected with TB in the Pham Ngoc Thach Hospital (PNTH; Vietnam) were analyzed by PURE-TB-LAMP. The performance of PURE-TB-LAMP was compared to those of the direct smear and culture method (Ogawa media) in terms of the positive ratios. Smear and culture tests for each sample were

15/25 60

34/34 100

**% positive**

100-200 7/7 100 20-99 6/8 75 1-19 2/10 20

**PURE-TB-LAMP (Positive/Total)**

centrifugation technique, which is quite tedious and time consuming.

**Culture(Ogawa)**

Positive Positive

Colony Counts

Table 1. Summary of clinical performance of PURE-TB-LAMP

conducted according to the standardized protocols in PNTH.

**3.2 Tuberculosis** 

the diagnosis of TB.

Negative

Positive

**Direct Smear** 

poverty-related disease (Walker et al., 2003).

*Trypanosoma brucei gambiense* or *Trypanosoma brucei rhodesiense* transmitted by tse-tse flies. The symptoms of HAT occur in 2 phases: hemolymphatic phase, followed by the neurological phase. If left untreated, the hemolymphatic phase permits the parasites to invade the central nervous system of the patient, resulting in fatal neurological symptoms such as coma and eventually death. Because no vaccine or preventative drug for HAT is available, and therapeutic drugs for HAT patients in the neurological phase causes severe side effects, a simple and sensitive diagnostic method is in high demand.

Fig. 4. Diagram of the procedures involved in the PURE–LAMP system

Thirty-five microliters of control blood spiked with cultured parasites (*P. falciparum*, from ATCC) was treated with PURE and tested by *P. falciparum*-specific and Pan-genus LAMP. Left and right tubes contain the same parasite numbers and are those of Pf-LAMP and Pg-LAMP, respectively.

To enhance sensitivity, the LAMP primers for HAT are designed on a multi-copy gene named RIME, which contains 80–250 copies in a parasite genome (Njiru et al., 2008). The efficacy of the PURE-HAT-LAMP assay was also evaluated by using uninfected control blood spike with cultured parasites. Positive results have been successfully obtained from samples with a parasite density of 100/ml, indicating that the overall analytical sensitivity of PURE-HAT-LAMP can be estimated to be approximately 100 parasites/ml blood. The sensitivities of the currently available smear microscopy tests have been reported to be between 100 and 10,000 parasites/ml blood (Chappuis., 2005). The sensitivity of PURE-HAT-LAMP has been found to be over 10 times higher than that of the simple Giemsastained smear microscopy test, which is one of the most common diagnostic tests in HAT endemic areas, and is almost comparable with that of the mini-anion-exchange centrifugation technique, which is quite tedious and time consuming.
